EVOLUTION OF ACYLSUGARS WITHIN THE SOLANUM GENUS By Paul D. Fiesel A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Biochemistry and Molecular Biology – Doctor of Philosophy 2023 ABSTRACT Plants synthesize a remarkable number of lineage- and tissue-specific specialized metabolites. These compounds exhibit diverse functions for plants, e.g., communication and defense, as well as for humans, e.g., medicine and food. The anti-insect and anti-microbial acylsugars are one class of specialized metabolites and accumulate in Solanaceae species. Despite being composed of the simple building blocks of sugar cores and acyl chains, acylsugars exhibit incredible structural diversity. This variation was previously demonstrated to impact plant pest mortality and oviposition. These factors suggest that characterizing the acylsugar diversity within Solanaceae species and understanding their biosynthesis can uncover how a biologically relevant trait has evolved. While acylsucroses are the most well-characterized acylsugar type, unusual acylinositols were characterized in three species of the large and megadiverse Solanum genus. In this study, the diversity and distribution of Solanum genus acylinositols were characterized and their biosynthetic pathway was investigated. I first characterized the trichome acylsugars of Clade II species Solanum melongena (brinjal eggplant) using liquid chromatography-mass spectrometry (LC-MS), gas chromatography (GC)-MS and nuclear magnetic resonance (NMR) spectroscopy, identifying eight unusual structures with inositol cores, inositol glycoside cores, and hydroxyacyl chains. LC-MS analysis of 31 Solanum DulMo clade, VANAns clade, and Clade II species revealed striking acylsugar diversity with some traits restricted to specific clades and species. Acylinositols were found in all three major clades while acylglucoses were restricted to the DulMo and VANAns species characterized. Unusual disaccharide sugar cores and medium- length hydroxyacyl chains were found to be widespread within the surveyed species. This investigation revealed inositol sugar cores as a predominant sugar core type and prompted an investigation into their biosynthesis. Utilizing an eggplant tissue-specific transcriptome and in vitro biochemistry, an acetyltransferase ACYLSUGAR ACYLTRANSFERASE 3-LIKE 1 (SmASAT3-L1) was characterized to act upon a triacylinositol glycoside. Analysis of S. melongena triacylinositol biosynthesis uncovered an in vitro pathway producing a triacylinositol identical to a plant triacylinositol, however, production of the correct products only occurred when accompanied by nonenzymatic acyl chain rearrangement. Using this pathway knowledge and previously developed transcriptomes and gene silencing methods, I determined that two other acylinositol-producing species, Solanum quitoense and Solanum nigrum, contain an analogous acylinositol biosynthetic pathway. These results support the hypothesis that there is a conserved pathway within two major Solanum clades, DulMo and Clade II, which evolved in part due to gene duplications and altered substrate specificity. This study not only highlights the enormous amount of plant chemical diversity but also the usefulness of comparative biochemistry to uncover evolutionary mechanisms underlying metabolic novelty. ACKNOWLEDGEMENTS This achievement and the work described in this dissertation would not have been possible without the guidance and patience from several people in my life. Of these, I first want to thank my advisor Dr. Robert Last for training me in his laboratory. He pushed me to be a better researcher, writer, and thinker. I am ever grateful for his training and the opportunity to do my specific project. I would also like to thank my committee members Dr. Patrick Edger, Dr. Michaela TerAvest, Dr. Daniel Jones, and Dr. Erich Grotewold. Their indispensable advice made my projects feasible, and their comments pushed me to look at my research in different ways. I am very fortunate to have had these mentors. I am immensely grateful for the guidance provided by former and current members of the Last Lab and the Solanaceae Specialized Metabolism Project. Many discussions during lunch, lab meetings, and over coffee guided my project and enabled the development of my molecular biology and biochemistry technical skills and knowledge. I will not try to name everyone who has helped but Dr. Rachel Kerwin, Dr. Bryan Leong, and Dr. Yann-Ru Lou were huge sources of help in my projects and this work could not have been done without it. Many people helped lead me to graduate school and to consider a career in science and analytical chemistry. Two of these were Dr. Jerry Cohen and Dr. Molly Tillmann which I was fortunate to be mentored by at the University of Minnesota. Under their patient guidance, I grew to love using analytical chemistry to understand plant biology. The skills gained in the Cohen lab were instrumental in the work presented here. Finally, I want to thank my family and friends for their support. The work presented here required a lot of time, and I thank my family and friends for their support and encouragement iv despite my time away from them. The support from my friends at Michigan State truly made my time in Michigan worthwhile. I am especially grateful for the support from my wife, Mariah, who moved across the country for me, made me take needed breaks, and supplemented my graduate stipend. All the support from those listed above really made this work possible. v TABLE OF CONTENTS CHAPTER 1: FRUITY, STICKY, STINKY, SPICY, BITTER, ADDICTIVE, AND DEADLY: EVOLUTIONARY SIGNATURES OF METABOLIC COMPLEXITY IN THE SOLANACEAE ...............................................................................................................................1 Abstract ........................................................................................................................................2 The Solanaceae: a phylogenetic framework for exploring metabolism .......................................3 Fruity: GWAS-enabled discovery of aroma variation during ripening ........................................8 Sticky: single-cell biochemical genetics reveals acylsugar metabolic complexity ....................12 Stinky: variations on a theme define terpene diversity across Solanum ....................................23 Spicy: lineage-specific biosynthesis of capsaicinoids in pepper ................................................29 Bitter: evolutionary signatures of glycoalkaloid biosynthesis in Solanum ................................31 Addictive and deadly: convergent and divergent evolution shapes nicotine and tropane alkaloid metabolism ...................................................................................................................38 Challenges and unexplored frontiers in Solanaceae metabolism ...............................................47 Conclusions ................................................................................................................................57 REFERENCES ..........................................................................................................................58 CHAPTER 2: TRADING ACYLS AND SWAPPING SUGARS: METABOLIC INNOVATIONS IN SOLANUM TRICHOMES ...........................................................................77 Abstract ......................................................................................................................................78 Introduction ................................................................................................................................79 Results and Discussion ...............................................................................................................82 Methods ....................................................................................................................................112 Acknowledgements ..................................................................................................................130 REFERENCES .........................................................................................................................132 APPENDIX ..............................................................................................................................142 CHAPTER 3: ACYLINOSITOL BIOSYNTHESIS WITHIN SOLANUM GLANDULAR TRICHOMES ..............................................................................................................................305 Abstract ....................................................................................................................................306 Introduction ..............................................................................................................................307 Results ......................................................................................................................................309 Discussion ................................................................................................................................319 Methods ....................................................................................................................................323 Acknowledgements ..................................................................................................................329 REFERENCES .........................................................................................................................331 APPENDIX .............................................................................................................................334 CHAPTER 4: CONCLUSIONS AND FUTURE DIRECTIONS ...............................................345 REFERENCES ........................................................................................................................350 vi CHAPTER 1: FRUITY, STICKY, STINKY, SPICY, BITTER, ADDICTIVE, AND DEADLY: EVOLUTIONARY SIGNATURES OF METABOLIC COMPLEXITY IN THE SOLANACEAE Works presented in this chapter have been published and are reproduced with permission from the Royal Society of Chemistry: *Fiesel, P. D., *Parks, H. M., Last, R. L., and Barry, C. S. (2022). Fruity, sticky, stinky, spicy, bitter, addictive, and deadly: evolutionary signatures of metabolic complexity in the Solanaceae. Nat. Prod. Rep., 2022, 39, 1438-1464. DOI: 10.1039/D2NP00003B *These authors contributed equally. 1 Abstract Plants collectively synthesize a huge repertoire of metabolites. General metabolites, also referred to as primary metabolites, are conserved across the plant kingdom and are required for processes essential to growth and development. These include amino acids, sugars, lipids, and organic acids. In contrast, specialized metabolites, historically termed secondary metabolites, are structurally diverse, exhibit lineage-specific distribution and provide selective advantage to host species to facilitate reproduction and environmental adaptation. Due to their potent bioactivities, plant specialized metabolites attract considerable attention for use as flavorings, fragrances, pharmaceuticals, and bio-pesticides. The Solanaceae (Nightshade family) consists of approximately 2700 species and includes crops of significant economic, cultural, and scientific importance: these include potato, tomato, pepper, eggplant, tobacco, and petunia. The Solanaceae has emerged as a model family for studying the biochemical evolution of plant specialized metabolism and multiple examples exist of lineage-specific metabolites that influence the senses and physiology of commensal and harmful organisms, including humans. These include, alcohols, phenylpropanoids, and carotenoids that contribute to fruit aroma and color in tomato (fruity), glandular trichome-derived terpenoids and acylsugars that contribute to plant defense (stinky & sticky, respectively), capsaicinoids in chili-peppers that influence seed dispersal (spicy), and steroidal glycoalkaloids (bitter) from Solanum, nicotine (addictive) from tobacco, as well as tropane alkaloids (deadly) from Deadly Nightshade that deter herbivory. Advances in genomics and metabolomics, coupled with the adoption of comparative phylogenetic approaches, resulted in deeper knowledge of the biosynthesis and evolution of these metabolites. This review highlights recent progress in this area and outlines opportunities 2 for – and challenges of-developing a more comprehensive understanding of Solanaceae metabolism. The Solanaceae: a phylogenetic framework for exploring metabolism Metabolism is a window into micro- and macro-evolutionary processes. Plant metabolic diversity is vast and collectively plants are hypothesized to synthesize ∼106 metabolites (Afendi et al., 2012). Many of these metabolites, including sugars, amino acids, fatty acids, and organic acids – referred to as general or primary metabolites – are conserved across the plant kingdom, and essential for growth and development. However, specialized metabolites (SM), also referred to in the literature as secondary metabolites, comprise the majority of plant metabolic complexity. Specialized metabolites are chemically diverse, display taxonomically restricted distribution, and are often synthesized in individual tissues or cell types. Plants evolved the capacity to synthesize specific classes of specialized metabolites to facilitate ecological adaptations. The advent of genomics, coupled with the ability to test the function of candidate genes in host species or heterologous systems, advanced our understanding of the biosynthesis and evolution of plant specialized metabolism (Fossati et al., 2014; Lau and Sattely, 2015; Nett et al., 2020). Although plant specialized metabolites exhibit considerable chemical complexity, they are ultimately derived from a pool of general metabolites formed through photosynthesis, glycolysis, the TCA cycle, amino acid metabolism, and the MEP-pathway (Vogt, 2010). General metabolites undergo transformations, including ligation and cyclization to generate scaffold molecules that are modified by glycosylation, acylation, methylation, prenylation, oxidation, and reduction to dramatically increase chemical complexity. In plants, the formation of these scaffold molecules and their subsequent decorations are catalyzed by large enzyme families formed by 3 repeated gene duplication followed by subfunctionalization, neofunctionalization, and gene loss to ultimately produce lineage-specific metabolites. The evolutionary mechanisms that create SM diversity are numerous but include co-option of general metabolism enzymes, evolution of catalytic promiscuity, enzyme compartment switching, the formation of biosynthetic gene clusters, and gene expression changes (Akiyama et al., 2021b; Itkin et al., 2013; Leong and Last, 2017; Schenck and Last, 2020; Sonawane et al., 2020). These evolutionary processes occur across different taxonomic scales, including inter-specific and intra-specific, to generate the chemical variation observed across the plant kingdom. The Solanaceae, or nightshade family, contains approximately 2700 documented species found on six continents, which collectively have evolved morphological and metabolic adaptations for nearly every environment (Särkinen et al., 2013). A single genus – the Solanum – accounts for nearly half of these species (Gagnon et al., 2022). Nightshades grow in environments ranging from deserts to rainforests, with growth habits that vary from epiphytes to trees. The family includes four major food crops (potato, tomato, pepper, and eggplant), a host of minor food crops (including tomatillo, naranjilla, tamarillo, and groundcherry) as well as the several ornamental crops (including petunia, salpiglossis, schizanthus, and brugmansia) and weed species (Jimson weed and bittersweet). In addition, several Solanaceae species are grown for their narcotic or medicinal properties (tobacco, corkwood tree, deadly nightshade, henbane, and Datura species). The Solanaceae family has become a model system for investigating biodiversity. The Solanaceae community concept was proposed nearly two decades ago, with the idea of using the nightshade family to connect genomics and biodiversity (Knapp et al., 2004). This concept envisioned harnessing Solanaceae natural diversity for evolutionary studies by creating the 4 necessary network of resources. One important tool was a detailed understanding of Solanaceae phylogenetic relationships (https://www.solanaceaesource.org). This framework provides a basis for evolutionary studies within the family. In parallel, the community-driven releases of the first tomato and potato genomes created a genomic foundation. These successful projects spawned numerous additional projects (e.g., SOL-100, Varitome Project, 100 Tomato Genomes Project), resulting in chromosome-scale genome assemblies draft genomes, pan-genomes, resequencing of numerous wild tomato species and cultivars, and an online database for genetic resources (Alonge et al., 2020; Barchi et al., 2021; Gao et al., 2019; Mueller et al., 2005; Sato et al., 2012; Song et al., 2019; Xu et al., 2011). As of early 2022, genome sequences are available for more than 30 Solanaceae species (https://plabipd.de/), and it seems likely that many more will follow over the next few years. These genomic tools are augmented by the availability of comprehensive germplasm resources, particularly for the major crop species of the Solanaceae. These resources allow genetic analysis of phenotypes of interest, facilitate genotype to phenotype comparisons and allow exploration of natural phenotypic diversity. The pioneering work of Charles Rick – and creation of seed stock centers (e.g., GRIN-Global and C. M. Rick Tomato Genetics Resource Center) provide access to crop and wild relative germplasm. Notably, connecting genotype to phenotype within tomato has been greatly accelerated by the development of the introgression lines (ILs) and backcrossed introgression lines (BILs) of wild tomato S. pennellii within a cultivated tomato background (Eshed and Zamir, 1995; Ofner et al., 2016). These ILs and BILs were instrumental in discovering genes underlying multiple phenotypes, including those related to metabolism (Fridman et al., 2004; Ofner et al., 2016; Schilmiller et al., 2012; Toal et al., 2018). In addition, the ability to perform RNA interference (RNAi), virus-induced gene silencing 5 (VIGS), and CRISPR/Cas9 tools in multiple Solanaceae species allows the functional characterization of candidate genes and a more precise connection of genotype and phenotype (Brooks et al., 2014; Liu et al., 2002; Schijlen et al., 2007; Van Eck, 2018). The Solanaceae has emerged as a model system for investigating the biosynthesis and evolution of specialized metabolism (Figure 1). Members of the family have evolved to synthesize several classes of bioactive and lineage-specific specialized metabolites, including phenylpropanoids, acylsugars, terpenes and distinct groups of alkaloids (Figure 2). These specialized metabolites are of interest because they influence fruit aroma and quality and are of potential use as biopesticides and pharmaceuticals. The development of genomic resources, coupled with the ability to survey metabolite variation across diverse germplasm, and to place the resulting data within a phylogenetic context, enabled elucidation of the biosynthesis and evolutionary trajectories of several major classes of Solanaceae SMs. Figure 1.1. Solanaceae as a model family for specialized metabolism evolution studies. The Solanaceae concept toolbox connects biodiversity, genetics, and evolutionary mechanisms to 6 PhysalisCapsicumJaltomataThelopodiumM CladePotato CladeLeptostemonumCyphomandraBrevantherumDaturaMandragoraAtropinaNicotianaBrunsfelsiaPetuniaSchizanthusGoetzeoideaeCestroideaeSalpiglossisConvulvulaceaeSolanumClade IISolanaceae2N2N4N Figure 1.1. (cont’d) each other. Chemical diversity informs metabolic pathway discovery, which in turn reveals evolutionary mechanisms underlying chemical diversity. Figure 1.2. Phylogenetic distribution of major Solanaceae specialized metabolite classes. The Solanaceae family produces specialized metabolites of multiple chemical classes. A simplified phylogeny of the Solanaceae family is shown based on prior determination of phylogenetic relationships (Gagnon et al., 2022; Särkinen et al., 2013). Major metabolite classes are mapped to the corresponding clades that produce high amounts of those metabolites and/or act as model species for studying their biosynthesis and evolution. Metabolites may not be distributed solely in the noted phylogenetic group. Additional information on metabolite distribution is provided throughout the text of this article. 7 PhysalisCapsicumJaltomataThelopodiumM CladePotato CladeLeptostemonumCyphomandraBrevantherumDaturaMandragoraAtropinaNicotianaBrunsfelsiaPetuniaSchizanthusGoetzeoideaeCestroideaeSalpiglossisConvulvulaceaeSolanumClade IISucrose sugarcoreGlucose sugarcoreInositol sugarcoreHOOOHOOOOOOHOOOOOOOS5:21(2,2,ai5,ai6,ai6)Salpiglossis sinuataOHOOOOOHOOOOOHOOOHOOHOOOSolanaceaeOHOOOOHOOOOHOOOHOHOOOOOHOOOOOOOOHOHOHOOOOOOOOOOOOHOOS5:25(ai5,ai5,ai5,ai7,malonyl)Petunia axillarisS4:17(2,i5,i5,ai5)Solanum lycopersicumG3:19(i4,ai5,i10)Solanum pennelliiG3:19(2,i4,i10)Solanum nigrumI3:17(i4,ai5,8)Solanum nigrumI4:24(2,2,10,10)Solanum quitoenseOHOOOOHOOOOHOOHOHOHOOOOOHOOOOOS3:19(i4,ai5,i10)Solanum pennellii Fruity: GWAS-enabled discovery of aroma variation during ripening The ripening of fleshy fruits is an agriculturally- and ecologically- important developmental process that makes fruits palatable and facilitates seed dispersal. Although fleshy fruits are highly diverse in morphology and flavor, ripening generally involves cell wall disassembly and associated softening, the conversion of starch into sugars, changes in color, and the biosynthesis of aroma volatiles. Fruit flavor and aroma is a complex species-specific quantitative trait involving the interaction between GM pathways, such as those influencing the accumulation of sugars and organic acids, as well as multiple SM pathways that yield aroma volatiles (Tieman et al., 2017). Tomato is the long-standing model crop species for investigating ripening mechanisms, including flavor and aroma biosynthesis. Recent progress in understanding the genetic and biochemical basis of tomato flavor was facilitated by large-scale genome sequencing and resequencing projects involving hundreds of phenotypically diverse cultivated tomato accessions and wild relatives. These studies revealed insights into the nature of the tomato pan-genome and sequence variation associated with crop domestication and improvement, including gene duplication, single nucleotide polymorphisms, insertion–deletions, and large-scale structural variants (Alonge et al., 2020; Gao et al., 2019; Tieman et al., 2017; Zhu et al., 2018). The development of these resources facilitates the identification of genetic variation underlying phenotypic traits via genome-wide association studies. Notably, this approach was successfully deployed for the identification of genetic components underlying variation in tomato fruit flavor and aroma, revealing how human selection for visible traits such as fruit size, yield, and color can lead to alternative outcomes and unintentionally influence SM pathways that contribute to fruit quality. 8 Several hundred volatiles are detectable in ripening tomato fruits, but consumer taste panels identified 33 metabolites associated with consumer liking and 37 correlated with flavor intensity (Tieman et al., 2017). These influential aroma volatiles are derived through diversion of general metabolites, including carotenoids, phenylalanine, isoleucine/leucine, and fatty acids into diverse SM pathways. Genetic variation is evident across tomato varieties and 13 fruit aroma volatiles are significantly reduced in a collection of 48 modern cultivars when compared to 236 heirloom tomato varieties. This work shows that breeding of modern varieties for traits such as yield, shelf-life, and disease resistance has inadvertently and negatively altered SM pathways that produce aroma volatiles associated with consumer preference (Tieman et al., 2017). Subsequent GWAS analyses performed using a panel of 398 diverse tomato accessions analyzed for 27 volatiles along with glucose, fructose, malic acid, and citric acid revealed the existence of 251 association signals for 20 traits, including 15 correlated with aroma volatile production. Among these associations are five loci that influence the production of carotenoid- derived volatiles. Two loci specifically influence the production of geranylacetone, which is formed by oxidative cleavage of the minor tomato fruit carotenoids phytoene, phytofluene, ζ- carotene, and neurosporene. A single locus specifically influences 6-methyl-5-hepten-2one (MHO) accumulation, which is derived from lycopene, the main carotenoid pigment in red- fruited tomato varieties. Two additional loci are associated with the production of both geranylacetone and MHO. Analysis of allele frequencies at these loci indicate that genetic complexity was progressively lost during breeding to the point where essentially only two allele combinations associated with accumulation of both volatiles persist in most modern cultivars. Analysis of MHO levels in genotypes with distinct allele combinations revealed that, as breeders selected for high lycopene in red-fruited varieties, they inadvertently selected favorable alleles 9 that increase MHO production. In contrast, the favorable alleles that promote geranylacetone accumulation are absent in modern cultivars (Tieman et al., 2017). GWAS also revealed the identity of loci important for producing lipid and phenylalanine- derived volatiles. Ripening tomato fruit accumulate C5 and C6 volatiles derived from the breakdown of linolenic and linoleic acid, which are released from glycerolipids such as triacylglycerol. GWAS analyses of the panel of 398 tomato accessions described above identified a chromosome 9-localized SNP that is significantly associated with the fatty acid derived volatiles Z-3-hexen-1-ol and hexyl alcohol (X. Li et al., 2020). This SNP lies within a metabolic QTL region known to influence lipid content in tomato fruit (Garbowicz et al., 2018). Solyc09g091050 (Sl-LIP8) was identified as a candidate gene close to this SNP and gene expression analysis revealed that accessions possessing the reference allele from the Heinz 1706 variety had increased levels of Z-3-hexen-1-ol and hexyl alcohol together with elevated Soly09g091050 transcripts. Confirmation that Sl-LIP8 is responsible for lipid-derived volatile synthesis was achieved through CRISPR/Cas9 gene editing and in vitro biochemical assays. The knock-out mutants showed reductions in two C5 (1-pentanol and 1-penten-3-ol) and three C6 (Z- 3-hexen-1-ol, E-2-hexen-1-ol, and hexyl alcohol) volatiles, while the recombinant enzyme catalyzed release of fatty acids from various glycerolipids (X. Li et al., 2020). The resultant free fatty acids undergo peroxidation at either the C9 or C13 positions in reactions catalyzed by 9- lipoxygenases and 13-lipoxygenases, respectively to yield aroma volatiles. The phenylalanine-derived volatiles guaiacol, eugenol, and methylsalicylate contribute to the aroma of tomato fruits and are associated with smoky and medicinal-like aromas, which are often negatively correlated with consumer liking (Zanor et al., 2009). Guaiacol, eugenol, and methylsalicylate accumulate in tomato fruits as diglycosides, and cleavage of the glycoside 10 groups leads to release of the volatiles in “smoky” cultivars. In contrast, in “non-smoky” varieties these metabolites exist as non-cleavable triglycosides resulting in reduced levels of volatile release (Tikunov et al., 2013). Formation of guaiacol, eugenol, and methylsalicylate triglycosides from their diglycoside precursors is catalyzed by the UDP-glucosyltransferase enzyme, NON-SMOKY GLYCOSYLTRANSFERASE1 (NSGT1). The NSGT1 gene resides at a locus on chromosome 9 that contains a second gene designated NSGT2. Both genes contain structural changes in “smoky” cultivars that are predicted to render them non-functional although the exact structure of the locus was unresolved (Tikunov et al., 2013). The recent development of 14 new reference tomato genomes assembled using Oxford Nanopore long read sequencing technology allowed the genome structure flanking the NSGT1 locus to be resolved. Five haplotypes were identified revealing evidence of intraspecific gene duplication and loss at an SM locus that was selected during crop improvement (Alonge et al., 2020). Haplotype I is proposed to be ancestral and contains predicted functional copies of NSGT1 and NSGT2. All other haplotypes contain coding sequence mutations in NSGT2. In addition, haplotypes IV and V also lack functional copies of NSGT1 and are therefore null mutations for both NSGT1 and NSGT2. Analysis of guaiacol levels across two GWAS panels and within an F2 population segregating for haplotype V and a functional copy of NSGT1 demonstrated that fruit guaiacol levels are reduced in individuals that contain a functional copy of NSGT1. Together, these data illustrate the combined power of genome sequences developed using long-read sequencing data and GWAS to investigate the evolution of loci associated with SM phenotypes, particularly when the variation is mediated by tandem gene duplication that may be unresolved in genome assemblies derived from short-read data. Overall, these studies 11 represent an example of fundamental science that provides opportunities to breed tomato varieties with favorable aroma volatile alleles. Sticky: single-cell biochemical genetics reveals acylsugar metabolic complexity Acylsugars are specialized metabolites produced in numerous plant families including the Solanaceae, Convolvulaceae, Geraniaceae, Martyniaceae, Rosaceae, Brassicaceae, and Caryophyllaceae (Asai et al., 2011, 2010; Asai and Fujimoto, 2011, 2010; Bah and Pereda- Miranda, 1996; Maldonado et al., 2006; Moghe et al., 2017; Ono et al., 2015; Pereda-Miranda et al., 1993; Wu et al., 2013). Many species across the Solanaceae produce acylsugars in hair-like Type I- and IV-glandular trichomes, while some species are documented to accumulate acylsugars in fruit pericarp or root exudates (Korenblum et al., 2020; Li et al., 2014; Maldonado et al., 2006; Nakashima et al., 2016). Acylsugars are composed of a sugar core, most commonly sucrose, and various fatty acids esterified to the core (Figure 1.3). Despite these simple components, variations in acylation position, chain length, chain branching pattern, and sugar core can result in hundreds of chromatographically separable acylsugars in a single species (Moghe et al., 2017). Solanaceae acylsugars are the most extensively characterized acylsugar type with more than 100 distinct NMR-resolved chemical structures (Bernal et al., 2018; Cao et al., 2015; Chortyk et al., 1997; Cicchetti et al., 2018; Ghosh et al., 2014; Hurney, 2018; Liu et al., 2017; Lou et al., 2021; Lybrand et al., 2020; Maldonado et al., 2006; C.-R. Zhang et al., 2016; C.-Y. Zhang et al., 2016). Acylsugars defend against microbes and insects; for example, deterring whitefly oviposition (Leckie et al., 2016), aphid settling (Goffreda et al., 1989), fungal growth (Luu et al., 2017), and mediating an ant-hornworm-tobacco interaction (Weinhold and Baldwin, 2011). 12 Figure 1.3. Phylogenetic distribution of acylsugar core types. (A) Simplified Solanaceae phylogeny with acylsugar core type placed on each lineage with characterized acylsugars. The phylogenetic tree is based upon previously published Solanaceae and Solanum trees (Gagnon et 13 PhysalisCapsicumJaltomataThelopodiumM CladePotato CladeLeptostemonumCyphomandraBrevantherumDaturaMandragoraAtropinaNicotianaBrunsfelsiaPetuniaSchizanthusGoetzeoideaeCestroideaeSalpiglossisConvulvulaceaeSolanumClade IISucrose sugarcoreGlucose sugarcoreInositol sugarcoreHOOOHOOOOOOHOOOOOOOS5:21(2,2,ai5,ai6,ai6)Salpiglossis sinuataOHOOOOOHOOOOOHOOOHOOHOOOSolanaceaeOHOOOOHOOOOHOOOHOHOOOOOHOOOOOOOOHOHOHOOOOOOOOOOOOHOOS5:25(ai5,ai5,ai5,ai7,malonyl)Petunia axillarisS4:17(2,i5,i5,ai5)Solanum lycopersicumG3:19(i4,ai5,i10)Solanum pennelliiG3:19(2,i4,i10)Solanum nigrumI3:17(i4,ai5,8)Solanum nigrumI4:24(2,2,10,10)Solanum quitoenseOHOOOOHOOOOHOOHOHOHOOOOOHOOOOOS3:19(i4,ai5,i10)Solanum pennellii Figure 1.3. (cont’d) al., 2022; Särkinen et al., 2013). (B) Characteristic acylsugar structures produced by Solanaceae species (Chortyk et al., 1997, 1993; Ghosh et al., 2014; Hurney, 2018; King et al., 1986; Liu et al., 2017; Lou et al., 2021; Lybrand et al., 2020; Maldonado et al., 2006; Moghe et al., 2017). Acylsugar nomenclature is given for each compound where the first letter represents the sugar core (S for sucrose, G for glucose, I for inositol); the first number represents the number of acylations; the number after the colon represents the number of carbons in acyl chains; and the individual acyl chains are listed inside parentheses (ai = anteiso, i = iso). Harnessing acylsugar genotypic diversity for tomato pathway determination Tomato acylsugar diversity was employed to uncover the acylsugar biosynthesis pathway within cultivated tomato, S. lycopersicum. Analysis of S. lycopersicum introgression lines carrying S. pennellii chromosomal segments was instrumental in identifying loci required for acylsugar biosynthesis (Schilmiller et al., 2010, 2012). The identification and subsequent validation of candidate genes was facilitated by trichome-specific transcriptome, in vitro enzyme assays, and in vivo gene VIGS knockdown and CRISPR/Cas9 knockout. These approaches uncovered the core acylsugar pathway in S. lycopersicum glandular trichomes. A series of evolutionarily related BAHD acyltransferases, named AcylSucrose AcylTransferase 1–4 (ASAT1-4), acylate sucrose sequentially to produce tetraacylsucroses consisting of acyl chains at R2, R3, R4, and R3’ (Fan et al., 2015; Schilmiller et al., 2015, 2012) (Figure 1.4). Each enzyme selectively acylates specific sucrose hydroxyls with varying promiscuity for acyl-CoA substrates. Documenting this pathway enabled discovery of mechanisms responsible for acylsugar diversity in wild tomato relatives. 14 Figure 1.4. Acylsucrose and acylglucose pathway diversity in Solanum species. The acylsucrose and acylglucose biosynthesis pathways for S. nigrum, S. lycopersicum and S. pennellii. All three biosynthetic pathways begin by acylating sucrose (Fan et al., 2015; Leong et 15 Figure 1.4. (cont’d) al., 2019; Lou et al., 2021; Schilmiller et al., 2015, 2012). Sequential acylations produce tetraacylsucroses, triacylsucroses, and diacylsucroses for S. lycopersicum, S. pennellii, and S. nigrum, respectively. S. pennellii triacylsucroses and S. nigrum diacylsucroses are cleaved by ASFF enzymes to form triacylglucoses and diacylglucoses, respectively (Leong et al., 2019; Lou et al., 2021). S. nigrum diacylglucose is acetylated by SnAGAT1 to form a triacylglucose (Lou et al., 2021). ASAT, acylsucrose acyltransferase; AGAT, acylglucose acyltransferase; ASFF, acylsugar fructofuranosidase; CoA, CoenzymeA. Intra- and inter-specific differences in tomato acylsugar structures result in part from differing ASAT activities. Comparative biochemical analysis of cultivated and wild tomato ASAT sequences uncovered amino acid residues responsible for specific activity differences. For example, the comparison of ASAT2 sequences and in vitro enzyme activities across tomato species revealed two mutations that impact acyl-CoA specificity. Residues Val/Phe408 and Ile/Leu44 influence the ability to use the structurally similar iC5-CoA and aiC5-CoA, respectively, without altering activity with nC12-CoA (Fan et al., 2015). Comparison of S. lycopersicum and S. habrochaites ASAT3 homologs revealed a Tyr/Cys41 residue change impacting the enzyme's ability to use nC12-CoA (Schilmiller et al., 2015). Characterization of S. habrochaites ASAT4 in accessions collected from Ecuador to Southern Peru revealed variations in acetylation patterns that were explained either by changes in ASAT4 expression or coding sequence mutations (Kim et al., 2012; Landis et al., 2021). The comparative biochemistry approach revealed differences in enzyme acyl donor specificity, which impacted acylsugar phenotypes. This approach also determined evolutionary changes in enzyme acyl acceptor specificity. S. pennellii LA0716 produces acylsucroses through a ‘flipped pathway’, resulting from changes in ASAT acyl acceptor specificity (Fan et al., 2017). While cultivated tomato produces acylsucroses with one furanose ring acylation (termed F-type acylsucroses), S. pennellii and some S. habrochaites accessions synthesize acylsucroses acylated exclusively on the pyranose 16 ring (Schilmiller et al., 2015). These ‘P-type’ acylsucroses are produced by alternate ASAT2 and ASAT3 homologs, which catalyze the third and second pathway steps, respectively. The published results suggest that S. pennellii ASAT2 likely evolved from an ancestral enzyme capable of acylating both mono- and diacylsucrose. Analogous sequence changes in ASAT3, potentiated by ASAT3 duplication, resulted in the neofunctionalized ASAT3 duplicate found in S. habrochaites and S. pennellii. This study revealed a remarkably small number of amino acid changes that caused a major change in pathway structure and product phenotypes in closely related species. The flipped S. pennellii pathway and recruitment of an invertase-like enzyme appear to have potentiated evolution of S. pennellii acylglucose synthesis (Figure 1.4). S. pennellii acylglucoses are synthesized from P-type acylsucroses by a neofunctionalized glycoside hydrolase 32 family (GH32) beta-fructofuranosidase, SpASFF1 (Leong et al., 2019). The modified SpASFF1 substrate binding site correlates with a derived P-type acylsucrose cleavage activity, yet the neofunctionalized enzyme does not act on the F-type acylsucrose produced by S. lycopersicum. In addition, SpASFF1 lacks activity with sucrose, associated with changes to the canonical sucrose binding pocket. Instead, the modified SpASFF1 substrate binding site correlates with a derived P-type acylsucrose cleavage activity, yet the neofunctionalized enzyme does not act on the F-type acylsucrose produced by S. lycopersicum. SpASFF1 specificity for P- type acylsucroses supports the hypothesis that P-type acylsucroses are required for acylglucose production. Indeed, cultivated tomato lines engineered to contain both the flipped pathway and SpASFF1 accumulate acylglucoses. This indicates that acylglucose biosynthesis requires both a neofunctionalized invertase and the S. pennellii flipped pathway. Finally, CRISPR/Cas9 deletion of SpASFF1 led to accumulation of only acylsucroses – without detectable acylglucoses – in S. 17 pennellii, reinforcing that the neofunctionalized invertase is necessary for acylglucose synthesis in the wild tomato. SpASFF1 invertase is an example of co-option of general metabolic enzyme to specialized metabolism into acylsugar biosynthesis – in this case resulting in different sugar core composition. The theme of GM enzymes recruitment to SM by gene duplication, changes in gene expression and enzyme structure and function also contribute to acyl chain type variation. For example, the duplicated and neofunctionalized isopropylmalate synthase gene, IPMS3, influences isoC5 acyl chain abundance (Ning et al., 2015). In contrast to the canonical Leu biosynthetic IPMS, IPMS3 expression is restricted to type I/IV glandular trichome tip cells, and the S. lycopersicum enzyme is insensitive to Leu-mediated feedback inhibition in vitro due to truncation of the C-terminal allosteric regulatory domain. Apparently, the lack of this domain frees the enzyme from Leu feedback regulation, enabling pathway diversion. IPMS3 allelic variation directly correlated with abundance of isoC5 and isoC4 acyl chains in wild S. pennellii accession acylsugars; accessions with majority isoC4 acyl chains were homozygous for a truncated, inactive IPMS3. In contrast, isoC5 acyl chains were abundant in accessions either heterozygous or homozygous for the unregulated IPMS3. These results reveal that acyl-CoA availability influences acylsugar acyl chain composition. Further evidence for this hypothesis was provided by identification of natural chain diversity associated with allelic diversity of two acyl-CoA biosynthesis genes (Fan et al., 2020). These trichome-expressed genes, an enoyl-CoA hydratase (AECH1) and acyl-CoA synthetase (AACS1), reside in a gene cluster syntenic to the chromosomal region containing ASAT1. The Solanaceae family shares the syntenic region, which was likely derived from a Solanaceae- specific polyploidy event. Silencing AECH1 and AACS1 in S. lycopersicum, S. pennellii, and the 18 more distantly related Solanum quitoense, reduced or eliminated medium length (10–12 carbons) acyl chains from acylsugars. Additionally, the presence of AECH1 and AACS1 correlates with natural variation in medium acyl chains. For example, in the short chain producing genera Petunia and Nicotiana, AECH1 and AACS1 are either missing or present as pseudogenes. These genes represent another example of how evolutionary changes in metabolic machinery impacted acylsugar composition. Genomics tools enable comparative biochemistry in non-model organisms Application of DNA sequencing, modern analytical chemistry, and reverse genetic tools such as VIGS and genome editing enabled documentation of additional acylsugar evolutionary mechanisms in non-model species. LC-MS screening and NMR-resolved structural analysis identified Solanaceae species that produce unique acylsugars with varying cores, acylation positions, and chain types (Hurney, 2018; Leong et al., 2020; Liu et al., 2017; Lou et al., 2021; Lybrand et al., 2020; Moghe et al., 2017). For example, extant members of early-diverging lineages produce acylsucroses with acylation patterns undocumented in cultivated and wild tomatoes. Additionally, acylated glucoses are detected in some species within the Petunia, Nicotiana, Datura, and Solanum genera (Chortyk et al., 1997, 1993; King and Calhoun, 1988; Lou et al., 2021; Matsuzaki et al., 1989). Within the large Solanum genus, myo-inositol sugar cores have been documented in S. lanceolatum, S. quitoense, and S. nigrum (Herrera-Salgado et al., 2005; Hurney, 2018; Leong et al., 2020; Lou et al., 2021). Evolution of acylsugar biosynthesis was investigated in four non-model species: Salpiglossis sinuata, Petunia axillaris, S. nigrum, and S. quitoense. Comparison of the enzymes and pathways in each species revealed features of long-term and clade-specific acylsugar traits. 19 Inferring early events in acylsugar evolution Investigations of two members of early diverging lineages, S. sinuata and P. axillaris, revealed acylsugar biosynthesis evolutionary changes occurring over tens of millions of years (Myr), well beyond the approximately 7 Myr of Solanum tomato clade history (Moghe et al., 2017; Nadakuduti et al., 2017; Särkinen et al., 2013). Despite similarity of acylation positions between tomato species, S. sinuata and Petunia acylsugars, a major shift occurred in the acylsugar biosynthetic pathway. The ancestral pathway found in S. sinuata and P. axillaris begins with a sucrose-acylating ancestral ASAT1, aASAT1, which is not found in tomato clade species. Another surprise is that the SlASAT1 and SlASAT2 orthologs, aASAT2 and aASAT3, respectively catalyze the second and third acylations. The first three acylations by the early evolving aASAT1-3 pathway produce triacylsucroses with the same three positions acylated as SlASAT1-3. Coinciding with this, aASAT2 and aASAT3 retained their selectivity for the R4 and R3 of sucrose, respectively, but shifted acyl acceptor specificity to free and monoacylsucrose, respectively. This activity shift correlates with aASAT1 loss in species with modern acylsugar biosynthesis pathways. Transcriptome and genome analyses suggest that the aASAT1 gene disappeared from the last common ancestor of the Capsicum and Solanum genera, ∼15–20 MYA. Identification of these ancestral acylsugar pathways support sucrose as the ancestral acyl acceptor. From these studies of early-diverging Solanaceae species, ASAT gene loss and neofunctionalizations were implicated in a changing acylsucrose pathway, analogous to those described above in the case of the S. pennellii flipped acylsucrose pathway. The ancestral and derived acylsucrose pathways provide insight into the evolutionary origins of acylsugars (Moghe et al., 2017). Lamiidae BAHD sequence homology, phylogenetics, and known whole genome duplication events all enabled inferences regarding early acylsugar 20 evolution. One hypothesis, based on sequence analysis, is that ASAT sequences derive from an alkaloid biosynthetic enzyme ancestor. Based on nonsynonymous mutation rates and historical polyploidy events, the clade containing ASAT1,2,3 appears to have arisen via an ancient whole genome duplication before the Solanaceae-Convolvulaceae split (∼50–65 MYA). Subsequent duplications prior to, and following the Solanaceae polyploidization, led to evolution of the ASATs and paralogs found in the ASAT1,2,3 clade. As described above, our model of acylsugar biosynthetic pathway evolution invokes loss of aASAT1, refinement of ASAT1 and ASAT2 activities, and recruitment of ASAT3 occurred later in Solanaceae diversification. Acylhexoses in non-model plants Metabolite profiling revealed that, like S. pennellii, black nightshade (Solanum nigrum) also produces acylglucoses, an observation that enabled discovery of convergent and new acylsugar enzyme activities. S. nigrum creates di- and triacylglucoses through a similar, yet distinct, pathway when compared to S. pennellii acylglucose biosynthesis (Lou et al., 2021) (Fig. 4). Both pathways proceed through a series of sucrose acylations, followed by action of an acylsugar fructofuranosidase. The S. nigrum invertase, SnASFF1, and SpASFF1 enzymes share similarities including a modified DDTK sucrose binding pocket, loss of canonical invertase activity cleaving sucrose, and neofunctionalized activity with acylsucroses. However, each ASFF1 enzyme resides in a distinct glycoside hydrolase subfamily 32 clade and cleaves different substrates: triacylsucroses by SpASFF1 and diacylsucroses by SnASFF1. SnAcylGlucoseAcetylTransferase1, SnAGAT1, catalyzes the third S. nigrum acylation, marking yet another distinction between S. nigrum and S. pennellii triacylglucose biosynthesis; this is the only enzyme to acylate an acylglucose described to date. As the two characterized Solanum 21 acylglucose biosynthetic pathways include distinct invertases, it is plausible that this mechanism evolved in other acylglucose-producing genera. In contrast to the detailed information available for acylsucrose and acylglucose biosynthesis, the pathway leading to acylinositol synthesis in the Solanum remains largely enigmatic. So far only one enzyme was demonstrated in acylinositol biosynthesis: the S. quitoense enzyme TriAcylInositolAcetylTransferase, SqTAIAT, acetylates triacylinositols to produce tetraacylinositols (Leong et al., 2020). SqTAIAT is the closest known S. quitoense homolog to the final enzyme in tomato acylsucrose biosynthesis, SlASAT4, indicating conservation of acetyltransferases across acylinositol and acylsucrose biosynthesis. Both enzymes acetylate triacylsugars differing in their sugar core. Similar enzymatic activity and high sequence similarity suggest a common evolutionary origin for acylinositol and acylsucrose biosynthesis. However, the initial steps of acylinositol biosynthesis remain unresolved. Further pathway elucidation in S. quitoense and S. nigrum may uncover the evolutionary innovations underlying acylinositol production. Into the depths with acylsugars It was recently shown that cultivated tomato accumulates acylsugars in roots and root exudates (Korenblum et al., 2020). Tomato root acylsugars structurally differ from those in trichomes, contrasting in acyl chain type, acyl chain number, and sugar core type. For example, six- and seven-carbon acyl chains and glucose sugar cores are only detected in the roots. These structural differences suggest evolutionary changes in the underlying biochemistry. One key observation is that characterized tomato trichome-expressed ASAT transcripts were not detected in root tissue, although they do express closely related homologs. These expression data suggest the hypothesis that roots produce acylsugars through an alternative pathway. In fact, expression 22 of two ASAT4 paralogs correlates with acylsugar abundance in roots. While the function of root acylsugars is unknown, different microbial communities systemically impacted root exudate acylsugar abundances (Korenblum et al., 2020). Investigating root acylsugar metabolism may unearth a root-specific acylsugar biosynthetic pathway among other tantalizing prospects. Stinky: variations on a theme define terpene diversity across Solanum Terpenoids are structurally diverse and are produced across all kingdoms of life, yet all are derived from the simple five-carbon isomers, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). These precursors are formed through either the mevalonate (MVA) or 2-C-methyl-D-erythritol-4-phosphate (MEP) pathways (Zhou and Pichersky, 2020a). Plants are unique in that they contain both the cytosolic MVA pathways and the plastid localized MEP pathway; having evolved to generate substantial flux towards DMAPP and IPP as well as create separate subcellular pools of these metabolites for different pathways (Zhou and Pichersky, 2020a). Terpenoids have diverse functions ranging from the production of photosynthetic pigments and ubiquinone in the electron transport chain to the production of several classes of plant hormones. However, most plant terpenoids are lineage-specific specialized metabolites with C10–C30 carbon skeletons that provide a fitness benefit to the host organism through signaling and defense (Zhou and Pichersky, 2020a). Plant terpenoid diversity is created at multiple levels. Firstly, small gene families produce cis and trans-prenyltransferases that initially condense a single molecule of DMAPP and IPP to form either geranyl diphosphate (GPP) (trans isomer) or neryl diphosphate (NPP) (cis isomer). These C10 metabolites can then be extended by five carbon units, through condensation with additional units of IPP, to yield trans- or cis-farnesyl diphosphate (E,E-FPP or Z,Z-FPP, C15), geranylgeranyl or nerylneryl diphosphate (GGPP or NNPP, C20), or longer chain prenyl 23 diphosphates (Zhou and Pichersky, 2020a). Short-chain prenyl diphosphates (C10–C20) are substrates for terpene synthases (TPS), which exist as moderately large gene families (up to ∼100 members) and catalyze the formation of hydrocarbon terpene skeletons via rearrangements and cyclization. TPS enzymes possess considerable catalytic potential. They frequently utilize more than one substrate, and catalysis by a single enzyme often generates multiple products (Karunanithi and Zerbe, 2019; Pazouki and Niinemets, 2016; Zhou and Pichersky, 2020a). These hydrocarbon terpene skeletons are often functionalized by the addition of hydroxyl groups, which provide targets for modifications such as epoxidation, methylation, acylation, and glycosylation, ultimately generating the vast complexity of terpenoids observed across the plant kingdom. The availability of a high-quality reference genome assembly for cultivated tomato (Solanum lycopersicum) facilitated what is likely the most comprehensive published catalogue of terpene scaffold biosynthesis in plants. The data highlight considerable chemical complexity with in vitro biochemical data revealing the potential to synthesize 53 known hydrocarbon terpene scaffolds plus several unidentified products. These terpenes arise through combined catalysis of seven cis-prenyltransferases and 10 trans-prenyltransferases that form C10, C15, and C20 prenyl diphosphates, together with 34 functional TPS enzymes (Akhtar et al., 2013; Zhou and Pichersky, 2020b). Consistent with the known catalytic promiscuity of TPS enzymes, many of the tomato TPSs can utilize more than one substrate, particularly the sesquiterpene synthases that use both E,E-FPP and Z,Z-FPP, and yield multiple products. In addition, considerable catalytic redundancy exists. For example, eight distinct TPSs catalyze the formation of the monoterpene β-myrcene. Individual CPT, TPT, and TPS enzymes are localized to the cytosol, plastids, as well as mitochondria, and the corresponding genes are differentially expressed across 24 tomato tissues: this highlights the spatial separation of terpene synthesis modules across tomato. Metabolite profiling of 13 tomato tissues identified 29 out of 53 terpenes in planta, suggesting that some terpenes are either below the limit of detection in tomato grown under standard cultural conditions or are further modified to produce more structurally complex metabolites. Genomic clustering is a key feature of terpene biosynthetic genes in plants (Boutanaev et al., 2015). These clusters generally consist of both paralogs and non-homologous genes encoding enzymes of terpene biosynthesis, creating a reservoir for the evolution of chemical novelty and facilitating the inheritance of SM modules that promote plant adaptation. Gene duplication within these clusters is often followed by pseudogenization and gene loss to create additional chemical variation. The majority of the 52 TPS loci in tomato, including 18 predicted pseudogenes, are located within gene clusters dispersed across the genome (Zhou and Pichersky, 2020b). In addition, the TPS gene clusters on chromosomes 6, 7, 8, and 12 also contain combinations of cis or trans prenyltransferases, cytochromes P450, methyltransferases, acyltransferases, and glycosyltransferases (Matsuba et al., 2013; Zhou and Pichersky, 2020b). While most of the potential terpene modifying enzymes within these clusters await functional characterization, a three-gene subcluster on chromosome 8 comprising SlTPS21-CYP71D51- SlCPT2 was demonstrated to synthesize (+)-lycosantalonol from NNPP (Zi et al., 2014). Along with the existence of the 18 TPS pseudogenes in the tomato genome, three TPS- related gene clusters on chromosomes 6, 8, and 12 also contain inactive cytochromes P450 genes (Zhou and Pichersky, 2020b). The high prevalence of pseudogenes within these tomato terpene biosynthetic gene clusters suggests that there is potential for considerable genetic variation. For example, a gene that is pseudogenized in one accession or species may be functional in another. Thus, variation in terpene-related gene clusters may exist between distinct accessions of S. 25 lycopersicum but also more likely across the genomes of diverse Solanaceae species. The increasing availability of high-quality chromosome scale reference genomes assembled from long-read sequencing will facilitate identification of additional gene clusters and future comparative evolutionary analysis of terpene biosynthesis across the Solanaceae. Within the Solanum genus, distinct evolutionary trajectories associated with trichome- derived terpene-related gene clusters are indeed apparent between cultivated tomato and wild relatives that diverged from a common ancestor approximately two-three million years ago (Särkinen et al., 2013). Notably, while limited terpene diversity exists in trichomes between cultivated tomato accessions, considerable variation is observed across distinct populations of Solanum habrochaites and between S. habrochaites and S. lycopersicum (Gonzales-Vigil et al., 2012). This genetic variation determines whether specific accessions preferentially synthesize monoterpenes (C10) or sesquiterpenes (C15), and results from differences at the cis- prenyltransferase 1 (CPT1) locus and associated TPS-e/f enzymes that are located within the chromosome 8 terpene gene cluster (Matsuba et al., 2013). For example, trichomes of cultivated tomato predominantly accumulate the monoterpene β-phellandrene, which is synthesized from NPP by neryl diphosphate synthase1 (NDPS1) (Schilmiller et al., 2009). While select monoterpene-producing accessions of S. habrochaites also contain an ortholog of NDPS1, a separate group of sesquiterpene producing accessions of S. habrochaites possess the C15- producing Z,Z-farnesyl diphosphate synthase (zFPS) at the CPT1 locus (Kang et al., 2014; Sallaud et al., 2009) (Fig. 5). Comparative sequence analysis, homology modeling, and site- directed mutagenesis revealed that the relative positioning of bulky aromatic amino acid residues within a hydrophobic cleft specifies substrate binding and prenyl-chain elongation between 26 CPT1 isoforms with NDPS1 and zFPS activity and that this contributes to intraspecific terpene variation in S. habrochaites (Kang et al., 2014). Figure 1.5. Terpenoid biosynthesis in the trichomes of Solanum habrochaites derived from cisoid substrates. NDPS1 catalyzes the condensation of a single molecule of DMAPP and IPP to form NPP (C10) (Schilmiller et al., 2009). In contrast, z,z-FPS catalyzes the formation of 2Z,6Z-FPP (C15) through sequential condensation of two molecules of IPP with a single molecule of DMAPP (Sallaud et al., 2009). In distinct NPP producing accessions of S. habrochaites the monoterpene synthases, ShPIS, ShLMS, and ShPHS1 catalyze the cyclization of NPP to form monoterpenes (Gonzales-Vigil et al., 2012). In a subset of 2Z,6Z-FPP forming accessions, the sesquiterpene synthase, ShSBS catalyzes the formation of endo-α-bergamotene and (+)-α-santalene (Gonzales-Vigil et al., 2012; Sallaud et al., 2009). These sesquiterpenes are converted to their corresponding acids by unknown enzymes. In a distinct subset of 2Z,6Z-FPP producing accessions, ShZIS catalyzes the formation of 7-epizingiberene, which is sequentially oxidized by ShCYP71D184 to 9-hydroxy-zingiberene and 9-hydroxy-10, 11-epoxy-zingiberene (Bleeker et al., 2012; Gonzales-Vigil et al., 2012; Zabel et al., 2021). In trichomes of cultivated 27 Figure 1.5. (cont’d) tomato, S. lycopersicum, only orthologs of NDPS1 and ShPHS1 are present resulting in the formation of β-phellandrene and δ-2-carene (Schilmiller et al., 2009). Thus, cisoid substrate derived terpene diversity is attenuated in S. lycopersicum in comparison to S. habrochaites. Abbreviations are as follows: DMAPP, dimethylallyl diphosphate; IPP, isopentenyl diphosphate; NPP, neryl diphosphate; 2Z,6Z-FPP, 2Z,6Z-farnesyl diphosphate; ShZIS, zingiberene synthase; ShSBS, santalene and bergamotene synthase; ShPIS, pinene synthase; ShLMS, limonene synthase; ShPHS1, β-phellandrene synthase. Together with divergent CPT1 enzymes, terpene diversity in S. habrochaites trichomes is also driven by natural variation in chromosome 8 cluster TPS-e/f subfamily members. S. lycopersicum, synthesizes a cocktail of monoterpenes in trichomes from NPP using the TPS-e/f enzyme, β-phellandrene synthase (SlPHS1/SlTPS20) (Schilmiller et al., 2009). PHS1 activity is conserved in some S. habrochaites accessions while others contain the TPS-e/f paralogs limonene synthase (ShLMS) and pinene synthase (ShPIS), which catalyze the formation of limonene and α-pinene from NPP, respectively (Gonzales-Vigil et al., 2012). In addition to this intraspecific variation in monoterpene biosynthesis, two additional groups of S. habrochaites accessions possess TPS-e/f enzymes that synthesize sesquiterpenes from Z,Z-FPP produced by zFPS: santalene and bergamotene synthase (ShSBS) catalyzes the formation of a mixture of santalene and bergamotene isomers (Gonzales-Vigil et al., 2012; Sallaud et al., 2009). In contrast, a distinct, yet closely related enzyme, zingiberene synthase (ShZIS) catalyzes the formation of 7-epizingiberene (Gonzales-Vigil et al., 2012) (Fig. 5). These sesquiterpene forming TPS-e/f enzymes are not present in S. lycopersicum and, to date, appear to be restricted to a subset of S. habrochaites accessions. Overall, together with variation at the CPT1 locus, these examples illustrate the evolutionary potential of SM associated gene clusters to create and maintain inter-specific and intra-specific chemical diversity. This relatively rapid intra-specific evolution of chemical variation in specific populations of plants may confer selective advantage against diverse biotic challenges. 28 The ability of trichomes of select S. habrochaites accessions to synthesize the sesquiterpenes santalene and bergamotene as well as 7-epizingiberene and their derivatives is known to confer increased tolerance to insect pests and pathogens when compared to trichomes that synthesize S. lycopersicum type monoterpenes (Bleeker et al., 2012, 2011; Coates et al., 1988; Frelichowski and Juvik, 2001). Santalene and bergamotene backbones are oxidized into sesquiterpene acids via unknown enzymes (Coates et al., 1988). In contrast, 7-epizingiberene is sequentially oxidized to a combination of 9-hydroxy-zingiberene and 9-hydroxy-10,11-epoxy- zingiberene in reactions catalyzed by the trichome-expressed cytochrome P450, ShCYP71D184 (Zabel et al., 2021) (Fig. 1.5). 9-Hydroxy-10,11-epoxy-zingiberene is particularly effective in bioactivity assays against whiteflies (Bemisia tabaci) and the microbial pathogens, Phytophthora infestans and Botrytis cinerea. ShCYP71D184 is encoded by the Sohab01g008670 locus and is therefore not located in the chromosome 8 TPS cluster responsible for the synthesis of the 7- epizingiberene substrate. The predicted ShCYP71D184 protein is 94% identical to its putative ortholog from S. lycopersicum SlCYP71D184/Solyc01g008670. The function of SlCYP71D184 is unknown but S. lycopersicum trichomes do not synthesize 7-epizingiberene and this enzyme is incapable of catalyzing the formation of 9-hydroxy-zingiberene and 9-hydroxy-10,11-epoxy- zingiberene. Although not completely understood, these data suggest that, like other loci that influence terpene biosynthesis in glandular trichomes of Solanum, genetic variation exists at the CYP71D184 locus that specifies chemical diversity. Spicy: lineage-specific biosynthesis of capsaicinoids in pepper Species within the Capsicum genus of the Solanaceae possess the capacity to synthesize a group of specialized metabolites known as capsaicinoids, including capsaicin, the principal determinant of pungency in chili peppers. These specialized metabolites are of culinary and 29 cultural importance but also possess applications as topical pain medications and show efficacy as anti-inflammatories, treatments for cancer and weight-loss, and possess anti-microbial activities (Duranova et al., 2022; Friedman et al., 2019; Spiller et al., 2008; Varghese et al., 2017). Capsaicinoids are synthesized within the placenta that surrounds the seeds of developing fruit and act as feeding deterrents for small mammals such as rodents, but not birds (Tewksbury and Nabhan, 2001). This deterrence is mediated by the mammalian vanilloid receptor 1 (VR1) ion channel that is localized to sensory nerve endings and responds to heat stimuli (Caterina et al., 2000). The ortholog of VR1 from birds does not respond to capsaicin and as such, birds, which are more efficient seed dispersers than small mammals, are unaffected by the pungency of pepper fruits (Jordt and Julius, 2002). The biosynthesis of capsaicinoids is not fully understood, particularly at the biochemical level and this pathway is yet to be reconstructed in a heterologous system. However, capsaicin biosynthesis is considered a derived trait within Capsicum, as species from the more ancient Andean clade of the genus are non-pungent (Carrizo García et al., 2016). Within Capsicum species, intra-specific variation exists resulting in loss of pungency (Carrizo García et al., 2016). Most notably, this intra-specific variation occurs in the major crop species Capsicum annuum and gives rise to both pungent and sweet pepper cultivars (Carrizo García et al., 2016). Capsaicin is synthesized through the condensation of vanillylamine, derived from the phenylpropanoid pathway, with 8-methyl-6-nonenoyl-CoA, produced through branched-chain amino acid metabolism and fatty acid synthesis (Kim et al., 2014). Genetic analyses identified loci associated with capsaicin accumulation and genes within the phenylpropanoid, branched-chain amino acid catabolism, and fatty acid synthesis pathways are among the candidates discovered (Han et al., 2018; Park et al., 2019; Tripodi et al., 2021). For example, loss of function alleles at 30 the AMT locus, which encodes an aminotransferase that catalyzes the formation of vanillylamine from vanillin, disrupts capsaicin biosynthesis (Lang et al., 2009; Tanaka et al., 2015; Weber et al., 2014). Similarly, mutation in a ketoacyl-ACP reductase (CaKR1), an enzyme involved in fatty acid biosynthesis, resulted in undetectable levels of capsaicin and 8-methyl-6-nonenoic acid, a precursor of 8-methyl-6-nonenoyl-CoA (Koeda et al., 2019). In addition, the BAHD acyltransferase capsaicin synthase, also known as Pun1, is associated with pungency in hot pepper and proposed to catalyze the condensation of vanillylamine with 8-methyl-6-nonenoyl- CoA to form capsaicin (Stewart Jr et al., 2005). A 2.5 kb deletion allele at this locus is present in non-pungent genotypes, although biochemical evidence supporting a direct role for this enzyme in capsaicin biosynthesis is lacking (Stewart Jr et al., 2005). Overall, these studies reveal genetic variation across Capsicum that has likely arisen due to domestication and selection. Bitter: evolutionary signatures of glycoalkaloid biosynthesis in Solanum Steroidal glycoalkaloids (SGAs) are bitter and toxic metabolites that occur in Solanum including the crop species tomato, potato, and eggplant. SGAs provide protection against herbivory as well as microbial pathogens and are proposed to function through the disruption of cell membranes and inhibition of cholinesterase activity (Roddick et al., 2001). In the United States, SGA levels are monitored in potato to maintain levels below an FDA-regulated threshold due to their toxicity (Dolan et al., 2010). Evolution and domestication shaped SGA diversity in Solanum; metabolite profiling and chemical structure elucidation reveal hundreds of SGAs that differ among members of the genus due to gene gain and loss between species (Gu et al., 2018; Iijima et al., 2013). For example, α-tomatine and esculeoside A accumulate in tomato while α- solasonine and α-solamargine are synthesized in eggplant. In contrast, domesticated potato synthesizes α-solanine and α-chaconine, while leptines, SGAs that display efficacy against 31 Colorado potato beetle (CPB), are found in wild potato species (Figure 1.6) (Akiyama et al., 2021b; Cárdenas et al., 2019; Paudel et al., 2017; Sánchez-Mata et al., 2010; Shinde et al., 2017). SGAs arise from the modification of cholesterol produced from the mevalonate pathway and are characterized by a nitrogen-containing 27-carbon core, which can undergo multiple glycosylations to form steroidal glycoalkaloids (Sonawane et al., 2016). Comparison of genomic sequences between species revealed that several biosynthetic steps of SGA formation in tomato, potato, and eggplant, encoded by GLYCOALKALOID METABOLISM (GAME) genes, are clustered within these genomes (Barchi et al., 2019; Itkin et al., 2013). 32 Figure 1.6. Steroidal glycoalkaloid biosynthesis in Solanum. CAS cyclizes 2,3-oxidosqualene from the mevalonate pathway to form cycloartenol a common metabolite in both phytosterol and cholesterol biosynthesis. Cycloartenol is converted to campesterol by a ten- 33 Figure 1.6. (cont’d) step pathway and through a nine-step pathway to form cholesterol (Sonawane et al., 2016). Following the production of cholesterol, five GAME enzymes are required to produce the spirosolane-type SGA core (Itkin et al., 2013). In tomato (red shaded box), GAME25 catalyzes the first of four steps resulting in tomatidine formation via the reduction of the spirosolane-type SGA core (Akiyama et al., 2019; Sonawane et al., 2018). Subsequent sugar additions by GAME1, GAME17, GAME18, and GAME2 result in the formation of α-tomatine (Itkin et al., 2013). GAME31, E8/Sl27DOX, GAME5, and an unknown acetyltransferase catalyze the fruit ripening associated formation of esculeoside A from α-tomatine (Akiyama et al., 2021a; Cárdenas et al., 2019; Kazachkova et al., 2021; Nakayasu et al., 2020; Szymański et al., 2020). In potato (yellow shading), the addition of solatriose and chacotriose moieties by sequential sugar additions to (22S,25S)-spirosol-5-en-3β-ol results in the formation of α- and β-solamarine, respectively (Akiyama et al., 2021b). The oxidization of α- and β-solamarine by DPS represents the first step in α-solanine and α-chaconine, Solanidane-type SGA, formation (Akiyama et al., 2021b). In S. chacoense, α-solanine and α-chaconine are oxidized by GAME32 to form leptinines, and leptine formation requires the acetylation at the GAME32 introduced oxidation (Cárdenas et al., 2019). The solasodine-type SGAs (α-solasonine and α-solamargine) are the main SGAs in eggplant (purple shading) and contain solatriose and chacotriose moieties at the C- 3 position, respectively. The biosynthetic mechanism leading to the stereochemical difference in spirosolane and solasodine cores remains uncharacterized (Akiyama et al., 2021b; Sánchez-Mata et al., 2010). Enzyme abbreviations are as follows: CAS, cycloartenol synthase; GAME, glycoalkaloid metabolism; SlS5αR2, steroid 5α-reductase 2; SGT, solanidine glycosyltransferase; DPS, dioxygenase for potato solanidane synthesis; E8/Sl27DOX, α-tomatine 27-hydroxylase; Gal, galactose; Glc, glucose; Xyl, xylose; Rha, Rhamnose. Formation of plant SGA sterol cores requires diversion of 2,3-oxidosqualene from the mevalonate pathway into cholesterol biosynthesis, and this biosynthetic pathway appears to have evolved from the duplication and divergence of genes involved in phytosterol biosynthesis, which leads to the production of brassinosteroids, an essential class of phytohormones (Sonawane et al., 2016). Cycloartenol synthase (CAS) converts 2,3-oxidosqualene into cycloartenol, and this metabolite is the branch point between cholesterol and phytosterol biosynthesis as it serves as a substrate for both SSR2 (sterol side chain reductase 2) and SMT1 (sterol C-24 methyltransferase) to form cycloartanol or 24-methylenecycloartanol, respectively (Sonawane et al., 2016). Cholesterol biosynthesis leads to the production of the SGAs and saponins in both glycosylated and aglycone forms (Sonawane et al., 2016). Elucidation of cholesterol biosynthesis in plants revealed five enzymes shared between the cholesterol and 34 phytosterol pathways (Sonawane et al., 2016). Phylogenetic analysis of enzymes specific to cholesterol biosynthesis suggests that C5-SD2 (sterol C-5(6) desaturase), 7-DR2 (7- dehydrocholesterol reductase), SMO3 (C-4 sterol methyl oxidase) and SMO4 likely arose from duplication and divergence of the phytosterol pathway genes, C5-SD1, 7-DR1, SMO1 and SMO2 (Sonawane et al., 2016). Presence-absence variation of genes involved in the conversion of dehydro-SGAs to dihydro-SGAs contributes to SGA diversity within Solanum. The first spirosolosane-type SGA formed, (22S, 25S)-spirosol-5-en-3β-ol, contains a Δ5,6 double bond (Akiyama et al., 2021b). In tomato, tomatidine is synthesized from a multistep process starting with the oxidation and isomerization of (22S, 25S)-spirosol-5-en-3β-ol to tomatid-4-en-3-one by GAME25, and the addition of four sugars (galactose, glucose, glucose, and xylose) to the C-3 position of tomatidine results in the production of tomatine, the major tomato SGA (Akiyama et al., 2019; Sonawane et al., 2020, 2018). Lack of a functional GAME25 is associated with the production of unsaturated SGAs, including α-solamargine, α-solasonine, and malonylsolamargine in S. melongena (eggplant) and expression of tomato GAME25 in eggplant results in the production of saturated SGAs (Sonawane et al., 2018). However, the mechanism underlying a lack of saturated SGA accumulation in domesticated potato is less clear. A putative GAME25 homolog is present in the genome of domesticated potato, and recombinant expression of the corresponding enzyme revealed the same activity as the tomato enzyme: 3β-hydroxyl group oxidation and isomerization of the double bond from the C-5,6 position. The potato GAME25 enzyme is active with unsaturated spirolosane- and solanidine-type SGAs although the corresponding saturated SGAs do not accumulate in domesticated potato (Sonawane et al., 2018). Overexpression of tomato GAME25 in potato hairy root cultures leads to accumulation of demissidine, a saturated 35 solanidine SGA found in wild potato. This suggests that the downstream enzymatic activities involved in the production of saturated SGAs exist in domesticated potato (Lee et al., 2019). However, the mechanism leading to the lack of saturated SGAs in domesticated potato remains unclear, and the in vivo function of the domesticated potato GAME25 and expression levels of the corresponding gene remain to be determined (Lee et al., 2019; Sonawane et al., 2018). While the initial steps of spirolosane-type SGA formation are conserved between tomato and potato, SGA biosynthesis diverges in potato to produce solanidine-type SGAs (Akiyama et al., 2021b). Potato contains two major solanidane-type SGAs, α-solanine and α-chaconine, which differ only in the identity of the C-3 sugar additions; solanine contains galactose with rhamnose and glucose additions while chaconine contains glucose with two rhamnose additions (Akiyama et al., 2021b). The 2-oxoglutarate dependent dioxygenase, DPS (Dioxygenase for Potato Solanidane synthesis), catalyzes solanidine ring formation via C-16 hydroxylation (Akiyama et al., 2021b). While both eggplant and tomato contain DPS homologs and each recombinant enzyme is capable of C-16 hydroxylation of spirolosane-type SGAs, the expression of the corresponding genes is low or undetectable in eggplant and tomato, which likely explains the lack of solanidine-type SGAs in these species (Akiyama et al., 2021b). The DPS genes are located on chromosome 1 within a syntenic block that is conserved in Solanum and contains additional SM-related genes, suggesting that the DPS genes evolved prior to speciation (Akiyama et al., 2021b). While some wild potato species, such as Solanum chacoense, produce leptines, solanidine-type SGAs that are effective at defending against CPB, domesticated potato does not produce these SGAs. Leptine formation requires the hydroxylation of solanidine-type SGAs by GAME32 and the subsequent acetylation by an unknown enzyme. Tomato and 36 domesticated potato lack a functional GAME32 homolog and the corresponding leptine SGAs (Cárdenas et al., 2019). Domestication and selection for non-bitter fruit to aid in seed dispersal influence SGA content in tomato during fruit ripening. The fruit ripening associated biosynthesis of esculeoside A from α-tomatine alleviates the bitter taste associated with SGAs (Cárdenas et al., 2019). The hydroxylation of α-tomatine at the C-23 position is the first committed step of fruit ripening associated SGA accumulation (i.e. esculeoside A), and is catalyzed by the 2-ODD enzyme, GAME31 (Cárdenas et al., 2019; Nakayasu et al., 2020). Esculeoside A formation requires an additional hydroxylation, followed by acetylation, and the glycosylation of acetoxy- hydroxytomatine by GAME5 (Akiyama et al., 2021a; Cárdenas et al., 2019; Szymański et al., 2020). The export of α-tomatine and α-tomatine derivatives out of the vacuole by a nitrate transporter 1/peptide transporter family (NPF) transporter, GORKY (meaning bitter in Russian), is essential for esculeoside A formation (Kazachkova et al., 2021). The sequestration of toxic SGAs to the vacuole likely prevents self-toxicity, and this is evidenced by the observation that tomato plants overexpressing GORKY (facilitating SGA export to the cytosol) displayed severe morphological phenotypes (Kazachkova et al., 2021). In contrast, fruit from the same overexpression lines did not display signs of self-toxicity suggesting that the conversion of toxic/bitter SGAs to esculeosides prevents self-toxicity (Kazachkova et al., 2021). The synteny of the metabolic gene clusters involved in SGA production among Solanum species highlights the common origin of the trait that diverged between species through loss or gain of function of individual genes to create SGA diversity. Several of the genes involved in spirolosane-type SGA formation are found clustered on potato, eggplant, and tomato chromosomes 7 and 12 (Barchi et al., 2019; Itkin et al., 2013). Tomato possesses two extra genes 37 in these clusters as potato and eggplant lack homologs of GAME17 and 18, two UDP- glucosyltransferases responsible for the consecutive additions of glucose to tomatidine galactoside during α-tomatine biosynthesis in tomato (Itkin et al., 2013). Current genomic resources show that pepper (Capsicum annuum) does not possess the chromosome 12 cluster or putative orthologs of GAME4 and GAME12 found within the cluster, and this absence likely results in the lack of SGAs in C. annuum (Barchi et al., 2019). The 2-ODD genes involved in solanidine, leptine, and esculeoside SGA biosynthesis are also clustered with additional 2-ODDs of unknown function (Cárdenas et al., 2019). Changes in gene expression (i.e. low expression of DPS tomato homolog) or the presence-absence of single genes (i.e. GAME32 presence in S. chacoense) contribute to SGA diversity in Solanum. Addictive and deadly: convergent and divergent evolution shapes nicotine and tropane alkaloid metabolism Several Solanaceae genera, including Datura, Atropa, Hyoscyamus, Mandragora, and Scopolia derive medicinal and toxic qualities from the biosynthesis of tropane alkaloids. Tropane alkaloids are characterized by an eight-membered, bicyclic, nitrogen-containing core and their synthesis is reported in 10 plant families, separated by ∼120 Mya of evolution (Kim et al., 2016). For example, the well-known narcotic cocaine is synthesized by Erythroxylum coca (Erythroxylaceae) while cochlearine is synthesized in Cochlearia officinalis (Brassicaceae). The Solanaceae family has emerged as a model system for studying tropane alkaloid biosynthesis, but comparative studies reveal instances of independent evolution of tropanes in distinct plant lineages (Brock et al., 2008; Jirschitzka et al., 2012). Scopolamine and hyoscyamine are tropane aromatic esters specific to the Solanaceae, and these compounds derive their medicinal properties from anticholinergic effects, blocking activity 38 of the neurotransmitter acetylcholine. Scopolamine is used to treat a variety of illnesses including motion sickness, drooling, and for palliative care in Parkinson's disease (Clissold and Heel, 1985; Mato et al., 2010; Pérez et al., 2011). Tropane aromatic ester production requires the biosynthesis of the tropane core as well as condensation of a phenyllactic acid moiety through an ester linkage (Qiu et al., 2020). Although the biosynthesis of the tropane core intermediate and polyhydroxylated derivates, known as calystegines, occurs in many genera of the Solanaceae, including Solanum, the biosynthesis of tropane aromatic esters is restricted to the genera described above, suggesting that not all species in the family possess the genes required for their synthesis (Nash et al., 1993). Due to their medicinal importance, considerable effort has focused on understanding the biosynthesis of hyoscyamine and scopolamine. Research leading to the elucidation of scopolamine biosynthesis spanned several decades, with progress driven by the available technologies of the time. Initially, approaches focused on feeding labeled forms of potential precursors to tropane producing plants and following incorporation of label into alkaloids (Kim et al., 2016). This resulted in identification of pathway precursors and intermediates, as well as the development of an overall framework of scopolamine biosynthesis. These efforts were followed by classical biochemical approaches to purify enzymes based on activity. Peptide sequencing of the resulting purified enzymes facilitated the design of oligonucleotide probes that were labeled and used to screen cDNA libraries to identify the corresponding clones. Confirmation of function was achieved through characterization of resulting recombinant enzymes expressed in E. coli. This led to the identification of several pathway genes, including hyoscyamine 6β-hydroxylase (H6H), tropinone reductase I/II (TRI and TRII), and putrescine N-methyltransferase (PMT). The development of expressed sequence tags in the mid-2000s, coupled with virus-induced gene 39 silencing (VIGS) for in vivo testing of function, led to the identification of littorine mutase, an enzyme that catalyzes the rearrangement of littorine into hyoscyamine aldehyde (Li et al., 2006). More recently, Atropa belladonna (Deadly Nightshade) emerged as a model for exploring tropane alkaloid biosynthesis following the development of a multi-tissue transcriptome assembly and the deployment of VIGS. These resources, coupled with synthetic biology, culminated in the identification of the missing steps in scopolamine formation. The first ring of the tropane core requires the conversion of ornithine, a non- proteinogenic amino acid, into putrescine by ornithine decarboxylase (ODC). Putrescine is then N-methylated by putrescine methyltransferase (PMT) and oxidized by methylputrescine oxidase (MPO). The N-methyl-Δ1-pyrrolinium cation forms through the spontaneous cyclization of N- methylaminobutanal, the product of MPO catalysis (Figure 1.7). PMT requires S-adenosyl-L- methionine (SAM) to N-methylate putrescine and shares high sequence similarity with spermidine synthase (SPDS), an enzyme involved in transferring the aminopropyl moiety from decarboxylated SAM (dcSAM) onto putrescine to form spermidine, a ubiquitous polyamine (Junker et al., 2013; Stenzel et al., 2006). It was hypothesized that PMT evolved from a gene duplication of SPDS and subsequent neofunctionalization, and although SPDS cannot catalyze putrescine N-methylation, mutation of a single SPDS amino acid, D103I, is sufficient to generate PMT activity (Junker et al., 2013). The pyrrole moiety of nicotine, a natural product produced in the Nicotiana genus of the Solanaceae, also requires N-methyl-Δ1-pyrrolinium cation biosynthesis. The biosynthetic steps leading to N-methyl-Δ1-pyrrolinium cation formation are conserved in Nicotiana, Solanum, and Petunia allowing the N-methyl-Δ1-pyrrolinium cation to act as a core for nicotine and tropane alkaloid biosynthesis found in Solanaceae and Convolvulaceae (Kajikawa et al., 2017; Xu et al., 2017). In contrast, the genes involved in the 40 formation of the pyridine ring in nicotine biosynthesis are Nicotiana-specific indicating that divergent evolution led to the formation of nicotine, likely through the duplication of the genes in the nicotinamide adenine dinucleotide (NAD) cofactor biosynthetic pathway (Xu et al., 2017). 41 Figure 1.7. Evolutionary trajectories of tropane and nicotine formation in distinct plant lineages. Comparison of tropane and nicotine alkaloid biosynthesis reveals examples of both convergent (cocaine biosynthesis in E. coca) and divergent (nicotine biosynthesis) evolution (Jirschitzka et al., 2012; Xu et al., 2017). Scopolamine (orange) and nicotine (purple) represent alternative fates of the N-methylpyrrolinium cation in different genera of the Solanaceae. The use 42 Figure 1.7. (cont’d) of an aldo-keto reductase enzyme (MecgoR) in the penultimate step of cocaine biosynthesis (blue) contrasts with catalysis by short-chain dehydrogenase/reductase (SDR) family enzymes (TRI and TRII) in scopolamine formation (green) (Jirschitzka et al., 2012). *Not shown is catalysis by a single, bifunctional SDR to produce both tropine and pseudotropine in Brassicaceae (Brock et al., 2008). Tropanol biosynthesis (green) is widely distributed across the Solanaceae compared to the biosynthesis of tropane aromatic esters such as scopolamine (orange) (Nash et al., 1993). Enzyme abbreviations are as follows: PMT2, Putrescine N- methyltransferase 2; MPO2, N-methylputrescine oxidase 2; PyKS, Polyketide Synthase; TRI, Tropinone reductase I; TRII, Tropinone Reductase II; MecgoR, Methylecgonone reductase. Formation of the tropane core in Solanaceae species requires a second cyclization event that yields tropinone, which possesses a ketone functional group at the carbon-3 position of the core (Figure 1.7). The first step in tropinone formation is catalyzed by a type III polyketide synthase, PYKS, which uses the N-methyl-Δ1-pyrrolinium cation and malonyl-Coenzyme A to form 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid (Bedewitz et al., 2018). Although PYKS can form 3-oxoglutaric acid without the N-methyl-Δ1-pyrrolinium cation and these two products can react non-enzymatically, the exact mechanism of 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid formation remains unclear (Huang et al., 2019; Nett et al., 2021). Tropinone synthase (CYP82M3) converts 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid to tropinone (Bedewitz et al., 2018). Although putative orthologs of PYKS and CYP82M3 are present in the genomes of several calystegine producing Solanaceae species including tomato, potato, and pepper, these genes are absent in Nicotiana spp.; this is consistent with the lack of detectable tropanes in these species (Bedewitz et al., 2018). In the Solanaceae, tropinone reductases I and II are members of the short-chain dehydrogenase/reductase superfamily (SDR) that catalyze the reduction of the ketone of tropinone to an alcohol to form tropine (3α-hydroxytropine) and pseudotropine (3β- hydroxytropine), respectively (Nakajima et al., 1993). TRI and TRII constitute a branch point in the tropane alkaloid biosynthetic pathway due to their stereospecificity: TRI leads to the 43 production of tropane aromatic esters, including hyoscyamine and scopolamine and TRII directs flux towards calystegine production. Biosynthesis of the principal aromatic tropane esters in the Solanaceae, littorine, hyoscyamine, and scopolamine, requires the diversion of phenylalanine into the tropane pathway through a two-step process that yields phenyllactic acid (Bedewitz et al., 2014; Qiu et al., 2018) (Figure 1.8). Identification of the aromatic aminotransferase (AbArAT4) responsible for conversion of phenylalanine into phenylpyruvate revealed the power of transcriptomics in Solanaceae tropane alkaloid enzyme discovery (Bedewitz et al., 2014). Analogous to bacterial aromatic amino acid biosynthesis, a cytosolic aromatic aminotransferase from petunia (Ph-PPY- AT) catalyzes the formation of phenylalanine from phenylpyruvate using tyrosine as an amino donor and yielding 4-hydroxyphenylpyruvate (Yoo et al., 2013). AbArAT4 is related to Ph-PPY- AT and utilizes the same four substrates, but the Atropa enzyme diverts phenylalanine into the tropane pathway by virtue of a ∼250-fold more active reverse reaction that yields phenylpyruvate and tyrosine. AbArAT4 is co-expressed in the roots with other tropane-related genes, and while silencing of this gene disrupts tropane alkaloid biosynthesis, it does not alter aromatic amino acid pools, further supporting its neofunctionalized and specific role in specialized metabolism (Bedewitz et al., 2014). Littorine biosynthesis requires the glycosylation of phenyllactate by a UDP-glucose dependent glycosyltransferase followed by the acylation of tropine. The serine carboxypeptidase-like (SCPL) acyltransferase (littorine synthase) acylates tropine using glycosylated phenyllactate as the acyl donor (Qiu et al., 2020). 44 Figure 1.8. Independent evolution of tropane aromatic ester formation in Solanaceae and Erythroxylaceae. Scopolamine biosynthesis requires the biosynthesis of D-phenyllactic acid via a two-step process mediated by ArAT4 and PPAR (Bedewitz et al., 2014; Qiu et al., 2018). D- Phenyllactic acid is glycosylated by UGT1 to form a glucose ester of phenyllactic acid, which is used, along with tropine, as substrate for littorine biosynthesis by Littorine Synthase, a serine carboxypeptidase-like acyltransferase (Qiu et al., 2020). Three enzymes, Littorine Mutase, HDH, and H6H, are required for the conversion of littorine to scopolamine (Hashimoto and Yamada, 1986; Li et al., 2006; Srinivasan and Smolke, 2020). In contrast, cocaine biosynthesis utilizes a BAHD acyl-transferase and coenzyme A donor to facilitate the transfer of a benzoyl moiety on to methylecgonine, the E. coca tropanol, to form cocaine (Schmidt et al., 2015). Enzyme abbreviations are as follows: ArAT4, aromatic amino acid transferase 4; PPAR, phenylpyruvic acid reductase; UGT1, UDP-glycosyltransferase 1; HDH, hyoscyamine dehydrogenase; H6H, 45 Figure 1.8. (cont’d) hyoscyamine-6-hydroxylase. Synthetic biology recently was utilized both to engineer scopolamine production in yeast and facilitate the discovery of the final missing enzyme in the pathway, which had eluded discovery using in planta experiments. The conversion of littorine to scopolamine requires four steps catalyzed by three enzymes (Figure 1.8). Littorine mutase, a cytochrome P450, catalyzes the rearrangement of littorine to hyoscyamine aldehyde (Li et al., 2006), which is converted to hyoscyamine by hyoscyamine aldehyde dehydrogenase. Finally, hyoscyamine-6-hydroxylase catalyzes the two-step hydroxylation and epoxidation of hyoscyamine to scopolamine (Hashimoto and Yamada, 1986). The production of scopolamine in yeast was achieved through the introduction of tropane alkaloid pathway genes from several species, including Datura stramonium, Datura metel, and Atropa belladonna (Srinivasan and Smolke, 2020). Optimization of scopolamine production in yeast required the elimination of several native genes to reduce the flow of tropane alkaloid intermediates into side products and the introduction of a transporter from Nicotiana tabacum to facilitate transport of tropine into the vacuole for esterification with phenyllactic acid (Srinivasan and Smolke, 2020). Notably, the introduction of the pathway into yeast revealed the dehydrogenase responsible for the reduction of hyoscyamine aldehyde into hyoscyamine, which had not previously been identified in planta (Srinivasan and Smolke, 2020). For example, silencing of this gene in A. belladonna did not result in a decrease in downstream tropane alkaloids, likely due to promiscuous enzymatic activity of other dehydrogenases (Qiu et al., 2021). Hence, reconstruction of the pathway in a genetic host where background activities were removed facilitated the identification of the final missing step in the scopolamine pathway. 46 Independent evolution of tropanes in distinct plant lineages Evidence for independent evolution of tropanes in distinct plant lineages is manifest at different steps throughout the pathway (Figures 1.7 and 1.8). While separate TRI and TRII enzymes reduce tropinone to tropine or pseudotropine in the Solanaceae, a single SDR enzyme catalyzes both reactions in C. officinalis, ultimately leading to tropine-derived cochlearine and pseudotropine-derived calystegines (Brock et al., 2008). In addition, while Solanaceae and Brassicaceae species utilize enzymes in the SDR family for the reduction of tropinone, the analogous reaction in E. coca cocaine biosynthesis, the reduction of methylecgonone to methylecgonine, is catalyzed by methylecgonone reductase (MecgoR) a member of the aldo-keto reductase family (Jirschitzka et al., 2012). Similarly, aromatic tropane ester biosynthesis is catalyzed by different classes of acyltransferases in the Solanaceae and Erythroxylaceae. Littorine formation is synthesized by an SCPL acyltransferase while cocaine synthase, which catalyzes the condensation of methylecgonine and benzoyl-CoA, is a member of the BAHD acyltransferase family (Schmidt et al., 2015). As additional tropane pathways in distinct plant lineages are elucidated it is likely that further examples of independent evolution will be discovered. Challenges and unexplored frontiers in Solanaceae metabolism There has been a rapid increase in understanding the biosynthesis and evolution of plant SM pathways during the last decade. Advances in genomics enabled gene–metabolite correlations in model and non-model species. These data – combined with development of methods to test gene function in diverse species, and transient expression in Nicotiana benthamiana, as well as engineering production in microbial systems – led to the elucidation of multiple plant SM pathways and identified regulators of known SM pathways (Fossati et al., 47 2014; Lau and Sattely, 2015; Y. Li et al., 2020; Nett et al., 2020; Srinivasan and Smolke, 2020). The widespread adoption of these approaches, coupled with phylogeny-guided comparative genomics and metabolomics, enabled exploration of the evolutionary trajectories of the exemplary Solanaceae SM pathways described here. However, despite advances in understanding Solanaceae SM biosynthesis and evolution, knowledge gaps persist related to specific aspects of these well-studied pathways and opportunities exist to develop a more comprehensive understanding of these pathways and networks. As evidenced through studies of acylsugar evolution, much can be learned through adopting a broader sampling strategy to include more phylogenetically diverse species that are typically less well studied (Lou et al., 2021; Moghe et al., 2017; Nadakuduti et al., 2017). Similar, phylogenetic-guided metabolite screening approaches could be adopted to assess chemical diversity in other SM classes as the foundation for exploring metabolite evolution using comparative genomics. For example, given the tremendous chemical variation observed in trichome-derived acylsugars across the Solanaceae, and that novel acylsugars were recently identified in root and root-exudates of tomato (Korenblum et al., 2020), it will be intriguing to determine whether comparable root acylsugar diversity exists across the family and if so, to assess how this diversity evolved. There are also several examples where the biosynthesis of exemplary SM pathways in the Solanaceae are not fully resolved. For example, the enzymes that catalyze the early steps in acylinositol biosynthesis in Solanum spp. are yet to be reported. Similarly, the majority of the enzymes involved in capsaicinoid biosynthesis and the final steps in nicotine biosynthesis await biochemical and functional characterization (Naves et al., 2019; Xu et al., 2017). In addition, although the biosynthesis of scopolamine is elucidated and the pathway reconstructed in yeast, 48 the steps leading to the biosynthesis of other classes of Solanaceae tropanes, including calystegines and schizanthines, are unknown (Christen et al., 2020; Kim et al., 2016). Comparative analyses of the evolution of SM-related gene clusters across the Solanaceae also remains under-explored. For example, as outlined in this review, terpene and SGA-related gene clusters exist in Solanum but variation across these clusters is mainly documented in a few model species, including tomato, potato, eggplant, and closely related wild species (Barchi et al., 2019; Itkin et al., 2013; Zhou and Pichersky, 2020b). Indeed, even for the comparatively well- studied terpenoid-related gene clusters of tomato, many of the enzymes that reside within these clusters, which may catalyze modifications of terpene scaffolds, remain uncharacterized. Furthermore, the extent of conservation of terpene and other SM gene clusters across the Solanaceae is unknown. As multiple chromosome scale genome assemblies of phylogenetically diverse Solanaceae species are available and others will likely be generated soon, charting the evolutionary trajectories of SM gene clusters and the metabolite variation they encode is now possible. Finally, it is also worth noting that the most extensively characterized Solanaceae SM pathways are those where the identities of the major metabolites were known for decades and their abundance is high in specific cell types or tissues, facilitating purification and structural elucidation. It is more challenging to identify unknown metabolites and purify metabolites that are of low abundance and technical challenges persist that impede a more comprehensive understanding of metabolism and bridging of the gap between genotype and phenotype. Challenges in the identification and annotation of SM enzymes Advances in DNA sequencing are making development of chromosome-scale genome assemblies more routine and recently several Solanaceae genomes were released, and the quality 49 of existing assemblies improved (Alonge et al., 2020; Barchi et al., 2021; Michael and VanBuren, 2020). These studies allow the gene complement of an organism to be determined. However, functional annotation of plant genomes remains incomplete, even for model species. The lack of accurate annotation is particularly problematic for large gene families encoding SM- related enzymes that catalyze common decorations of scaffold molecules, including cytochromes P450, 2-oxoglutarate dependent dioxygenases, glycosyltransferases, and acyltransferases. SM- related enzymes are often catalytically promiscuous and encoded by genes that evolved rapidly through duplication and associated subfunctionalization, neofunctionalization, and gene loss (Weng et al., 2012). Thus, annotation of SM enzymes based solely on sequence similarity, predicted orthology, or synteny is often misleading. This concept is clearly illustrated by examples identified through studying the evolution of acylsugar and terpene biosynthesis in Solanum glandular trichomes. These studies reveal how activity can be altered by a few amino acid differences in closely related enzymes from sister species, or diverse accessions within a species (Fan et al., 2015; Kang et al., 2014; Zabel et al., 2021). Hence, empirical determination of enzyme function remains imperative. Although characterization of enzyme activities is often technically challenging, time consuming, and limited by substrate availability, medium and high- throughput methods based on microtiter plates and microfluidics are utilized for screening natural and computationally designed enzymes and such methods could potentially be adapted for screening the activity of plant SM-related enzymes (Bunzel et al., 2018). As documented throughout this review, co-expression is a powerful approach for predicting membership of genes in metabolic pathways, particularly when there is a priori knowledge about enzymes from the target pathway. Elucidation of the pathway leading to scopolamine biosynthesis, described above, is an excellent example of the use of co-expression 50 analyses to identify candidate genes co-expressed in roots. However, when results of co- expression analysis are ambiguous or multiple candidate genes are identified, as is often the case when investigating large SM-related gene families, additional filtering and refinement of gene candidates may be required prior to time-consuming functional studies. In such cases, comparative genomic analysis such as synteny or gene-cluster analysis – together with phylogenetic analysis to determine whether gene candidates exhibit lineage-specific distribution or arose through a recent duplication event – provide opportunities for refining candidate gene lists (Jacobowitz and Weng, 2020). Outside of tomato, there is a lack of publicly available transcriptome data, including data from diverse tissues, environmental perturbations, and treatments. This limits novel metabolite pathway discovery in diverse Solanaceae species and reduces the resolution of studies investigating the phylogenetic distribution and evolution of SM pathways. Furthermore, plant SM pathways are often restricted to specific cell types, and therefore the general focus on whole tissue sampling for transcriptome analysis can be limiting (Courdavault et al., 2014; Leong et al., 2019; Onoyovwe et al., 2013). The recent development of single-cell and single-nucleus transcriptome analyses holds great promise for increasing the resolution of transcriptome data and refining candidate gene lists to facilitate the identification, characterization, and cellular localization of Solanaceae SM pathways (Ryu et al., 2019; Seyfferth et al., 2021). Machine learning is another promising approach to distinguish GM and SM-related enzymes without prior knowledge of pathway membership or gene–metabolite correlation information. Multiple features including gene expression, transcriptional network analysis, rate of evolution, and duplication mechanism allowed creation of statistical models that can distinguish GM from SM genes in Arabidopsis. In agreement with the established characteristics 51 of SM genes, machine learning models revealed that relative to GM genes, SM genes tend to be less conserved, tandemly duplicated, more narrowly expressed, and expressed at lower levels (Moore et al., 2019). The prediction models also facilitated the classification of 1220 enzyme encoding genes of unknown function as putatively SM-related. Similar machine learning strategies were deployed in tomato to predict gene association with SM or GM pathways and to determine if gene expression data can predict metabolic pathway membership (Moore et al., 2020; Wang et al., 2021). These approaches show potential to build high-quality models but are limited by the quality of the input data, including mis-annotations and the low number of functionally validated reference genes in tomato. These current limitations suggest that application of machine learning for de novo prediction of novel SM pathways in tomato is not yet possible at high accuracy. Furthermore, additional functional annotation, including the development of more comprehensive genome and transcriptome data, will be needed to apply machine learning approaches to predict SM pathway membership in additional members of the Solanaceae. Indeed, models predicting whether a tomato gene is associated with specialized versus general metabolism were improved when a transfer learning strategy was employed that utilized data from Arabidopsis models to filter tomato annotations that disagreed with Arabidopsis (Moore et al., 2020). This represents a promising approach to using comparative genomics data in specialized metabolic enzyme identification. Challenges in the identification and annotation of plant metabolites. Estimates suggest that ∼106 metabolites are synthesized across species of the plant kingdom, collectively (Afendi et al., 2012). While we have deep knowledge of well-studied classes of plant metabolites, opportunities and challenges for improving metabolome annotation remain. Several factors make separation and annotation of metabolites challenging: for example, 52 their diverse chemical composition, chemical properties (polarity and hydrophilicity/hydrophobicity), and the orders of magnitude concentration range in which they occur in biological samples (Last et al., 2007; Perez de Souza et al., 2021). Improvements in analytical techniques, particularly liquid-chromatography coupled with high-resolution mass- spectrometry (LC-HRMS) based metabolite profiling, allows the detection of >103 metabolites within a single plant extract at high mass accuracy. However, a single extraction solvent and chromatographic separation method are generally selected for individual experiments, leading to unavoidable bias in the types of metabolites that are extracted and resolved and therefore an under-representation of the metabolome (Perez de Souza et al., 2021). Furthermore, most metabolites in a plant extract are uncharacterized and many are of low abundance. In such cases, annotation can be challenging. This is particularly true for specialized metabolites that are formed from diverse metabolic precursors, possess multiple chemical modifications, and frequently exist as positional or structural isomers that may be difficult to resolve. For example, even though tomato fruit ripening is one of the most extensively studied plant biological processes, a large component of this metabolome remains unannotated. In a recent study, untargeted metabolomics of tomato fruit at two different developmental stages identified >1000 semi-lipophilic metabolites but only ∼170 metabolites were annotated with some degree of confidence, suggesting that the bulk of the tomato fruit metabolome remains unresolved (Szymański et al., 2020). Metabolite databases containing spectra derived from tandem mass- spectrometry of known metabolites are expanding and are useful for identifying unknown metabolites (Horai et al., 2010; Tsugawa et al., 2019; Wang et al., 2016). However, given the vast diversity of plant metabolites and their frequent lineage-specific distribution, populating and 53 curating such databases requires substantial research funding, effort, and community engagement. As with spatially resolved or single cell transcriptomics, the ability to obtain spatially resolved metabolome data through mass spectrometry imaging of plant tissues represents an exciting development that will enhance understanding of metabolism. Specifically, this technology will further refine the ability to detect gene–metabolite correlations and allow the detection of metabolites that may be restricted to individual cell types and therefore fall below the limit of detection in an extract prepared from a complex tissue sample (Sumner et al., 2015). Mass spectrometry imaging has been utilized for investigating the spatial distribution of metabolites in tomato fruit, including investigating the influence of genetic perturbation on SGA accumulation (Dong et al., 2020). Similarly, the spatial separation of SGAs and acylsugars were demonstrated in tomato roots (Korenblum et al., 2020). As improved MSI technologies develop and increase in availability, they will undoubtedly be more widely adopted for exploring diverse aspects of Solanaceae metabolism. Integration of genetic variation with metabolomics is a powerful approach to expand understanding of SM metabolic networks and bridge the gap between genotype and phenotype. As described above, both GWAS and metabolite QTL (mQTL) approaches were used to identify genomic regions and genes that influence specialized metabolism in diverse tissues of tomato. In particular, the S. lycopersicum x S. pennellii introgression line and the related backcross introgression line (BIL) populations were foundational to improving understanding of the loci that influence metabolism within the tomato clade (Alseekh et al., 2020; Cárdenas et al., 2019; Garbowicz et al., 2018; Schilmiller et al., 2010; Szymański et al., 2020). Approaches that harness natural variation are limited to species where it is possible to develop inter-specific genetic 54 populations or sufficient genetic variation is present within a species, to facilitate GWAS. Although not currently as extensively characterized as the genetic resources for tomato, germplasm panels and genetic populations, including introgression lines, are being developed and characterized for the three additional major food crops of the Solanaceae; potato, pepper, and eggplant (Gramazio et al., 2017; Hirsch et al., 2013; Tripodi et al., 2021). In some cases, these genetic resources are being utilized to investigate metabolic diversity via targeted and untargeted metabolomics and refinement of these efforts should facilitate linking genotype to phenotype (Levina et al., 2021; Sulli et al., 2021). An alternative, less frequently utilized, approach to harness genetic variation to interrogate metabolism is to combine untargeted metabolite profiling with targeted disruption or over-expression of known enzymes or transcription factors (Tzin et al., 2012; Zhang et al., 2015). This approach, while more targeted than a strategy incorporating genome-wide genetic variation, can be utilized in any species where genetic manipulation is feasible and has significant potential to increase understanding of plant SM networks. For example, disruption of an SM enzyme will result in reduction of metabolites downstream of the enzyme, while the abundance of metabolites upstream of the target enzyme can increase. This approach also allows detection of alternate fates for pathway metabolites that accumulate due to gene disruption, revealing the existence of biosynthetically linked metabolites. Referred to as “silent metabolism” this component of the metabolome is likely substantial and certainly under-explored, including for engineering of novel products (Lewinsohn and Gijzen, 2009). Furthermore, as SM enzymes possess increased tendency for catalytic promiscuity, untargeted metabolite profiling of lines disrupted in an enzyme of interest may reveal the existence of previously uncharacterized catalytic activities. 55 While purification and structural elucidation of metabolites by NMR is a cornerstone of SM pathway discovery, it is time-consuming and typically represents a major bottleneck. This is especially problematic for metabolites that are of low abundance or co-purify with other compounds. Recent structural elucidation of acyl-hexoses from S. nigrum was achieved using a combination of LC-MS, GC-MS, and 2D-NMR approaches from crude and partially purified extracts without purification to homogeneity (Lou et al., 2021). Similar approaches should be adaptable to resolve the structures of other metabolites present in semi-purified plant extracts. The recent adoption of microcrystal electron diffraction (MicroED) for structural elucidation, including absolute stereochemistry, of mixtures of small organic molecules also shows great promise for structural elucidation of plant specialized metabolites (Gruene et al., 2018; Jones et al., 2018). MicroED can be used to resolve the structures of nanocrystals of ∼100 nm (∼10−15 g) and thus is potentially more suitable for low abundance metabolites than NMR, which typically requires hundreds of micrograms to milligram quantities of purified compound. Application of this technology to specialized metabolite discovery was recently demonstrated through a combined genome-mining, synthetic biology, and MicroED analysis that elucidated the biosynthesis and structures of several 2-pyrridone metabolites from fungi (Kim et al., 2021). Similarly, synthetic biology can be utilized to engineer production of plant SMs in heterologous systems for subsequent purification and structural elucidation. This strategy was effectively demonstrated by the synthesis of gram scale quantities of the triterpene β-amyrin by vacuum infiltration of N. benthamiana co-expressing a feedback insensitive variant of HMG-CoA reductase and oat β-amyrin synthase (Reed et al., 2017). Subsequent experiments combining co- expression of these enzymes with triterpene decorating cytochrome P450s from multiple species facilitated the production of novel non-natural triterpenes at sufficient scale to allow purification 56 and structural determination by NMR. N. benthamiana is widely used for transient expression of candidate genes and as demonstrated above, represents a readily scalable platform to produce metabolites for purification and subsequent structural elucidation. Conclusions Advances in genomics and metabolomics continue to enable greater understanding of SM pathway biosynthesis and evolution. This review focused on the catalytic steps of five well- studied SM classes that show varying degrees of lineage-specific distribution across the Solanaceae. This genetic variation, coupled with high abundance, and often restricted distribution in specific tissue or cell types, facilitated both purification and structural elucidation of these diverse metabolites as well as the identification of the enzymes responsible for their biosynthesis. For example, acylsugar and terpene biosynthesis in glandular trichomes, nicotine and tropane alkaloid biosynthesis in roots, and capsaicinoid biosynthesis in pepper fruit placenta. These studies reveal examples of both intra- and inter-specific variation as well as convergent evolution that has shaped the metabolic landscape across the Solanaceae. However, only a small fraction of the metabolome and the genes responsible for its formation are resolved. Thus, many opportunities exist to expand understanding of known pathways as well as identify novel pathways that will enable a network level understanding of metabolism across the Solanaceae and identify target molecules for agricultural and medicinal applications. 57 REFERENCES Afendi, F.M., Okada, T., Yamazaki, M., Hirai-Morita, A., Nakamura, Y., Nakamura, K., Ikeda, S., Takahashi, H., Altaf-Ul-Amin, Md., Darusman, L.K., Saito, K., Kanaya, S., 2012. KNApSAcK family databases: integrated metabolite–plant species databases for multifaceted plant research. Plant Cell Physiol. 53, e1. https://doi.org/10.1093/pcp/pcr165 Akhtar, T.A., Matsuba, Y., Schauvinhold, I., Yu, G., Lees, H.A., Klein, S.E., Pichersky, E., 2013. The tomato cis–prenyltransferase gene family. Plant J. 73, 640–652. https://doi.org/10.1111/tpj.12063 Akiyama, R., Lee, H.J., Nakayasu, M., Osakabe, K., Osakabe, Y., Umemoto, N., Saito, K., Muranaka, T., Sugimoto, Y., Mizutani, M., 2019. Characterization of steroid 5α- reductase involved in α-tomatine biosynthesis in tomatoes. Plant Biotechnol. 36, 253– 263. https://doi.org/10.5511/plantbiotechnology.19.1030a Akiyama, R., Nakayasu, M., Umemoto, N., Kato, J., Kobayashi, M., Lee, H.J., Sugimoto, Y., Iijima, Y., Saito, K., Muranaka, T., Mizutani, M., 2021a. Tomato E8 encodes a C-27 hydroxylase in metabolic detoxification of α-tomatine during fruit ripening. Plant Cell Physiol. 62, 775–783. https://doi.org/10.1093/pcp/pcab080 Akiyama, R., Watanabe, B., Nakayasu, M., Lee, H.J., Kato, J., Umemoto, N., Muranaka, T., Saito, K., Sugimoto, Y., Mizutani, M., 2021b. The biosynthetic pathway of potato solanidanes diverged from that of spirosolanes due to evolution of a dioxygenase. Nat. Commun. 12, 1300. https://doi.org/10.1038/s41467-021-21546-0 Alonge, M., Wang, X., Benoit, M., Soyk, S., Pereira, L., Zhang, L., Suresh, H., Ramakrishnan, S., Maumus, F., Ciren, D., Levy, Y., Harel, T.H., Shalev-Schlosser, G., Amsellem, Z., Razifard, H., Caicedo, A.L., Tieman, D.M., Klee, H., Kirsche, M., Aganezov, S., Ranallo-Benavidez, T.R., Lemmon, Z.H., Kim, J., Robitaille, G., Kramer, M., Goodwin, S., McCombie, W.R., Hutton, S., Van Eck, J., Gillis, J., Eshed, Y., Sedlazeck, F.J., van der Knaap, E., Schatz, M.C., Lippman, Z.B., 2020. Major Impacts of Widespread Structural Variation on Gene Expression and Crop Improvement in Tomato. Cell 182, 145-161.e23. https://doi.org/10.1016/j.cell.2020.05.021 Alseekh, S., Ofner, I., Liu, Z., Osorio, S., Vallarino, J., Last, R.L., Zamir, D., Tohge, T., Fernie, A.R., 2020. Quantitative trait loci analysis of seed-specialized metabolites reveals seed- specific flavonols and differential regulation of glycoalkaloid content in tomato. Plant J. 103, 2007–2024. https://doi.org/10.1111/tpj.14879 Asai, T., Fujimoto, Y., 2011. 2-Acety-1-(3-glycosyloxyoctadecanoyl)glycerol and dammarane triterpenes in the exudates from glandular trichome-like secretory organs on the stipules and leaves of Cerasus yedoensis. Phytochem. Lett. 4, 38–42. https://doi.org/10.1016/j.phytol.2010.11.001 Asai, T., Fujimoto, Y., 2010. Cyclic fatty acyl glycosides in the glandular trichome exudate of Silene gallica. Phytochemistry 71, 1410–1417. https://doi.org/10.1016/j.phytochem.2010.05.008 58 Asai, T., Hara, N., Fujimoto, Y., 2010. Fatty acid derivatives and dammarane triterpenes from the glandular trichome exudates of Ibicella lutea and Proboscidea louisiana. Phytochemistry 71, 877–894. https://doi.org/10.1016/j.phytochem.2010.02.013 Asai, T., Sakai, T., Ohyama, K., Fujimoto, Y., 2011. n-Octyl α-L-rhamnopyranosyl-(1→2)-β-D- glucopyranoside derivatives from the glandular trichome exudate of Geranium carolinianum. Chem. Pharm. Bull. (Tokyo) 59, 747–752. https://doi.org/10.1248/cpb.59.747 Bah, M., Pereda-Miranda, R., 1996. Detailed FAB-mass spectrometry and high resolution NMR investigations of tricolorins A-E, individual oligosaccharides from the resins of Ipomoea tricolor (Convolvulaceae). Tetrahedron 52, 13063–13080. https://doi.org/10.1016/0040- 4020(96)00789-2 Barchi, L., Pietrella, M., Venturini, L., Minio, A., Toppino, L., Acquadro, A., Andolfo, G., Aprea, G., Avanzato, C., Bassolino, L., Comino, C., Molin, A.D., Ferrarini, A., Maor, L.C., Portis, E., Reyes-Chin-Wo, S., Rinaldi, R., Sala, T., Scaglione, D., Sonawane, P., Tononi, P., Almekias-Siegl, E., Zago, E., Ercolano, M.R., Aharoni, A., Delledonne, M., Giuliano, G., Lanteri, S., Rotino, G.L., 2019. A chromosome-anchored eggplant genome sequence reveals key events in Solanaceae evolution. Sci. Rep. 9, 11769. https://doi.org/10.1038/s41598-019-47985-w Barchi, L., Rabanus-Wallace, M.T., Prohens, J., Toppino, L., Padmarasu, S., Portis, E., Rotino, G.L., Stein, N., Lanteri, S., Giuliano, G., 2021. Improved genome assembly and pan- genome provide key insights into eggplant domestication and breeding. Plant J. 107, 579–596. https://doi.org/10.1111/tpj.15313 Bedewitz, M.A., Góngora-Castillo, E., Uebler, J.B., Gonzales-Vigil, E., Wiegert-Rininger, K.E., Childs, K.L., Hamilton, J.P., Vaillancourt, B., Yeo, Y.-S., Chappell, J., DellaPenna, D., Jones, A.D., Buell, C.R., Barry, C.S., 2014. A root-expressed l-phenylalanine:4- hydroxyphenylpyruvate aminotransferase is required for tropane alkaloid biosynthesis in Atropa belladonna. Plant Cell 26, 3745–3762. https://doi.org/10.1105/tpc.114.130534 Bedewitz, M.A., Jones, A.D., D’Auria, J.C., Barry, C.S., 2018. Tropinone synthesis via an atypical polyketide synthase and P450-mediated cyclization. Nat. Commun. 9, 5281. https://doi.org/10.1038/s41467-018-07671-3 Bernal, C.-A., Castellanos, L., Aragón, D.M., Martínez-Matamoros, D., Jiménez, C., Baena, Y., Ramos, F.A., 2018. Peruvioses A to F, sucrose esters from the exudate of Physalis peruviana fruit as α-amylase inhibitors. Carbohydr. Res. 461, 4–10. https://doi.org/10.1016/j.carres.2018.03.003 Bleeker, P.M., Diergaarde, P.J., Ament, K., Schütz, S., Johne, B., Dijkink, J., Hiemstra, H., de Gelder, R., de Both, M.T.J., Sabelis, M.W., Haring, M.A., Schuurink, R.C., 2011. Tomato-produced 7-epizingiberene and R-curcumene act as repellents to whiteflies. Phytochemistry 72, 68–73. https://doi.org/10.1016/j.phytochem.2010.10.014 59 Bleeker, P.M., Mirabella, R., Diergaarde, P.J., VanDoorn, A., Tissier, A., Kant, M.R., Prins, M., de Vos, M., Haring, M.A., Schuurink, R.C., 2012. Improved herbivore resistance in cultivated tomato with the sesquiterpene biosynthetic pathway from a wild relative. Proc. Natl. Acad. Sci. U.S.A. 109, 20124–20129. https://doi.org/10.1073/pnas.1208756109 Boutanaev, A.M., Moses, T., Zi, J., Nelson, D.R., Mugford, S.T., Peters, R.J., Osbourn, A., 2015. Investigation of terpene diversification across multiple sequenced plant genomes. Proc. Natl. Acad. Sci. U.S.A. 112, E81–E88. https://doi.org/10.1073/pnas.1419547112 Brock, A., Brandt, W., Dräger, B., 2008. The functional divergence of short-chain dehydrogenases involved in tropinone reduction. Plant J. 54, 388–401. https://doi.org/10.1111/j.1365-313X.2008.03422.x Brooks, C., Nekrasov, V., Lippman, Z.B., Van Eck, J., 2014. Efficient gene editing in tomato in the first generation using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated9 System. Plant Physiol. 166, 1292–1297. https://doi.org/10.1104/pp.114.247577 Bunzel, H.A., Garrabou, X., Pott, M., Hilvert, D., 2018. Speeding up enzyme discovery and engineering with ultrahigh-throughput methods. Curr. Opin. Struct. Biol. 48, 149–156. https://doi.org/10.1016/j.sbi.2017.12.010 Cao, C.-M., Wu, X., Kindscher, K., Xu, L., Timmermann, B.N., 2015. Withanolides and sucrose esters from Physalis neomexicana. J. Nat. Prod. 78, 2488–2493. https://doi.org/10.1021/acs.jnatprod.5b00698 Cárdenas, P.D., Sonawane, P.D., Heinig, U., Jozwiak, A., Panda, S., Abebie, B., Kazachkova, Y., Pliner, M., Unger, T., Wolf, D., Ofner, I., Vilaprinyo, E., Meir, S., Davydov, O., Gal- on, A., Burdman, S., Giri, A., Zamir, D., Scherf, T., Szymanski, J., Rogachev, I., Aharoni, A., 2019. Pathways to defense metabolites and evading fruit bitterness in genus Solanum evolved through 2-oxoglutarate-dependent dioxygenases. Nat. Commun. 10, 5169. https://doi.org/10.1038/s41467-019-13211-4 Carrizo García, C., Barfuss, M.H.J., Sehr, E.M., Barboza, G.E., Samuel, R., Moscone, E.A., Ehrendorfer, F., 2016. Phylogenetic relationships, diversification and expansion of chili peppers (Capsicum, Solanaceae). Ann. Bot. 118, 35–51. https://doi.org/10.1093/aob/mcw079 Caterina, M.J., Leffler, A., Malmberg, A.B., Martin, W.J., Trafton, J., Petersen-Zeitz, K.R., Koltzenburg, M., Basbaum, A.I., Julius, D., 2000. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 288, 306–313. https://doi.org/10.1126/science.288.5464.306 Chortyk, O.T., Kays, S.J., Teng, Q., 1997. Characterization of insecticidal sugar esters of Petunia. J. Agric. Food Chem. 45, 270–275. https://doi.org/10.1021/jf960322f 60 Chortyk, O.T., Severson, R.F., Cutler, H.C., Sisson, V.A., 1993. Antibiotic activities of sugar esters isolated from selected Nicotiana species. Biosci. Biotechnol. Biochem. 57, 1355– 1356. https://doi.org/10.1271/bbb.57.1355 Christen, P., Cretton, S., Humam, M., Bieri, S., Muñoz, O., Joseph-Nathan, P., 2020. Chemistry and biological activity of alkaloids from the genus Schizanthus. Phytochem. Rev. 19, 615–641. https://doi.org/10.1007/s11101-018-9598-5 Cicchetti, E., Duroure, L., Le Borgne, E., Laville, R., 2018. Upregulation of skin-aging biomarkers in aged NHDF cells by a sucrose ester extract from the agroindustrial waste of Physalis peruviana calyces. J. Nat. Prod. 81, 1946–1955. https://doi.org/10.1021/acs.jnatprod.7b01069 Clissold, S.P., Heel, R.C., 1985. Transdermal hyoscine (scopolamine). Drugs 29, 189–207. https://doi.org/10.2165/00003495-198529030-00001 Coates, R.M., Denissen, J.F., Juvik, J.A., Babka, B.A., 1988. Identification of alpha-santalenoic and endo-beta-bergamotenoic acids as moth oviposition stimulants from wild tomato leaves. J. Org. Chem. 53, 2186–2192. https://doi.org/10.1021/jo00245a012 Courdavault, V., Papon, N., Clastre, M., Giglioli-Guivarc’h, N., St-Pierre, B., Burlat, V., 2014. A look inside an alkaloid multisite plant: the Catharanthus logistics. Curr. Opin. Plant Biol., SI: Physiology and metabolism 19, 43–50. https://doi.org/10.1016/j.pbi.2014.03.010 Dolan, L.C., Matulka, R.A., Burdock, G.A., 2010. Naturally occurring food toxins. Toxins 2, 2289–2332. https://doi.org/10.3390/toxins2092289 Dong, Y., Sonawane, P., Cohen, H., Polturak, G., Feldberg, L., Avivi, S.H., Rogachev, I., Aharoni, A., 2020. High mass resolution, spatial metabolite mapping enhances the current plant gene and pathway discovery toolbox. New Phytol. 228, 1986–2002. https://doi.org/10.1111/nph.16809 Duranova, H., Valkova, V., Gabriny, L., 2022. Chili peppers (Capsicum spp.): the spice not only for cuisine purposes: an update on current knowledge. Phytochem. Rev. 21, 1379–1413. https://doi.org/10.1007/s11101-021-09789-7 Eshed, Y., Zamir, D., 1995. An introgression line population of Lycopersicon pennellii in the cultivated tomato enables the identification and fine mapping of yield-associated QTL. Genetics 141, 1147–1162. https://doi.org/10.1093/genetics/141.3.1147 Fan, P., Miller, A.M., Liu, X., Jones, A.D., Last, R.L., 2017. Evolution of a flipped pathway creates metabolic innovation in tomato trichomes through BAHD enzyme promiscuity. Nat. Commun. 8, 2080. https://doi.org/10.1038/s41467-017-02045-7 Fan, P., Miller, A.M., Schilmiller, A.L., Liu, X., Ofner, I., Jones, A.D., Zamir, D., Last, R.L., 2015. In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network. Proc. Natl. Acad. Sci. U.S.A. https://doi.org/10.1073/pnas.1517930113 61 Fan, P., Wang, P., Lou, Y.R., Leong, B.J., Moore, B.M., Schenck, C.A., Combs, R., Cao, P., Brandizzi, F., Shiu, S.H., Last, R.L., 2020. Evolution of a plant gene cluster in solanaceae and emergence of metabolic diversity. eLife 9, 1–26. https://doi.org/10.7554/eLife.56717 Fossati, E., Ekins, A., Narcross, L., Zhu, Y., Falgueyret, J.-P., Beaudoin, G.A.W., Facchini, P.J., Martin, V.J.J., 2014. Reconstitution of a 10-gene pathway for synthesis of the plant alkaloid dihydrosanguinarine in Saccharomyces cerevisiae. Nat. Commun. 5, 3283. https://doi.org/10.1038/ncomms4283 Frelichowski, J.E., Jr., Juvik, J.A., 2001. Sesquiterpene carboxylic acids from a wild tomato species affect larval feeding behavior and survival of Helicoverpa zea and Spodoptera exigua (Lepidoptera: Noctuidae). J. Econ. Entomol. 94, 1249–1259. https://doi.org/10.1603/0022-0493-94.5.1249 Fridman, E., Carrari, F., Liu, Y.-S., Fernie, A.R., Zamir, D., 2004. Zooming in on a quantitative trait for tomato yield using interspecific introgressions. Science 305, 1786–1789. https://doi.org/10.1126/science.1101666 Friedman, J.R., Richbart, S.D., Merritt, J.C., Brown, K.C., Denning, K.L., Tirona, M.T., Valentovic, M.A., Miles, S.L., Dasgupta, P., 2019. Capsaicinoids: multiple effects on angiogenesis, invasion and metastasis in human cancers. Biomed. Pharmacother. 118, 109317. https://doi.org/10.1016/j.biopha.2019.109317 Gagnon, E., Hilgenhof, R., Orejuela, A., McDonnell, A., Sablok, G., Aubriot, X., Giacomin, L., Gouvêa, Y., Bragionis, T., Stehmann, J.R., Bohs, L., Dodsworth, S., Martine, C., Poczai, P., Knapp, S., Särkinen, T., 2022. Phylogenomic discordance suggests polytomies along the backbone of the large genus Solanum. Am. J. Bot. 109, 580–601. https://doi.org/10.1002/ajb2.1827 Gao, L., Gonda, I., Sun, H., Ma, Q., Bao, K., Tieman, D.M., Burzynski-Chang, E.A., Fish, T.L., Stromberg, K.A., Sacks, G.L., Thannhauser, T.W., Foolad, M.R., Diez, M.J., Blanca, J., Canizares, J., Xu, Y., van der Knaap, E., Huang, S., Klee, H.J., Giovannoni, J.J., Fei, Z., 2019. The tomato pan-genome uncovers new genes and a rare allele regulating fruit flavor. Nat. Genet. 51, 1044–1051. https://doi.org/10.1038/s41588-019-0410-2 Garbowicz, K., Liu, Z., Alseekh, S., Tieman, D., Taylor, M., Kuhalskaya, A., Ofner, I., Zamir, D., Klee, H.J., Fernie, A.R., Brotman, Y., 2018. Quantitative trait loci analysis identifies a prominent gene involved in the production of fatty acid-derived flavor volatiles in tomato. Mol. Plant 11, 1147–1165. https://doi.org/10.1016/j.molp.2018.06.003 Ghosh, B., Westbrook, T.C., Jones, A.D., 2014. Comparative structural profiling of trichome specialized metabolites in tomato (Solanum lycopersicum) and S. habrochaites: acylsugar profiles revealed by UHPLC/MS and NMR. Metabolomics 10, 496–507. https://doi.org/10.1007/s11306-013-0585-y Goffreda, J.C., Mutschler, M.A., Avé, D.A., Tingey, W.M., Steffens, J.C., 1989. Aphid deterrence by glucose esters in glandular trichome exudate of the wild tomato, 62 Lycopersicon pennellii. J. Chem. Ecol. 15, 2135–2147. https://doi.org/10.1007/BF01207444 Gonzales-Vigil, E., Hufnagel, D.E., Kim, J., Last, R.L., Barry, C.S., 2012. Evolution of TPS20- related terpene synthases influences chemical diversity in the glandular trichomes of the wild tomato relative Solanum habrochaites. Plant J. 71, 921–935. https://doi.org/10.1111/j.1365-313X.2012.05040.x Gramazio, P., Prohens, J., Plazas, M., Mangino, G., Herraiz, F.J., Vilanova, S., 2017. Development and genetic characterization of advanced backcross materials and an introgression line population of Solanum incanum in a S. melongena background. Front. Plant Sci. 8. Gruene, T., Wennmacher, J.T.C., Zaubitzer, C., Holstein, J.J., Heidler, J., Fecteau-Lefebvre, A., De Carlo, S., Müller, E., Goldie, K.N., Regeni, I., Li, T., Santiso-Quinones, G., Steinfeld, G., Handschin, S., van Genderen, E., van Bokhoven, J.A., Clever, G.H., Pantelic, R., 2018. Rapid structure determination of microcrystalline molecular compounds using electron diffraction. Angew. Chem. Int. Ed. 57, 16313-16317. https://doi.org/10.1002/anie.201811318 Gu, X.-Y., Shen, X.-F., Wang, L., Wu, Z.-W., Li, F., Chen, B., Zhang, G.-L., Wang, M.-K., 2018. Bioactive steroidal alkaloids from the fruits of Solanum nigrum. Phytochemistry 147, 125–131. https://doi.org/10.1016/j.phytochem.2017.12.020 Han, K., Lee, H.-Y., Ro, N.-Y., Hur, O.-S., Lee, J.-H., Kwon, J.-K., Kang, B.-C., 2018. QTL mapping and GWAS reveal candidate genes controlling capsaicinoid content in Capsicum. Plant Biotechnol. J. 16, 1546–1558. https://doi.org/10.1111/pbi.12894 Hashimoto, T., Yamada, Y., 1986. Hyoscyamine 6β-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, in alkaloid-producing root cultures. Plant Physiol. 81, 619–625. https://doi.org/10.1104/pp.81.2.619 Herrera-Salgado, Y., Garduño-Ramírez, M.L., Vázquez, L., Rios, M.Y., Alvarez, L., 2005. Myo- inositol-derived glycolipids with anti-inflammatory activity from Solanum lanceolatum. J. Nat. Prod. 68, 1031–1036. https://doi.org/10.1021/np050054s Hirsch, C.N., Hirsch, C.D., Felcher, K., Coombs, J., Zarka, D., Van Deynze, A., De Jong, W., Veilleux, R.E., Jansky, S., Bethke, P., Douches, D.S., Buell, C.R., 2013. Retrospective view of North American potato (Solanum tuberosum L.) breeding in the 20th and 21st centuries. G3: Genes Genomes Genet. 3, 1003–1013. https://doi.org/10.1534/g3.113.005595 Horai, H., Arita, M., Kanaya, S., Nihei, Y., Ikeda, T., Suwa, K., Ojima, Y., Tanaka, Kenichi, Tanaka, S., Aoshima, K., Oda, Y., Kakazu, Y., Kusano, M., Tohge, T., Matsuda, F., Sawada, Y., Hirai, M.Y., Nakanishi, H., Ikeda, K., Akimoto, N., Maoka, T., Takahashi, H., Ara, T., Sakurai, N., Suzuki, H., Shibata, D., Neumann, S., Iida, T., Tanaka, Ken, Funatsu, K., Matsuura, F., Soga, T., Taguchi, R., Saito, K., Nishioka, T., 2010. 63 MassBank: a public repository for sharing mass spectral data for life sciences. J. Mass Spectrom. 45, 703–714. https://doi.org/10.1002/jms.1777 Huang, J.-P., Fang, C., Ma, X., Wang, L., Yang, J., Luo, J., Yan, Y., Zhang, Y., Huang, S.-X., 2019. Tropane alkaloids biosynthesis involves an unusual type III polyketide synthase and non-enzymatic condensation. Nat. Commun. 10, 4036. https://doi.org/10.1038/s41467-019-11987-z Hurney, S.M., 2018. Strategies for profiling and discovery of acylsugar specialized metabolites (Ph.D.). Michigan State University, United States -- Michigan. Iijima, Y., Watanabe, B., Sasaki, R., Takenaka, M., Ono, H., Sakurai, N., Umemoto, N., Suzuki, H., Shibata, D., Aoki, K., 2013. Steroidal glycoalkaloid profiling and structures of glycoalkaloids in wild tomato fruit. Phytochemistry 95, 145–157. https://doi.org/10.1016/j.phytochem.2013.07.016 Itkin, M., Heinig, U., Tzfadia, O., Bhide, A.J., Shinde, B., Cardenas, P.D., Bocobza, S.E., Unger, T., Malitsky, S., Finkers, R., Tikunov, Y., Bovy, A., Chikate, Y., Singh, P., Rogachev, I., Beekwilder, J., Giri, A.P., Aharoni, A., 2013. Biosynthesis of antinutritional alkaloids in Solanaceous crops is mediated by clustered genes. Science 341, 175–179. https://doi.org/10.1126/science.1240230 Jacobowitz, J.R., Weng, J.-K., 2020. Exploring uncharted territories of plant specialized metabolism in the postgenomic era. Annu. Rev. Plant Biol. 71, 631–658. https://doi.org/10.1146/annurev-arplant-081519-035634 Jirschitzka, J., Schmidt, G.W., Reichelt, M., Schneider, B., Gershenzon, J., D’Auria, J.C., 2012. Plant tropane alkaloid biosynthesis evolved independently in the Solanaceae and Erythroxylaceae. Proc. Natl. Acad. Sci. U.S.A. 109, 10304–10309. https://doi.org/10.1073/pnas.1200473109 Jones, C.G., Martynowycz, M.W., Hattne, J., Fulton, T.J., Stoltz, B.M., Rodriguez, J.A., Nelson, H.M., Gonen, T., 2018. The cryoEM method microED as a powerful tool for small molecule structure determination. ACS Cent. Sci. 4, 1587–1592. https://doi.org/10.1021/acscentsci.8b00760 Jordt, S.-E., Julius, D., 2002. Molecular basis for species-specific sensitivity to “hot” chili peppers. Cell 108, 421–430. https://doi.org/10.1016/S0092-8674(02)00637-2 Junker, A., Fischer, J., Sichhart, Y., Brandt, W., Draeger, B., 2013. Evolution of the key alkaloid enzyme putrescine N-methyltransferase from spermidine synthase. Front. Plant Sci. 4. Kajikawa, M., Sierro, N., Kawaguchi, H., Bakaher, N., Ivanov, N.V., Hashimoto, T., Shoji, T., 2017. Genomic insights into the evolution of the nicotine biosynthesis pathway in tobacco. Plant Physiol. 174, 999–1011. https://doi.org/10.1104/pp.17.00070 Kang, J.-H., Gonzales-Vigil, E., Matsuba, Y., Pichersky, E., Barry, C.S., 2014. Determination of residues responsible for substrate and product specificity of Solanum habrochaites short- 64 chain cis-prenyltransferases. Plant Physiol. 164, 80–91. https://doi.org/10.1104/pp.113.230466 Karunanithi, P.S., Zerbe, P., 2019. Terpene synthases as metabolic gatekeepers in the evolution of plant terpenoid chemical diversity. Front. Plant Sci. 10. Kazachkova, Y., Zemach, I., Panda, S., Bocobza, S., Vainer, A., Rogachev, I., Dong, Y., Ben- Dor, S., Veres, D., Kanstrup, C., Lambertz, S.K., Crocoll, C., Hu, Y., Shani, E., Michaeli, S., Nour-Eldin, H.H., Zamir, D., Aharoni, A., 2021. The GORKY glycoalkaloid transporter is indispensable for preventing tomato bitterness. Nat. Plants 7, 468–480. https://doi.org/10.1038/s41477-021-00865-6 Kim, J., Kang, K., Gonzales-Vigil, E., Shi, F., Daniel Jones, A., Barry, C.S., Last, R.L., 2012. Striking natural diversity in glandular trichome acylsugar composition is shaped by variation at the acyltransferase2 locus in the wild tomato Solanum habrochaites. Plant Physiol. 160, 1854–1870. https://doi.org/10.1104/pp.112.204735 Kim, L.J., Ohashi, M., Zhang, Z., Tan, D., Asay, M., Cascio, D., Rodriguez, J.A., Tang, Y., Nelson, H.M., 2021. Prospecting for natural products by genome mining and microcrystal electron diffraction. Nat. Chem. Biol. 17, 872–877. https://doi.org/10.1038/s41589-021- 00834-2 Kim, N., Estrada, O., Chavez, B., Stewart, C., D’Auria, J.C., 2016. Tropane and granatane alkaloid biosynthesis: a systematic analysis. Molecules 21, 1510. https://doi.org/10.3390/molecules21111510 Kim, S., Park, M., Yeom, S.-I., Kim, Y.-M., Lee, J.M., Lee, H.-A., Seo, E., Choi, J., Cheong, K., Kim, K.-T., Jung, K., Lee, G.-W., Oh, S.-K., Bae, C., Kim, S.-B., Lee, H.-Y., Kim, S.-Y., Kim, M.-S., Kang, B.-C., Jo, Y.D., Yang, H.-B., Jeong, H.-J., Kang, W.-H., Kwon, J.-K., Shin, C., Lim, J.Y., Park, J.H., Huh, J.H., Kim, J.-S., Kim, B.-D., Cohen, O., Paran, I., Suh, M.C., Lee, S.B., Kim, Y.-K., Shin, Y., Noh, S.-J., Park, J., Seo, Y.S., Kwon, S.-Y., Kim, H.A., Park, J.M., Kim, H.-J., Choi, S.-B., Bosland, P.W., Reeves, G., Jo, S.-H., Lee, B.-W., Cho, H.-T., Choi, H.-S., Lee, M.-S., Yu, Y., Do Choi, Y., Park, B.-S., van Deynze, A., Ashrafi, H., Hill, T., Kim, W.T., Pai, H.-S., Ahn, H.K., Yeam, I., Giovannoni, J.J., Rose, J.K.C., Sørensen, I., Lee, S.-J., Kim, R.W., Choi, I.-Y., Choi, B.- S., Lim, J.-S., Lee, Y.-H., Choi, D., 2014. Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species. Nat. Genet. 46, 270–278. https://doi.org/10.1038/ng.2877 King, R.R., Calhoun, L.A., 1988. 6 2,3-Di-O- and 1,2,3-tri-O-acylated glucose esters from the glandular trichomes of Datura metel. Phytochemistry 27, 3761–3763. https://doi.org/10.1016/0031-9422(88)83013-9 King, R.R., Pelletier, Y., Singh, R.P., Calhoun, L.A., 1986. 3,4-Di-O-isobutyryl-6-O- caprylsucrose: the major component of a novel sucrose ester complex from the type B glandular trichomes of Solanum berthaultii Hawkes (Pl 473340). J. Chem. Soc. Chem. Commun. 1078–1079. https://doi.org/10.1039/C39860001078 65 Knapp, S., Bohs, L., Nee, M., Spooner, D.M., 2004. Solanaceae—a model for linking genomics with biodiversity. Comp. Funct. Genomics 5, 285–291. https://doi.org/10.1002/cfg.393 Koeda, S., Sato, K., Saito, H., Nagano, A.J., Yasugi, M., Kudoh, H., Tanaka, Y., 2019. Mutation in the putative ketoacyl-ACP reductase CaKR1 induces loss of pungency in Capsicum. Theor. Appl. Genet. 132, 65–80. https://doi.org/10.1007/s00122-018-3195-2 Korenblum, E., Dong, Y., Szymanski, J., Panda, S., Jozwiak, A., Massalha, H., Meir, S., Rogachev, I., Aharoni, A., 2020. Rhizosphere microbiome mediates systemic root metabolite exudation by root-to-root signaling. Proc. Natl. Acad. Sci. U.S.A. 117, 3874– 3883. https://doi.org/10.1073/pnas.1912130117 Landis, J.B., Miller, C.M., Broz, A.K., Bennett, A.A., Carrasquilla-Garcia, N., Cook, D.R., Last, R.L., Bedinger, P.A., Moghe, G.D., 2021. Migration through a major Andean ecogeographic disruption as a driver of genetic and phenotypic diversity in a wild tomato species. Mol. Biol. Evol. 38, 3202–3219. https://doi.org/10.1093/molbev/msab092 Lang, Y., Kisaka, H., Sugiyama, R., Nomura, K., Morita, A., Watanabe, T., Tanaka, Y., Yazawa, S., Miwa, T., 2009. Functional loss of pAMT results in biosynthesis of capsinoids, capsaicinoid analogs, in Capsicum annuum cv. CH-19 Sweet. Plant J. 59, 953–961. https://doi.org/10.1111/j.1365-313X.2009.03921.x Last, R.L., Jones, A.D., Shachar-Hill, Y., 2007. Towards the plant metabolome and beyond. Nat. Rev. Mol. Cell Biol. 8, 167–174. https://doi.org/10.1038/nrm2098 Lau, W., Sattely, E.S., 2015. Six enzymes from mayapple that complete the biosynthetic pathway to the etoposide aglycone. Science 349, 1224–1228. https://doi.org/10.1126/science.aac7202 Leckie, B.M., D’Ambrosio, D.A., Chappell, T.M., Halitschke, R., Jong, D.M.D., Kessler, A., Kennedy, G.G., Mutschler, M.A., 2016. Differential and synergistic functionality of ]acylsugars in suppressing oviposition by insect herbivores. PLOS ONE 11, e0153345. https://doi.org/10.1371/journal.pone.0153345 Lee, H.J., Nakayasu, M., Akiyama, R., Kobayashi, M., Miyachi, H., Sugimoto, Y., Umemoto, N., Saito, K., Muranaka, T., Mizutani, M., 2019. Identification of a 3β-hydroxysteroid dehydrogenase/ 3-ketosteroid reductase involved in α-tomatine biosynthesis in tomato. Plant Cell Physiol. 60, 1304–1315. https://doi.org/10.1093/pcp/pcz049 Leong, B.J., Hurney, S.M., Fiesel, P.D., Moghe, G.D., Jones, A.D., Last, R.L., 2020. Specialized metabolism in a nonmodel nightshade: trichome acylinositol biosynthesis. Plant Physiol. 183, 915–924. https://doi.org/10.1104/pp.20.00276 Leong, B.J., Last, R.L., 2017. Promiscuity, impersonation and accommodation: evolution of plant specialized metabolism. Curr. Opin. Struct. Biol. 47, 105–112. https://doi.org/10.1016/j.sbi.2017.07.005 66 Leong, B.J., Lybrand, D.B., Lou, Y.-R., Fan, P., Schilmiller, A.L., Last, R.L., 2019. Evolution of metabolic novelty: a trichome-expressed invertase creates specialized metabolic diversity in wild tomato. Sci. Adv. 5, eaaw3754. https://doi.org/10.1126/sciadv.aaw3754 Levina, A.V., Hoekenga, O., Gordin, M., Broeckling, C., De Jong, W.S., 2021. Genetic analysis of potato tuber metabolite composition: genome-wide association studies applied to a nontargeted metabolome. Crop Sci. 61, 591–603. https://doi.org/10.1002/csc2.20398 Lewinsohn, E., Gijzen, M., 2009. Phytochemical diversity: the sounds of silent metabolism. Plant Sci. 176, 161–169. https://doi.org/10.1016/j.plantsci.2008.09.018 Li, C., Wang, Z., Jones, A.D., 2014. Chemical imaging of trichome specialized metabolites using contact printing and laser desorption/ionization mass spectrometry. Anal. Bioanal. Chem. 406, 171–182. https://doi.org/10.1007/s00216-013-7444-6 Li, R., Reed, D.W., Liu, E., Nowak, J., Pelcher, L.E., Page, J.E., Covello, P.S., 2006. Functional genomic analysis of alkaloid biosynthesis in Hyoscyamus niger reveals a cytochrome P450 involved in littorine rearrangement. Chem. Biol. 13, 513–520. https://doi.org/10.1016/j.chembiol.2006.03.005 Li, X., Tieman, D., Liu, Z., Chen, K., Klee, H.J., 2020. Identification of a lipase gene with a role in tomato fruit short-chain fatty acid-derived flavor volatiles by genome-wide association. Plant J. 104, 631–644. https://doi.org/10.1111/tpj.14951 Li, Y., Chen, Yang, Zhou, L., You, S., Deng, H., Chen, Ya, Alseekh, S., Yuan, Y., Fu, R., Zhang, Z., Su, D., Fernie, A.R., Bouzayen, M., Ma, T., Liu, M., Zhang, Y., 2020. MicroTom metabolic network: rewiring tomato metabolic regulatory network throughout the growth cycle. Mol. Plant 13, 1203–1218. https://doi.org/10.1016/j.molp.2020.06.005 Liu, X., Enright, M., Barry, C.S., Jones, A.D., 2017. Profiling, isolation and structure elucidation of specialized acylsucrose metabolites accumulating in trichomes of Petunia species. Metabolomics 13, 85. https://doi.org/10.1007/s11306-017-1224-9 Liu, Y., Schiff, M., Dinesh-Kumar, S.P., 2002. Virus-induced gene silencing in tomato. Plant J. 31, 777–786. https://doi.org/10.1046/j.1365-313X.2002.01394.x Lou, Y.-R., Anthony, T.M., Fiesel, P.D., Arking, R.E., Christensen, E.M., Jones, A.D., Last, R.L., 2021. It happened again: convergent evolution of acylglucose specialized metabolism in black nightshade and wild tomato. Sci. Adv. 7, eabj8726. https://doi.org/10.1126/sciadv.abj8726 Luu, V.T., Weinhold, A., Ullah, C., Dressel, S., Schoettner, M., Gase, K., Gaquerel, E., Xu, S., Baldwin, I.T., 2017. O-Acyl sugars protect a wild tobacco from both native fungal pathogens and a specialist herbivore. Plant Physiol. 174, 370–386. https://doi.org/10.1104/pp.16.01904 67 Lybrand, D.B., Anthony, T.M., Jones, A.D., Last, R.L., 2020. An integrated analytical approach reveals trichome acylsugar metabolite diversity in the wild tomato Solanum pennellii. Metabolites 10, 1–25. https://doi.org/10.3390/metabo10100401 Maldonado, E., Torres, F.R., Martínez, M., Pérez-Castorena, A.L., 2006. Sucrose esters from the fruits of Physalis nicandroides var. attenuata. J. Nat. Prod. 69, 1511–1513. https://doi.org/10.1021/np060274l Mato, A., Limeres, J., Tomás, I., Muñoz, M., Abuín, C., Feijoo, J.F., Diz, P., 2010. Management of drooling in disabled patients with scopolamine patches. Br. J. Clin. Pharmacol. 69, 684–688. https://doi.org/10.1111/j.1365-2125.2010.03659.x Matsuba, Y., Nguyen, T.T.H., Wiegert, K., Falara, V., Gonzales-Vigil, E., Leong, B., Schäfer, P., Kudrna, D., Wing, R.A., Bolger, A.M., Usadel, B., Tissier, A., Fernie, A.R., Barry, C.S., Pichersky, E., 2013. Evolution of a complex locus for terpene biosynthesis in Solanum. Plant Cell 25, 2022–2036. https://doi.org/10.1105/tpc.113.111013 Matsuzaki, T., Shinozaki, Y., Suhara, S., Ninomiya, M., Shigematsu, H., Koiwai, A., 1989. Isolation of glycolipids from the surface lipids of Nicotiana bigelovii and their distribution in Nicotiana species. Agric. Biol. Chem. 53, 3079–3082. https://doi.org/10.1271/bbb1961.53.3079 Michael, T.P., VanBuren, R., 2020. Building near-complete plant genomes. Curr. Opin. Plant Biol. 54, 26–33. https://doi.org/10.1016/j.pbi.2019.12.009 Moghe, G.D., Leong, B.J., Hurney, S.M., Jones, A.D., Last, R.L., 2017. Evolutionary routes to biochemical innovation revealed by integrative analysis of a plant-defense related specialized metabolic pathway. eLife 6, 1–33. https://doi.org/10.7554/eLife.28468 Moore, B.M., Wang, P., Fan, P., Lee, A., Leong, B., Lou, Y.-R., Schenck, C.A., Sugimoto, K., Last, R., Lehti-Shiu, M.D., Barry, C.S., Shiu, S.-H., 2020. Within- and cross-species predictions of plant specialized metabolism genes using transfer learning. Silico Plants 2, diaa005. https://doi.org/10.1093/insilicoplants/diaa005 Moore, B.M., Wang, P., Fan, P., Leong, B., Schenck, C.A., Lloyd, J.P., Lehti-Shiu, M.D., Last, R.L., Pichersky, E., Shiu, S.-H., 2019. Robust predictions of specialized metabolism genes through machine learning. Proc. Natl. Acad. Sci. U.S.A. 116, 2344–2353. https://doi.org/10.1073/pnas.1817074116 Mueller, L.A., Tanksley, S.D., Giovannoni, J.J., van Eck, J., Stack, S., Choi, D., Kim, B.D., Chen, M., Cheng, Z., Li, C., Ling, H., Xue, Y., Seymour, G., Bishop, G., Bryan, G., Sharma, R., Khurana, J., Tyagi, A., Chattopadhyay, D., Singh, N.K., Stiekema, W., Lindhout, P., Jesse, T., Lankhorst, R.K., Bouzayen, M., Shibata, D., Tabata, S., Granell, A., Botella, M.A., Giuliano, G., Frusciante, L., Causse, M., Zamir, D., 2005. The omato Sequencing Project, the first cornerstone of the International Solanaceae Project (SOL). Comp. Funct. Genomics 6, 153–158. https://doi.org/10.1002/cfg.468 68 Nadakuduti, S.S., Uebler, J.B., Liu, X., Jones, A.D., Barry, C.S., 2017. Characterization of trichome-expressed BAHD acyltransferases in Petunia axillaris reveals distinct acylsugar assembly mechanisms within the Solanaceae. Plant Physiol. 175, 36–50. https://doi.org/10.1104/pp.17.00538 Nakajima, K., Hashimoto, T., Yamada, Y., 1993. Two tropinone reductases with different stereospecificities are short-chain dehydrogenases evolved from a common ancestor. Proc. Natl. Acad. Sci. U.S.A. 90, 9591–9595. https://doi.org/10.1073/pnas.90.20.9591 Nakashima, T., Wada, H., Morita, S., Erra-Balsells, R., Hiraoka, K., Nonami, H., 2016. Single- cell metabolite profiling of stalk and glandular cells of intact trichomes with internal electrode capillary pressure probe electrospray ionization mass spectrometry. Anal. Chem. 88, 3049–3057. https://doi.org/10.1021/acs.analchem.5b03366 Nakayasu, M., Akiyama, R., Kobayashi, M., Lee, H.J., Kawasaki, T., Watanabe, B., Urakawa, S., Kato, J., Sugimoto, Y., Iijima, Y., Saito, K., Muranaka, T., Umemoto, N., Mizutani, M., 2020. Identification of α-tomatine 23-hydroxylase involved in the detoxification of a bitter glycoalkaloid. Plant Cell Physiol. 61, 21–28. https://doi.org/10.1093/pcp/pcz224 Nash, R.J., Rothschild, M., Porter, E.A., Watson, A.A., Waigh, R.D., Waterman, P.G., 1993. Calystegines in Solanum and Datura species and the death’s-head hawk-moth (Acherontia atropus). Phytochemistry, The International Journal of Plant Biochemistry 34, 1281–1283. https://doi.org/10.1016/0031-9422(91)80016-T Naves, E.R., Silva, L. de Á., Sulpice, R., Araújo, W.L., Nunes-Nesi, A., Peres, L.E.P., Zsögön, A., 2019. Capsaicinoids: pungency beyond Capsicum. Trends Plant Sci. 24, 109–120. https://doi.org/10.1016/j.tplants.2018.11.001 Nett, R.S., Dho, Y., Low, Y.-Y., Sattely, E.S., 2021. A metabolic regulon reveals early and late acting enzymes in neuroactive Lycopodium alkaloid biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 118, e2102949118. https://doi.org/10.1073/pnas.2102949118 Nett, R.S., Lau, W., Sattely, E.S., 2020. Discovery and engineering of colchicine alkaloid biosynthesis. Nature 584, 148–153. https://doi.org/10.1038/s41586-020-2546-8 Ning, J., Moghe, G.D., Leong, B., Kim, J., Ofner, I., Wang, Z., Adams, C., Jones, A.D., Zamir, D., Last, R.L., 2015. A feedback-insensitive isopropylmalate synthase affects acylsugar composition in cultivated and wild tomato. Plant Physiol. 169, 1821–1835. https://doi.org/10.1104/pp.15.00474 Ofner, I., Lashbrooke, J., Pleban, T., Aharoni, A., Zamir, D., 2016. Solanum pennellii backcross inbred lines (BILs) link small genomic bins with tomato traits. Plant J. 87, 151–160. https://doi.org/10.1111/tpj.13194 Ono, M., Takigawa, A., Muto, H., Kabata, K., Okawa, M., Kinjo, J., Yokomizo, K., Yoshimitsu, H., Nohara, T., 2015. Antiviral activity of four new resin glycosides Calysolins XIV– XVII from Calystegia soldanella against herpes simplex virus. Chem. Pharm. Bull. (Tokyo) 63, 641–648. https://doi.org/10.1248/cpb.c15-00307 69 Onoyovwe, A., Hagel, J.M., Chen, X., Khan, M.F., Schriemer, D.C., Facchini, P.J., 2013. Morphine biosynthesis in opium poppy involves two cell types: sieve elements and laticifers. Plant Cell 25, 4110–4122. https://doi.org/10.1105/tpc.113.115113 Park, M., Lee, J.-H., Han, K., Jang, S., Han, J., Lim, J.-H., Jung, J.-W., Kang, B.-C., 2019. A major QTL and candidate genes for capsaicinoid biosynthesis in the pericarp of Capsicum chinense revealed using QTL-seq and RNA-seq. Theor. Appl. Genet. 132, 515–529. https://doi.org/10.1007/s00122-018-3238-8 Paudel, J.R., Davidson, C., Song, J., Maxim, I., Aharoni, A., Tai, H.H., 2017. Pathogen and pest responses are altered due to RNAi-mediated knockdown of GLYCOALKALOID METABOLISM 4 in Solanum tuberosum. Mol. Plant-Microbe Interactions 30, 876–885. https://doi.org/10.1094/MPMI-02-17-0033-R Pazouki, L., Niinemets, Ü., 2016. Multi-substrate terpene synthases: their occurrence and physiological significance. Front. Plant Sci. 7. Pereda-Miranda, R., Mata, R., Anaya, A.L., Wickramaratne, D.B.M., Pezzuto, J.M., Kinghorn, A.D., 1993. Tricolorin A, major phytogrowth inhibitor from Ipomoea tricolor. J. Nat. Prod. 56, 571–582. https://doi.org/10.1021/np50094a018 Perez de Souza, L., Alseekh, S., Scossa, F., Fernie, A.R., 2021. Ultra-high-performance liquid chromatography high-resolution mass spectrometry variants for metabolomics research. Nat. Methods 18, 733–746. https://doi.org/10.1038/s41592-021-01116-4 Pérez, L.M., Farriols, C., Puente, V., Planas, J., Ruiz, I., 2011. The use of subcutaneous scopolamine as a palliative treatment in Parkinson’s disease. Palliat. Med. 25, 92–93. https://doi.org/10.1177/0269216310381662 Qiu, F., Yan, Y., Zeng, J., Huang, J.-P., Zeng, L., Zhong, W., Lan, X., Chen, M., Huang, S.-X., Liao, Z., 2021. Biochemical and metabolic insights into hyoscyamine dehydrogenase. ACS Catal. 11, 2912–2924. https://doi.org/10.1021/acscatal.0c04667 Qiu, F., Yang, C., Yuan, L., Xiang, D., Lan, X., Chen, M., Liao, Z., 2018. A phenylpyruvic acid reductase is required for biosynthesis of tropane alkaloids. Org. Lett. 20, 7807–7810. https://doi.org/10.1021/acs.orglett.8b03236 Qiu, F., Zeng, J., Wang, J., Huang, J.-P., Zhou, W., Yang, C., Lan, X., Chen, M., Huang, S.-X., Kai, G., Liao, Z., 2020. Functional genomics analysis reveals two novel genes required for littorine biosynthesis. New Phytol. 225, 1906–1914. https://doi.org/10.1111/nph.16317 Reed, J., Stephenson, M.J., Miettinen, K., Brouwer, B., Leveau, A., Brett, P., Goss, R.J.M., Goossens, A., O’Connell, M.A., Osbourn, A., 2017. A translational synthetic biology platform for rapid access to gram-scale quantities of novel drug-like molecules. Metab. Eng. 42, 185–193. https://doi.org/10.1016/j.ymben.2017.06.012 70 Roddick, J.G., Weissenberg, M., Leonard, A.L., 2001. Membrane disruption and enzyme inhibition by naturally-occurring and modified chacotriose-containing Solanum steroidal glycoalkaloids. Phytochemistry 56, 603–610. https://doi.org/10.1016/S0031- 9422(00)00420-9 Ryu, K.H., Huang, L., Kang, H.M., Schiefelbein, J., 2019. Single-cell RNA sequencing resolves molecular relationships among individual plant cells. Plant Physiol. 179, 1444–1456. https://doi.org/10.1104/pp.18.01482 Sallaud, C., Rontein, D., Onillon, S., Jabès, F., Duffé, P., Giacalone, C., Thoraval, S., Escoffier, C., Herbette, G., Leonhardt, N., Causse, M., Tissier, A., 2009. A novel pathway for sesquiterpene biosynthesis from Z,Z-farnesyl pyrophosphate in the wild tomato Solanum habrochaites. Plant Cell 21, 301–317. https://doi.org/10.1105/tpc.107.057885 Sánchez-Mata, M.-C., Yokoyama, W.E., Hong, Y.-J., Prohens, J., 2010. α-Solasonine and α- solamargine contents of gboma (Solanum macrocarpon L.) and scarlet (Solanum aethiopicum L.) eggplants. J. Agric. Food Chem. 58, 5502–5508. https://doi.org/10.1021/jf100709g Särkinen, T., Bohs, L., Olmstead, R.G., Knapp, S., 2013. A phylogenetic framework for evolutionary study of the nightshades (Solanaceae): a dated 1000-tip tree. BMC Evol. Biol. 13, 214. https://doi.org/10.1186/1471-2148-13-214 The Tomato Genome Consortium, 2012. The tomato genome sequence provides insights into fleshy fruit evolution. Nature 485, 635–641. https://doi.org/10.1038/nature11119 Schenck, C.A., Last, R.L., 2020. Location, location! cellular relocalization primes specialized metabolic diversification. FEBS J. 287, 1359–1368. https://doi.org/10.1111/febs.15097 Schijlen, E.G.W.M., de Vos, C.H.R., Martens, S., Jonker, H.H., Rosin, F.M., Molthoff, J.W., Tikunov, Y.M., Angenent, G.C., van Tunen, A.J., Bovy, A.G., 2007. RNA interference silencing of chalcone synthase, the first step in the flavonoid biosynthesis pathway, leads to parthenocarpic tomato fruits. Plant Physiol. 144, 1520–1530. https://doi.org/10.1104/pp.107.100305 Schilmiller, A., Shi, F., Kim, J., Charbonneau, A.L., Holmes, D., Daniel Jones, A., Last, R.L., 2010. Mass spectrometry screening reveals widespread diversity in trichome specialized metabolites of tomato chromosomal substitution lines. Plant J. 62, 391–403. https://doi.org/10.1111/j.1365-313X.2010.04154.x Schilmiller, A.L., Charbonneau, A.L., Last, R.L., 2012. Identification of a BAHD acetyltransferase that produces protective acyl sugars in tomato trichomes. Proc. Natl. Acad. Sci. U.S.A. 109, 16377–16382. https://doi.org/10.1073/pnas.1207906109 Schilmiller, A.L., Moghe, G.D., Fan, P., Ghosh, B., Ning, J., Jones, A.D., Last, R.L., 2015. Functionally divergent alleles and duplicated loci encoding an acyltransferase contribute to acylsugar metabolite diversity in Solanum trichomes. Plant Cell 27, 1002–17. https://doi.org/10.1105/tpc.15.00087 71 Schilmiller, A.L., Schauvinhold, I., Larson, M., Xu, R., Charbonneau, A.L., Schmidt, A., Wilkerson, C., Last, R.L., Pichersky, E., 2009. Monoterpenes in the glandular trichomes of tomato are synthesized from a neryl diphosphate precursor rather than geranyl diphosphate. Proc. Natl. Acad. Sci. U.S.A. 106, 10865–10870. https://doi.org/10.1073/pnas.0904113106 Schmidt, G.W., Jirschitzka, J., Porta, T., Reichelt, M., Luck, K., Torre, J.C.P., Dolke, F., Varesio, E., Hopfgartner, G., Gershenzon, J., D’Auria, J.C., 2015. The last step in cocaine biosynthesis is catalyzed by a BAHD acyltransferase. Plant Physiol. 167, 89– 101. https://doi.org/10.1104/pp.114.248187 Seyfferth, C., Renema, J., Wendrich, J.R., Eekhout, T., Seurinck, R., Vandamme, N., Blob, B., Saeys, Y., Helariutta, Y., Birnbaum, K.D., De Rybel, B., 2021. Advances and opportunities in single-cell transcriptomics for plant research. Annu. Rev. Plant Biol. 72, 847–866. https://doi.org/10.1146/annurev-arplant-081720-010120 Shinde, B.A., Dholakia, B.B., Hussain, K., Panda, S., Meir, S., Rogachev, I., Aharoni, A., Giri, A.P., Kamble, A.C., 2017. Dynamic metabolic reprogramming of steroidal glycol- alkaloid and phenylpropanoid biosynthesis may impart early blight resistance in wild tomato (Solanum arcanum Peralta). Plant Mol. Biol. 95, 411–423. https://doi.org/10.1007/s11103-017-0660-2 Sonawane, P.D., Heinig, U., Panda, S., Gilboa, N.S., Yona, M., Kumar, S.P., Alkan, N., Unger, T., Bocobza, S., Pliner, M., Malitsky, S., Tkachev, M., Meir, S., Rogachev, I., Aharoni, A., 2018. Short-chain dehydrogenase/reductase governs steroidal specialized metabolites structural diversity and toxicity in the genus Solanum. Proc. Natl. Acad. Sci. U.S.A. 115, E5419–E5428. https://doi.org/10.1073/pnas.1804835115 Sonawane, P.D., Jozwiak, A., Panda, S., Aharoni, A., 2020. ‘Hijacking’ core metabolism: a new panache for the evolution of steroidal glycoalkaloids structural diversity. Curr. Opin. Plant Biol. 55, 118–128. https://doi.org/10.1016/j.pbi.2020.03.008 Sonawane, P.D., Pollier, J., Panda, S., Szymanski, J., Massalha, H., Yona, M., Unger, T., Malitsky, S., Arendt, P., Pauwels, L., Almekias-Siegl, E., Rogachev, I., Meir, S., Cárdenas, P.D., Masri, A., Petrikov, M., Schaller, H., Schaffer, A.A., Kamble, A., Giri, A.P., Goossens, A., Aharoni, A., 2016. Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism. Nat. Plants 3, 1–13. https://doi.org/10.1038/nplants.2016.205 Song, B., Song, Y., Fu, Y., Kizito, E.B., Kamenya, S.N., Kabod, P.N., Liu, H., Muthemba, S., Kariba, R., Njuguna, J., Maina, S., Stomeo, F., Djikeng, A., Hendre, P.S., Chen, X., Chen, W., Li, X., Sun, W., Wang, S., Cheng, S., Muchugi, A., Jamnadass, R., Shapiro, H.-Y., Van Deynze, A., Yang, H., Wang, J., Xu, X., Odeny, D.A., Liu, X., 2019. Draft genome sequence of Solanum aethiopicum provides insights into disease resistance, drought tolerance, and the evolution of the genome. GigaScience 8, giz115. https://doi.org/10.1093/gigascience/giz115 72 Spiller, F., Alves, M.K., Vieira, S.M., Carvalho, T.A., Leite, C.E., Lunardelli, A., Poloni, J.A., Cunha, F.Q., de Oliveira, J.R., 2008. Anti-inflammatory effects of red pepper (Capsicum baccatum) on carrageenan- and antigen-induced inflammation. J. Pharm. Pharmacol. 60, 473–478. https://doi.org/10.1211/jpp.60.4.0010 Srinivasan, P., Smolke, C.D., 2020. Biosynthesis of medicinal tropane alkaloids in yeast. Nature 585, 614–619. https://doi.org/10.1038/s41586-020-2650-9 Stenzel, O., Teuber, M., Dräger, B., 2006. Putrescine N-methyltransferase in Solanum tuberosum L., a calystegine-forming plant. Planta 223, 200–212. https://doi.org/10.1007/s00425- 005-0077-z Stewart Jr, C., Kang, B.-C., Liu, K., Mazourek, M., Moore, S.L., Yoo, E.Y., Kim, B.-D., Paran, I., Jahn, M.M., 2005. The Pun1 gene for pungency in pepper encodes a putative acyltransferase. Plant J. 42, 675–688. https://doi.org/10.1111/j.1365-313X.2005.02410.x Sulli, M., Barchi, L., Toppino, L., Diretto, G., Sala, T., Lanteri, S., Rotino, G.L., Giuliano, G., 2021. An eggplant recombinant inbred population allows the discovery of metabolic QTLs controlling fruit nutritional quality. Front. Plant Sci. 12. Szymański, J., Bocobza, S., Panda, S., Sonawane, P., Cárdenas, P.D., Lashbrooke, J., Kamble, A., Shahaf, N., Meir, S., Bovy, A., Beekwilder, J., Tikunov, Y., Romero de la Fuente, I., Zamir, D., Rogachev, I., Aharoni, A., 2020. Analysis of wild tomato introgression lines elucidates the genetic basis of transcriptome and metabolome variation underlying fruit traits and pathogen response. Nat. Genet. 52, 1111–1121. https://doi.org/10.1038/s41588- 020-0690-6 Tanaka, Y., Sonoyama, T., Muraga, Y., Koeda, S., Goto, T., Yoshida, Y., Yasuba, K., 2015. Multiple loss-of-function putative aminotransferase alleles contribute to low pungency and capsinoid biosynthesis in Capsicum chinense. Mol. Breed. 35, 142. https://doi.org/10.1007/s11032-015-0339-9 Tewksbury, J.J., Nabhan, G.P., 2001. Directed deterrence by capsaicin in chillies. Nature 412, 403–404. https://doi.org/10.1038/35086653 Tieman, D., Zhu, G., Resende, M.F.R., Lin, T., Nguyen, C., Bies, D., Rambla, J.L., Beltran, K.S.O., Taylor, M., Zhang, B., Ikeda, H., Liu, Z., Fisher, J., Zemach, I., Monforte, A., Zamir, D., Granell, A., Kirst, M., Huang, S., Klee, H., 2017. A chemical genetic roadmap to improved tomato flavor. Science 355, 391–394. https://doi.org/10.1126/science.aal1556 Tikunov, Y.M., Molthoff, J., de Vos, R.C.H., Beekwilder, J., van Houwelingen, A., van der Hooft, J.J.J., Nijenhuis-de Vries, M., Labrie, C.W., Verkerke, W., van de Geest, H., Viquez Zamora, M., Presa, S., Rambla, J.L., Granell, A., Hall, R.D., Bovy, A.G., 2013. NON-SMOKY GLYCOSYLTRANSFERASE1 prevents the release of smoky aroma from tomato fruit. Plant Cell 25, 3067–3078. https://doi.org/10.1105/tpc.113.114231 73 Toal, T.W., Ron, M., Gibson, D., Kajala, K., Splitt, B., Johnson, L.S., Miller, N.D., Slovak, R., Gaudinier, A., Patel, R., de Lucas, M., Provart, N.J., Spalding, E.P., Busch, W., Kliebenstein, D.J., Brady, S.M., 2018. Regulation of root angle and gravitropism. G3: Genes Genomes Genet. 8, 3841–3855. https://doi.org/10.1534/g3.118.200540 Tripodi, P., Rabanus-Wallace, M.T., Barchi, L., Kale, S., Esposito, S., Acquadro, A., Schafleitner, R., van Zonneveld, M., Prohens, J., Diez, M.J., Börner, A., Salinier, J., Caromel, B., Bovy, A., Boyaci, F., Pasev, G., Brandt, R., Himmelbach, A., Portis, E., Finkers, R., Lanteri, S., Paran, I., Lefebvre, V., Giuliano, G., Stein, N., 2021. Global range expansion history of pepper (Capsicum spp.) revealed by over 10,000 genebank accessions. Proc. Natl. Acad. Sci. U.S.A. 118, e2104315118. https://doi.org/10.1073/pnas.2104315118 Tsugawa, H., Nakabayashi, R., Mori, T., Yamada, Y., Takahashi, M., Rai, A., Sugiyama, R., Yamamoto, H., Nakaya, T., Yamazaki, M., Kooke, R., Bac-Molenaar, J.A., Oztolan-Erol, N., Keurentjes, J.J.B., Arita, M., Saito, K., 2019. A cheminformatics approach to characterize metabolomes in stable-isotope-labeled organisms. Nat. Methods 16, 295– 298. https://doi.org/10.1038/s41592-019-0358-2 Tzin, V., Malitsky, S., Zvi, M.M.B., Bedair, M., Sumner, L., Aharoni, A., Galili, G., 2012. Expression of a bacterial feedback-insensitive 3-deoxy-d-arabino-heptulosonate 7- phosphate synthase of the shikimate pathway in Arabidopsis elucidates potential metabolic bottlenecks between primary and secondary metabolism. New Phytol. 194, 430–439. https://doi.org/10.1111/j.1469-8137.2012.04052.x Van Eck, J., 2018. Genome editing and plant transformation of solanaceous food crops. Curr. Opin. Biotechnol., Food biotechnology • Plant biotechnology 49, 35–41. https://doi.org/10.1016/j.copbio.2017.07.012 Varghese, S., Kubatka, P., Rodrigo, L., Gazdikova, K., Caprnda, M., Fedotova, J., Zulli, A., Kruzliak, P., Büsselberg, D., 2017. Chili pepper as a body weight-loss food. Int. J. Food Sci. Nutr. 68, 392–401. https://doi.org/10.1080/09637486.2016.1258044 Vogt, T., 2010. Phenylpropanoid biosynthesis. Mol. Plant 3, 2–20. https://doi.org/10.1093/mp/ssp106 Wang, M., Carver, J.J., Phelan, V.V., Sanchez, L.M., Garg, N., Peng, Y., Nguyen, D.D., Watrous, J., Kapono, C.A., Luzzatto-Knaan, T., Porto, C., Bouslimani, A., Melnik, A.V., Meehan, M.J., Liu, W.-T., Crüsemann, M., Boudreau, P.D., Esquenazi, E., Sandoval- Calderón, M., Kersten, R.D., Pace, L.A., Quinn, R.A., Duncan, K.R., Hsu, C.-C., Floros, D.J., Gavilan, R.G., Kleigrewe, K., Northen, T., Dutton, R.J., Parrot, D., Carlson, E.E., Aigle, B., Michelsen, C.F., Jelsbak, L., Sohlenkamp, C., Pevzner, P., Edlund, A., McLean, J., Piel, J., Murphy, B.T., Gerwick, L., Liaw, C.-C., Yang, Y.-L., Humpf, H.-U., Maansson, M., Keyzers, R.A., Sims, A.C., Johnson, A.R., Sidebottom, A.M., Sedio, B.E., Klitgaard, A., Larson, C.B., Boya P, C.A., Torres-Mendoza, D., Gonzalez, D.J., Silva, D.B., Marques, L.M., Demarque, D.P., Pociute, E., O’Neill, E.C., Briand, E., Helfrich, E.J.N., Granatosky, E.A., Glukhov, E., Ryffel, F., Houson, H., Mohimani, H., Kharbush, 74 J.J., Zeng, Y., Vorholt, J.A., Kurita, K.L., Charusanti, P., McPhail, K.L., Nielsen, K.F., Vuong, L., Elfeki, M., Traxler, M.F., Engene, N., Koyama, N., Vining, O.B., Baric, R., Silva, R.R., Mascuch, S.J., Tomasi, S., Jenkins, S., Macherla, V., Hoffman, T., Agarwal, V., Williams, P.G., Dai, J., Neupane, R., Gurr, J., Rodríguez, A.M.C., Lamsa, A., Zhang, C., Dorrestein, K., Duggan, B.M., Almaliti, J., Allard, P.-M., Phapale, P., Nothias, L.-F., Alexandrov, T., Litaudon, M., Wolfender, J.-L., Kyle, J.E., Metz, T.O., Peryea, T., Nguyen, D.-T., VanLeer, D., Shinn, P., Jadhav, A., Müller, R., Waters, K.M., Shi, W., Liu, X., Zhang, L., Knight, R., Jensen, P.R., Palsson, B.Ø., Pogliano, K., Linington, R.G., Gutiérrez, M., Lopes, N.P., Gerwick, W.H., Moore, B.S., Dorrestein, P.C., Bandeira, N., 2016. Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking. Nat. Biotechnol. 34, 828–837. https://doi.org/10.1038/nbt.3597 Wang, P., Moore, B.M., Uygun, S., Lehti-Shiu, M.D., Barry, C.S., Shiu, S.-H., 2021. Optimising the use of gene expression data to predict plant metabolic pathway memberships. New Phytol. 231, 475–489. https://doi.org/10.1111/nph.17355 Weber, N., Ismail, A., Gorwa-Grauslund, M., Carlquist, M., 2014. Biocatalytic potential of vanillin aminotransferase from Capsicum chinense. BMC Biotechnol. 14, 25. https://doi.org/10.1186/1472-6750-14-25 Weinhold, A., Baldwin, I.T., 2011. Trichome-derived O-acyl sugars are a first meal for caterpillars that tags them for predation. Proc. Natl. Acad. Sci. U.S.A. 108, 7855–7859. https://doi.org/10.1073/pnas.1101306108 Weng, J.-K., Philippe, R.N., Noel, J.P., 2012. The rise of chemodiversity in plants. Science 336, 1667–1670. https://doi.org/10.1126/science.1217411 W. Sumner, L., Lei, Z., J. Nikolau, B., Saito, K., 2015. Modern plant metabolomics: advanced natural product gene discoveries, improved technologies, and future prospects. Nat. Prod. Rep. 32, 212–229. https://doi.org/10.1039/C4NP00072B Wu, Q., Cho, J.-G., Lee, D.-S., Lee, D.-Y., Song, N.-Y., Kim, Y.-C., Lee, K.-T., Chung, H.-G., Choi, M.-S., Jeong, T.-S., Ahn, E.-M., Kim, G.-S., Baek, N.-I., 2013. Carbohydrate derivatives from the roots of Brassica rapa ssp. campestris and their effects on ROS production and glutamate-induced cell death in HT-22 cells. Carbohydr. Res. 372, 9–14. https://doi.org/10.1016/j.carres.2012.09.015 Xu, S., Brockmöller, T., Navarro-Quezada, A., Kuhl, H., Gase, K., Ling, Z., Zhou, W., Kreitzer, C., Stanke, M., Tang, H., Lyons, E., Pandey, P., Pandey, S.P., Timmermann, B., Gaquerel, E., Baldwin, I.T., 2017. Wild tobacco genomes reveal the evolution of nicotine biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 114, 6133–6138. https://doi.org/10.1073/pnas.1700073114 The Potato Genome Sequencing Consortium, 2011. Genome sequence and analysis of the tuber crop potato. Nature 475, 189–195. https://doi.org/10.1038/nature10158 75 Yoo, H., Widhalm, J.R., Qian, Y., Maeda, H., Cooper, B.R., Jannasch, A.S., Gonda, I., Lewinsohn, E., Rhodes, D., Dudareva, N., 2013. An alternative pathway contributes to phenylalanine biosynthesis in plants via a cytosolic tyrosine:phenylpyruvate aminotransferase. Nat. Commun. 4, 2833. https://doi.org/10.1038/ncomms3833 Zabel, S., Brandt, W., Porzel, A., Athmer, B., Bennewitz, S., Schäfer, P., Kortbeek, R., Bleeker, P., Tissier, A., 2021. A single cytochrome P450 oxidase from Solanum habrochaites sequentially oxidizes 7-epi-zingiberene to derivatives toxic to whiteflies and various microorganisms. Plant J. 105, 1309–1325. https://doi.org/10.1111/tpj.15113 Zanor, M.I., Rambla, J.-L., Chaïb, J., Steppa, A., Medina, A., Granell, A., Fernie, A.R., Causse, M., 2009. Metabolic characterization of loci affecting sensory attributes in tomato allows an assessment of the influence of the levels of primary metabolites and volatile organic contents. J. Exp. Bot. 60, 2139–2154. https://doi.org/10.1093/jxb/erp086 Zhang, C.-R., Khan, W., Bakht, J., Nair, M.G., 2016. New antiinflammatory sucrose esters in the natural sticky coating of tomatillo (Physalis philadelphica), an important culinary fruit. Food Chem. 196, 726–732. https://doi.org/10.1016/j.foodchem.2015.10.007 Zhang, C.-Y., Luo, J.-G., Liu, R.-H., Lin, R., Yang, M.-H., Kong, L.-Y., 2016. 1H NMR spectroscopy-guided isolation of new sucrose esters from Physalis alkekengi var. franchetii and their antibacterial activity. Fitoterapia 114, 138–143. https://doi.org/10.1016/j.fitote.2016.09.007 Zhang, Y., Butelli, E., Alseekh, S., Tohge, T., Rallapalli, G., Luo, J., Kawar, P.G., Hill, L., Santino, A., Fernie, A.R., Martin, C., 2015. Multi-level engineering facilitates the production of phenylpropanoid compounds in tomato. Nat. Commun. 6, 8635. https://doi.org/10.1038/ncomms9635 Zhou, F., Pichersky, E., 2020a. More is better: the diversity of terpene metabolism in plants. Curr. Opin. Plant Biol., Physiology and Metabolism 55, 1–10. https://doi.org/10.1016/j.pbi.2020.01.005 Zhou, F., Pichersky, E., 2020b. The complete functional characterisation of the terpene synthase family in tomato. New Phytol. 226, 1341–1360. https://doi.org/10.1111/nph.16431 Zhu, G., Wang, S., Huang, Z., Zhang, S., Liao, Q., Zhang, C., Lin, T., Qin, M., Peng, M., Yang, C., Cao, X., Han, X., Wang, X., Knaap, E. van der, Zhang, Z., Cui, X., Klee, H., Fernie, A.R., Luo, J., Huang, S., 2018. Rewiring of the fruit metabolome in tomato breeding. Cell 172, 249-261.e12. https://doi.org/10.1016/j.cell.2017.12.019 Zi, J., Matsuba, Y., Hong, Y.J., Jackson, A.J., Tantillo, D.J., Pichersky, E., Peters, R.J., 2014. Biosynthesis of lycosantalonol, a cis-prenyl derived diterpenoid. J. Am. Chem. Soc. 136, 16951–16953. https://doi.org/10.1021/ja508477e 76 CHAPTER 2: TRADING ACYLS AND SWAPPING SUGARS: METABOLIC INNOVATIONS IN SOLANUM TRICHOMES Works presented in this chapter have been published: Fiesel, P. D., Kerwin, R.A., Jones, A.D., Last, R.L., 2023. Trading acyls and swapping sugars: metabolic innovations in Solanum trichomes. bioRxiv. https://doi.org/10.1101/2023.06.05.542877 77 Abstract Solanaceae (nightshade family) species synthesize a remarkable array of clade- and tissue-specific specialized metabolites. Protective acylsugars, one such class of structurally diverse metabolites, are produced from sugars and acyl-Coenzyme A esters by acylsugar acyltransferases in glandular trichomes. We characterized trichome acylsugars of the Clade II species Solanum melongena (brinjal eggplant) using liquid chromatography-mass spectrometry (LC-MS), gas chromatography-MS and nuclear magnetic resonance (NMR) spectroscopy. This led to the identification of eight unusual structures with inositol cores, inositol glycoside cores, and hydroxyacyl chains. LC-MS analysis of 31 species in the megadiverse Solanum genus revealed striking acylsugar diversity with some traits restricted to specific clades and species. Acylinositols were found throughout each clade while acylglucoses were restricted to DulMo and VANAns species. Medium length hydroxyacyl chains were found in many species. Analysis of tissue-specific transcriptomes and interspecific acylsugar acetylation differences led to characterization of the S. melongena Acylsugar AcylTransferase 3-Like 1 (SmASAT3-L1; SMEL4.1_12g015780) enzyme. This enzyme is distinct from previously characterized acylsugar acetyltransferases, which are in the ASAT4 clade, and is a functionally divergent ASAT3. This study provides a foundation for investigating the evolution of diverse Solanum acylsugar structures and harnessing this diversity in breeding and synthetic biology. 78 Introduction Plants are remarkable synthetic chemists, producing a multitude of structurally complex specialized metabolites that differ from the products of general, or primary, metabolism in their lineage-specific distribution and tissue or cell type-specific biosynthesis. In contrast to the negative selection against changes in primary metabolism (for example, amino acids, energy metabolism intermediates and vitamin cofactors), less constrained evolution of specialized metabolism led to accumulation of hundreds of thousands of taxonomically restricted metabolites in broad classes. Specialized metabolites play many critical roles such as in abiotic and biotic stress adaptation (Agati and Tattini, 2010; De Moraes et al., 2001; Landry et al., 1995), pollinator attraction (Kretschmar and Baumann, 1999) and mediation of interactions with beneficial and pathogenic microbes (Yu et al., 2021). These diverse and bioactive small molecules have historical and modern uses in human medicine, including the anticancer alkaloids vinblastine and paclitaxel, antimalarial artemisinin, and painkillers such as morphine. Acylsugars are specialized metabolites produced across the Solanaceae (nightshade) family, aiding in defense against herbivores, fungi, and bacteria (Goffreda et al., 1989; Leckie et al., 2016; Luu et al., 2017; Weinhold and Baldwin, 2011). In Type I- and IV-glandular trichomes, BAHD-type AcylSugar AcylTransferase (ASAT) enzymes assemble acylsugars from the basic building blocks of sugars, often sucrose, and short- to medium-length acyl chains derived from acyl-CoAs (Fan et al., 2015; Lou et al., 2021; Moghe et al., 2017; Schilmiller et al., 2012; Schilmiller et al., 2015). Despite their simple components, acylsugars exhibit remarkable chemical diversity arising from variations in sugar core composition and acyl chain length, branching pattern, position, and number (Fan et al., 2019; Fiesel et al., 2022; Ghosh et al., 2014a; Hurney, 2018; Lou et al., 2021; Moghe et al., 2017; Schenck et al., 2022). For example, 79 acylsucroses, composed of a sucrose disaccharide core, accumulate in cultivated tomato (Solanum lycopersicum) trichomes, while acylsucroses and acylglucoses have been observed in the trichomes of wild tomato species, Solanum pennellii. Acylsugar structural variation also impacts biological activity; for example, differential oviposition deterrence was demonstrated from naturally derived acylsugar mixtures (Leckie et al., 2016). Solanaceae acylsugars have become an exemplary model to study evolution of a diverse, biologically relevant trait in a plant family with genomic and phylogenetic resources. Utilization of this model revealed gene duplication, neofunctionalization, co-option, and loss involved in acylsugar evolution (Fan et al., 2020, 2017; Leong et al., 2019; Moghe et al., 2017). For example, neofunctionalization of an invertase-like enzyme, AcylSucrose FructoFuranosidase 1 (ASFF1) (Leong et al., 2019) and functional divergence of core ASAT enzymes is responsible for differences in sugar core type and acyl chain positions between cultivated tomato S. lycopersicum and wild tomato S. pennellii acylsugars (Fan et al., 2017). Identifying mechanisms of acylsugar evolution across the Solanum requires a detailed understanding of acylsugar diversity and biosynthesis, which is lacking for many species. Specialized metabolism diversification is often driven by gene duplication and subsequent sequence divergence. In fact, specialized metabolism genes have higher duplication rates than general metabolism genes (Moore et al., 2019). Duplicates often arise through whole genome duplications and localized tandem duplications and can exhibit lower rates of selection leading to genes and gene products with new functions, localizations, and regulation (Panchy et al., 2016). For example, the tandem duplication of N-methyltransferases and cytochrome P450s led to evolution of the alkaloid caffeine and the benzoxazinoid DIMBOA, respectively (Dutartre et al., 2012; Denoeud et al., 2014). 80 Nearly half of the Solanaceae falls into the large (>1200 species) and phenotypically diverse Solanum genus. This genus is split into several major clades, including Potato, Regmandra, DulMo, VANAns, and Clade II; the Potato clade contains cultivated tomato and potato and their wild relatives while Clade II contains the cultivated brinjal eggplant, Solanum melongena, and other ‘spiny Solanums’ (Bohs, 2004; Bohs and Olmstead, 1997; Gagnon et al., 2022; Levin et al., 2006; PBI Solanum Project, 2022; Särkinen et al., 2013; Stern et al., 2011; Tepe et al., 2016; Weese and Bohs, 2007) (Gagnon et al., 2022). To date, documentation of acylsugar diversity largely focused on a handful of species within the Potato clade, including cultivated tomato and its close relatives. These efforts identified at least 38 nuclear magnetic resonance (NMR) spectroscopy-resolved acylsugar structures and liquid chromatography mass spectrometry (LC-MS) supported annotations of many more (Fan et al., 2017; Ghosh et al., 2014a; Lybrand et al., 2020; Schilmiller et al., 2016). While limited acylsugar screening outside of the Potato clade was reported, novel structural variants not observed among cultivated tomato relatives were identified. For example, acylsugars with myo-inositol sugar cores (i.e., acylinositols) were characterized in three species: S. nigrum, from the DulMo clade, and S. lanceolatum and S. quitoense, from Clade II (Herrera-Salgado et al., 2005; Hurney, 2018; Leong et al., 2020; Lou et al., 2021). These discoveries highlight the benefits of a more comprehensive description of Solanum acylsugars within the well-developed phylogenetic framework. Here we report analysis of Solanum acylsugar chemical diversity in species from the relatively unexplored Solanum clades DulMo, VANAns, and Clade II, which together comprise ~1000 Solanum species. We first established the Clade II brinjal eggplant, S. melongena, as a reference species. Eggplant is a major worldwide fruit crop with extensive genomic, transcriptomic, and germplasm resources (Barchi et al., 2021; Li et al., 2021; Mennella et al., 81 2010). We characterized eggplant trichome acylinositols, acylinositol glycosides, and acylsugars with unusual hydroxylated acyl chains using electrospray ionization LC-quadrupole time of flight-MS (ESI LC-QToF-MS) and NMR. These atypical structures likely reflect altered biochemistry from the cultivated tomato acylsucrose pathway. Moving out from this model organism framework, LC-MS phylogenetic screening of 31 Clade II, DulMo, and VANAns species, including S. melongena, led to the identification of remarkable acylsugar structural variation. LC-MS features with characteristics of acylinositols were found in 25 of the 26 acylsugar-producing species, suggesting one or a small number of evolutionary origins. In contrast, acylglucoses were detected in DulMo and VANAns species, but not in any tested Clade II species. As a first step towards unraveling the molecular basis underlying the extensive acylsugar diversity, we utilized interspecific acylsugar differences and an eggplant tissue-specific transcriptome to identify an acylinositol biosynthetic enzyme, S. melongena Acylsugar AcylTransferase 3-Like 1 (SmASAT3-L1; SMEL4.1_12g015780), responsible for acetylating a triacylinositol glycoside acyl acceptor. SmASAT3-L1 exhibits a different acyl-CoA specificity than previously characterized ASAT3 homologs, highlighting how gene duplication and functional divergence created acylsugar metabolic novelty in this part of the Solanum clade. Results and Discussion Eggplant glandular trichomes accumulate acylsugar-like compounds We began our investigation of Solanum acylsugar diversity with the brinjal eggplant S. melongena due to its economic importance, genomic resources, and phylogenetic position within the monophyletic Eastern Hemisphere Spiny clade of Clade II (formerly known as the ‘Old World spiny clade’) (Gagnon et al., 2022). Using eight eggplant accessions (Table S2.37), we observed glandular trichomes on hypocotyls, cotyledons, and the first three true leaves of young 82 eggplants, which resemble the acylsugar-producing structures found in other Solanum species (Figure 2.2) (Leong et al., 2020; Lou et al., 2021; Schilmiller et al., 2012). In contrast, we observed only non-glandular stellate trichomes on leaves and stems of mature eggplants (Figure S2.12), which are unlikely to accumulate and synthesize acylsugars or other specialized metabolites (Levin, 1973; Wagner, 1991). We analyzed surface metabolite extracts from young and mature eggplant tissues using LC-QToF-MS coupled with collision-induced dissociation (CID) in negative and positive ion mode, and annotated acylsugars based on relative mass defect, molecular adduct ion masses, retention times, and ions present in CID mass spectra. Briefly, acylsugars were annotated from masses of fatty acid carboxylate fragment ions, as well as fragment ions corresponding to stepwise losses of acyl chains from the pseudomolecular ion to a sugar core fragment ion. The acylsugar annotation methods and confidence criteria are explained in detail in the Methods. This analysis revealed abundant acylsugars in extracts from young, glandular trichome-producing eggplant tissues, but not from mature, non-glandular trichome producing tissues. We annotated 38 acylsugars in young eggplant extracts from eight accessions, including 16 acylhexoses and 22 acyldisaccharides (Table 2.1). LC-MS based acylsugar annotations are described using a modified shorthand nomenclature (Leong et al., 2020) as follows: UX:Y:Z(A,B,C,D), where U – as a single or multi-letter designation – represents the sugar core, X represents the number of acyl chains, Y represents the sum of acyl chain carbons, Z represents the number of unsaturated bonds in the acyl chains (when present), and A-D represent the number of carbon atoms in the individual acyl chains. For example, the eggplant acylhexose I3:18(4,4,10) consists of a myo-inositol core with three acyl chains with a total of 18 carbon atoms. Curiously, eggplant acyldisaccharides contain an atypical pentose-hexose core, as 83 evidenced by a fragment ion mass of m/z 293.09 in negative-ion mode, corresponding to a fully deacylated sugar core minus a proton. Further evidence was provided by positive mode CID, which promotes glycosidic bond cleavage, yielding a fragment ion corresponding to the neutral loss of an unacylated pentose ring. All acylhexoses annotated with medium to high confidence formed abundant fragment ions in positive mode but few fragment ions in negative mode as illustrated in Figure S2.1; this pattern is characteristic of acylinositols found in S. quitoense and S. nigrum, but has not been observed for acylglucoses, suggesting that all detected S. melongena acylhexoses are acylinositols (Hurney, 2018; Leong et al., 2020; Lou et al., 2021). The eggplant acylsugars contained three to four acylations, all on the hexose core, including one medium eight-carbon (C8) to C14 acyl chain and two to three short C4 or C5 acyl chains. The medium acyl chains (C8, C10, C12, and C14) were either iso-branched or straight as revealed by GC-MS acyl chain analysis (Figure S2.85). Additionally, we identified hydroxylated C12, C14, and C16 acyl chains not previously reported in Solanaceae acylsugars. Although we did not observe large differences between the eight eggplant accessions, eggplant acylsugars differ in chain length, functional groups, and acyl chain composition from the reported Solanum acylinositols (Herrera- Salgado et al., 2005; Hurney, 2018; Lou et al., 2021). Table 2.1. Summary of annotated acylsugars detected in S. melongena leaf surface extracts. PH = pentose-hexose; AI = arabinose-inositol, I = inositol. RT = retention time; m/zacc = theoretical monoisotopic formate adduct mass; m/zex = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Compound number is listed for NMR characterized compounds (Figure 2.2). Percent total peak area calculated by dividing acylsugar peak area by total acylsugar peak area. Acylsugars composing ≥1% of total acylsugar peak area are bolded. Percent peak area calculated from 15 S. melongena leaf surface extracts. Annotation method is described in Materials and Methods. Acylsugars are sorted by number of sugar moieties and then by elution order. # d n u o p m o C Annotation confidence level RT (min) Name Average percent peak area (%) Δm (ppm) m/zacc m/zex Neutral molecule chemical formula 84 0.203 0.0246 0.0453 0.0851 17.1 0.114 0.324 0.901 42.5 1.12 0.964 0.329 0.0428 0.0447 0.127 0.00422 0.0389 0.130 0.0356 0.0107 0.2 -4.6 0.0 8.2 3.2 1.9 679.3177 679.3146 623.2915 623.2916 9.2 1.9 1.8 0.7 -3.5 2.5 high medium medium medium high medium medium medium medium medium 1 2 medium medium medium medium medium medium medium medium medium medium medium medium 1.98 C23H40O12 553.2496 553.2496 2.19 C25H40O14 609.2395 609.2445 2.23 C25H42O13 595.2602 595.2621 2.54 C27H44O14 637.2708 637.2720 2.61 C27H46O13 2.66 C27H46O13 2.93 C28H46O14 651.2864 651.2924 2.96 C28H48O13 637.3071 637.3083 3.11 C29H48O14 665.3021 665.3033 3.27 C29H48O14 665.3021 665.3026 3.61 C29H50O13 651.3228 651.3205 3.74 C29H50O13 651.3228 651.3245 3.76 C30H50O14 3.83 C30H50O14 4.22 C31H52O14 693.3334 693.3350 4.57 C31H52O14 693.3334 693.3351 4.68 C31H52O14 693.3334 693.3349 4.85 C31H52O14 693.3334 693.3379 5.05 C31H54O13 679.3541 679.3554 5.23 C31H54O13 679.3541 679.3552 7.54 C33H58O13 707.3854 707.3879 7.85 C33H58O13 707.3854 707.3892 Table 2.1. (cont’d) Acyldisaccharides PH2:12(4,8) PH4:14(2,4,4,4) PH3:14(2,4,8) PH4:16(2,2,4,8) PH3:16(4,4,8) AI3:16(4,4,8) PH4:17(2,2,4,9) PH3:17(4,5,8) PH4:18(2,4,4,8) AI4:18(2,4,4,8) PH3:18(4,4,10) PH3:18(4,4,10) PH4:19(2,4,5,8) PH4:19(2,4,5,8) PH4:20(4,4,4,8) PH4:20(2,4,4,10) PH4:20(2,4,4,10) PH4:20(2,4,4,10) PH3:20(4,4,12) PH3:20(4,4,12) PH3:22(4,4,14) PH3:22(4,4,14) Acylhexoses I3:16(4,4,8) I3:20(4,4,12-OH) I3:20(4,4,12-OH) I4:18(2,4,4,8) I3:18(4,4,10) I3:18(4,4,10) I3:20(2,4,14-OH) I3:20(2,4,14-OH) I3:22(4,4,14-OH) I3:22(4,4,14-OH) I3:20(4,4,12) I3:20(4,4,12) I3:24(4,4,16-OH) I3:24(4,4,16-OH) I3:22(4,4,14) I3:22(4,4,14) Although LC-MS provided information about sugar core mass, it did not reveal the sugar core 3.27 C22H38O9 491.2492 491.2492 4.16 C26H46O10 563.3068 563.3071 4.32 C26H46O10 563.3068 563.3068 4.22 C24H40O10 533.2598 533.2604 519.2805 519.2802 4.70 C24H42O9 4.88 519.2805 519.2794 C24H42O9 4.68 C26H46O10 563.3068 563.3082 4.90 C26H46O10 563.3068 563.3077 6.06 C28H50O10 591.3381 591.3385 6.34 C28H50O10 591.3381 591.3380 547.3118 547.3119 6.81 C26H46O9 7.09 547.3118 547.3127 C26H46O9 8.51 C30H54O10 619.3694 619.3707 8.85 C30H54O10 619.3694 619.3683 575.3431 575.3435 9.34 575.3431 575.3433 9.68 medium medium medium medium high medium medium medium high high high medium medium medium high high -0.1 0.5 0.0 1.1 -0.5 -2.1 2.5 1.6 0.7 -0.1 0.1 1.7 2.0 -1.8 0.7 0.3 2.3 2.5 2.1 6.6 1.9 1.6 3.5 5.3 C28H50O9 C28H50O9 7 8 4 5 6 3 3.99 0.0808 0.500 0.0248 10.6 0.693 0.0588 0.298 4.96 10.6 2.24 0.105 0.0473 0.0355 0.986 0.722 structure, prompting analysis using gas chromatography-mass spectrometry (GC-MS) of 85 derivatized S. melongena acylsugar cores. When free sugar cores, produced by metabolite extract saponification, were derivatized to form alditol acetates, GC-MS peaks corresponding to derivatized myo-inositol and glucose were detected, supporting the presence of acylinositols (Figure 2.1A). The detection of glucose might have resulted from other compounds in the leaf surface metabolite extracts. The disaccharide sugar core composition was determined by hydrolyzing the saponified sugar cores with formic acid to cleave the glycosidic linkage. Comparison of the hydrolyzed and unhydrolyzed samples revealed a peak corresponding to arabinose only in the hydrolyzed plant samples (Figure 2.1B). Taken together, the results of saponification with and without hydrolysis, followed by derivatization, confirmed identification of myo-inositol sugar cores, and identified the pentose moiety of the hexose-pentose disaccharide core as arabinose. 86 Figure 2.1. Identification of S. melongena acylsugar core composition through GC-MS analysis of alditol acetate sugar derivatives. S. melongena acylsugars collected from surface extracts were first saponified to remove acyl chains, and then, with or without acid hydrolysis to break the glycosidic linkages, sugar cores were derivatized to alditol acetates. (A) Alditol acetate derivatization of saponified S. melongena acylsugars yield a peak that comigrated with that of a myo-inositol standard. The traces displayed are GC-MS total ion chromatograms (TICs) normalized to the highest ion count in the selected traces. (B) Alditol acetate pentose derivatives 87 AB11.8011.9012.0012.1012.2012.3012.4012.5012.6012.7012.80%0100RelativeAbundanceMannoseRetention time (min)Saponified hydrolyzed sugar coresSaponified sugar coresmyo-inositolGlucoseGalactose8.959.009.059.109.159.209.259.309.359.409.459.509.559.609.659.70%0100RelativeAbundanceRiboseXyloseArabinoseRetentionTime (min)Saponified hydrolyzed sugar coresSaponified sugar cores Figure 2.1. (cont’d) of saponified and hydrolyzed acylsugar cores comigrate with that of a derivatized arabinose standard. The traces displayed are GC-MS TICs. NMR analysis of eight eggplant acylsugars While MS analysis provided valuable information about acyl chain number and length, sugar core mass, as well as acyl chain number, complementary information about acyl chain branch structure, sugar core stereochemistry, and acyl chain positions was obtained using NMR. We purified and resolved the structures of eight abundant eggplant acylsugars from accession PI 555598 using a combination of 1D and 2D NMR experiments (Figure 2.2). All eight structures are newly described, and because atomic connections were determined by NMR and MS data, the proposed structures meet Metabolomics Standards Initiative level 1 criteria for metabolite identification (Sumner et al., 2007). A series of NMR experiments confirmed that the acylhexose and acyldisaccharide sugar cores are myo-inositol and 4-O-β-arabinopyranosyl myo-inositol, respectively (Figure 2.2C,D). We assigned all sugar ring proton signals of each sugar core’s spin system with total correlation spectroscopy (TOCSY). Inferences from correlation spectroscopy (COSY) data then identified the order of ring protons and identified pyranose and cyclitol ring structures for the pentose and hexose rings, respectively. Relative stereochemistry at ring positions was subsequently determined through comparison of spin-spin splitting (multiplicities and coupling constants) referenced to expected patterns inferred from chemical principles and previously reported acylinositols (Hurney, 2018; Leong et al., 2020). The disaccharide glycosidic linkage position was determined by heteronuclear multiple bond correlation (HMBC) correlations to be at position 4 and position 1 of myo-inositol and arabinose, respectively. The arabinose β anomeric configuration was inferred from the anomeric carbon 1JCH (162 Hz) as revealed by a coupled- heteronuclear single quantum coherence (coupled-HSQC) experiment of the free disaccharide 88 sugar core after saponification. Our sugar core assignments identifying this unusual disaccharide agreed with the previous GC-MS sugar core results, supporting the efficacy of the sugar core GC-MS identification method. This disaccharide differs only in the pentose moiety identity from a recently reported acylated 4-O-ß-xylopyranosyl myo-inositol in the Solanum Clade II species S. quitoense (Hurney, 2018). To the best of our knowledge, this is the first report of acylated 4-O-ß- arabinosyl myo-inositol sugars. Acyl chain positions, branching patterns, and hydroxyl positions were also resolved through integration of different NMR experiments. We found that all acyl chains were confined to myo-inositol, consistent with the LC-MS results. All eight acylsugars are decorated with two short iso-branched iC4 acyl chain esters at positions 1 and 2, and one medium C8 to C14 acyl chain ester at position 3 (Figure 2.2). Compound 2 (Figure 2.2C), the only tetraacylated acylsugar identified in eggplant, additionally carried an acetylation at position 6 (Figure 2.2C). The medium-length acyl chains at position 3 were resolved as straight (nC14) or terminally iso- branched (iC8, iC10, iC12, iC14) based on signals characteristic of protons near the branched carbons. Strikingly, we identified peculiar hydroxylated straight and iso-branched 3- hydroxytetradecanoate acyl chains, 3-OH-nC14 and 3-OH-iC14, in compounds 7 and 8, respectively (Figure 2.2D). We assigned hydroxylation positions of 3-OH-nC14 and 3-OH-iC14 to the third acyl carbon based on a downfield shifted signal in the 1H NMR spectrum at 3.92 ppm, corresponding to one hydrogen at that position. In contrast, the non-hydroxylated medium acyl chains observed in compounds 1-6 (Figure 2.2C,D), carry two hydrogen atoms at the third acyl carbon, and these have a characteristic signal near 1.50 ppm. We believe this is the first report of 3-OH-nC14 and 3-OH-iC14 chains in Solanaceae acylsugars. While 3-OH-nC14 chains are observed in the acylsugar-like bacterial Lipid A glycolipids, the hydroxylation position 89 differentiates these eggplant chains from hydroxyacyl chains in castor bean (Ricinus communis) seed oil, Silene gallica gallicasides, Ibicella lutea fatty acid glycosides, and Convolvulaceae resin glycosides (Asai et al., 2010; Asai and Fujimoto, 2010; Bah and Pereda-Miranda, 1996; Smith, 1971). Figure 2.2. Profiling of S. melongena acylsugars. (A) LC-MS base peak intensity (BPI) chromatogram of S. melongena acylsugars. Peaks characterized by NMR are indicated with their 90 Figure 2.2. (cont’d) compound numbers from Panels C and D. Metabolites were collected from seedling tissue with glandular trichomes similar to the ones displayed in the closeup photo of a S. melongena hypocotyl. (B) Annotation of HMBC experiments used to identify acylation positions of specific acyl chains. At the top, I3:18(4,4,10) is displayed in the chair conformation along with hydrogens important for determining acylation position. HMBC NMR spectrum showing couplings between hydrogens and carbonyl carbons are shown. (C) NMR-characterized acyldisaccharide structures 1 and 2, with inositol ring carbons numbered. (D) NMR- characterized acylinositol monosaccharide structures 3-8. The OH-tetradecanoate acyl chain has R stereochemistry To gain insight into the biosynthetic origin of hydroxylated acyl chains, we tested chirality of the 3-OH-nC14 acyl chain of compound 7 as this chain has available commercial standards. Two-step chiral derivatization and GC-MS analysis was performed on compound 7 and two commercial standards, (3R)-OH-nC14 and the corresponding racemate (3R/S)-OH- nC14. Fatty acids were converted to ethyl esters to aid volatilization, followed by esterification of the acyl chain hydroxyl group with the Mosher acid (R)-(−)-α-methoxy-α- (trifluoromethyl)phenylacetate (R-MPTA) to distinguish stereoisomers by GC separation of the diastereomeric derivatives. GC-MS analysis of the derivatized (3R/S)-OH-nC14 standard compounds yielded two peaks at retention times of 42.21 and 42.40 minutes, while one peak at 42.40 minutes was obtained for (3R)-OH-nC14. We observed expected fragment ions corresponding to the MPTA fragment ion, fatty acid ethyl ester, and fatty acid which supported peak assignment (Figure S2.5). Thus (3S)-OH-nC14 and (3R)-OH-nC14 elute at 42.21 and 42.40 minutes, respectively. The compound 7 acyl chain was assigned as (3R)-OH-nC14 as its derivatized 3-OH-tetradecanoate acyl chain eluted at 42.40 minutes. The (3R)-OH-nC14 chain hydroxyl position and stereochemistry have implications for the metabolic pathway leading to its biosynthesis. As fatty acid hydroxylases typically act on the acyl chain terminal region, they are unlikely to catalyze C3 hydroxylation of a C14 fatty acid (Pinot and Beisson, 2011). Of the other well-characterized pathways, only mitochondrial and 91 plastidial fatty acid metabolism mechanisms produce (3R)-OH-acyl-thioester intermediates. The (3R)-OH-nC14 chain hydroxyl position and stereochemistry have implications for the metabolic pathway leading to its biosynthesis. As fatty acid hydroxylases typically act on the acyl chain terminal region, they are unlikely to catalyze C3 hydroxylation of a C14 fatty acid (Pinot and Beisson, 2011). Of the other well-characterized pathways, only mitochondrial and plastidial fatty acid metabolism mechanisms produce (3R)-OH-acyl-thioester intermediates. Consistent with this result, previous work identified two trichome-expressed, mitochondrial enzymes, Acylsugar Enoyl-CoA Hydratase 1 (AECH1) and Acylsugar Acyl-CoA Synthetase 1 (AACS1), and one trichome-expressed, plastidial enzyme, a beta-ketoacyl-(acyl-carrier-protein) reductase (SpKAR1), involved in medium C10 and C12 acyl chain production in cultivated tomato and its wild relative, S. pennellii (Fan et al., 2020; Ji et al., 2022). We hypothesize that acylsugar hydroxyacyl chains evolved through one of three scenarios. First, changes in acyl-CoA substrate availability, possibly through the actions of thioesterases or acyl-CoA synthetases, enabled a promiscuous ASAT to esterify hydroxyacyl chains to acylinositols. Alternatively, substrate specificity of an existing acylinositol ASAT expanded to utilize available hydroxyacyl-CoAs. Like this second hypothesis, amino acid substitutions between copies of ASAT2 from different tomato species impacted the accumulation of differently branched five carbon acyl chains on acylsugars (Fan et al., 2015). A third possibility is that a ‘new’ acyltransferase, unrelated to characterized ASATs and capable of utilizing hydroxyacyl-CoA substrates, was co-opted into acylinositol biosynthesis. Future genetic and biochemical studies should reveal the mechanisms behind (3R)-OH acyl chain production, potentially providing new approaches to engineer unique acylsugars with hydroxyacyl chains and hydroxylated fatty acids for polymeric building blocks. Acylsugar structures impact their 92 function, thus we hypothesize that engineered tomato producing these unusual hydroxyacyl chains on their acylsugars may exhibit different defense capabilities than the wildtype acylsucroses. Figure 2.3. The 3-OH-n14 acyl chain on Compound 7, I3:22(i4,i4,n14-OH), is in the R configuration as revealed by GC-MS using a derivatization method that separates hydroxyacyl chain enantiomers. Hydroxyacyl chains analyzed by GC-SIM- MS after ethyl transesterification and derivation to their diastereomeric (S)-MPTA form. Fragment ions with m/z of 189, 209, and 255 were monitored, and relative abundance was normalized to the highest peak. Yellow and green traces represent derivatives from commercial (3R/S)- and (3R)-OH-14:0 standards. The main derivatized 3-OH-14:0 acyl chain from Compound 7 (Figure 2.2), shown with the blue trace, comigrates with the peak corresponding to the R enantiomer. Enormous Solanum acylsugar diversity revealed by LC-MS metabolite screening To date, most acylsugar screening across the Solanum has focused on cultivated tomato and its Potato clade relatives (Fan et al., 2017; Ghosh et al., 2014a; Lybrand et al., 2020; Schilmiller et al., 2016), which represent a small fraction of the species diversity in this genus (PBI Solanum Project, 2022). Though limited in number, studies profiling non-Potato clade species, including S. nigrum (Lou et al., 2021), S. lanceolatum (Herrera-Salgado et al., 2005), S. quitoense (Hurney, 2018; Leong et al., 2020), and S. melongena (this work) demonstrate acylsugar diversity across the Solanum is vast, and largely uncharacterized. To address this knowledge gap, we analyzed tissue surface extracts from 30 additional Solanum species, 93 including 23 Clade II, five DulMo clade, and two VANAns clade members (listed in Table S2.37). While NMR is the gold standard for structural elucidation, it is prohibitively time- consuming for a largescale metabolite diversity survey. LC-MS coupled with CID provides substantial structural information and is compatible with high-throughput screening. To strike a balance between data quality and quantity, we performed LC-MS-CID screening on all 30 species and annotated acylsugars as described for S. melongena. Twenty-five of the 30 analyzed species produced detectable acylsugars from visible surface glandular trichomes, and species lacking acylsugars had no or few observable glandular trichomes in the tissues analyzed. Targeted analysis of leaf surface extracts from 31 species, including S. melongena, and fruit surface extracts from two species, uncovered previously unreported acylsugars, inter-species acylsugar differences, and identified the distributions of unusual acylsugar chemical traits in the Solanum genus (Figure 2.5; Tables 2.1,S2.1-26). Although we did not observe intraspecific acylsugar variation, acylsugars varied enormously between species (Figure 2.4; Tables 2.1,S2.1- 26). 94 Figure 2.4. Solanum species produce dramatically different leaf surface metabolite profiles. The seven species display morphological differences, as demonstrated by the flower and fruit 95 Figure 2.4. (cont’d) images, and they also exhibit diverse metabolite profiles as demonstrated by base peak intensity LC-MS chromatograms from their leaf surface metabolites. Most of the peaks shown consist of acylsugars with varied sugar cores and acyl chain types and numbers. Acylsugar profiles for these species are detailed in Tables S2.4, S2.7, S2.17, S2.20, and S2.24-26. 96 Figure 2.5. Distribution of acylsugar traits within the Solanum genus. Traits were mapped onto a previously published Supermatrix maximum likelihood phylogeny (Gagnon et al., 2022). The reported polytomy between Clade II, DulMO, VANANs, Regmandra, and Potato clades is 97 Figure 2.5. (cont’d) not indicated (Gagnon et al., 2022). Acylsugar trait data from this report included annotations based upon LC-MS and NMR analysis (Figure 2.2, Tables 2.1, S2.2-26), while data from previous studies were limited to those with NMR structural data (Ghosh et al., 2014b; Herrera- Salgado et al., 2005; Hurney, 2018; King et al., 1986; Leong et al., 2020, 2019; Lou et al., 2021; Lybrand et al., 2020). (A) Distribution of sugar core types within the Solanum genus. Inositol and glucose cores determined by characteristic LC-MS negative mode electrospray ionization (ESI) fragmentation (Figures S2.1 and S2.7). (B) Distribution of species with acylsugars that coelute with eggplant acylsugars containing the same acyl chains and sugar core. (C) Distribution of hydroxyacyl chains and glycosylated hydroxyacyl chains. Identification of diverse acyl chain types in Solanum acylsugars Our survey of Solanum species uncovered acylsugars with a surprising diversity of acyl chain lengths, functional groups, and combinations of chain types and positions. We identified C2 to C18 acyl chains, including unsaturated and hydroxylated forms, based on analysis of fatty acid fragment carboxylate ions in negative mode and neutral losses in both negative and positive mode (Table S2.1). Acyl chain compositions differed between acylsugars within and between species: acylinositols with only medium C8 to C14 acyl chains were observed in 5 species (Figure 2.5, Tables S2.13-15, S2.20, S2.22, S2.24), while acylsugars with two short C4 to C6 acyl chains and one medium C8 to C14 acyl chain were observed in 17 species, an acylation pattern also observed in eggplant and wild tomato (Figures 2.2; Table 2.1) (Lybrand et al., 2020). Acylsugars with unsaturated acyl chains, including C5:1, C18:1, C18:2, and C18:3, were only detected in S. torvum (Table S2.17). To the best of our knowledge, this is the first report of unsaturated C18 chains in Solanaceae acylsugars. In contrast, hydroxylated medium-length acyl chains were surprisingly common: we detected acylsugars bearing hydroxylated acyl chains in three DulMo clade species and 25 Clade II species (Figure 2.5C). The additional hydroxyl group present on these hydroxylated acyl chains is susceptible to further modifications, including glycosylations and acylations, which would result in even greater acylsugar diversity. 98 Indeed, we obtained evidence for glycosylated acyl chains in three Clade II species, S. prinophyllum, S. sisymbriifolium, S. lasiophyllum, and one DulMo species, S. dulcamara. Several lines of evidence lead us to conclude that these species accumulate acylsugars with glycosyl groups attached via ether linkages to medium hydroxyacyl chains esterified to the hexose core. First, the compounds fragmented abnormally under negative mode CID: the glycosyl group was observed as a neutral loss from the [M-H]- ion, producing a [sugar – H]- fragment ion (Figure S2.2). This contrasts with extensive observations that CID spectra of other characterized acyldisaccharides do not exhibit negative mode disaccharide glycosidic linkage cleavage (Figures S2.1 and S2.8) (Ghosh et al., 2014; Hurney, 2018; Lou et al., 2021). Further evidence that the glycosylation is not on the primary acylated hexose ring was obtained by subjecting acylsugar extracts to saponification, which induces ester linkage breakage while retaining ether linkages. Upon analyzing saponified acylsugar extracts by LC-MS, we detected pentose and hexose sugars with C14 or C16 acyl chains rather than glycosylated hexoses, indicating that the glycosyl group is connected to a medium hydroxyacyl chain by an ether linkage that is not cleaved during alkaline saponification (Figure S2.3). We named these compounds glycohydroxyacylhexoses and annotated them by modifying the conventional acylsugar nomenclature where the molecule in Figure S2.2 is named H3:22(4,4,14-O-p) with 14- O-p representing the pentosylated (p) C14 hydroxyacyl chain (14-O). To our knowledge, this is the first report of acylhexoses with glycosylated hydroxyacyl chains in Solanaceae acylsugars. Resin glycosides produced by Convolvulaceae family species contain similar hydroxyacyl chain glycosidic linkages. However, in resin glycosides, hydroxyacyl chains are connected to the oligosaccharide core at two points – by an ester and ether linkage, respectively – forming a 99 macrocyclic structure not observed in the Solanum glycohydroxyacylhexoses (Bah and Pereda- Miranda, 1996; Kruse et al., 2022; Pereda-Miranda et al., 1993). The unusual glycohydroxyacylhexoses raises questions regarding their biosynthetic and evolutionary origins. As these compounds are found in different lineages (Eastern Hemisphere Spiny, Dulcamaroid, and Sisymbriifolium sections) – and closely related species lack detectable glycohydroxyacylhexoses – this trait appears to be highly labile (Figure 4C). Because we observe the cognate, non-glycosylated acylinositol in each of these species, we hypothesize that glycohydroxyacylhexoses consist of a myo-inositol hexose core. Further analysis of acylsugars from additional species may identify other types of modified acylsugar hydroxyacyl chains in Solanum acylsugars. Sixteen species accumulate acylsugars with the same molecular masses, sugar core masses, and acyl chain complements as the NMR-characterized eggplant acylinositols, suggesting they may be identical. To test this hypothesis, we mixed purified eggplant NMR- resolved standards with metabolite extracts from each species and performed liquid co- chromatography. Coelution provided one line of evidence that the eggplant-like acylsugars detected in 11 Clade II species are identical to characterized eggplant acylinositols. In contrast, the eggplant-like acylsugars from five other species, including four DulMo clade members and one Clade II member, eluted separately from characterized eggplant acylinositols, indicating they are acylation positional isomers or acyl chain branching isomers (Figure S2.4). Examples of acylinositol positional isomers were described between the DulMo clade species S. nigrum and the Clade II species S. quitoense and S. melongena, providing precedent that the non-coeluting compounds might be positional isomers of the eggplant acylsugars (Figure 2.2) (Leong et al., 2020; Lou et al., 2021). Our data reveal a phylogenetic pattern of acylinositol distribution, in 100 which Clade II species generally accumulate eggplant-like acylinositol isomers whereas DulMo clade species accumulate noneggplant-like acylinositol isomers (Figure 2.5B). While the eggplant-like isomers coelute with eggplant acylsugars further analysis with complementary analytical methods is needed to confirm whether these are structurally identical across species. Acylhexoses and acyldisaccharides accumulate throughout Clade II, DulMo, and VANAns Solanum species Acylhexoses were detected in all analyzed acylsugar-producing species which we annotated as acylglucoses or acylinositols based on characteristic fragmentation behavior under negative ion mode MS conditions (Leong et al., 2020; Lou et al., 2021). Identification of acylglucoses was based on observation of a sugar core fragment ion of m/z 143.03 (C6H7O4-) and stepwise acyl chain losses at the lowest collision energy level (0 V) (Figure S2.7). In contrast, acylinositol annotation was based on an absence of acyl chain fragment ions at 0 V and MSe CID negative mode functions (Figure S2.1). Using these rules, we identified acylinositols in all acylsugar-producing species tested except for S. americanum (Tables 2.1, S2.1-26). In contrast, acylglucoses were not observed in any Clade II species tested, but were common among the DulMo and VANAns clades, present in all species with characterized acylsugars except S. dulcamara (Tables S2.1, S2.18, S2.19, S2.22, S2.23, S2.2520; Figure 2.5A; Lou et al., 2021). In contrast to the acylhexose distribution, acyldisaccharides were detected in two DulMo species and 14 Clade II species. Based on negative [M-H]- fragment masses and positive mode fragmentation of the glycosidic linkage, the acyldisaccharides were composed of hexose-hexose, pentose-hexose, or deoxyhexose-hexose sugar cores (Table S2.1, Figures S2.1 and S2.8). Though complete structural information cannot be obtained from MS fragmentation alone, we hypothesize that these acyldisaccharides are composed of glycosylated inositol cores based on 101 two lines of evidence. First, the majority of species with detectable acyldisaccharides also accumulate cognate acylinositols with the same acyl chain lengths (leaf and fruit surface extracts from 10 of 15 and two of two different species, respectively), suggesting a shared inositol- containing core structure (Tables S2.1, S2.2-8, S2.11-16, S2.19, S2.21, S2.25). Second, acylinositol glycosides with varying sugar core sizes and stereochemistries were described in S. lanceolatum (Herrera-Salgado et al., 2005), S. quitoense (Hurney, 2018), and S. melongena (Figure 2.2), suggesting that the species in this study may accumulate similar compounds. The ratio of acylhexose to acyldisaccharide peak number and abundance differs across the surveyed species; for example, S. mammosum only accumulated detectable acylinositols while S. anguivi primarily accumulated acyldisaccharides that comprised 80% of the total acylsugar peak area (Tables S2.20 and S2.3). This is reminiscent of varied acylhexose accumulation observed in trichomes of evolutionarily distant Solanum species: S. nigrum only contains detectable acylinositol monosaccharides (Lou et al., 2021), while S. pennellii accessions have mixtures of acylglucoses and acylsucroses ranging from 41-95% of total acylsugars as acylglucoses. (Lybrand et al., 2020; Shapiro et al., 1994). Sticky fruits accumulate acylinositols We investigated the chemical basis of sticky fruit surface substances previously reported in botanical species descriptions of two Clade II species, S. acerifolium and S. atropurpureum (Nee, 2022a, 2022b, 1991). LC-MS analysis of fruit surface extracts revealed similar acylsugar profiles between the two species, with each extract containing what appeared to be the same 128 acylsugars. These fruit acylsugars were distinct from the 22 and 19 trichome acylsugars identified in S. acerifolium and S. atropurpureum, respectively, as evidenced by their LC elution and MS characteristics (Tables S2.13-16; Figure 2.6). Seventy-seven fruit acylsugars were 102 identified as acylinositols with two or three medium C8 to C14 acyl chains, including one to three hydroxyacyl chains. The remaining 51 fruit acylsugars are acyldisaccharides containing an unusual deoxyhexose-hexose core not previously reported in Solanaceae acylsugars. In contrast, S. acerifolium and S. atropurpureum trichome acylsugars, composed of a pentose-hexose disaccharide core decorated with short C5 or C6 acyl chains, were not detected in fruit surface extracts. Figure 2.6. S. atropurpureum trichomes and fruit exhibit dramatically different metabolite profiles. The top LC-MS base peak intensity (BPI) chromatogram displays metabolites from a leaf surface extract, while the bottom LC-MS base peak intensity chromatogram displays metabolites from a fruit surface extract. The leaf surface extract acylsugars elute from 2.2 to 7.6 min (Table S2.16), while fruit surface extract acylsugars elute from 4.6 to 18.4 min (Table S2.14). S. mammosum and S. capsicoides, which are closely related to S. acerifolium and S. atropurpureum, do not produce sticky fruit surfaces, suggesting this trait evolved in the common ancestor of S. acerifolium and S. atropurpureum, perhaps as recently as 2 Mya (Särkinen et al., 103 2013). Fruit surface acylsugars were described previously within the Physalis genus and likely represent an independent evolutionary origin from the S. acerifolium and S. atropurpureum fruit acylsugars (Bernal et al., 2018; Cao et al., 2015; Cicchetti et al., 2018; Maldonado et al., 2006). Considering that, like eggplant, S. acerifolium and S. atropurpureum fruits are glabrous (Nee, 2022a, 2022b), some other organ synthesizes the fruit surface acylsugars. Structures similar to Solanum fernadesii petiolar resin glands (Sampaio et al., 2021), Hypericum androsaemum microscopic fruit glands (Caprioli et al., 2016), and fennel and chamomile fruit secretory ducts (vittae) (Zizovic et al., 2007) may be involved in S. acerifolium and S. atropurpureum fruit acylsugar secretion. Future work will be needed to understand the cellular, biosynthetic and evolutionary relationships between trichome and fruit acylsugars. Sugar core evolution across the Solanaceae Our screening revealed acylinositols are broadly distributed across Clade II, DulMo, and VANAns (Figure 2.5A, Table S2.1). Outside of this study, acylinositols were only reported in Solanum species, including two Clade II members, S. quitoense and S. lanceolatum, and one DulMo clade member, S. nigrum. The acylinositol distribution suggests that acylinositol biosynthesis arose one or more times within the Solanum genus. However, our ability to elucidate the likely number of acylinositol origins is impeded by the lack of resolution of the phylogenetic relationships between the major Solanum clades, including Clade II, DulMo, VANAns, Potato, and Regmandra (Gagnon et al., 2022). Further biochemical and genetic analyses of acylinositol biosynthesis may reveal how this trait evolved and whether the pathway is conserved between Clade II, DulMo, and VANAns species. Acylglucoses were reported to be sporadically present across the Solanaceae, including in Salpiglossis sinuata, Petunia, Nicotiana spp., and Datura, as well as S. nigrum and S. pennellii, 104 members of the DulMo and Potato clades, respectively (Castillo et al., 1989; Chortyk et al., 1997; Fiesel et al., 2022; Fobes et al., 1985; Hurney, 2018; King and Calhoun, 1988; Lou et al., 2021; Matsuzaki et al., 1989; Schenck et al., 2022; Van Dam and Hare, 1998). We observed acylglucoses in members of the DulMo and VANAns clades, but not in Clade II. This spotty distribution suggests that acylglucose biosynthesis 1) arose once within the Solanaceae then underwent repeated losses or 2) arose independently several times in different Solanaceae clades. Recent work in S. pennellii and S. nigrum revealed that acylglucoses are synthesized from acylsucroses by a neofunctionalized invertase-like enzyme, AcylSucrose FructoFuranosidase 1 (ASFF1) (Leong et al., 2019; Lou et al., 2021). Interestingly, S. nigrum and S. pennellii employ non-orthologous ASFF1 enzymes, providing support for multiple acylglucose biosynthesis origins. Elucidation of acylglucose biosynthesis outside of the Solanum clade will be needed to resolve the evolutionary history of these metabolites. Varied acyldisaccharides were observed across DulMo and Clade II, and we predict they are biosynthesized from acylinositols. Within the Potato clade as well as outside the Solanum genus, acylsucroses are the dominant acyldisaccharide in the reported species and tissues (Fiesel et al., 2022). In contrast, no acylsucroses were detected in any species screened. However, acylsucrose biosynthesis likely persists in DulMo and VANAns species considering that acylglucoses were detected in all but one DulMo and VANAns species and that acylsucroses can be intermediates in acylglucose biosynthesis (Leong et al., 2019; Lou et al., 2021). The lack of both acylglucoses and acylsucroses in the analyzed Clade II species suggests a loss of acylsucrose biosynthesis in Clade II. Rather, our LC-MS data are consistent with the hypothesis that the acyldisaccharides observed in this study consist of glycosylated inositol cores. Recent in vitro biochemistry and in vivo genetic evidence in S. quitoense suggests that acylinositols are the 105 precursors to acylinositol disaccharides in Clade II Solanum species (Leong et al., 2022, 2019). In this scenario, acylinositols would be converted to their cognate glycosides by one or more glycosyltransferases, with different sugars added either by a conserved promiscuous glycosyltransferase or multiple glycosyltransferases. S. quitoense and S. melongena, with their acylinositol glycosides bearing different glycosyl moieties, are promising systems for addressing the biochemical origins of these unusual Solanum acyldisaccharides. Figure 2.7. SmASAT3-L1 acetylates AI3:16 to produce AI4:18. (A) LC-MS analysis of AI3:16(4,4,8) and AI4(2,4,4,8) in S. aethiopicum (red) and S. melongena (purple) leaf surface extracts. S. aethiopicum does not produce detectable levels of AI4:18. (B) S. melongena Clade III 106 Figure 2.7. (cont’d) BAHDs. Shown is a clade III BAHD tree subset from a phylogeny including 106 predicted BAHDs (PF002458) in the eggplant Smel_V4.1 reference genome, published reference BAHD sequences for clades I-VII, characterized ASAT sequences from other Solanaceae species, and the SaASAT3 and SaASAT3-L1 candidates from S. aethiopicum (see Figure S2.11 for full phylogeny). The maximum likelihood tree was inferred from amino acid sequences using the Jones-Taylor-Thornton algorithm with seven rate categories in IQ-TREE v2.1.3. Values at nodes indicate bootstrap support calculated from 100,000 ultrafast bootstrap replicates. Heatmap shows absolute transcript abundance (log2 TPM) across trichomes, trichomeless hypocotyls, and roots from 7-day-old eggplant seedlings. Expression data for non-eggplant sequences not included in heatmap. Color gradient provides a visual marker to rank the transcript abundance from high (purple) to low (white) or absent (grey). ASAT, acylsugar acyltransferase. log2TPM, log2 transformed transcripts per million. (C) Of the seven tested enzymes, only SmASAT3-L1 acetylates AI3:16(4,4,8) to form AI4:18(2,4,4,8). The extracted ion chromatograms on the left display products from forward enzyme assays and the formate adduct of AI4:18, m/z 665.30. The extracted ion chromatograms on the right display products from reverse enzyme assays and the formate adduct of AI3:16, m/z 623.29. (D) SmASAT3-L1 acetylates AI3:16 in vitro to produce AI4:18. Forward assay extracted ion chromatograms display the formate adduct of AI4:18, m/z 665.30, and reverse assay extracted ion chromatograms display the formate adduct of AI3:16, m/z 623.29. The reverse assay chromatograms display peaks for both AI3:16 and AI4:18 due to in source fragmentation of remaining AI4:18. S. aethiopicum is defective in expression of a trichome acylsugar acyltransferase enzyme We began analyzing the biochemical mechanisms underlying the observed Solanum acylsugar diversity in seven of 23 members from the Eggplant clade and related Anguivi grade, two small subclades within Clade II that includes brinjal eggplant and its close relatives. In contrast to the remaining six analyzed species, S. aethiopicum (scarlet or Ethiopian eggplant) does accumulate detectable AI3:16(i4,i4,i8) (Compound 1) but does not accumulate the acetylated form, AI4:18(2,i4,i4,i8) – the highest abundance S. melongena acylsugar (Compound 2; Figure S2.6, Tables S2.1, S2.3, S2.4). We hypothesized that the absence of AI4:18(2,i4,i4,i8) in S. aethiopicum is due either to mutations causing loss-of-function or loss of expression of an acetylating enzyme in this species. The availability of S. melongena and S. aethiopicum genomic sequences (Barchi et al., 2021; Song et al., 2019) provided the opportunity to seek a trichome expressed acetyltransferase responsible for producing AI4:18 and the mechanism behind the lack of detectable AI4:18 in S. aethiopicum. 107 To identify suitable enzyme candidates, we sequenced transcriptomes of isolated trichomes, trichome-depleted shaved hypocotyls, and whole roots collected from 7-day-old eggplant seedlings and performed differential gene expression analysis. Of the 23,251 eggplant genes expressed in at least one of the three tissues, 745 were significantly enriched (log2 fold- change >2, p-value < 0.05) in trichomes compared to hypocotyls and roots, including 20 BAHDs (Supplemental datafile 1). We selected for further testing seven BAHDs that were abundantly expressed in trichomes (TPM > 200) and homologous to characterized Solanum ASATs (Figure 2.7B) (D’Auria, 2006; Fan et al., 2015; Leong et al., 2022, 2020; Lou et al., 2021). We expressed each of the seven candidates in Escherichia coli and tested for acetylation of Compound 1, AI3:16(4,4,8). An SlASAT3 outparalog (Koonin, 2005) SMEL4.1_12g015780, ACYLSUGAR ACYLTRANSFERASE3-LIKE1 (SmASAT3-L1) was the only enzyme to exhibit forward activity converting Compound 1, AI3:16(4,4,8), to Compound 2, AI4:18(2,4,4,8) (Figure 2.7C). As shown in Figure S2.14, LC-MS analysis of the SmASAT3-L1 in vitro assay product revealed that it had identical molecular masses and elution times to plant-derived AI4:18(2,i4,i4,i8) (Compound 1) providing evidence that SmASAT3-L1 acetylates AI3:16(i4,i4,i8). Characterization of reverse activities of the seven BAHD acyltransferases (Leong et al., 2020; Lou et al., 2021), in which an acyl chain can be removed from an acylsugar by incubating it with the enzyme and free Coenzyme A, confirmed that SmASAT3-L1 was the only candidate enzyme that removed an acetyl chain from AI4:18(2,4,4,8) (Figures 2.7D and S2.14). Taken together, these results indicate that trichome-expressed SmASAT3-L1 encodes an acylinositol acetyltransferase responsible for AI4:18(2,4,4,8) biosynthesis. We tested the hypothesis that S. aethiopicum trichome extracts lack detectable acetylated AI4:18(2,4,4,8) due to a defect in SmASAT3-L1 ortholog expression. Indeed, reverse 108 transcription (RT)-PCR comparing isolated trichome and trichomeless hypocotyl cDNA revealed that SaASAT3-L1 (GenBank ID: OQ547782) transcript abundance was undetectable in S. aethiopicum trichomes (Figures S2.10). This result implicates the defect in ASAT3-L1 enzyme gene expression as responsible for lack of AI4:18 in S. aethiopicum. The interspecific variation in ASAT3-L1 expression is reminiscent of the ASAT4 expression differences among accessions of the wild tomato Solanum habrochaites. In this case, a subgroup of S. habrochaites accessions possessed a functional ASAT4 copy, but its low levels of gene expression correlated with reduced accumulation of acetylated acylsugars in these accessions (Kim et al., 2012). Figure 2.8. Characterized in vitro acyl-CoA usage by ASATs. The maximum likelihood phylogeny was inferred from amino acid sequences using a time-reversible algorithm specifying a plant-specific empirical substitution matrix, invariant sites, and four rate categories (Q.plant+I+G4) in IQ-TREE v2.1.3. Bootstrap support was calculated from 100,000 ultrafast bootstrap replicates; values >=95 indicate strong support. Other than SmASAT3-L1, enzyme activities were described previously (Fan et al., 2017, 2015; Leong et al., 2022, 2020; Lou et al., 2021; Moghe et al., 2017; Nadakuduti et al., 2017; Schenck et al., 2022; Schilmiller et al., 2015; Schilmiller et al., 2012). Only positive activities are displayed, and the blank squares may not indicate a lack of activity. 109 Natural variation potentiates novel activities and uses Until recently, it appeared that acylsucroses dominate acylsugar profiles of Solanaceae species with a small number of documented examples of acylglucose- and acylinositol-producing Solanum species (Fiesel et al., 2022; Fobes et al., 1985; Herrera-Salgado et al., 2005; Hurney, 2018; King and Calhoun, 1988; Leong et al., 2020; Liu et al., 2017; Lou et al., 2021; Moghe et al., 2017; Schenck et al., 2022). Our chemotaxonomic survey of more than two dozen Solanum species from the sparsely sampled Clade II, DulMo, and VANAns clades revealed widespread occurrence of glucose-, inositol-, and non-sucrose disaccharide-based acylsugars decorated with unusual acyl chains, including medium-length (C12-C16) hydroxyacyl chains, glycosylated hydroxyacyl chains, and unsaturated chains. Considering that this work and previously published analyses together cover <3% of Solanum species, this is only the ‘tip of the iceberg’ of trichome acylsugar diversity within this large genus. Adding to interspecific variation we observe here, the recent identification of acylsugars in cultivated tomato root exudates supports the value of metabolite screening in additional tissue types as well as species (Korenblum et al., 2020). Knowledge of Solanum acylsugar diversity provides a framework for investigating the molecular basis of metabolic pathway evolution. For example, the widespread occurrence of acylglucoses across the DulMo clade leads us to hypothesize that the neofunctionalized invertase responsible for S. nigrum acylglucose synthesis (Lou et al., 2021) is shared by other DulMo clade species. Similarly, documented acylsugar variation between cultivated eggplant, S. melongena, and its relative, S. aethiopicum, led to the discovery of a terminal acylsugar acetyltransferase in eggplant, SmASAT3-L1. The characteristics of ASAT3-L1, the enzyme absent from the S. aethiopicum trichome transcriptome, in turn reveals the dynamic nature of plant specialized metabolism. First, SmASAT3-L1 is unique among characterized ASATs in its 110 acyl acceptor specificity, acetylating an acylinositol glycoside rather than an acylated sucrose, glucose, or myo-inositol. Second, acetyl-CoA donor activity was previously only described for a subset of Clade III BAHDs, which include ASAT4 and ASAT5 clade members (Figure 2.8). This breaks a pattern seen across the Solanaceae, including trichome ASATs from species of early evolving lineages, Petunia axillaris and Salpiglossis sinuata (Moghe et al., 2017; Nadakuduti et al., 2017), Nicotiana attenuata (Schenck et al., 2022), as well as cultivated and wild tomatoes (Kim et al., 2012; Schilmiller et al., 2012). This theme was also observed in trichomes of two other Solanum plants, more closely related to the species screened in this study: the Clade II S. quitoense SqTAIAT (Leong et al., 2020) and DulMo S. nigrum SnAGAT1 (Lou et al., 2021). Both enzymes reside outside of the ASAT3 clade, and thus are phylogenetically distinct from SmASAT3-L1. SmASAT3-L1 characteristics and those of recently published S. quitoense enzymes (Leong et al., 2022, 2020) also suggest that acylinositols are synthesized through one or more pathway(s) distinct from that of acylsucroses. Existing genetic resources in S. nigrum, S. quitoense, and S. melongena can be utilized to explore when and how acylinositols arose and to identify the underlying mechanisms behind acylation position and chain type differences between these species. The remarkably varied structures of acylsugars, and their associated physical properties, strongly suggest that they have evolved distinct bioactivities, likely in coevolutionary arms races between plants and their insect and microbial pests. For example, hydrophobic acylsugars with longer acyl chains and few free hydroxyl groups may disrupt membranes, reminiscent of less polar triterpene saponin variants (Augustin et al., 2011; Baumann et al., 2000). Variation in acyl chain linkage chemistries also likely influence acylsugar mode of action. For example, ester linked short chains acylsucroses are digested by Manduca sexta (hawkmoth) larvae and the 111 volatile organic acids released into the environment, attracting ant predators (Weinhold and Baldwin, 2011). In contrast, longer chain hydroxylated and ether-linked glycohydroxyacyl chains observed in the study are likely to have quite different metabolic fates, perhaps persisting in the digestive systems of herbivores and alternative metabolism in microbes. Uncovering the enzymes responsible for synthesis of the wide variety of natural acylsugars will enable transgenic production of isogenic variants and rigorous testing of their ecological activities. Similarly, access to a wider variety of often promiscuous ASAT BAHD acyltransferases (Moghe et al., 2023) creates opportunities for producing novel acylsugars with a wide range of physical properties and bioactivities (Schenck et al., 2022). Hydroxyacyl chains also present promising opportunities to expand upon natural acylsugar diversity through synthetic biology and/or synthetic chemistry. The hydroxyl acts as a reactive chemical handle which was exploited in nature to create glycohydroxyacylhexoses. We can further modify the hydroxyacyl chains to add unusual sugars, acyl chains, and aromatic groups through promiscuous enzymes or synthetic chemistry. These structural changes would likely impact biological activities and add another asset to developing more pest resistant plants. Characterizing more natural acylsugar diversity will further enable synthetic biology approaches to create completely new acylsugar structures. Methods Plant material and growth conditions Solanum spp. seeds were obtained from the sources described in Table S2.37. Seeds were treated with 10% (v/v) bleach for 10 min while being rocked at 24 rpm with a GyroMini nutating mixer (Labnet, Edison, NJ, USA), and then rinsed 5-6 times with distilled water. Unless otherwise noted, seeds were germinated on Whatman filter paper (MilliporeSigma, Burlington, MA, USA) 112 at 28°C and in the dark. Germinated seedlings were transferred to peat pots (Jiffy, Zwijndrecht, Netherlands), and grown at 25°C, 16/8-h day/night light cycle, and ~70 μmol m-2s-2 photosynthetic photon flux density with cool white fluorescent bulbs. Mature plants were grown in controlled environment growth chambers or in a greenhouse. The growth chamber conditions consisted of 25°/18°C day/night temperatures, 16/8-h day/night light cycle, and ~100 μmol m-2s-2 photosynthetic photon flux density under LED bulbs. The greenhouse conditions consisted of a 16/8-h day/night light cycle achieved with supplemental sodium iodide lighting. Plants were fertilized weekly with 0.5X Hoagland’s solution. Surface metabolite extractions Surface metabolites were extracted as described previously (Leong et al., 2019; Lou and Leong, 2019). Briefly, 0.1-1 g of leaf, stem, hypocotyl, or cotyledon tissue was collected into a 1.5 mL screw-cap tube (Dot Scientific, Inc., Burton, MI, USA), 1 mL extraction solvent (3:3:2 acetonitrile:isopropanol:water, 0.1% formic acid, 10 µM propyl 4-hydroxybenzoate (internal standard)) was added and the sample was rocked at 24 rpm by a GyroMini nutating mixer for two min. After extraction, the supernatant was transferred into a glass 2 mL autosampler vial (Restek, Bellefonte, PA, USA) and sealed with a 9 mm cap containing a PTFE/silicone septum (J.G. Finneran, Vineland, NJ, USA). For fruit surface metabolite extractions, we modified the protocol by placing 1-2 fruit into a 15 mL polypropylene conical tube (Corning Inc., Corning, NY, USA) containing 5 mL extraction solvent then proceeded to the nutation step above. Bulk eggplant acylsugar extraction and purification for NMR analysis Surface metabolites were bulk extracted from approximately 1000 S. melongena PI 555598 seedlings. Seeds were treated with 10% (v/v) bleach (Clorox, Oakland, CA, USA) for 10 min while being gently rocked at 24 rpm with a GyroMini nutating mixer and subsequently 113 rinsed 5-6 times with distilled water. Seeds were sown in moist SUREMIX soil (Michigan Grower Products, Galesburg, MI, USA) in Perma-Next plant trays, 22 x 11 x 2.5 inches (Growers Supply Company, Dexter, MI, USA), covered with a humidity dome, 22 x 11 x 3 inches (Growers Supply Company), then transferred to the growth chamber immediately. Seedlings were harvested when 2-3 true leaves were observed, approximately one week, by cutting them at the base and placing them into two 2L beakers each containing 1L 100% acetonitrile. Surface metabolites were extracted with gentle agitation with a metal spatula for five min at room temperature. Plant material and sediment was removed by vacuum filtration through a Büchner funnel lined with Whatman filter paper (MilliporeSigma). Solvent was removed in vacuo by rotary evaporation and dried residue was dissolved in 20 mL of acetonitrile with 0.1% formic acid. Solvent was removed again using a vacuum centrifuge and the dried residue was dissolved in 1 mL of acetonitrile:water:formic acid (80:20:0.001). The semi-preparative LC method is described in detail in Table S2.32 and is described in brief here. S. melongena acylsugars were separated with a Waters 2795 HPLC (Waters Corporation, Milford, MA, USA) equipped with an Acclaim 120 C18 HPLC column (4.6 x 150 mm, 5 μm; Thermo Fisher Scientific, Waltham, MA, USA). Solvent A was water with 0.1% formic acid and Solvent B was acetonitrile. The reverse-phase linear gradient was as follows: 5% B at 0 min, 60% B at 2 min, 80% B at 40 min, 100% B at 42 min, 5% B at 42.01 min, held at 5% B until 44 min. Flow rate was 1.5 mL/min, injection volume was 100 µL, and the column temperature was 40℃. Fractions were collected automatically at 0.25 min intervals by a 2211 Superrac fraction collector (LKB Bromma, Stockholm, Sweden) and assessed for acylsugar presence and purity by LC-MS analysis, as described below. Column fractions were collected in the same tubes for each method run which worked to pool the fractions between each run. 114 LC-MS acylsugar analysis Acylsugars were analyzed by LC-MS with each method described below and in Tables S2.27-31. For each LC method, the mobile phase consisted of aqueous 10 mM ammonium formate, adjusted to pH 2.8 with formic acid (Solvent A) and 100% acetonitrile (Solvent B). The flow rate was maintained at 0.3 mL/min. Acylsugar extracts were analyzed with a 22 min LC gradient using a Waters Acquity UPLC coupled to a Waters Xevo G2-XS QToF mass spectrometer (Waters Corporation, Milford, MA) equipped with electrospray ionization (ESI) operating in positive (ESI+) and negative (ESI-) modes. 10 µL acylsugar extracts were separated on an Acquity UPLC BEH C18 column (10 cm x 2.1 mm, 130 Å, 1.7 µm; Waters), kept at 40℃, using a binary solvent gradient. The 22 min linear gradient was as follows: 5% B at 0 min, 60% B at 2 min, 100% B at 16 min, held at 100% B until 20 min, 5% B at 20.01 min, held at 5% B until 22 min. Acylsugar extracts were analyzed with both ESI-, and ESI+. For ESI-, the following parameters were used: capillary voltage, 2 kV; sampling cone voltage, 35 V; source temperature, 100°C; desolvation temperature 350°C; cone gas flow, 50 L/Hr; desolvation gas flow, 600 L/Hr. For ESI+, the following parameters were used: capillary voltage, 3 kV; sampling cone voltage, 35 V; source temperature, 100°C; desolvation temperature 300°C; cone gas flow, 50 L/Hr; desolvation gas flow, 600 L/Hr. Acylsugars were fragmented in either MSE or data-dependent acquisition (DDA) MS/MS modes as described previously (Lou et al., 2021; Lybrand et al., 2020). DDA survey and MS/MS functions acquired over m/z 50 to 1500 with scan times of 0.2 s. Ions selected for MS/MS were fragmented with a ramped collision energy where voltage varies based on ion mass. Collision energies followed a ramp with the voltage changing linearly for ions between the low mass setting (m/z 50, 15 to 30 V) and high mass setting (m/z 1500, 30 to 60 V). To increase mass 115 accuracy, lock mass correction was performed during data collection, with leucine enkephalin as the reference. Semi-preparative LC fractions were analyzed with direct infusion and a 14-min LC gradient using a LC-20ADvp ternary pump (Shimadzu, Kyoto, Japan) coupled to a Waters Xevo G2-XS QToF mass spectrometer (Waters Corporation). The direct infusion method quickly screened fractions for acylsugar presence while the 14-min LC method tested fraction purity. 10 µL of acylsugar fractions were injected into an Ascentis Express C18 HPLC column (10 cm x 2.1 mm, 2.7 µm; Supelco, Bellefonte, PA, USA), kept at 40℃. The 14-min gradient was as follows: 5% B at 0 min, 60% B at 2 min, 100% B at 10 min held until 12 min, 5% B at 12.01 min, and held at 5% until 14 min. Fractions were analyzed under ESI+ with the following parameters: capillary voltage, 3 kV; sampling cone voltage, 40 V; source temperature, 100°C; desolvation temperature 350°C; cone gas flow, 20 L/hr; desolvation gas flow, 500 L/hr. For direct infusion analysis, ions were acquired from m/z 50 to 1500 with a scan time of 0.1 s and one acquisition function with 0 V collision potential. For 14-min LC analysis, ions were acquired from m/z 50 to 1200 with a scan time of 0.1 s and three acquisition functions with different collision potentials (0, 25, 60 V). Lock mass calibration referenced to the leucine enkephalin [M+H]+ ion was applied during data acquisition. Enzyme assays products were analyzed with a 7-min gradient using Waters Acquity UPLC coupled to a Waters Xevo G2-XS QToF mass spectrometer (Waters Corporation, Milford, MA) equipped with electrospray ionization (ESI) operating in negative (ESI-) mode. Reaction products were separated with an Ascentis Express C18 HPLC column (10 cm x 2.1 mm, 2.7 µm; Supelco), kept at 40℃, using a binary solvent gradient. The 7 min linear gradient was as follows: 5% B at 0 min, 60% B at 1 min, 100% B at 5 min, held at 100% B until 6 min, 5% B at 6.01 min, 116 held at 5% B until 7 min. The ESI- parameters described for acylsugar extract analysis were used. Ions were acquired from m/z 50 to 1200 with a scan time of 0.1 s and three acquisition functions with different collision potentials (0, 25, 60 V). Lock mass calibration referenced to the leucine enkephalin [M+H]- ion was applied during data acquisition. For coelution analysis between enzymatically- and plant-produced AI3:16 and AI4:18, a 24-min linear gradient was used with an Acquity UPLC BEH C18 column (10 cm x 2.1 mm, 130 Å, 1.7 µm; Waters), kept at 40⁰C, on the same instrument used for enzyme assay analysis. The binary solvent, linear gradient was as follows: 5% B at 0 min, 60% B at 18 min, 100% B at 18.01 min, held at 100% B until 22 min, 5% B at 22.01 min, held at 5% B until 24 min. ESI- parameters and MS method from the above enzyme assay analysis were used. Saponified acylsugars were analyzed using a Waters Acquity UPLC coupled to a Waters Xevo G2-XS QToF mass spectrometer (Waters Corporation, Milford, MA). 10 µL of saponified acylsugars were injected into either an Acquity UPLC BEH C18 column (10 cm x 2.1 mm, 130 Å, 1.7 µm; Waters) or an Acquity UPLC BEH Amide column (10 cm x 2.1 mm, 130 Å, 1.7 µm; Waters), kept at 40℃. Samples were analyzed on the C18 column with the 22-min method described above. To detect free sugars resulting from saponification, samples were analyzed on the BEH amide column with a 9 min method which was as follows: 95% B at 0 min held until 1 min, 60% B at 6 min, 5% B at 7 min, 95% B at 7.01 min, held at 95% B until 9 min. Both methods used the ESI- parameters and MS acquisition parameters described for enzyme assays. Acylsugar annotation We inferred acylsugar structures utilizing negative and positive mode MS and MS/MS collision-induced dissociation (CID) as previously described (Ghosh et al., 2014a; Hurney, 2018; Leong et al., 2019; Lou et al., 2021; Lybrand et al., 2020). We employed modified confidence 117 criteria developed by Lou and coworkers (Lou et al., 2021). Putative acylsugars only meeting criterion A were annotated as low confidence. Putative acylsugars meeting criteria A-D were annotated as medium confidence. We also added two additional criteria, G and H, described below. Acylsugars meeting criteria A-F and either G or H were annotated with highest confidence. Confidence levels for each reported acylsugar are specified in their species-specific annotation table (Tables S2.1-26). (G) NMR structural determination. Acylsugars from S. melongena were further characterized by NMR experiments in which sugar core relative stereochemistry, acyl chain positions, and acyl chain branching were resolved. (H) Coelution with NMR characterized compounds. Acylsugars with the same acyl chains and sugar core size were analyzed by coelution with S. melongena acylsugars. If acylsugars coeluted with an NMR characterized peak and shared the same acyl chains and fragmentation patterns, they met this criterion and were annotated with high confidence. This criterion was employed for acylsugars from species other than S. melongena. Acylsugar metabolomic analysis To collect accurate formate adduct masses and relative abundances from putative acylsugars, negative mode LC-MS raw data from metabolite extracts were analyzed with Progenesis QI software v3.0 (Waters, Milford, MA, USA) using retention time alignment, peak detection, adduct grouping, and deconvolution. Lock mass correction was performed during data collection. We used the following analysis parameters: peak picking sensitivity, default; retention time range, 0-22 min; adducts detected, M+FA-H, M+Cl, M-H, 2M-H, and 2M+FA-H. Average percent peak abundance was calculated for each putative acylsugar by dividing its raw 118 abundance by the sum of the total acylsugar raw abundance and then averaging that value across all samples for a species. NMR analysis of acylsugars Purified acylsugars were dissolved in acetonitrile-d3 (99.96 atom % D; MilliporeSigma, Burlington, MA, USA) and transferred to Kontes NMR tubes (MilliporeSigma, Burlington, MA, USA). Samples were analyzed at the Michigan State University Max T. Rogers NMR Core with a Varian Inova 600 MHz spectrometer (Agilent , Santa Clara, CA, USA) equipped with a Nalorac 5 mm PFG switchable probe, a DirectDrive2 500 MHz spectrometer (Agilent, Santa Clara, CA, USA) equipped with a OneNMR probe, Bruker Avance NEO 800 MHz spectrometer (Bruker, Billerica, MA, USA) equipped with a 5 mm helium cryogenic HCN probe, or a Bruker Avance NEO 600 MHz spectrometer equipped with a 5 mm nitrogen cryogenic HCN Prodigy probe. All acylsugars were analyzed with a series of 1D (1H, and 13C) and 2D (gCOSY, TOCSY, gHSQC, gHMBC, gH2BC, J-resolved) experiments. Resulting spectra were referenced to acetonitrile-d3 (𝛿H = 1.94 and 𝛿C = 118.70 ppm). NMR spectra were processed and analyzed with MestReNova 12.0 software (MestreLab, A Coruña, Spain). For full NMR metadata, see Tables S2.47-50. Sugar core composition analysis S. melongena PI 555598 acylsugar extracts were dried down in vacuo with centrifugation (Savant, ThermoFisher Scientific). Dried acylsugar extracts were dissolved in 1 mL of methanol. One mL of 3 M ammonium hydroxide was added, and the solution was mixed vigorously. This initial saponification reaction proceeded at room temperature for 48 hours. Solvent was removed by vacuum centrifugation. Saponified sugar cores were reduced and derivatized with acetate groups as previously described (Sassaki et al., 2008). Sugars were dissolved in 50 µL of 1 M 119 ammonium hydroxide for 15 min at room temperature. Addition of 50 µL 20 mg/mL NaBH4 and incubation at 100°C for 10 min converted aldoses and ketoses to polyols. Excess sodium borohydride was quenched with 100 µL 1M trifluoroacetic acid. Two volumes of methanol were added, and the sample was dried down by a stream of nitrogen gas. Dried down reaction products were redissolved in 200 µL of methanolic 0.5 M HCl and heated at 100°C for 15 min in a closed ½ dram vial, 12 x 35 mm (Kimble Chase, Vineland, NJ, USA). Solvent was removed by a stream of nitrogen gas. To acetylate the sugar cores, residue was dissolved in 200 µL of pyridine:acetic anhydride (1:1) and incubated at 100°C for 30 min. Acetylated polyols were then dried down in vacuo in a vacuum centrifuge and redissolved in 100 µL hexane for GC-MS analysis. To determine the composition of disaccharide sugar cores, saponified sugars were hydrolyzed prior to derivatization. Dried down sugars were redissolved in 98 µL water in a ½ dram vial, 12 x 35 mm (Kimble Chase). After addition of 102 µL of 88% formic acid, the vial was sealed and heated at 100℃ for 15 hours in a heat block. Hydrolyzed sugars were dried down in vacuo with a vacuum centrifuge and derivatized to alditol acetates following the method described above. Hydroxyl acyl chain stereochemistry analysis We derivatized and prepared hydroxyacyl chains as previously described (Jenske and Vetter, 2007). The commercial standards (3R)-OH-tetradecanoate (Cayman Chemical, Ann Arbor, MI, USA) and (3R/S)-OH-tetradecanoate (Cayman Chemical, Ann Arbor, MI, USA) were first derivatized to their respective ethyl ester derivatives. 600 µg of each fatty acid was dissolved in 0.5 mL of 0.5 M ethanolic KOH and incubated at 80°C for 5 min. After the solution was cooled on ice, 1 mL of ethanolic BF3 was added, and reactions were incubated at 80°C for 120 five min. Two-phase partitioning with hexane and saturated sodium chloride yielded ethyl ester derivatives in the hexane partition. The 3-OH-nC14 acyl chain from I3:22(iC4,iC4,3-OH-nC14) was derivatized to an ethyl ester following a previously published method (Fan et al., 2020; Ning et al., 2015). Half of the purified I3:22(iC4,iC4,3-OH-nC14) was dissolved in acetonitrile with 0.1% formic acid and transferred to a 1.5 mL microcentrifuge tube. The purified acylsugar was dried down in vacuo with vacuum centrifugation. The resulting dry, purified I3:22(iC4,iC4,3-OH-nC14) was dissolved in 300 µL of 21% (w/w) sodium ethoxide in ethanol (MilliporeSigma, Burlington, MA, USA). The reaction proceeded at room temperature for 30 min with constant rocking at 24 rpm by a GyroMini nutating mixer (Labnet, Edison, NJ, USA) and occasional vortexing. 400 µL hexane with 55 ng/µL tetradacane (MilliporeSigma, Burlington, MA, USA) as an internal standard was then added followed by vigorous vortexing. 500 µL of aqueous saturated sodium chloride was added to the hexane-ethanol mixture, and vortexed. The hexane layer was pipetted to another tube and two more two-phase partitions with saturated sodium chloride were completed. The final hexane layer containing the acyl chain ethyl esters was extracted and dried down by a stream of nitrogen gas. The resulting fatty acid ethyl esters from the commercial standards and purified compound were derivatized with (R)-(-)-ɑ-methoxy-ɑ-trifluoromethylphenylacetyl chloride ((R)- (-)-MTPA-Cl; MilliporeSigma, Burlington, MA, USA) following a previously published procedure (Jenske and Vetter, 2007). Dried fatty acid ethyl esters were redissolved in 400 µL pyridine and 15 µL of (R)-(-)-MPTA-Cl. The reaction proceeded at room temperature for two hours, after which, 5 mL of water and tert-butyl methyl ether (TBME) were added along with solid K2CO3 (one spatula tip). The TBME phase was collected after three successive phase 121 separations with 5 mL of water. TBME was evaporated to 1 mL and subjected to GC-MS analysis. GC-MS analysis All GC-MS analyses employed an Agilent 5890 GC and an Agilent 5975 single quadrupole MS equipped with a FactorFour VF-5ms column (30 m x 0.25 mm, 0.25 um; Agilent) and 10 m EZ-Guard column (Agilent, Santa Clara, CA, USA). Helium was used as the carrier gas with a constant flow rate of 1 mL/min. Electron energy was set at 70 eV. MS source and quadrupole were maintained at 230°C and 150°C, respectively. Parameters specific to each analysis are listed below. For analysis of sugar core alditol acetate derivatives, we followed previously published GC-MS parameters (Sassaki et al., 2008). Inlet temperature was maintained at 275°C. GC oven temperature was held at 60°C for one min and then was ramped at a rate of 40°C/min to 180°C. Oven temperature was then ramped to 240°C at a rate of 5°C/min and held for three min. Total run time was 19 min. The MS detector transfer line was maintained at 280°C. Split ratio was 10:1 with a split flow rate of 10 mL/min. A three min solvent delay was used. MS data was collected in full scan mode, m/z 50-600. For analysis of MPTA fatty acid ethyl ester derivatives, we followed previously published GC-MS parameters (Jenske and Vetter, 2007). GC oven temperature was held at 60°C for 1.5 min and then was ramped at a rate of 40°C/min to 180°C. 180°C was held for two min after which the oven temperature was ramped to 230°C at a rate of 2°C/min. After 230°C was held for nine min, the oven temperature was ramped to 300°C at 10°C/min and held for 7.5 min. Total run time was 55 min. A 5-min solvent delay was applied. The MS detector transfer line 122 was maintained at 280°C. Selective ion monitoring detected the ions m/z 189, 209, and 255 with a dwell time of 50 ms. Sample collection and transcriptome sequencing We sequenced 18 transcriptomes: six biological replicates each of trichomes isolated from hypocotyls (trichomes), hypocotyls stripped of their trichomes (stripped hypocotyls), and whole roots (Supplemental data file 1). We sampled trichomes and stripped hypocotyls from 7- day-old S. melongena 67/3 seedlings following methods developed for root hair cell isolation (Bucher et al., 1997) with modifications. We grew lawns of eggplant seedlings in soil flats as described above for bulk leaf surface metabolite extraction. At 7 days post germination, we removed roots and cotyledons from seedlings, then transferred hypocotyls to liquid nitrogen in a plastic 2 L Dewar flask. Frozen hypocotyls were gently stirred with a glass rod for 20 minutes to physically shear trichomes from hypocotyls. After confirming under a dissecting scope that a sample of three hypocotyls had been stripped bare, we filtered trichomes into a 2 L glass beaker by slowly pouring the contents of the Dewar flask through a 500 μm wire mesh sieve (MilliporeSigma, Burlington, MA, USA). To maximize trichome recovery, stripped hypocotyls were returned to the Dewar, rinsed with liquid nitrogen, and filtered through the 500 μm sieve six more times. Stripped hypocotyls were divided into six pre-weighed 50 mL conical tubes, quickly weighed, then transferred to -80⁰C for storage. Trichomes were subsequently filtered through a 150 μm sieve (MilliporeSigma, Burlington, MA, USA) into a 500 mL beaker to increase sample purity then transferred to a 50 mL conical tube to allow the excess liquid nitrogen to evaporate. Finally, filtered trichomes were divided into six pre-weighed 2 mL screw- cap tubes (Dot Scientific, Inc., Burton, MI, USA), quickly weighed, then transferred to -80⁰C for storage. 123 We extracted total RNA using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s directions, measured RNA concentration using a Qubit 1.0 instrument (Thermo Fisher Scientific, Waltham, MA, USA) with the RNA HS assay, and the samples were processed by the Michigan State University Research Technology Support Facility Genomics Core (East Lansing, MI, USA) for library preparation and high-throughput sequencing. The core checked RNA quality using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA), constructed sequencing libraries using an Illumina Stranded mRNA Prep kit (Illumina, Inc. San Diego, CA, USA), and sequenced all 18 libraries on a NovaSeq 6000 (Illumina, Inc. San Diego, CA) using a single S4 flow cell lane, producing 150-base pair paired- end reads (Supplemental datafile 1). The raw fastq files are available to download from the Sequence Read Archive database under BioProject PRJNA935765. Transcriptome alignment and differential gene expression analysis To prepare the 150-bp paired-end sequences for alignment, we employed Trimmomatic (Bolger et al., 2014) to trim adapters and low-quality bases, then filter reads shorter than 75 bp, removing an average of 3.7% of sequences (range: 3.3% - 4.2%). We mapped the resulting 75– 150-bp (average length: 149 bp) paired-end RNAseq reads separately to the eggplant V4.1 and HQ reference genomes using STAR in two-pass mode, which enhances splice junction discovery and mapping sensitivity (Dobin et al., 2013; Dobin and Gingeras, 2015). Using this approach, an average of 79.2% (range: 60.1 - 87.1%) and 82.2% (range: 62.0 - 90.7%) of reads mapped to a unique genomic location in V4.1 and HQ, respectively (Supplemental datafile 1). We filtered the resulting transcriptome alignments according to best practices as defined by the Genome Analysis Toolkit (GATK) (DePristo et al., 2011; Van der Auwera et al., 2013). Briefly, we removed optical and PCR duplicates with MarkDuplicates from the Picard toolkit 124 (http://broadinstitute.github.io/picard), parsed reads into exon segments and removed intron- spanning bases using SplitNCigarReads from GATK (McKenna et al., 2010). Finally, we selected unique alignments by eliminating reads with a mapping quality score below Q60 with the view command from SAMtools (Li et al., 2009). To generate raw read counts, we used the HTSeq command, htseq-count with the nonunique parameter set to all (Anders et al., 2015). Raw read counts generated by HTSeq-count were used to perform differential gene expression analysis in edgeR (Robinson et al., 2010). To restrict comparisons to expressed genes, only transcripts with at least one read count-per-million (CPM) in at least one sample were retained for further analysis. This filtering step removed 11,665 (33.4%) and 14,544 (39.8%) annotated genes in the V4.1 and HQ eggplant genomes, respectively, yielding 23,251 and 22,024 expressed transcripts for differential gene expression analysis. Next, we normalized transcript abundances across the 18 transcriptomes in our eggplant V4.1 and HQ alignments using the default trimmed mean of M-values (TMM) method in the calcNormFactors function, then performed multidimensional scaling (MDS) with the plotMDS function to compare global gene expression profiles (Figure S2.13). This showed that our samples cluster tightly by tissue (i.e., trichome, shaved hypocotyl, or root), with no obvious differences between V4.1 and HQ alignments. To test differences in gene expression across tissues, we implemented a generalized linear model (GLM) using a quasi-likelihood (QL) approach: we generated an experimental design matrix specifying the three tissues (i.e., trichomes, trichomeless hypocotyls, and roots) with the model.matrix function, then used the glmQLFit function to fit our data to a QL-GLM model. To identify genes with a log2 fold-change (FC) > 2 between tissues, we used the glmTreat function, which performs threshold hypothesis testing, a rigorous statistical approach that evaluates variance and magnitude to detect expression differences greater than the specified 125 value (e.g., log2 FC > 2), then applies false discovery rate (FDR) p-value corrections. Genes were classified as significantly differentially expressed between two tissues if log2 FC > 2 and FDR-corrected p-value < 0.05. We calculated absolute transcript abundance for all expressed genes as transcripts per million (TPM) with the calculateTPM function in scateR (McCarthy et al., 2017). Phylogenetic analyses Characterized ASAT enzymes fall into clade III of the BAHD family (Fan et al., 2017, 2015; Leong et al., 2022, 2020; Lou et al., 2021; Moghe et al., 2017; Nadakuduti et al., 2017; Schenck et al., 2022; Anthony L Schilmiller et al., 2015; Schilmiller et al., 2012). To identify ASAT candidates in eggplant, we searched for sequences containing the Pfam Transferase domain (PF02458) (Finn et al., 2010), associated with all characterized catalytically active BAHD proteins. The PF02458 HMM profile was obtained from the Pfam website (http://pfam.sanger.ac.uk/) and queried against the V4.1 and HQ eggplant proteomes using the hmmsearch tool from HMMER v3.2.1 (hmmer.org), revealing 106 and 108 putative BAHD sequences, respectively. Using MAFFT v7.471 in E-INS-i mode, we built multiple sequence alignments (MSA) of amino acid sequences from four sources: 1) V4.1 or HQ eggplant PF02458 hits, 2) published reference sequences for clades I-VII (Moghe et al., 2023), 3) characterized ASAT sequences from other Solanaceae species, and 4) the SaASAT3 and SaASAT3-L1 candidates from S. aethiopicum (Katoh and Standley, 2013). The E-INS-i algorithm implements local alignment with a generalized affine gap cost (Altschul, 1998), which aligns conserved regions (e.g., the BAHD transferase domain) and essentially ignores nonconserved regions. Phylogenetic reconstruction was performed using IQ-TREE v2.1.3 (Minh et al., 2020). The ModelFinder tool was implemented to identify the best maximum likelihood model for 126 estimating evolutionary relationships (Kalyaanamoorthy et al., 2017), leading to selection of Jones-Taylor-Thornton (JTT)+F with seven rate categories and Q.plant+F with seven rate categories for the V4.1 and HQ MSAs, respectively. Phylogenetic trees were inferred by maximum likelihood using the chosen model, and branch support was obtained from 100,000 ultrafast bootstrap iterations (Hoang et al., 2018). The resulting BAHD phylogenies were visualized using the ggtree package in R (Yu et al., 2017). To generate the clade III BAHD heatmap-tree, we used the viewClade function in ggtree to subset the phylogeny and used gheatmap to visualize transcript abundance (log2 TPM) for the eggplant BAHDs. BAHD acyltransferase cloning, expression, and purification Candidate ASATs were cloned into pET28b(+) (MilliporeSigma, Burlington, MA, USA). Open reading frames from genes were either synthesized by Twist Biosciences (South San Francisco, CA, USA) with or without codon-optimization for E. coli expression (Table S2.35) or amplified from genomic DNA or cDNA with primers listed in Table S2.36. Q5 2X Hotstart master mix (New England Biolabs, Ipswich, MA, USA) was used for cloning PCRs. The source of cloned DNA and whether a gene was codon optimized is described in Table S2.35. Amplified genes were purified by agarose gel electrophoresis and extraction with the Monarch DNA Gel Extraction Kit (New England Biolabs, Ipswich, MA, USA). Both the synthesized genes and PCR amplified genes were then inserted into a doubly digested BamHI/XhoI pET28b(+) through Gibson assembly using the 2X Gibson Assembly Master Mix (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. The constructs using synthesized genes were transformed into BL21(DE3) (MilliporeSigma, Burlington, MA, USA) and constructs with PCR amplified genes were transformed into BL21 Rosetta(DE3) cells (MilliporeSigma, Burlington, MA, USA). Constructs were verified with colony PCR and Sanger sequencing using 127 T7 terminator and promoter primers (Table S2.36). Sanger sequencing was completed by the Michigan State University Research Technology Support Facility Genomics Core (East Lansing, MI, USA). Protein expression occurred as previously described (Leong et al., 2022; Lou et al., 2021). Briefly, 50 mL cultures of picked transformation colonies were grown overnight at 37°C, shaking at 225 rpm in Luria-Bertani (LB) media (Neogen, Lansing, MI, USA) supplemented with 1% glucose (w/v). Fifteen mL of the overnight cultures were inoculated into 1 L of fresh LB medium, which was incubated at 37°C shaking at 225 rpm until an OD600 of 0.5 was reached. The cultures were incubated on ice for 25 min, after which, isopropylthio-β-galactoside was added to a final concentration of 50-500 µM. Then, cultures were incubated at 16°C shaking at 180 rpm overnight for 16 hours before cells were harvested by centrifugation at 4,000 rpm for 10 minutes at 4°C. S. melongena BAHDs were purified as previously described (Leong et al., 2020) with the following modifications. The extraction buffer contained 10 mM imidazole, the wash buffer contained 20 mM imidazole, and the elution buffer contained 500 mM imidazole. Protein eluent was concentrated with 30-kD Amicon Ultra centrifugal filter units (MilliporeSigma). Enzyme assays Enzyme assays were conducted in 100 mM sodium phosphate buffer at pH 6. For forward assays, acetyl-CoA (MilliporeSigma, Burlington, MA, USA) was added to a final concentration of 0.1 mM, and for reverse assays free CoA (MilliporeSigma, Burlington, MA, USA) was added to a final concentration of 1 mM. Purified AI3:16 and AI4:18 substrates were dried down using a vacuum centrifuge and redissolved in ethanol:water:formic acid (1:1:0.001). One microliter of the prepared acylsugars were used as acyl acceptors. Six microliters of enzyme 128 were added to a final volume of 60 µL. For negative controls, 6 µL of enzyme that was heat inactivated at 95°C for 10 minutes was substituted in place of untreated enzyme. Assays were incubated at 30°C for 30 minutes after which 120 µL of acetonitrile:isopropanol:formic acid (1:1:0.001) with 1.5 µM telmisartan (MilliporeSigma, Burlington, MA, USA) stop solution was added. Reactions were then spun at 17,000 x g for 10 minutes to remove precipitate. Supernatant was placed in autosampler vials and analyzed by LC-MS. RT-PCR of S. aethiopicum BAHDs We employed semi-quantitative RT-PCR to test S. aethiopicum BAHD expression in glandular trichomes with cDNA and genomic DNA (gDNA). Total RNA was isolated with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), including an on-column DNase digestion (Qiagen, Hilden, Germany), from shaved hypocotyls from accession S. aethiopicum PI 666075 and glandular trichomes from accessions PI 666075 and Grif 14165 isolated as described above for S. melongena glandular trichomes. RNA was quantified with a Nanodrop 2000c instrument (Thermo Fisher Scientific, Waltham, MA, USA). We synthesized cDNA using 10 ng of RNA and SuperScript III Reverse Transcriptase (Invitrogen, Waltham, MA, USA). gDNA was isolated with the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from young leaf tissue collected from mature S. aethiopicum PI 666075 plants. PCR reactions (25 µL) were set up with GoTaq Green Master Mix (Promega, Madison, WI, USA), 200 nM of forward and reverse primers (Table S2.36), and 1 µL of cDNA or gDNA. PCR was performed under these conditions: 2 min at 95°C followed by 22, 30, and 35 cycles of 30s at 95°C, 30s at 58°C, and 1 min at 72°C. We identified the putative S. aethiopicum orthologs of the S. melongena candidate BAHDs by querying S. aethiopicum annotated transcripts (Song et al., 2019) with S. melongena candidate ASAT DNA sequences using BLASTn. SaASAT3 and SaASAT3-L1 (GenBank 129 accession: OQ547782) were not annotated in the S. aethiopicum genome and were identified by querying S. aethiopicum scaffolds with BLASTn and then identifying open reading frames with Geneious software v9.1.8 (Dotmatics, Boston, MA, USA). The S. aethiopicum ef1α gene was identified by querying a putative S. melongena ef1α, SMEL4.1_06g005890, against the S. aethiopicum scaffolds. S. melongena acyl chain composition analysis S. melongena acyl chain composition was assessed with a previously developed esterification and GC-MS methods (Fan et al., 2020; Ning et al., 2015; Schenck et al., 2022). Acyl chain identifies were determined through authentic reference standards for nC8, nC10, nC12, and nC14 fatty acid ethyl esters (MilliporeSigma). The QuanLynx function of MassLynx v4.1 (Waters Corporation) integrated peaks from extracted ion chromatograms. Extracted ion chromatograms for the medium acyl chains iC8, nC8, iC10, nC10, iC12, nC12, iC14, and nC14 were generated for the m/z of 88 with a mass window of m/z of 0.50. Extracted ion chromatograms for the short chains iC4, aiC5, and iC5 were generated for with the m/z values of 71, 102, and 101, respectively, with a mass window of m/z of 0.50. Accession numbers Sequence data in this article can be found in the GenBank/EMBL data libraries under accession number SaASAT3-L1; OQ547782. Acknowledgements I thank Dr. Christopher T. Martine for providing Australian Solanum ssp. seeds, Dr. Joyce van Eck for providing Solanum prinophyllum seeds, Dr. Guiseppe Leonardo Rotino and the Consiglio per la ricercar in agricoltura e l’analisi dell’economia agraria for providing S. melongena 67/3 and 305E40 seeds, and USDA-GRIN for providing Solanum ssp. seeds. I 130 acknowledge the Michigan State University RTSF Mass Spectrometry and Metabolomics Core Facilities for LC-MS analysis support. I also acknowledge Dr. Daniel Holmes and Dr. Li Xie at the Michigan State University Max T. Rogers NMR Facility for experimental design and data analysis support. I also acknowledge members of the Last lab for helpful feedback. 131 REFERENCES Agati, G., Tattini, M., 2010. Multiple functional roles of flavonoids in photoprotection. New Phytol. 186, 786–793. Altschul, S.F., 1998. Generalized affine gap costs for protein sequence alignment. Proteins Struct. Funct. Bioinforma. 32, 88–96. https://doi.org/10.1002/(SICI)1097- 0134(19980701)32:1<88::AID-PROT10>3.0.CO;2-J Anders, S., Pyl, P.T., Huber, W., 2015. HTSeq—a Python framework to work with high- throughput sequencing data. Bioinformatics 31, 166–169. https://doi.org/10.1093/bioinformatics/btu638 Asai, T., Fujimoto, Y., 2010. Cyclic fatty acyl glycosides in the glandular trichome exudate of Silene gallica. Phytochemistry 71, 1410–1417. https://doi.org/10.1016/j.phytochem.2010.05.008 Asai, T., Hara, N., Fujimoto, Y., 2010. Fatty acid derivatives and dammarane triterpenes from the glandular trichome exudates of Ibicella lutea and Proboscidea louisiana. Phytochemistry 71, 877–894. https://doi.org/10.1016/j.phytochem.2010.02.013 Aubriot, X., Knapp, S., Syfert, M.M., Poczai, P., Buerki, S., 2018. Shedding new light on the origin and spread of the brinjal eggplant (Solanum melongena L.) and its wild relatives. Am. J. Bot. 105, 1175–1187. https://doi.org/10.1002/ajb2.1133 Augustin, J.M., Kuzina, V., Andersen, S.B., Bak, S., 2011. Molecular activities, biosynthesis and evolution of triterpenoid saponins. Phytochemistry 72, 435–457. https://doi.org/10.1016/j.phytochem.2011.01.015 Bah, M., Pereda-Miranda, R., 1996. Detailed FAB-mass spectrometry and high resolution NMR investigations of tricolorins A-E, individual oligosaccharides from the resins of Ipomoea tricolor (Convolvulaceae). Tetrahedron 52, 13063–13080. https://doi.org/10.1016/0040- 4020(96)00789-2 Barchi, L., Rabanus-Wallace, M.T., Prohens, J., Toppino, L., Padmarasu, S., Portis, E., Rotino, G.L., Stein, N., Lanteri, S., Giuliano, G., 2021. Improved genome assembly and pan- genome provide key insights into eggplant domestication and breeding. Plant J. 107, 579–596. https://doi.org/10.1111/tpj.15313 Baumann, E., Stoya, G., Völkner, A., Richter, W., Lemke, C., Linss, W., 2000. Hemolysis of human erythrocytes with saponin affects the membrane structure. Acta Histochem. 102, 21–35. https://doi.org/10.1078/0065-1281-00534 Bernal, C.-A., Castellanos, L., Aragón, D.M., Martínez-Matamoros, D., Jiménez, C., Baena, Y., Ramos, F.A., 2018. Peruvioses A to F, sucrose esters from the exudate of Physalis peruviana fruit as α-amylase inhibitors. Carbohydr. Res. 461, 4–10. https://doi.org/10.1016/j.carres.2018.03.003 132 Bohs, L., 2004. Major clades in Solanum based on ndhF sequence data. Monogr. Syst. Bot. 27– 49. Bohs, L., Olmstead, R.G., 1997. Phylogenetic relationships in Solanum (Solanaceae) based on ndhF sequences. Syst. Bot. 22, 5–17. https://doi.org/10.2307/2419674 Bolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120. https://doi.org/10.1093/bioinformatics/btu170 Cao, C.-M., Wu, X., Kindscher, K., Xu, L., Timmermann, B.N., 2015. Withanolides and sucrose esters from Physalis neomexicana. J. Nat. Prod. 78, 2488–2493. https://doi.org/10.1021/acs.jnatprod.5b00698 Caprioli, G., Iannarelli, R., Cianfaglione, K., Fiorini, D., Giuliani, C., Lucarini, D., Papa, F., Sagratini, G., Vittori, S., Maggi, F., 2016. Volatile profile, nutritional value and secretory structures of the berry-like fruits of Hypericum androsaemum L. Food Res. Int. 79, 1–10. https://doi.org/10.1016/j.foodres.2015.11.021 Castillo, M., Connolly, J.D., Ifeadike, P., Labbé, C., Rycroft, D.S., Woods, N., 1989. Partially acylated glucose and sucrose derivatives from Salpiglossis sinuata (Solanaceae). J. Chem. Res. Synop. 398–399. Chortyk, O.T., Kays, S.J., Teng, Q., 1997. Characterization of insecticidal sugar esters of Petunia. J. Agric. Food Chem. 45, 270–275. https://doi.org/10.1021/jf960322f Cicchetti, E., Duroure, L., Le Borgne, E., Laville, R., 2018. Upregulation of skin-aging biomarkers in aged NHDF cells by a sucrose ester extract from the agroindustrial waste of Physalis peruviana calyces. J. Nat. Prod. 81, 1946–1955. https://doi.org/10.1021/acs.jnatprod.7b01069 D’Auria, J.C., 2006. Acyltransferases in plants: a good time to be BAHD. Curr. Opin. Plant Biol. 9, 331–340. https://doi.org/10.1016/j.pbi.2006.03.016 De Moraes, C.M., Mescher, M.C., Tumlinson, J.H., 2001. Caterpillar-induced nocturnal plant volatiles repel conspecific females. Nature 410, 577–580. https://doi.org/10.1038/35069058 Denoeud, F., Carretero-Paulet, L., Dereeper, A., Droc, G., Guyot, R., Pietrella, M., Zheng, C., Alberti, A., Anthony, F., Aprea, G., Aury, J.-M., Bento, P., Bernard, M., Bocs, S., Campa, C., Cenci, A., Combes, M.-C., Crouzillat, D., Da Silva, C., Daddiego, L., De Bellis, F., Dussert, S., Garsmeur, O., Gayraud, T., Guignon, V., Jahn, K., Jamilloux, V., Joët, T., Labadie, K., Lan, T., Leclercq, J., Lepelley, M., Leroy, T., Li, L.-T., Librado, P., Lopez, L., Muñoz, A., Noel, B., Pallavicini, A., Perrotta, G., Poncet, V., Pot, D., Priyono, null, Rigoreau, M., Rouard, M., Rozas, J., Tranchant-Dubreuil, C., VanBuren, R., Zhang, Q., Andrade, A.C., Argout, X., Bertrand, B., de Kochko, A., Graziosi, G., Henry, R.J., Jayarama, null, Ming, R., Nagai, C., Rounsley, S., Sankoff, D., Giuliano, G., Albert, V.A., Wincker, P., Lashermes, P., 2014. The coffee genome provides insight into the 133 convergent evolution of caffeine biosynthesis. Science 345, 1181–1184. https://doi.org/10.1126/science.1255274 DePristo, M.A., Banks, E., Poplin, R., Garimella, K.V., Maguire, J.R., Hartl, C., Philippakis, A.A., del Angel, G., Rivas, M.A., Hanna, M., McKenna, A., Fennell, T.J., Kernytsky, A.M., Sivachenko, A.Y., Cibulskis, K., Gabriel, S.B., Altshuler, D., Daly, M.J., 2011. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat. Genet. 43, 491–498. https://doi.org/10.1038/ng.806 Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., Gingeras, T.R., 2013. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21. https://doi.org/10.1093/bioinformatics/bts635 Dobin, A., Gingeras, T.R., 2015. Mapping RNA-seq reads with STAR. Curr. Protoc. Bioinforma. 51, 11.14.1-11.14.19. https://doi.org/10.1002/0471250953.bi1114s51 Dutartre, L., Hilliou, F., Feyereisen, R., 2012. Phylogenomics of the benzoxazinoid biosynthetic pathway of Poaceae: gene duplications and origin of the Bx cluster. BMC Evol. Biol. 12, 64. https://doi.org/10.1186/1471-2148-12-64 Fan, P., Leong, B.J., Last, R.L., 2019. Tip of the trichome: evolution of acylsugar metabolic diversity in Solanaceae. Curr. Opin. Plant Biol. 49, 8–16. https://doi.org/10.1016/j.pbi.2019.03.005 Fan, P., Miller, A.M., Liu, X., Jones, A.D., Last, R.L., 2017. Evolution of a flipped pathway creates metabolic innovation in tomato trichomes through BAHD enzyme promiscuity. Nat. Commun. 8, 2080. https://doi.org/10.1038/s41467-017-02045-7 Fan, P., Miller, A.M., Schilmiller, A.L., Liu, X., Ofner, I., Jones, A.D., Zamir, D., Last, R.L., 2015. In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network. Proc. Natl. Acad. Sci. U.S.A https://doi.org/10.1073/pnas.1517930113 Fan, P., Wang, P., Lou, Y.R., Leong, B.J., Moore, B.M., Schenck, C.A., Combs, R., Cao, P., Brandizzi, F., Shiu, S.H., Last, R.L., 2020. Evolution of a plant gene cluster in Solanaceae and emergence of metabolic diversity. eLife 9, 1–26. https://doi.org/10.7554/eLife.56717 Fiesel, P.D., Parks, H.M., Last, R.L., Barry, C.S., 2022. Fruity, sticky, stinky, spicy, bitter, addictive, and deadly: evolutionary signatures of metabolic complexity in the Solanaceae. Nat. Prod. Rep. 39, 1438–1464. https://doi.org/10.1039/D2NP00003B Fobes, J.F., Mudd, J.B., Marsden, M.P.F., 1985. Epicuticular lipid accumulation on the leaves of Lycopersicon pennellii (Corr.) D’Arcy and Lycopersicon esculentum Mill. Plant Physiol. 77, 567–570. https://doi.org/10.1104/pp.77.3.567 Gagnon, E., Hilgenhof, R., Orejuela, A., McDonnell, A., Sablok, G., Aubriot, X., Giacomin, L., Gouvêa, Y., Bragionis, T., Stehmann, J.R., Bohs, L., Dodsworth, S., Martine, C., Poczai, 134 P., Knapp, S., Särkinen, T., 2022. Phylogenomic discordance suggests polytomies along the backbone of the large genus Solanum. Am. J. Bot. 109, 580–601. https://doi.org/10.1002/ajb2.1827 Ghosh, B., Westbrook, T.C., Jones, A.D., 2014a. Comparative structural profiling of trichome specialized metabolites in tomato (Solanum lycopersicum) and S. habrochaites: acylsugar profiles revealed by UHPLC/MS and NMR. Metabolomics 10, 496–507. https://doi.org/10.1007/s11306-013-0585-y Goffreda, J.C., Mutschler, M.A., Avé, D.A., Tingey, W.M., Steffens, J.C., 1989. Aphid deterrence by glucose esters in glandular trichome exudate of the wild tomato, Lycopersicon pennellii. J. Chem. Ecol. 15, 2135–2147. https://doi.org/10.1007/BF01207444 Herrera-Salgado, Y., Garduño-Ramírez, M.L., Vázquez, L., Rios, M.Y., Alvarez, L., 2005. Myo- inositol-derived glycolipids with anti-inflammatory activity from Solanum lanceolatum. J. Nat. Prod. 68, 1031–1036. https://doi.org/10.1021/np050054s Hoang, D.T., Chernomor, O., von Haeseler, A., Minh, B.Q., Vinh, L.S., 2018. UFBoot2: improving the ultrafast bootstrap approximation. Mol. Biol. Evol. 35, 518–522. https://doi.org/10.1093/molbev/msx281 Hurney, S.M., 2018. Strategies for profiling and discovery of acylsugar specialized metabolites (Ph.D.). Michigan State University, United States -- Michigan. Jenske, R., Vetter, W., 2007. Highly selective and sensitive gas chromatography–electron- capture negative-ion mass spectrometry method for the indirect enantioselective identification of 2- and 3-hydroxy fatty acids in food and biological samples. J. Chromatogr. A 1146, 225–231. https://doi.org/10.1016/j.chroma.2007.01.102 Ji, W., Mandal, S., Rezenom, Y.H., McKnight, T.D., 2022. Specialized metabolism by trichome- enriched Rubisco and fatty acid synthase components. Plant Physiol. kiac487. https://doi.org/10.1093/plphys/kiac487 Kalyaanamoorthy, S., Minh, B.Q., Wong, T.K.F., von Haeseler, A., Jermiin, L.S., 2017. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat. Methods 14, 587–589. https://doi.org/10.1038/nmeth.4285 Katoh, K., Standley, D.M., 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780. https://doi.org/10.1093/molbev/mst010 Kim, J., Kang, K., Gonzales-Vigil, E., Shi, F., Daniel Jones, A., Barry, C.S., Last, R.L., 2012. Striking natural diversity in glandular trichome acylsugar composition is shaped by variation at the acyltransferase2 locus in the wild tomato Solanum habrochaites. Plant Physiol. 160, 1854–1870. https://doi.org/10.1104/pp.112.204735 135 King, R.R., Calhoun, L.A., 1988. 6 2,3-Di-O- and 1,2,3-tri-O-acylated glucose esters from the glandular trichomes of Datura metel. Phytochemistry 27, 3761–3763. https://doi.org/10.1016/0031-9422(88)83013-9 King, R.R., Pelletier, Y., Singh, R.P., Calhoun, L.A., 1986. 3,4-Di-O-isobutyryl-6-O- caprylsucrose: the major component of a novel sucrose ester complex from the type B glandular trichomes of Solanum berthaultii Hawkes (Pl 473340). J. Chem. Soc. Chem. Commun. 1078–1079. https://doi.org/10.1039/C39860001078 Koonin, E.V., 2005. Orthologs, paralogs, and evolutionary genomics. Annu. Rev. Genet. 39, 309–338. https://doi.org/10.1146/annurev.genet.39.073003.114725 Korenblum, E., Dong, Y., Szymanski, J., Panda, S., Jozwiak, A., Massalha, H., Meir, S., Rogachev, I., Aharoni, A., 2020. Rhizosphere microbiome mediates systemic root metabolite exudation by root-to-root signaling. Proc. Natl. Acad. Sci. U.S.A. 117, 3874– 3883. https://doi.org/10.1073/pnas.1912130117 Kretschmar, J.A., Baumann, T.W., 1999. Caffeine in Citrus flowers. Phytochemistry 52, 19–23. https://doi.org/10.1016/S0031-9422(99)00119-3 Kruse, L.H., Bennett, A.A., Mahood, E.H., Lazarus, E., Park, S.J., Schroeder, F., Moghe, G.D., 2022. Illuminating the lineage-specific diversification of resin glycoside acylsugars in the morning glory (Convolvulaceae) family using computational metabolomics. Hortic. Res. 9, uhab079. https://doi.org/10.1093/hr/uhab079 Landry, L.G., Chapple, CCS., Last, R.L., 1995. Arabidopsis mutants lacking phenolic sunscreens exhibit enhanced ultraviolet-B injury and oxidative damage. Plant Physiol. 109, 1159– 1166. https://doi.org/10.1104/pp.109.4.1159 Leckie, B.M., D’Ambrosio, D.A., Chappell, T.M., Halitschke, R., De Jong, D.M., Kessler, A., Kennedy, G.G., Mutschler, M.A., 2016. Differential and synergistic functionality of acylsugars in suppressing oviposition by insect herbivores. PLoS ONE. https://doi.org/10.1371/journal.pone.0153345 Leong, B.J., Hurney, S., Fiesel, P., Anthony, T.M., Moghe, G., Jones, A.D., Last, R.L., 2022. Identification of BAHD acyltransferases associated with acylinositol biosynthesis in Solanum quitoense (naranjilla). Plant Direct 6, e415. https://doi.org/10.1002/pld3.415 Leong, B.J., Hurney, S.M., Fiesel, P.D., Moghe, G.D., Jones, A.D., Last, R.L., 2020. Specialized metabolism in a nonmodel nightshade: trichome acylinositol biosynthesis. Plant Physiol. 183, 915–924. https://doi.org/10.1104/pp.20.00276 Leong, B.J., Lybrand, D.B., Lou, Y.R., Fan, P., Schilmiller, A.L., Last, R.L., 2019. Evolution of metabolic novelty: a trichome-expressed invertase creates specialized metabolic diversity in wild tomato. Sci. Adv. 5, 1–14. https://doi.org/10.1126/sciadv.aaw3754 Levin, D.A., 1973. The role of trichomes in plant defense. Q. Rev. Biol. 48, 3–15. 136 Levin, R.A., Myers, N.R., Bohs, L., 2006. Phylogenetic relationships among the “spiny solanums” (Solanum subgenus Leptostemonum, Solanaceae). Am. J. Bot. 93, 157–169. https://doi.org/10.3732/ajb.93.1.157 Li, D., Qian, J., Li, Weiliu, Yu, N., Gan, G., Jiang, Y., Li, Wenjia, Liang, X., Chen, R., Mo, Y., Lian, J., Niu, Y., Wang, Y., 2021. A high-quality genome assembly of the eggplant provides insights into the molecular basis of disease resistance and chlorogenic acid synthesis. Mol. Ecol. Resour. 21, 1274–1286. https://doi.org/10.1111/1755-0998.13321 Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R., 1000 Genome Project Data Processing Subgroup, 2009. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079. https://doi.org/10.1093/bioinformatics/btp352 Liu, X., Enright, M., Barry, C.S., Jones, A.D., 2017. Profiling, isolation and structure elucidation of specialized acylsucrose metabolites accumulating in trichomes of Petunia species. Metabolomics 13, 85. https://doi.org/10.1007/s11306-017-1224-9 Lou, Y.-R., Anthony, T.M., Fiesel, P.D., Arking, R.E., Christensen, E.M., Jones, A.D., Last, R.L., 2021. It happened again: convergent evolution of acylglucose specialized metabolism in black nightshade and wild tomato. Sci. Adv. 7, eabj8726. https://doi.org/10.1126/sciadv.abj8726 Lou, Y.-R., Leong, B., 2019. Leaf surface acylsugar extraction and LC-MS profiling - v1.0. Luu, V.T., Weinhold, A., Ullah, C., Dressel, S., Schoettner, M., Gase, K., Gaquerel, E., Xu, S., Baldwin, I.T., 2017. O-acyl sugars protect a wild tobacco from both native fungal pathogens and a specialist herbivore. Plant Physiol. 174, 370–386. https://doi.org/10.1104/pp.16.01904 Lybrand, D.B., Anthony, T.M., Jones, A.D., Last, R.L., 2020. An integrated analytical approach reveals trichome acylsugar metabolite diversity in the wild tomato Solanum pennellii. Metabolites 10, 1–25. https://doi.org/10.3390/metabo10100401 Maldonado, E., Torres, F.R., Martínez, M., Pérez-Castorena, A.L., 2006. Sucrose esters from the fruits of Physalis nicandroides var. attenuata. J. Nat. Prod. 69, 1511–1513. https://doi.org/10.1021/np060274l Matsuzaki, T., Shinozaki, Y., Suhara, S., Ninomiya, M., Shigematsu, H., Koiwai, A., 1989. Isolation of glycolipids from the surface lipids of Nicotiana bigelovii and their distribution in Nicotiana species. Agric. Biol. Chem. 53, 3079–3082. https://doi.org/10.1271/bbb1961.53.3079 McCarthy, D.J., Campbell, K.R., Lun, A.T.L., Wills, Q.F., 2017. Scater: pre-processing, quality control, normalization and visualization of single-cell RNA-seq data in R. Bioinformatics 33, 1179–1186. https://doi.org/10.1093/bioinformatics/btw777 137 McKenna, A., Hanna, M., Banks, E., Sivachenko, A., Cibulskis, K., Kernytsky, A., Garimella, K., Altshuler, D., Gabriel, S., Daly, M., DePristo, M.A., 2010. The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303. https://doi.org/10.1101/gr.107524.110 Mennella, G., Rotino, G.L., Fibiani, M., D’Alessandro, A., Francese, G., Toppino, L., Cavallanti, F., Acciarri, N., Lo Scalzo, R., 2010. Characterization of health-related compounds in eggplant (Solanum melongena L.) lines derived from introgression of allied species. J. Agric. Food Chem. 58, 7597–7603. https://doi.org/10.1021/jf101004z Minh, B.Q., Schmidt, H.A., Chernomor, O., Schrempf, D., Woodhams, M.D., von Haeseler, A., Lanfear, R., 2020. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol. Biol. Evol. 37, 1530–1534. https://doi.org/10.1093/molbev/msaa015 Moghe, G., Kruse, L.H., Petersen, M., Scossa, F., Fernie, A.R., Gaquerel, E., D’Auria, J.C., 2023. BAHD company: the ever-expanding roles of the BAHD acyltransferase gene family in plants. Annu. Rev. Plant Biol. 74, annurev-arplant-062922-050122. https://doi.org/10.1146/annurev-arplant-062922-050122 Moghe, G.D., Leong, B.J., Hurney, S.M., Jones, A.D., Last, R.L., 2017. Evolutionary routes to biochemical innovation revealed by integrative analysis of a plant-defense related specialized metabolic pathway. eLife 6, 1–33. https://doi.org/10.7554/eLife.28468 Moore, B.M., Wang, P., Fan, P., Leong, B., Schenck, C.A., Lloyd, J.P., Lehti-Shiu, M.D., Last, R.L., Pichersky, E., Shiu, S.-H., 2019. Robust predictions of specialized metabolism genes through machine learning. Proc. Natl. Acad. Sci. U.S.A. 116, 2344–2353. https://doi.org/10.1073/pnas.1817074116 Nadakuduti, S.S., Uebler, J.B., Liu, X., Jones, A.D., Barry, C.S., 2017. Characterization of trichome-expressed BAHD acyltransferases in Petunia axillaris reveals distinct acylsugar assembly mechanisms within the Solanaceae. Plant Physiol. 175, 36–50. https://doi.org/10.1104/pp.17.00538 Nee, M., 2022a. Solanum acerifolium [Dunal ] [WWW Document]. URL https://solanaceaesource.myspecies.info/taxonomy/term/105367/descriptions (accessed 10.3.22). Nee, M., 2022b. Solanum atropurpureum [Schrank ] [WWW Document]. URL https://solanaceaesource.myspecies.info/taxonomy/term/105749/descriptions (accessed 10.3.22). Nee, M., 1991. Synopsis of Solanum section Acanthophora: a group of interest for glycoalkaloids, in: Solanaceae III, taxonomy, chemistry, evolution. Royal Botanic Gardens for the Linnean Society of London, Kew, Richmond, Surrey, UK, pp. 257–266. Ning, J., Moghe, G.D., Leong, B., Kim, J., Ofner, I., Wang, Z., Adams, C., Jones, A.D., Zamir, D., Last, R.L., 2015. A feedback-insensitive isopropylmalate synthase affects acylsugar 138 composition in cultivated and wild tomato. Plant Physiol. 169, 1821–1835. https://doi.org/10.1104/pp.15.00474 Panchy, N., Lehti-Shiu, M., Shiu, S.-H., 2016. Evolution of Gene Duplication in Plants. Plant Physiol. 171, 2294–2316. https://doi.org/10.1104/pp.16.00523 PBI Solanum Project, 2022. Solanaceae Source [WWW Document]. URL https://solanaceaesource.org (accessed 10.3.22). Pereda-Miranda, R., Mata, R., Anaya, A.L., Wickramaratne, D.B.M., Pezzuto, J.M., Kinghorn, A.D., 1993. Tricolorin A, major phytogrowth inhibitor from Ipomoea tricolor. J. Nat. Prod. 56, 571–582. https://doi.org/10.1021/np50094a018 Pinot, F., Beisson, F., 2011. Cytochrome P450 metabolizing fatty acids in plants: characterization and physiological roles. FEBS J. 278, 195–205. https://doi.org/10.1111/j.1742-4658.2010.07948.x Robinson, M.D., McCarthy, D.J., Smyth, G.K., 2010. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139– 140. https://doi.org/10.1093/bioinformatics/btp616 Sampaio, V. da S., Coutinho, Í.A.C., Särkinen, T., Loiola, M.I.B., 2021. Secretory and ecological function of petiolar glands in Solanum fernandesii: first description of resin glands in the genus Solanum. Aust. J. Bot. 70, 32–41. https://doi.org/10.1071/BT21001 Särkinen, T., Bohs, L., Olmstead, R.G., Knapp, S., 2013. A phylogenetic framework for evolutionary study of the nightshades (Solanaceae): a dated 1000-tip tree. BMC Evol. Biol. 13, 214. https://doi.org/10.1186/1471-2148-13-214 Sassaki, G.L., Souza, L.M., Serrato, R.V., Cipriani, T.R., Gorin, P.A.J., Iacomini, M., 2008. Application of acetate derivatives for gas chromatography–mass spectrometry: novel approaches on carbohydrates, lipids and amino acids analysis. J. Chromatogr. A 1208, 215–222. https://doi.org/10.1016/j.chroma.2008.08.083 Schenck, C.A., Anthony, T.M., Jacobs, M., Jones, A.D., Last, R.L., 2022. Natural variation meets synthetic biology: promiscuous trichome-expressed acyltransferases from Nicotiana. Plant Physiol. kiac192. https://doi.org/10.1093/plphys/kiac192 Schilmiller, A.L., Charbonneau, A.L., Last, R.L., 2012. Identification of a BAHD acetyltransferase that produces protective acyl sugars in tomato trichomes. Proc. Natl. Acad. Sci. U.S.A. 109, 16377–16382. https://doi.org/10.1073/pnas.1207906109 Schilmiller, A.L., Gilgallon, K., Ghosh, B., Jones, A.D., Last, R.L., 2016. Acylsugar acylhydrolases: carboxylesterase-catalyzed hydrolysis of acylsugars in tomato trichomes. Plant Physiol. 170, 1331–1344. https://doi.org/10.1104/pp.15.01348 Schilmiller, A.L., Moghe, G.D., Fan, P., Ghosh, B., Ning, J., Jones, A.D., Last, R.L., 2015. Functionally divergent alleles and duplicated Loci encoding an acyltransferase contribute 139 to acylsugar metabolite diversity in Solanum trichomes. Plant Cell 27, 1002–17. https://doi.org/10.1105/tpc.15.00087 Shapiro, J.A., Steffens, J.C., Mutschler, M.A., 1994. Acylsugars of the wild tomato Lycopersicon pennellii in relation to geographic distribution of the species. Biochem. Syst. Ecol. 22, 545–561. https://doi.org/10.1016/0305-1978(94)90067-1 Smith, C.R., 1971. Occurrence of unusual fatty acids in plants. Prog. Chem. Fats Other Lipids 11, 137–177. https://doi.org/10.1016/0079-6832(71)90005-X Song, B., Song, Y., Fu, Y., Kizito, E.B., Kamenya, S.N., Kabod, P.N., Liu, H., Muthemba, S., Kariba, R., Njuguna, J., Maina, S., Stomeo, F., Djikeng, A., Hendre, P.S., Chen, X., Chen, W., Li, X., Sun, W., Wang, S., Cheng, S., Muchugi, A., Jamnadass, R., Shapiro, H.-Y., Van Deynze, A., Yang, H., Wang, J., Xu, X., Odeny, D.A., Liu, X., 2019. Draft genome sequence of Solanum aethiopicum provides insights into disease resistance, drought tolerance, and the evolution of the genome. GigaScience 8, giz115. https://doi.org/10.1093/gigascience/giz115 Stern, S., Agra, M. de F., Bohs, L., 2011. Molecular delimitation of clades within New World species of the “spiny solanums” (Solanum subg. Leptostemonum). TAXON 60, 1429– 1441. https://doi.org/10.1002/tax.605018 Sumner, L.W., Amberg, A., Barrett, D., Beale, M.H., Beger, R., Daykin, C.A., Fan, T.W.-M., Fiehn, O., Goodacre, R., Griffin, J.L., Hankemeier, T., Hardy, N., Harnly, J., Higashi, R., Kopka, J., Lane, A.N., Lindon, J.C., Marriott, P., Nicholls, A.W., Reily, M.D., Thaden, J.J., Viant, M.R., 2007. Proposed minimum reporting standards for chemical analysis. Metabolomics 3, 211–221. https://doi.org/10.1007/s11306-007-0082-2 Tepe, E.J., Anderson, G.J., Spooner, D.M., Bohs, L., 2016. Relationships among wild relatives of the tomato, potato, and pepino. TAXON 65, 262–276. https://doi.org/10.12705/652.4 Van Dam, N.M., Hare, J.D., 1998. Biological activity of Datura wrightii glandular trichome exudate against Manduca sexta larvae. J. Chem. Ecol. 24, 1529–1549. https://doi.org/10.1023/A:1020963817685 Van der Auwera, G.A., Carneiro, M.O., Hartl, C., Poplin, R., del Angel, G., Levy-Moonshine, A., Jordan, T., Shakir, K., Roazen, D., Thibault, J., Banks, E., Garimella, K.V., Altshuler, D., Gabriel, S., DePristo, M.A., 2013. From FastQ data to high-confidence variant calls: the genome analysis toolkit best practices pipeline. Curr. Protoc. Bioinforma. 43, 11.10.1- 11.10.33. https://doi.org/10.1002/0471250953.bi1110s43 Wagner, G.J., 1991. Secreting glandular trichomes: more than just hairs. Plant Physiol. 96, 675– 679. https://doi.org/10.1104/pp.96.3.675 Weese, T.L., Bohs, L., 2007. A three-gene phylogeny of the genus Solanum (Solanaceae). Syst. Bot. 32, 445–463. 140 Weinhold, A., Baldwin, I.T., 2011. Trichome-derived O-acyl sugars are a first meal for caterpillars that tags them for predation. Proc. Natl. Acad. Sci. U.S.A. 108, 7855–7859. https://doi.org/10.1073/pnas.1101306108 Yu, G., Smith, D.K., Zhu, H., Guan, Y., Lam, T.T.-Y., 2017. ggtree: an r package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods Ecol. Evol. 8, 28–36. https://doi.org/10.1111/2041-210X.12628 Yu, P., He, X., Baer, M., Beirinckx, S., Tian, T., Moya, Y.A.T., Zhang, X., Deichmann, M., Frey, F.P., Bresgen, V., Li, C., Razavi, B.S., Schaaf, G., von Wirén, N., Su, Z., Bucher, M., Tsuda, K., Goormachtig, S., Chen, X., Hochholdinger, F., 2021. Plant flavones enrich rhizosphere Oxalobacteraceae to improve maize performance under nitrogen deprivation. Nat. Plants 7, 481–499. https://doi.org/10.1038/s41477-021-00897-y Zizovic, I., Stamenić, M., Orlović, A., Skala, D., 2007. Supercritical carbon dioxide extraction of essential oils from plants with secretory ducts: mathematical modelling on the micro- scale. J. Supercrit. Fluids 39, 338–346. https://doi.org/10.1016/j.supflu.2006.03.009 141 APPENDIX Figure S2.1. Annotation of I3:18(4,4,10) and AI3:16(4,4,8) using negative and positive mode MS and CID MS/MS fragmentation. (A) I3:18(4,4,10) negative and positive mode MS and MS/MS fragmentation. (B) AI3:16(4,4,8) negative and positive MS and MS/MS fragmentation. For each compound, ESI- MS (top panels) display the formate adduct accurate mass which was used to determine chemical formulas. ESI- MS/MS (second from the top panels) exhibit acyl chain carboxylate fragment ions for both compounds and stepwise loss of acyl chains for only AI3:16. ESI+ MS/MS exhibit stepwise losses of acyl chains for both compounds. Positive mode CID of AI3:16 also produces a fragment ion (m/z 429.2492) corresponding to the neutral loss of the pentose moiety. This indicates that all acyl chains reside on the hexose ring. The acylsugar LC-MS annotation method is described in the Materials and Methods. fa = fatty acid; k = ketene. 142 Figure S2.2. Negative mode MS/MS fragmentation of H3:22(4,4,16-O-p) from S. sisymbriifolium results in neutral loss of a pentose moiety. This neutral loss of a sugar group (C5H10O5 plus formic acid) from the [M+formate]- ion m/z 751.4025 to form m/z 555.3522 is not observed with negative CID of other acyldisaccharides (Figures S2.1 and S2.8) suggesting an unusual glycosidic linkage on the acyl chain. fa = fatty acid; k = ketene; 16-O-p = pentosylated 16 carbon acyl chain. 143 Figure S2.3. Acylsugar saponification produces free glycosylated fatty acids in four Solanum species supporting the identification of glycohydroxyacyl hexoses. All traces are extracted ion LC/MS chromatograms for pentosylated hydroxymyristic acid, m/z 375.24±0.05 (retention time 2.90 min) from saponified leaf surface extracts. S. melongena does not contain detectable glycosylated fatty acids and acts as a negative control. Vertical scale is normalized to the largest signal within the displayed region (values in the upper right of each chromatogram). 144 Figure S2.4. Coelution analysis reveals that five Solanum species accumulate acylsugar positional or branching isomers that differ from S. melongena acylsugars. Extracted ion chromatograms were generated for combined signals of m/z 591.34 and 519.28 corresponding to I3:22(4,4,14-OH) and I3:18(4,4,10) [M+formate]- adducts. The yellow traces (bottom row) represent S. melongena, the blue traces represent the five other Solanum species (S. prinophylum, S. abutiloides, S. dulcamara, S. villosum, and S. scabrum), and the middle row green traces represent acylsugar mixtures between S. melongena and the species in the trace below. The mixed samples allow for corrections of retention time drift that may occur between samples. 145 Figure S2.5. GC-MS full scan mass spectra of 3-OH-14:0 ethyl ester stereoisomers as their MPTA derivatives. The mass spectra contain ions at m/z 189, 209, and 255 corresponding to the expected fragmentation of the derivatized fatty acid. EE = ethyl ester. Peak numbers correspond to the peaks displayed in Figure 2.3. 146 Figure S2.6. Phylogenetic analysis of Eggplant Clade and Anguivi Grade species reveals S. aethiopicum is the only analyzed acylsugar-producing species to not accumulate detectable AI4:18(2,4,4,8). AI3:16(4,4,8) and AI4:18(2,4,4,8) presence and absence in surface extracts as detected by LC-MS are plotted on a phylogeny of the Eggplant Clade and Anguivi Grade. Species that either do not accumulate detectable acylsugars (S. macrocarpon and S. virginianum) or were not analyzed did not have AI3:16 and AI4:18 presence plotted. The phylogeny was modified from a previously published version (Aubriot et al., 2018). Full acylsugar profiles are detailed Tables 2.1, S2.2-7. 147 Figure S2.7. Negative and positive mode MS and CID MS/MS fragmentation of G3:15(2,5,8) from S. americanum. Acylglucoses fragment characteristically in negative mode MS and MS/MS functions producing fragment ions corresponding to stepwise acyl chain loss. This is in stark contrast to acylinositol negative mode fragmentation which does not produce 148 Figure S2.7. (cont’d) major fragment ions corresponding to stepwise acyl chain loss (Figure S2.1). This fragmentation difference distinguishes the two sugar cores and enables their annotation by LC-MS. G3:15 formate and ammonium adduct ions were selected by data-dependent acquisition software and fragmented with a ramped collision energy detailed in the Materials and Methods. fa = fatty acid; k = ketene. 149 Figure S2.8. Negative and positive mode MS and CID MS/MS fragmentation of dHH3:36(10,12-OH,14-OH) from S. atropurpureum fruit and HH3:18(2,8,8) from S. heteropodium. Formate and ammonium adduct ions were selected by data-dependent acquisition software and fragmented with a ramped collision energy detailed in the Materials and Methods. Under negative mode CID MS/MS, each compound produces fragment ions corresponding to stepwise acyl chain loss down to a free sugar core fragment ion. In contrast, under positive mode CID MS/MS each compound produces fragment ions corresponding to neutral loss of a sugar moiety. (A) dHH3:36(10,12-OH,14-OH) MS and MS/MS fragmentation. Positive mode CID produces a fragment ion (m/z 569.4069) corresponding to a neutral loss of a deoxyhexose acylated with a ten carbon acyl chain. dHH = deoxyhexose-hexose (B) HH3:18(2,8,8) MS and MS/MS fragmentation. Positive mode CID produces a fragment ion (m/z 331.1836) corresponding to a neutral loss of a hexose acylated with an eight carbon acyl chain. HH = hexose-hexose; fa = fatty acid; k = ketene. 150 Figure S2.9. S. aethiopicum RT-PCR primers validated with gDNA controls. Soaet10024792 primers amplified a region containing an intron resulting in the larger amplicon length. 151 EF1αSaASAT3 primer pair 1SaASAT3-L1 primer pair 1Soaet10022603Soaet10024792Soaet10024793Soaet10043742SoaetASAT3 primer pair 2 SoaetASAT3-L1 primer pair 2500 bp1000 bpgDNA #1 gDNA #3 gDNA #2 gDNA #1 gDNA #3 gDNA #2 500 bp1000 bp Figure S2.10. Semi-quantitative RT-PCR analysis of S. aethiopicum BAHD expression in glandular trichomes. Elongation factor1α (EF1α) was used as a positive control. P1 = primer pair 1; P2 = primer pair 2; S = shaved hypocotyl; T1 = PI 666075 glandular trichomes; T2 = Grif 14165 glandular trichomes. 152 Figure S2.11. S. melongena BAHD tree. Shown is a phylogeny including 106 predicted BAHDs (PF002458) in the eggplant Smel_V4.1 reference genome, published reference BAHD sequences for clades I-VII, characterized ASAT sequences from other Solanaceae species, and the SaASAT3 and SaASAT3-L1 candidates from S. aethiopicum. The maximum likelihood tree was inferred from amino acid sequences using the Jones-Taylor-Thornton algorithm with seven rate categories in IQ-TREE v2.1.3. Bootstrap support calculated from 100,000 ultrafast bootstrap replicates and values >=95 indicate strong support. Clade III BAHDs are highlighted in purple, with a purple dot marking the node. Branch lengths represent substitution rates as denoted by scale bar. 153 Figure S2.12. S. melongena produces single stalked glandular trichomes on seedling above ground tissue but stellate nonglandular trichomes on non-seedling above ground tissue. (A) Close up photo of a S. melongena hypocotyl displaying glandular trichomes with similar morphology to the acylsugar-producing S. lycopersicum Type I/IV trichomes (Luckwill, 1943; Schilmiller et al., 2012). (B) Close up photo of a S. melongena leaf from a reproductive stage plant displaying non-glandular stellate trichomes. Leaf surface metabolite extracts from this tissue do not have detectable acylsugars by LC-MS. 154 Figure S2.13. MDS plot of S. melongena RNAseq data. Genome-wide expression patterns across eggplant trichomes, trichomeless hypocotyls, and roots. Depicted is a multidimensional scaling (MDS) plot demonstrating that our 18 eggplant RNAseq samples cluster tightly by tissue identity. Distance between points illustrates expression differences between pairs of samples, calculated as leading (i.e., largest absolute) log2 fold-change (FC) in two dimensions, dim 1 and dim 2. Along the y-axis (dim 2), samples are separated into three tissue-specific groups. Along the x-axis (dim 1), samples are separated into two groups representing aerial (trichomes and trichomeless hypocotyls) and subterranean (roots) tissues, respectively. Colors and shapes represent the three tissues. T, trichomes (green circles); H, trichomeless hypocotyls (yellow squares); R, roots (brown triangles). 155 Figure S2.14. SmASAT3-L1 forward and reverse assay produced AI4:18 and AI3:16, respectively, coelute with plant produced AI4:18 and AI3:16. (A) Forward assay produced AI4:18 (top) coelutes with AI4:18 from a S. melongena 555598 leaf surface extract (bottom). The extracted ion chromatograms display the formate adduct of AI4:18, m/z 665.30. (B) Reverse assay produced AI3:16 (top) coelutes with AI3:16 from a S. melongena 555598 leaf surface extract (bottom). The extracted ion chromatograms display the formate adduct of AI3:16, m/z 623.20. 156 Table S2.1. Sugar cores and acyl chains annotated in Solanum Clade II, VANAns, and DulMo acylsugars. All acylsugars were annotated from leaf surface extracts except for the combined S. acerifolium and S. atropurprueum fruit data. Table includes S. nigrum and S. quitoense acylsugar traits from published reports (Hurney, 2018; Leong et al., 2020; Lou et al., 2021). Acylsugar profiles are described further in Tables S2.1-26. Major Clade Minor Clade Species Mono- Clade II EHS Solanum melongena Solanum incanum Solanum aethiopicum Solanum anguivi Solanum lichtensteinii Solanum linnaeanum Solanum richardii Solanum heteropodium Solanum sejuntum Solanum melanospermum Solanum dioicum Solanum prinophyllum Solanum lasiophyllum Torva Sisymbriifolium Solanum torvum Solanum sisymbriifolium Lasiocarpa Acanthophora VANAns Brevantherum Archaeasolanum Solanum quitoense Solanum capsioides Solanum acerifolium/ atropurpureum Solanum acerifolium/ atropurpureum fruit Solanum mammosum Solanum abutiloides Solanum laciniatum I I I I I I I I I I I I I I I I I I I I I I Sugar core Di- PH PH PH PH PH PH PH, HH HH PH PH PH, HH, GlNac-I Non-modified 2,4,5,8,10,12,14 2,4,5,8,10,12,14 4,5,8,10,12,14 2,4,5,8,10,12,14 2,4,5,8,10,12,14 2,4,5,8,10,12,14 2,4,5,8,10,12,14 2,4,5,8,10,12,14 2,4,5,8,9,10,12 4,5,8,9,10,11,12 4,5,8,9,10,11,12 4,5,10,12,14 2,4,8,10,12,14 2,4,5,10,11,12, 13,14,16,17,18 4,5 2,10,12 6,8,9,10,12 Acyl chain hydroxylated unsaturated 12,14 12,14 12,14 12,14 12,14 12,14 12,14 12,14 12,14 14 14 12,14 12,14 14,15,16 12,14 5:1,18:1,18:2,18:3 PH 2,5,6,8,10 12,14,15,16 8,9,10,11 4,5,6,8 2,3,4,5,12,14,16 6,8 10,12,13,14 6,12,14,16 12,14 dHH 157 Table S2.1. (cont’d) DulMo Dulcamaroid Morelloid Solanum duclamara Solanum nigrum Solanum americanum Solanum scabrum Solanum villosum I I, G G I, G I, G PH 4 2,4,5,8,9,10 2,4,5,6,8,9,10 4,5,8,9,10 14 10 HH 2,4,5,10 12,14,16 158 Table S2.2. Summary of annotated acylsugars detected in S. incanum trichome extracts. PH = pentose-hexose; AI = arabinose-inositol; I = inositol. RT = retention time; Annotation method and confidence level criteria are described within the Methods. m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI and was averaged over two samples. Acylsugars are sorted by number of sugar moieties and then by elution order. Confidence level RT (min) Chemical formula m/z acc m/z exp Δm(ppm) Average acylsugar abundance (%) Name Acyldisaccharides PH3:14(2,4,8) PH4:16(2,2,4,8) PH3:16(4,4,8) AI3:16(4,4,8) PH3:17(4,5,8) AI4:18(2,4,4,8) PH3:18(4,4,10) PH3:18(4,4,10) PH4:19(2,4,5,8) PH4:19(2,4,5,8) PH3:20(4,4,12) PH3:20(4,4,12) medium medium medium high medium high medium medium medium medium medium medium Acylhexoses I3:20(4,4,12-OH) medium I3:20(4,4,12-OH) medium I3:20(2,4,14-OH) medium high I3:18(4,4,10) I3:18(4,4,10) medium I3:20(2,4,14-OH) medium I3:22(4,4,14-OH) I3:22(4,4,14-OH) I3:20(4,4,12) I3:22(4,4,14) high high high high I3:22(4,4,14) high 0.207 1.44 15.3 0.159 63.1 0.00728 0.151 0.284 0.000206 0.0318 0.0301 0.0450 0.208 6.21 0.164 0.131 5.83 2.74 2.85 0.900 0.174 C25H42O13 C27H44O14 C27H46O13 C27H46O13 C28H48O13 C29H48O14 C29H50O13 C29H50O13 C30H50O14 C30H50O14 C31H54O13 C31H54O13 C26H46O10 C26H46O10 C26H46O10 C24H42O9 C24H42O9 C26H46O10 C28H50O10 C28H50O10 C26H46O9 C28H50O9 2.23 2.54 2.61 2.66 2.96 3.33 3.52 3.61 3.76 3.83 5.05 5.23 4.16 4.32 4.68 4.70 4.88 4.90 6.06 6.34 6.71 9.34 9.68 595.2607 595.2621 637.2713 637.2720 2.4 1.1 623.2920 623.2919 -0.2 637.3077 637.3083 665.3026 665.3026 651.3233 651.3239 651.3233 651.3231 679.3183 679.3186 679.3546 679.3554 679.3546 679.3552 563.3073 563.3071 563.3073 563.3068 563.3073 563.3082 519.2811 519.2802 519.2811 519.2794 563.3073 563.3077 591.3386 591.3385 591.3386 591.3380 547.3124 547.3119 575.3437 575.3435 1.0 0.0 0.9 -0.3 0.5 1.2 0.9 -0.4 -0.9 1.6 -1.7 -3.3 0.7 -0.2 -1.0 -1.0 -0.3 -0.7 C28H50O9 575.3437 575.3433 159 Table S2.3. Summary of annotated acylsugars detected in S. anguivi trichome extracts. PH = pentose-hexose; AI = arabinose-inositol; I = inositol. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI and was averaged over six samples. Acylsugars are sorted by number of sugar moieties and then by elution order. Confidence level RT (min) Chemical formula m/z acc m/z exp Δm(ppm) Average acylsugar abundance (%) Name Acyldisaccharides PH4:16(2,2,4,8) PH3:16(4,4,8) AI3:16(4,4,8) PH3:17(4,5,8) AI4:18(2,4,4,8) PH4:19(2,4,5,8) PH4:19(2,4,5,8) PH3:20(4,4,12) PH3:20(4,4,12) medium medium high medium high medium medium medium medium high Acylhexoses I3:20(4,4,12-OH) medium I3:20(4,4,12-OH) medium I3:18(4,4,10) I3:20(2,4,14-OH) medium I3:18(4,4,10) medium I3:20(2,4,14-OH) medium I3:22(4,4,14-OH) I3:22(4,4,14-OH) I3:20(4,4,12) I3:22(4,4,14) high high high high I3:22(4,4,14) high 2.54 2.58 2.63 2.93 3.33 3.70 3.77 4.98 5.15 4.14 4.32 4.68 4.68 4.88 4.90 5.98 6.24 6.71 9.25 9.56 C27H44O14 C27H46O13 C27H46O13 C28H48O13 C29H48O14 C30H50O14 C30H50O14 C31H54O13 C31H54O13 C26H46O10 C26H46O10 C24H42O9 C26H46O10 C24H42O9 C26H46O10 C28H50O10 C28H50O10 C26H46O9 C28H50O9 C28H50O9 637.2713 637.2720 1.1 623.2920 623.2919 0.97 637.3077 637.3083 665.3026 665.3026 679.3183 679.3186 679.3546 679.3554 679.3546 679.3552 563.3073 563.3071 563.3073 563.3068 519.2811 519.2802 563.3073 563.3082 519.2811 519.2794 563.3073 563.3077 591.3386 591.3385 591.3386 591.3380 547.3124 547.3119 575.3437 575.3435 575.3437 575.3433 1.0 0.0 1.1 1.2 0.9 -0.4 -0.9 -1.7 1.6 -3.3 0.7 -0.2 -1.0 -1.0 -0.3 -0.7 2.73 16.8 0.0921 59.8 0.147 0.00021 0.0262 0.023 0.0461 6.35 0.393 0.0955 0.132 5.78 4.37 2.53 0.594 0.134 160 Table S2.4. Summary of annotated acylsugars detected in S. aethiopicum trichome extracts. Annotation method and confidence level criteria are described within the Methods. PH = pentose-hexose; AI = arabinose-inositol; I = inositol. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI and was averaged over 25 samples. Acylsugars are sorted by number of sugar moieties and then by elution order. Confidence level RT (min) Chemical formula m/z acc m/z exp Δm(ppm) Average acylsugar abundance (%) Name Acyldisaccharides PH3:16(4,4,8) AI3:16(4,4,8) PH3:18(4,4,10) PH3:18(4,4,10) PH3:20(4,4,12) PH3:20(4,4,12) medium high medium medium medium medium Acylhexoses I3:16(4,4,8) medium I3:20(4,4,12-OH) medium I3:20(4,4,12-OH) medium I3:18(4,4,10) I3:18(4,4,10) I3:22(4,4,14-OH) I3:20(4,8,8) I3:22(4,4,14-OH) I3:20(4,4,12) I3:20(4,4,12) I3:22(4,8,10) I3:22(4,4,14) high medium high medium high high medium medium high I3:22(4,4,14) high 47.8 3.47 3.35 0.191 0.179 1.29 0.590 1.44 15.3 0.596 15.8 0.369 5.73 1.81 0.0694 0.261 1.71 0.139 C27H46O13 C27H46O13 C29H50O13 C29H50O13 C31H54O13 C31H54O13 C22H38O9 C26H46O10 C26H46O10 C24H42O9 C24H42O9 C28H50O10 C26H46O9 C28H50O10 C26H46O9 C26H46O9 C28H50O9 C28H50O9 2.61 2.66 3.49 3.59 4.98 5.15 3.28 4.09 4.27 4.70 4.87 6.01 6.21 6.26 6.84 7.11 8.58 9.41 9.75 623.2920 623.2919 1.1 651.3233 651.3239 651.3233 651.3231 679.3546 679.3554 679.3546 679.3552 491.2498 491.2492 563.3073 563.3071 563.3073 563.3068 519.2811 519.2802 519.2811 519.2794 591.3386 591.3385 547.3124 547.3122 591.3386 591.338 547.3124 547.3119 547.3124 547.3127 575.3437 575.3439 575.3437 575.3435 0.9 -0.3 1.2 0.9 -1.3 -0.4 -0.9 -1.7 -3.3 -0.2 -0.3 -1.0 -1.0 0.6 0.3 -0.3 -0.7 C28H50O9 575.3437 575.3433 161 Table S2.5. Summary of annotated acylsugars detected in S. lichtensteinii trichome extracts. PH = pentose-hexose; AI = arabinose-inositol; I = inositol. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI and was averaged over four samples. Acylsugars are sorted by number of sugar moieties and then by elution order. Confidence level RT (min) Chemical formula m/z acc m/z exp Δm(ppm) Average acylsugar abundance (%) Name Acyldisaccharides PH3:14(2,4,8) PH4:16(2,2,4,8) AI3:16(4,4,8) PH3:17(4,5,8) AI4:18(2,4,4,8) PH3:18(4,4,10) PH3:18(4,4,10) PH4:19(2,4,5,8) PH3:20(4,4,12) PH3:20(4,4,12) medium medium high medium high medium medium medium medium medium Acylhexoses I3:20(4,4,12-OH) medium I3:20(4,4,12-OH) medium I3:20(2,4,14-OH) medium I3:18(4,4,10) high medium I3:18(4,4,10) I3:20(2,4,14-OH) medium I3:22(4,4,14-OH) I4:22(4,4,4,10) I3:20(4,8,8) I4:22(4,4,4,10) I3:22(4,4,14-OH) I3:20(4,4,12) I3:20(4,4,12) I3:22(4,8,10) I3:22(4,4,14) high medium medium medium high high medium medium high I3:22(4,4,14) high 2.23 2.58 2.66 2.94 3.29 3.48 3.59 3.76 5.21 5.45 4.16 4.32 4.68 4.70 4.88 4.92 6.08 6.15 6.23 6.34 6.36 6.84 7.13 8.58 9.41 9.76 C25H42O13 C27H44O14 C27H46O13 C28H48O13 C29H48O14 C29H50O13 C29H50O13 C30H50O14 C31H54O13 C31H54O13 C26H46O10 C26H46O10 C26H46O10 C24H42O9 C24H42O9 C26H46O10 C28H50O10 C28H48O10 C26H46O9 C28H48O10 C28H50O10 C26H46O9 C26H46O9 C28H50O9 C28H50O9 595.2607 595.2621 637.2713 637.2720 623.2920 623.2919 637.3077 637.3083 665.3026 665.3026 651.3233 651.3239 651.3233 651.3231 679.3183 679.3186 679.3546 679.3554 679.3546 679.3552 563.3073 563.3071 563.3073 563.3068 563.3073 563.3082 519.2811 519.2802 519.2811 519.2794 563.3073 563.3077 591.3386 591.3385 589.3229 589.3227 547.3124 547.3122 589.3229 589.3237 591.3386 591.3380 547.3124 547.3119 547.3124 547.3127 575.3437 575.3439 575.3437 575.3435 C28H50O9 575.3437 575.3433 2.4 1.1 -0.2 1.0 0.0 0.9 -0.3 0.5 1.2 0.9 -0.4 -0.9 1.6 -1.7 -3.3 0.7 -0.2 -0.3 -0.3 1.3 -1.0 -1.0 0.6 0.3 -0.3 -0.7 1.03 4.64 6.35 0.0256 31.6 2.13 4.47 0.0866 0.0609 0.00228 0.00268 0.0794 0.459 30.4 0.448 0.527 3.00 4.37 0.0428 0.0209 8.85 0.567 0.125 0.0877 0.345 0.232 162 Table S2.6. Summary of annotated acylsugars detected in S. linnaeanum trichome extracts. PH = pentose-hexose; AI = arabinose-inositol; I = inositol. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI and was averaged over seven samples. Acylsugars are sorted by number of sugar moieties and then by elution order. Confidence level RT (min) Chemical formula m/z acc m/z exp Δm(ppm) Average acylsugar abundance (%) Name Acyldisaccharides PH3:14(2,4,8) PH4:16(2,2,4,8) AI3:16(4,4,8) PH3:17(4,5,8) AI4:18(2,4,4,8) PH4:19(2,4,5,8) medium medium high medium high medium Acylhexoses I3:20(4,4,12-OH) medium I3:20(4,4,12-OH) medium I3:20(2,4,14-OH) medium high I3:18(4,4,10) medium I3:18(4,4,10) I3:20(2,4,14-OH) medium I3:22(4,4,14-OH) I3:20(4,8,8) I3:22(4,4,14-OH) I3:20(4,4,12) I3:20(4,4,12) I3:22(4,8,10) I3:22(4,4,14) high medium high high medium medium high I3:22(4,4,14) high 2.23 2.58 2.66 2.94 3.29 3.76 4.16 4.32 4.68 4.70 4.88 4.92 6.10 6.23 6.36 6.84 7.13 8.58 9.41 9.76 C25H42O13 C27H44O14 C27H46O13 C28H48O13 C29H48O14 C30H50O14 595.2607 595.2621 637.2713 637.2720 623.2920 623.2919 637.3077 637.3083 665.3026 665.3026 679.3183 679.3186 C26H46O10 C26H46O10 C26H46O10 C24H42O9 C24H42O9 C26H46O10 C28H50O10 C26H46O9 C28H50O10 C26H46O9 C26H46O9 C28H50O9 C28H50O9 563.3073 563.3071 563.3073 563.3068 563.3073 563.3082 519.2811 519.2802 519.2811 519.2794 563.3073 563.3077 591.3386 591.3385 547.3124 547.3122 591.3386 591.3380 547.3124 547.3119 547.3124 547.3127 575.3437 575.3439 575.3437 575.3435 C28H50O9 575.3437 575.3433 2.4 1.1 -0.2 1.0 0.0 0.5 -0.4 -0.9 1.6 -1.7 -3.3 0.7 -0.2 -0.3 -1.0 -1.0 0.6 0.3 -0.3 -0.7 2.28 1.64 26.8 0.365 56.4 0.385 0.00207 0.0248 0.0482 4.71 0.235 0.216 0.763 0.371 4.37 1.13 0.0103 0.000535 0.108 0.130 163 Table S2.7. Summary of annotated acylsugars detected in S. richardii trichome extracts. PH = pentose-hexose; AI = arabinose-inositol; I = inositol. Annotation method and confidence level criteria are described within the Methods section. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI and was averaged over two samples. Acylsugars are sorted by number of sugar moieties and then by elution order. Name Confidenc e level RT (min) Chemical formula m/z acc m/z exp Average acylsugar abundance (%) Δm (ppm) Acyldisaccharides HH3:16(4,4,8) medium PH3:21(4,4,13-O) medium medium PH3:16(4,4,8) high AI3:16(4,4,8) medium HH4:18(2,4,4,8) medium PH3:17(4,5,8) medium HH3:18(4,4,10) AI4:18(2,4,4,8) high PH3:20(4,4,12- OH) HH3:19(4,5,10) PH3:18(4,4,10) PH3:18(4,4,10) PH4:19(2,4,5,8) PH3:19(4,5,10) PH3:19(4,5,10) PH4:20(4,4,4,8) PH4:20(2,4,4,10) PH3:20(4,8,8) PH4:21(4,4,5,8) PH4:21(4,4,5,8) PH3:22(4,4,14- OH) PH3:20(4,4,12) PH3:22(4,4,14- OH) PH4:21(2,4,5,10) PH3:20(4,4,12) PH3:21(5,8,8) PH3:21(4,5,12) PH3:21(4,5,12) PH3:22(4,8,10) PH3:22(4,8,10) medium medium medium medium medium medium medium medium medium medium medium medium medium medium medium medium medium medium medium medium medium medium 2.39 2.48 2.61 2.66 2.87 2.89 3.09 3.24 3.46 3.47 3.49 3.61 3.70 4.02 4.10 4.18 4.55 4.57 4.78 4.85 4.85 5.05 5.07 5.20 5.27 5.28 5.84 6.08 6.48 6.71 C28H48O14 C32H54O14 C27H46O13 C27H46O13 C30H50O15 C28H48O13 C30H52O14 C29H48O14 C31H54O14 C31H54O14 C29H50O13 C29H50O13 C30H50O14 C30H52O13 C30H52O13 C31H52O14 C31H52O14 C31H54O13 C32H54O14 C32H54O14 651.3233 651.3233 679.3183 665.3390 665.3390 693.3339 693.3339 679.3582 707.3496 707.3496 C33H58O14 C31H54O13 723.3809 679.3546 723.3809 707.3496 679.3546 693.3703 693.3703 693.3703 707.3859 707.3859 C33H58O14 C32H54O14 C31H54O13 C32H56O13 C32H56O13 C32H56O13 C33H58O13 C33H58O13 164 653.3026 707.3496 653.3024 707.3477 623.2920 623.2916 695.3132 637.3077 681.3339 665.3026 695.3104 637.3091 681.3330 665.3047 -0.3 -2.7 -0.6 -4.0 2.2 -1.3 3.2 695.3496 695.3482 -2.0 651.3205 651.3205 679.3146 665.3364 665.3364 693.3358 693.3358 679.3520 707.3531 707.3531 723.3819 679.3520 723.3819 707.3477 679.3574 693.3682 693.3682 693.3735 707.3858 707.3858 -4.3 -4.3 -5.4 -3.9 -3.9 2.7 2.7 -9.1 4.9 4.9 1.4 -3.8 1.4 -2.7 4.1 -3.0 -3.0 4.6 -0.1 -0.1 0.284 0.0361 4.49 0.407 2.51 0.653 24.0 0.449 15.9 0.482 3.75 4.98 0.156 1.27 5.63 0.842 0.0192 0.0465 0.458 0.317 0.369 0.544 0.0756 0.199 0.0945 0.0213 0.791 0.0243 7.31 C33H58O13 7.62 C33H58O13 707.3859 707.3859 707.3858 707.3858 -0.1 -0.1 563.3073 563.3073 563.3073 519.2811 519.2811 563.3073 533.2967 591.3386 547.3124 591.3386 547.3124 605.3542 605.3542 547.3124 605.3542 605.3542 575.3437 575.3437 575.3437 589.3593 575.3437 589.3593 647.4012 589.3593 589.3593 647.4012 603.3750 603.3750 563.3071 563.3068 563.3082 519.2802 519.2794 563.3077 533.2962 591.3385 547.3122 591.3380 547.3119 605.3545 605.3549 547.3127 605.3543 605.3551 575.3439 575.3447 575.3435 589.3587 575.3433 589.3594 647.4017 589.3596 589.3596 647.4021 603.3762 603.3745 -0.4 -0.9 1.6 -1.7 -3.3 0.7 -0.9 -0.2 -0.3 -1.0 -1.0 0.4 1.1 0.6 0.2 1.4 0.3 1.7 -0.3 -1.0 -0.7 0.2 0.7 0.5 0.5 1.3 2.0 -0.8 0.280 0.0837 0.186 1.06 0.00899 12.0 0.193 0.00280 2.71 3.27 0.820 4.98 1.03 0.279 0.0493 0.0675 0.437 0.105 1.46 0.0232 1.29 0.255 0.0984 0.00569 0.101 0.0538 0.0173 0.217 0.0465 0.0394 medium medium Table S2.7. (cont’d) PH3:22(4,4,14) PH3:22(4,4,14) Acylhexoses I3:20(4,4,12-OH) I3:20(4,4,12-OH) I3:20(2,4,14-OH) I3:18(4,4,10) I3:18(4,4,10) I3:20(2,4,14-OH) I3:19(4,5,10) I3:22(4,4,14-OH) I3:20(4,8,8) I3:22(4,4,14-OH) I3:20(4,4,12) I3:23(4,5,14-OH) I3:23(4,5,14-OH) I3:20(4,4,12) I3:23(4,5,14-OH) I3:23(4,5,14-OH) I3:22(4,8,10) H3:22 I3:22(4,4,14) I3:23(5,8,10) I3:22(4,4,14) H3:23 I3:26(4,8,14-OH) I3:23(4,5,14) H3:23 I3:26(4,8,14-OH) H3:24 medium medium medium high medium medium medium high medium high high medium medium medium medium medium medium low high medium high low medium medium low medium low 4.16 C26H46O10 4.32 C26H46O10 4.68 C26H46O10 C24H42O9 4.70 4.88 C24H42O9 4.90 C26H46O10 C25H44O9 5.30 6.01 C28H50O10 6.23 C26H46O9 6.28 C28H50O10 C26H46O9 6.84 6.86 C29H52O10 6.98 C29H52O10 C26H46O9 7.13 7.14 C29H52O10 7.27 C29H52O10 C28H50O9 8.58 C28H50O9 8.86 C28H50O9 9.41 C29H52O9 9.49 C28H50O9 9.75 9.76 C29H52O9 10.21 C32H58O10 10.30 C29H52O9 10.46 C29H52O9 10.54 C32H58O10 11.04 C30H54O9 H3:24 low 11.11 C30H54O9 165 Table S2.8. Summary of annotated acylsugars detected in S. heteropodium trichome extracts. HH = hexose-hexose; I = inositol. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI. Acylsugars are sorted by number of sugar moieties and then by elution order. Hexose Hexose Confidence level RT (min) Chemical formula m/z acc m/z exp Δm(ppm) Average acylsugar abundance (%) 2 2 8 8 8 medium 8 medium 3.29 3.35 C30H52O14 C30H52O14 681.3339 681.3350 1.6 4.40 medium medium medium medium medium medium medium medium medium low medium medium 3.04 5.43 5.68 6.34 7.06 7.24 7.77 8.02 8.80 9.74 10.43 11.40 C21H38O8 463.2549 463.2542 C25H44O9 533.2967 533.2961 C28H50O10 591.3386 591.3421 C26H46O9 547.3124 547.3122 C29H52O10 605.3542 605.3549 C27H48O9 561.3280 561.3297 C27H48O9 561.3280 561.3284 C30H54O10 619.3699 619.3728 C28H50O9 575.3437 575.3446 C29H52O9 589.3593 589.3610 C29H52O9 589.3593 589.3615 C30H54O9 603.3750 603.3764 -1.6 -1.1 5.9 -0.3 1.2 3.1 0.7 4.8 1.5 2.8 3.8 2.3 3.83 47.3 0.0311 32.4 0.296 0.610 3.97 0.0987 2.04 0.0636 3.09 2.02 Name Acyldisaccharides HH3:18(2,8,8) HH3:18(2,8,8) Acylhexoses I2:15(5,10) I3:19(4,5,10) I3:22(5,5,12-OH) I3:20(5,5,10) I3:23(4,5,14-OH) I3:21(4,5,12) I3:21(4,5,12) I3:24(5,5,14-OH) I3:22(5,5,12) H3:23 I3:23(4,5,14) I3:24(5,5,14) 166 Table S2.9. Summary of annotated acylsugars detected in S. sejunctum trichome extracts. PH = pentose-hexose; I = inositol. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI. Acylsugars are sorted by number of sugar moieties and then by elution order. Name Confidence level RT (min) Chemical formula m/z acc m/z exp Δm(ppm) Average acylsugar abundance (%) Acyldisaccharides HH4:22(2,4,4,12-OH) medium HH4:23(2,4,5,12-OH) medium HH4:24(2,5,5,12-OH) medium HH4:24(2,4,4,14-OH) medium HH4:25(2,4,5,14-OH) medium HH4:25(2,4,5,14-OH) medium 3.61 C34H58O16 767.3707 767.3769 4.14 C35H60O16 781.3863 781.3919 4.85 C36H62O16 795.4020 795.4096 5.23 C36H62O16 795.4020 795.4080 6.01 C37H64O16 809.4176 809.4241 6.10 C37H64O16 809.4176 809.4251 Acylhexoses I2:12(4,8) H2:13 I2:14(5,9) I2:14(4,10) H2:15 I3:16(4,4,8) I3:16(4,4,8) I3:16(4,4,8) I3:17(4,5,8) I3:17(4,4,9) I3:17(4,4,9) I3:18(4,5,9) I3:18(4,4,10) H3:21:[O1] I3:19(5,5,9) H3:19 I3:19(4,5,10) I3:22(4,4,14-OH) I3:20(4,5,11) I3:22(4,4,14-OH) I3:20(5,5,10) I3:20(4,8,8) I3:20(4,8,8) I3:20(4,4,12) I3:23(4,5,14-OH) medium low medium medium low medium medium medium medium medium medium medium high low medium low medium medium medium high medium medium medium high medium 491.2493 C18H32O8 C19H34O8 C22H40O8 C20H36O8 C21H38O8 C22H38O9 C22H38O9 C22H38O9 C23H40O9 C23H40O9 C23H40O9 C24H42O9 C24H42O9 421.2079 421.2071 2.19 435.2230 435.2226 2.38 449.2392 449.2384 2.58 449.2392 449.2384 2.73 463.2543 463.2542 3.04 491.2498 3.22 491.2498 3.31 491.2498 491.2484 3.41 505.2654 505.2656 3.69 505.2654 505.2649 3.79 505.2654 505.2648 3.92 519.2811 519.2804 4.37 519.2811 519.2810 4.75 4.92 C27H48O10 577.3229 577.3254 533.2967 533.2984 5.10 5.30 533.2967 533.3000 533.2967 533.2961 5.47 5.71 C28H50O10 591.3386 591.3421 6.11 547.3124 547.3174 6.23 C28H50O10 591.3386 591.3395 547.3124 547.3122 6.34 547.3124 547.3131 6.52 6.63 547.3124 547.3119 547.3124 547.3129 6.91 7.12 C29H52O10 605.3542 605.3549 C26H46O9 C26H46O9 C26H46O9 C26H46O9 C25H44O9 C25H44O9 C25H44O9 C26H46O9 167 8.1 7.2 9.6 7.5 8.1 9.2 -1.9 -1.1 -1.7 -1.8 -0.3 -1.0 -2.9 0.3 -0.9 -1.2 -1.4 -0.1 4.3 3.3 6.2 -1.1 5.9 9.2 1.6 -0.3 1.2 -0.9 0.9 1.2 0.593 1.19 0.488 2.29 2.43 1.05 0.572 1.62 0.420 1.93 0.438 3.26 0.654 0.180 16.5 0.339 9.84 13.9 0.767 0.169 0.0507 13.8 0.187 0.0317 6.86 0.626 0.452 0.231 0.992 10.2 0.1 1.20 3.1 2.1 561.3284 561.3280 0.0689 0.388 C27H48O9 C27H48O9 C27H48O9 C27H48O9 C27H48O9 C27H48O9 561.3280 561.3297 561.3280 561.3292 561.3280 561.3280 561.3280 561.3280 Table S2.9. (cont’d) low H3:21 medium I3:21(5,8,8) medium I3:21(5,8,8) medium I3:21(4,5,12) medium I3:21(4,5,12) I3:21(4,5,12) medium I3:24(5,5,14-OH) medium medium I3:22(5,8,9) medium I3:22(5,8,9) medium I3:22(5,8,9) low H3:24:[O1] C28H50O9 medium I3:22(4,8,10) C28H50O9 medium I3:22(4,8,10) C28H50O9 medium I3:22(4,8,10) C29H52O9 medium I3:23(5,8,10) C29H52O9 medium I3:23(5,8,10) C29H52O9 medium I3:23(4,9,10) medium 10.43 C29H52O9 I3:23(4,9,10) 7.22 7.34 7.44 7.53 7.77 7.83 2.14 8.09 C30H54O10 619.3699 619.3728 0.311 575.3437 575.3449 8.29 0.291 575.3437 575.3453 8.42 0.0206 575.3437 575.3491 8.66 8.75 C30H54O10 619.3699 619.3776 12.4 0.0323 1.07 8.88 0.340 9.05 0.132 9.53 0.575 9.73 0.423 9.88 0.296 9.96 575.3437 575.3446 575.3437 575.3450 575.3437 575.3469 589.3593 589.3610 589.3593 589.3600 589.3593 589.3602 589.3593 C28H50O9 C28H50O9 C28H50O9 1.5 2.2 5.5 2.8 1.1 1.5 4.8 2.1 2.9 9.3 0.521 0.7 589.3615 3.8 0.195 I3:23(4,9,10) medium 10.51 C29H52O9 589.3593 168 Table S2.10. Summary of annotated acylsugars detected in S. melanospermum trichome extracts. I = inositol. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI. Acylsugars are sorted by number of sugar moieties and then by elution order. Name Confidence level RT (min) Chemical formula m/z acc m/z exp Δm(ppm) Average acylsugar abundance (%) medium low medium medium low medium medium medium medium medium medium high low medium medium medium low medium Acylhexoses I2:12(4,8) H2:13 I2:14(4,10) I2:14(4,10) H2:15 I3:16(4,4,8) I3:16(4,4,8) I3:17(4,5,8) I3:17(4,4,9) I3:17(4,5,8) I3:18(4,5,9) I3:18(4,4,10) H3:21:[O1] I3:19(5,5,9) I3:19(5,5,9) I3:19(4,5,10) H3:22:[O1] I3:20(4,5,11) I3:22(4,4,14-OH) high I3:20(5,5,10) I3:20(4,8,8) I3:20(4,8,8) I3:20(4,4,12) I3:23(4,5,14-OH) medium medium I3:21(5,8,8) medium I3:21(4,8,9) medium I3:21(4,8,9) medium I3:21(4,5,12) medium I3:21(4,5,12) I3:21(4,5,12) medium I3:24(5,5,14-OH) medium medium I3:22(5,8,9) medium medium medium high 2.19 2.36 2.58 2.73 3.04 3.31 3.41 3.69 3.79 3.90 4.38 4.75 4.92 5.10 5.30 5.47 5.71 6.13 6.23 6.34 6.52 6.63 6.91 7.12 7.22 7.34 7.46 7.52 7.77 7.83 8.07 8.29 421.2079 421.2071 435.2230 435.2226 449.2392 449.2384 449.2392 449.2384 463.2543 463.2542 491.2498 491.2493 491.2498 491.2484 505.2654 505.2656 505.2654 505.2649 505.2654 505.2648 519.2811 519.2804 519.2811 519.2810 577.3229 577.3254 533.2967 533.2984 533.2967 533.3000 533.2967 533.2961 591.3386 591.3421 547.3124 547.3174 591.3386 591.3395 547.3124 547.3122 547.3124 547.3131 547.3124 547.3119 547.3124 547.3129 605.3542 605.3549 561.3280 561.3297 561.3280 561.3292 561.3280 561.3280 561.3280 561.3267 561.3280 561.3284 561.3280 561.3267 619.3699 619.3728 575.3437 575.3449 C18H32O8 C19H34O8 C20H36O8 C20H36O8 C21H38O8 C22H38O9 C22H38O9 C23H40O9 C23H40O9 C23H40O9 C24H42O9 C24H42O9 C27H48O10 C25H44O9 C25H44O9 C25H44O9 C25H44O9 C26H46O9 C28H50O10 C26H46O9 C26H46O9 C26H46O9 C26H46O9 C29H52O10 C27H48O9 C27H48O9 C27H48O9 C27H48O9 C27H48O9 C27H48O9 C30H54O10 C28H50O9 169 -1.9 -1.1 -1.7 -1.8 -0.3 -1.0 -2.9 0.3 -0.9 -1.2 -1.4 -0.1 4.3 3.3 6.2 -1.1 5.9 9.2 1.6 -0.3 1.2 -0.9 0.9 1.2 3.1 2.1 0.1 -2.3 0.7 -2.3 4.8 2.1 1.19 3.67 0.980 3.82 0.850 4.95 2.74 0.282 23.0 1.25 11.7 14.5 0.0992 0.200 0.0972 14.1 0.0242 0.0651 0.952 0.882 0.827 1.04 1.02 1.42 0.124 0.555 2.75 0.528 0.251 0.339 Table S2.10. (cont’d) I3:22(5,8,9) medium I3:22(5,8,9) medium I3:22(4,8,10) medium I3:22(4,8,10) medium I3:22(4,8,10) medium I3:23(5,8,10) medium I3:23(5,8,10) medium I3:23(5,8,10) medium I3:23(4,9,10) medium I3:23(4,9,10) medium 8.42 8.64 8.90 9.05 9.53 9.73 9.89 9.99 10.41 10.49 2.9 9.3 1.5 2.2 5.5 2.8 1.1 1.5 3.8 0.595 0.0313 1.48 1.21 0.0664 0.594 0.702 0.960 0.0883 C28H50O9 575.3437 C28H50O9 575.3437 C28H50O9 575.3437 C28H50O9 575.3437 C28H50O9 575.3437 C29H52O9 589.3593 C29H52O9 589.3593 C29H52O9 589.3593 C29H52O9 575.3453 575.3491 575.3446 575.3450 575.3469 589.3610 589.3600 589.3602 589.3593 589.3615 C29H52O9 170 Table S2.11. Summary of annotated acylsugars detected in S. lasiophyllum trichome extracts. PH = pentose-hexose; AI = arabinose-inositol; I = inositol. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI. Acylsugars are sorted by number of sugar moieties and then by elution order. Confidence level low low medium high medium medium low Name Acylsugars with two sugar groups PH3:16 PH3:20:[O1] H3:20(4,4,12-O-P) AI4:18(2,4,4,8) H3:21(4,5,12-O-P) PH3:18(4,4,10) PH3:22:[O1] H3:22:1(4,4,14-O-P) medium H3:22:1(4,4,14-O-P) medium PH3:23:[O1] PH3:23:[O1] PH3:20(4,4,12) PH3:20(4,4,12) PH3:21(4,5,12) PH3:21(4,5,12) PH3:22 PH3:22(4,4,14) PH3:22(4,4,14) PH3:23 PH3:23 Acylhexoses H3:20:1 H3:19:[O1] H3:20:[O1] I3:20(4,4,12-OH) H3:18 I3:18(4,4,10) H3:21:[O1] I3:21(4,5,12-OH) H3:19 I3:19(4,5,10) I3:22(5,5,12-OH) low low low medium low high low medium low medium low low low medium medium medium medium low medium medium low low RT (min) Chemical formula m/z acc m/z exp Δm(ppm) Average acylsugar abundance (%) 2.66 2.94 3.04 3.29 3.42 3.72 3.96 4.14 4.31 4.77 4.97 5.23 5.47 6.01 6.28 7.26 7.55 7.89 8.52 8.85 3.04 3.42 4.09 4.25 4.57 4.75 4.70 4.90 5.27 5.47 5.65 C27H46O13 623.2920 623.2928 C31H54O14 695.3496 695.3484 C31H54O14 695.3496 695.3496 C29H48O14 665.3026 665.3027 C32H56O14 709.3652 709.3644 C29H50O13 651.3233 651.3225 C33H58O14 723.3809 723.3826 C33H58O14 723.3809 723.3814 C33H58O14 723.3809 723.3803 C34H60O14 737.3965 737.3961 C34H60O14 737.3965 737.3948 C31H54O13 679.3546 679.3543 C31H54O13 679.3546 679.3535 C32H56O13 693.3703 693.3704 C32H56O13 693.3703 693.3682 C33H58O13 707.3859 707.3885 C33H58O13 707.3859 707.3887 C33H58O13 707.3859 707.3896 C34H60O13 721.4010 721.4040 C34H60O13 721.4010 721.4055 C26H44O9 545.2962 545.2958 C25H44O10 549.2911 549.2910 C26H46O10 563.3073 563.3091 C26H46O10 563.3073 563.3062 519.2811 519.2818 C24H42O9 C24H42O9 519.2811 519.2789 C27H48O10 577.3229 577.3228 C27H48O10 577.3229 577.3201 533.2967 533.2962 C25H44O9 533.2967 533.2953 C25H44O9 C28H50O10 591.3386 591.3384 171 1.3 -1.7 -0.1 0.2 -1.1 -1.2 2.3 0.7 -0.8 -0.5 -2.3 -0.4 -1.6 0.2 -3.0 3.6 3.9 5.2 4.1 6.1 -0.7 -0.2 3.1 -2.0 1.4 -4.3 -0.1 -4.9 -0.9 -2.7 -0.4 0.00437 0.0804 17.5 0.0519 11.9 0.262 0.0254 0.210 1.20 0.102 0.496 0.0678 2.78 0.0502 3.10 0.00458 0.00894 0.0176 0.0104 0.0157 0.0604 0.116 0.0152 10.9 0.0123 2.42 0.0417 16.3 0.0159 3.89 0.0596 Table S2.11. (cont’d) I3:22(4,4,14-OH) I3:22(4,4,14-OH) H3:20 H3:20 I3:20(4,4,12) I3:23(4,5,14-OH) I3:23(4,5,14-OH) I3:21(4,5,12) I3:21(4,5,12) H3:22 H3:22 H3:22 H3:23 H3:23 high high low low high medium medium medium medium low low low low low 5.98 6.24 6.31 6.65 6.94 6.79 7.09 7.52 7.81 8.83 9.25 9.6 10.16 10.51 C28H50O10 C28H50O10 C26H46O9 C26H46O9 C26H46O9 C29H52O10 C29H52O10 C27H48O9 C27H48O9 C28H50O9 C28H50O9 C28H50O9 C29H52O9 591.3386 591.3386 547.3124 547.3124 547.3124 605.3542 605.3542 561.3280 561.3280 575.3437 575.3437 575.3437 589.3593 591.3380 591.3364 547.3143 547.3106 547.3115 605.3541 605.3530 561.3270 561.3273 575.3454 575.3438 575.3441 589.3612 C29H52O9 589.3593 589.3598 -1.1 -3.7 3.5 -3.3 -1.7 -0.2 -2.0 -1.8 -1.3 3.0 0.2 0.6 3.2 0.8 0.0963 2.66 0.00880 0.117 11.3 0.112 1.43 0.130 12.1 0.0155 0.0365 0.0878 0.0238 0.0882 172 Table S2.12. Summary of annotated acylsugars detected in S. prinophyllum trichome extracts. PH = pentose-hexose; AI = arabinose-inositol; I = inositol. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI and was averaged over three samples. Acylsugars are sorted by number of sugar moieties and then by elution order. Confidence level low low low low low low low low low medium medium low Name Acylsugars with two sugar groups PH3:16 PH4:18 PH4:18 H3:20(4,4,12-O-P) medium PH3:20:[O1] PH3:20:[O1] PH3:20:[O1] PH3:20:[O1] H3:21(4,5,12-O-P) medium PH3:21:[O1] PH3:21:[O1] PH3:18(4,4,10) PH3:18(4,4,10) HH3:22:[O1] H3:22(4,4,14-O-H) medium H3:22(4,4,14-O-P) medium H3:22(4,4,14-O-P) medium PH3:22:[O1] PH3:22:[O1] HH3:23:[O1] H4:24(4,4,4,12-O- P) PH3:23:[O1] H3:23(4,5,14-O-P) medium H3:23(4,5,14-O-P) medium medium PH3:20(4,4,12) PH3:20(4,4,12) medium H4:26(4,4,4,14-O- P) H4:26(4,4,4,14-O- P) medium low low low low medium medium RT (min) Chemical formula m/z acc m/z exp Δm(ppm) 2.73 3.09 3.29 3.04 2.91 3.16 3.26 3.41 3.44 3.58 3.72 3.61 3.72 3.66 3.81 4.14 4.32 4.52 4.93 4.39 4.53 4.77 4.86 4.99 5.23 5.47 C27H46O13 C29H48O14 C29H48O14 C31H54O14 C31H54O14 C31H54O14 C31H54O14 C31H54O14 C32H56O14 C32H56O14 C32H56O14 C29H50O13 C29H50O13 C34H60O15 C34H60O15 C33H58O14 C33H58O14 C33H58O14 C33H58O14 C35H62O15 C35H60O15 C34H60O14 C34H60O14 C34H60O14 C31H54O13 C31H54O13 623.2920 665.3026 665.3026 695.3496 695.3496 695.3496 695.3496 695.3496 709.3652 709.3652 709.3652 651.3233 651.3233 753.3914 753.3914 723.3809 723.3809 723.3809 723.3809 767.4065 623.2906 665.3060 665.3008 695.3479 695.3481 695.3539 695.3529 695.3473 709.3628 709.3660 709.3667 651.3224 651.3233 753.3915 753.3916 723.3812 723.3813 723.3813 723.3809 767.4082 765.3914 737.3965 765.3905 737.3969 737.3965 373.3950 679.3546 679.3546 679.3534 679.3530 -2.2 5.2 -2.8 -2.4 -2.1 6.1 4.7 -3.3 -3.3 1.1 2.1 -1.4 0.0 0.2 0.3 0.4 0.6 0.5 -0.1 2.2 -1.2 0.6 -2.0 -1.8 -2.4 Average acylsugar abundance (%) 0.545 0.000629 0.263 3.17 0.00561 0.00359 0.00365 0.0759 0.342 0.00176 0.0147 0.814 2.13 0.0227 2.79 1.03 26.6 0.0691 0.0230 0.0734 0.399 0.0224 3.71 0.0507 2.28 6.31 C37H64O15 793.4227 793.4241 1.8 0.0528 6.56 C37H64O15 793.4227 793.4224 -0.3 2.74 173 Table S2.12. (cont’d) H4:27(4,4,5,14-O- P) H4:27(4,4,5,14-O- P) PH3:22(4,4,14) PH3:22(4,4,14) PH3:23 PH3:23 Acylhexoses H4:17 H4:17 H5:19 H3:20:[O1] H4:18 I3:20(4,4,12-OH) H3:20:[O1] H3:20:[O1] I3:18(4,4,10) I3:18(4,4,10) H3:18 H4:19 I3:21(4,5,12-OH) H3:21:[O1] H5:20 H3:21:[O1] H3:21:[O1] H3:19 H3:19 H5:21 H5:21 I3:22(4,4,14-OH) I3:22(4,4,14-OH) I3:20(4,4,12) I3:20(4,4,12) H5:22 I3:23(4,5,14-OH) I3:23(4,5,14-OH) H5:22 H3:24:[O1] H3:21 H3:21 I3:22(4,4,14) I3:22(4,4,14) I3:23(4,5,14) medium medium medium medium low low low low low low low medium low low medium medium low low medium low low low low low low low low medium medium medium medium low medium medium low low low low medium medium medium 7.31 C38H66O15 807.4384 807.4380 -0.5 0.559 7.42 7.55 7.89 8.88 9.23 C38H66O15 C33H58O13 C33H58O13 C34H60O13 C34H60O13 807.4384 707.3859 707.3859 721.4010 721.4010 807.4381 707.3847 707.3848 721.4012 721.4043 3.36 3.62 3.72 4.09 4.14 4.25 4.57 4.73 4.57 4.75 4.82 4.90 4.85 5.10 5.35 5.35 5.45 5.27 5.48 6.03 6.15 5.98 6.24 6.65 6.94 6.78 6.84 7.13 7.76 8.09 7.79 8.24 9.25 9.60 10.16 519.2442 519.2442 563.2704 563.3073 533.2598 563.3073 563.3073 563.3073 519.2811 519.2811 519.2811 547.2755 577.3229 577.3229 575.2704 577.3229 577.3229 533.2962 533.2962 589.2860 589.2860 591.3386 591.3386 547.3124 547.3124 603.3017 605.3542 605.3542 617.3173 619.3694 561.3275 561.3275 575.3437 575.3437 589.3593 519.2452 519.2429 563.2710 563.3091 533.2593 563.3067 563.3103 563.3094 519.2793 519.2792 519.2800 547.2741 577.3212 577.3206 575.2746 577.3239 577.3223 533.2965 533.2955 589.2847 589.2858 591.3359 591.3388 547.3111 547.3107 603.3004 605.3510 605.3529 617.3167 619.3694 561.3265 561.3271 575.3418 575.3424 589.3596 C23H38O10 C23H38O10 C25H42O11 C26H46O10 C24H40O10 C26H46O10 C26H46O10 C26H46O10 C24H42O9 C24H42O9 C24H42O9 C25H42O10 C27H48O10 C27H48O10 C26H42O11 C27H48O10 C27H48O10 C25H44O9 C25H44O9 C27H44O11 C27H44O11 C28H50O10 C28H50O10 C26H46O9 C26H46O9 C28H46O11 C29H52O10 C29H52O10 C29H48O11 C30H54O10 C27H48O9 C27H48O9 C28H50O9 C28H50O9 C29H52O9 174 -0.4 -1.7 -1.6 0.2 4.5 1.9 -2.4 1.1 3.1 -0.9 -1.1 5.3 3.8 -3.4 -3.7 -2.1 -2.6 -3.0 -3.9 7.4 1.7 -1.0 0.6 -1.4 -2.2 -0.4 -4.6 0.3 -2.4 -3.1 -2.2 -5.4 -2.2 -1.0 0.0 -1.8 -0.7 -3.4 -2.2 0.5 0.0593 0.144 2.21 0.0991 0.00364 0.00412 0.495 0.0123 0.00276 0.669 4.23 0.00220 0.00130 0.0608 0.166 0.000654 0.104 0.292 0.00116 0.00229 0.00318 0.00678 0.00792 0.0139 0.0454 0.0146 1.10 24.8 0.0169 1.23 0.354 0.0488 4.39 0.359 0.0434 0.0522 0.0134 0.947 9.19 0.0258 Table S2.12. (cont’d) I3:23(4,5,14) I3:23(4,5,14) I3:23(4,5,14) I3:23(4,5,14) H3:24 H3:24 medium medium medium medium low low H3:24 low 10.24 10.50 10.59 10.94 11.57 11.90 12.03 C29H52O9 C29H52O9 C29H52O9 C29H52O9 C30H54O9 C30H54O9 589.3593 589.3584 -1.6 0.917 589.3593 603.3744 603.3744 589.3585 603.3768 603.3781 -1.4 4.0 6.1 0.8 0.0307 0.0141 0.00236 0.00432 C30H54O9 603.3744 603.3749 175 Table S2.13. Summary of annotated acylsugars detected in S. acerifolium fruit surface metabolite extracts. dHH = deoxyhexose-hexose; I = inositol. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI and was averaged over three samples. Acylsugars are sorted by number of sugar moieties and then by elution order. Name Confidence level RT (min) Chemical formula m/z acc m/z exp Average acylsugar abundance (%) Δm (ppm) low low low low low low low low low low low low low low Acyldisaccharides dHH3:30:[O2] dHH3:30:[O2] dHH3:30:[O2] dHH3:30:[O2] dHH3:34:[O3] dHH3:28:[O1] dHH3:31:[O2] dHH3:31:[O2] dHH3:31:[O2] dHH3:31:[O2] dHH3:29:[O1] dHH3:32:[O2] dHH3:32:[O2] dHH3:32:[O2] dHH3:30(8,10,12-OH) medium dHH3:32:[O2] dHH3:30(8,10,12-OH) medium dHH3:30(8,10,12-OH) medium dHH3:33:[O2] dHH3:33:[O2] dHH3:34:[O3] dHH3:30:[O1] dHH3:33:[O2] dHH3:33:[O2] dHH3:31:[O1] dHH3:31(9,10,12-OH) medium dHH3:31:[O1] dHH3:34(10,12- OH,12-OH) low low low low low low low medium low low 9.33 9.47 9.76 10.04 10.49 10.51 10.54 10.93 11.46 11.62 11.69 11.75 11.89 12.10 12.52 12.58 12.75 12.83 12.86 13.10 13.12 13.35 13.38 13.66 13.58 13.83 13.93 C42H76O15 C42H76O15 C42H76O15 C42H76O15 C46H84O16 C40H72O14 C43H78O15 C43H78O15 C43H78O15 C43H78O15 C41H74O14 C44H80O15 C44H80O15 C44H80O15 C42H76O14 C44H80O15 C42H76O14 C42H76O14 C45H82O15 C45H82O15 C46H84O16 C42H76O14 C45H82O15 C45H82O15 C43H78O14 C43H78O14 C43H78O14 865.5161 865.5161 865.5161 865.5161 937.5736 821.4899 879.5317 879.5317 879.5317 879.5317 835.5055 893.5474 893.5474 893.5474 849.5217 893.5474 849.5217 849.5217 907.5630 907.5630 937.5736 849.5217 907.5630 907.5630 863.5374 863.5374 863.5374 865.5191 865.5189 865.5192 865.5199 937.5772 821.4919 879.5347 879.5352 879.5345 879.5359 835.5074 893.5501 893.5503 893.5503 849.5240 893.5506 849.5236 849.5236 907.5658 907.5663 937.5783 849.5259 907.5666 907.5667 863.5402 863.5398 863.5404 3.5 3.3 3.6 4.4 3.8 2.5 3.4 4.0 3.2 4.7 2.3 3.0 3.3 3.3 2.7 3.6 2.2 2.2 3.1 3.6 5.0 4.9 4.0 4.0 3.2 2.7 3.4 0.00889 0.0104 0.00865 0.00563 0.0342 0.0754 0.0571 0.0462 0.0217 0.00233 0.205 0.787 0.0700 0.624 0.0289 0.0411 1.81 0.809 0.492 0.0112 0.00531 0.0743 0.244 0.0553 0.763 0.0824 13.94 C46H84O15 921.5792 921.5827 3.8 2.79 dHH3:34(10,12- OH,12-OH) medium 14.12 C46H84O15 921.5792 921.5827 3.8 3.64 176 Table S2.13. (cont’d) dHH3:34(10,12-OH,12- OH) dHH3:31:[O1] dHH3:32(10,10,12-OH) dHH3:33:[O2] dHH3:34(10,12-OH,12- OH) dHH3:35(10,12-OH,13- OH) dHH3:32:[O1] dHH3:32(10,10,12-OH) dHH3:32(10,10,12-OH) dHH3:35(10,12-OH,13- OH) dHH3:32(10,10,12-OH) dHH3:35:[O2] dHH3:35:[O2] dHH3:36:[O2] dHH3:33(10,11,12-OH) dHH3:36(10,12-OH,14- OH) dHH3:36(10,12-OH,14- OH) dHH3:31 dHH3:33:[O1] dHH3:33:[O1] dHH3:36:[O2] dHH3:36:[O2] Acylhexoses I2:20(8,12-OH) I2:20(8,12-OH) I2:20(8,12-OH) I2:21(9,12-OH) I2:21(9,12-OH) I2:22(10,12-OH) I2:22(10,12-OH) I2:22(10,12-OH) I3:28(8,10-OH,10-OH) I3:28(8,10-OH,10-OH) H2:24:[O1] I3:28(8,10-OH,10-OH) H3:26:[O1] H2:22 H3:29:[O3] H2:24:[O1] H3:29:[O3] medium low medium low 14.39 C46H84O15 14.44 C43H78O14 14.52 C44H80O14 14.54 C45H82O15 921.5792 863.5374 877.5530 907.5630 921.5822 863.5412 877.5554 907.5673 3.2 4.5 2.7 4.7 medium 14.56 C46H84O15 921.5792 921.5820 3.0 medium low medium medium medium medium low low low medium 14.87 C47H86O15 14.59 C44H80O14 14.76 C44H80O14 14.91 C44H80O14 15.06 C47H86O15 15.26 C44H80O14 15.32 C47H86O15 15.49 C47H86O15 15.50 C45H82O14 15.63 C45H82O14 935.5949 877.5530 877.5530 877.5530 935.5949 877.5530 935.5949 935.5949 891.5687 891.5687 935.5978 877.5552 877.5554 877.5555 935.5980 877.5553 935.5984 935.5980 891.5716 891.5715 3.1 2.5 2.7 2.8 3.3 2.7 3.7 3.3 3.2 3.1 0.902 0.0126 0.144 0.0188 0.418 1.67 0.615 2.17 0.125 1.89 0.0865 0.154 0.702 0.491 1.08 medium 15.74 C48H88O15 949.6105 949.6145 4.2 4.31 medium low low low low low medium medium medium medium medium medium medium medium medium medium low medium low low low low low 15.89 C48H88O15 16.07 C43H78O13 16.10 C45H82O14 16.18 C45H82O14 16.17 C48H88O15 16.30 C48H88O15 C26H48O9 4.68 C26H48O9 5.07 C26H48O9 5.36 C27H50O9 5.62 C27H50O9 6.39 C28H52O9 6.71 C28H52O9 7.22 C28H52O9 7.59 C34H62O11 8.80 C34H62O11 9.07 C30H56O9 9.27 C34H62O11 9.35 C32H58O10 9.83 C28H52O8 9.90 C35H64O12 9.96 10.23 C30H56O9 10.24 C35H64O12 177 949.6105 847.5419 891.5687 891.5687 949.6105 949.6105 549.3280 549.3280 549.3280 563.3437 563.3437 577.3593 577.3593 577.3593 691.4274 691.4274 605.3901 691.4274 647.4007 561.3639 705.4425 605.3901 705.4425 949.6148 847.5442 891.5722 891.5722 949.6140 949.6136 549.3287 549.3324 549.3284 563.3449 563.3435 577.3592 577.3610 577.3591 691.4288 691.4287 605.3907 691.4281 647.4013 561.3644 705.4443 605.3909 705.4437 4.5 2.7 3.9 3.9 3.7 3.3 1.3 8.0 0.7 2.2 -0.4 -0.1 2.9 -0.3 2.1 1.8 1.0 1.0 1.1 1.0 2.5 1.3 1.6 5.36 0.0769 0.0542 0.338 0.311 0.00156 0.0000280 0.00164 0.000590 0.00811 0.0353 0.000368 0.117 0.00171 0.00257 0.0128 0.00749 0.0114 0.00318 0.00203 0.0120 0.0104 Table S2.13. (cont’d) H3:29:[O3] H3:28:[O1] I3:28(8,10,10-OH) I3:28(8,8,12-OH) H3:31:[O2] H3:31:[O2] H4:33:[O3] H3:34:[O3] H3:31:[O2] I3:31(9,10-OH,12-OH) I3:31(9-OH,10,12-OH) I3:34(10-OH,12-OH,12- OH) H3:31:[O2] H3:29:[O1] H3:34:[O3] I3:32(10,10-OH,12-OH) I3:29(8,9,12-OH) I3:29(8,9,12-OH) H3:29:[O1] I3:32(10,10-OH,12-OH) I3:32(10,10-OH,12-OH) I3:32(10,10-OH,12-OH) H3:30:[O1] I3:30(9,10,10-OH) I3:30(8,10,12-OH) H3:30:[O1] H3:33:[O2] I3:33(9,12-OH,12-OH) H3:33:[O2] I3:30(9,10,10-OH) I3:33(9,10-OH,14-OH) I3:31(9,10,12-OH) I3:31(9,10,12-OH) I3:31(8,11,12-OH) H3:31:[O1] H3:31:[O1] I3:34(10,12-OH,12-OH) I3:34(10,12-OH,12-OH) I3:32(10,10,12-OH) I3:32(10,10,12-OH) I3:32(10,10,12-OH) I3:35(10,12-OH,13-OH); I3:35(9,12-OH,14-OH); I3:35(11,12-OH,12-OH) low low medium medium low low low low low medium medium medium low low low medium medium medium low medium medium medium low medium medium low low medium low medium medium medium medium medium low low medium medium medium medium medium 10.38 C35H64O12 11.85 C34H62O10 12.19 C34H62O10 12.21 C34H62O10 12.22 C37H68O11 12.29 C37H68O11 12.25 C39H70O13 12.45 C40H74O12 12.48 C37H68O11 12.53 C37H68O11 12.68 C37H68O11 12.70 C40H74O12 12.88 C37H68O11 12.88 C35H64O10 12.90 C40H74O12 13.25 C38H70O11 13.28 C35H64O10 13.40 C35H64O10 13.51 C35H64O10 13.53 C38H70O11 13.61 C38H70O11 13.81 C38H70O11 13.86 C36H66O10 14.20 C36H66O10 14.28 C36H66O10 14.40 C36H66O10 14.24 C39H72O11 14.47 C39H72O11 14.60 C39H72O11 14.52 C36H66O10 14.71 C39H72O11 14.76 C37H68O10 15.14 C37H68O10 15.20 C37H68O10 15.34 C37H68O10 15.47 C37H68O10 15.35 C40H74O11 15.57 C40H74O11 15.61 C38H70O10 15.92 C38H70O10 16.03 C38H70O10 705.4425 675.4325 675.4325 675.4325 705.4436 675.4330 675.4328 675.4328 1.6 0.7 0.4 0.4 0.0124 0.0142 1.17 733.4744 733.4750 0.8 0.0468 791.4798 791.5162 791.4830 791.5167 4.0 0.6 0.00832 0.00458 733.4744 733.4749 0.6 733.4744 733.4750 0.8 791.5162 733.4744 689.4481 791.5162 747.4900 791.5174 733.4756 689.4495 791.5176 747.4908 1.5 1.6 2.1 1.7 1.1 0.529 0.183 0.739 0.00716 0.0132 0.0346 0.121 689.4481 689.4488 1.1 1.07 689.4481 689.4493 1.7 0.0133 747.7900 747.4910 1.3 747.4900 703.4638 747.4910 703.4649 1.3 1.6 3.47 0.433 0.104 703.4638 703.4651 1.9 3.37 761.5057 761.5073 2.1 0.0356 761.5057 761.5067 1.3 2.83 703.4638 761.5057 717.4794 703.4642 761.5067 717.4808 0.6 1.3 2.0 0.235 0.436 0.0398 717.4794 717.4803 1.3 4.22 775.5213 775.5213 731.4951 731.4951 731.4951 775.5237 775.5228 731.4966 731.4967 731.4962 3.1 2.0 2.1 2.2 1.5 13.8 3.86 0.103 4.64 3.41 medium 16.17 C41H76O11 789.5370 789.5390 2.5 6.29 178 Table S2.13. (cont’d) I3:32(10,10,12-OH) I3:35(10,12-OH,13-OH); I3:35(11,12-OH,12-OH) I3:33(10,11,12-OH) H3:36:[O2] I3:33(10,11,12-OH) H3:36:[O2] I3:36(10,12-OH,14-OH) I3:33(10,11,12-OH) I3:33(10,11,12-OH) I3:36(10,12-OH,14-OH) I3:34(10,10,14-OH) I3:34(10,12,12-OH) I3:34(10,10,14-OH) I3:37(11,12-OH,14-OH); I3:37(10,13-OH,14-OH) I3:34(10,12,12-OH) H3:37:[O2] H3:35:[O1] medium 16.27 C38H70O10 731.4951 731.4960 1.2 0.127 medium medium low medium low medium medium medium medium medium medium medium medium medium low low 16.37 C41H76O11 16.37 C39H72O10 16.44 C42H78O11 16.65 C39H72O10 16.77 C42H78O11 16.90 C42H78O11 16.77 C39H72O10 17.00 C39H72O10 17.06 C42H78O11 17.08 C40H74O10 17.33 C40H74O10 17.46 C40H74O10 17.58 C43H80O11 17.71 C40H74O10 17.81 C43H80O11 18.06 C41H76O10 789.5370 745.5107 803.5526 745.5107 789.5381 745.5095 803.5539 745.5114 1.4 -1.6 1.6 0.9 0.870 0.0392 0.0906 1.80 803.5526 803.5538 1.5 6.28 745.5107 745.5107 803.5526 759.5264 759.5264 759.5264 817.5683 759.5264 817.5683 773.5415 745.5111 745.5100 803.5540 759.5285 759.5273 759.5273 817.5699 759.5327 817.5713 773.5436 0.6 -0.9 1.7 2.7 1.1 1.2 1.9 8.3 3.7 2.7 4.2 0.767 0.0247 1.12 0.0276 1.70 0.849 0.896 0.0111 0.0343 0.209 0.0120 H3:38:[O2] low 18.39 C44H82O11 831.5834 831.5869 179 Table S2.14. Summary of annotated acylsugars detected in S. atropurpureum fruit surface metabolite extracts. dHH = deoxyhexose-hexose; I = inositol. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI and was averaged over three samples. Acylsugars are sorted by number of sugar moieties and then by elution order. Name Acyldisaccharides dHH3:30:[O2] dHH3:30:[O2] dHH3:30:[O2] dHH3:30:[O2] dHH3:34:[O3] dHH3:28:[O1] dHH3:31:[O2] dHH3:31:[O2] dHH3:31:[O2] dHH3:31:[O2] dHH3:29:[O1] dHH3:32:[O2] dHH3:32:[O2] dHH3:32:[O2] dHH3:30(8,10,12-OH) dHH3:32:[O2] dHH3:30(8,10,12-OH) dHH3:30(8,10,12-OH) dHH3:33:[O2] dHH3:33:[O2] dHH3:34:[O3] dHH3:30:[O1] dHH3:33:[O2] dHH3:33:[O2] dHH3:31:[O1] dHH3:31(9,10,12-OH) dHH3:31:[O1] dHH3:34(10,12-OH,12- OH) dHH3:34(10,12-OH,12- OH) dHH3:34(10,12-OH,12- OH) Confidence level RT (min) Chemical formula m/z acc m/z exp Average acylsugar abundance (%) Δm (ppm) low low low low low low low low low low low low low low medium low medium medium low low low low low low low medium low 9.33 9.47 9.76 10.04 10.49 10.51 10.54 10.93 11.46 11.62 11.69 11.75 11.89 12.1 12.52 12.58 12.75 12.83 12.86 13.10 13.12 13.35 13.38 13.66 13.58 13.83 13.93 C42H76O15 C42H76O15 C42H76O15 C42H76O15 C46H84O16 C40H72O14 C43H78O15 C43H78O15 C43H78O15 C43H78O15 C41H74O14 C44H80O15 C44H80O15 C44H80O15 C42H76O14 C44H80O15 C42H76O14 C42H76O14 C45H82O15 C45H82O15 C46H84O16 C42H76O14 C45H82O15 C45H82O15 C43H78O14 C43H78O14 C43H78O14 865.5161 865.5161 865.5161 865.5161 937.5736 821.4899 879.5317 879.5317 879.5317 879.5317 835.5055 893.5474 893.5474 893.5474 849.5217 893.5474 849.5217 849.5217 907.5630 907.5630 937.5736 849.5217 907.5630 907.5630 863.5374 863.5374 863.5374 865.5191 865.5189 865.5192 865.5199 937.5772 821.4919 879.5347 879.5352 879.5345 879.5359 835.5074 893.5501 893.5503 893.5503 849.5240 893.5506 849.5236 849.5236 907.5658 907.5663 937.5783 849.5259 907.5666 907.5667 863.5402 863.5398 863.5404 3.5 3.3 3.6 4.4 3.8 2.5 3.4 4.0 3.2 4.7 2.3 3.0 3.3 3.3 2.7 3.6 2.2 2.2 3.1 3.6 5.0 4.9 4.0 4.0 3.2 2.7 3.4 0.00889 0.0104 0.00865 0.00563 0.0342 0.0754 0.0571 0.0462 0.0217 0.00233 0.205 0.787 0.0700 0.624 0.0289 0.0411 1.81 0.809 0.492 0.0112 0.00531 0.0743 0.244 0.0553 0.763 0.0824 medium 13.94 C46H84O15 921.5792 921.5827 3.8 2.79 medium 14.12 C46H84O15 921.5792 921.5827 3.8 3.64 medium 14.39 C46H84O15 921.5792 921.5822 3.2 0.902 180 low low medium medium low low low medium low Table S2.14. (cont’d) dHH3:31:[O1] dHH3:32(10,10,12-OH) medium dHH3:33:[O2] dHH3:34(10,12-OH,12- OH) dHH3:35(10,12-OH,13- OH) dHH3:32:[O1] dHH3:32(10,10,12-OH) medium dHH3:32(10,10,12-OH) medium dHH3:35(10,12-OH,13- medium OH) dHH3:32(10,10,12-OH) medium dHH3:35:[O2] dHH3:35:[O2] dHH3:36:[O2] dHH3:33(10,11,12-OH) medium dHH3:36(10,12-OH,14- OH) dHH3:36(10,12-OH,14- OH) dHH3:31 dHH3:33:[O1] dHH3:33:[O1] dHH3:36:[O2] dHH3:36:[O2] Acylhexoses I2:20(8,12-OH) I2:20(8,12-OH) I2:20(8,12-OH) I2:21(9,12-OH) I2:21(9,12-OH) I2:22(10,12-OH) I2:22(10,12-OH) I2:22(10,12-OH) I3:28(8,10-OH,10-OH) I3:28(8,10-OH,10-OH) H2:24:[O1] I3:28(8,10-OH,10-OH) H3:26:[O1] H2:22 H3:29:[O3] H2:24:[O1] H3:29:[O3] H3:29:[O3] H3:28:[O1] medium medium medium medium medium medium medium medium medium medium low medium low low low low low low low medium low low low low low 14.44 14.52 14.54 C43H78O14 C44H80O14 C45H82O15 863.5374 877.5530 907.5630 863.5412 877.5554 907.5673 4.5 2.7 4.7 0.0126 0.144 0.0188 14.56 C46H84O15 921.5792 921.5820 3.0 0.418 14.87 14.59 14.76 14.91 15.06 15.26 15.32 15.49 15.50 15.63 C47H86O15 C44H80O14 C44H80O14 C44H80O14 C47H86O15 C44H80O14 C47H86O15 C47H86O15 C45H82O14 C45H82O14 935.5949 877.5530 877.5530 877.5530 935.5949 877.5530 935.5949 935.5949 891.5687 891.5687 935.5978 877.5552 877.5554 877.5555 935.5980 877.5553 935.5984 935.5980 891.5716 891.5715 3.1 2.5 2.7 2.8 3.3 2.7 3.7 3.3 3.2 3.1 1.67 0.615 2.17 0.125 1.89 0.0865 0.154 0.702 0.491 1.08 15.74 C48H88O15 949.6105 949.6145 4.2 4.31 15.89 16.07 16.10 16.18 16.17 16.30 4.68 5.07 5.36 5.62 6.39 6.71 7.22 7.59 8.80 9.07 9.27 9.35 9.83 9.90 9.96 10.23 10.24 10.38 11.85 C48H88O15 C43H78O13 C45H82O14 C45H82O14 C48H88O15 C48H88O15 C26H48O9 C26H48O9 C26H48O9 C27H50O9 C27H50O9 C28H52O9 C28H52O9 C28H52O9 C34H62O11 C34H62O11 C30H56O9 C34H62O11 C32H58O10 C28H52O8 C35H64O12 C30H56O9 C35H64O12 C35H64O12 C34H62O10 181 949.6105 847.5419 891.5687 891.5687 949.6105 949.6105 549.3280 549.3280 549.3280 563.3437 563.3437 577.3593 577.3593 577.3593 691.4274 691.4274 605.3901 691.4274 647.4007 561.3639 705.4425 605.3901 705.4425 705.4425 675.4325 949.6148 847.5442 891.5722 891.5722 949.6140 949.6136 549.3287 549.3324 549.3284 563.3449 563.3435 577.3592 577.3610 577.3591 691.4288 691.4287 605.3907 691.4281 647.4013 561.3644 705.4443 605.3909 705.4437 705.4436 675.4330 4.5 2.7 3.9 3.9 3.7 3.3 1.3 8.0 0.7 2.2 -0.4 -0.1 2.9 -0.3 2.1 1.8 1.0 1.0 1.1 1.0 2.5 1.3 1.6 1.6 0.7 5.36 0.0769 0.0542 0.338 0.311 0.00240 0.000190 0.00185 0.000724 0.00690 0.0459 0.00147 0.114 0.00667 0.00908 0.0146 0.0236 0.0312 0.00698 0.00406 0.00850 0.0243 0.0265 0.0149 medium low low low medium medium low low low low low medium medium Table S2.14. (cont’d) I3:28(8,10,10-OH) I3:28(8,8,12-OH) H3:31:[O2] H3:31:[O2] H4:33:[O3] H3:34:[O3] H3:31:[O2] I3:31(9,10-OH,12-OH) I3:31(9-OH,10,12-OH) I3:34(10-OH,12-OH,12- OH) H3:31:[O2] H3:29:[O1] H3:34:[O3] I3:32(10,10-OH,12-OH) medium medium I3:29(8,9,12-OH) medium I3:29(8,9,12-OH) low H3:29:[O1] I3:32(10,10-OH,12-OH) medium I3:32(10,10-OH,12-OH) medium I3:32(10,10-OH,12-OH) medium H3:30:[O1] I3:30(9,10,10-OH) I3:30(8,10,12-OH) H3:30:[O1] H3:33:[O2] I3:33(9,12-OH,12-OH) H3:33:[O2] I3:30(9,10,10-OH) I3:33(9,10-OH,14-OH) I3:31(9,10,12-OH) I3:31(9,10,12-OH) I3:31(8,11,12-OH) H3:31:[O1] H3:31:[O1] I3:34(10,12-OH,12-OH) medium I3:34(10,12-OH,12-OH) medium medium I3:32(10,10,12-OH) medium I3:32(10,10,12-OH) I3:32(10,10,12-OH) medium I3:35(10,12-OH,13-OH); I3:35(9,12-OH,14-OH); I3:35(11,12-OH,12-OH) medium medium I3:32(10,10,12-OH) low medium medium low low medium low medium medium medium medium medium low low 12.19 12.21 12.22 12.29 12.25 12.45 12.48 12.53 12.68 12.70 12.88 12.88 12.90 13.25 13.28 13.40 13.51 13.53 13.61 13.81 13.86 14.20 14.28 14.40 14.24 14.47 14.60 14.52 14.71 14.76 15.14 15.20 15.34 15.47 15.35 15.57 15.61 15.92 16.03 C34H62O10 C34H62O10 C37H68O11 C37H68O11 C39H70O13 C40H74O12 C37H68O11 C37H68O11 C37H68O11 C40H74O12 C37H68O11 C35H64O10 C40H74O12 C38H70O11 C35H64O10 C35H64O10 C35H64O10 C38H70O11 C38H70O11 C38H70O11 C36H66O10 C36H66O10 C36H66O10 C36H66O10 C39H72O11 C39H72O11 C39H72O11 C36H66O10 C39H72O11 C37H68O10 C37H68O10 C37H68O10 C37H68O10 C37H68O10 C40H74O11 C40H74O11 C38H70O10 C38H70O10 C38H70O10 675.4325 675.4325 675.4328 675.4328 0.4 0.4 1.97 733.4744 733.4750 0.8 0.0632 791.4798 791.5162 791.4830 791.5167 4.0 0.6 0.0230 0.00353 733.4744 733.4749 0.6 0.670 733.4744 733.4750 0.8 0.220 791.5162 733.4744 689.4481 791.5162 747.4900 791.5174 733.4756 689.4495 791.5176 747.4908 1.5 1.6 2.1 1.7 1.1 0.838 0.00960 0.00802 0.0400 0.207 689.4481 689.4488 1.1 1.45 689.4481 689.4493 1.7 0.0144 747.4900 747.4910 1.3 4.45 747.4900 703.4638 747.4910 703.4649 1.3 1.6 0.544 0.0700 703.4638 703.4651 1.9 4.23 761.5057 761.5073 2.1 0.0449 761.5057 761.5067 1.3 2.33 703.4638 761.5057 717.4794 703.4642 761.5067 717.4808 0.6 1.3 2.0 0.253 0.339 0.0209 717.4794 717.4803 1.3 4.44 775.5213 775.5213 731.4951 731.4951 731.4951 775.5237 775.5228 731.4966 731.4967 731.4962 3.1 2.0 2.1 2.2 1.5 10.7 3.36 0.0746 6.14 3.42 16.17 16.27 C41H76O11 C38H70O10 789.5370 731.4951 789.5390 731.4960 2.5 1.2 3.76 0.164 182 Table S2.14. (cont’d) I3:35(10,12-OH,13-OH); I3:35(11,12-OH,12-OH) medium medium I3:33(10,11,12-OH) low H3:36:[O2] medium I3:33(10,11,12-OH) H3:36:[O2] low I3:36(10,12-OH,14-OH) medium medium I3:33(10,11,12-OH) I3:33(10,11,12-OH) medium I3:36(10,12-OH,14-OH) medium medium I3:34(10,10,14-OH) medium I3:34(10,12,12-OH) I3:34(10,10,14-OH) medium I3:37(11,12-OH,14-OH); I3:37(10,13-OH,14-OH) medium medium I3:34(10,12,12-OH) low H3:37:[O2] low H3:35:[O1] H3:38:[O2] low 16.37 16.37 16.44 16.65 16.77 16.90 16.77 17.00 17.06 17.08 17.33 17.46 17.58 17.71 17.81 18.06 18.39 C41H76O11 C39H72O10 C42H78O11 C39H72O10 C42H78O11 C42H78O11 C39H72O10 C39H72O10 C42H78O11 C40H74O10 C40H74O10 C40H74O10 C43H80O11 C40H74O10 C43H80O11 C41H76O10 789.5370 745.5107 803.5526 745.5107 789.5381 745.5095 803.5539 745.5114 1.4 -1.6 1.6 0.9 0.557 0.0156 0.0595 1.91 803.5526 803.5538 1.5 5.37 745.5107 745.5107 803.5526 759.5264 759.5264 759.5264 817.5683 759.5264 817.5683 773.5415 745.5111 745.5100 803.5540 759.5285 759.5273 759.5273 817.5699 759.5327 817.5713 773.5436 0.6 -0.9 1.7 2.7 1.1 1.2 1.9 8.3 3.7 2.7 4.2 0.539 0.0175 0.883 0.0237 1.84 0.625 0.534 0.0137 0.0143 0.155 0.00855 C44H82O11 831.5834 831.5869 183 Table S2.15. Summary of annotated acylsugars detected in S. acerifolium trichome extracts. PH = pentose-hexose; I = inositol. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI and was averaged over two samples. Acylsugars are sorted by number of sugar moieties and then by elution order. Confidence level RT (min) Chemical formula m/z acc m/z exp Δm (ppm) Name Acyldisaccharide s PH3:16 PH3:17 PH3:18 PH4:18 PH4:19(2,5,6,6) Acylhexoses H3:17 H2:18:[O1] H3:17 H2:19:[O1] H2:19:[O1] I3:18(6,6,6) I2:20(6,14-OH) H3:19 I2:21(5,14-OH) I2:21(6,15-OH) I3:20(6,6,8) I3:20(6,6,8) I2:22(6,16-OH) H3:21 low low low low medium low low low low low medium medium low medium medium medium medium medium low 2.21 2.28 2.50 2.66 2.81 3.16 3.41 3.79 4.05 4.20 4.49 4.92 5.35 5.80 5.98 6.30 6.38 7.19 7.42 Average acylsugar abundanc e (%) 0.0143 1.45 0.00475 0.0468 8.82 0.00823 0.272 0.0456 0.253 0.00233 29.2 46.7 3.29 0.156 0.246 1.62 7.76 0.106 8.2 1.4 -8.4 8.0 -0.1 2.4 4.3 4.1 5.2 2.6 -1.7 -1.6 -0.4 5.3 4.5 -0.8 7.6 C27H46O13 C28H48O13 C33H50O10 C29H48O14 C30H50O14 623.2915 623.2966 637.3071 637.3080 651.3381 651.3326 665.3021 665.3074 679.3183 679.3183 505.2649 505.2661 521.2962 521.2984 505.2649 505.2670 535.3118 535.3146 535.3118 535.3132 519.2811 519.2802 549.3280 549.3271 533.2967 533.2965 563.3437 563.3467 563.3437 563.3462 C23H40O9 C24H44O9 C23H40O9 C25H46O9 C25H46O9 C24H42O9 C26H48O9 C25H44O9 C27H50O9 C27H50O9 C26H46O9 C26H46O9 C28H52O9 577.3593 577.3588 C27H48O9 561.3275 561.3317 184 547.3124 547.3127 0.5 Table S2.16. Summary of annotated acylsugars detected in S. atropurpureum trichome extracts. PH = pentose-hexose; I = inositol. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI. Acylsugars are sorted by number of sugar moieties and then by elution order. Confidence level RT (min) Chemical formula m/z acc m/z exp Δm(ppm ) Name Acyldisaccharide s PH3:16 PH3:17 PH3:18 PH4:18 PH4:19(2,5,6,6) Acylhexoses H3:17 H2:18:[O1] H3:17 H2:19:[O1] H2:19:[O1] I3:18(6,6,6) I2:20(6,14-OH) H3:19 I2:21(5,14-OH) I2:21(6,15-OH) I3:20(6,6,8) I3:20(6,6,8) I2:22(6,16-OH) H3:21 low low low low medium low low low low low medium medium low medium medium medium medium medium low 2.21 2.28 2.50 2.66 2.81 3.16 3.41 3.79 4.05 4.20 4.49 4.92 5.35 5.80 5.98 6.30 6.38 7.19 7.42 Average acylsugar abundanc e (%) 0.0143 1.45 0.00475 0.0468 8.82 0.00823 0.272 0.0456 0.253 0.00233 29.2 46.7 3.29 0.156 0.246 1.62 7.76 0.106 8.2 1.4 -8.4 8.0 -0.1 2.4 4.3 4.1 5.2 2.6 -1.7 -1.6 -0.4 5.3 4.5 -0.8 7.6 C27H46O13 C28H48O13 C33H50O10 C29H48O14 C30H50O14 623.2915 623.2966 637.3071 637.3080 651.3381 651.3326 665.3021 665.3074 679.3183 679.3183 505.2649 505.2661 521.2962 521.2984 505.2649 505.2670 535.3118 535.3146 535.3118 535.3132 519.2811 519.2802 549.3280 549.3271 533.2967 533.2965 563.3437 563.3467 563.3437 563.3462 C23H40O9 C24H44O9 C23H40O9 C25H46O9 C25H46O9 C24H42O9 C26H48O9 C25H44O9 C27H50O9 C27H50O9 C26H46O9 C26H46O9 C28H52O9 577.3593 577.3588 C27H48O9 561.3275 561.3317 185 547.3124 547.3127 0.5 Table S2.17. Summary of annotated acylsugars detected in S. torvum trichome extracts. I = inositol. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI and was averaged over eight samples. Acylsugars are sorted by elution order. Name Confidence level RT (min) Chemical formula m/z acc m/z exp Δm(ppm) Acylhexoses low H3:17:1 medium I3:17:1(2,5:1,10) medium I2:16(2,14) medium I2:17:1(5:1,12) medium I2:16(2,14) low H3:17 medium I3:18:1(2,5:1,11) low H2:16 low H3:18:1 medium I2:16(2,14) medium I2:17(5,12) medium I2:18:1(5:1,13) medium I3:18(2,5,11) medium I2:18:1(5:1,13) medium I3:19:1(2,5:1,12) low H2:18 medium I3:18(2,4,12) low H2:18 medium I3:19:1(2,5:1,12) medium I2:19:1(5:1,14) medium I2:18(2,16) low H2:18 medium I3:19(2,5,12) low I3:19 medium I2:18(2,16) medium I2:19(5,14) medium I2:23:3(5:1,18:2) I3:25:4(2,5:1,18:3) medium medium I3:21:1(2,5:1,14) medium I3:21:1(2,5:1,14) low H3:20 medium I3:21:1(2,5:1,14) medium I3:25:3(2,5,18:3) 3.37 3.48 3.48 3.82 3.90 3.97 4.02 4.07 4.20 4.24 4.39 4.45 4.62 4.65 5.08 5.12 5.13 5.35 5.48 5.70 5.71 5.75 5.86 6.21 6.34 6.52 6.89 7.11 7.37 7.42 7.85 7.89 7.97 503.2498 503.2506 503.2498 503.2482 477.2705 477.2699 489.2705 489.2671 477.2705 477.2679 505.2649 505.2628 517.2654 517.2625 477.2705 477.2665 517.2600 517.2638 477.2705 477.2689 491.2862 491.2851 503.2862 503.2852 519.2811 519.2802 503.2862 503.2859 531.2811 531.2800 505.3013 505.3010 519.2811 519.2805 505.3013 505.3037 531.2811 531.2798 517.3018 517.3002 505.3018 505.3004 505.3018 505.2999 533.2967 533.2955 533.2967 533.2963 505.3018 505.3012 519.3175 519.3173 569.3331 569.3329 609.3280 609.3270 559.3124 559.3114 547.3124 547.3126 559.3124 559.3118 611.3437 611.3425 C23H38O9 C23H38O9 C22H40O8 C23H40O8 C22H40O8 C23H40O9 C24H40O9 C22H40O8 C24H40O9 C22H40O8 C23H42O8 C24H42O8 C24H42O9 C24H42O8 C25H42O9 C24H44O8 C24H42O9 C24H44O8 C25H42O9 C25H44O8 C24H44O8 C24H44O8 C25H44O9 C25H44O9 C24H44O8 C25H46O8 C29H48O8 C31H48O9 C27H46O9 C27H46O9 C26H46O9 C27H46O9 C31H50O9 186 1.7 -3.1 -1.3 -7.0 -5.5 -4.1 -5.6 -8.4 7.4 -3.3 -2.3 -2.0 -1.7 -0.5 -2.2 -0.6 -1.2 4.8 -2.4 -3.2 -2.7 -3.7 -2.3 -0.7 -1.1 -0.4 -0.4 -1.6 -1.7 0.4 -1.1 -1.9 Average acylsugar abundance (%) 0.00119 0.143 0.000158 0.822 0.00411 0.0400 0.154 0.00973 0.000868 0.0408 0.323 0.0603 0.0571 0.00213 3.43 0.0223 0.0377 0.000392 0.0943 7.6 0.189 0.00340 2.44 0.0213 0.0858 3.33 0.187 0.288 16.4 0.663 1.83 0.0341 Table S2.17. (cont’d) medium I2:21:1(5:1,16) medium I2:20(4,16) medium I3:21(2,5,14) I3:25:3(2,5:1,18:2) medium medium I3:21(2,5,14) medium I2:20(2,18) low I3:25:3 low H3:21 medium I2:21(5,16) low H3:22 low H5:23 medium I3:25:2(2,5,18:2) low H3:22 medium I3:23:1(2,5:1,16) medium I3:22(2,4,16) I3:23:1(2,5:1,16) medium I3:25:2(2,5:1,18:1) medium medium I3:22(2,4,16) I2:23:1(5:1,18) medium I3:25:2(2,5:1,18:1) medium medium I3:24:1(2,4,18:1) medium I3:24:1(2,5:1,17) medium I3:23(2,5,16) low H4:26 medium I3:24:1(2,5:1,17) medium I3:23(2,5,16) low H3:25:1 low H3:23 medium I3:24:1(2,5:1,17) low I4:25:1 medium I2:23(5,18) low H3:24 low H3:25:1 low H4:25 low H3:25:1 medium I3:24(2,4,18) medium I3:25:1(2,5:1,18) medium I3:24(2,4,18) low H4:25 medium I3:25:1(2,5:1,18) medium I3:25(2,5,18) I3:25(2,5,18) medium 8.20 8.27 8.38 8.64 8.77 9.01 9.03 9.20 9.23 9.41 9.53 9.61 9.76 10.06 10.23 10.52 10.52 10.56 10.77 10.89 10.92 11.04 11.11 11.27 11.37 11.44 11.54 11.67 11.79 12.00 12.00 12.05 12.12 12.18 12.29 12.40 12.60 12.81 12.83 12.98 13.56 13.78 545.3331 545.3311 C27H48O8 533.3331 533.3322 C26H48O8 561.3280 561.3269 C27H48O9 611.3437 611.3430 C31H50O9 561.3280 561.3268 C27H48O9 533.3331 533.3332 C26H48O8 611.3437 611.3447 C31H50O9 561.3280 561.3291 C27H48O9 547.3488 547.3477 C27H50O8 575.3437 575.3425 C28H50O9 C29H48O10 601.3224 601.3216 613.3593 613.3581 C31H52O9 575.3437 575.3426 C28H50O9 587.3437 587.3437 C29H50O9 575.3437 575.3427 C28H50O9 587.3437 587.3425 C29H50O9 613.3593 613.3580 C31H52O9 575.3437 575.3442 C28H50O9 573.3644 573.3639 C29H52O8 613.3593 613.3545 C31H52O9 601.3593 601.3604 C30H52O9 601.3593 601.3588 C30H52O9 C29H52O9 589.3593 589.3588 C32H56O10 645.3850 645.3848 601.3593 601.3594 C30H52O9 589.3593 589.3579 C29H52O9 615.3744 615.3765 C31H54O9 589.3593 589.3556 C29H52O9 601.3593 601.3599 C30H52O9 629.3542 629.3531 C32H56O9 575.3801 575.3794 C29H54O8 603.3744 603.3755 C30H54O9 C31H54O9 615.3744 615.3695 C31H54O10 631.3694 631.3722 615.3744 615.3746 C31H54O9 603.3750 603.3746 C30H54O9 615.3750 615.3743 C31H54O9 603.3750 603.3758 C30H54O9 C31H54O10 631.3694 631.3695 615.3750 615.3739 C31H54O9 617.3906 617.3911 C31H56O9 C31H56O9 617.3906 617.3913 187 -3.6 -1.7 -1.9 -1.1 -2.1 0.2 1.7 1.9 -2.1 -2.1 -1.4 -2.0 -1.9 0.1 -1.7 -2.1 -2.1 0.8 -0.8 -7.9 1.7 -0.9 -0.9 -0.3 0.2 -2.4 3.3 -6.2 1.0 -1.7 -1.2 1.8 -8.0 4.5 0.3 -0.7 -1.1 1.4 0.3 -1.7 0.8 1.1 3.85 0.109 12.3 1.54 0.304 0.215 0.0362 0.0119 0.653 0.0247 0.0806 0.175 0.0242 15.5 0.739 2.45 1.42 0.0256 1.37 0.0757 0.00330 0.0669 5.67 0.0191 0.215 0.311 0.190 0.0136 0.0212 0.539 0.242 0.00517 0.0282 0.00570 0.0483 0.0115 8.35 0.626 0.0244 2.56 1.74 0.0999 Table S2.18. Summary of annotated acylsugars detected in S. scabrum trichome extracts. H = hexose; G = glucose; I = inositol. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI and was averaged over six samples. Acylsugars are sorted by elution order. Name Acylhexoses I3:18:OH(4,4,10- OH) G2:13(4,9) I3:18:OH(4,4,10- OH) G2:13(4,9) I3:18:OH(4,4,10- OH) G2:13(4,9) H2:14 I3:16(4,4,8) G2:13(4,9) I3:16(4,4,8) H2:13 H4:18:[O1] I3:16(4,4,8) G2:14(4,10) H3:13 G2:14(4,10) I3:17(4,5,8) G2:14(4,10) I3:17(4,5,8) I3:17(4,4,9) G2:15(5,10) G2:15(5,10) I3:18(4,4,10) G2:15(5,10) I3:18(4,4,10) G3:16(2,4,10) I3:18(4,4,10) G3:16(2,4,10) H3:21:[O1] I3:19(4,5,10) G3:16(2,4,10) Confidence level RT (min) Chemical formula m/z acc m/z exp Average acylsugar abundance (%) Δm (ppm) medium medium medium medium medium medium low medium medium medium low low medium medium low medium medium medium medium medium medium medium medium medium medium medium medium medium low medium medium 2.86 C24H42O10 C19H34O8 2.86 535.2760 435.2236 535.2780 435.2248 2.94 C24H42O10 C19H34O8 2.94 535.2760 435.2236 535.2768 435.2235 3.02 C24H42O10 C19H34O8 3.04 C20H36O8 3.04 C22H38O9 3.06 C19H34O8 3.14 C22H38O9 3.22 3.24 C19H34O8 3.27 C25H42O11 C22H38O9 3.31 C20H36O8 3.46 C20H34O9 3.49 C20H36O8 3.61 C23H40O9 3.69 C20H36O8 3.76 C23H40O9 3.77 C23H40O9 3.94 C21H38O8 3.99 C21H38O8 4.18 C24H42O9 4.37 C21H38O8 4.38 C24H42O9 4.57 C22H38O9 4.70 C24H42O9 4.73 4.87 C22H38O9 4.90 C27H48O10 C25H44O9 5.07 C22H38O9 5.08 188 535.2760 435.2236 449.2392 491.2498 435.2236 491.2498 435.2236 563.2704 491.2498 449.2392 463.2179 449.2392 505.2654 449.2392 505.2654 505.2654 463.2549 463.2549 519.2811 463.2549 519.2811 491.2498 519.2811 491.2498 577.3229 533.2967 491.2498 535.2760 435.2233 449.2421 491.2497 435.2233 491.2497 435.2237 563.2726 491.2498 449.2388 463.2217 449.2389 505.2652 449.2391 505.2658 505.2652 463.2545 463.2544 519.2806 463.2543 519.2812 491.2510 519.2804 491.2492 577.3237 533.2967 491.2494 3.8 2.8 1.5 -0.3 0.0 -0.6 6.4 -0.1 -0.6 -0.1 0.2 4.0 -0.1 -1.0 8.1 -0.7 -0.3 -0.2 0.8 -0.5 -0.9 -1.1 -0.9 -1.2 0.1 2.5 -1.3 -1.1 1.3 -0.1 -0.8 0.0251 0.0166 0.0712 0.116 0.168 0.192 0.00312 0.150 0.0890 3.58 0.00505 0.0772 7.44 1.37 0.0219 3.20 3.11 1.14 11.5 2.95 1.52 4.32 3.33 2.54 9.13 0.0287 15.8 0.562 0.143 0.221 0.464 Table S2.18. (cont’d) H4:19 H4:19 I3:19(4,5,10) I3:19(4,5,10) H4:19 G3:17(2,5,10) G3:17(2,5,10) G3:17(2,5,10) I4:20 I4:20 I3:20(5,5,10) I3:20(5,5,10) I4:20(2,4,4,10) I3:20(5,5,10) I4:20(2,4,4,10) G2:18(8,10) H3:20 G2:18(8,10) I4:21(2,4,5,10) G2:18(8,10) I4:21(2,4,5,10) H4:22 H4:22 H4:22 H4:22 low low medium medium low medium medium medium low low medium medium medium medium medium medium low medium medium medium medium low low low low 5.10 C25H42O10 5.27 C25H42O10 C25H44O9 5.27 5.47 C25H44O9 5.48 C25H42O10 C23H40O9 5.48 C23H40O9 5.68 5.89 C23H40O9 6.01 C26H44O10 6.08 C26H44O10 C26H46O9 6.11 6.34 C26H46O9 6.34 C26H44O10 C26H46O9 6.56 6.58 C26H44O10 C24H44O8 6.67 C26H46O9 6.91 6.94 C24H44O8 7.20 C27H46O10 7.26 C24H44O8 7.46 C27H46O10 8.20 C28H48O10 8.45 C28H48O10 8.68 C28H48O10 8.72 C28H48O10 547.2760 547.2760 533.2967 533.2967 547.2760 505.2654 505.2654 505.2654 561.2916 561.2916 547.3124 547.3124 561.2916 547.3124 561.2916 505.3018 547.3124 505.3018 575.3073 505.3018 575.3073 589.3229 589.3229 589.3229 589.3229 547.2757 547.2760 533.2969 533.2967 547.2763 505.2687 505.2654 505.2651 561.2918 561.2919 547.3123 547.3120 561.2913 547.3123 561.2916 505.3030 547.3124 505.3018 575.3072 505.3023 575.3074 589.3247 589.3237 -0.5 0.0 0.4 0.0 0.5 6.6 -0.1 -0.7 0.4 0.5 -0.2 -0.7 -0.5 -0.1 0.0 2.3 0.0 0.1 -0.1 0.9 0.2 3.0 1.4 589.3274 7.7 1.97 3.27 8.48 18.0 0.270 0.0125 0.378 0.473 0.276 0.227 0.484 1.72 1.47 0.330 3.45 0.0517 0.108 0.153 1.76 0.102 5.40 0.0549 0.222 0.0189 H4:22 low 9.06 C28H48O10 589.3229 589.3277 8.1 0.00728 189 Table S2.19. Summary of annotated acylsugars detected in S. villosum trichome extracts. HH = hexose-hexose; G = glucose; I = inositol. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI and was averaged over six samples. Acylsugars are sorted by number of sugar moieties and then by elution order. Name Confidence level RT (min) Chemical formula m/z acc m/z exp Δm(ppm) low Acyldisaccharides HH4:22(2,4,4,12-OH) medium HH3:22:[O1] HH4:23(2,4,5,12-OH) medium HH4:23(2,4,5,12-OH) medium medium HH3:22(4,4,14-OH) HH4:24(2,5,5,12-OH) medium HH4:24(2,4,4,14-OH) medium HH4:24(2,4,4,14-OH) medium HH4:25(2,4,5,14-OH) medium HH4:25(2,4,5,14-OH) medium HH4:26(2,5,5,14-OH) medium 781.3863 781.3881 3.61 C34H58O16 767.3707 767.3722 3.99 C34H60O15 753.3914 753.3972 4.12 C35H60O16 4.19 C35H60O16 4.35 C34H60O15 753.3914 753.3937 4.82 C36H62O16 795.4020 795.4037 5.01 C36H62O16 795.4020 795.4045 5.21 C36H62O16 795.4020 795.4036 5.98 C37H64O16 809.4176 809.4196 6.06 C37H64O16 809.4176 809.4196 6.93 C38H66O16 823.4333 823.4355 Acylhexoses G2:14(4,10) G2:14(4,10) G2:14(4,10) G2:15(5,10) G2:15(5,10) I3:20(4,4,12-OH) G2:15(5,10) G3:16(2,4,10) G3:16(2,4,10) I3:21(4,5,12-OH) G3:16(2,4,10) G3:17(2,5,10) G3:17(2,5,10) I3:22(5,5,12-OH) G3:17(2,5,10) I3:22(4,4,14-OH) I3:22(4,4,14-OH) I3:23(4,5,14-OH) I3:23(4,5,14-OH) medium medium medium medium medium medium medium medium medium medium medium medium medium medium medium medium medium medium medium C21H38O8 C22H38O9 C22H38O9 C20H36O8 C20H36O8 C20H36O8 C21H38O8 C21H38O8 449.2392 449.2384 3.46 449.2392 449.2385 3.61 449.2392 449.2383 3.76 463.2549 463.2542 3.97 4.19 463.2549 463.2541 4.24 C26H46O10 563.3073 563.3073 463.2549 463.2542 4.37 491.2498 491.2493 4.68 4.85 491.2498 491.2490 4.90 C27H48O10 577.3229 577.3226 491.2498 491.2492 5.05 505.2654 505.2657 5.45 5.63 505.2654 505.2649 5.68 C28H50O10 591.3386 591.3388 5.86 505.2654 505.2659 5.95 C28H50O10 591.3386 591.3394 6.21 C28H50O10 591.3386 591.3387 6.79 C29H52O10 605.3542 605.3556 7.09 C29H52O10 605.3542 605.3542 C22H38O9 C23H40O9 C23H40O9 C23H40O9 190 2.0 7.7 2.3 3.1 2.2 3.1 2.0 2.5 2.4 2.7 -1.7 -1.6 -2.1 -1.6 -1.7 0.0 -1.5 -1.1 -1.6 -0.5 -1.2 0.7 -1.0 0.4 0.9 1.4 0.1 2.3 0.0 Average acylsugar abundance (%) 3.07 0.00373 3.04 0.0268 0.907 0.0509 3.25 2.07 0.900 0.690 9.06 13.4 2.70 5.10 8.85 1.91 1.61 0.484 4.68 4.62 1.13 0.0569 2.25 0.659 0.315 0.145 12.8 0.0467 14.0 Table S2.19. (cont’d) I3:24(5,5,14-OH) I3:24(4,4,16-OH) medium medium 8.04 8.70 C30H54O10 619.3699 619.3701 C30H54O10 619.3699 619.3708 0.2 1.4 2.06 0.0296 191 Table S2.20. Summary of annotated acylsugars detected in S. mammosum trichome extracts. H = hexose; I = inositol. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI and was averaged over six samples. Name Acylhexoses I3:16(4,6,6-OH) I3:16(4,6,6-OH) H3:17:[O2] I3:14(4,4,6) I3:14(4,4,6) H3:17:[O2] H3:18:[O1] I3:15(4,5,6) I3:18(6,6,6-OH) I3:18(6,6,6-OH) I3:16(4,6,6) I3:16(4,6,6) H2:18:[O2] I3:16(4,6,6) H2:18:[O2] I3:16(4,6,6) H2:18:[O2] H3:16 I3:17(5,6,6) H2:18:[O2] H3:20:[O1] I3:17(5,6,6) H3:20:[O1] H3:20:[O1] H2:18:[O2] H3:17 H2:18:[O2] H3:20:[O1] H3:20:[O1] H2:18:[O2] H3:20:[O1] I3:18(6,6,6) Confidence level RT (min) Chemical formula m/z acc m/z exp Average acylsugar abundance (%) Δm (ppm) medium medium low medium medium low low medium medium medium medium medium low medium low medium low low medium low low medium low low low low low low low low low medium 507.2447 507.2447 521.2598 507.2439 507.2439 521.2613 -1.6 -1.6 2.8 0.0311 0.159 0.00197 463.2185 463.2170 -3.2 0.912 521.2598 535.2760 477.2341 521.2613 535.2746 477.2326 2.9 -2.6 -3.2 535.2760 535.2740 -3.7 491.2498 491.2476 -4.5 537.2911 491.2498 537.2911 491.2498 537.2911 491.2498 505.2654 537.2911 563.3068 505.2654 537.2908 491.2474 537.2893 491.2472 537.2912 491.2482 505.2647 537.2920 563.3092 505.2638 -0.5 -4.9 -3.4 -5.3 0.2 -3.3 -1.5 1.7 4.3 -3.1 0.00722 0.0659 0.211 1.27 6.93 0.00314 1.42 0.0610 0.317 0.0155 0.0866 0.951 0.0182 0.00402 0.394 563.3068 563.3077 1.7 0.0312 537.2911 505.2654 537.2911 563.3068 563.3068 537.2911 563.3068 519.2811 537.2910 505.2646 537.2911 563.3071 563.3063 537.2904 563.3055 519.2794 -0.1 -1.5 -0.1 0.6 -0.8 -1.3 -2.2 -3.2 0.0171 0.0270 0.0844 0.0140 0.0508 0.313 0.112 7.78 2.28 2.36 2.45 2.46 2.53 2.59 2.66 2.71 2.87 2.96 3.08 3.13 3.13 3.22 3.29 3.33 3.42 3.44 3.56 3.56 3.59 3.66 3.66 3.72 3.66 3.77 3.82 3.85 3.96 3.99 4.07 4.09 C22H38O10 C22H38O10 C23H40O10 C20H34O9 C20H34O9 C23H40O10 C24H42O10 C21H36O9 C24H42O10 C24H42O10 C22H38O9 C22H38O9 C24H44O10 C22H38O9 C24H44O10 C22H38O9 C24H44O10 C22H38O9 C23H40O9 C24H44O10 C26H46O10 C23H40O9 C26H46O10 C26H46O10 C24H44O10 C23H40O9 C24H44O10 C26H46O10 C26H46O10 C24H44O10 C26H46O10 C24H42O9 192 low low medium medium medium low medium medium low low low low low low low low low low low low low Table S2.20. (cont’d) H1:18:[O2] H3:20:[O1] I3:18(6,6,6) I3:18(6,6,6) I3:18(6,6,6) H1:18:[O2] I3:18(6,6,6) I3:18(6,6,6) H3:19 H2:20:[O2] H3:24:[O2] H3:19 H3:24:[O2] H3:19 H3:19 H2:20:[O2] H3:24:[O2] H3:19 H2:20:[O2] H3:24:[O2] H3:19 I3:24(4,6-OH,14-OH) medium H3:19 I3:24(4,6-OH,14-OH) medium medium I3:20(6,6,8) low H2:20:[O2] medium I3:22(4,6,12-OH) medium I3:20(6,6,8) medium I3:20(6,6,8) medium I3:22(4,6,12-OH) medium I3:20(6,6,8) low H2:20:[O2] medium I3:20(6,6,8) medium I3:22(4,6,12-OH) medium I3:20(6,6,8) low H3:23:[O2] low H3:20 low H3:23:[O2] low H3:20 H3:26:[O2] low I3:26(6,6-OH,14-OH) medium H3:24:[O1] H3:24:[O1] low low low 523.3118 563.3068 519.2811 523.3114 563.3067 519.2800 -0.8 -0.1 -2.0 0.0205 0.0287 4.33 519.2811 519.2797 -2.6 2.33 523.3118 519.2811 519.2811 533.2962 565.3224 635.3648 533.2962 635.3648 523.3113 519.2804 519.2797 533.2951 565.3214 635.3685 533.2956 635.3679 -1.0 -1.3 -2.7 -2.0 -1.8 5.9 -1.1 4.9 0.720 0.943 0.134 0.0879 0.137 0.00256 0.0279 0.00194 533.2962 533.2954 -1.5 0.0745 565.3224 635.3648 533.2962 565.3224 635.3648 533.2962 635.3648 533.2962 635.3648 547.3124 565.3224 591.3386 565.3229 635.3667 533.2957 565.3225 635.3659 533.2962 635.3652 533.2973 635.3635 547.3108 565.3219 591.3375 0.9 3.1 -0.8 0.2 1.7 0.1 0.7 2.0 -2.1 -2.9 -0.9 -1.9 0.0218 0.00373 0.0537 0.0932 0.00490 0.0200 0.0180 0.00827 0.0580 1.02 0.317 0.438 547.3124 547.3109 -2.7 1.02 591.3386 547.3124 565.3224 547.3124 591.3386 547.3124 621.3122 547.3124 621.3122 547.3124 663.3961 663.3961 619.3699 619.3699 591.3369 547.3105 565.3212 547.3107 591.3382 547.3105 621.3100 547.3119 621.3065 547.3124 663.3985 663.3953 619.3707 619.3723 -2.8 -3.5 -2.1 -3.1 -0.7 -3.5 -3.6 -1.0 -9.2 0.1 3.6 -1.2 1.3 3.9 0.204 1.69 0.732 1.31 0.0119 1.03 0.0248 0.0711 0.402 0.0123 0.00455 0.0455 0.00225 0.00812 4.12 C24H46O9 4.19 C26H46O10 4.20 C24H42O9 4.35 C24H42O9 4.43 C24H42O9 4.45 C24H46O9 4.55 C24H42O9 4.73 C24H42O9 4.79 C25H44O9 4.82 C26H48O10 4.85 C30H54O11 4.93 C25H44O9 5.01 C30H54O11 5.03 C25H44O9 5.10 C25H44O9 5.05 C26H48O10 5.20 C30H54O11 5.21 C25H44O9 5.23 C26H48O10 5.28 C30H54O11 5.32 C25H44O9 5.41 C30H54O11 5.43 C25H44O9 5.55 C30H54O11 5.69 C26H46O9 5.71 C26H48O10 5.73 C28H50O10 5.78 C26H46O9 5.84 C26H46O9 5.90 C28H50O10 5.93 C26H46O9 5.93 C26H48O10 6.03 C26H46O9 6.10 C28H50O10 6.13 C26H46O9 6.31 C29H52O11 6.52 C26H46O9 6.54 C29H52O11 6.74 C26H46O9 6.74 C32H58O11 6.98 C32H58O11 7.01 C30H54O10 7.14 C30H54O10 193 Table S2.20. (cont’d) I3:26(6,6-OH,14-OH) medium 7.22 C32H58O11 I3:26(6,6-OH,14-OH) medium 7.39 C32H58O11 I3:26(6,6-OH,14-OH) medium 7.45 C32H58O11 I3:24(6,6,12-OH) medium 7.41 C30H54O10 I3:26(6,6-OH,14-OH) medium 7.62 C32H58O11 medium I3:24(6,6,12-OH) 7.67 C30H54O10 medium I3:24(4,6,14-OH) 7.70 C30H54O10 medium I3:24(6,6,12-OH) 7.77 C30H54O10 medium I3:24(4,6,14-OH) 7.94 C30H54O10 medium I3:24(4,6,14-OH) 8.00 C30H54O10 medium I3:24(4,6,14-OH) 8.25 C30H54O10 medium I3:24(4,6,14-OH) 8.45 C30H54O10 low H4:24:[O2] 8.53 C30H52O12 low H4:24:[O2] 8.68 C30H52O12 medium I3:25(5,6,14-OH) 8.91 C31H56O10 low H4:24:[O2] 9.01 C30H52O12 medium I3:25(5,6,14-OH) 9.12 C31H56O10 medium I3:25(5,6,14-OH) 9.23 C31H56O10 medium I3:25(5,6,14-OH) 9.43 C31H56O10 medium I3:26(6,6,14-OH) 9.63 C32H58O10 9.95 C32H58O10 medium I3:26(6,6,14-OH) medium 10.16 C32H58O10 I3:26(6,6,14-OH) medium 10.26 C32H58O10 I3:26(6,6,14-OH) 10.30 C30H54O9 low H3:24 medium 10.46 C32H58O10 I3:26(6,6,14-OH) low H3:24 10.49 C30H54O9 medium 10.67 C32H58O10 I3:26(6,6,14-OH) 10.79 C30H54O9 low H3:24 10.84 C33H60O10 low H3:27:[O1] low H3:27:[O1] 11.00 C33H60O10 medium 11.02 C32H58O10 I3:26(6,6,14-OH) 11.02 C30H54O9 low H3:24 11.13 C33H60O10 low H3:27:[O1] 11.22 C30H54O9 low H3:24 11.24 C32H58O10 low H3:26:[O1] 11.32 C33H60O10 low H3:27:[O1] 11.52 C33H60O10 low H3:27:[O1] 11.54 C30H54O9 low H3:24 11.70 C33H60O10 low H3:27:[O1] low H3:24 11.79 C30H54O9 medium 11.90 C34H62O10 I3:28(6,8,14-OH) 11.98 C33H60O10 low H3:27:[O1] medium 12.18 C34H62O10 I3:28(6,8,14-OH) 663.3961 663.3951 663.3961 663.3954 619.3699 663.3961 619.3699 619.3687 663.3966 619.3687 619.3699 619.3695 619.3699 619.3688 619.3699 619.3699 649.3435 649.3435 633.3855 649.3435 619.3694 619.3689 649.3463 649.3442 633.3862 649.3422 633.3855 633.3844 633.3855 647.4012 647.4012 647.4012 647.4012 603.3744 647.4012 603.3744 647.4012 603.3744 661.4163 661.4163 647.4012 603.3744 661.4163 603.3744 647.4012 661.4163 661.4163 603.3744 661.4163 603.3744 675.4325 661.4163 675.4325 633.3841 647.3994 647.4005 647.4003 647.4011 603.3759 647.4011 603.3742 647.3997 603.3730 661.4147 661.4165 647.4004 603.3742 661.4175 603.3753 647.4026 661.4163 661.4172 603.3751 661.4183 603.3791 675.4324 661.4178 675.4319 194 -1.5 -1.1 -2.0 0.7 -1.9 -0.6 -1.7 -0.9 -1.7 4.3 1.0 1.1 -2.0 -1.7 -2.2 -2.7 -1.1 -1.3 -0.2 2.4 -0.1 -0.3 -2.3 -2.3 -2.5 0.3 -1.2 -0.4 1.7 1.4 2.2 0.0 1.3 1.1 3.0 7.7 -0.1 2.3 -0.9 0.240 0.294 0.728 0.00643 0.943 0.787 2.84 8.80 1.10 0.0131 0.00273 0.0998 0.601 0.514 0.0939 0.643 7.29 4.08 14.9 0.00245 8.24 0.0595 1.36 0.199 0.00106 0.0338 0.335 0.105 0.0277 0.0311 0.0177 0.118 0.0351 0.0286 0.00867 0.00366 0.0360 0.0124 0.497 low low Table S2.20. (cont’d) H3:27:[O1] low I3:28(6,8,14-OH) medium I3:28(6,8,14-OH) medium I3:28(6,8,14-OH) medium H3:26 I3:28(6,8,14-OH) medium I3:28(6,6,16-OH) medium I3:28(6,8,14-OH) medium H3:26 I3:28(6,6,16-OH) medium I3:28(6,8,14-OH) medium H3:29:[O1] H3:26 H3:26 H3:29:[O1] H3:29:[O1] H3:26 H3:29:[O1] H3:26 H3:26 H3:30:[O1] H3:30:[O1] H3:30:[O1] low low low low low low low low low low low low H3:30:[O1] low 12.20 12.37 12.48 12.66 12.73 12.77 12.78 12.86 12.93 12.94 12.95 13.10 13.16 13.25 13.21 13.41 13.43 13.64 13.64 13.95 14.11 14.39 14.59 14.82 C33H60O10 C34H62O10 C34H62O10 C34H62O10 C32H58O9 C34H62O10 C34H62O10 C34H62O10 C32H58O9 C34H62O10 C34H62O10 C35H64O10 C32H58O9 C32H58O9 C35H64O10 C35H64O10 C32H58O9 C35H64O10 C32H58O9 C32H58O9 C36H66O10 C36H66O10 C36H66O10 661.4163 675.4325 675.4325 675.4325 631.4057 661.4161 675.4318 675.4313 675.4315 631.4097 675.4325 675.4317 675.4325 631.4057 675.4314 631.4078 675.4325 675.4319 689.4476 689.4518 631.4057 631.4074 689.4476 689.4476 631.4057 689.4476 631.4057 631.4057 703.4633 703.4633 703.4633 689.4431 689.4500 631.4091 689.4484 631.4058 631.4076 703.4660 703.4641 703.4639 C36H66O10 703.4633 703.4640 -0.2 -1.0 -1.8 -1.5 6.3 -1.1 -1.7 3.3 -0.9 6.1 2.7 -6.5 3.5 5.3 1.2 0.1 2.9 3.9 1.2 0.9 1.1 0.00133 0.622 1.45 1.69 0.00272 1.31 0.275 0.0237 0.330 0.000248 0.0283 0.000322 0.00342 0.00984 0.00345 0.00120 0.00200 0.0126 0.0772 0.174 0.113 195 Table S2.21. Summary of annotated acylsugars detected in S. abutiloides trichome extracts. HH = hexose-hexose; I = inositol. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI and was averaged over six samples. Acylsugars are sorted by number of sugar moieties and then by elution order. Name Hexose Hexose Confidence level RT (min) Chemical formula m/z acc m/z exp Δm(ppm) Acyldisaccharides HH4:22(2,4,4,12-OH) 4 4 12-OH HH3:22:[O1] HH4:23(2,4,5,12-OH) 4 5 12-OH HH4:23(2,4,5,12-OH) 4 5 12-OH HH3:22:[O1] HH4:24(2,5,5,12-OH) 5 5 12-OH HH3:23:[O1] HH4:24(2,4,4,14-OH) 4 4 14-OH HH3:23:[O1] HH4:24(2,4,4,14-OH) 4 4 14-OH 12 HH4:22(2,4,4,12) 12 HH4:22(2,4,4,12) HH4:25(2,4,5,14-OH) 4 5 14-OH HH3:24(5,5,14-OH) 5 5 14-OH HH4:25(2,4,5,14-OH) 4 5 14-OH HH4:25(2,4,5,14-OH) 4 5 14-OH HH4:25(2,4,5,14-OH) 4 5 14-OH HH4:23 HH4:26(2,5,5,14-OH) 5 5 14-OH HH4:23 HH3:22 4 4 4 4 2 2 2 2 2 2 2 2 2 2 2 2 2 medium low medium medium low medium low medium low medium medium medium medium medium medium medium medium low medium low low C34H58O16 C34H60O15 C35H60O16 C35H60O16 C34H60O15 C36H62O16 C35H62O15 C36H62O16 C35H62O15 C36H62O16 C34H58O15 C34H58O15 C37H64O16 C36H64O15 C37H64O16 C37H64O16 C37H64O16 C35H60O15 C38H66O16 C35H60O15 C34H60O14 3.55 3.99 4.12 4.18 4.35 4.85 4.99 5.01 5.08 5.23 5.35 5.75 5.75 5.82 5.84 6.03 6.10 6.59 6.67 6.73 6.86 196 Average acylsugar abundance (%) 4.43 0.0131 7.24 0.100 2.86 0.233 0.122 0.110 7.59 767.3707 767.3713 753.3909 753.3934 781.3863 781.3873 753.3909 753.3923 795.4020 795.4035 767.4065 767.4084 795.4020 795.4037 767.4065 767.4085 795.4020 795.4038 0.7 3.3 1.3 1.8 1.9 2.4 2.1 2.6 2.2 751.3782 751.3772 -1.3 0.0624 809.4176 809.4194 781.4227 781.4238 809.4176 809.4195 809.4176 809.4190 809.4176 809.4189 765.3909 765.3928 823.4333 823.4359 765.3909 765.3935 737.3960 737.3980 2.2 1.5 2.3 1.7 1.6 2.5 3.2 3.5 2.7 0.0615 0.166 0.0312 7.48 3.94 0.0558 0.00791 0.0148 0.0124 4 4 4 4 14 2 14 2 5 5 5 5 Table S2.21. (cont’d) HH4:26(2,5,5,14-OH) 5 5 14-OH 2 12 2 HH4:24(2,5,5,12) HH4:24(2,5,5,12) 12 2 HH3:23 HH4:24(2,4,4,14) HH3:23 HH4:24(2,4,4,14) HH4:25 HH3:24 HH4:25 HH4:25 HH4:25 HH3:24 HH4:26(2,5,5,14) HH3:25 HH3:25 HH4:26(2,4,4,16) HH3:26 HH4:27(2,4,5,16) HH4:28(2,5,5,16) Acylhexoses H3:20:[O1] I3:20(4,4,12-OH) H3:21:[O1] I3:21(4,5,12-OH) H3:21:[O1] I3:21(3,4,14-OH) I3:22(5,5,12-OH) I3:22(4,4,14-OH) I3:22(4,4,14-OH) 14 2 5 5 medium medium medium low medium low medium low low low low low low medium low low medium low medium medium low medium low medium low medium medium medium medium 6.98 7.58 7.67 7.76 7.89 7.92 8.22 8.87 8.88 9.00 9.20 9.33 9.55 10.32 10.51 10.66 10.91 11.62 11.89 12.88 4.09 4.20 4.73 4.93 5.20 5.47 5.71 5.96 6.24 C38H66O16 C36H62O15 C36H62O15 C35H62O14 C36H62O15 C35H62O14 C36H62O15 C37H64O15 C36H64O14 C37H64O15 C37H64O15 C37H64O15 C36H64O14 C38H66O15 C37H66O14 C37H66O14 C38H66O15 C38H68O14 C39H68O15 C40H70O15 C26H46O10 C26H46O10 C27H48O10 C27H48O10 C27H48O10 C27H48O10 C28H50O10 C28H50O10 C28H50O10 823.4333 823.4347 779.4071 779.4096 751.4116 779.4071 751.4116 779.4071 793.4222 765.4273 793.4222 793.4222 793.4222 765.4273 807.4384 779.4429 779.4429 807.4384 793.4586 821.4540 835.4697 563.3082 563.3082 577.3229 577.3229 577.3229 577.3229 591.3386 591.3386 591.3386 751.4138 779.4098 751.4151 779.4084 793.4263 765.4299 793.4275 793.4240 793.4242 765.4292 807.4399 779.4446 779.4455 807.4393 793.4610 821.4553 835.4715 563.3087 563.3070 577.3254 577.3229 577.3249 577.3232 591.3383 591.3386 591.3382 1.7 3.1 2.9 3.5 4.6 1.7 5.2 3.5 6.7 2.3 2.5 2.5 1.8 2.2 3.4 1.2 3.1 1.6 2.2 0.8 -2.2 4.4 0.0 3.5 0.4 -0.5 0.0 -0.7 197 4.10 0.0122 0.0299 0.00798 0.00580 0.392 0.00312 0.0115 0.00124 0.329 0.0986 0.0408 0.0982 0.124 0.0186 0.487 0.0592 0.838 0.166 0.00549 2.33 0.00462 5.10 0.00201 0.0295 1.68 0.398 15.2 Table S2.21. (cont’d) I3:23(4,5,14-OH) H3:20 I3:23(4,5,14-OH) I3:24(5,5,14-OH) H3:21 I3:24(5,5,14-OH) I3:24(4,4,16-OH) I3:22(4,4,14) I3:22(4,4,14) I3:22(4,4,14) H3:25:[O1] I3:23(4,5,14) I3:23(4,5,14) I3:23(4,5,14) I3:23(4,5,14) H3:26:[O1] H3:24 I3:24(5,5,14) H3:24 I3:24(4,4,16) H3:25 I3:25(4,5,16) I3:25(4,5,16) I3:26(5,5,16) medium low medium medium low medium medium medium medium medium low medium medium medium medium low low medium low medium low medium medium medium 6.87 6.96 7.17 7.85 7.90 8.14 8.83 8.97 9.28 9.63 9.83 10.19 10.30 10.55 10.63 10.82 11.32 11.62 11.96 12.29 12.86 13.08 13.18 14.01 C29H52O10 C26H46O9 C29H52O10 C30H54O10 C27H48O9 C30H54O10 C30H54O10 C28H50O9 C28H50O9 C28H50O9 C31H56O10 C29H52O9 C29H52O9 C29H52O9 C29H52O9 C32H58O10 C30H54O9 C30H54O9 C30H54O9 C30H54O9 C31H56O9 C31H56O9 C31H56O9 C32H58O9 605.3542 547.3118 605.3542 619.3699 561.3275 619.3699 619.3699 575.3437 575.3437 575.3437 633.3850 589.3593 589.3593 605.3541 547.3125 605.3538 619.3713 561.3288 619.3703 619.3700 575.3448 575.3452 575.3433 633.3855 589.3603 589.3607 589.3593 589.3590 647.4007 603.3750 603.3750 603.3750 603.3750 617.3906 647.4018 603.3750 603.3750 603.3782 603.3746 617.3928 617.3906 617.3905 -0.2 1.2 -0.7 2.3 2.3 0.7 0.1 1.9 2.6 -0.7 0.8 1.6 2.4 -0.5 1.8 0.0 0.0 5.4 -0.7 3.6 -0.2 631.4063 631.4061 -0.3 0.183 0.0286 20.8 0.00615 0.0183 7.98 0.182 0.00372 0.00499 0.369 0.297 0.00181 0.00319 0.789 0.0446 0.000515 0.201 0.000502 0.832 0.000501 1.73 0.422 198 Table S2.22. Summary of annotated acylsugars detected in S. laciniatum trichome extracts. H = hexose; G = glucose; I = inositol. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI and was averaged over two samples. Acylsugars are sorted by elution order. Confidence level Name Acylhexoses medium I2:12(6,6) I2:12(6,6) medium G2:12(6,6) medium G2:12(6,6) medium medium I2:14(6,8) medium I2:14(6,8) G2:14(6,8) medium G2:14(6,8) medium G2:14(6,8) medium medium I2:16(8,8) medium I2:16(8,8) I2:16(8,8) medium I3:18(6,6,6) medium I3:18(6,6,6) medium I3:18(6,6,6) medium G2:16(8,8) medium G2:16(8,8) medium I3:18(6,6,6) medium G2:16(8,8) medium I3:18(6,6,6) medium G3:18(6,6,6) medium G3:18(6,6,6) medium I3:20(6,6,8) medium G3:18(6,6,6) medium I3:20(6,6,8) medium H3:22:OH G3:18(6,6,6) medium I3:20(6,6,8) medium I3:20(6,6,8) medium I3:20(6,6,8) medium I3:20(6,6,8) medium I3:20(6,6,8) medium low RT (min) Chemical formula m/z acc m/z exp Δm(ppm) Average acylsugar abundance (%) 2.15 2.28 2.56 2.63 2.81 2.87 3.33 3.42 3.54 3.70 3.82 3.96 4.20 4.32 4.49 4.53 4.70 4.85 4.87 4.93 5.65 5.82 5.95 6.11 6.18 6.24 6.28 6.30 6.39 6.56 6.67 6.81 421.2079 421.2066 C18H32O8 421.2079 421.2055 C18H32O8 421.2079 421.2061 C18H32O8 421.2079 421.2056 C18H32O8 449.2392 449.2364 C20H36O8 449.2392 449.2371 C20H36O8 449.2392 449.2371 C20H36O8 449.2392 449.2372 C20H36O8 449.2392 449.2377 C20H36O8 477.2705 477.2695 C22H40O8 477.2705 477.2686 C22H40O8 477.2705 477.2693 C22H40O8 519.2811 519.2806 C24H42O9 519.2811 519.2797 C24H42O9 519.2811 519.2793 C24H42O9 477.2705 477.2731 C22H40O8 477.2705 477.2711 C22H40O8 519.2811 519.2825 C24H42O9 477.2705 477.2730 C22H40O8 519.2811 519.2804 C24H42O9 519.2811 519.2816 C24H42O9 519.2811 519.2794 C24H42O9 547.3124 547.3114 C26H46O9 519.2811 519.2817 C24H42O9 C26H46O9 547.3124 547.3120 C28H50O10 591.3386 591.3395 519.2811 519.2798 C24H42O9 547.3124 547.3120 C26H46O9 547.3124 547.3109 C26H46O9 547.3124 547.3129 C26H46O9 547.3124 547.3144 C26H46O9 547.3124 547.3146 C26H46O9 199 -3.1 -5.6 -4.2 -5.5 -6.2 -4.6 -4.6 -4.4 -3.3 -2.0 -4.1 -2.5 -1.0 -2.6 -3.4 5.5 1.3 2.8 5.2 -1.3 0.9 -3.2 -1.9 1.2 -0.8 1.5 -2.6 -0.8 -2.7 0.9 3.7 4.1 0.275 3.48 3.33 1.07 6.41 10.3 6.86 8.35 0.471 0.423 1.96 1.26 0.531 8.23 20.5 0.0945 0.505 0.327 0.327 0.226 0.519 1.96 0.498 0.951 1.23 NA 5.52 0.751 1.05 0.439 0.167 0.315 Table S2.22. (cont’d) G3:20(6,6,8) medium G3:20(6,6,8) medium I3:22(6,8,8) medium G3:20(6,6,8) medium I3:22(6,8,8) medium G3:20(6,6,8) medium I3:22(6,8,8) medium G3:20(6,6,8) medium I3:22(6,8,8) medium 7.77 7.97 8.04 8.07 8.20 8.29 8.38 8.52 8.57 C26H46O9 547.3124 547.3124 C26H46O9 547.3124 547.3121 C28H50O9 575.3437 575.3485 C26H46O9 547.3124 547.3134 C28H50O9 575.3437 575.3430 C26H46O9 547.3124 547.3107 C28H50O9 575.3437 575.3426 C26H46O9 547.3124 547.3109 C28H50O9 575.3437 575.3425 -0.1 -0.5 8.4 1.8 -1.2 -3.1 -1.9 -2.7 -2.0 0.640 1.24 0.0682 0.301 0.574 1.96 1.30 3.70 1.86 200 Table S2.23. Summary of annotated acylsugars detected in S. dulcamara trichome extracts. I = inositol. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI and was averaged over six samples. Acylsugars are sorted by number of sugar moieties. Confidenc e level RT (min) Chemical formula m/z acc m/z exp Δm(ppm ) Average acylsugar abundanc e (%) medium 4.31 C33H58O1 4 723.380 9 723.377 7 -4.5 32.5 Name Glycohydroxyacylhexoses H3:22(4,4,14-O-P) Acylhexoses I3:22(4,4,14-OH) medium 6.24 C28H50O1 0 591.338 6 591.337 2 -2.4 67.5 201 Table S2.24. Summary of annotated acylsugars detected in S. capsicoides trichome extracts. I = inositol. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI and was averaged over four samples. Acylsugars are sorted by number of sugar moieties and then by elution order. Name Confidence level RT (min) Chemical formula m/z acc m/z exp Δm(ppm) low low low low low medium low medium low low low low low low low low low low low Acylhexoses H3:16:[O1] H3:18:[O1] H3:18:[O1] H3:19:[O1] H3:19:[O1] I2:20(8,12-OH) H3:20:[O1] I2:20(8,12-OH) H3:20:[O1] H3:20:[O1] H2:21:[O1] H3:25:[O1] H3:25:[O1] H3:22 H3:22 H3:22 H3:22 H3:26:[O1] H3:26:[O1] I3:26(6,8,12-OH) medium I3:26(6,8,12-OH) medium H3:24 I3:26(6,8,12-OH) medium medium I3:24(8,8,8) low H3:26:[O1] medium I3:24(8,8,8) medium I3:24(8,8,8) medium I3:24(8,8,8) low H3:28:[O1] low H3:28:[O1] low H3:28:[O1] low H3:28:[O1] low 507.2447 535.2760 535.2760 549.2911 549.2911 549.3280 563.3073 549.3280 563.3073 563.3073 563.3431 633.3492 633.3492 575.3437 575.3437 575.3437 575.3437 647.4012 647.4012 647.4012 647.4012 603.3750 647.4012 603.3750 647.4012 603.3750 603.3750 603.3750 661.4163 661.4163 661.4163 675.4325 507.2429 535.2745 535.2753 549.2964 549.2956 549.3275 563.3076 549.3269 563.3070 563.3062 563.3449 633.3499 633.3480 575.3450 575.3440 575.3430 575.3440 647.4023 647.4009 647.3995 647.4001 603.3760 647.4003 603.3744 647.4024 603.3745 603.3746 603.3746 661.4176 661.4166 661.4169 675.4342 2.81 3.89 4.05 4.64 4.72 5.21 5.23 5.40 5.78 6.06 6.12 6.65 6.93 8.17 8.40 8.60 8.75 9.84 9.93 10.08 10.30 10.30 10.44 10.54 10.67 10.74 10.82 11.02 11.04 11.22 11.32 11.44 C22H38O10 C24H42O10 C24H42O10 C25H44O10 C25H44O10 C26H48O9 C26H46O10 C26H48O9 C26H46O10 C26H46O10 C27H50O9 C30H52O11 C30H52O11 C28H50O9 C28H50O9 C28H50O9 C28H50O9 C32H58O10 C32H58O10 C32H58O10 C32H58O10 C30H54O9 C32H58O10 C30H54O9 C32H58O10 C30H54O9 C30H54O9 C30H54O9 C33H60O10 C33H60O10 C33H60O10 C34H62O10 202 -3.6 -2.8 -1.4 9.6 8.3 -0.9 0.5 -2.0 -0.5 -2.0 3.1 1.1 -1.9 2.3 0.5 -1.3 0.6 1.8 -0.5 -2.6 -1.7 1.6 -1.5 -1.0 1.9 -0.8 -0.7 -0.7 2.0 0.5 0.9 2.6 Average acylsugar abundance (%) 0.945 0.228 10.4 0.00628 0.00459 0.0181 0.00358 0.170 0.00217 0.891 0.00696 0.0120 1.21 0.00327 0.0386 0.0511 0.0192 0.0118 0.0690 0.727 1.68 0.00824 4.26 0.164 0.0185 0.168 0.383 0.950 0.0378 0.153 0.154 0.0119 low low low low Table S2.24. (cont’d) low H3:28:[O1] low H3:28:[O1] low H3:28:[O1] H3:28:[O1] low I3:28(8,8,12-OH) medium I3:28(8,8,12-OH) medium I3:28(8,8,12-OH) medium H3:28:[O1] H3:28:[O1] H3:29:[O1] I3:29(8,9,12-OH) medium I3:29(8,9,12-OH) medium I3:29(8,9,12-OH) medium I3:29(8,9,12-OH) medium H3:29:[O1] I3:30(8,10,12- OH) medium I3:30(8,8,14-OH) medium I4:34(6,8,8,12- OH) I3:30(8,10,12- OH) I3:30(8,10,12- OH) medium I3:30(8,8,14-OH) medium I3:30(8,8,14-OH) medium I4:34(6,8,8,12- OH) I4:34(6,8,8,12- OH) I3:30(8,10,12- OH) medium I3:30(8,8,14-OH) medium H3:30:[O1] H3:32:[O1] I3:32(8,12,12- OH) I3:32(8,12,12- OH) I3:32(8,12,12- OH) I3:32(8,12,12- OH) low low medium medium medium medium medium medium medium medium H3:32:[O1] low 11.50 11.65 11.72 11.89 12.12 12.38 12.58 12.78 12.93 12.98 13.14 13.23 13.34 13.40 13.71 14.19 14.24 C33H60O10 C34H62O10 C33H60O10 C34H62O10 C34H62O10 C34H62O10 C34H62O10 C34H62O10 C34H62O10 C35H64O10 C35H64O10 C35H64O10 C35H64O10 C35H64O10 C35H64O10 661.4163 675.4325 661.4163 675.4325 675.4325 675.4325 675.4325 675.4325 675.4325 689.4481 661.4176 675.4327 661.4209 675.4325 675.4319 675.4321 675.4316 675.4321 675.4318 689.4486 689.4481 ###### 689.4481 ###### 689.4481 689.4500 C36H66O10 C36H66O10 703.4638 703.4638 703.4631 703.4631 14.31 C40H72O11 773.5057 773.5054 2.0 0.3 6.9 0.0 -0.9 -0.6 -1.4 -0.5 -1.1 0.7 4.0 4.0 2.7 -1.0 -1.0 -0.4 14.42 C36H66O10 703.4638 703.4631 -0.9 0.0851 0.0240 0.00834 0.160 5.56 22.5 26.9 0.173 0.390 0.0478 0.964 2.61 0.0743 0.189 1.03 0.327 2.40 14.50 14.50 14.57 C36H66O10 C36H66O10 C36H66O10 703.4638 ###### 0.3 2.68 14.51 C40H72O11 773.5057 773.5064 14.65 C40H72O11 773.5057 773.5067 14.65 14.75 15.04 15.92 C36H66O10 C36H66O10 C36H66O10 C38H70O10 703.4638 ###### 703.4638 731.4951 703.4645 731.4949 16.07 C38H70O10 731.4951 731.4948 16.22 C38H70O10 731.4951 731.4947 16.30 C38H70O10 731.4951 731.4947 16.47 16.72 C38H70O10 731.4951 731.4949 C38H70O10 731.4951 731.4967 0.9 1.3 1.0 1.0 -0.3 -0.4 -0.5 -0.5 -0.3 2.2 1.32 0.763 3.46 0.0685 0.221 0.494 0.777 1.54 2.34 0.0316 203 Table S2.25. Summary of annotated acylsugars detected in S. americanum trichome extracts. PH = pentose-hexose. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI and was averaged over three samples. Acylsugars are sorted by number of sugar moieties and then by elution order. Name Acyldisaccharides PH5:18(2,2,4,4,6) PH5:19(2,2,4,5,6) PH5:20(2,2,5,5,6) Acylhexoses H2:13 H2:13 G2:14(5,9) G2:14(5,9) G2:14(4,10) G2:14(4,10) G3:15(2,5,8) H2:15 G3:15(2,5,8) G2:15(5,10) G2:15(5,10) G3:16(2,4,10) G3:16(2,4,10) G3:17(2,5,10) G3:17(2,5,10) Hexose Pentose Confidence level RT (min) Chemical formula m/z acc m/z exp Δm(ppm) 2 4 6 2 5 6 2 5 6 2 2 2 4 medium 4 medium 5 medium low low medium medium medium medium medium low medium medium medium medium medium medium medium 2.48 2.73 2.99 2.93 3.06 3.33 3.48 3.61 3.76 3.77 3.99 4.02 4.18 4.39 4.77 5.07 5.55 5.91 C29H46O15 679.2819 679.2800 C30H48O15 693.2975 693.2964 C31H50O15 707.3132 707.3127 C19H34O8 435.2230 435.2220 C19H34O8 435.2230 435.2227 C20H36O8 449.2392 449.2397 C20H36O8 449.2392 449.2417 C20H36O8 449.2392 449.2384 C20H36O8 449.2392 449.2392 C21H36O9 477.2341 477.2333 C21H38O8 463.2549 463.2572 C21H36O9 477.2341 477.2327 C21H38O8 463.2549 463.2534 C21H38O8 463.2549 463.2528 C22H38O9 491.2498 491.2482 C22H38O9 491.2498 491.2485 C23H40O9 505.2654 505.2636 C23H40O9 505.2654 505.2636 -2.8 -1.6 -0.8 -2.3 -0.8 1.2 5.7 -1.8 0.1 -1.6 5.0 -3.0 -3.2 -4.4 -3.2 -2.6 -3.6 -3.6 204 Average acylsugar abundance (%) 8.58 41.6 6.31 0.543 0.439 0.00860 0.0187 0.152 0.231 2.17 0.0339 6.22 5.06 4.79 0.509 1.29 7.09 15.0 Table S2.26. Summary of annotated acylsugars detected in S. sisymbriifolium trichome extracts. I = inositol. Annotation method and confidence level criteria are described within the Methods. RT = retention time; m/z acc = theoretical monoisotopic formate adduct mass; m/z exp = experimental formate adduct mass; Δm (ppm) = mass measurement error in parts per million. Acylsugar abundance was reported by Progenesis QI and was averaged over four samples. Acylsugars are sorted by number of sugar moieties and then by elution order. Name Acylsugars with two sugar groups H22:3(4,4,14-O-h) H22:3(4,4,14-O-p) H22:3(4,4,14-O-p) H23:3(4,5,14-O-h) H23:3(4,5,14-O-p) H24:3(4,4,16-O-p) PH23:3[O1] PH23:3[O1] H24:3(4,4,16-O-p) H24:3(4,4,16-O-p) H24:3(4,4,16-O-p) H23:3(4,5,14-O-p) H24:3(5,5,14-O-p) H24:3(4,4,16-O-p) H24:3(5,5,14-O-p) H24:3(4,5,15-O-p) H24:3(4,4,16-O-p) H24:3(4,4,16-O-p) H25:3(5,5,15-O-p) H25:3(4,5,16-O-p) Hexose Ring 2 Confidence level RT (min) Chemical formula m/z acc m/z exp Average acylsugar abundance (%) Δm (ppm) 4 4 4 4 4 4 4 4 4 4 5 4 5 4 4 4 5 4 4 4 4 5 5 4 4 4 4 5 5 4 5 5 4 4 5 5 14-OH 14-OH 14-OH 14-OH 14-OH 16-OH 16-OH 16-OH 16-OH 14-OH 14-OH 16-OH 14-OH 15-OH 16-OH 16-OH 15-OH 16-OH 3.82 4.32 4.53 5.01 5.23 5.40 5.40 5.48 5.51 5.60 5.62 5.75 5.80 6.01 6.03 6.05 6.34 6.65 6.94 7.26 C34H60O15 C33H58O14 C33H58O14 C34H60O14 C34H60O14 C35H62O14 C34H60O14 C34H60O14 C35H62O14 C35H62O14 C36H64O15 C34H60O14 C35H62O14 C35H62O14 C35H62O14 C35H62O14 C35H62O14 C35H62O14 C36H64O14 C36H64O14 753.3914 753.3945 723.3809 723.3822 723.3809 723.3813 737.3965 737.3977 737.3965 737.3970 751.4122 751.4135 737.3965 737.3989 737.3965 737.3989 751.4122 751.4137 751.4122 751.4136 781.4227 781.4244 737.3965 737.3977 751.4122 751.4141 4.1 1.8 0.5 1.7 0.7 1.7 3.3 3.2 2.0 1.9 2.2 1.6 2.5 0.00568 0.0295 0.272 0.0440 0.367 0.000185 0.00241 0.00774 0.00176 0.00600 0.133 0.0733 0.0206 751.4122 751.4133 1.4 0.172 751.4122 751.4130 751.4122 751.4129 765.4278 765.4300 765.4278 765.4293 1.1 1.0 2.9 2.0 1.39 5.47 0.0439 1.97 medium medium medium medium medium medium low low medium medium medium medium medium medium medium medium medium medium medium medium 205 Table S2.26. (cont’d) H4:27(4,4,5,14-O-p) H25:3(4,5,16-O-p) H25:3(4,5,16-O-p) H4:27(4,4,5,14-O-p) H4:27(4,4,5,14-O-p) PH4:26:[O1] PH4:26:[O1] H4:28(4,5,5,14-O-p) H3:26(5,5,16-O-p) H4:26(4,4,4,14-O-p) H3:26(5,5,16-O-p) H3:26(5,5,16-O-p) H4:27(4,4,5,14-O-p) H4:26(4,4,4,14-O-p) H4:27(4,4,5,14-O-p) H3:26(5,5,16-O-p) H4:28(4,5,5,14-O-p) PH4:29[O1] H4:27(4,4,5,14-O-p) H3:26(4,4,18-O-p) H4:27(4,4,5,14-O-p) H4:29(4,4,5,16-O-p) H3:27(5,5,17-O-p) H4:27(4,4,5,14-O-p) PH4:27[O1] H4:29(4,4,5,16-O-p) PH4:27[O1] PH3:27:[O1] PH4:27:[O1] H4:29(4,4,5,16-O-p) H4:28(4,5,5,14-O-p) 4 4 4 4 4 4 5 4 5 5 4 4 5 5 4 5 4 4 4 4 4 5 4 5 5 4 4 5 5 4 5 5 4 4 5 5 4 5 5 4 4 5 5 5 14-OH 16-OH 16-OH 14-OH 14-OH 14-OH 16-OH 14-OH 16-OH 16-OH 14-OH 14-OH 16-OH 14-OH 14-OH 18-OH 14-OH 16-OH 14-OH 16-OH 16-OH 14-OH 5 5 5 5 4 5 5 4 5 4 5 5 4 4 5 medium medium medium medium medium low low medium medium medium medium medium medium medium medium medium medium low medium medium medium medium medium medium low medium low low low medium medium 206 7.34 7.45 7.57 7.50 7.64 8.07 8.18 8.20 8.24 8.35 8.45 8.53 8.57 8.64 8.83 8.90 9.07 9.10 9.16 9.23 9.35 9.45 9.53 9.63 9.75 9.76 9.84 9.91 9.95 9.96 10.11 C38H66O15 C36H64O14 C36H64O14 C38H66O15 C38H66O15 C37H64O15 C37H64O15 C39H68O15 C37H66O14 C37H64O15 C37H66O14 C37H66O14 C38H66O15 C37H64O15 C38H66O15 C37H66O14 C39H68O15 C40H70O15 C38H66O15 C37H66O14 C38H66O15 C40H70O15 C38H68O14 C38H66O15 C38H66O15 C40H70O15 C38H66O15 C38H68O14 C38H66O15 C40H70O15 C39H68O15 779.4451 765.4288 807.4384 807.4408 765.4278 765.4278 807.4384 807.4411 807.4384 807.4428 793.4227 793.4260 793.4227 793.4264 821.4540 821.4557 779.4435 779.4446 793.4227 793.4244 779.4435 779.4435 807.4384 807.4421 793.4227 793.4244 807.4384 807.4416 779.4435 779.4458 821.4540 821.4560 835.4697 835.4722 807.4384 807.4406 779.4435 779.4451 807.4384 807.4399 835.4697 835.4724 793.4591 793.4604 807.4384 807.4397 807.4384 807.4409 835.4697 835.4718 807.4384 807.4411 793.4591 793.4613 807.4384 807.4411 835.4697 835.4718 821.4540 821.4563 3.0 1.3 3.3 5.5 4.2 4.7 2.1 1.4 2.1 2.0 4.5 2.1 4.0 3.0 2.4 3.0 2.7 2.1 1.8 3.3 1.6 1.6 3.1 2.5 3.4 2.8 3.3 2.5 2.8 0.0241 7.73 0.00673 0.000622 0.222 0.0169 0.0124 0.782 0.475 4.02 0.0198 0.143 0.00803 0.0308 0.737 0.146 1.87 0.205 1.18 0.0353 0.106 0.133 0.0203 2.88 0.00800 0.0316 0.0143 1.02 2.86 Table S2.26. (cont’d) H3:27(4,5,18-O-p) H4:28(4,5,5,14-O-p) PH4:30:[O1] PH4:28:[O1] H4:30(4,5,5,16-O-p) H4:30(4,5,5,16-O-p) PH4:28:[O1] H4:30(4,5,5,16-O-p) H4:28(4,5,5,14-O-p) H4:29(5,5,5,14-O-p) H4:29(5,5,5,14-O-p) H4:29(4,4,5,16-O-p) H4:31(5,5,5,16-O-p) PH4:31:[O1] H4:29(4,4,5,16-O-p) PH4:31:[O1] H4:29(4,4,5,16-O-p) PH4:30:[O1] PH4:31:[O1] H4:30(4,5,5,16-O-p) PH4:31:[O1] H4:30(4,5,5,16-O-p) H4:30(4,5,5,16-O-p) H4:31(5,5,5,16-O-p) H4:31(5,5,5,16-O-p) H4:31(5,5,5,16-O-p) PH4:31:[O1] H4:31(4,5,5,17-O-p) Acylhexoses I3:22(4,4,14-OH) 5 5 4 4 4 5 5 5 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 4 5 4 5 5 5 5 5 5 18-OH 14-OH 16-OH 16-OH 16-OH 14-OH 14-OH 14-OH 16-OH 16-OH 16-OH 16-OH 16-OH 16-OH 16-OH 16-OH 16-OH 5 5 5 5 5 5 5 5 5 5 4 5 5 5 5 5 medium medium low low medium medium low medium medium medium medium medium medium low medium low medium low low medium low medium medium medium medium medium low medium 10.23 10.32 10.32 10.63 10.63 10.72 10.76 10.89 10.94 11.06 11.26 11.37 11.49 11.56 11.69 11.77 11.89 11.96 12.12 12.25 12.35 12.53 12.75 13.08 13.38 13.56 13.93 14.11 C38H68O14 C39H68O15 C41H72O15 C39H68O15 C41H72O15 C41H72O15 C39H68O15 C41H72O15 C39H68O15 C40H70O15 C40H70O15 C40H70O15 C42H74O15 C42H74O15 C40H70O15 C42H74O15 C40H70O15 C41H72O15 C42H74O15 C41H72O15 C42H74O15 C41H72O15 C41H72O15 C42H74O15 C42H74O15 C42H74O15 C42H74O15 C42H74O15 793.4591 793.4611 821.4540 821.4565 849.4853 849.4886 821.4540 821.4569 849.4853 849.4878 849.4853 849.4880 821.4540 821.4564 849.4853 849.4876 821.4540 821.4561 835.4697 835.4716 835.4697 835.4711 835.4697 835.4711 2.5 3.0 3.8 3.5 3.0 3.1 2.9 2.7 2.6 2.2 1.7 1.7 0.389 1.16 0.0656 0.136 2.47 1.12 0.185 0.452 3.46 1.64 0.533 0.375 863.5010 863.5027 2.0 2.04 835.4697 835.4718 863.5010 863.5040 835.4697 835.4723 849.4853 849.4883 863.5010 863.5022 849.4853 849.4874 863.5010 863.5015 849.4853 849.4872 849.4853 849.4878 863.5010 863.5036 863.5010 863.5038 863.5010 863.5034 863.5010 863.5039 863.5010 863.5040 2.5 3.5 3.1 3.5 1.4 2.5 0.6 2.2 2.9 3.1 3.2 2.7 3.4 3.4 8.99 0.0933 7.66 0.165 0.0229 0.543 0.00472 11.8 6.60 0.139 6.18 2.76 0.272 0.150 0.0334 high 6.24 C28H50O10 591.3382 591.3387 0.9 207 Table S2.26. (cont’d) I3:23(4,5,14-OH) I3:24(4,4,16-OH) I3:24(5,5,14-OH) I3:25:[O1] I3:25(4,5,16-OH) I3:24(4,4,16-OH) I3:24(4,4,16-OH) I3:25(4,5,16-OH) I3:25:[O1] I3:25(4,5,16-OH) I3:25(4,5,16-OH) I3:26(5,5,16-OH) I3:26(5,5,16-OH) I3:26(5,5,16-OH) medium medium medium low medium medium medium medium low medium medium medium medium medium 7.14 7.54 8.12 8.22 8.33 8.45 8.79 9.12 9.25 9.43 9.78 10.43 10.78 11.44 C29H52O10 C30H54O10 C30H54O10 C31H56O10 C31H56O10 C30H54O10 C30H54O10 C31H56O10 C31H56O10 C31H56O10 C31H56O10 C32H58O10 C32H58O10 605.3542 605.3546 619.3699 619.3713 619.3699 619.3701 633.3855 633.3860 633.3855 633.3861 619.3699 619.3710 619.3699 619.3696 633.3855 633.3872 633.3855 633.3870 633.3855 633.3863 633.3855 633.3855 647.4012 647.4029 647.4012 647.4016 C32H58O10 647.4012 647.4018 0.7 2.2 0.4 0.8 1.0 1.8 -0.4 2.6 2.4 1.3 -0.1 2.6 0.6 1.0 0.121 0.00224 0.112 0.00561 0.00424 0.0161 1.90 0.00658 0.000808 0.0264 2.36 0.00809 1.28 0.0258 208 Table S2.27. 22 min C18 acylsugar analysis LC-MS method. Flow (mL/min) %A %B Initial 1 16 20 20.01 22 0.3 0.3 0.3 0.3 0.3 0.3 95 40 0 0 95 95 Column: Acquity UPLC BEH amide (10 cm x 2.1 mm, 130 Å, 1.7 µm) 5 60 A: 10 mM ammonium formate in water, pH 2.8 100 B: Acetonitrile 100 5 5 209 Table S2.28. 14 min C18 LC-MS method for analysis of purified acylsugars. Flow (mL/min) % A %B Initial 2 10 12 12.01 14 0.3 0.3 0.3 0.3 0.3 0.3 95 40 0 0 95 95 Column: Ascentis Express C18 HPLC column (10 cm x 2.1 mm, 2.7 µm) 5 60 A: 10 mM ammonium formate in water, pH 2.8 100 B: Acetonitrile 100 5 5 210 Table S2.29. 7 min C18 LC-MS method for analysis of enzyme assays. Flow (mL/min) %A %B Initial 1 5 6 6.01 7 0.3 0.3 0.3 0.3 0.3 0.3 95 40 0 0 95 95 Column: Ascentis Express C18 HPLC column (10 cm x 2.1 mm, 2.7 µm) 5 60 A: 10 mM ammonium formate in water, pH 2.8 100 B: Acetonitrile 100 5 5 211 Table S2.30. 24 min C18 LC-MS method for analysis AI3:16 and AI4:18 coelution. Flow (mL/min) %A %B Initial 18 18.01 22 22.01 24 0.3 0.3 0.3 0.3 0.3 0.3 95 40 0 0 95 95 Column: Acquity UPLC BEH amide (10 cm x 2.1 mm, 130 Å, 1.7 µm) 5 60 A: 10 mM ammonium formate in water, pH 2.8 100 B: Acetonitrile 100 5 5 212 Table S2.31. 9 min C18 LC-MS method for analysis saponified acylsugars. Flow (mL/min) %A %B Initial 6 7 7.01 9 0.3 0.3 0.3 0.3 0.3 5 60 95 5 5 Column: Acquity UPLC BEH amide (10 cm x 2.1 mm, 130 Å, 1.7 µm) 95 40 A: 10 mM ammonium formate in water, pH 2.8 5 B: Acetonitrile 95 95 213 Table S2.32. 54 min C18 LC-MS method for separation and purification of S. melongena acylsugars. Flow (mL/min) %A %B Initial 2 40 42 42.01 44 1 1 1 1 1 1 95 40 20 0 95 95 Column: Acclaim 120 C18 HPLC column (4.6 x 150 mm, 5 μm) 5 60 A: water + 0.1% Formic acid 80 B: Acetonitrile 100 5 5 214 Table S2.33. Sugar core composition GC-MS method information. Inlet temp Transfer line temp Helium gas flow rate Split ratio 280°C 280°C 10 mL/min 10:1 Temp gradient (°C/min) initial 1 4 16 19 Temp (°C) 60 60 180 240 240 0 40 5 0 215 Table S2.34. Hydroxylated acyl chain stereochemistry GC-MS method information. Inlet temp Transfer line temp Helium gas flow rate Split ratio 250°C 280°C 1 mL/min NA Temp gradient (°C/min) initial Temp (°C) 60 60 180 180 230 230 300 300 1.5 4.5 6.5 31.5 40.5 47.5 55 0 40 0 2 0 10 0 216 Table S2.35. S. melongena ASAT candidate gene cloning information. Gene ID Gene name SMEL_05g005070 SMEL_06g025230 SMEL_07g013870 SMEL_07g013880 SMEL_08g013890 SMEL_12g015770 SMEL_12g015780 Source of cloned DNA synthesized gene fragment synthesized gene fragment synthesized gene fragment cDNA synthesized gene fragment Species source for DNA sequence Solanum melongena Solanum melongena Solanum melongena Solanum melongena Solanum melongena Solanum melongena Solanum melongena Accession source for DNA sequence Codon optimized (y/n) 67/3 67/3 67/3 PI 555598 67/3 67/3 PI 452123 Y Y N N Y N N SmASAT3- L1 gDNA gDNA 217 SMEL_07g013870_F SMEL_07g013880_F SMEL_07g013880_R Table S2.36. Oligonucleotides used in this study. Sequence Sequence name agcatgactggtggacagcaaatgggtcggATGGCTTCATCACAGATTCTATCA ATCCAC gccggatctcagtggtggtggtggtggtgcTTATATTGGGTGAGCAAACTCAAG GAGTTG agcatgactggtggacagcaaatgggtcggATGGCTGCATCACGATTTGCTTTG ATTTCC gccggatctcagtggtggtggtggtggtgcTTAAAGACCCAAACTTGGAGAAGC AAATTC agcatgactggtggacagcaaatgggtcggATGGTAGCATCAAGAATTGTGTCT AAAAAG gccggatctcagtggtggtggtggtggtgcTCACAGACATGTAGTATCTTTGATT TTGAC agcatgactggtggacagcaaatgggtcggATGGCATCATCAAGAATTATGTCT AGAAAG gccggatctcagtggtggtggtggtggtgcTTATTCCGATGACCAACCAACCGG AGAAGC SMEL_07g013870_R SmASAT3-L1_F SmASAT3_R SmASAT3_F SmASAT3-L1_R Soaet10022603_RT_F CCAAACCAACACCTCCAAAC Soaet10022603_RT_R TCAACTCCACCATCATCATCTC Soaet10024792_RT_F ACAAGGTTGCGGATGGATATAG Soaet10024792_RT_R TGAACCTGTTGCGGAGTTT Soaet10024793_RT_F CCATCTTCTCCAGTATCGTCTTT Soaet10024793_RT_R ACATCATCGTCGTCCCTTTC Soaet10043742_RT_F CGCGATAGGAGATGCAAGTAG Soaet10043742_RT_R CGTCTCCCTAGCACATTCTTT Soaet_ASAT3_RT_1_ F Soaet_ASAT3_RT_1_ R Soaet_ASAT3_RT_2_ F Soaet_ASAT3_RT_2_ R Soaet_ASAT3L1_RT_ 1_F CGTGATTCTGGTGGAGCATTTA CTTGCTAGGGTCACTCTTATGG CCTCACTCCTCCTTCACTTAGA TCACCATTTGCCTTCTTCTACC GACGCCACGTGTCAAGAATA 218 Description Sequence used for cloning into pET28b with BamHI/XhoI site Sequence used for cloning into pET28b with BamHI/XhoI site Sequence used for cloning into pET28b with BamHI/XhoI site Sequence used for cloning into pET28b with BamHI/XhoI site Sequence used for cloning into pET28b with BamHI/XhoI site Sequence used for cloning into pET28b with BamHI/XhoI site Sequence used for cloning into pET28b with BamHI/XhoI site Sequence used for cloning into pET28b with BamHI/XhoI site Primer for RT-PCR of Soaet10022603 Primer for RT-PCR of Soaet10022603 Primer for RT-PCR of Soaet10024792 Primer for RT-PCR of Soaet10024792 Primer for RT-PCR of Soaet10024793 Primer for RT-PCR of Soaet10024793 Primer for RT-PCR of Soaet10043742 Primer for RT-PCR of Soaet10043742 Primer for RT-PCR of SaASAT3 Primer for RT-PCR of SaASAT3 Primer for RT-PCR of SaASAT3 Primer for RT-PCR of SaASAT3 Primer for RT-PCR of SaASAT3-L1 Table S2.36. (cont’d) Soaet_ASAT3L1_RT_1_R Soaet_ASAT3L1_RT_2_F Soaet_ASAT3L1_RT_2_R TGCAGGAGATGGTTTGGAATC ACAATCGGGCGTCTTCAAA CGGAGAAGCAAACCGTAGAA Soaet10001586_EF1α_F Soaet10001586_EF1α_R CTGACTGTGCTGTCCTGATTAT AGCTTCATGGTGCATCTCTAC T7_promoter TAATACGACTCACTATAGGG T7_terminator GCTAGTTATTGCTCAGCGG Primer for RT-PCR of SaASAT3-L1 Primer for RT-PCR of SaASAT3-L1 Primer for RT-PCR of SaASAT3-L1 Primer for RT-PCR of Soaet10001586 (EF1α) Primer for RT-PCR of Soaet10001586 (EF1α) Primer for Sanger sequencing and colony PCR of pET28b plasmids Primer for Sanger sequencing and colony PCR of pET28b plasmids 219 Table S2.37. Plant material metadata. Surface metabolites from all listed accessions were analyzed by LC-MS. USDA-GRIN = United States Department of Agriculture - Germplasm Resources Information Network; CREA = Consiglio per la ricerca in agricoltura e l'analisi dell'economia agrarian. Species Accession Name Origin Source Major Clade Acylsugars detected (y/n) Solanum abutiloides Solanum acerifolium Solanum aethiopicum Solanum aethiopicum Solanum aethiopicum Solanum aethiopicum Solanum aethiopicum Solanum aethiopicum Solanum aethiopicum PI 305325 GUADALUPE 3 Colombia Mansfield, Missourri, United States Baker Creek Heirloom Seeds Clade II USDA-GRIN Clade II Grif 14165 G-2563 Brazil USDA-GRIN Clade II PI 194166 Former Serbia and Montengro USDA-GRIN Clade II PI 247828 NSUA Congo USDA-GRIN Clade II PI 374695 India USDA-GRIN Clade II PI 424859 W-1790 Brazil USDA-GRIN Clade II PI 636107 CGN 18558 United Kingdom USDA-GRIN Clade II PI 666075 CGN 17454 Japan Solanum anguivi PI 180485 Solanum anguivi PI 183357 Solanum anguivi PI 194789 10842 11195 11747 India India India Thailand Colombia PI 319855 PI 305320 Solanum anguivi Solanum atropurpureum Solanum capsicoides Solanum capsicoides Solanum capsicoides Solanum carolinense PI 196300 2930 Nicaragua USDA-GRIN Clade II PI 370043 India USDA-GRIN Clade II PI 390818 W-C 1203 Peru Bath Township, Michigan, United States USDA-GRIN Beal Botanical Garden Clade II Clade II 220 USDA-GRIN USDA-GRIN USDA-GRIN USDA-GRIN USDA-GRIN Clade II Clade II Clade II Clade II Clade II USDA-GRIN Clade II y y y y y y y y y y y y y y y y y n Table S2.37. (cont’d) Solanum elaeagnifolium PI 346963 Solanum incanum PI 196043 PI 381155 PI 678385 PI 645685 PI 388846 PI 388847 PI 441915 PI 305323 PI 370045 PI 413675 305E40 67/3 PI 441908 PI 491260 Solanum incanum Solanum lasiophyllum Solanum lichtensteinii Solanum linnaeanum Solanum linnaeanum Solanum macrocarpon Solanum mammosum Solanum mammosum Solanum mammosum Solanum melanospermum Solanum melongena Solanum melongena Solanum melongena Solanum melongena Solanum melongena Solanum melongena Solanum melongena Solanum melongena Mexico Ethiopia Delhi, India 1196 9624 PLB 294 B and T World Seeds No. 16471 B and T World Seeds No. 442206 WL-74 WL-85 Italy Italy BGH 841 Brazil Colombia India USDA-GRIN USDA-GRIN USDA-GRIN USDA-GRIN USDA-GRIN USDA-GRIN USDA-GRIN USDA-GRIN USDA-GRIN USDA-GRIN 1738 Colombia 4216 Bing Bong Terminal NT, Australia USDA-GRIN Dr. Christopher T. Martine, Bucknell University Montanaso Lombardo, Italy CREA Montanaso Lombardo, Italy CREA BGH 5008 Brazil Tsakinik Greece USDA-GRIN USDA-GRIN Clade II Clade II Clade II Clade II Clade II Clade II Clade II Clade II Clade II Clade II Clade II Clade II Clade II Clade II Clade II Clade II Clade II Clade II Clade II Clade II n y y y y y y n y y y y y y y y y y y y PI 555598 LIAO JIAO 1 HAO Beijing, China USDA-GRIN PI 560904 SEVEN LEAVES China PI 639117 Grif 14479 Sri Lanka USDA-GRIN USDA-GRIN PI 666079 Thai Green Iowa, United States USDA-GRIN 221 Table S2.37. (cont’d) Solanum melongena Solanum prinophyllum Solanum pseudocapsicum PI 452123 Tonda di Manfredonia Italy Grif 16422 Red Giant Solanum richardii PI 500922 Zambia Solanum rostratum PI 420997 1300 Netherlands USDA-GRIN Dr. Joyce van Eck, Boyce Thompson Institute USDA-GRIN USDA-GRIN USDA-GRIN Solanum rostratum PI 675066 LBJWC-0110 United States Barrk Sandstone Walk, Kakadu, NT, Australia USDA-GRIN Dr. Christopher T. Martine, Bucknell University Santa Rosa, California, United States Trade Winds Fruit Clade II Solanum sejunctum Solanum sibundoyensis Solanum sisymbriifolium Solanum torvum PI 358311 India USDA-GRIN Fort Myers, Florida, United States Top Tropicals Solanum virginianum Grif 16940 B and T World Seeds No. 27142 Solanum virginianum PI 381293 PLB 161 Solanum virginianum PI 390213 Solanum americanum PI 268152 Solanum dulcamara PI 643457 India Japan United States Sheffield's Seed Co. Lot No. 9804 Georgia, United States Solanum retroflexum PI 634755 WONDERBERRY Wyoming, United States Solanum retroflexum PI 636106 WONDERBERRY Wyoming, United States Solanum scabrum Solanum villosum Richfield, Minnesota, United States Solanum laciniatum PI 337284 Solanum laciniatum PI 337310 Hungary New Zealand 222 USDA-GRIN USDA-GRIN USDA-GRIN USDA-GRIN USDA-GRIN USDA-GRIN USDA-GRIN New York Botanical Garden Experimental Farm Network USDA-GRIN USDA-GRIN Clade II not tested Clade II Clade II Clade II Clade II Clade II Clade II Clade II Clade II Clade II Clade II Clade II DulMo DulMo DulMo DulMo DulMo DulMo VANAns VANAns y n y n n y n y y n n n y y n n y y y y Table S2.38. AI3:16(i4,i4,i8) chemical shifts and coupling constants. AI3:16(i4,i4,i8) Molecular Formula: C27H46O13 Instrument: Agilent 500 MHz DDR2 NMR solvent: CD3CN Fractions: 32-37 InChI Key: LLNCXHBBKCPKDQ-XSTJKNNJSA-N SMILES: O[C@H]1[C@H](O)[C@H](O)CO[C@H]1O[C@@H]2[ C@@H](OC(CCCCC(C)C)=O)[C@@H](OC(C(C)C)=O) [C@@H](OC(C(C)C)=O)[C@H](O)[C@H]2O Carbon # (Group) 1(CH) -1(CO) -2(CH) -3,4(CH3) -1(CO) -2(CH) -3,4(CH3) -1(CO) -2(CH2) -3(CH2) -4(CH2) -5(CH2) -6(CH) -7,8(CH3) 2(CH) 3(CH) 4(CH) 5(CH) 6(CH) 1′(CH) 2′(CH) 3′(CH) 4′(CH) 5′(CH2) 1H (𝛿, ppm) 4.86 (dd, J = 2.91, 10.24 Hz) 2.61 (hept, J = 6.97 Hz) 1.08 (d, J = 6.97 Hz) 1.06 (d, J = 6.97 Hz) 5.50 (t, J = 2.93 Hz) 2.47 (hept, J = 6.98 Hz) 1.20 (d, J = 6.97 Hz) 4.99 (dd, J = 3.04, 10.22 Hz) 2.26 (m) 1.53 (m) 1.29 (m) 1.19 (m) 1.55 (m) 0.84 (d, J = 6.60 Hz) 3.85 (t, J = 10.00 Hz) 3.47 (t, J = 9.30 Hz) 3.78 (t, J = 9.85 Hz) 4.26 (d, J = 7.01 Hz) 3.41 (dd, J = 7.0, 9.36 Hz) 3.47 (dd, J = 3.4, 9.3 Hz) 3.73 (m) 3.88 (dd, J = 2.41, 12.58 Hz) 3.54 (dd, J = 1.54, 12.63 Hz) a – 13C signals not resolved in 2D spectra. 223 13C (𝛿, ppm) 70.92 176.00 33.80 18.02 18.24 68.10 or 68.08a 175.88 33.69 or 33.64a 18.41, 18.40 70.16 172.71 33.69 or 33.64a 24.53 26.57 38.34 27.57 21.89 80.60 72.49 70.66 104.17 71.10 72.49 68.10 or 68.08a 66.13 Figure S2.15. AI3:16(4,4,8) 1H NMR. 224 Figure S2.16. AI3:16(4,4,8) 13C NMR. 225 Figure S2.17. AI3:16(4,4,8) 1H-1H COSY. 226 Figure S2.18. AI3:16(4,4,8) J-resolved. 227 Figure S2.19. AI3:16(4,4,8) 1H-13C HSQC. 228 Figure S2.20. AI3:16(4,4,8) 1H-13C HMBC with apodization optimized for correlations between acyl chain carbonyl carbon and C2 proton(s). 229 Figure S2.21. AI3:16(4,4,8) 1H-13C HMBC with apodization optimized for correlations between acyl chain carbonyl carbon and sugar ring protons. 230 Table S2.39. AI4:18(2,i4,i4,i8) chemical shifts and coupling constants. AI4:18(2,i4,i4,i8) Molecular Formula: C29H48O14 Instrument: Agilent 500 MHz DDR2 and Varian 600 MHz Inova NMR solvent: CD3CN Fractions: 44-52 InChI Key: JIKVNSLWQPFKKW-INTPKTGNSA-N SMILES: O[C@H]1[C@H](O)[C@H](O)CO[C@H]1O[C@@H]2[C @@H](OC(CCCCC(C)C)=O)[C@@H](OC(C(C)C)=O)[C @@H](OC(C(C)C)=O)[C@H](OC(C)=O)[C@H]2O Carbon # (Group) 1(CH) -1(CO) -2(CH) -3,4(CH3) 2(CH) -1(CO) -2(CH) -3,4(CH3) 3(CH) -1(CO) -2(CH2) -3(CH2) -4(CH2) -5(CH2) -6(CH) -7,8(CH3) 4(CH) 5(CH) 6(CH) -1(CO) -2(CH3) 1′(CH) 2′(CH) 3′(CH) 4′(CH) 5′(CH2) 1H(𝛿, ppm) 5.03 (dd, J = 2.9, 10.5 Hz) 2.45 (hept, J = 7.0 Hz) 1.07 (d, J = 7.0 Hz) 5.54 (t, J = 2.97 Hz) 2.69 (hept, J = 6.94 Hz) 1.21 (t, J = 6.80 Hz) 5.05 (dd, J = 2.9, 10.5 Hz) 2.29 (m) 1.53 (m) 1.30 (m) 1.19 (m) 1.55 (m) 0.89 (d, J = 6.6 Hz) 3.94 (t, J = 9.7 Hz) 3.67 (t, J = 9.48 Hz) 5.35 (t, J = 10.10 Hz) 2.02 (s) 4.25 (d, J = 6.98 Hz) 3.43 (dd, J = 7.00, 9.36 Hz) 3.47 (dd, J = 3.4, 9.3 Hz) 3.78 (m) 3.93 (dd, J = 2.5, 12.5 Hz) 3.61 (dd, J = 1.5, 12.7 Hz) 231 13C (𝛿, ppm) 68.88 175.51 33.67 17.85 67.87 175.79 33.78 18.46 69.72 172.68 33.60 24.52 26.54 38.32 27.56 21.88 80.93 70.22 71.07 169.75 20.08 104.22 71.07 72.80 68.12 66.24 Figure S2.22. AI4:18(2,4,4,8) 1H NMR. 232 Figure S2.23. AI4:18(2,4,4,8) 13C NMR. 233 Figure S2.24. AI4:18(2,4,4,8) 1H-1H COSY. 234 Figure S2.25. AI4:18(2,4,4,8) J-resolved. 235 Figure S2.26. AI4:18(2,4,4,8) 13C DEPT. 236 Figure S2.27. AI4:18(2,4,4,8) 1H-13C HSQC. 237 Figure S2.28. AI4:18(2,4,4,8) 1H-13C H2BC. 238 Figure S2.29. AI4:18(2,4,4,8) 1H-13C HMBC. 239 Table S2.40. I3:18(i4,i4,i10) Chemical shifts and coupling constants. I3:18(i4,i4,i10) Molecular Formula: C24H42O9 Instrument: Agilent 500 MHz DDR2 NMR solvent: CD3CN Fractions: 71-74 InChI Key: IRUZHFMTUQKAJJ-QANWUKQVSA-N SMILES: O[C@H]1[C@H](O)[C@@H](OC(CCCCCCC(C)C)=O)[C@@ H](OC(C(C)C)=O)[C@@H](OC(C(C)C)=O)[C@@H]1O 1H (𝛿, ppm) 13C (𝛿, ppm) 4.80 (dd, J = 2.95, 7.15 Hz) 2.52 (hept, J = 6.98 Hz) 1.09, 1.11 (d, J = 7.01 Hz) 5.47 (t, J = 2.98 Hz) 2.64 (hept, J = 6.97 Hz) 1.18 (d, J = 6.95 Hz) 4.80 (dd, J = 2.95, 7.15 Hz) 2.26 (m) 1.56 (m) 1.36-1.25 (m) 1.18 (m) 1.54 (hept, J = 6.7 Hz) 0.89 (d, J = 6.6 Hz) 3.75 (t, J = 9.67 Hz) 3.34 (t, J = 9.29 Hz) 3.73 (t, J = 9.67 Hz) 71.31 or 71.33a 176.01 33.68 or 33.70a 18.01, 18.24 68.16 179.89 33.85 18.35, 18.42 71.31 or 71.33a 172.80 33.68 or 33.70a 24.55 29.27(5), 28.73(4), 26.96(6) 38.70 27.74 21.93 70.49 or 70.51a 70.49 or 70.51a 74.33 Carbon # (Group) 1(CH) -1(CO) -2(CH) -3,4(CH3) 2(CH) -1(CO) -2(CH) -3,4(CH3) 3(CH) -1(CO) -2(CH2) -3(CH2) -4-6(CH2) -7(CH2) -8(CH) -9,10(CH3) 4(CH) 5(CH) 6(CH) a – 13C signals not resolved in 2D spectra. 240 Figure S2.30. I3:18(i4,i4,i10) 1H NMR. 241 Figure S2.31. I3:18(i4,i4,i10) 13C NMR. 242 Figure S2.32. I3:18(i4,i4,i10) 1H-1H COSY. 243 Figure S2.33. I3:18(i4,i4,i10) 1H-1H TOCSY. 244 Figure S2.34. I3:18(i4,i4,i10) J-resolved. 245 Figure S2.35. I3:18(i4,i4,i10) 1H-13C HSQC. 246 Figure S2.36. I3:18(i4,i4,i10) 1H-13C H2BC. 247 Figure S2.37. I3:18(i4,i4,i10) 1H-13C HMBC. 248 Table S2.41. I3:20(i4,i4,i12) chemical shifts and coupling constants. I3:20(i4,i4,i12) Molecular Formula: C26H46O9 Instrument: Agilent 500 MHz DDR2 NMR solvent: CD3CN Fractions: 105-107 InChI Key: WFZVKWMBYUFKOS-WQRAYAPSSA-N SMILES: O[C@H]1[C@H](O)[C@@H](OC(CCCCCCCCC(C)C)=O)[C@ @H](OC(C(C)C)=O)[C@@H](OC(C(C)C)=O)[C@@H]1O Carbon # (Group) 1(CH) 2(CH) 3(CH) -1(CO) -2(CH) -3,4(CH3) -1(CO) -2(CH) -3,4(CH3) -1(CO) -2(CH2) -3(CH2) -4-8(CH2) -9(CH2) -10(CH) -11,12(CH3) 1H (𝛿, ppm) 4.80 or 4.79 (dd, J = 3.1, 10.1 Hz) 2.51 (hept, J = 6.92 Hz) 1.11, 1.09 (d, J = 7.0 Hz) 5.46 (t, J = 3.01 Hz) 2.62 (hept, J = 6.95 Hz) 1.18 (d, J = 7.0 Hz) 4.80 or 4.79 (dd, J = 3.1, 10.1 Hz) 2.26 (m) 1.54 (m) 1.33-1.27 (m) 1.19 (m) 1.54 (m) 0.88 (d, J = 6.71 Hz) 3.75 or 3.73 (t, J = 9.9 Hz) 3.33 (t, J = 9.30 Hz) 3.75 or 3.73 (t, J = 9.9 Hz) 13C (𝛿, ppm) 71.33 or 71.31a 157.99 33.69 or 33.67a 18.21, 17.99 68.18 175.87 33.84 18.39, 18.32 71.33 or 71.31a 172.77 33.69 or 33.67a 24.53 28.68(4), 28.97(5), 29.18(6), 29.57 (7), 27.10(8) 38.76 27.73 21.91 70.53 or 70.51a 74.36 70.53 or 70.51a 4(CH) 5(CH) 6(CH) a – 13C signals not resolved in 2D spectra. 249 Figure S2.38. I3:20(i4,i4,i12) 1H NMR. 250 Figure S2.39. I3:20(i4,i4,i12) 13C NMR. 251 Figure S2.40. I3:20(i4,i4,i12) 1H-1H COSY. 252 Figure S2.41. I3:20(i4,i4,i12) 1H-1H TOCSY. 253 Figure S2.42. I3:20(i4,i4,i12) J-resolved. 254 Figure S2.43. I3:20(i4,i4,i12) 1H-13C HSQC. 255 Figure S2.44. I3:20(i4,i4,i12) 1H-13C H2BC. 256 Figure S2.45. I3:20(i4,i4,i12) 1H-13C HMBC with apodization optimized for correlations between acyl chain carbonyl carbon and C2 proton(s). 257 Figure S2.46. I3:20(i4,i4,i12) 1H-13C HMBC with apodization optimized for correlations between acyl chain carbonyl carbon and sugar ring protons. 258 Table S2.42. I3:22(i4,i4,i14) chemical shifts and coupling constants. I3:22(i4,i4,i14) Molecular Formula: C28H50O9 Instrument: Varian 600 MHz Inova, Bruker Avance NEO 600 MHz NMR, Bruker Avance NEO 800 MHz NMR solvent: CD3CN Fractions: 139-141 InChI Key: FPKCJTQXNJKTGE-KTKTTYTDSA-N SMILES: O[C@H]1[C@H](O)[C@@H](OC(CCCCCCCCCCC(C)C) =O)[C@@H](OC(C(C)C)=O)[C@@H](OC(C(C)C)=O)[C @@H]1O Carbon # (Group) 1(CH) 2(CH) 3(CH) -1(CO) -2(CH) -3,4(CH3) -1(CO) -2(CH) -3,4(CH3) -1(CO) -2(CH2) -3(CH2) -4(CH2) -5-10(CH2) -11(CH2) -12(CH) -13,14(CH3) 1H (𝛿, ppm) 4.80 (dt, J = 3.25, 10.23 Hz) 2.52 (hept, J = 6.96 Hz) 1.11 (dd, J = 6.97, 13.05 Hz) 5.47 (t, J = 2.98 Hz) 2.64 (hept, J = 6.96 Hz) 1.19 (dd, J = 2.59, 6.96 Hz) 4.80 (dt, J = 3.25, 10.23 Hz) 2.27 (m) 1.55 (m) 1.29 (m) 1.30 (m) 1.19 (m) 1.54 (m) 0.89 (d, J = 6.64 Hz) 3.74 (td, J = 8.24, 9.85, 9.90 Hz) 3.34 (t, J = 9.31 Hz) 3.74 (td, J = 8.24, 9.85, 9.90 Hz) 13C (𝛿, ppm) 71.34 or 71.31 176.03 or 175.89a 33.85 18.40 – 18.00 68.18 176.03 or 175.89a 33.70 18.40 – 18.00 71.34 or 71.31a 172.81 33.67 24.53 28.68 29.63, 29.39, 29.36, 29.32, 29.16, 28.98 38.78 27.74 21.92 70.53 or 70.50a 74.36 70.53 or 70.50a 4(CH) 5(CH) 6(CH) a – 13C signals not resolved in 2D spectra. 259 Figure S2.47. I3:22(i4,i4,i14) 1H NMR. 260 Figure S2.48. I3:22(i4,i4,i14) 13C NMR. 261 Figure S2.49. I3:22(i4,i4,i14) 1H-1H COSY. 262 Figure S2.50. I3:22(i4,i4,i14) 13C DEPT-Q. 263 Figure S2.51. I3:22(i4,i4,i14) 1H-13C HSQC. 264 Figure S2.52. I3:22(i4,i4,i14) 1H-13C HMBC. 265 Table S2.43. I3:22(i4,i4,n14) chemical shifts and coupling constants. I3:22(i4,i4,n14) Molecular Formula: C28H50O9 Instrument: Bruker Avance NEO 800 MHz NMR NMR solvent: CD3CN Fractions: 144-146 InChI Key: ZHGFFHULNGWPKL-KTKTTYTDSA-N SMILES: O[C@H]1[C@H](O)[C@@H](OC(CCCCCCCCCCCCC)= O)[C@@H](OC(C(C)C)=O)[C@@H](OC(C(C)C)=O)[C@ @H]1O Carbon # (Group) 1(CH) 2(CH) 3(CH) -1(CO) -2(CH) -3,4(CH3) -1(CO) -2(CH) -3,4(CH3) -1(CO) -2(CH2) -3(CH2) -4(CH2) -5-12(CH2) -13(CH2) -14(CH3) 1H (𝛿, ppm) 4.80 (ddd, J = 2.98, 4.60, 10.20 Hz) 2.64 (hept, J = 6.97 Hz) 1.18 (dd, J = 3.48, 6.98 Hz) 5.46 (t, J = 2.97 Hz) 2.52 (hept, J = 6.97 Hz) 1.11 (dd, J = 6.98, 17.92 Hz) 4.80 (ddd, J = 2.98, 4.60, 10.20 Hz) 2.26 (m) 1.55 (m) 1.29 (m) 1.30 (m) 1.33 (m) 0.91 (m) 3.74 (q, J = 10.23 Hz) 3.34 (t, J = 9.33 Hz) 3.74 (q, J = 10.23 Hz) 13C (𝛿, ppm) 71.89 or 71.87a 176.56 34.41 18.96, 18.90 68.72 176.43 34.26 or 34.23a 18.79, 18.56 71.89 or 71.87a 173.34 34.26 or 34.23a 25.10 32.22 29.95, 29.93, 29.89, 29.73, 29.65, 29.55, 29.24 22.97 13.96 71.07 or 71.05a 74.90 71.07 or 71.05a 4(CH) 5(CH) 6(CH) a – 13C signals not resolved in 2D spectra. 266 Figure S2.53. I3:22(i4,i4,n14) 1H NMR. 267 Figure S2.54. I3:22(i4,i4,n14) 13C NMR. 268 Figure S2.55. I3:22(i4,i4,n14) 1H-1H COSY. 269 Figure S2.56. I3:22(i4,i4,n14) 1H-1H TOCSY. 270 Figure S2.57. I3:22(i4,i4,n14) J-resolved. 271 Figure S2.58. I3:22(i4,i4,n14) 1H-13C HSQC. 272 Figure S2.59. I3:22(i4,i4,n14) 1H-13C H2BC. 273 Figure S2.60. I3:22(i4,i4,n14) 1H-13C HMBC. 274 Table S2.44. I3:22(i4,i4,3-OH-i14) chemical shifts and coupling constants. I3:22(i4,i4,3-OH-i14) Molecular Formula: C28H50O10 Instrument: Agilent 500 MHz DDR2 NMR solvent: CD3CN Fractions: 95-97 InChI Key: REKBIFNRGXIEET-GBYYZRSBSA-N SMILES: O[C@H]1[C@H](O)[C@@H](OC(CC(O)CCCCCCCCC(C) C)=O)[C@@H](OC(C(C)C)=O)[C@@H](OC(C(C)C)=O)[C @@H]1O Carbon # (Group) 1(CH) 1H (𝛿, ppm) 4.82 (dd, J = 2.94, 10.21 Hz) 2(CH) 3(CH) -1(CO) -2(CH) -3,4(CH3) -1(CO) -2(CH) -3,4(CH3) -1(CO) -2(CH2) -3(CHOH) -4(CH2) -5-9(CH2) -10(CH2) -11(CH2) -12(CH) -13,14(CH3) 4(CH) 5(CH) 6(CH) a – 13C signals not resolved in 2D spectra. 2.63 (hept, J = 7.04, 7.04, 6.96, 6.96, 6.96 Hz) 1.18 (dd, J = 2.73, 6.97 Hz) 5.48 (t, J = 2.97 Hz) 2.51 (hept, J = 7.00 Hz) 1.11 (dd, J = 7.00, 12.57 Hz) 4.86 (dd, J = 2.94, 10.18 Hz) 2.46 (dd, J = 4.25, 15.29 Hz) 2.30 (dd, J = 8.44, 15.32 Hz) 3.92 (m) 1.41 (m) 1.29 (m) 1.29 (m) 1.18 (m) 1.53 (hept, J = 6.72 Hz) 0.89 (d, J = 6.64 Hz) 3.76 (td, J = 5.22, 9.71, 9.76 Hz) 3.36 (t, J = 9.31 Hz) 3.76 (td, J = 5.22, 9.71, 9.76 Hz) 13C (𝛿, ppm) 71.34 175.98 33.88 18.33, 18.40 68.27 176.01 33.70 18.02, 18.22 71.50 171.16 42.14 67.75 36.53 25.25, 29.26, 29.32, 29.38,29.65 27.18 38.7 27.75 21.89 70.66 or 70.54a 74.15 70.66 or 70.54a 275 Figure S2.61. I3:22(i4,i4,3-OH-i14) 1H NMR. 276 Figure S2.62. I3:22(i4,i4,3-OH-i14) 13C NMR. 277 Figure S2.63. I3:22(i4,i4,3-OH-i14) 1H-1H COSY. 278 Figure S2.64. I3:22(i4,i4,3-OH-i14) 1H-1H TOCSY. 279 Figure S2.65. I3:22(i4,i4,3-OH-i14) 1H-13C HSQC. 280 Figure S2.66. I3:22(i4,i4,3-OH-i14) 1H-13C H2BC. 281 Figure S2.67. I3:22(i4,i4,3-OH-i14) 1H-13C HMBC. 282 Table S2.45. I3:22(i4,i4,(3R)-OH-n14) chemical shifts and coupling constants. I3:22(i4,i4,(3R)-OH-n14) Molecular Formula: C28H50O10 Instrument: Agilent 500 MHz DDR2 NMR solvent: CD3CN Fractions: 101-103 InChI Key: KJJFWJBFXVGTFN-KGWGJSIPSA-N SMILES: O[C@H]1[C@H](O)[C@@H](OC(C[C@H](O)CCCCCCCCC CC)=O)[C@@H](OC(C(C)C)=O)[C@@H](OC(C(C)C)=O)[C @@H]1O Carbon # (Group) 1(CH) 1H (𝛿, ppm) 4.82 (dd, J = 2.96, 10.21 Hz) 2(CH) 3(CH) -1(CO) -2(CH) -3,4(CH3) -1(CO) -2(CH) -3,4(CH3) -1(CO) -2(CH2) -3(CHOH) -4(CH2) -5-12(CH2) -13(CH2) -14(CH3) 4(CH) 5(CH) 6(CH) a – 13C signals not resolved in 2D spectra. 2.63 (hept, J = 7.04, 7.04, 6.96, 6.96, 6.96 Hz) 1.18 (dd, J = 2.73, 6.97 Hz) 5.48 (t, J = 2.97 Hz) 2.51 (hept, J = 7.00 Hz) 1.11 (d, J = 7.00 Hz) 4.86 (dd, J = 2.95, 10.17 Hz) 2.30 (dd, J = 8.44, 15.32 Hz) 2.46 (dd, J = 4.25, 15.29 Hz) 3.92 (m) 1.42 (m) 1.41-1.30 (m) 1.30 (m) 0.91(m) 3.76 (m) 3.36 (t, J = 9.32 Hz) 3.76 (m) 13C (𝛿, ppm) 71.34 175.98 33.88 18.33, 18.40 68.27 176.01 33.71 18.02, 18.22 71.50 171.16 42.14 67.75 36.53 29.41, 29.38, 29.35, 29.25, 29.11, 25.30, 22.42 31.67 13.42 70.54 or 70.66a 74.16 70.54 or 70.66a 283 Figure S2.68. I3:22(i4,i4,3-OH-n14) 1H NMR. 284 Figure S2.69. I3:22(i4,i4,3-OH-n14) 13C NMR. 285 Figure S2.70. I3:22(i4,i4,3-OH-n14) 1H-1H COSY. 286 Figure S2.71. I3:22(i4,i4,3-OH-n14) 1H-1H TOCSY. 287 Figure S2.72. I3:22(i4,i4,3-OH-n14) 1H-13C HSQC. 288 Figure S2.73. I3:22(i4,i4,3-OH-n14) 1H-13C HMBC. 289 Table S2.46. 4-O-β-arabinosyl-myo-inositol chemical shifts and coupling constants. 4-O-β-arabinosyl-myo-inositol derived from saponified AI4:18(2,4,4,8) Molecular Formula: C11H20O10 Instrument: Agilent 500 MHz DDR2 NMR solvent: D2O InChI Key: ZTUXXEBTGKCWOB-YFYAPIRNSA-N SMILES: O[C@H]1[C@@H]([C@@H](CO[C@H]1O[C@@H]2[ C@H]([C@H]([C@H]([C@@H]([C@H]2O)O)O)O)O)O )O Carbon # (Group) 1(CH) 2(CH) 3(CH) 4(CH) 5(CH) 6(CH) 1′(CH) 2′(CH) 3′(CH) 4′(CH) 5′(CH2) 1H (𝛿, ppm) 3.50 (dd, J = 3.3, 9.7 Hz) 3.86 (t, J = 2.95 Hz) 3.53 (dd, J = 2.9, 9.8 Hz) 3.60 (t, J = 10.1 Hz) 3.19 (t, J = 9.2 Hz) 3.44 (t, J = 10.4 Hz) 4.37 (d, J = 7.61 Hz) 3.43 (t, J = 9.8 Hz) 3.48 (dd, J = 1.7, 13.5 Hz) 3.75 (m) 3.77 (dd, J = 2.38, 13.4 Hz); 3.48 (dd, J = 1.7, 13.4 Hz) 13C (𝛿, ppm) 72.18 or 72.12a 71.96 70.69 81.93 72.48 72.18 or 72.12a 103.72 71.18 72.18 or 72.12a 68.19 66.18 a – 13C signals not resolved in 2D spectra. 290 Figure S2.74. 4-O-arabinopyranosyl myo-inositol derived from saponified AI4:18(2,4,4,8) 1H NMR. 291 Figure S2.75. 4-O-arabinopyranosyl myo-inositol derived from saponified AI4:18(2,4,4,8) 13C NMR. 292 Figure S2.76. 4-O-arabinopyranosyl myo-inositol derived from saponified AI4:18(2,4,4,8) 1H-1H COSY. 293 Figure S2.77. 4-O-arabinopyranosyl myo-inositol derived from saponified AI4:18(2,4,4,8) 1H-1H TOSCY. 294 Figure S2.78. 4-O-arabinopyranosyl myo-inositol derived from saponified AI4:18(2,4,4,8) J- resolved. 295 Figure S2.79. 4-O-arabinopyranosyl myo-inositol derived from saponified AI4:18(2,4,4,8) 1H-13C HSQC. 296 Figure S2.80. 4-O-arabinopyranosyl myo-inositol derived from saponified AI4:18(2,4,4,8) 1H-13C H2BC. 297 Figure S2.81. 4-O-arabinopyranosyl myo-inositol derived from saponified AI4:18(2,4,4,8) 1H-13C HMBC. 298 Figure S2.82. 4-O-arabinopyranosyl myo-inositol derived from saponified AI4:18(2,4,4,8) 1H-13C coupled HSQC. 299 Table S2.47. NMR metadata for the Agilent DDR2 500 MHz instruments. Facility supervisor: Analyst Instrument location Facility instrument title Manufacturer Field frequency lock Additional solute Solvent Chemical shift standard Dr. Daniel Holmes Paul D. Fiesel MSU Max T. Rogers NMR Facility Ahriman and Ormuzd Agilent Acetonitrile-d3; D2O None CD3CN: 600 μL; D2O: 600 μL CH3CN-d3 (δH = 1.94 and δC = 118.70 ppm); H2O- d2 (δH = 4.36 ppm) None Agilent DDR2 500 MHz with 7600AS 96 autosamplers 42.7288, -84.4745 499.91 MHz OneNMR Probe with Protune accessory for hands-off tuning VnmrJ 4.2A Kontes NMR tube, 8 in, Temperature @ 298K, no spinning Concentration standard Instrument Geographic location of instrument Magnet Probe Acquisition software Sample details 300 Table S2.48. NMR metadata for the Varian Inova 600 MHz instrument. Facility supervisor: Analyst Instrument location Facility instrument title Manufacturer Field frequency lock Additional solute Solvent Chemical shift standard Concentration standard Instrument Geographic location of instrument Magnet Probe Dr. Daniel Holmes Paul D. Fiesel MSU Max T. Rogers NMR Facility Sobek Varian Acetonitrile-d3 None CD3CN: 600 μL CH3CN-d3 (δH = 1.94 and δC = 118.70 ppm) None Varian Inova 600 MHz 42.7288, -84.4745 599.77 MHz Nalorac 5 mm PFG switchable probe pretuned for 1H, 13C VnmrJ 4.2A Kontes NMR tube, 8 in, Temperature @ 298K, no spinning Acquisition software Sample details 301 Table S2.49. NMR metadata for the Bruker Avance NEO 600 MHz instrument. Facility supervisor: Analyst Instrument location Manufacturer Field frequency lock Additional solute Solvent Chemical shift standard Concentration standard Instrument Geographic location of instrument Magnet Probe Acquisition software Sample details Dr. Daniel Holmes Paul D. Fiesel MSU Max T. Rogers NMR Facility Bruker Acetonitrile-d3 None CD3CN: 600 μL CH3CN-d3 (δH = 1.94 and δC = 118.70 ppm) None Bruker Avance NEO 600 MHz NMR with shielded magnet 42.7288, -84.4745 600.32 MHz 5 mm nitrogen cryogenic HCN Prodigy probe TopSpin 4.1.1 Kontes NMR tube, 8 in, Temperature @ 298K, no spinning 302 Table S2.50. NMR metadata for the Bruker Avance NEO 800 MHz instrument. Facility supervisor: Analyst Instrument location Manufacturer Field frequency lock Additional solute Solvent Chemical shift standard Concentration standard Instrument Geographic location of instrument Magnet Probe Acquisition software Sample details Dr. Daniel Holmes Paul D. Fiesel MSU Max T. Rogers NMR Facility Bruker Acetonitrile-d3 None CD3CN: 600 μL CH3CN-d3 (δH = 1.94 and δC = 118.70 ppm) None Bruker Avance NEO 800 MHz NMR with shielded magnet 42.7288, -84.4745 800.33 MHz 5 mm helium cryogenic HCN probe TopSpin 4.1.1 Kontes NMR tube, 8 in, Temperature @ 298K, no spinning 303 Figure S2.83. Acyl chain composition of S. melongena acylsugars. Relative abundance of acyl chains shown between three S. melongena leaf surface extracts. Straight acyl chains were identified with authentic reference standards and iso-branched chains were identified with NIST mass spectral library searches. Hydroxylated acyl chains were not included in this analysis and only detected acyl chains are included. 304 ACYLINOSITOL BIOSYNTHESIS WITHIN SOLANUM GLANDULAR TRICHOMES CHAPTER 3: 305 Abstract Plants synthesize a diverse array of lineage- and tissue-specific compounds called specialized metabolites. These exhibit diverse bioactivities involved in plant defense and communication as well as human medicine and foods. Acylsugars, one class of specialized metabolites, are produced within glandular trichomes of Solanaceae (nightshade) family plants and act as anti-insect and anti-fungal molecules. The vast acylsugar structural diversity observed serves as an excellent model for investigating plant metabolic evolution. Acylinositols, one type of acylsugar, are produced in multiple major Solanum genus clades and exhibit enormous structural diversity and unique structures with inositol core and chain glycosylations and hydroxyacyl chains. The observation that acylinositols differ in core structure and acylchains from well-characterized acylsucroses, suggested that their biosynthetic pathways are distinct. Considering this, understanding acylinositol biosynthesis can provide insights into their evolution and enables study of their biological roles. In this study, a brinjal eggplant, Solanum melongena, tissue-specific transcriptome was utilized to uncover an in vitro pathway capable of synthesizing a triacylinositol with chromatographic and mass spectral characteristics identical to a plant-produced triacylinositol. This pathway knowledge was transferred to study trichome acylinositol metabolism in Solanum quitoense, a South American fruit crop, identifying an enzyme capable of catalyzing the third inositol acylation. Utilizing a previously developed transcriptome and transient gene silencing protocol, I provided evidence that Solanum nigrum, a species residing in a different Solanum major clade than S. melongena and S. quitoense, utilizes a similar acylinositol biosynthetic pathway. This study highlights the usefulness of comparative biochemistry to uncover evolutionary mechanisms underlying metabolic novelty. 306 Introduction Acylsugars exhibit enormous chemical diversity as documented in Chapter 2, and this diversity presumably impacts their biological activities against insects, fungi, and microbes. In fact, multiple reports demonstrate that acylsugar structural differences can influence multiple mechanisms of pest deterrence. For example, swapping sugar cores and acyl chains had differential impacts against the mortality of the arthropod species Cacopsylla pyricola, Manduca sexta, Manduca nicotianae, and Tetranychus urticae (Puterka et al., 2003). Additionally, the relationship between acylsugar structure and biological activity is not always consistent between different arthropod species (Leckie et al., 2016; Puterka et al., 2003). This acylsugar structure- function relationship is still underdeveloped, and tools such as near isogenic plant lines, differing only in the introduced transgenic genes, with varied acylsugar components can help clarify this association. Understanding the biosynthesis of unusual acylsugar traits can enable development of these tools. Acylinositols were identified throughout multiple Solanum major clades including Clade II, DulMo, VANAns, and Potato clades (Chapter 2; Kerwin et al., unpublished) and these metabolites exhibit multiple atypical traits such as hydroxylated medium length acyl chains and glycosylation. From the phylogenetic distribution of acylinositols, we can infer that this trait arose near the Solanum crown node, ~14 mya, however, the lack of resolution between the major Solanum clades limits our ability to infer the number of evolutionary origins (Gagnon et al., 2022). Uncovering acylinositol biosynthetic enzymes and pathways from members within different major clades would provide insight into acylinositol evolutionary history and enable testing of their biological function. 307 Inositol acetyltransferases were recently identified to create tetraacylated compounds in the brinjal eggplant, Solanum melongena, and the South American fruit crop lulo, Solanum quitoense (Leong et al., 2020). S. melongena SmASAT3-L1 catalyzes the R4 acetylation of AI3:16(i4,i4,i8), while S. quitoense SqTAIAT acetylates I3:22(2,10,10) and I3:24(2,10,12) at R6. Both enzymes are likely orthologous or paralogous to ASATs in the S. lycopersicum acylsucrose pathway, specifically SlASAT3 and SlASAT4, suggesting the acylinositol pathway shares biosynthetic enzymes with acylsucrose biosynthesis. In contrast, the first steps of trichome acylinositol biosynthesis remain unclear. S. quitoense gene knockdown experiments implicated the genes SqASAT1H, SqASAT3H, and SqASAT4H to be involved in producing acylinositols (Leong et al., 2022). In vitro enzyme assays suggest the pathway begins with SqASAT1H, an SlASAT1 outparalog (Koonin, 2005), which acylates myo-inositol with a C10 or C12 medium acyl chain. SqASAT4H and SqASAT3H, enzymes homologous to SlASAT4 and SlASAT3 can further acylate the resultant monoacylinositol with an acetyl chain and another medium acyl chain, respectively. However, the resulting enzymatically-produced triacylinositol did not coelute with plant-produced triacylinositols by LC-MS suggesting one or more pathway components are missing. Here I propose an in vitro triacylinositol biosynthetic pathway for trichomes of the brinjal eggplant and identify a new enzyme as putatively involved in S. quitoense acylinositol biosynthesis. Utilizing the recently generated eggplant tissue-specific transcriptome (Chapter 2), we tested S. melongena candidate acyltransferases for forward acylating activity starting with myo-inositol and reverse activity with purified triacylinositols. Multiple one pot enzyme combinations of SmASAT1-L, SmASAT3, and SmASAT3-L7 synthesized I2:8(i4,i4). A fourth enzyme, SmASAT3-INOSITOL (SmASAT3-I), acylated I2:8(i4,i4) with medium length acyl 308 chains. However, the in vitro triacylinositol products coeluted by LC-MS with plant-produced triacylinositol only under conditions that promote pH-induced acyl chain rearrangement. I transferred this pathway knowledge to S. quitoense and identified SqASAT3-I, a trichome- expressed homolog of SmASAT3-I. VIGS and reverse enzyme assays supported the hypothesis that SqAST3-I performs the third acylation in S. quitoense acylinositol pathway. I then tested if the more distantly related species S. nigrum shared a similar acylinositol pathway to S. melongena and S. quitoense by reducing expression of SnASAT1-L and SnASAT3 mRNA with VIGS. Indeed, knockdown of SnASAT1-L and SnASAT3 expression decreased acylinositol accumulation suggesting that the three species share a similar pathway and acylinositol biosynthesis originated in their shared common ancestor. Results S. melongena in vitro acylinositol pathway discovery I tested the seven S. melongena candidate enzymes identified in Chapter 2 for activity acylating myo-inositol with both short and medium length acyl-CoAs (iC4, nC8, nC10, nC12, nC14), chain lengths related to chains found in S. melongena acylsugars. We hypothesized that the SlASAT1 homolog SMEL_07g013870, SmASAT1-LIKE (SmASAT1-L), would catalyze this first acylation as it shares 92% amino acid identity to SqASAT1H which acylates myo- inositol with nC10 and nC12-CoAs (Leong et al., 2022). Indeed, SmASAT1-L was a candidate enzyme that showed activity acylating myo-inositol with the medium acyl chains nC8-, nC10-, nC12-, and nC14-CoAs (Figure S3.5). I also tested the seven candidate enzymes for activity with the short chain iC4-CoA and myo-inositol and found that both SmASAT1-L and the ortholog of SlASAT3, SMEL_12g015770 (SmASAT3), were active (Figure S3.4), suggesting that the pathway could begin with either short or medium chain acylation. Testing the hypothesis that the 309 pathway begins with medium chain acylation, addition of either the SmASAT3 paralog SmASAT3-LIKE7 (SmASAT3-L7; SMEL_07g013880) or another SmASAT3 paralog SmASAT3-L1 to ‘one-pot’ assays with SmASAT1-L produced I2:12(i4,n10) (Figure S3.6). The I2:12 products created by the ASAT3 paralogs did not coelute suggesting different positions are acylated (Figure S3.6). After heat-inactivation, the addition of SmASAT3 and iC4-CoA to the above one-pot assays, now a two-step assay, produced I3:18(i4,i4,n10) (Figure S3.7). However, this triacylinositol did not coelute with any plant-produced I3:18(i4,i4,10) product (Figure S3.7), indicating different positions are acylated. Testing the hypothesis that the pathway begins with iC4 acylation, we found that multiple two enzyme combinations in one-pot assays could produce the diacylinositol I2:8(i4,i4), with the combinations of SmASAT1-L+SmASAT3, SmASAT1- L+SmASAT3-L7, and SmASAT3+SmASAT3-L7 exhibiting the highest activity (Figure 3.1). However, further addition of candidate enzymes and medium chain CoAs to one-pot and multistep assays failed to add a medium acyl chain to create a triacylinositol. Figure 3.1. Multiple two enzyme combinations of SmASATs produce the diacylinositol I2:8(i4,i4). Products were formed in one-pot assays supplied with myo-inositol and iC4-CoA buffered at pH 8.0 and were analyzed with the 7 min I2:8 LC inlet method. Extracted ion 310 Figure 3.1. (cont’d) chromatograms display the formate adduct of I2:8, 365.145 m/z. Taking advantage of the ability of BAHD acyltransferases to act in the reverse direction (Leong et al., 2020; Lou et al., 2021; Schenck et al., 2022), I sought an acyltransferase capable of removing an acyl chain from purified triacylinositols and found evidence that a BAHD distantly related to characterized ASATs catalyzes a third acylation. These reactions are useful because observed activity confirms an enzyme is acting upon a ‘correct’ position, information not gleaned in the forward assays. I tested the candidate enzymes for reverse activity against the in vivo products I3:18(i4,i4,n10) and I3:22(i4,i4,i14-OH) and found SMEL_06g025230 catalyzed the removal of the medium acyl chain from all three triacylinositols. In contrast, SMEL_08g018390 removed the iC10 chain from I3:18(i4,i4,i10), producing I2:8(i4,i4) at a ~1000-fold lower signal intensity than the de-acylated product formed by SMEL_06g025230 (Figure 3.2). Phylogenetic analysis revealed that SMEL_06g025230 resides within a clade of 16 S. melongena BAHDs with no characterized ASATs (Chapter 2). With no close homology for naming this enzyme, we named this enzyme SmASAT3-INOSITOL (SmASAT3-I) based on the hypothesis that it performs third acylation in the pathway and that it acts upon inositol esters. 311 Figure 3.2. SmASAT3-I catalyzes the removal of medium acyl chains from purified S. melongena triacylinositols. (A) Reaction scheme for the removal of the iC10 chain from I3:18(i4,i4,i10) observed in (C). (B) Reaction scheme for the removal of the 3-OH-iC14 chain from I3:18(i4,i4,3-OH-i14) observed in (D). (C) LC-MS analysis of reverse in vitro assays supplied with I3:18(i4,i4,i10). Extracted ion chromatograms (EIC) display the formate adduct of I2:8(i4,i4), 365.145 m/z. (D) LC-MS analysis of reverse in vitro assays supplied with I3:22(i4,i4,3-OH-i14). In this set of assays, only the two enzymes with activity in panel C were used. Extracted ion chromatograms (EIC) display the formate adduct of I2:8(i4,i4), 365.145 m/z. Considering that SmASAT3-I did not demonstrate measurable activity acylating I2:8(i4,i4) generated by SmASAT3-L1 and SmASAT3-L7, we tested for acyl chain position 312 differences between the forward- and reverse-produced I2:8 based upon LC-MS coelution (Figure 3.3A). Indeed, the two products do not coelute, consistent with the hypothesis that different inositol positions are acylated considering identical acyl chains are present. This result is reminiscent of S. quitoense acylinositol enzymes that produce triacylinositols acylated at positions different than plant-produced triacylinositols (Leong et al., 2022). A possible role for intramolecular rearrangement in acylinositol biosynthesis The positional differences led us to consider two hypotheses: first, an untested enzyme is involved and second, that acyl chain rearrangement produces I2:8 acylated at the ‘correct’ position, which is subsequently acted upon by SmASAT3-I. Because intramolecular nonenzymatic acyl chain rearrangement was described previously, and occurs at higher rates with increasing pH values from 6-8 and greater (Fan et al., 2015; Leong et al., 2022; Lou et al., 2021), I investigated this hypothesis by testing if the forward assay I2:8 rearranges at pH 8.0 and if so, whether SmASAT3-I can act upon one of the rearranged isomer(s). As hypothesized, one pot forward assays with SmASAT1-L and SmASAT3 produced one I2:8(i4,i4) isomer at pH 6.0 and multiple isomers at pH 8.0, with one pH 8.0 isomer coeluting with I2:8 generated from the reverse reaction of SmASAT3-I against I3:22(i4,i4,3-OH-i14) (Figure 3.3A). SmASAT3-I and nC14-CoA were then added to the diacylinositol-producing forward assays after a heat-enzyme inactivation step. One and two peaks corresponding to I3:22(i4,i4,n14) were detected in these assays conducted at pH 6.0 and 8.0, respectively (Figure 3.3B), and the peak exclusive to the pH 8.0 reaction coeluted with plant produced I3:22(i4,i4,n14) (Figure 3.3B and S3.3). This result is consistent with the hypothesis that acyl chain rearrangement is involved in acylinositol biosynthesis. 313 Figure 3.3. Analysis of di- and triacylinositol in vitro assay products at pH 6.0 or pH 8.0. (A) LC-MS analysis of I2:8(i4,i4) in vitro assay products created in one-pot reactions with SmASAT1-L and SmASAT3-L7 at pH 6.0 or pH 8.0. SmASAT3-I reverse assay product generated from I3:22(i4,i4,3-OH-i14). Forward assay products generated with SmASAT1-L and SmASAT3-L7 at pH 6.0 (top) and pH 8.0 (middle) with a heat inactivation following the first reaction. (B) LC-MS analysis of I3:22(i4,i4,n14) in vitro assay products generated in a two-step assay with SmASAT1-L, SmASAT3-L7, and SmASAT3-I. The first step included SmASAT1-L, SmASAT3-L7, and iC4-CoA generating I2:8(i4,i4). After a heat inactivation step, SmASAT3-I and nC14-CoA were added generating I3:22(i4,i4,n14). Reactions were conducted at pH 6.0 (middle) or pH 8.0 (bottom) and their products were compared to purified I3:22(i4,i4,n14) (top). Evidence for S. quitoense ASAT3-I homolog in acylinositol biosynthesis The identification of an eggplant acyltransferase not orthologous or paralogous to previously characterized ASATs suggested a possible explanation for the incomplete S. quitoense in vitro acylinositol pathway and led us to hypothesize the involvement of a SmASAT3-I homolog in synthesis of S. quitoense acylinositols. A tBLASTn search of 314 SmASAT3-I against the S. quitoense transcriptome (Moghe et al., 2017) identified the homologous transcript c25595_g1 (SqASAT3-INOSITOL, SqASAT3-I). This transcript was >340-fold enriched in trichome compared with shaved petiole, and highly expressed in glandular trichomes at 12,000 average reads (Figure 3.4A) (Moghe et al., 2017). I tested the hypothesis that SqASAT3-I is involved in acylinositol biosynthesis with in vitro ‘reverse’ assays. Consistent with this hypothesis, the heterologously expressed SqASAT3-I gene product catalyzed the removal of a nC10 acyl chain from I3:22(2,10,10) to create I2:12(2,10) (Figure 3.5). Similarly, SqASAT3-I gene expression knockdown, validated by qPCR (Figure S3.1), resulted in a statistically significant decrease in I3:22(2,10,10) and increase in I2:12(2,10) abundance (Figure 3.4C-F). These results support the hypothesis that SqASAT3-I is the third acylating enzyme in S. quitoense acylinositol biosynthesis. 315 Figure 3.4. VIGS of ASAT3-I in S. quitoense. (A) The transcript c25595_g1 (SqASAT3-I) is highly expressed in glandular trichomes, similar to previously characterized S. quitoense ASATs (Leong et al., 2022, 2020). Expression data, rounded to two significant figures are derived from Moghe et al. (2017) and describe expression in isolated glandular trichomes and trichome-less petioles. (B) Plants with silenced phytoene desaturase gene expression were used as visual markers for when tissue from the experimental plants should be collected. (C-F) Acylsugar analysis of c25595_g1-targeted and empty vector plants. (C-D) Comparison of I2:12(2,10) LC- MS response between c25595_g1 targeted plants and empty vector control plants with (C) and 316 Figure 3.4. (cont’d) (D) representing data from two independent experiments. (E-F) Comparison of total acylsugar LC-MS response between c25595_g1 targeted plants and empty vector control plants with (E) and (F) representing data from two independent experiments. Acylsugar peak area in (C-F) were normalized to the internal standard telmisartan and leaf tissue dry weight (DW). ***P < 0.001; ****P < 0.0001. Statistical comparisons were conducted with Welch’s two-sample t test. For experiment one (C, E), c25595_P1 targeted leaf samples, n = 28; empty vector leaf samples n = 16. For experiment two (D, F), c25595_P1 targeted leaf samples, n = 42; c25595_P2 targeted leaf samples, n = 48; empty vector leaf samples, n = 40. Figure 3.5. SqASAT3-I acts in the reverse direction to remove a C10 acyl chain from I3:22(2,10,10). Both extracted ion chromatograms display the formate adduct of I2:12(2,10), 421.21 m/z. (A) LC-MS analysis of SqASAT3-I reverse reaction products when supplied with free CoA and I3:22(2,10,10). (B) The I2:12(2,10) SqASAT3-I reverse reaction product in (A) coelutes with the plant-produced I2:12(2,10). Knockdown of ASAT1-L and ASAT3 homologs in S. nigrum supports a shared acylinositol pathway I tested if the S. nigrum acylinositol pathway is similar to that in S. quitoense and S. melongena by knocking down gene expression of S. nigrum ASAT1-L and ASAT3 homologs using an established and highly effective VIGS method (Lou et al., 2021). The S. nigrum homologs of SmASAT1-L and SmASAT3 were identified with BLAST searches yielding the transcripts c64578_g1, SnASAT1-L, and c71009_g1, SnASAT3, both of which were highly expressed and enriched in glandular trichome tissue compared to shaved petioles (Table 3.1). 317 SnASAT1-L and SnASAT3 gene expression knockdown, validated by qPCR (Figure S3.2), resulted in a statistically significant decrease in the ratio of acylinositols to acylglucoses (Figure 3.6). Noting that S. nigrum resides in DulMo, a different major clade than the Clade II members S. melongena and S. quitoense (Gagnon et al., 2022), these collective results support a acylinositol pathway shared between the Solanum major clades of DulMo and Clade II with a common evolutionary origin. Table 3.1. Tissue-specific expression of characterized S. nigrum ASATs, and ASAT1-L and ASAT3. Expression data are derived from Moghe et al., (2017). The genes SnASAT1, SnASAT2, and SnAGAT1 were previously characterized (Lou et al., 2021). Transcript name Average trichome reads SnASAT1 SnASAT2 SnAGAT1 SnASAT1-L SnASAT3 2382 1416 3408 1397 1815 Average shaved petiole reads 11 17 31 11 61 Figure 3.6. VIGS of ASAT1-L and ASAT3 in S. nigrum. The ratio of acylinositol and acylglucose responses were compared between ASAT targeted plants, SnASAT1-L and 318 Figure 3.6. (cont’d) SnASAT3, and empty vector control plants. To calculate the acylinositol to acylglucose ratio, acylsugar peak area was normalized to the internal standard telmisartan. Values for acylinositols (I3:16, I3:17, I3:18) and acylglucoses (G2:12, G2:13, G2:14, G2:15, G3:14, G3:15, G3:16, G3:17) were then summed. ****P < 0.0001. Statistical comparisons were conducted with Welch’s two-sample t test. Samples from 16 plants were collected for each VIGS vector. Discussion Aided by extensive available genetic resources and remarkable small molecule diversity, the Solanaceae family is a model system for studying specialized metabolism evolution. Harnessing the Solanaceae family helped uncover evolutionary mechanisms and biosynthetic pathways underlying terpene, steroidal glycoalkaloid, tropane alkaloid, and acylsugar diversity (Fiesel et al., 2022). In acylsugar biosynthesis, gene loss, duplication, and neofunctionalization were demonstrated to drive acylsugar phenotypic differences (Fan et al., 2017; Schilmiller et al., 2015). Application of this system uncovered the putative S. melongena, S. quitoense, and S. nigrum acylinositol pathways and identified familiar mechanisms underlying acylsugar pathway evolution. Here, I describe evidence for an in vitro triacylinositol pathway using S. melongena enzymes, which produces a triacylinositol that coelutes with a plant-produced triacylinositol. I transferred this putative pathway to two non-model species, taking advantage of available transcriptomic and gene silencing resources. In S. quitoense, I identified a previously undescribed enzyme, SqASAT3-I, which can perform the third acylation in vitro. In S. nigrum, I obtained genetic evidence that SnASAT1-L and SnASAT3 are involved in acylinositol biosynthesis suggesting existence of a biosynthetic pathway shared between S. melongena, S. quitoense, and S. nigrum. 319 Non-enzymatic rearrangement may play a role in acylinositol biosynthesis Non-enzymatic acyl chain rearrangement is required for the outlined S. melongena in vitro pathway, and I hypothesize that this rearrangement occurs in planta based on three lines of reasoning. First, of the seven S. melongena BAHDs with the highest trichome gene expression, no enzyme produced detectable levels of I1:4 from the reverse assay-produced I2:8(i4,i4). Additionally, acyl chain rearrangement occurs at pH values >7 which fall within reported plant cytosolic pH values (Moseyko and Feldman, 2001; Shen et al., 2013), although, it is important to note that glandular trichomes are atypical plant cells and could have an atypical acidic cytosolic pH which limits acyl chain rearrangement. While unusual, non-enzymatic reactions including intramolecular rearrangement were described in other natural product pathways such as in the synthesis of the bacterially-derived cladoniamides D-G and siderophore compounds (Bouthillette et al., 2022; Du et al., 2014; Wuest et al., 2009). Further work characterizing the site of acylsugar biosynthesis and the site’s pH may provide evidence supporting this rearrangement hypothesis. SqASAT3-I catalyzes the third acylation in the proposed S. quitoense acylinositol pathway Two lines of evidence support the hypothesis that SqASAT3-I catalyzes the medium chain acylation of diacylinositols. VIGS-mediated SqASAT3-I gene expression reduction led to decreased acylsugar accumulation and an increase in the accumulation of the diacylinositol I2:12(2,10). Additionally, heterologously expressed SqASAT3-I protein catalyzed the reverse reaction to remove a C10 chain from purified I3:22(2,10,10) to produce I2:12(2,10). These data are consistent with the hypothesis that SqASAT3-I is the third acylating enzyme in S. quitoense acylinositol biosynthesis. 320 Acylinositol biosynthesis is distinct from acylsucrose biosynthesis and evolved through multiple evolutionary mechanisms My results and the previous S. quitoense acylinositol investigations (Fiesel et al., 2023; Leong et al., 2022, 2020) suggest acylinositols arose through multiple evolutionary changes creating a distinctly different pathway from characterized acylsucrose pathways. Our data are consistent with the hypothesis that gene duplication generated additional ASAT1 copies, resulting in the duplicate ASAT1-L exhibiting divergent inositol acylating activity from ASAT1 (Leong et al., 2022; Lou et al., 2021). The relaxed selection upon ASAT1-L may have enabled the enzyme’s acyl acceptor specificity to shift to another common sugar, myo-inositol. This story of duplication leading to altered acylsugar phenotypes is common. For example, the wild tomato Solanum pennellii LA0716 ‘flipped’ acylsucrose pathway evolved in part because of a duplicated ASAT3 copy (Fan et al., 2017). This ASAT3 duplicate exhibits altered acyl acceptor specificity to acylate monoacylsucroses as well as the 2-position on the pyranose sucrose ring rather than the 3’-position on the furanose sucrose ring. These changes as well as SpASAT2 protein sequence changes resulted in production of acylsucroses acylated at different positions than the cultivated tomato acylsucroses. In our model, acylinositol biosynthesis includes a co-opted Clade III BAHD, ASAT3-I, which is phylogenetically distinct from other characterized ASATs. Previous phylogenetic analysis revealed that ASAT3-I resides within a clade consisting of 16 S. melongena BAHDs and CaPun1, capsaicin synthase, which is the only characterized gene in this BAHD subgroup (Chapter 2; Moghe et al., 2023). CaPun1 is predicted to catalyze a nitrogen acylation unusual for Clade III BAHDs (Moghe et al., 2023; Stewart Jr et al., 2005). ASAT3-I also exhibits atypical acylating activity but with an OH-C14 acyl chain (Figure 3.2), suggesting that the co-option of 321 ASAT3-I was involved in the evolution of acylsugar hydroxyacyl chains. Analysis of enzymes related to ASAT3-I and CaPun1 may identify further atypical enzymatic activities underlying plant specialized metabolites. The proposed pathways provide a starting point for investigating the enormous interspecific acylinositol structural differences identified in Chapter 2. In fact, analysis of only two species – S. melongena and S. quitoense – led me to document significant divergences in their proposed biosynthetic pathways. For example, unlike the proposed S. melongena pathway, the proposed S. quitoense pathway begins with a medium chain acylation and produces acylsugars with only C2, C10, and C12 acyl chains. Acylinositol structural differences with unidentified biochemical mechanisms include the contrasting acylinositol disaccharide core structures between S. melongena and S. quitoense and acylation position differences between S. melongena and S. nigrum. Investigating these differences equipped with the transcriptomic resources and gene knockdown/knockout techniques for these three species will better our understanding of how acylsugars evolve (Chapter 2) (Hurney, 2018; Lou et al., 2021). Synthetic biology application of acylinositol pathway enzymes The acylinositol biosynthetic enzymes discovered here and previously described (Leong et al., 2022, 2020) will be useful synthetic biology tools in understanding the how different sugar cores impact acylsugar biological activities as well as generating new acylsugars not observed in plants. Our current understanding of how different acylsugar traits impact plant defense can be expanded with tools created through synthetic biology. For example, transgenic production of near-isogenic lines with varied acylsugar structures will accelerate the study of the relationship between acylsugar structures and their protective functions. Additionally, acylsugar biosynthetic enzymes can be mixed and matched to produce new acylsugars not observed in plants potentially 322 producing compounds with varied bioactivities against different plant pests. This approach has been validated in vitro and its utilization in planta may increase plant resilience against pests (Schenck et al., 2022). Methods Gene cloning, heterologous protein expression and purification All S. melongena candidate genes were cloned into pET28b as described in Chapter 2. SqASAT3-I was cloned into pET28b(+) (MilliporeSigma, Burlington, MA, USA) for protein expression. The SqASAT3-I coding sequence was amplified from S. quitoense cDNA with primers listed in Table S3.1 and Q5 2X Hotstart master mix (New England Biolabs, Ipswich, MA, USA), and the PCR amplicon was purified by agarose gel separation and extraction with the Monarch DNA Gel Extraction Kit (New England Biolabs). Using 2X Gibson Assembly Master Mix (New England Biolabs), the c25595_g1 PCR amplicon was inserted into a doubly digested BamHI/XhoI pET28b(+) through Gibson assembly according to the manufacturer’s instructions. The construct was transformed into BL21 Rosetta(DE3) cells (MilliporeSigma) and verified by colony PCR and Sanger sequencing using T7 promoter and terminator primers (Table S3.1). Michigan State University Research Technology Support Facility Genomics Core (East Lansing, MI, USA) performed the Sanger sequencing. The remaining S. melongena and S. quitoense candidate genes were cloned into pET28b as described previously (Chapter 2; Leong et al., 2022, 2020). Candidate enzymes were expressed as previously described (Chapter 2). In brief, 50 mL LB cultures with 1% glucose (w/v) were inoculated from transformation colonies and incubated overnight at 37⁰C and 225 rpm. Secondary 1 L cultures were inoculated with 15 mL of the overnight culture and were incubated at 37⁰C and 225 rpm. After an OD600 of 0.5 was reached, 323 the cultures were chilled on ice for 25 min and isopropylthio-β-galactoside was added to a final concentration of 50 µM, except for SmASAT3-L7 which had a final concentration of 25 µM. Cultures were then incubated for 16 hours at 16⁰C and 180 rpm. Cells were harvested by centrifugation at 4,000 rpm for 10 minutes at 4⁰C. S. melongena BAHDs were purified as described in Chapter 2. S. quitoense BAHDs were purified following the protocol described for SaASAT3-L1 in Chapter 2. Enzyme assays Enzyme assays were conducted similar to those in Chapter 2 and in Leong et al., (2022). All assays were buffered in 100 mM sodium phosphate buffer at pH 6.0 or 8.0 as noted. For forward assays, acyl-CoAs (MilliporeSigma) were supplied at a final concentration of 0.1 mM each, myo-inositol was added to a final concentration of 10 mM. For reverse assays, free CoA (MilliporeSigma, Burlington, MA, USA) was added to a final concentration of 1 mM. Purified acylsugar substrates were dried down using a vacuum centrifuge and redissolved in ethanol:water:formic acid (1:1:0.001). One microliter of the prepared acylsugars were used as acyl acceptors. Six microliters of each enzyme were added to a final volume of 60 µL. For negative controls, 6 µL of enzyme that was heat inactivated at 95°C for 10 minutes was substituted in place of untreated enzyme. In general, assays were incubated at 30°C for 30 minutes, unless noted otherwise. For two-step assays, the first step substrates were incubated at 30°C for 30 minutes, then heat-inactivated at 65C for 10 minutes. Then, the second step reagents were added and incubated at 30°C for 30 minutes. After incubation, 120 µL of acetonitrile:isopropanol:formic acid (1:1:0.001) with 1.5 µM telmisartan (MilliporeSigma) stop solution was added. Reactions were then spun at 17,000 x g for 10 minutes to remove precipitate. Supernatant was placed in autosampler vials and analyzed by LC-MS. 324 VIGS analysis Constructs for VIGS targeting c25595_g1, SnASAT1-L, and SnASAT3 were assembled by adaptation of a previously published method (Dong et al., 2007). pTRV2-LIC was linearized through PstI digestion and subsequent purification from a 1% agarose gel using the New England Biolabs Monarch gel purification kit. The c25595_g1 VIGS target sequences were PCR amplified from S. quitoense cDNA using primers with adaptors for ligation (Table S3.1). The SnASAT1-L and SnASAT3 target sequences were PCR amplified from S. nigrum leaf cDNA using primers with adaptors for ligation (Table S3.1). In separate 5 µL reactions, The PCR amplicons and linearized vector were incubated with 1X NEB 2.1 reaction buffer, 5 mM dNTP (dATP for PCR product; dTTP for linearized pTRV2-LIC), and T4 polymerase (New England Biolabs) for 30 min at 22⁰C followed by 20 min at 70⁰C. One µL and 2 µL of the pTRV2-LIC and PCR amplicon reactions, respectively, were mixed together and incubated for 2 min at 65⁰C followed by 10 min at 22⁰C. The constructs were then transformed into chemically competent cells, Top10 (ThermoFisher Scientific, Waltham, MA, USA) following manufacturer’s protocol. S. quitoense seedlings were inoculated following a modified protocol (Leong et al., 2022; Velásquez et al., 2009). S. quitoense seeds were sterilized with 10% bleach (v/v) for 10 minutes while being rocked at 24 rpm with a GyroMini nutating mixer (Labnet, Edison, NJ, USA), and subsequently rinsed with distilled, sterile water 5-6 times. Seeds were soaked in sterile 1000 ppm Giberellin A3 (GoldBio, St. Louis, MO, USA) overnight and then transferred to Whatman filter paper (MilliporeSigma). Seeds were stored in the dark at room temperature for 7 days and then transferred to peat pots (Jiffy, Zwijndrecht, Netherlands). Peat pots were incubated at 25°C, 16/8-h day/night light cycle, and ~70 μmol m-2s-2 photosynthetic photon flux density with cool white fluorescent bulbs. Peat pots were covered with a humidity dome, 22 x 11 x 3 inches 325 (Growers Supply Company) for the first five days. When seedlings had one true leaf and were about an inch tall, inoculation cultures were prepared following Velásquez et al. (2009) with modifications described by Leong et al. (2022). Two independent VIGS experiments were conducted with the c25595_P1 construct used in the first experiment and both c25595_P1 and c25595_P2 constructs used in the second experiment. Gene expression of SnASAT1-L and SnASAT3 in S. nigrum was silenced following a previously developed protocol (Lou et al., 2021) which adapted a published vacuum infiltration protocol (Hartl et al., 2008). Acylsugar abundances in VIGS leaf surface extracts were quantified by LC-MS with the QuanLynx function in MassLynx v4.1 (Waters Corporation, Milford, MA, USA) as previously described (Lou et al., 2021; Lybrand et al., 2020). In short, QuanLynx generated extracted ion chromatograms for the formate adduct of each acylsugar with a mass window of m/z of 0.05. Peak areas were integrated and normalized to the peak area of the telmisartan internal standard to create acylsugar response values. For the S. quitoense VIGS data, the acylsugar response values were then normalized to the dry weight of extracted tissue for each sample. Acylsugar extractions Acylsugars were extracted from VIGS plants as previously described (Leong et al., 2019; Lou and Leong, 2019). Briefly, half of a leaf was placed into a 1.5 mL tube (Dot Scientific, Inc., Burton, MI, USA) containing 1 mL extraction solvent (3:3:2 acetonitrile:isopropanol:water, 0.1% formic acid, 1 µM telmisartan (internal standard) (MilliporeSigma)). The extractions were rocked at 24 rpm for 2 min with a GyroMini nutating mixer (Labnet). Solvent was extracted and placed in a 2 mL glass autosampler vial (Restek, Bellefonte, PA, USA) and sealed with a 9 mm cap with a PTFE/silicone septum (J.G. Finneran, Vineland, NJ, USA). 326 VIGS qPCR analysis qPCR analysis was conducted following a modified protocol (Leong et al., 2022, 2020; Lou et al., 2021). RNA was extracted from VIGS leaf samples with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) with on-column DNase digestion (Qiagen) following the manufacturer's instructions. RNA was quantified with a Nanodrop 2000c instrument (ThermoFisher Scientific). cDNA was synthesized from 1 µg of RNA and SuperScript II Reverse Transcriptase for S. nigrum samples and SuperScript III Reverse Transcriptase for S. quitoense samples. S. quitoense cDNA was diluted 100-fold, and S. nigrum cDNA was then diluted 40-fold. S. quitoense qPCRs used SYBR Power Green PCR Master Mix (ThermoFisher Scientific) while S. nigrum qPCRs used SYBR Green PCR Master Mix (ThermoFisher Scientific). RT_c25595_1_F/R, RT_SnASAT1-L_1_F/R, RT_SnASAT3_1/2_F/R, RT_Sq_ACTIN_1_F/R, RT_Sq_EF1A_1_F/R, RT_Sn_ACTIN1_F/R, and RT_Sn_ACTIN3_F/R primers were used to detect c25595_g1, SnASAT1-L, SnASAT3, SqACTIN, SqEF1α, SnACTIN1, and SnACTIN3 transcripts, respectively, at a final concentration of 200 nM (Table S3.1). Primers had amplification efficiencies within 90-110%. Michigan State University Research Technology Support Facility Genomics Core (East Lansing, MI, USA) carried out the qPCRs using a QuantStudio 7 Flex Real-Time PCR System (Applied Bio-systems). The following temperature cycling conditions were used: 50⁰C for 2 min, 95⁰C for 10 min, and 40 cycles of 95⁰C for 14 s and 60⁰C for 1 min. Relative expression values for c25595_g1, SnASAT1-L, and SnASAT3 were calculated with the ΔΔCT method and normalized to the geometric mean of housekeeping gene transcript levels, ACTIN and EF1α for S. quitoense and ACTIN1 and ACTIN3 for S. nigrum, and the mean expression values from empty vector plants. Three to four technical replicates were used for all qPCRs. 327 LC-MS analysis In vitro and in planta acylsugars were analyzed by LC-MS with the methods described below. For all analyses, 10 µL of sample were injected into a column kept at 40⁰C, using binary solvent gradients with a flow rate of 0.3 mL/min, unless otherwise noted. VIGS plant extracts were analyzed with a 7-min LC gradient using a LC-20ADvp ternary pump (Shimadzu, Kyoto, Japan) coupled to a Waters Xevo G2-XS QToF mass spectrometer (Waters Corporation) and equipped with an Ascentis Express C18 HPLC column (10 cm x 2.1 mm, 2.7 µm; Supelco). The gradient with a flow rate of 0.4 mL/min was as follows: 5% B at 0 min, 60% B at 1 min, 100% B at 5 min, held at 100% B until 6 min, 5% B at 6.01 min, held at 5% B until 7 min. Ions were acquired from m/z 50 to 1200 with a scan time of 0.1 s and three acquisition functions with different collision potentials (0, 25, 60 V). Lock mass calibration referenced to the leucine enkephalin [M+H]- ion was applied during data acquisition. The ESI- parameters were as follows: capillary voltage, 2 kV; sampling cone voltage, 60 V; source temperature, 100°C; desolvation temperature 350°C; cone gas flow, 50 L/Hr; desolvation gas flow, 600 L/Hr. Enzyme assay products were analyzed with a Waters Acquity UPLC coupled to a Waters Xevo G2-XS QToF mass spectrometer (Waters Corporation) equipped with electrospray ionization (ESI) source. All enzyme assays used binary solvent gradients with 100% acetonitrile as solvent A and 0.1% formic acid in water as solvent B. Enzyme assay products were analyzed with a general 7-min gradient described in Chapter 2 and Table S2.29, unless noted otherwise. The I2:8(i4,i4) enzyme assays shown in Figure 3.1 and 3.6 used a modified 7-min gradient as follows: 2% B at 0 min, 30% B at 3 min, 99% B at 4 min, held at 99% B until 5 min, 2% B at 5.01 min, held at 2% B until 7 min. The I1:4(i4) enzyme assays shown in Figure S3.4 were 328 analyzed with a 9-min gradient using an Acquity BEH Amide column (10 cm x 2.1 mm, 130 Å, 1.7 µm; Waters) which was as follows: 95% B at 0 min held until 1 min, 60% B at 6 min, 5% B at 7 min, 95% B at 7.01 min, held at 95% B until 9 min. For coelution analysis between enzymatically- and plant-produced I3:18(4,4,10), a 14- min linear gradient was used with an Acquity UPLC BEH C18 column (10 cm x 2.1 mm, 130 Å, 1.7 µm; Waters), kept at 40⁰C, on the same instrument used for enzyme assay analysis. The binary solvent, linear gradient was as follows: 5% B at 0 min, 40% B at 2 min, 90% B at 10 min, 100% B at 10.01 min, held at 100% B until 12 min, 5% B at 12.01 min, held at 5% B until 14 min. For coelution analysis between enzymatically- and plant-produced I3:22(i4,i4,n14), a 14- min linear gradient was used with an Acquity UPLC BEH C18 column (10 cm x 2.1 mm, 130 Å, 1.7 µm; Waters), kept at 40⁰C, on the same instrument used for enzyme assay analysis. The binary solvent, linear gradient was as follows: 5% B at 0 min, 60% B at 2 min, 70% B at 8 min, 100% at 10 min, held at 100% B until 12 min, 5% B at 12.01 min, held at 5% B until 14 min. For all enzyme assays, the following ESI- parameters were used: capillary voltage, 2 kV; sampling cone voltage, 35 V; source temperature, 100°C; desolvation temperature 350°C; cone gas flow, 50 L/Hr; desolvation gas flow, 600 L/Hr. Ions were acquired from m/z 50 to 1200 with a scan time of 0.1 s and three acquisition functions with different collision potentials (0, 25, 60 V). Lock mass calibration referenced to the leucine enkephalin [M+H]- ion was applied during data acquisition. Acknowledgements I acknowledge Dr. Yann-Ru Lou for her indispensable collaboration with the S. nigrum VIGS experiments. I also acknowledge Dr. Bryan J. Leong for his work cloning SqASAT3-I into 329 pET28b. I acknowledge the Michigan State University RTSF Mass Spectrometry and Metabolomics Core Facilities for LC-MS analysis support. 330 REFERENCES Bouthillette, L.M., Aniebok, V., Colosimo, D.A., Brumley, D., MacMillan, J.B., 2022. Nonenzymatic reactions in natural product formation. Chem. Rev. 122, 14815–14841. https://doi.org/10.1021/acs.chemrev.2c00306 Dong, Y., Burch-Smith, T.M., Liu, Y., Mamillapalli, P., Dinesh-Kumar, S.P., 2007. A ligation- independent cloning tobacco rattle virus vector for high-throughput virus-induced gene silencing identifies roles for NbMADS4-1 and -2 in floral development. Plant Physiol. 145, 1161–1170. https://doi.org/10.1104/pp.107.107391 Du, Y.-L., Williams, D.E., Patrick, B.O., Andersen, R.J., Ryan, K.S., 2014. Reconstruction of cladoniamide biosynthesis reveals nonenzymatic routes to bisindole diversity. ACS Chem. Biol. 9, 2748–2754. https://doi.org/10.1021/cb500728h Fan, P., Miller, A.M., Liu, X., Jones, A.D., Last, R.L., 2017. Evolution of a flipped pathway creates metabolic innovation in tomato trichomes through BAHD enzyme promiscuity. Nat. Commun. 8, 2080. https://doi.org/10.1038/s41467-017-02045-7 Fan, P., Miller, A.M., Schilmiller, A.L., Liu, X., Ofner, I., Jones, A.D., Zamir, D., Last, R.L., 2015. In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network. Proc. Natl. Acad. Sci. U.S.A. 113, E239-248. https://doi.org/10.1073/pnas.1517930113 Fiesel, P.D., Kerwin, R.E., Jones, A.D., Last, R.L., 2023. Trading acyls and swapping sugars: metabolic innovations in Solanum trichomes. bioRxiv https://doi.org/10.1101/2023.06.05.542877 Fiesel, P.D., Parks, H.M., Last, R.L., Barry, C.S., 2022. Fruity, sticky, stinky, spicy, bitter, addictive, and deadly: evolutionary signatures of metabolic complexity in the Solanaceae. Nat. Prod. Rep. 39, 1438–1464. https://doi.org/10.1039/D2NP00003B Gagnon, E., Hilgenhof, R., Orejuela, A., McDonnell, A., Sablok, G., Aubriot, X., Giacomin, L., Gouvêa, Y., Bragionis, T., Stehmann, J.R., Bohs, L., Dodsworth, S., Martine, C., Poczai, P., Knapp, S., Särkinen, T., 2022. Phylogenomic discordance suggests polytomies along the backbone of the large genus Solanum. Am. J. Bot. 109, 580–601. https://doi.org/10.1002/ajb2.1827 Hartl, M., Merker, H., Schmidt, D.D., Baldwin, I.T., 2008. Optimized virus-induced gene silencing in Solanum nigrum reveals the defensive function of leucine aminopeptidase against herbivores and the shortcomings of empty vector controls. New Phytol. 179, 356– 365. https://doi.org/10.1111/j.1469-8137.2008.02479.x Hurney, S.M., 2018. Strategies for profiling and discovery of acylsugar specialized metabolites (Ph.D.). Michigan State University, United States -- Michigan. Koonin, E.V., 2005. Orthologs, paralogs, and evolutionary genomics. Annu. Rev. Genet. 39, 309–338. https://doi.org/10.1146/annurev.genet.39.073003.114725 331 Leckie, B.M., D’Ambrosio, D.A., Chappell, T.M., Halitschke, R., De Jong, D.M., Kessler, A., Kennedy, G.G., Mutschler, M.A., 2016. Differential and synergistic functionality of acylsugars in suppressing oviposition by insect herbivores. PLoS ONE 11, e0153345. https://doi.org/10.1371/journal.pone.0153345 Leong, B.J., Hurney, S., Fiesel, P., Anthony, T.M., Moghe, G., Jones, A.D., Last, R.L., 2022. Identification of BAHD acyltransferases associated with acylinositol biosynthesis in Solanum quitoense (naranjilla). Plant Direct 6, e415. https://doi.org/10.1002/pld3.415 Leong, B.J., Hurney, S.M., Fiesel, P.D., Moghe, G.D., Jones, A.D., Last, R.L., 2020. Specialized metabolism in a nonmodel nightshade: trichome acylinositol biosynthesis. Plant Physiol. 183, 915–924. https://doi.org/10.1104/pp.20.00276 Leong, B.J., Lybrand, D.B., Lou, Y.-R., Fan, P., Schilmiller, A.L., Last, R.L., 2019. Evolution of metabolic novelty: a trichome-expressed invertase creates specialized metabolic diversity in wild tomato. Sci. Adv. 5, eaaw3754. https://doi.org/10.1126/sciadv.aaw3754 Lou, Y.-R., Anthony, T.M., Fiesel, P.D., Arking, R.E., Christensen, E.M., Jones, A.D., Last, R.L., 2021. It happened again: convergent evolution of acylglucose specialized metabolism in black nightshade and wild tomato. Sci. Adv. 7, eabj8726. https://doi.org/10.1126/sciadv.abj8726 Lou, Y.-R., Leong, B., 2019. Leaf surface acylsugar extraction and LC-MS profiling - v1.0. Lybrand, D.B., Anthony, T.M., Jones, A.D., Last, R.L., 2020. An integrated analytical approach reveals trichome acylsugar metabolite diversity in the wild tomato Solanum pennellii. Metabolites 10, 1–25. https://doi.org/10.3390/metabo10100401 Moghe, G., Kruse, L.H., Petersen, M., Scossa, F., Fernie, A.R., Gaquerel, E., D’Auria, J.C., 2023. BAHD company: the ever-expanding roles of the BAHD acyltransferase gene family in plants. Annu. Rev. Plant Biol. 74, annurev-arplant-062922-050122. https://doi.org/10.1146/annurev-arplant-062922-050122 Moghe, G.D., Leong, B.J., Hurney, S.M., Jones, A.D., Last, R.L., 2017. Evolutionary routes to biochemical innovation revealed by integrative analysis of a plant-defense related specialized metabolic pathway. eLife 6, 1–33. https://doi.org/10.7554/eLife.28468 Moseyko, N., Feldman, L.J., 2001. Expression of pH-sensitive green fluorescent protein in Arabidopsis thaliana. Plant Cell Environ. 24, 557–563. https://doi.org/10.1046/j.1365- 3040.2001.00703.x Puterka, G.J., Farone, W., Palmer, T., Barrington, A., 2003. Structure-function relationships affecting the insecticidal and miticidal activity of sugar esters. J. Econ. Entomol. 96, 636– 644. https://doi.org/10.1093/jee/96.3.636 Schenck, C.A., Anthony, T.M., Jacobs, M., Jones, A.D., Last, R.L., 2022. Natural variation meets synthetic biology: Promiscuous trichome-expressed acyltransferases from Nicotiana. Plant Physiol. kiac192. https://doi.org/10.1093/plphys/kiac192 332 Schilmiller, A.L., Moghe, G.D., Fan, P., Ghosh, B., Ning, J., Jones, A.D., Last, R.L., 2015. Functionally divergent alleles and duplicated Loci encoding an acyltransferase contribute to acylsugar metabolite diversity in Solanum trichomes. Plant Cell 27, 1002–17. https://doi.org/10.1105/tpc.15.00087 Shen, J., Zeng, Y., Zhuang, X., Sun, L., Yao, X., Pimpl, P., Jiang, L., 2013. Organelle pH in the Arabidopsis endomembrane system. Mol. Plant 6, 1419–1437. https://doi.org/10.1093/mp/sst079 Stewart Jr, C., Kang, B.-C., Liu, K., Mazourek, M., Moore, S.L., Yoo, E.Y., Kim, B.-D., Paran, I., Jahn, M.M., 2005. The Pun1 gene for pungency in pepper encodes a putative acyltransferase. Plant J. 42, 675–688. https://doi.org/10.1111/j.1365-313X.2005.02410.x Velásquez, A.C., Chakravarthy, S., Martin, G.B., 2009. Virus-induced gene silencing (VIGS) in Nicotiana benthamiana and tomato. JoVE J. Vis. Exp. e1292. https://doi.org/10.3791/1292 Wuest, W.M., Sattely, E.S., Walsh, C.T., 2009. Three siderophores from one bacterial enzymatic assembly line. J. Am. Chem. Soc. 131, 5056–5057. https://doi.org/10.1021/ja900815w 333 APPENDIX Figure S3.1. Relative transcript abundance of SqASAT3-I in VIGS plants. (A) SqASAT3-I relative transcript abundance from leaf three (left) and leaf four (right). c25595 P1, n = 16; empty 334 Figure S3.1. (cont’d) vector, n = 8. (B) SqASAT3-I relative transcript abundance from plants with two different gene fragments targeting SqASAT3-I, c25595 P1 (left) and c25595 P2 (right). c25595 P1, n = 12; c25595 P2, n = 12; empty vector, n = 20. **P < 0.01, ****P <0.0001. Statistical comparisons were conducted with Welch’s two-sample t test. 3-4 technical replicates were used for each sample. 335 Figure S3.2. Relative transcript abundance of SnASAT1-L and SnASAT3 in VIGS plants. SnASAT1-L was targeted in plants with two different constructs, SnASAT1-L P1 (top left) and SnASAT1-L P2 (top right). SnASAT3 was targeted in plants with two different constructs, SnASAT3 P1 (bottom left) and SnASAT3 P2 (bottom right). Top left: SnASAT1-L P1, n = 16; empty vector, n = 15. Top right: SnASAT1-L P2, n = 7; empty vector, n = 16. Bottom left: 336 Figure S3.2. (cont’d) SnASAT3 P1, n = 16; empty vector, n = 16. Bottom right: SnASAT3 P2, n = 14; empty vector, n = 16. *P < 0.05, ****P <0.0001. Statistical comparisons were conducted with Welch’s two- sample t test. 3-4 technical replicates were used for each sample. 337 Figure S3.3. Coelution analysis of I3:22(i4,i4,n14) in vitro assay product. Extracted ion chromatograms display the formate adduct of I3:22, 575.35 m/z. The enzymatically-produced I3:22 was synthesized in a two-step enzyme reaction with SmASAT1-L, SmASAT3-L7, myo- inositol, and iC4-CoA in the first step. After a heat-inactivation, SmASAT3-I and nC14-CoA were added for the second step incubation. 338 Figure S3.4. SmASAT1-L and SmASAT3 acylate myo-inositol with iC4-CoA. Extracted ion chromatograms display the chloride adduct of I1:4(i4), 285.074 m/z. Products were analyzed with the 9 min BEH amide LC method. Assays were conducted at pH 8.0. 339 Figure S3.5. SmASAT1-L acylates myo-inositol with medium length acyl chains. nC8- (top left), nC10- (top right), nC12- (bottom left), nC14-CoAs (bottom left) were supplied to SmASAT1-L (purple traces) or boiled SmASAT1-L (gray traces) and myo-inositol. The top left chromatogram displays the formate adduct for I1:8(nC8), 351.17 m/z. The top right chromatogram displays the formate adduct for I1:10(nC10), 379.20 m/z. The bottom left chromatogram displays the formate adduct for I1:12(nC12), 407.22 m/z. The bottom right chromatogram displays the formate adduct for I1:14(nC14), 435.26 m/z. All reactions were conducted at pH 6.0. 340 Figure S3.6. I2:14(i4,n10) formed in one-pot assays with the enzyme combinations of SmASAT1-L+SmASAT3-L7 and SmASAT1-L+SmASAT3-L1. The forward assays were conducted at pH 6.0 and were analyzed with the general 7-min enzyme assay LC gradient. Extracted ion chromatograms display the formate adduct of I2:14(i4,n10), 449.24 m/z. 341 Figure S3.7. In two-step assays, SmASAT3 and SmASAT3-L1 acylate I2:14(i4,n10) to produce I3:18(i4,i4,n10) that does not coelute with any plant-produced I3:18. The first step included SmASAT1-L, SmASAT3-L7, iC4-CoA, and iC10-CoA generating I2:14(i4,n10). After a heat inactivation step, iC4-CoA and SmASAT3 or SmASAT3-L1 were added generating I3:18(i4,i4,n10). Reactions were conducted at pH 6.0 or pH 8.0 and their products were compared to I3:18 from a S. melongena plant extract (top). The largest peak in the plant extract is the NMR characterized I3:18(i4,i4,i10) and the smaller peak is likely I3:18(i4,i4,n10). This annotation of the smaller peak is supported by two pieces of evidence: acylinositols with straight medium acyl chains tend to elute after their isomers with iso-branched medium acyl chains (Chapter 2), and GC-MS analysis of acyl chain composition detected nC10 acyl chains (Chapter 2). Extracted ion chromatograms display the formate adduct of I3:18, 519.28 m/z. 342 Table S3.1. Oligonucleotides used in this study. Sequence name c25595_g1_F c25595_g1_F Sequence agcatgactggtggacagcaaatgggtcggATGTCATGTGTATATAAA GCTAGCTTCTTT gccggatctcagtggtggtggtggtggtgcAAATAATGTTCACCTTAG TAACGTTTTGAA T7_promoter TAATACGACTCACTATAGGG GCTAGTTATTGCTCAGCGG GCCAAGGGTCCATGTAACAAG TGAAGAAGTTGCTCGTCACG CGGATCCCGTAGAGATCAAA CATCCAAAGTCCATGTCACG GCCTTCTTCTACCCCAAACC TTCGGACATTGAACGATTGA AATGAGTGCGTGCATGTCAC CTGTAGCGGCCCAATCATTAATG GTGTGATGGTTGGCATGGGG GGTGTTCCTCAGGGGCAACA CTCAAGGCTGAGCGTGAACG GCACAGTCAGCCTGAGAGGT ACAATTGGTGCTGAGCGTTT TTTCAGGTGGAGCAACAACC TCACAGAGCGTGGTTACTCG CTGCTTCCATTCCGATCATT T7_terminator RT_c25595_1_F RT_c25595_1_R RT_SnASAT1-L_1_F RT_SnASAT1-L_1_R RT_SnASAT3_1_F RT_SnASAT3_1_R RT_SnASAT3_2_F RT_SnASAT3_2_R RT_Sq_ACTIN_1_F RT_Sq_ACTIN_1_R RT_Sq_EF1A_1_F RT_Sq_EF1A_1_R RT_Sn_ACTIN1_F RT_Sn_ACTIN1_R RT_Sn_ACTIN3_F RT_Sn_ACTIN3_R Sq_c25595_g1_i1_VIG S_01_F Sq_c25595_g1_i1_VIG S_01_R Description Sequence used for cloning into pET28b with BamHI/XhoI site Sequence used for cloning into pET28b with BamHI/XhoI site Primer for Sanger sequencing and colony PCR of pET28b plasmids Primer for Sanger sequencing and colony PCR of pET28b plasmids For qPCR analysis of c25595 expression For qPCR analysis of c25595 expression For qPCR analysis of SnASAT1-L expression For qPCR analysis of SnASAT1-L expression For qPCR analysis of SnASAT3 expression For qPCR analysis of SnASAT3 expression For qPCR analysis of SnASAT3 expression For qPCR analysis of SnASAT3 expression For qPCR analysis of SqActin expression For qPCR analysis of SqActin expression For qPCR analysis of SqEF1alpha expression For qPCR analysis of SqEF1alpha expression For qPCR analysis of SnActin1 expression For qPCR analysis of SnActin1 expression For qPCR analysis of SnActin3 expression For qPCR analysis of SnActin3 expression CGACGACAAGACCCTCATGGGCTGGAAGTCGAAGA Sequence used for cloning into pTRV2 GAGGAGAAGAGCCCTGCTAATGCAGCCCAATCCCT Sequence used for cloning into pTRV2 343 Table S3.1. (cont’d) Sequence used for cloning into pTRV2 Sq_c25595_g1_i1_VIGS_02_F CGACGACAAGACCCTTTCCTTTGGACCATCGCCAA Sq_c25595_g1_i1_VIGS_02_R GAGGAGAAGAGCCCTTTTGGGGGTAGCTGAGGTGA Sequence used for cloning into pTRV2 344 CHAPTER 4: CONCLUSIONS AND FUTURE DIRECTIONS 345 The Solanaceae family continues to be a rich resource for understanding metabolic evolution. In this dissertation, an enormous amount of acylsugar diversity was revealed within the Solanum genus making use of available germplasm. Utilizing this diversity helped uncover pieces of the acylinositol biosynthetic pathway, revealing familiar evolutionary mechanisms of gene duplication and loss leading to metabolic diversity. This research greatly expands our knowledge of plant diversity and provides exciting opportunities for uncovering further metabolic evolution. The acylsugar chemical analyses presented in Chapter 2 expand and redefine our understanding of acylsugar structural diversity. Acylsucroses were thought to be the predominant acylsugar type in contrast to the sporadically observed acylglucoses and rarely observed acylinositols, as described in Chapter 1 (Fiesel et al., 2022). Contrasting with this paradigm, I found acylinositols were widespread with the megadiverse Solanum genus, being detected in multiple major Solanum clades representing ~1100 of the c. 2700 Solanaceae species. In fact, I hypothesized that the largest Solanum clade, Clade II, lost acylsucrose biosynthesized as supported by a lack of detectable acylsucroses in this clade. Considering that acylsugars have been studied for decades, how did these widespread acylinositols go overlooked? A few reasons play a role in this. First, prior work in the Solanaceae has been biased by the tractable acylsucrose-producer cultivated tomato and other readily available ornamental plants such as Nicotiana, Salpiglossis, and Petunia species. In addition, the most cultivated acylinositol- producer, S. melongena, hardly produces glandular trichomes (Figure S2.12). This broad study of Solanum acylsugars not only showed that acylinositols are widespread but also uncovered 18 acyl chains and two new sugar cores not previously observed. With 33 acyl chains (Chapter 2, Lou et al., 2021; Leong et al., 2020; Herrera-Salgado et al., 2005) and six inositol acylation 346 positions, there are 1.3 billion theoretically possible acylinositol structures outnumbering the previously estimated 820 million theoretically possible acylsucrose structures (Fan et al., 2019). This acylinositol estimate does not even account for glycosylated acylinositols and glycohydroxyacylhexoses, demonstrating that this study greatly expands our knowledge of acylsugar structural diversity. Plants synthesize a tremendous amount of chemical diversity, yet characterizing this remains technically challenging and time consuming. This was exemplified by the analysis of Solanum acylsugars in Chapter 2 in which more than a hundred acylsugars could be identified in a single species. Because acylsugar isomers were not always resolved into distinct peaks, our reported acylsugar numbers are almost certainly an underestimate of the true diversity. Incorporating orthogonal techniques such as ion mobility can increase compound separation without compromising on acquisition time. Along with chromatographic resolution limitations, compound identification continues to be a major bottleneck in plant metabolomic studies (Chaleckis et al., 2019; da Silva et al., 2015). In the work described in Chapter 2, acylsugars were identified manually, a time-intensive method. Other software methods exist but exhibit significant limitations. For example, MS/MS database queries can accurately identify compounds, but many databases lack plant metabolites. Other methods such as the machine learning based CANOPUS bin compounds into classes (Dührkop et al., 2021), but do not provide specific compound annotations. The scientific community must address these data collection and analysis limitations in order to enhance our ability to characterize plant chemical diversity. Our understanding of plant chemistry is also limited by available germplasm. The Solanum acylsugar survey covered many of the minor clades composing Clade II, VANAns, and DulMo clades, yet many clades were unrepresented (e.g. Micracantha, Crinitum, Erythrotrichum, 347 and Thomasiifolium) as restricted by obtainable germplasm. Expanding access to diverse germplasm is key to understanding plant diversity, however navigating the complex legal and ethical constraints of importing plant germplasm remains difficult (Buck and Hamilton, 2011; Prathapan et al., 2018; Secretariat of the Convention on Biological Diversity, 2011). While this issue is beyond the scope of this dissertation, as a scientific community we should work collaboratively with international research groups to characterize and understand plant diversity (Pearce et al., 2020). With the acylsugar characterizations in Chapter 2 and available genetic resources, pieces of the acylinositol pathway were discovered revealing familiar evolutionary themes. Gene duplication was repeatedly observed as an important driver in acylsugar structure evolution likely due to relaxed selection on duplicated genes leading to altered enzymatic activities (Carretero-Paulet and Fares, 2012; Moghe et al., 2014; Ohno, 2013; Panchy et al., 2016). This was observed within the tomato clade in which ASAT3 duplicated and neofunctionalized to acylate a different sucrose hydroxyl (Fan et al., 2017). Similarly, a duplicated ASAT1, ASAT1- L, with myo-inositol acylating activity led to the evolution of acylinositols within the Solanum genus. In addition, gene loss impacted acylsugar phenotypes both in the tomato clade, e.g. ASAT4 and IPMS (Kim et al., 2012; Ning et al., 2015), and the eggplant clade, ASAT3-L1 (Chapter 3). More recently, we observed the co-option of “new” acyltransferases, i.e acyltransferases not orthologous or paralogous to characterized ASATs, with unique acylating activities. Two of these “new” acyltransferases are SnAGAT1 which represents the first reported ASAT to acylate glucose (Lou et al., 2021) and SmASAT3-I which exhibits unique activity with hydroxyacyl chains (Chapter 4). 348 The investigation of 31 out of more than 1200 Solanum species reported a tremendous amount of acylsugar diversity, indicating we are just scratching the surface of plant metabolic diversity. Expanding this analysis to different tissue types, specialized metabolite classes, and additional Solanum clades promise to uncover further chemical diversity and provide insights into plant metabolic evolution. One exciting prospect lies in addressing the evolution, diversity, and phylogenetic distribution of the recently identified root acylsugars through a phylogenetically-guided metabolomic analysis (Korenblum et al., 2020; Kerwin et al., unpublished). Commensurate with our expanded understanding of Solanum acylsugar diversity described in Chapter 2 comes many exciting opportunities to uncover how these compounds are produced and evolved. Exploring acylinositol glycoside structural variation and differences in acyl chain structural and positional differences between S. melongena and S. nigrum, as documented in Chapter 2 and previous reports (Hurney, 2018; Lou et al., 2021), are both exciting avenues for future work. Additionally, acylsugar-like compounds were reported to be produced outside of the Solanaceae family (Liu et al., 2019; Moghe et al., 2023), and their unknown biosynthetic pathways introduce many research opportunities. Of the non-Solanaceae acylsugars, investigation of the Asteraceae Inula ssp. produced acylinositols would provide further insights into acylinositol evolution and biosynthesis (Sun et al., 2021; Wang et al., 2013; Wu et al., 2015; Zou et al., 2008). This dissertation advanced our understanding of acylsugar diversity, biosynthesis, and evolution, and I expect this work will serve as a foundation for other researchers. 349 REFERENCES Buck, M., Hamilton, C., 2011. The Nagoya Protocol on access to genetic resources and the fair and equitable sharing of benefits arising from their utilization to the convention on biological diversity. Review of European Community & International Environmental Law 20, 47–61. https://doi.org/10.1111/j.1467-9388.2011.00703.x Carretero-Paulet, L., Fares, M.A., 2012. Evolutionary dynamics and functional specialization of plant paralogs formed by whole and small-scale genome duplications. Molecular Biology and Evolution 29, 3541–3551. https://doi.org/10.1093/molbev/mss162 Chaleckis, R., Meister, I., Zhang, P., Wheelock, C.E., 2019. Challenges, progress and promises of metabolite annotation for LC–MS-based metabolomics. Current Opinion in Biotechnology, Analytical Biotechnology 55, 44–50. https://doi.org/10.1016/j.copbio.2018.07.010 da Silva, R.R., Dorrestein, P.C., Quinn, R.A., 2015. Illuminating the dark matter in metabolomics. Proceedings of the National Academy of Sciences 112, 12549–12550. https://doi.org/10.1073/pnas.1516878112 Dührkop, K., Nothias, L.-F., Fleischauer, M., Reher, R., Ludwig, M., Hoffmann, M.A., Petras, D., Gerwick, W.H., Rousu, J., Dorrestein, P.C., Böcker, S., 2021. Systematic classification of unknown metabolites using high-resolution fragmentation mass spectra. Nat Biotechnol 39, 462–471. https://doi.org/10.1038/s41587-020-0740-8 Fan, P., Leong, B.J., Last, R.L., 2019. Tip of the trichome: evolution of acylsugar metabolic diversity in Solanaceae. Curr. Opin. Plant Biol. 49, 8–16. https://doi.org/10.1016/j.pbi.2019.03.005 Fan, P., Miller, A.M., Liu, X., Jones, A.D., Last, R.L., 2017. Evolution of a flipped pathway creates metabolic innovation in tomato trichomes through BAHD enzyme promiscuity. Nat Commun 8, 2080. https://doi.org/10.1038/s41467-017-02045-7 Herrera-Salgado, Y., Garduño-Ramírez, M.L., Vázquez, L., Rios, M.Y., Alvarez, L., 2005. Myo- inositol-derived glycolipids with anti-inflammatory activity from Solanum lanceolatum. J. Nat. Prod. 68, 1031–1036. https://doi.org/10.1021/np050054s Hurney, S.M., 2018. Strategies for profiling and discovery of acylsugar specialized metabolites (Ph.D.). Michigan State University, United States -- Michigan. Kim, J., Kang, K., Gonzales-Vigil, E., Shi, F., Daniel Jones, A., Barry, C.S., Last, R.L., 2012. Striking natural diversity in glandular trichome acylsugar composition is shaped by variation at the acyltransferase2 locus in the wild tomato Solanum habrochaites. Plant Physiology 160, 1854–1870. https://doi.org/10.1104/pp.112.204735 Korenblum, E., Dong, Y., Szymanski, J., Panda, S., Jozwiak, A., Massalha, H., Meir, S., Rogachev, I., Aharoni, A., 2020. Rhizosphere microbiome mediates systemic root 350 metabolite exudation by root-to-root signaling. P. N. A. S. U.S.A. 117, 3874–3883. https://doi.org/10.1073/pnas.1912130117 Leong, B.J., Hurney, S.M., Fiesel, P.D., Moghe, G.D., Jones, A.D., Last, R.L., 2020. Specialized metabolism in a nonmodel nightshade: trichome acylinositol biosynthesis. Plant Physiol. 183, 915–924. https://doi.org/10.1104/pp.20.00276 Liu, Y., Jing, S.-X., Luo, S.-H., Li, S.-H., 2019. Non-volatile natural products in plant glandular trichomes: chemistry, biological activities and biosynthesis. Nat. Prod. Rep. 36, 626–665. https://doi.org/10.1039/C8NP00077H Lou, Y.-R., Anthony, T.M., Fiesel, P.D., Arking, R.E., Christensen, E.M., Jones, A.D., Last, R.L., 2021. It happened again: Convergent evolution of acylglucose specialized metabolism in black nightshade and wild tomato. Science Advances 7, eabj8726. https://doi.org/10.1126/sciadv.abj8726 Moghe, G., Irfan, M., Sarmah, B., 2023. Dangerous sugars: Structural diversity and functional significance of acylsugar-like defense compounds in flowering plants. Curr. Opin. Plant Biol. 73, 102348. https://doi.org/10.1016/j.pbi.2023.102348 Moghe, G.D., Hufnagel, D.E., Tang, H., Xiao, Y., Dworkin, I., Town, C.D., Conner, J.K., Shiu, S.-H., 2014. Consequences of whole-genome triplication as revealed by comparative genomic analyses of the wild radish Raphanus raphanistrum and three other Brassicaceae species. Plant Cell 26, 1925–1937. https://doi.org/10.1105/tpc.114.124297 Ning, J., Moghe, G.D., Leong, B., Kim, J., Ofner, I., Wang, Z., Adams, C., Jones, A.D., Zamir, D., Last, R.L., 2015. A feedback-insensitive isopropylmalate synthase affects acylsugar composition in cultivated and wild tomato. Plant Physiol. 169, 1821–1835. https://doi.org/10.1104/pp.15.00474 Ohno, S., 2013. Evolution by gene duplication. Springer Science & Business Media. Panchy, N., Lehti-Shiu, M., Shiu, S.-H., 2016. Evolution of gene duplication in plants. Plant Physiol. 171, 2294–2316. https://doi.org/10.1104/pp.16.00523 Pearce, T.R., Antonelli, A., Brearley, F.Q., Couch, C., Campostrini Forzza, R., Gonçalves, S.C., Magassouba, S., Morim, M.P., Mueller, G.M., Nic Lughadha, E., Obreza, M., Sharrock, S., Simmonds, M.S.J., Tambam, B.B., Utteridge, T.M.A., Breman, E., 2020. International collaboration between collections-based institutes for halting biodiversity loss and unlocking the useful properties of plants and fungi. PLANTS, PEOPLE, PLANET 2, 515–534. https://doi.org/10.1002/ppp3.10149 Prathapan, K.D., Pethiyagoda, R., Bawa, K.S., Raven, P.H., Rajan, P.D., 172 CO- SIGNATORIES FROM 35 COUNTRIES, 2018. When the cure kills—CBD limits biodiversity research. Science 360, 1405–1406. https://doi.org/10.1126/science.aat9844 Secretariat of the Convention on Biological Diversity, 2011. Nagoya Protocol on access to genetic resources and the fair and equitable sharing of benefits arising from their 351 utilization to the convention on biological diversity : text and annex. (Report). Secretariat of the Convention on Biological Diversity. https://doi.org/10.25607/OBP-789 Sun, C.-P., Jia, Z.-L., Huo, X.-K., Tian, X.-G., Feng, L., Wang, C., Zhang, B.-J., Zhao, W.-Y., Ma, X.-C., 2021. Medicinal Inula species: phytochemistry, biosynthesis, and bioactivities. Am. J. Chin. Med. 49, 315–358. https://doi.org/10.1142/S0192415X21500166 Wang, C., Zhang, X., Wei, P., Cheng, X., Ren, J., Yan, S., Zhang, W., Jin, H., 2013. Chemical constituents from Inula wissmanniana and their anti-inflammatory activities. Arch. Pharm. Res. 36, 1516–1524. https://doi.org/10.1007/s12272-013-0143-1 Wu, J., Tang, C., Yao, S., Zhang, L., Ke, C., Feng, L., Lin, G., Ye, Y., 2015. Anti-inflammatory inositol derivatives from the whole plant of Inula cappa. J. Nat. Prod. 78, 2332–2338. https://doi.org/10.1021/acs.jnatprod.5b00135 Zou, Z.-M., Xie, H.-G., Zhang, H.-W., Xu, L.-Z., 2008. Inositol angelates from the whole herb of Inula cappa. Fitoterapia 79, 393–394. https://doi.org/10.1016/j.fitote.2007.11.031 352