IDENTIFICATION AND FUNCTIONAL ANALYSIS OF PROTEINS INVOLVED IN AUXIN REGULATED PLANT GROWTH By Jie Li A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Biochemistry and Molecular Biology - Doctor of Philosophy 2016 ABSTRACT IDENTIFICATION AND FUNCTIONAL ANALYSIS OF PROTEINS INVOLVED IN AUXIN REGULATED PLANT GROWTH By Jie Li During growth, plants integrate cell division with cell elongation and cell differentiation. We used proteomics to identify proteins from auxin (indole-3-acetic acid; IAA) -induced rapidly growing corn (Zea mays) coleoptiles to find candidates controlling this growth as well as the underlying cell wall and cuticle biosynthesis. Of 86 proteins identified, 15 showed a predicted association with cell wall/cuticle biosynthesis or trafficking machinery; the analysis also revealed four proteins of unknown function. Parallel real-time PCR indicated that the steady-state mRNA levels of genes with a known or predicted role in cell-wall biosynthesis increase upon auxin-treatment. Importantly, genes encoding two of the hypothetical proteins (ARRP1 and Hypothetical protein 3, a putative endoglucanase) also showed higher levels of mRNA; their gene expression was directly correlated with coleoptile and leaf growth. This suggested a role of these two novel proteins in the regulation of processes related to cell and organ expansion and thus plant growth. These data are described in chapter 2. Chapter 3 focuses on ARRP1 (Auxin rapid response protein 1), one of the proteins of unknown function associated with cell and organ growth. Upon auxin treatment ARRP1 mRNA and protein increased within 30 minutes Œ prior to measurable growth and increased expression of genes encoding cell wall biosynthetic enzymes, suggesting an intracellular signaling function. This was further supported by the localization of ARRP1 at the cell periphery as well as in the nucleus. Arabidopsis mutants lacking the ARRP1 homologue grew normally under standard conditions, but showed delayed growth and delayed flowering when germinated in the dark and transferred to light. This phenotype that could be reversed by expressing ZmARRP1 in the Arabidopsis mutant. ZmARRP1 expression was also induced by red and blue light, indicating a possible role in phytochrome and cryptochrome signaling at the interface with the auxin response. In situ hybridization demonstrated that ARRP1 mRNA is enriched in the vasculature of growing corn tissues. In addition, the corn ARRP1 interacted with the cytoplasmic domain of both Arabidopsis and Zea mays VH1/BRL2 which plays a role in the signaling of vascular development. Based on the fact that ARRP1 protein is involved in light-induced growth responses as well as with components of auxin signaling, we propose that it may integrate several environmental and hormonal signals affecting plant development. Chapter 4 describes the initiation of a series of experiments investigating addtional proteins that interact with ARRP1 to elucidate other components of the signaling pathway that use ARRP1. Using in vitro pull-down assays and mass spectrometry, I identified 32 putative ARRP1-interacting proteins in the developing corn leaf. Some of these proteins appear to be involved in cell wall biosynthesis, including a beta-glucosidase homologue. Others are predicted to be signal molecules, including a tetratricopeptide repeat domain containing protein and a CobW/P47K family protein. Using semiquantitative RT-PCR, I showed that several of these genes have a similar expression pattern compared to ARRP1, suggesting a function in the same signaling/response path. IVAcknowledgement Since I first came to the US in the summer of 2009, I have had the most productive and pleasant six years so far in my life and have met some of the most important people to me here in MSU. I was very lucky to be a graduate student with my advisor Prof. Susanne Hoffmann-Benning. She is a role model as an enthusiastic scientist. She trusted me and gave me plenty of freedom to explore the project according to my own pace and interest. She supported me during numerous hard times when I encountered failure and guide me through these darkness. She taught me to work independently and to think critically. I have learned so much from her in the past six years, and works cannot express my appreciation towards her as a guide and a mentor. I want to thank my committee members: Drs. Thomas Sharkey, Robert Last, Michael Garavito, and Jonathon Walton. They provided a lot of constructive suggestions to my research that better refined my projects and my writing. They also encouraged me and offer useful resources. I also want to thank my lab mates, especially Banita Tamot, who taught me many techniques and offer numerous resources. Lastly, I want to thank my wife, my parents and my friends for years of support. VTABLEOFCONTENTSLISTOFFIGURES––––––––––––––––––––––––––––––––––––––––––––––––......VIILISTOFTABLES–––––––––––––––––––––––––––––––––––––––––––––––––––.IXCHAPTER1INTRODUCTION:AUXININDUCEDPLANTGROWTHANDTHEINTEGRATIONOFAUXINWITHLIGHTANDOTHERHORMONES....................................................................................................................11.1Phytohormonesasessentialregulatorsofplantgrowth....................................................................21.1.1Thestructureandfunctionofauxin............................................................................................31.1.2Auxinbiosynthesis.......................................................................................................................51.1.3Auxintransport............................................................................................................................61.1.4Auxinperception..........................................................................................................................71.2Thecorncoleoptileasamodeltoexamineauxininducedgrowth....................................................91.2.1Auxininducedacidgrowth........................................................................................................101.2.2Auxinactivationofionchannels................................................................................................111.2.3Proteinsynthesisisimportantformaintainingcellelongation.................................................121.3Integrationofauxinsignalingwithotherhormonesandlight.........................................................131.3.1Crosstalkbetweenauxinandotherhormones..........................................................................131.3.2Crosstalkbetweenauxinsignalingandlight..............................................................................151.3.2.2Lightaffectsauxintransport...................................................................................................171.3.2.3Lightandauxinsignalingpathwayshavecommongenetargets...........................................18REFERENCES............................................................................................................................................25CHAPTER2:CONTRIBUTIONOFPROTEOMICSINTHEIDENTIFICATIONOFNOVELPROTEINSASSOCIATEDWITHPLANTGROWTH................................................................................................................................372.1Abstract.............................................................................................................................................382.2Introduction......................................................................................................................................392.3MaterialsandMethods.....................................................................................................................442.3.1Plantmaterialandgrowthconditions.......................................................................................442.3.2Sampleharvest..........................................................................................................................442.3.3Proteincollectionandidentification.........................................................................................442.3.4RNAextraction,quantitative&qualitativeanalysis..................................................................462.4ResultsandDiscussion......................................................................................................................482.4.1Proteomicsanalysis...................................................................................................................482.4.2Expressionstudies......................................................................................................................542.4.3Genesencodingproteinswithapossibleroleincutinbiosynthesis.........................................542.4.4Genesencodingproteinswithapossibleroleincellwallbiosynthesis....................................562.4.5Hypotheticalproteins................................................................................................................582.5Conclusions.......................................................................................................................................60REFERENCES............................................................................................................................................87CHAPTER3:ARRP1INTHEREGULATIONOFCELLEXPANSIONGROWTH..................................................953.1Abstract.............................................................................................................................................963.2Introduction......................................................................................................................................97VI3.3MethodandMaterials....................................................................................................................1013.3.1Plantmaterialandgrowthconditions.....................................................................................1013.3.2Sequenceanalysisandbioinformaticsanalysis.......................................................................1013.3.3Hormoneandlightresponseanalyses.....................................................................................1023.3.4TotalRNAisolationandquantification....................................................................................1023.3.5RecombinantARRP1andVH1kinasedomainconstructforproteininteractionessays,purification,andantibodyproduction..............................................................................................1033.3.6Proteinextraction,andwesternblotanalysis.........................................................................1043.3.7IdentificationandverificationofTDNAinsertionlines..........................................................1053.3.8Transientexpressionandstabletransgenictransformation...................................................1053.3.9Invitrocoimmunoprecipitation..............................................................................................1063.3.10Yeasttwohybridassay..........................................................................................................1073.3.11Insituhybridization...............................................................................................................1073.4ResultsandDiscussion....................................................................................................................1083.4.1ARRP1encodesaproteinwithaDUF538conserveddomain.................................................1083.4.2SequencealignmentofZmARRP1anditsArabidopsishomologuesAt1g56580andAt1g09310..........................................................................................................................................................1093.4.3ARRP1expressioniscorrelatedwithcelldivisionandcellelongation....................................1103.4.4ARRP1expressioniscontrolledbyauxinandlight..................................................................1113.4.5AtARRP1isrequiredintheinitiationofplantgrowthanddark/lighttransition.....................1133.4.6ZmARRP1interactswithBRL2.................................................................................................1153.4.7ARRP1andVH1/BRL2areexpressedinoverlappingpatterns................................................1173.4.8SubcellularlocalizationofARRP1............................................................................................1173.5Conclusions:....................................................................................................................................119REFERENCES..........................................................................................................................................140CHAPTER4:CONCLUSIONSANDOUTLOOKS............................................................................................1484.1ContributionofProteomicsintheIdentificationofNovelProteinsAssociatedwithPlantGrowth...............................................................................................................................................................1494.2ARRP1intheregulationofcellexpansiongrowth..........................................................................1504.3Futureperspective..........................................................................................................................1524.3.1QuestionsremainingaboutARRP1function...........................................................................1524.3.2IdentificationofARRP1interactingpartners...........................................................................1534.3.3Confirmationofproteinproteininteraction...........................................................................1564.3.4ExpressionstudyofthreeconfirmedARRP1bindingpartners..............................................1574.3.5EvaluationoftheTDNAknockoutmutants...........................................................................158REFERENCES..........................................................................................................................................171APPENDICES..............................................................................................................................................174APPENDIXA:STUDYOFAPUTATIVEENDO(1,3)(1,4)GLUCANASE.................................................175APPENDIXB:REFERENCES...................................................................................................................192 VIILISTOFFIGURESFigure1.1Chemicalstructureofthemostcommonphytohormones(a)and2,4Dichlorophenoxyaceticacid(b),asyntheticauxinusedasherbicide..............................................................................................22Figure1.2PotentialIAAbiosyntheticpathwaysareintegratedwiththeeffectoftheR:FRlightratioonIAAbiosynthesis..........................................................................................................................................23Figure1.3Overviewofthecellularauxinmachinery.................................................................................24Figure2.1Transmissionelectronmicrographofacrosssectionthroughacorncoleoptileepidermiscelltreatedwith30MIAAfor1hour.............................................................................................................61Figure2.2Flowchartofexperimentalsetupforcoleoptiletreatmentandharvest..................................62Figure2.3Auxininducedcorncoleoptilerapidlygrowth...........................................................................63Figure2.4aSDSPAGEgelcomparisonofbandingpatterns.......................................................................64Figure2.4bGroupingofproteinsenhancedintheepidermisascomparedtothecoleoptileanduponauxininductionbasedontheirpredictedfunction/involvementincellularprocesses............................65Figure2.5DifferentialexpressionofauxininducedgenesinthecorncoleoptileusingquantitativeRTPCR..............................................................................................................................................................66Figure2.6SemiquantitativeanalysisoftheexpressionlevelsofgenesencodingUDPgdh,UDPgp,Hyp4,Hyp3,PLTP,andRGPincorncoleoptiles(a)andleaf(b)afterthetransitionofdarktolight..................67Figure2.7ExpressionlevelsofgenesencodingHyp3(a)andHyp4(b)invariouscornseedlingorgansasdeterminedbyRTPCR................................................................................................................................69FigureS2.1PCRproductsobtainedfrominduced(I)andcontrol(C)coleoptileepidermis.......................72Figure3.1TheARRP1proteinbelongstoaDUF538superfamily.............................................................120Figure3.2IllustrationofthedistributionoftheDUF538conserveddomaininplants............................121Figure3.3ARRP1levelsthroughoutthecornseedlings...........................................................................122Figure3.4ARRP1expressionindifferentleafzones................................................................................123Figure3.5ResponseofARRP1infivedaysoldcorncoleoptilestoAuxintreatment...............................124Figure3.6ARRP1expressionincornleavesinresponsetodifferentlightconditions............................125Figure3.7ExpressionlevelsofAtARRP11(a)andAtARRP12(b)invariousArabidopsisthalianaorgans,asdeterminedbyRTPCR..........................................................................................................................127VIIIFigure3.8Singleknockoutmutantsofatarrp11andatarrp12showstrichomeswithsmallerbranchlengthandvariablenumberofbranches..................................................................................................128Figure3.9ARRP1interactswithBRL2homologuesfromArabidopsisandmaize....................................130Figure3.10SubcellularlocalizationofARRP1...........................................................................................132Figure3.11InsituhybridizationshowsthatZmARRP1isexpressedintheyoungleavesandenrichedinthevascularbundles.................................................................................................................................133Figure3.12AmodelforproposedfunctionofARRP1..............................................................................134FigureS3.1ExpressionandpurificationofARRP1andwesternblottinganalysis....................................137FigureS3.2ARRP1mRNAlevelsincorncoleoptileareunchangedbycytokininorbrassinosteroid......138FigureS3.3Y2HanalysisofZmARRP1withAtBRL2..................................................................................139Figure4.1aSDSPAGEgelcomparisonofpulldownassay.......................................................................159Figure4.1bGroupingofproteinsidentifiedbypulldownassaybasedontheirpredictedfunction/involvementincellularprocesses..............................................................................................160Figure4.2YeasttwohybridanalysesforconfirmingtheproteinproteininteractionofARRP1anditspartners.....................................................................................................................................................161Figure4.3SemiquantitativeanalysesoftranscriptionlevelsofgenesencodingASRL1(a,b),Annexin1(c,d)andTPR1(e,f)indifferentcornseedlingorgansandalongtheyoungleaf .......................................162Figure5.1ProteinsequencealignmentofHYP3(a),AtHYP3andAtHYP32(b)......................................187Figure5.2EnzymaticanalysisofHYP3(grey),Endo(1,3)(1,4)glucanase(red),andglucanase(blue)usinglichenin(a),barleyglucan(b),carboxymethylcellulose(c),andxyloglucan(d)assubstrates...188Figure5.3Phenotypingofathyp31,athyp32andZmHYP3overexpressionlines..................................190Figure5.4SubcellularlocalizationofHYP3...............................................................................................191 IXLISTOFTABLESTable2.1:Auxinregulatedepidermalproteinswithfunctionsrelatedtosecretorypathway,cellwallorcuticlemetabolismwithcomparisonofRTPCRtomicroarraydata..........................................................71TableS2.1:Primersusedforquantitative(a)andsemiquantitative(b)RTPCR.......................................73TableS2.2:Proteinsidentifiedintheouterepidermisofcorncoleoptiles;samplesinboldwerefrombandswhichappearedinducedupontreatmentwithauxin.....................................................................74TableS2.3:StatisticalanalysisofRTPCRdata............................................................................................85TableS3.1Primersforthepaper..............................................................................................................135TableS3.2Cisregulatoryelementwithin1KbARRP1promotor.............................................................136Table4.1ARRP1interactingproteinswithapossiblenovelornotfullyelucidatedroleindownstreamsignaltransduction....................................................................................................................................166TableS4.1ProteinsidentifiedintheARRP1pulldownassay...................................................................167 1 CHAPTER 1 INTRODUCTION: AUXIN INDUCED PLANT GROWTH AND THE INTEGRATION OF AUXIN WITH LIGHT AND OTHER HORMONES 2With the increase in the world population comes the need for improved efficiency in the use of arable land. When faced with similar problems in the mid- and late- 20th century the first green revolution led by Norman Borlaug dramatically changed the field of agriculture (McKersie, 2015). During that time period, new chemical fertilizers and synthetic herbicides and pesticides were created and utilized. In addition, the generation of high-yielding crop varieties and the improvement of agricultural management techniques increased the agricultural yield and the nutritional intake of billions and has reduced child mortality and malnourishment of infants around the world (Khush, 1999). It also supported overall economic growth in many Asia countries over the years. However, due to the current climate change, population growth, and changing food preferences, global crop production now encounters a new crisis. To solve these problems and provide food security for the foreseeable future, we need crops that have greater yield or are more tolerant to biotic and abiotic stresses. 1.1 Phytohormones as essential regulators of plant growth To adapt to the continuously changing environment, plants evolved a complex phytohormone network. Phytohormones are small signaling molecules that are essential in the regulation of plant growth, development, reproduction, and survival. Their concentrations are very low, but control a broad variety of biological mechanisms. They not only orchestrate intrinsic developmental programs but also convey environmental inputs and drive adaptive responses to a wide variety of biotic and abiotic stresses (Kende and Zeevaart, 1997; Santner et al., 2009; Santner and Estelle, 2009; Wolters and Jurgens, 2009; Depuydt and Hardtke, 2011). 3Auxin, gibberellin (GA), cytokinin, abscisic acid (ABA) and ethylene are generally referred to as the five ‚classic™ plant hormones (Kende and Zeevaart, 1997). More recently, several additional compounds have been recognized as hormones, including brassinosteroids, jasmonate, salicylic acid, nitric oxide and strigolactones (Santner and Estelle, 2009) (Fig. 1.1a). Moreover, we now know that plants also use peptide hormones, such as Flowering locus T, CLAVATA3, or Phytosulfokine to regulate various growth responses (Corbesier et al., 2007; Lin et al., 2007; Tamaki et al., 2007; Jun et al., 2008; Sauter, 2015). During growth, the plant integrates cell division with cell elongation and cell differentiation. Based on the phenotype of mutants with disrupted hormone biosynthesis or perception, auxin, gibberellins, cytokinin and brassinosteroids are considered essential for growth (Depuydt and Hardtke, 2011). Cytokinins regulate cell proliferation while gibberellins promote cell elongation; auxin is involved in both processes (Werner et al., 2001; Sauer et al., 2013; Claeys et al., 2014). Brassinostoids promote cell expansion, yet their role in cell division remains unclear (Nakaya et al., 2002; Hardtke et al., 2007). 1.1.1 The structure and function of auxin Auxins were the first of the major phytohormones to be discovered. The term ‚auxin™ originates from the Greek word ‚auxein™, which means to enlarge/grow. In 1880, the first paper about the effect of auxin, ‚The Power of Movement in Plants™, was published by Charles and Francis Darwin (1880). They noted a messenger that is transmitted in the downstream direction from the tip of the coleoptile and causes its bending towards the light. In 1934, this messenger, auxin, was first isolated from Avena 4sativa and structurally identified as indole-3-acetic acid (IAA) (Kögl F, 1934). IAA is the most abundant endogenous auxin and is able to fulfill most of the auxin functions. Now auxin is the generic name for a group of important molecules in plants. In addition to IAA, there are three more natural compounds with auxin activity, indole-3-butyric acid (IBA) (Ludwig-Muller and Epstein, 1991), 4-chloroindole-3-acetic acid (4-CI-IAA) (Engvild, 1985), and phenylacetic acid (PAA) (Okamoto I, 1967; Fig. 1.1a). At high concentrations, auxins are toxic affect mainly dicots over monocot species, such as grasses and cereal crops. Because of these properties many synthetic compounds with auxin-like activity were developed and used as herbicides; one example, 2,4- dichlorophenoxyacetic acid (2,4-D) is one of the world™s most widely used herbicide (Grossmann, 2010) (Fig. 1.1b). Auxin was classically defined as the stimulator to promote elongation in coleoptile and stem sections, but also rooting (Went, 1934). Since then, auxin was shown to be essential for multiple aspects of plant development. It controls cell division by directly or indirectly influencing both transcriptional and posttranscriptional regulation of cell cycle machinery components such as CDKA , CYCD3, KRP1 and KRP2 (Hemerly et al., 1993; Himanen et al., 2002; Menges et al., 2006; Perrot-Rechenmann, 2010). It also regulates cell elongation by altering cell wall plasticity. It controls cell differentiation as it affects root and shoot architecture (Gallavotti, 2013), leaf abscission (Wetmore and Jacobs, 1953), leaf venation, and fruit formation (de Jong et al., 2009). It mediates plant responses to pathogens but also to light, gravity and other abiotic factors (Woodward and Bartel, 2005; Kazan and Manners, 2009; Vanneste and Friml, 2009; Wang et al., 2010). Auxin function requires several components: gradients of auxin 5concentration between tissues (I) and different cells (II) both of which are based on auxin biosynthesis and polar transport; and (III) auxin perception and signaling within the cell (Grones and Friml, 2015). 1.1.2 Auxin biosynthesis Auxin biosynthesis can occur via either the tryptophan (Trp)-independent or Trp-dependent pathways (Fig. 1.2) (Korasick et al., 2013). The Trp-dependent pathways are better characterized and more important for plant development (Sugawara et al., 2009; Korasick et al., 2013). Based on major intermediates, the Trp-dependent pathway can be separated into four pathways, which synthesize auxin via the intermediates indole-3-acetaldoxime (IAOx), indole-3-acetamide (IAM), tryptamine (TAM), or indol-3-ylpyruvic acid (IPA), respectively. The IPA pathway appears to be the main route for the IAA biosynthesis in both maize and Arabidopsis. It consists of two steps: a Trp aminotransferase (TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS: TAA) converts Trp to IPA, followed by the conversion of IPA to IAA by a flavin monooxygenase of the YUCCA (YUC) family (Korasick et al., 2013). Overexpression of YUC family genes in Arabidopsis leads to auxin overproduction phenotypes, but plants overexpressing TAA genes are indistinguishable from wild type, suggesting that YUC is probably the rate-limiting step of IPA pathway. The Trp-deficient mutant of Arabidopsis, exhibits no difference in free IAA levels, but accumulates amide- and ester- linked IAA conjugate, when compared to wild type, indicating that the Trp-independent pathway also contributes to IAA biosynthesis (Wright et al., 1991; Normanly, 2010). In addition, the use of labeled Trp-precursors in feeding assays showed that the percent isotopic incorporation into IAA is greater than 6that of tryptophan, and supports the concept of Trp-independent auxin biosynthesis (Normanly et al., 1993). However no important intermediates or enzymes associated for this pathway have been identified so far. This route is postulated to originate from either indole or indole-3-glycerol phosphate (Ouyang et al., 2000). 1.1.3 Auxin transport Auxin is synthesized mainly in the shoot apex, the tip of the coleoptile, and young leaves and then translocated to other plant tissues or cells. This movement occurs via polar auxin transport, employing specific carriers for movement across the membrane as well as through bulk flow in the phloem (Goldsmith et al., 1974; Hoad, 1995). The polar transport and establishment of localized auxin maxima are crucial for plant development. This process regulates embryonic development, stem cell maintenance, root and shoot architecture, and tropic growth responses (Peer et al., 2011). IAA moves among plant tissues through a combination of membrane diffusion and carrier-mediated transport (Friml, 2003). Three classes of carrier proteins, which facilitate auxin uptake and efflux, have been identified in plants: AUXIN RESISTANT1/LIKE AUX1 (AUX1/LAX) uptake permeases, ATP Binding Cassette subfamily B (ABCB/PGP) transporters, and PIN-FORMED (PIN) carrier proteins (Fig. 1.3) (Benjamins and Scheres, 2008; Petrasek and Friml, 2009; Zazimalova et al., 2010). IAA can enter cells via lipophilic diffusion in its protonated form (IAAH) or through proton symporters of the AUX1/LAX family in its anionic form (IAA-) (Ugartechea-Chirino et al., 2010; Della Rovere et al., 2015). ABCB4 appears to contribute to auxin uptake in root epidermal cells when auxin levels are low (Santelia et al., 2005; Terasaka et al., 2005). Once inside the cell, IAA exists predominantly in its anionic form, necessitating carrier mediated transport to exit cell. 7PIN proteins and a subset of ABCB/PGP transporters mediate this tissue-specific cellular auxin export (Zazimalova et al., 2010). PIN proteins are integral membrane proteins essential for polarized auxin movement. Each member of the PIN family displays a unique tissue-specific expression pattern, and pin mutants generally exhibit growth phenotypes that are consistent with the loss of directional auxin transport in the corresponding tissue (Chen et al., 1998; Galweiler et al., 1998; Muller et al., 1998; Friml et al., 2002; Friml et al., 2002). Among them, PIN3 has a lateral localization in the cells of the shoot endodermis, in the root columella, and in pericycle cells and appears to function in the lateral redistribution of auxin that is involved in photo- and gravitropic growth (Friml et al., 2002). Dramatic progress in understanding the mechanism and regulation of auxin transport has made it clear that this process is essential in almost all plant growth stages and environmental responses (Peer et al., 2011). 1.1.4 Auxin perception To trigger a biological process, auxin must be perceived by the plant. To date at least two auxin receptors are known. The most well-studied auxin receptor is the F-box protein TRANSPORT INHIBITOR RESISTANT1 (TIR1), which was identified as a subunit of the ubiquitin E3 ligase (Ruegger et al., 1998; Dharmasiri et al., 2005; Kepinski and Leyser, 2005) (Fig. 1.3). Crystallographic studies showed that IAA fits into the core of a ring-like structure formed by TIR1 (Tan et al., 2007). Binding of IAA promotes the interaction between TIR1 and proteins of the AUXIN/INDOLE ACETIC ACID (Aux/IAA) family, a group of proteins that regulates auxin-induced gene expression and development (Reed, 2001). The interaction with TIR1 triggers the ubiquitination of Aux/IAA proteins, which designates them for degradation by the 26S 8proteasome (Kepinski and Leyser, 2004, 2005): At low, basal auxin concentration, Aux/IAA protein form an inhibitory complex with AUXIN RESPONSE FACTOR (ARF) family transcription factors that bind to the AUXIN RESPONSIVE ELEMENT (ARE) in the promotors of auxin response genes and repress their transcription. An increase in auxin levels leads to TIR1-Aux-IAA interaction and the proteasome-mediated degradation of Aux/IAAs, which in turn allows for a gradually increasing number of functionally active ARF proteins and the transcriptional activation of auxin regulons (Quint and Gray, 2006; Weijers and Friml, 2009; Sauer et al., 2013; Salehin et al., 2015). Although conceptually straightforward, this system of auxin perception is functionally quite complex. Arabidopsis thaliana has five TIR1 members, termed AFB1-AFB5, which all bind auxin (Calderon Villalobos et al., 2012). There are also 29 members of Aux/IAA family that share a common structure of four domains. Since Aux/IAAs directly participate in the receptorŒligand interaction, they can be seen as auxin co-receptors (Tan et al., 2007). Several experiments suggest that plants form different TIR/AFBŒAux/IAA co-receptor pairs with distinct auxin affinities. In addition, plants also have 23 ARF genes, which generate additional receptor-transcription factor combinations (Wilmoth et al., 2005; Korasick et al., 2014). This diversity contributes to the great variety of auxin functions (Vernoux et al., 2011; Enders and Strader, 2015). In addition to the chain of events initiated by the binding of auxin to TIR1 in the nucleus, two more auxin perception modes have been proposed. AUXIN BINDING PROTEIN1 (ABP1) is the longest known putative auxin receptor. It was first isolated form maize, but remains an enigma (Jones and Venis, 1989). ABP1 is mainly localized 9in the endoplasmic reticulum (ER). However, it is secreted to some extent into the extracellular space and appears to be active as an auxin receptor there as well (Napier et al., 2002). Experimental evidence suggests that ABP1 mediates the rapid auxin response close to the plasma membrane, such as the rapid cell elongation response (Sauer and Kleine-Vehn, 2011). Several papers also indicated that ABP1 is also involved in the coleoptile elongation as a component of the auxin-light-signaling network (David et al., 2007; Ljung, 2013). However, a recent study suggests that ABP1 is not required for auxin signaling and plant development (Gao et al., 2015). An additional recently identified candidate for an auxin receptor is the S-Phase Kinase-Associated Protein 2A (SKP2A), which is the regulator of the cell cycle and is involved with the degradation of at least two cell cycle factors, DPB and E2FC (Jurado et al., 2010). Further studies are needed to reveal more details of the auxin-SKP2A pathway. 1.2 The corn coleoptile as a model to examine auxin-induced growth Maize (Zea mays L.) plays an important agronomic role as feed, food, and source of bioethanol. Auxin-induced cell elongation of Maize coleoptiles is one of the fastest and longest-known phytohormone responses known. Frits Went employed the famous experiments using excised oat coleoptiles and auxin-containing agar blocks as a tool to study the fiauxin-hypothesisfl of coleoptile elongation (Went, 1927). Since then corn coleoptiles have been widely used as a model system to study the regulation of cell growth and tropisms (Haga and Iino, 1998). 101.2.1 Auxin induced acid growth Although auxin-induced coleoptile growth has been studied for almost one century, the biochemical basis behind this response is still not fully understood. Several studies suggested that one of the first steps is an auxin-induced pH change in the apoplastic space via rapid activation of proton secreting plasma membrane H+-ATPase (Rayle and Cleland, 1970; Hager et al., 1971; Cosgrove, 1997; Hager, 2003; Cosgrove, 2005; Takahashi et al., 2012). This fiAcid-growth theoryfl was first proposed by Hager (1971). In his theory, he suggests that the auxin-induced acidification of the apoplast is the prerequisite for cell elongation. The lower pH will activate so-called fiwall-loosening proteinsfl, thus mediating a shifting of cellulose microfibrils and subsequent expansion growth.However, several publications showed that the pH established by IAA is only 4.8-5.0 which causes almost no enhancement of growth compared with the water control. Growth that is mediated by the fungal phytotoxin fusicoccin (FC) can be inhibited by neutral buffers infiltrated into the outer epidermal wall, but a substantial growth response occurs after addition of IAA under identical conditions, suggesting IAA and H+ act via separate mechanisms (Kutschera, 1994, 2001, 2006; Niklas and Kutschera, 2012; Visnovitz et al., 2012; Burdach et al., 2014; Rudnicka et al., 2014). The details of the fiAcid-growth theoryfl still need further investigation, but it is clear that auxin-induced proton secretion is insufficient to cause the rapid growth phenotype. Wall-loosening proteins include expansins, xyloglucan endotransglucosylases/ hydrolases (XTH). Expansins are able to loosen bonds between cellulose and hemicellulose fibrils in the cell wall (Cosgrove et al., 2002; Cosgrove, 2015). By regulating xyloglucan turnover, XTHs also cause an enhancement of the cell wall 11plasticity (Brummell and Hall, 1987; Cosgrove, 1997; Takahashi et al., 2012). Together, these changes lead to the initiation of turgor-driven cell elongation. 1.2.2 Auxin activation of ion channels To study auxin induced rapid growth, coleoptiles were pulled off the primary leaf and, after removal of the tips, shaken in distilled water to remove residual auxin. Upon application of auxin the coleoptile growth rate is constant during the first four hours. But two hours later, the elongation rate slows down and completely ceases after one day. In 1981, Stevenson and Cleland showed that this cessation of coleoptile elongation in water can be prevented by the addition of absorbable solutes, such as sucrose or potassium chloride (KCl), suggesting that the uptake of osmolytes which maintain the turgor pressure is also important for auxin-induced growth (Stevenson and Cleland, 1981; Cosgrove and Cleland, 1983). Moreover, by importing K+ ions, the most abundant cation in plants, cells can easily balance the charge of the secreted protons. This importance of K+ for auxin-induced H+ pumping had already been suggested in the 1970s (Haschke and Luttge, 1975; Nelles, 1977). In maize coleoptiles, auxin-induced cell elongation strongly depends on the availability of K+ in the bathing medium and their uptake through potassium-selective ion channels: auxin-induced plant growth could be inhibited by addition of K+ channel blockers or removal of K+ ions from the medium (Claussen et al., 1997). Thus, studies of K+ channels in maize coleoptiles were initiated (Philippar et al., 1999). Maize has at least two K+ channels, ZMK1 and ZMK2 (Bauer et al., 2000; Deeken et al., 2000; Deeken et al., 2002). ZMK1 is mainly localized in cortex and epidermal cells. Its transcription is induced by auxin treatment. In addition, electrophysiological studies suggest that the auxin-induced acidification activates K+ 12transport via ZMK1 (Philippar et al., 1999; Philippar et al., 2006). Using patch clamp studies, K+ channel activation can be seen 10 min after auxin stimulation, which strongly suggests the involvement of K+ uptake via ZMK1 in cell elongation (Philippar et al., 1999; Thiel, 1999; Philippar et al., 2006). One possible explanation to the 10 min delay is that the initial rise in growth rate is due to acidification of the apoplast and subsequent cell wall loosening. K+ uptake via ZMK1 might thus be required for sustained growth, characterized by the need to import osmotically active substances. 1.2.3 Protein synthesis is important for maintaining cell elongation In 1963, it was demonstrated that several antibiotics, including chloramphenicol, puromycin, and p-fluorophenylalanine, which suppress protein biosynthesis can also inhibit the elongation of coleoptiles and mesocotyls (Noodén and Thimann, 1963). In addition, using dactinomycin, a specific inhibitor of RNA synthesis, Key and Ingle showed that IAA-induced growth is significantly diminished when transcription is inhibited (Key and Ingle, 1964). As a result, the concept of growth-limiting proteins (GLPs) emerged and was corroborated by several subsequent experiments. Parallel to the suppression of protein biosynthesis, cell wall loosening and cell elongation are both inhibited by cycloheximide (CHX), a protein synthesis inhibitor (Cleland, 1971; Cleland and Haughton, 1971). Time course studies further suggested that the biosynthesis of several GLPs plays a key role in auxin-mediated elongation. Using electron microscopy, osmiophilic particles (OPs) were found between the plasma membrane and the outer epidermal wall of corn coleoptiles (OEW; Kutschera et al., 1987). These particles were only seen in the auxin-induced growth tissues, not in FC or acid mediated ones. Closer investigation revealed that these particles are 13secreted only to the outer wall of expanding epidermal cells, and were seen in a variety growing tissues/plants regardless of the growth-inducing signal (Kutschera and Kende, 1989; Hoffmann-Benning et al., 1994). The inhibition of protein synthesis by CHX diminished not only the appearance of OPs but also plant elongation. Monensin, is an ionophore that inhibits the secretory pathway. It causes the same growth phenotype as CHX treatment and prevents the formation of OPs (Hoffmann-Benning et al., 1994). In addition, enzyme-gold labeling confirmed that they are, at least in part, proteinateous. However despite the importance of the secretion of OPs for cell elongation, their exact composition remains unclear. Their localization and appearance suggested that they might transport cell wall or cuticle components or biosynthetic enzymes. 1.3 Integration of auxin signaling with other hormones and light 1.3.1 Crosstalk between auxin and other hormones Plant development is largely postembryonic, which allows for adaptive responses to different environmental stimuli, such as light, nutrition, and temperature. Mounting evidence suggests that these external cues target the metabolism, distribution, or perception of plant hormones (Kende and Zeevaart, 1997; Hardtke et al., 2007; Santner et al., 2009; Depuydt and Hardtke, 2011; Enders and Strader, 2015; Singh and Savaldi-Goldstein, 2015). Plant hormones are very important endogenous mediators that control and coordinate numerous plant developmental processes. The final developmental output is determined by a complex network in which different hormonal pathways interact with each other as direct, indirect, and co-regulated cross talk. For direct cross talk, multiple hormonal pathways regulate the same target and jointly control the expression of a common gene or modulate the activity of the same protein. During 14indirect cross talk, one hormone affects the perception, sensitivity, or availability of another hormone. If hormones regulate the same process via independent pathways that is called co-regulation (Chandler, 2009). Peng et al. (2009) examined direct auxin crosstalk, by analyzing the Arabidopsis Hormone Database and found that 17% of all hormone-regulated genes were under control of more than one hormone. For example, in the brevix radix mutant (brx), both, brassinosteroids levels in roots, as well as the transcription of auxin-responsive genes are repressed, suggesting that several BR-responsive genes are coregulated by auxin (Mouchel et al., 2006). In addition, application of exogenous auxin affects the expression of 25% of BR-upregulated genes. Indirect auxin crosstalk is the most common interaction mechanism between auxin and other hormones. For example, auxin enhances ethylene biosynthesis by stimulating the activity of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase. Auxin also regulates the metabolism of GA through the induction of several GA oxidase genes. In addition, several genes in the auxin signaling pathway are also regulated by other hormones. One example is the AUX/IAA gene SHORT HYPOCOTYL2 (SHY2), whose transcriptional level in the root meristem transition zone is controlled by B-type Arabidopsis response regulator (ARR) transcription factors ARR1 and ARR2. Both also act downstream of cytokinin signaling (Dello Ioio et al., 2007; Dello Ioio et al., 2008). Furthermore, auxin distribution is affected by gibberellin, brassinosteroids, and cytokinin (Depuydt and Hardtke, 2011). In Arabidopsis, brassinosteroids induce the expression of PIN1 and PIN2 in the root and repress the transcription of PIN3, PIN4 and PIN7 in seedlings (Li et al., 2005; Mouchel et al., 2006). In contrast, cytokinins inhibit PIN 15transcription through activation of SHY2, which is a potential ARFs repressor (Vieten et al., 2005; Pernisova et al., 2009). 1.3.2 Crosstalk between auxin signaling and light Light, which provides energy for photosynthesis as well as information about seasonal timing and local habitat conditions, is vital for plant growth and development. Plants have developed sophisticated light signaling networks that detect and respond to changes in light duration, intensity and spectral quality. There are four major groups of light receptors, including the red and far-red light absorbing phytochromes (phy), the blue-light absorbing cryptochromes (cry), phototropins, and a ZTL-like F-box protein (Somers et al., 2004; Halliday et al., 2009). These receptors collectively control important aspects of plant development such as flowering and germination, tuberization and bud dormancy, and adaptive responses to microclimate and local habitat. Many of these light responses are achieved by linking to auxin signaling. Photomorphogenesis is a complex process that refers to various developmental responses of plants to light signals (Whitelam et al., 1998). It depends on the action of several photoreceptors that interact with endogenous developmental programs. One key player is the phytochrome system. Phytochromes are a family of red/far-red responsive photoreceptors that constitute the most important regulator of photomorphogenesis, including de-etiolation, germination, shade avoidance, circadian rhythm and flowering (Yun-Jeong Han, 2007). Arabidopsis seedlings with an abundance of auxin show longer hypocotyls and reduced cotyledon expansion, similar to the phenotype which can be seen in dark-grown, etiolated seedlings and phytochrome deficient mutants (Vanderhoef and Briggs, 1978; Sawers et al., 2002; 16Hoecker et al., 2004; Nagashima et al., 2008; Halliday et al., 2009). In contrast, plants with deficiencies in auxin biosynthesis display shorter hypocotyls that mirror enhanced photomorphogenesis/de-etiolation. Corn seedling development is similarly regulated by auxin and light. Light inhibits mesocotyl and coleoptile elongation by controlling the auxin supply from the coleoptiles (Vanderhoef and Briggs, 1978; Barker-Bridgers M, 1998). Together these phenomena suggest that light signals are closely tied to auxin homeostasis. In fact, through linking to the auxin system, light is able to control plant growth and development in response to the frequent changes in the light conditions. By regulating both SUR2, a suppressor, and TAA1, an enhancer of IAA biosynthesis, light controls auxin levels in planta (Mikkelsen et al., 2004; Lau et al., 2008; Tao et al., 2008) (Fig. 1.2). SUR2 is a cytochrome P450 monooxygenase CYP83B1, which catalyzes the first step of indole glucosinolate biosynthesis thus converting IAOx to an inactive metabolite rather than IAA (Fig. 1.2). Disruption of SUR2 function in Arabidopsis mutants blocks the metabolic route from indole-3-acetaldoxime to glucosinolate, enhancing IAA production (Hoecker et al., 2004). TAA1, a tryptophan aminotransferase, converts tryptophan to IAA. Active phyB reduces IAA levels by coordinated activation of SUR2, and repression of the TAA1 transcription level (Stepanova et al., 2008; Tao et al., 2008). In the taa1 mutant, plants are unable to respond to low R:FR ratios, due to the reduced IAA levels. Phytochromes control not only auxin biosynthesis but also auxin availability. Auxin levels can be modulated by the GH3 gene family of enzymes, which catalyze the conjugation of IAAs to amino acids for storage or degradation. By regulating the transcription of several GH3-type genes, phyA and phyB impose a strong influence on 17active auxin levels. Plants with suppressed GH3 gene transcription show a dark specific elongated hypocotyl phenotype, while plants with elevated GH3 levels display an enhanced photomorphogenic seedling phenotype. Via these mechanisms, environmental signals can exert the fine-tuning of auxin availability (Nakazawa et al., 2001;Tanaka et al., 2002; Takase et al., 2003; Takase et al., 2004). Phytochrome photoreceptors may also directly control auxin signaling. For instance, it has been shown that a recombinant oat phyA can interact with, and phosphorylate, the recombinant Arabidopsis auxin receptor SHY2/IAA3, as well as AXR3/IAA17, IAA1, IAA9 in vitro (Colon-Carmona et al., 2000). Native phyB was also shown to interact with recombinant SHY2/IAA3 and AXR3/IAA17. These studies provide a model explaining how light could exert direct control on Aux/IAA protein activity. 1.3.2.2 Light affects auxin transport Auxin is a mobile morphogen. Reed showed that both shoot and root development can be significantly changed by N-1-naphylphtalamic acid (NPA), a polar auxin transporter inhibitor (Benning, 1986; Heyn et al., 1987; Reed et al., 1998), which alters the auxin distribution between shoot and root. This mechanism seems to be used by light to control seedling growth and development. In phytochrome and cryptochrome receptor mutants, the inhibition of hypocotyl growth caused by NPA was reduced, suggesting that auxin polar transport requires phytochrome and cryptochrome action (Salisbury et al., 2007). In addition, loss of phyA and phyB leads to a reduction of auxin transport between shoot and root, an accumulation of auxin in the shoot, and causes simultaneous elongation of the hypocotyl, reduction of primary root growth, and lateral root production (Bhalerao et al., 2002; Canamero et al., 2006; Salisbury et al., 2007). 18Light regulates auxin movement by modifying the abundance and distribution of auxin transporters (PGPs and PINs). Several recent studies showed that the PIN3 transcription is highly correlated with phytochrome Pfr levels. phyB activation reduces PIN3 mRNA level, and phyB inactivation or loss lead to an increase of PIN3 transcripts (Friml et al., 2002). In addition, the phyA, phyB, cry1, and cry2 light receptors regulate the PGP19 (P-glycoprotein 19) protein level within the upper portion of the hypocotyl collectively (Nagashima et al., 2008). This interferes directly with the auxin-transport activity of PGP-1. At the same time, light signaling can also moderate auxin distribution by directly regulating PIN1, PIN2 and PIN7 protein function (Laxmi et al., 2008). Light controls the intracellular localization of PIN2 via the ELONGATED HYPOCPTYL 5 (HY5) branch of blue-light receptor pathway, thus maintaining its plasma membrane localization, and reducing vacuolar targeting for protein turnover (Oyama et al., 1997; Osterlund et al., 2000). Together, these results indicate that light regulates plant growth partially by changing the intracellular distribution of PIN proteins and, as a consequence, the auxin distribution within the plant. 1.3.2.3 Light and auxin signaling pathways have common gene targets Light not only controls auxin levels and distribution but also directly integrates with auxin signal transduction. Upon perception of auxin, the transcription of three gene families, Aux/IAA, SAUR, and GH3, is rapidly induced. Aux/IAA proteins are repressors of auxin response factors. They have been reported to be involved with a range of processes, such as cotyledon expansion, hypocotyl and primary root elongation, and lateral root formation (Reed, 2001; Sauer et al., 2013). Although the function of SAUR 19proteins still remains unknown, they have been implicated in a calmodulin (CaM) -auxin signal transduction pathway (Yang and Poovaiah, 2000; Li et al., 2015). By controlling the auxin levels, the GH3 proteins, a group of enzymes that conjugate free IAA with amino acids, have a strong effect on the auxin signaling pathway (Staswick et al., 2005; Park et al., 2007). Using transcriptomic analysis, Tepperman has shown that during seedling de-etiolation these gene families are regulated by phytochrome. In addition, genes with altered expression under low R:FR-ratio are highly correlated with auxin-related genes (Tepperman et al., 2001; Tepperman et al., 2006). Together these results suggest that phytochrome signaling operates closely with auxin perception. These transcriptional links between light- and auxin-regulated gene expression were reinforced by mutant analysis. The shy2/iaa3 mutant, which was isolated as a suppressor of the phytochrome chromophore-deficient mutant hy2, showed a constitutively photomorphogenic phenotype (Tian et al., 2002). A mild photomorphogenic phenotype was also seen in a dominant mutation of AXR2/IAA7 that stabilizes IAA7 by reducing the affinity for TIR1. This suggests that turnover of Aux/IAA is important for photomorphogenesis . In plants, the shade avoidance syndrome demonstrates the strongest connection between auxin and light signaling. By investigating the biochemical principles behind this phenomenon, a number of PHYTOCHROME RAPIDLY REGULATED or PAR genes, which play a key role in coordinating light and auxin signaling, have been identified. Some of the PAR genes are members of the bHLH family of transcription factors, such as PIL1, PAR1, PAR2, and HFR1 (Salter et al., 2003; Sessa et al., 2005; Roig-Villanova et al., 2006; Roig-Villanova et al., 2007). PAR1 and PAR2 suppress 20auxin-induced expression of Small Auxin-Up RNA gene15 (SAUR15) and SAUR68. PAR1, PAR2, and HFR1 have been demonstrated to play an important role in linking auxin signaling and the physiological response to low R:FR-ratio light. Other PAR genes, including ATHB2, ATHB4, and HAT1, 2, 3, belong to the HD-zip class-II subfamily of transcription factors (Steindler et al., 1999; Sawa et al., 2002; Ciarbelli et al., 2008). Recently, ATHB4 protein has been shown to participate in shade avoidance as well. It can suppress the transcription of SAUR15 and SAUR68. In addition, elevated ATHB4 activities reduce the hypocotyl response to exogenous auxin (Sorin et al., 2009). In addition, the expression of HAT2 is significantly induced not only by low R:FR ratio light but also by auxin application (Sawa et al., 2002). The bZip transcription factors HY5 (LONG HYPOCOTYL 5) and HYH (HY5 HOMOLOG) play prominent roles in light and auxin signaling (Holm et al., 2002). Light abolishes the activity of the COP1 E3 ligase, which targets HY5 for degradation, and leads to an accumulation of HY5 protein (Sibout et al., 2006). By binding to G-box motifs in promoter sequences, HY5 and HYH induce the transcription of light-related genes (Ang et al., 1998; Chattopadhyay et al., 1998; Holm et al., 2002); HY5 and HYH are also proposed as negative regulators of auxin signaling pathway. They directly regulate AXR2/IAA7 and SRL/IAA14 gene expression. In addition, loss of HY5 causes a repression of the transcription of both genes (Cluis et al., 2004; Sibout et al., 2006). Therefore, it seems that light modulates the expression of both auxin-signaling components and modulators of auxin responsiveness. In this thesis I compared proteins from either the intact corn coleoptile or the peeled outer epidermis of both, auxin-treated rapidly growing or non-treated slow 21growing coleoptiles, using one-dimensional SDS-Polyacrylamide gel-electrophoresis (SDS-PAGE) followed by LC-ESI-MS/MS to identify proteins involved in expansion growth. Of 86 proteins identified, four were novel proteins of unknown function. I showed that one of the proteins of unknown function, ARRP1, with a DUF538 domain, is expressed in above ground tissues and enriched in the area where cell division and cell elongation growth take place. Upon auxin-treatment ARRP1 mRNA and protein increase within 30 minutes Œ prior to measurable growth and to the expression of genes encoding cell wall biosynthetic enzymes. ARRP1 expression could be induced by red and, to a lesser extent, blue light, indicating a possible role in phytochrome and cryptochrome signaling. In situ hybridization showed that ARRP1 was expressed in the vasculature of growing tissues. This is consistent with our finding that corn ARRP1 interacts with the cytoplasmic domain of Arabidopsis VH1/BRL2, which plays a role in vascular development. Arabidopsis plants lacking the ARRP1 homologue will grow normally under standard conditions, but show delayed growth, delayed flowering, and reduced yield, when germinated in the dark and transferred to light, a process that can be reversed by complementing the Arabidopsis mutant with the maize gene. These findings together with the localization of ARRP1 in cytoplasm and nucleus suggest a possible function in the light/auxin signaling path. 22(b)(a)Figure 1.1 Chemical structure of the most common phytohormones (a) and 2,4-Dichlorophenoxyacetic acid (b), a synthetic auxin used as herbicide (Kende and Zeevaart, 1997; Santner et al., 2009; Santner and Estelle, 2009; Wolters and Jurgens, 2009; Depuydt and Hardtke, 2011). (b)2,4-Dichlorophenoxyacetic acid 23Figure 1.2 Potential IAA biosynthetic pathways are integrated with the effect of the R:FR light ratio on IAA biosynthesis. Pathways for which the enzymes have been identified are indicated by solid arrows; dashed arrows indicate pathways for which enzymes have not yet been identified and that may consist ofsingle or multiple steps. 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37 CHAPTER 2: CONTRIBUTION OF PROTEOMICS IN THE IDENTIFICATION OF NOVEL PROTEINS ASSOCIATED WITH PLANT GROWTH This chapter was published in Journal of Proteome Research (2013): Contributionofproteomicsintheidentificationofnovelproteinsassociatedwithplantgrowth J Li, TJ Dickerson, S Hoffmann-Benning Journal of proteome research 12 (11), 4882-4891 382.1 Abstract The need to produce sufficient food for an increasing world population during today™s climate changes puts urgency into our understanding of how plants grow under adverse environmental conditions. Since the epidermis is not only the interface between the plant and the environment, but also a growth limiting tissue, understanding the initiation and regulation of its expansion growth is essential for addressing this issue. We used mass spectrometry to identify proteins from auxin-induced rapidly-growing corn (Zea mays) coleoptiles to find possible candidates controlling this growth as well as the underlying cell wall and cuticle biosynthesis. Of 86 proteins identified, fifteen showed a predicted association with cell wall/cuticle biosynthesis or trafficking machinery; four were proteins of unknown function. As expected, real-time PCR indicated that the steady-state mRNA levels of genes with a known or predicted role in cell wall biosynthesis increases upon treatment with auxin. Importantly, genes encoding two of the hypothetical proteins also show higher levels of mRNA; additionally, their gene expression is down regulated as coleoptile growth ceases and up regulated in expanding leaves. This suggests a major role of those novel proteins in the regulation of processes related to cell and organ expansion and thus plant growth. 392.2 Introduction As the world population grows, our need for food and fuel increases drastically. The green revolution, occurring from the 1930s to 1960s, addressed these issues through the development of high-yielding varieties, the distribution of hybridized seeds, synthetic fertilizers and pesticides, and a modernization of management techniques and irrigation systems (Borlaug, 2000). Today, the amount of arable land is limited and often there is competition between food and fuel crops. In addition, changes in the global climate may reduce future yields. To continue to provide sufficient food and fuel, we need plants that show either accelerated growth or have a higher grain or cell wall yield or quality and are more resistant to biotic and abiotic stresses. One proposed approach towards the solution of this problem is to better understand the regulation and the processes necessary for plant growth. The corn coleoptile is a good model system for these studies (Haga and Iino, 1998). It is a protective sheath surrounding the first leaf of monocotyledons as the mesocotyl grows and pushes the seedling through and out of the soil. At the same time the coleoptile also elongates though cell expansion. Once it reaches the surface and perceives light, coleoptile growth ceases and the leaves emerge. This expansion growth can be mimicked by treating the coleoptile with the growth hormone auxin which leads to rapid cell expansion and elongation of the entire organ. If this rapidly growing tissue is split longitudinally it will bend outward, suggesting that it is the outer epidermis, which mechanically limits the growth (Schopfer, 2006). Whether this is indeed the case has been debated for decades (Peters and Tomos, 2000; for reviews see Schopfer, 2006; Kutschera and Niklas, 2007; Savaldi-Goldstein, 2007). 40Examination of the cell ultrastructure of rapidly growing tissues has shown the appearance of osmiophilic particles (OPs; Fig. 2.1) between the plasma membrane and the outer epidermal wall of rapidly growing tissues in a variety of plants (Robards, 1969; Oleson, 1980; Kutschera et al., 1987; Kutschera and Kende, 1989; Hoffmann-Benning et al., 1994a). Mostly, they are thought to be connected with cell-wall biosynthesis or loosening. Vaughn (2002) had shown a strong correlation of osmiophilic particles with an increased pectin deposition during weed dodder attachment to the host. Yet, while he could identify the pectin content of the newly formed wall, he reported no labeling of the particles themselves. Closer investigation of the osmiophilic particles has revealed that they are in part proteinaceous and are secreted only to the outer wall of expanding epidermal cells. Inhibition of the secretory pathway with monensin reduces not only the appearance of the osmiophilic particles but also organ growth and cutin synthesis (Hoffmann-Benning et al., 1994a, Hoffmann-Benning and Kende, 1994b). Upon inhibition of o-glycosylation the appearance of osmiophilic particles, synthesis and transport of proteins into cell wall, and concomitant growth were inhibited, suggesting that glycosylation and secretion are essential steps in auxin-induced expansion growth (Edelmann, 1995). The key differences between cells of the outer epidermis as compared to cells of the inner parenchyma are the larger diameter of the outer epidermal wall and its superimposition with the cuticle. The localization and appearance of the OPs suggests that they could transport cell wall or cuticle components or enzymes for cell-wall or cuticle biosynthesis. 41The plant cell wall and the cuticle play an important role in plant growth and development and form a barrier against pathogen infection. The cuticle provides the outermost barrier between a land plant and its aerial environment. It limits non-stomatal water loss and gas exchange (Baker et al., 1982; Riederer and Schreiber, 2001; Ristic and Jenks, 2002; Vogg et al., 2004; Aharoni et al., 2004), protects the plant from biotic and non-biotic stressors (Schweizer et al., 1996; Cameron et al., 2006; Bessire et al., 2007; Chassot et al., 2007), and controls proper organ fusion and development (Lolle et al., 1998; Sieber et al., 2000; Kurdyukov et al., 2006). The cutin matrix is a polymer which is mostly composed of C16 and C18 hydroxy and epoxy fatty acids, but also contains small amounts of phenolic acids and other aromatic monomers (Riley and Kolattukudy, 1975; Kolattukudy, 1996; Nawrath, 2002, 2006; Heredia, 2003). Depending on the composition of the epicuticular wax, the cuticle can be spiky to discourage feeding or slippery to protect from small herbivores or to catch insects in carnivorous plants (Riedel et al., 2003; Riedel et al., 2007). As the plant cell expands the cuticle has to grow with it. Earlier publications had shown that the rate of cutin biosynthesis increased during gibberellic acid-induced stem extension in peas while the composition did not appear to vary significantly in pea (Bowen and Walton, 1988). Yet in rapidly growing deepwater rice internodes, both the rate of cutin synthesis as well as its composition changed (Hoffmann-Benning and Kende, 1994b). Likewise, the composition of the cuticular wax in Kalanchoe changed during leaf expansion (Van Maarseveen et al., 2009). Several attempts are in progress to better characterize cutin biosynthesis and deposition and its relation to organ expansion (Suh et al., 2005; Kunst and Samuels, 2006; Samuels et al., 2008; Matas et al., 2011). 42The cell wall is a dynamic structure which gives the cell and the complete organism its structure and stability but is also essential for cell division and differentiation, cell and organ expansion, and for the response to environmental stimuli and pathogen infection. (Cosgrove et al., 2002; Cosgrove, 2005; Huckelhoven, 2007; Anderson et al., 2010). It consists of an intertwined network of polysaccharides, lignin, and proteins (Carpita and Gibeaut, 1993). Up to 95% of its mass is composed of polysaccharides in the form of cellulose, hemicellulose and pectin. Only 5-10% of the cell wall mass is composed of proteins (Cassab and Varner, 1988). These cell wall proteins (CWPs) can be (i) structural components of the cell wall, (ii) enzymes involved in cell wall biosynthesis, modification or turnover, or (iii) they can play a role in generating signals within and transmitting them through the cell wall, or they can have as of yet unknown functions. While cell wall polysaccharides lend strength to the cell wall, they also confer rigidity and thus constrain cell expansion. Thus, cell expansion must consist of cell wall loosening as well as synthesis. This wall loosening can be accomplished through the activites of proteins such as expansins, endo-(1,4)--D-glucanases, and xyloglucan endoglucosylases/hydrolases (Cosgrove, 2005; Humphrey et al., 2007). Consequently, cells have to retain the ability to monitor changes in cell wall composition and integrity as well as signal those changes and regulate the biosynthesis machinery (Steinwand and Kieber, 2010). Of the estimated number of CWPs, approximately one fourth, or four hundred, have been identified in Arabidopsis. Many contain posttranslational modifications, like hydroxylation, glycosylation or phosphorylation. These CWPs have to be transported to the apoplast and, most likely, pass through the secretory system. However, some 43CWPs seem to be lacking predicted signal peptides and may use alternative pathways (for a review see Jamet et al., 2008). In addition, while the polysaccharide composition of the cell wall of grasses is well known, it is different from that of dicotyledonous plants in that it contains less pectin and xyloglucan and more heteroxylan and compounds like mixed-linked glucans (MLGs), which are of high nutritional importance. The majority of CWPs related to cell wall synthesis have been characterized in dicotyledonous plants, while their nature in grasses remains largely speculative (Burton et al., 2005, 2008; for a review see Fincher, 2009). Our objective here was to better understand the changes that occur during expansion growth as it relates to regulation of biosynthetic processes in cereal grasses. Using corn coleoptiles and auxin-induced growth as a model system, we used a proteomics approach to identify proteins present in the epidermis of rapidly-growing corn coleoptiles and analyzed their expression pattern. Our approach allowed for the identification and characterization of two novel proteins, whose gene expression is correlated with growth and who have a possible role in cell wall or cutin biosynthesis in maize. Based on homology searches one could function in the early regulation/initiation of cell expansion growth while the second protein may be directly involved in the synthesis of mixed-linked glucans. As a result, they could be important for the production of biomass in agriculture. 442.3 Materials and Methods 2.3.1 Plant material and growth conditions Zea mays Great Lakes 4758 hybrid seeds were rinsed thoroughly to remove fungicides and shaken in water for up to one hour to speed germination. Kernels were planted in a standard soil mixture containing equal parts of Bacto Soil (Michigan Pear Company, Houston), medium vermiculite and perlite. They were grown in the dark at room temperature. Unless indicated otherwise, coleoptiles were harvested 4.5 days after planting. 2.3.2 Sample harvest Coleoptiles were pulled off the primary leaf and cut to a length of 10 mm after removal of the tips. All coleoptiles (40 total per experiment) were shaken in the dark in distilled water for 1 hour to remove residual auxin. Half were transferred into fresh water (control) and half into an auxin (3x10-5 M indole acetic acid - IAA; Sigma-Aldrich; rapidly-growing sample). The samples were covered while shaking for 4-5 hours. Coleoptile length was measured at the end of the incubation period to confirm auxin action and induced growth. To collect the epidermis, twenty coleoptiles were carefully peeled, the resulting epidermis immediately dipped into liquid nitrogen and stored at -80°C (Fig. 2.2 & 2.3) for further experiments. 2.3.3 Protein collection and identification Tissue from 20 coleoptiles per treatment was ground in 50 mM Tris/HCL (pH 8), 5% glycerol V/V, 0.1 M KCl and centrifuged at 10,000g for 2 min. Identical amounts of proteins from the supernatant were loaded onto a Bio-Rad Polyacrylamide15% Tris-HCl Ready Gel. Protein bands (indicated with asterisks and arrows in Fig. 2.4a) were 45excised and digested with trypsin (Shevchenko et al., 1996). Each protein band was analyzed from at least two different gels from independent sample preparations according to Guelette et al. (2012): The digested peptides were extracted into 60% acetonitrile / 1% TFA. Liquid chromatography / mass spectrometry was performed on a Capillary LC system (Waters, Milford, MA) coupled to a LCQ DECA ion trap mass spectrometer (Thermofinnigan, San Jose, CA) equipped with a nanospray ionization source. The sample was trapped onto a Peptide Cap Trap (Michrom BioResources, Auburn, CA) and flushed onto a 5 cm x 75 µm ID picofrit column packed with 5 µm ProteoPep C18 material (New Objective, Woburn, MA). Peptides were eluted with a gradient of 2% to 95% ACN in 0.1% formic acid at a flow rate of 200 nl per minute for 60 minutes. Peak lists for MS2 were generated by Bioworks and spectra searched using SEQUEST or MASCOT against Zea mays and Arabidopsis databases. Carbamidomethyl cysteine was set as Þxed modiÞcation (+57.0215 Da), and oxidation of Met (+15.9949 Da) and phosphorylation of Ser, Thr, and Tyr (+79.96633 Da) was allowed. Up to two missed tryptic sites were permitted. Peptide tolerance was set to 2.5 Da, and MS/MS tolerance was set to 0.8 Da. Positive identiÞcations were conÞrmed by individually comparing MS/MS spectra. Positive identiÞcation required at least two unique peptides per protein, counting only peptides with signiÞcant scores (95% conÞdence per peptide; >2.0 for SEQUEST). Database searches using individual tryptic fragments were performed using the BLAST searches at NCBI (http://www.ncbi.nlm.nih.gov/blast). 462.3.4 RNA extraction, quantitative & qualitative analysis RNA was extracted from equal amounts of tissue using a Qiagen RNeasy Mini-Kit. The RNA was treated with 1 µl/100 ml of 10 Units/µl Roche RNase-free DNase I for 1 hour at 37°C followed by RNA cleanup using the same kit. Quantification was determined using the Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies Inc., now Thermo Scientific; Wilmington, DE). RNA quality was confirmed using 260/280 nm ratio. Quantitative real-time PCR analysis was performed in 96-well plates on an ABI7000 instrument (Bio-Rad) using the 2×SYBR Green PCR core reagent (Applied Biosystems). Reactions were performed in a volume of 30 µl containing 2 µl cDNA, 0.1 mM forward and reverse primers, and 15 µl 2×SYBR Green master mix reagent. Each sample had three biological duplicates. The amplification conditions of all genes were: 95 for 3 min; 40 cycles of 95 for 10 s, 63 for 30 s, and 72 for 30 s; and a final extension at 72 for 5 min. At the end of the amplification, a dissociate stage was added to verify the amplification specificity, and the fluorescence was monitored. The threshold values were obtained using the automated setting of the instrument software, and the Cq (quantification cycle) value was also automatically calculated by the software. The data, expressed as Cq values, were imported into a Microsoft Excel applet for successive analysis. All the primers used were designed using the free Primer 3 software with the exception of the 18S primers and are shown in Table S2.1b. 18S was used to generate the stand curve and as a control. qPCR was performed with three biological replicates. Each biological replicate was repeated three times. 47For RT-PCR 400 ng mRNA was reverse transcribed using Omniscript Reverse Transcriptase (Qiagen). All Primers used for RT-PCR are listed in the Table S2.1a. PCR products from 6-8 biological replicates were visualized with a 1% agarose gel and quantified using pixel counts. The 18S signal for each sample was used for normalization. Data were quantiÞed using a Student™s t test (Table S2.3). Using a linear regression analysis on the data conÞrmed that the average expression level of these genes is signiÞcantly decreased with time in all cases (p value <0.023) The identity of the PCR products was confirmed by ligation into Promega™s pGEMT-easy vector system followed by transformation into Stratagene™s XL10-Gold Ultracompetent Cells. The Research Technology Support Facility at Michigan State University synthesized all oligonucleotides and performed all automated DNA sequencing. 482.4 Results and Discussion 2.4.1 Proteomics analysis To identify proteins with a possible role in cell expansion growth corn coleoptiles were treated with water (control) or 3 x 10-5 M Indole acetic acid (IAA; rapidly growing) for four hours. Proteins from either the intact corn coleoptile or the peeled outer epidermis were extracted and separated using one-dimensional SDS-Polyacrylamide gel electrophoresis (SDS-PAGE; Fig. 2.4a). The comparison between auxin-treated and control tissues was expected to reveal proteins involved in auxin-induced expansion growth. Comparison between coleoptile and epidermal tissue would indicate epidermis-specific roles. The comparison consistently revealed nine protein bands that were increased in the outer epidermis as compared to the total coleoptile (at. 75, 55, 50, 40, 25, 21, 19, 17, 9, 8 kDa, respectively; Fig. 2.4a as indicated with an asterisk). Quantification of the gel bands revealed increases of up to 30% above the band intensity in control lines. Several of these as well as six additional bands were increased in the auxin-induced rapidly growing sample (116, 56, 24, 17, 16, 9 kDa; indicated with arrows). Since the outer epidermis is discussed as the growth-limiting tissue (Peters and Tomos, 2000; Schopfer, 2006; Kutschera and Niklas, 2007; Savaldi-Goldstein, 2007) and is distinguished from the rest of the coleoptile tissue by containing the cuticle as well as a much thicker cell wall, these proteins could potentially play an important role in the regulation of organ growth as well as the associated cell wall and cutin biosynthesis. In-gel digestion (Shevchenko et al., 1996) followed by LC-ESI-MS/MS revealed 86 proteins, 66 of were derived from bands induced in the epidermis of rapidly growing coleoptiles (see table S2.2). The proteins were categorized by their predicted 49function (Fig. 2.4b). Eighteen of these proteins are involved in protein biosynthesis (eight ribosomal proteins, five translation initiation or elongation factors) or are chaperones (five). The rise in this group of proteins is not surprising since an increase in protein synthesis and their proper folding will be necessary to synthesize building blocks and provide energy to maintain cell and organ expansion. One of them, cyclophilin, is known to be increased in germinating seedlings and growing tissues as well as in response to various stress factors and plant hormones (Marivet et al., 1994; Cho and Kim, 2008). Fifteen proteins are involved in carbon metabolism and transport while others play a role in cell division and/or are part of the cytoskeleton (11). Eight proteins play a role in pathogen or stress response and an additional 19 proteins belong to no specific subgroup. Of particular interest are proteins with a possible novel or not fully elucidated role in cell expansion and organ growth and the necessary cell wall and cutin biosynthesis: They could function in cell wall biosynthesis/ secretory pathway (I), lipid metabolism/cutin biosynthesis (II) or are previously uncharacterized hypothetical proteins with an either regulatory or synthetic function (III). Three groups of identified proteins fall into that category: Proteins with a Possible Role in Secretory Pathway or Cell-Wall Biosynthesis. Not surprisingly, several of the identified proteins are possibly related to the cell wall or the secretory pathway (sec 15, calreticulin, translationally controlled tumor-protein like; see table 1): One protein which is secreted is the translationally controlled tumor protein like protein (TCTP). It was originally identified as a calcium-regulated gene involved in 50the gravitropic bending of maize roots (Kim, J.-Y. and Poovaiah, B.W.; NCBI; submitted Sept. 2002) and belongs to the TCTP superfamily. Arabidopsis plants impaired in TCTP expression displayed a dwarf phenotype due to a reduced cell size and a reduced sensitivity to auxin (Berkowitz et al., 2008). This suggests that plant TCTPs may act in an early control of cell division and cell elongation rather than in cell wall synthesis. The reversibly glycosylated polypeptide (RGP) is highly similar to the Golgi-associated se-wap41. RGPs are delivered to the cell wall/plasmodesmata via the Golgi apparatus (Sagi et al., 2005). They are now considered UDP-Ara mutases that catalyze the interconversion of UDP-L-arabinopyranose and UDP-L-arabinofuranose, which are important in heteroxylan biosynthesis. Similarly, UDP-glucose pyrophosphorylase (UDPgp) and UDP-glucose dehydrogenase (UDPgdh), two enzymes with a function in the synthesis of cell-wall precursors, were found in our sample: UDPgp catalyzes the reversible conversion of UDP-glucose and pyrophosphate to glucose-1-phosphate and UTP. UDPgdh converts UDP-glucose to UDP-glucuronic acid, which is the precursor for UDP-galacturonic acid, UDP-xylose, UDP-arabinose, and other precursors for both the pectin and the hemicellulose components of cell walls.The identiÞcation of known cell-wall biosynthesis protein conÞrms the suitability of our approach for the identiÞcation of proteins associated with cell expansion and growth. Proteins Associated with Lipid Metabolism. We detected several proteins associated with lipid metabolism and, thus a possible affiliation with cutin biosynthesis such as a lipoxygenase, an acyl transferase, phospholipase D, and several lipid transfer proteins (LTPs). The acyl transferase may function in either cutin or phytoalexin and 51thus indirectly in lignin biosynthesis. Plant lipoxygenases belong to a large group of enzymes that catalyze the oxygenation of free polyunsaturated fatty acids, typically forming biologically active molecules, which are called oxylipins (Porta et al., 2002). Phospholipase D functions in the production of phosphatidic acid, a lipid involved in many aspects of stress signaling (Welti et al., 2002; McDermott et al., 2004; Testerink and Munnik, 2005; Lu et al., 2013). Rather than a biosynthetic, these proteins likely have a regulatory function (Feussner et al., 2002). A second induced lipid-related protein is an acyltransferase homologue with a high similarity to N-hydroxycinnamoyl /benzoyltransferase and a possible function in phytoalexin biosynthesis and, indirectly, lignin biosynthesis (Marchler-Bauer et al., 2007). Of the three identified lipid transfer proteins (LTPs), one is categorized as a non-specific lipid transfer protein, while the other two are grouped as two distinct phospholipid transfer proteins. Plant lipid transfer proteins are small cationic peptides of 7 to 10 kDa. They have the ability to bind phospholipids and other lipids and transfer them between a donor and a membrane in vitro (Kader, 1996). LTPs have a signal sequence and are typically transferred through the secretory pathway into the apoplast (Tsuboi et al., 1992; Thoma et al., 1993; Pyee et al., 1994; Carvalho et al., 2004). However, they have also been found in other cellular compartments and expanding hypocotyls (Irshad et al., 2008). Again, the finding of several proteins with a possible role in cell expansion confirms the usefulness of our approach for finding novel candidates: 52Hypothetical Proteins with, As of yet, Unknown Function. We were able to identify four hypothetical proteins with, as of yet, unknown function. Hypothetical protein 1 (HP1) appears to be similar to an Arabidopsis protein which is associated with the mitochondria. Hypothetical protein 2 (HP2) has similarity to MATH domain containing proteins. Proteins within this group include extracellular metallo proteases which are anchored to the membrane. Hypothetical protein 3 (Hyp3) is a 22 kDa protein with a similarity to endo-1,3-1,4-beta-D-glucanases. It could potentially be involved in the extension of mixed-linked glucans, a nutritionally important group of unusual cell wall polysaccharides mostly limited to cereal grasses (Rieder and Samuelsen, 2012). They contain stretches of (1,4)--D-glucans with interspersed (1,3)--glycosyl groups, which introduce fikinksfl in the polysaccharide chains that lead to an increased solubility and flexibility (Fincher, 2009). Their exact composition varies between different species and tissues and increases during phases of rapid cell elongation (Carpita et al., 2001; Gibeaut et al., 2005; McCann et al., 2007). While enzymes involved in the depolymerisation of (1,3;1,4)--D-glucans are well characterized, scientists are just starting to identify some of the genes including members of the CslF and H gene families that may contribute to (1,3;1,4)--D-glucan synthesis (Burton and Fincher, 2012). These genes largely control the synthesis of polymers in the Golgi apparatus (Carpita and McCann, 2010), however, the enzymes necessary for linkage and expansion of larger chains are yet to be identified. Expression and enzymatic activity of the barley homologue are induced in response to auxin (Kotake et al., 2002). It was hypothesized that endo-1,3-1,4--D-53glucanases act via simultaneous hydrolysis and incorporation of new glucans into the cell wall during plant growth (Hoson et al., 1992; Thomas et al., 2000). The exact function of hypothetical protein 3 remains to be determined. Initial experiments are described in the appendix of this thesis. Hypothetical protein 4 (Hyp4) contains a DUF538 domain. Its Arabidopsis homologue has been found to interact with VIT, a receptor modulator protein that is known to interact with brassinosteroid-receptor-like (BRL) proteins and predicted to play a role in signaling.70 However, the role of Hyp 4-like/DUF538 proteins in the signaling path remains unknown. The two latter hypothetical proteins are of particular interest because they may be new factors in the regulation or synthesis of cell wall or cuticle as they relate to growth. Several studies looking at gene expression in growing Arabidopsis stems (Suh et al., 2005), corn epidermis (Nakazono et al., 2003), and CW proteins in Arabidopsis hypocotyls (Irshad et al., 2008) or epidermal membranes of rye (Deng et al., 2012) have identified a large number of genes/proteins with a possible role in CW synthesis or growth, yet, of the 12 proteins we investigated further (see Table 1), only lipoxygenase, wall associated kinase, UDPgdh, and LTPs have been found in one or more of those studies. The homologues to the newly identified hypothetical proteins Hyp3 and Hyp4 have not been described. This shows that despite the 1-D gel electrophoresis, the selection of bands that are intensified in the growing epidermis, led to the discovery of additional proteins. 542.4.2 Expression studies Protein identification of 1D SDS-PAGE bands often yields more than one protein per band. Hence it is difficult to quantitatively assess their individual abundance. In addition, some less abundant proteins may escape discovery. Despite these shortfalls, our proteomics data still lead to the identification of four novel proteins of unknown function. To examine the differential expression of the genes encoding our proteins of interest, quantitative RT-PCR analysis was performed for lipoxygenase, UDPgdh, UDPgp, TCTP, PLTP, ATH, RGP, two hypothetical proteins (Hyp3 & 4) as well as two proteins that were identified with only one tryptic fragment. The latter were included because a) the fragment had been identified with a good score in two independent experiments, b) the mass of the protein band corresponded to the molecular weight of the protein band, and c) the literature suggests that they are involved in cell wall biosynthesis (sec15-homologue: S15L; fiber dTDP-glucose 4-6 dehydratase: FDG). Except for Hyp 3 and Hyp 4, these proteins are expected to be involved in cell expansion growth and, as a result, their gene expression should be increase also. Hence, they could serve as positive controls. Steady-state mRNA levels in epidermal peels from slow versus rapidly growing coleoptiles were determined using quantitative PCR (Fig. 2.5; Fig. S2.1 visualizes abundance of RT-PCR products): 2.4.3 Genes encoding proteins with a possible role in cutin biosynthesis:We had detected several proteins associated with lipid metabolism: A lipoxygenase, an acyltransferase homologue (ATH), and three distinct lipid transfer proteins. While the expression of genes encoding ATH and PLTP was induced slightly to 150-200%, expression of the lipoxygenase gene was reduced to ca 50%, of the 55expression in control samples. The reduction in lipoxygenase gene expression in rapidly growing coleoptiles correlates with the findings of Kutschera et al. (2010) who had observed an increase in gene expression during cessation of rye coleoptile growth. Thus, expression of the lipoxygenase gene appears to be inversely correlated with growth. The function of the corn ATH is currently not known. Li et al. (2008) had identified an acyl-CoA: diacylglycerol acyltransferase, which is expressed in the Arabidopsis stem and is necessary for stem wax ester biosynthesis. However, the corn protein identified here shows little similarity to the acyltransferase found in Arabidopsis and, hence, may have a very different function in plant growth. LTPs have long been candidates for wax transport through the cell wall. They are abundant in the epidermis, are secreted into the apoplast and can bind fatty acids (Samuels et al., 2008). Yet, the large number of LTPs in plants has made it difficult to identify their individual roles. Experimental evidence points to a wide array of possible functions for lipid transfer proteins: cutin synthesis (Pyee et al., 1994; Han et al., 2001; Kunst and Samuel, 2003; Samuels et al., 2008; Lee et al., 2009), -oxidation, embryogenesis, allergenics, pollen adherence, plant signaling and plant defense (for a review see Carvalho and Gomes 2007). Several of their functions appear to be epidermis specific (Tchang et al. 1988, Arondel et al. 1991, Clark and Bohnert 1999). Auxin-mediated regulation of gene expression of acyltransferase (a,b), lipoxygenase (b), and several LTP homologs (a, b, c), has been previously shown in the epidermis of corn coleoptiles (a; Nakazono et al., 2003) and Arabidopsis stems (b; Suh et al., 2005 ) using microarray analysis and in a cell wall proteomics experiment in Arabidopsis 56hypocotyls (c; Irshad et al., 2008). We only see a small up regulation of gene expression for PLTP (1.7 fold) and ATH (1.8 fold). To further investigate and confirm the role of PLTP in cell expansion growth, we determined its gene expression in coleoptiles that were moved from dark to light. When coleoptiles are exposed to light their growth ceases within a few days. PLTP gene expression was reduced in parallel to coleoptile growth cessation further indicating a role in cell expansion (Fig 2.6). 2.4.4 Genes encoding proteins with a possible role in cell wall biosynthesis Since cell wall synthesis is necessary to maintain cell expansion growth, it would be expected that expression of genes encoding cell wall biosynthesis proteins is increased. We monitored the expression of several putative cell-wall biosynthesis genes in response to auxin as well as during the light-induced cessation of coleoptile growth. Indeed, quantitative RT- PCR showed that expressionof UDPgp and UDPgdh, was increased approximately two-fold, while FDG was increased almost threefold and RGP was increased fivefold upon treatment with auxin (Fig. 2.5; Table 2.1; sup. Fig. 2.1). RGP is highly similar to the Golgi-associated se-wap41. It was previously isolated from maize mesocotyl cell wall and found to localize to plasmodesmata (Epel et al., 2005). RGPs have been shown to auto-glycosylate reversibly (Dhugga et al., 1991; Dhugga et al., 1997; Delgado et al., 1998; Testasecca et al., 2004). They are delivered to the cell wall/ plasmodesmata via the Golgi apparatus (Sagi et al., 2005) and have been shown to be involved in pollen development (Drakakaki et al., 2006) and cell wall (xyloglucan/ hemicellulose) biosynthesis in pea, cotton, and Arabidopsis (Delgado et al., 1998; Zhao and Liu, 2002). They are homologues to UDP-glucose:protein transglucosylases, a group of proteins also involved in cell wall biosynthesis (UPTGs; 57Bocca et al., 1999). Several Arabidopsis RGPs function as UDP-Ara mutases with a role in heteroxylan synthesis (Rautengarten et al., 2011). UDPgps catalyze the conversion of UTP and glucose-1-phosphate to UDP-glucose and pyrophosphate. UDPgp acts at the intersection between multiple pathways: it can provide the substrate for sucrose biosynthesis or participate in sucrose breakdown, it can be involved in the synthesis for the carbohydrate moiety of glycolipids and glycoproteins and it provides a substrate (UDP-glucose), which is directly or indirectly used for cell wall biosynthesis (for a review see Kleczkowski et al. 2004). Arabidopsis ugp1/ugp2 double mutants showed little to no change in growth and cell wall composition but displayed reduced seed formation (Meng et al. 2009). In poplar, on the other hand, it was found up regulated in late cell expansion and secondary cell wall formation (Hertzberg et al. 2001). A third protein from our list, UDPgdh, converts UDP-glucose to UDP-glucuronic acid, which is then converted to other precursors for both the pectin and the hemicellulose components of cell walls (Mohnen 2002; Seifert 2004). Localization studies in Arabidopsis led to the suggestion of an alternative UDP-glucuronic acid synthesis pathway via inositol in rapidly expanding hypocotyls (Seitz et al. 2000). However, Kärkönen et al. (2005) showed that while a reduction of the enzyme activity in transposon-tagged mutants showed a reduced pentose content in the cell wall, it had no impact on plant growth and development. Yet, it appears that at least one of the two isoforms of this enzyme in maize is necessary for polysaccharide biosynthesis. Quantitative RT-PCR showed that expression of all four cell wall related genes was increased upon treatment with auxin (Fig. 2.5). 58To further confirm the correlation with growth, the expression of all three genes was monitored during the growth cessation of coleoptiles in response to light. UDPgdh and RGP showed a 50% reduction in gene expression within five days (Fig. 2.6). This finding was confirmed when the expression of the Arabidopsis homologue for the maize RGP (At5g16510) was studied in silico using the Bio-Array Resource for Arabidopsis Functional Genomics (http://bar.utoronto.ca/) - it indicated co-expression with UDP-glucose dehydrogenase (UDPgdh). Finding UDPgp, UDPgdh and RGP increased in our rapidly growing samples and the fact that their expression is correlated with coleoptile growth confirms the known importance of these proteins for cell wall synthesis and as factors controlling growth. 2.4.5 Hypothetical proteins The question remained whether the gene expression for two of the hypothetical proteins is associated with growth. Quantitative PCR showed that the Hyp3 and Hyp4 gene expression in response to auxin treatment was increased 2.5 and 1.7 fold, respectively (Fig. 2.5). To confirm the relevance of the two hypothetical proteins for cell and organ expansion we investigated their expression levels in several growing versus non-growing plant tissues using semi-quantitative PCR (Fig. 2.6 and 2.7). Hyp 3 expression was higher in the epidermis than in total coleoptile tissue (Fig. 2.7a), suggesting the epidermis as predominant locus of action. As seedlings were moved to the light, the coleoptile stopped expanding while leaf expansion increased. In parallel, Hyp3 expression was reduced to 68% of the dark expression within two days of the transition to light and to 40% of the dark expression levels after 5 days in the light (Fig. 2.6 & 2.7a; p<0.02). In leaves exposed to light, Hyp3 expression increased almost two 59fold as the leaf expanded (Fig. 2.7a). This suggests that it plays a role in the growth-controlling processes within the epidermis. Hyp4 expression is induced 1.7-fold in auxin-treated coleoptiles and reduced by 50% in light-exposed, slow growing coleoptiles (Fig. 2.6 & 2.7b). It, too, showed reduced expression as the coleoptile growth ceased and increased expression levels in expanding leaves. Together with the observation that its expression is higher in coleoptile and leaves than in the coleoptile epidermis, this suggests that it may participate in more general, possibly regulatory or signaling processes during growth. Both, Hyp3 and Hyp4 expression is increased in response to auxin. More importantly, their expression levels are reduced as coleoptile growth ceases and increased during leaf expansion. This suggests that they, indeed, play a role in cell expansion growth, possibly with Hyp3 in a synthetic and Hyp4 a regulatory function. 602.5 Conclusions Our goal was the identification of novel proteins with a role in cell and organ growth. Using rapidly growing corn coleoptiles as a model system and a combination of proteomics and PCR analysis, we have been able to identify 15 proteins with known or suggested roles in trafficking or cutin- or cell wall- biosynthesis, including four novel proteins of unknown function. For nine of those, the up-regulation is apparent at the mRNA level. The expression of the genes encoding two of the novel and uncharacterized proteins, Hyp 3 and Hyp 4 is increased in rapidly growing coleoptiles and expanding leaves but reduced in coleoptiles that have ceased growing. Our findings suggest they are indeed associated with cell (and plant) growth. They provide new and additional tools for the understanding of whole plant growth and can now be studied for their effect on whole plant growth and their usefulness in the increased production of biomass. 61Figure 2.1 Transmission electron micrograph of a cross section through a corn coleoptile epidermis cell treated with 30 M IAA for 1 hour. The osmiophilic particles (arrows) are present between the plasma membrane and the cell wall and within a secretory vesicle. The size bar corresponds to 200 nm. CWPMER62Figure 2.2 Flow chart of experimental setup for coleoptile treatment and harvest. 63Figure 2.3 Auxin induced corn coleoptile rapidly growth. Corn seedlings consist of a coleoptile, mesocotyl, primary leaf, and root (a). Size difference of auxin-depleted coleoptiles after incubation in water (slow-growing, left) or auxin (rapidly-growing, right) for four hours (b). Measurements indicate coleoptile length after four hours in either water (control) or 30M IAA. Data are mean and SE obtained from 45 coleoptiles. c) Preparation of epidermal peel as used in our experiments. 64Figure 2.4a SDS-PAGE gel comparison of banding patterns. The marked bands were selected for LC-ESI-MS because they showed differences between the treatments (Co-Control, IAA-auxin; arrow) and/or the sample site (coleoptile, epidermis; asterisk) 65Figure 2.4b Grouping of proteins enhanced in the epidermis as compared to the coleoptile and upon auxin-induction based on their predicted function/ involvement in cellular processes: (A) 11% Secretory pathway, cell wall and cuticle associated. (B) 13% Carbon metabolism/ETC. (C) 7% Structural/cell division. (D) 7% Others-oxidoreducatase, mutases, etc. (E) 7% Unknown/hypothetical proteins. (F) 23% Protein synthesis/degradation. (G) 16% Transcription/translation, splicing 66012 3 4 5 6 7GeneRatio of Induced/ ControlFigure 2.5 Differential expression of auxin-induced genes in the corn coleoptile using quantitative RT-PCR. fiInducedfl: coleoptiles were incubated in 30M IAA for four hours; fiControlfl: coleoptiles incubated in water for four hours. STD:18S mRNA; Lipo: Lipoxygenase; UPDgd: UDP-glucose dehydrogenase; UDPgP: UDP-glucose pyrophosphorylase; TCTP: translationally-controlled tumor protein; PLTP: phospholipid transfer protein; RGP: reversibly glycosylated protein; ATH: Acyl transferase; HP3/4: hypothetical proteins 3 and 4; S15L: sec 15-like protein; FDG: Fiber dTDP-glucose 4-6 dehydratase; Values are the mean and standard error for 9 replicates (three biological, three technical replicates, each). 67Figure 2.6 Semiquantitative analysis of the expression levels of genes encoding UDPgdh, UDPgp, Hyp 4, Hyp 3, PLTP, and RGP in corn coleoptiles (a) and leaf (b) after the transition of dark to light. Bars represent the mean ± S.E. of three biological replicates. Statistical analysis (t test) revealed signiÞcant reduction compared with the day 0 time point (p < 0.1: *, p < 0.01:**). 00.20.40.60.811.21.41.61.8UDPgdhUDPgpHyp4Hyp3PLTPRGPRatioofGene/18SExpressioninColeoptilesDay0Day2Day4Day5a***********************6800.511.522.5UDPgdUDPgpHP4HP3PLTPRGPRatioofGene/18SExpressioninLeavesDay0Day2Figure 2.6 (cont™d) b***69Figure 2.7 Expression levels of genes encoding Hyp 3 (a) and Hyp 4 (b) in various corn seedling organs as determined by RT-PCR. fiEpidermisfl corresponds to the epidermis of coleoptiles of seedlings germinated and grown in the dark for five days; Material for coleoptiles was harvested either after five days in the dark (dark) or 1, 2, or 5 days after transition of the 5-day-old, dark-grown seedling to light. Leaf samples were taken either from the leaf within the coleoptile of the 5-day-old, dark-grown seedling (undeveloped leaf) or two days after transition to light (2 days light). Bars represent the mean +/- S.E. of 3 biological replicates. 70Figure 2.7 (cont™d) 71Table 2.1: Auxin-regulated epidermal proteins with functions related to secretory pathway, cell wall or cuticle metabolism with comparison of RT-PCR to microarray data. Arrows indicate induction or reduction; U: no significant difference to control; NA: not analyzed: no oligonucleotides present on the microarray chip. Similar proteins which are up-regulated in the epidermis of top stems in Arabidopsis (Suh et al., 2005). ProteinAccess#MWqPCRPossibleFunctionSec15likeBAB93424105VesicletraffickingLipoxygenase1AAF7620798RegulatoroflipidbasedbiochemicalandsignaltransductionpathwaysWallassociatedkinase4like1AAF1850974ATPbinding/calciumionbindingproteinkinaseUDPglucosedehydrogenaseAAK1619465ConversionofUDPglucosetoUDPglucuronicacidUDPglucosepyrophosphorylaseAAP8631758ReversibleproductionofUDPglucoseandpyrophosphate(PPi)fromGlc1PandUTPAcyltransferasehomologBAA9345356PossiblyligninbiosynthesisReversiblyglycosylatedpolypeptideAAM6502041Biosynthesisofhemicellulose,mayuseUDPglucoseassubstrateHypotheticalprotein#3CAC2195529possible(1,3;1,4)DendoglucanaseHypotheticalprotein#4BU57199522Unknown;similartoriceNP_00106738withDUF538FiberdTDPglc46dehydrataseAAR0766021UnknownTCTPlikeAAN4068619Cabinding,presentinsecretionsPhospholipidtransferproteinAAAB0644312Lipidtransfer72Figure S2.1 PCR products obtained from induced (I) and control (C) coleoptile epidermis. The table (b) displays mean and standard error of the pixilation intensity comparisons between the gene of interest and the control 18S for 6-8 replicates [(IAA/18S)/ (Co/18S)]. A: RGP/ reversibly glycosylated protein; B: Hyp 4; C: Lipoxygenase; D: PLTP ; E: TCTP/ translationally-controlled tumor protein; F: UDPgp/ UDP-glucose pyrophosphorylase ; G: WAK/ wall associated kinase; H: Sec15-like; I: FDP/ Fiber dTDP-glucose 4-6 dehydratase; J: UDPgdh/ UDP-glucose dehydrogenase; K: ATH/ Acyl transferase; L: Hyp 3; 18S 73Table S2.1: Primers used for quantitative (a) and semiquantitative (b) RT-PCR; a Protein PrimersAccess # CDSReversibly glycosylated polypeptide F™ ATCTCATTGGCCCTGCTATG R™ GTCTTGACTCCCAGGCTCAG AY087476 Translationally controlled tumor-like protein F™ GCCGTGAAGGTGGTTGATAT R™ GCTTTCTCTGGCTCCAACAC AF548024 Lipoxygenase F™ CAACACCTCCGACACGAAGG R™ GGGGAAACGCAAACAATCTA AF271894 Phospholipid transfer protein F™ CCTGCAACTGCCTCAAGAAC R™ AGTCGGTGGAGGTGCTGAT ZMU66105 UDP-Glucose- pyrophosporylase F™ GCTCAAGGTTTCTCCCAGTG R™ TGAAGGGTTAGCTGGATTCG AY260746 Sec 15 F™ TGAATGTCCTTGCATTTGGA R™ GTGCCATGAAGCTCACAGAA AP003764 Acyltransferase F™ CTACTTCGGCAACCTCATCC R™ TACTCCTCCAGCCTCCTGGT AB026495 UDP-Glucose dehydrogenase F™ GATCCTCACCACCAACCTGT R™ CGTAAGCCACCTCAGTCACA AC079887 Fiber dTDP-glucose 4-6-dehydratase F™ GTTCAAAGTCTCCGCTCTGC R™ ACCCTACGATGTTCGACTGG AY378100 Wall-associated kinase-like 4 F™ CAGACTCACCTCTTCAGCTACG R™ GTTGTTGTACAGCCGCTTC T24D18 Hypothetical Protein 2 F™ GGAGCTTCTCATCTGGGTCA R™ GTTCCACTTCCTCTGGGACA DQ246015 (mRNA) Hypothetical Protein 3 F™ CACGAGGTTCCCTACTACGC R™ TCAGTTGAGGTGCTTGTTGAA AX053124 Hypothetical Protein 4 F™ GGGTAAGCTCCACTGCAAGG R™ GTTCTTGTCCCCCTCCTCCT BU571995 b Protein Primers Amplicon size Reversibly glycosylated polypeptide F™ GGTGCTGACTTTGTGCGTGG R™ CCCACATGTCGTCGTAGCG ~303 bps Translationally controlled tumor-like protein F™ CCTATCTCCTATCTGGCG R™ GGTTGCACCCTCGACACCC ~393 bps Lipoxygenase F™ CATCGGCGAGTTCCTCGG R™ GGGAGCTCCTTGGTGCCG ~530 bps Phospholipid transfer protein F™ CGGCCATCAGCTGCGGGC R™ GGAGGTGCTGATGGTGTAGG ~ 260 bps UDP-Glucose- pyrophosporylase F™ CGCTGGCTGACGTTAAGGG R™ GGATGGACTTGAACCGAGC ~ 490 bps Sec 15 F™ CTTCTCACGGATCACTGG R™ GCCCACATCTCTGAAAGG ~250 bps Acyltransferase F™ GCAGCAGGCTTATGCTGC R™ GATCTGGAATGGGGGCAG ~154 bps UDP-Glucose dehydrogenase F™ GAAGAGTAGCCACACACCG R™ GCTGTTATGGGCGTTGGTC ~300 bps Fiber dTDP-glucose 4-6-dehydratase F™ GCTCCCACCCTAGAACTTCC R™ GAGCTTCTGCTATGTCGCCG ~235 bps Wall-associated kinase-like 4 F™ GTTGTTGTACAGCCGCTTC R™ CCATGGTGTTGTTCACTGC ~456 bps Hypothetical Protein 4 F™ GCAAGGTGGACCACTACTTCG R™ CGTCCCTGGGAACGACACC ~279 bps 74Table S2.2: Proteins identified in the outer epidermis of corn coleoptiles; samples in bold were from bands which appeared induced upon treatment with auxin. Protein Identification Access # Trp frag. MW Localization/Function Sequest/Sequence Chargestate(Z)ASR1AAG28426 2 98 Carbon-oxygen lyase; cytosol; hydrolase 3.43 (R)SGVTATDLVLTVTQM*LR Z=2 3.25 (K)AFEDADSLGLTGHER Z=2Elongation factor 2 (putative) rice NP_001046972 NP_001052057 (At1g45332) 2 94 Elongation factor; GTP-binding; protein biosynthesis; 3.23 (K)AFLPVIESFGFSSQLR Z=2 3.03(R)LYMEARPLEEGLAEAIDDGRZ=2Phospholipase D maize NP_001105686 =Q43270 (At3g15730) 5 92 Phosphoric diester hydrolase; Ca-binding; 3.04 (K)VTLYQDAHVPDNFVPR Z=2 2.91 (R)TLDTVHHDDFHQPNFEGGSIK Z=3 2.76 (R)DSEIAM*GAYQPYHLATR Z=2 2.48 (R)NPDDSGSFVQDLQISTM*FTHHQK Z=3 2.30 (R)YDTQYHSLFR Z=2 Lipoxygenase maize NP_001105003 =AAF76207 =LOX (At1g55020) 9 98 Key regulator of lipid-based biochemical and signal transduction pathways 4.26 (R)GDGTLVPVAIELSLPELR Z=2 3.09 (K)LYEGGIQLPK Z=2 3.06 (K)LEGLTVQQALHGNR Z=2 2.90 (K)YGDHTSTITAEHIEK Z=2 2.59 (R)GMAVADPSSPYK Z=2 2.31 (R)VNSLEGNFIYATR Z=2 70 (K)DWNFTEQALPDDLIK Z=2 63 (K)YGDHTSTIR Z=2 Methionine synthase maize AAL33589 =LOC541942 16 84 Cytosol, catalyze the formation of methionine by the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine 3.63 (K)YGAGIGPGVYDIHSPR Z=2 3.54 (K)KLNLPILPTTTIGSFPQTVELR Z=2 3.30 (R)GTQTLGLVTSAGFPAGK Z=2 3.17 (R)YLFAGVVDGR Z=2 3.13 (K)YIPSNTFSYYDQVLDTTAMALGAVPER Z=2 2.88 (K)GMLTGPVTILNWSFVR Z=2 2.84 (K)ALAGQKDEAYFAANAAAQASR Z=2 2.73 (K)YTEVKPALTNMVSAAK Z=2 2.70 (K)ISEEEYVTAIKEEINK Z=2 2.68 (R)KYTEVKPALTNMVSAAK Z=2 2.63 (R)IPSAEEIADR Z=2 2.57 (K)ALGVDTVPVLVGPVSYLLLSKPAK Z=3 251(K)GM*LTGPVTILNWSFVRZ275Table S2.2 (cont™d) 2.21 (R)VLNTGSPITVPVGR Z=2 2.18 (R)IINVIGEPIDEK Z=2 UDP-glucose pyrophosphorylase poplar AAP86317 (At5g17310) 4 58 Stress-responsive, provides substrate for the reversibly glycosylated polypeptide 3.93 (K)VLQLETAAGAAIR Z=2 2.77 (R)ANPANPSIELGPEFK Z=2 2.25 (K)LDILLAQGK Z=1 2.06 (K)YSNSNIEIHTFNQSQYPR Z=2 Acyltransferase homolog petunia BAA93453 3 56 Phospholipid biosynthesis 3.06 (R)RPLLAVQFTK Z=2 2.56 (R)VRLELPASAEAHEK Z=2 2.06 (R)GVPLSLQPIHDR Z=2 Adenosylhomocysteinase-like pro CD439905 4 53 Linked to the cyclin-mediated regulation of the cell cycle 3.01 (K)IPDPEST DNAEFK Z=2 2.69 (K)VAVVCGYGDVGK Z=2 2.63 (R)LVGVSEETTTGVK Z=2 3.30 (R)WVFPETNTGILVLAEGR Z=2 Glyceraldehyde 3-P- dehydrogenase maize XP_479895 CAA33620 P08735 8 53 Oxidoreductase 5.11 (K)VIHDNFGIIEGLMTTVHAITATQK Z=3 4.11 (K)VIHDNFGIIEGLM*TTVHAITATQK Z=3 3.17 (R)VPTVDVSVVDLTVR Z=2 3.15 (R)NPEEIPWGEAGAEYVVESTGVFTDKDK Z=2 3.00 (K)TLLFGEKPVTVFGIR Z=2 2.92 (K)AGIALNDHFVK Z=2 2.66 (K)YDTVHGQWK Z=2 Acid invertase maize CAD91358 2 53 Sucrose cleavage 3.27 (R)VLVDHSIVESFAQGGR Z=2 2.51 (K)GLDGSLATHFCQDESR Z=2 Beta-7 tubulin maize AAA19708 (At2g29550) 16 51 Structural protein/involved in cell divison 133 (K)GHYTEGAELIDSVLDVVR Z=2 92 (R)AVLMDLEPGTMDSVR Z=2 80 (K)EVDEQM*INVQNK Z=2 78 (K)YAGDSDLQLER Z=2 * 76 (R)VSEQFTAMFR Z=2 72 (R)KLAVNLIPFPR Z=2 64 (R)YLTASAMFR Z=2 64 (K)LAVNLIPFPR Z=2 60 (R)LHFFMVGFAPLTSR Z=2 57 (R)FPGQLNSDLR Z=2 52 (K)NMMCAADPR Z=2 51 (R)RVSEQFTAMFR Z=2 40 (K)SSVCDIPPIGLK Z=2 * 40 (K)NSSYFVEWIPNNVK Z=2 37 (K)IREEYPDR Z=2 76Table S2.2 (cont™d) Beta-5 tubulin maize CAA52720 17 50 Structural protein/involved in cell divison 133 (K)GHYTEGAELIDSVLDVVR Z=2 105 (R)ALTVPELTQQMWDAK Z=2 92 (R)AVLMDLEPGTMDSVR Z=2 80 (K)EVDEQM*INVQNK Z=2 76 (R)VSEQFTAMFR Z=2 72 (R)KLAVNLIPFPR Z=2 69 (R)YVGTSDLQLER Z=2 * 64 (R)YLTASAMFR Z=2 64 (K)LAVNLIPFPR Z=2 60 (R)LHFFMVGFAPLTSR Z=2 57 (R)FPGQLNSDLR Z=2 52 (K)NMMCAADPR Z=2 51 (R)RVSEQFTAMFR Z=2 46 (K)LTTPSFGDLNHLISATMSGVTCCLR Z=3 Beta-6 tubulin maize AAA20186 (At5g23860) 18 50 Structural protein/involved in cell divison 133 (K)GHYTEGAELIDSVLDVVR Z=2 105 (R)ALTVPELTQQMWDAK Z=2 92 (R)AVLMDLEPGTMDSVR Z=2 80 (K)EVDEQM*INVQNK Z=2 76 (R)VSEQFTAMFR Z=2 72 (R)KLAVNLIPFPR Z=2 65 (R)EILHIQGGQCGNQIGAK Z=2 * 64 (R)YLTASAMFR Z=2 64 (K)LAVNLIPFPR Z=2 60 (R)LHFFMVGFAPLTSR Z=2 57 (R)FPGQLNSDLR Z=2 52 (K)NMMCAADPR Z=2 51 (R)RVSEQFTAMFR Z=2 50 (K)SSVCDIPPR Z=2 46 (K)LTTPSFGDLNHLISATMSGVTCCLR Z=3 43 (R)YTGNSDLQLER Z=2 * 40 (K)NSSYFVEWIPNNVK Z=2 37 (K)IREEYPDR Z=2 * Elongation factor 1 maize BAA08249 (At1g07920) 4 50 Translation elongation factor; protein synthesis 72 (K)IGGIGTVPVGR Z=2 60 (K)YYCTVIDAPGHR Z=2 58 (R)VETGVIKPMVVTFGPTGLTTEVK Z=3 43(K)STTTGHLIYK Z=2Alpha1-tubulin maize CAA33734 At1g63740 11 50 Structural protein 123 (R)FDGALNVDVNEFQTNLVPYPR Z=2 100 (K)TIGGGDDAFNTFFSETGAGK Z=2 98 (R)LVSQVISSLTASLR Z=2 77Table S2.2 (cont™d) 89 (K)CGINYQPPSVVPGGDLAK Z=2 * 88 (R)AVFVDLEPTVIDEVR Z=2 * 79 (R)TIQFVDWCPTGFK Z=2 75 (K)DVNAAVATIK Z=2 74 (R)IHFMLSSYAPVISAEK Z=2 43 (R)QLFHPEQLISGK Z=2 42 (K)AYHEQLSVAEITNSAFEPSSMMAK Z=2 Alpha-tubulin #3 Maize CAA44861 (At5g19970) 7 50 Structural protein 123 (R)FDGALNVDVNEFQTNLVPYPR Z=2 98 (R)LVSQVISSLTASLR Z=2 98 (R)AIFVDLEPTVIDEVR Z=2 79 (R)TIQFVDWCPTGFK Z=2 75 (K)DVNAAVATIK Z=2 43(R)QLFHPEQLISGK Z=2Alpha-tubulin #5 Maize CAA44862 At5g19780 6 50 Structural protein 105 (R)LISQIISSLTTSLR Z=2 * 98 (R)AIFVDLEPTVIDEVR Z=2 * 89 (K)CGINYQPPSVVPGGDLAK Z=2 75 (K)DVNAAVATIK Z=2 74 (R)IHFMLSSYAPVISAEK Z=2 43 (R)QLFHPEQLISGK Z=2 Tublin alpha-2/alpha-4 chain Arabidopsis At1g04820 6 50 Structural protein 98 (R)LVSQVISSLTASLR Z=2 similar to maize -tubulin 1 88 (R)AVFVDLEPTVIDEVR Z=2 79 (R)TIQFVDWCPTGFK Z=2 74 (R)IHFMLSSYAPVISAEK Z=2 43 (R)QLFHPQLISGK Z=2 UDP-glucosyltransferase BX9 Maize AAL57038 (At5g05800) 4 50 Detoxification of benzoxazinoids in maize; signal transduction and carbohydrate transport and metabolism in Arabidopsis 99 (K)LVPTATASLHGVVQADR Z=2 82 (R)GFESGALPDGVEDEVR Z=2 45 (R)TDLTDLVDLIK Z=2 40(K)ALSVPVFAVAPLNK Z=2UDP-glucosyltransferase BX8 Maize AAL57037 (At5g05900) 3 49 Detoxification of benzoxazinoids in maize; signal transduction and carbohydrate transport and metabolism in Arabidopsis 116 (R)LSALLSAADGEAGEAGGR Z=2 65 (R)GVGITVFHTAGAR Z=2 63(K)VGTEVAGDQLERZ=2Enolase Maize CAA39454 (At2g36530) 7 48 2-phospho-D-glycerate hydrolase; activity increase twice its initial level after 48 hours of anaerobiosis; Catalyzes the conversion of phosphoglercerate to phosphopyruvate, only dehydration step in glycolytic pathway 2.63/ 92 (K)VNQIGSVTESIEAVR Z=2 2.25 (R)GNPTVEVDVGLSDGSYAR Z=2 78Table S2.2 (cont™d) 2.16/ 119 (R)GAVPSGASTGIYEALELR Z=2 2.02 (K)IPLYQHIANLAGNK Z=2 133 (R)IEEELGDAAVYAGAK Z=2 69 (K)AVSNVNNIIGPAIVGK Z=2 Translation EF-TuM AAG32661 (At4g02930) 3 48 Elongation factor; mitochondrial protein synthesis 79 (R)GSALSALQGNNDEIGK Z=2 62 (K)TGEDVEILGLAQTGPLK Z=2 60(K)AVAFDEIDKAPEEK Z=2Translation initiation factor eIF- 4A Maize AAB67607 (At3g13920) 8 47 Protein synthesis 95 (K)FYNVLIEELPANVADLL Z=2 87 (R)GIYAYGFEKPSAIQQR Z=2 77 (R)KGVAINFVTR Z=2 74 (K)GLDVIQQAQSGTGK Z=2 61 (K)RDELTLEGIK Z=2 57 (K)VHACVGGTSVR Z=2 55 (R)DHTVSATHGDMDQNTR Z=2 50 (R)SRDHTVSATHGDMDQNTR Z=2 S-adenosylmethionine synthetase At1g02500 At3g17390 3 43 Synthesis of AdoMet by donating a methyl group 71 (R)FVIGGPHGDAGLTGR Z=2 52(K)VLVNIEQQSPDIAQGVHGHFTKZ=3Pyruvate dehydrogenase E1 alpha subunit maizeAAC72195 (At1g59900) 4 43 Oxidative decarboxylation of pyruvate 81 (R)DVTTTPAELVTFFR Z=2 75 (K)ESSMPDTSELFTNVYK Z=2 43 (R)TRDEISGVR Z=2 38 (R)YHGHSM*SDPGSTYR Z=3 Calreticulin maizeCAA54975 3 43 Ca-binding protein (coats vesicles) 2.98 (K)SGTLFDNIIITDDPALAK Z=2 2.29 IGIELWQVK Z=2 Hypothetical protein 1 At3g15000 2 43 Mitochondrial; unknown function; similar to At1g53260 74 (K)TLAQVVGSEEEAR Z=2 Adenine nucleotide translocator maize CAA33743 (At4g28390) (At3g08580) 11 42 Mitochondrial ADP/ATP carrier protein 1 or 2 93 (R)AIAGAGVLSGYDQLQILFFGK Z=2 70 (R)TIKDEGFSSLWR Z=2 59 (R)QFDGLVDVYR Z=2 56 (K)SSLDAFKQILK Z=2 54 (R)MMMTSGEAVK Z=2 51 (R)MMM*TSGEAVK Z=2 50 (R)YFPTQALNFAFK Z=2 47 (K)LLIQNQDEMIK Z=2 38 (R)MM*M*TSGEAVK Z=2 32(K)LLIQNQDEM*IK Z=2Phytase CAA11391 4 4Phosphoric monoester hydrolase in developing seed 79Table S2.2 (cont™d) maize 5.48 (R)AHYDALAVADPAANVEGLAAEASEYK Z=3 3.83 (K)VADETTKPAIQEDGAESK Z=2 2.84 (K)GEEFEDLDDTQKLEVYNSIIVESGR Z=2 2.18 (K)LLDDSMAAR Z=2 Reversibly glycosylated polpeptide maize AAB49896 (At5g15650) 4 41 Role in biosynthesis of hemicullulosic polysaccharides(cell wall), may use UDP-glucose as substrate; high similarity to Golgi-associated proteins se- wap41: Isolated from maize mesocotyl cell wall; immunolocalizes to plasmodesmata; -1,4- glucan-protein synthase 2.25 (R)EGAHTAVSHGLWLNIPDYDAPTQLVKPK Z=2 2.16 (K)NAAYIGTPGK Z=1 Herbicide safener binding protein/o-methyltransferase maize AAC12715 (At4g35160) 15 40 Protects against injury from chloroacetabulude & thiocarbamate herbicides 7.15 (132) (R)HGGAASAAELVTALSLPSTK Z=2 4.00 (67) (K)LVLHHLTDEECVK Z=2 3.47 (R)CAVELGIPTAIYR Z=2 2.83 (R)YIEAGIGLAEWFK Z=1 3.59 (R)QRDEKEWSELFTK Z=2 2.99 (76) (K)LLAASGVFTVDK Z=2 2.94 (R)ISPVSYLLVDGIPHEDKMNKTALVLTCTSTR Z=2 2.92 (R)YIEAGIGLAEWFK 2.39 (K)IIATKPADGAKMINYVEGDMFSFIPPAQTVVLK Z=2 2.27 (K)CTVLAPPK Z=2 2.23 (K)AQADIWR Z=2 2.12 (R)DEKEWSELFTK Z=1 2.05 (K)IIATKPADGAK Z=2 42 (K)LLAQCR Z=2 Fructose bisphosphate aldolase maize CAA31366 (At2g36460) 6 39 Gluconeogensis, glycolysis, pentose phosphate pathway 3.25 (K)ALNEHHVLLEGTLLKPNMVTPGSDSK Z=3 2.59 (K)GTIEVVGTDKETTTQGHDDLGKR Z=2 2.48 (K)VDKGTIEVVGTDKETTTQGHDDLGKR Z=3 2.27 (K)GILAADESTGTIGKR Z=2 56 (K)GDAADTESLHVK Z=2Actin 2/7 maize AAB40106 (At5g09810) 3 37 Protein binding/structural protein 2.89 (R)VAPEEHPVLLTEAPLNPK Z=2 101 (K)SYELPDGQVITIGAER Z=2 68(R)TTGIVLDSGDGVSHTVPIYEGYALPHAILRZ=3Probable Glyceraldehyde-3- phosphate dehydrogenase maize CAA30151 (At3g04120) 9 37 Probable Glyceraldehyde-3-phosphate dehydrogenase C1; Glycolysis/ gluconeogenesis 104 (R)VPTVDVSVVDLTVR Z=2 71 (K)TLLFGDKPVTVFGIR Z=2 68 (K)YIHDNFGIVEGLMTTVHAITATQK Z=3 * 58 (K)HSDITLK Z=2 * 53 (K)VIHDNFGIIEGLMTTVHAITATQK Z=3 * 80Table S2.2 (cont™d) 48 (K)KVISAPSK Z=2 * 47 (K)AGIALNDHFVK Z=2 * 39 (K)YDTVHGHWK Z=2 42 (K)GASYEDIKK Z=2 Glyceraldehyde-3-phosphate dehydrogenase GAPC3 maize AAA87579 (At113440) 12 36 Glycolysis/ gluconeogenesis 127 (K)GIMGYVEEDLVSTDFTGDSR Z=2 113 (R)VPTVDVSVVDLTVR Z=2 98 (K)GIMGYVEEDLVSTDFTGDSR Z=2 78 (K)AGIALNDHFIK Z=2 58 (K)TLLFGEKPVTVFGIR Z=2 56 (R)SSIFDAK Z=2 * 55 (K)TLLFGEKPVTVFGIR Z=2 51 (K)YDTVHGQWK Z=2 46 (K)KVVISAPSK Z=2 40 (R)FGIVEGLMTTVHSITATQK Z=3 * 40(K)GASYEEIKKZ=2Adenosine kinase Maize CAB40376 (At5g03300) 6 36 Phosphate transfer using ATP or GTP as donor 4.19 (90) (R)IAVITQGADPVVVAEDGK Z=2 2.36 (82) (K)VLPYADYIFGNETEAK Z=1(2) 2.05 (R)GWETENIEEIALK Z=1 77 (K)LNNAILAEEK Z=2 55 (K)LVDTNGAGDAFVGGFLSQLVLGK Z=3 43 (K)DKFGEEMKK Z=2 Malate dehydrogenase [NAD] At1g53240 7 36 Mitochondrial; carbon fixation, carbon metabolism 84 (R)TQDGGTEVVEAK Z=2 69 (K)LFGVTTLDVVR Z=2 64 (R)DDLNFNINAGIVK Z=2 64 (K)KLFGVTTLDRVVR Z=2 60 (K)R TQDGGTEVVEAK Z=2 59 (K)VAILGAAGGIGQPLALLMK Z=2 43 (K)RTQDGGTEVVEAK Z=2 Malate dehydrogenase maize AAB64290 (At5g43330) (At1g04410) 10 36 NADP; oxidoreductase, cytoplasmic 3.89 (K)SQASALEAHAAPNCK Z=2 3.50 (K)VAILGAAGGIGQPLSLLMK Z=2 96 (K)MELVDAAFPLLK Z=2 94 (K)VLVVANPANTNALILK Z=2 86 (K)MDATAQELTEEK Z=2 65 (R)KKMDATAQELTEEK Z=2 57 (K)KM*DATAQELTEEK Z=2 51 (R)ALGQISER Z=2 44(K)IVQGLPIDEFSR Z=2Hypothetical protein 2 At3g58290 2 32 MATH-domain-containing proteins: extracellular metalloproteases which are anchored to the membrane and are capable of cleaving growth factors, extracellular matrixproteins,andbiologicallyactivepeptides81Table S2.2 (cont™d) 43 (R)LKLNRALEK Z=2 39 (K)LIIGAR Z=240S ribosomal protein S4 maize AAB66899 (At5g58420) 5 30 Protein synthesis 101 (K)FDVGNVVM*VTGGR Z=2 77 (K)GSFETIHVEDSLGHQFATR Z=3 62 (R)EVISILM*QR Z=2 44 (R)LHPIRDEDAK Z=2 40S ribosomal protein S3A At3g04840 At1g431702 30 Protein synthesis 55 (R)LRAEDVQGR Z=2 46 (R)VLAHTQIR Z=2 Voltage-dependent anion channel protein 1a maizeAAD56651 (At3g01280) 2 30 Mitochondrial potassium transporter; porin 87 (K)KADLILGEIQSQIK Z=2 45(K)GDNLTGAYYHK Z=2Voltage-dependent anion channel protein 1b maize AAD56652 (At3g01280) 2 30 Mitochondrial potassium transporter; porin 45 (K)GDNLTGAYYHK Z=2 41 (K)KADLIFSEIHSQIK Z=2 Voltage-dependent anion channel protein 2 maize AAD56653 (At5g15090) 3 29 Mitochondrial anion channel; porin 144 (K)AIAVGADAAFDTSSGDLTK Z=2 90 (R)KDEAIFNEIQSQLK Z=2 49(K)HNNVTVDVKZ=2Putative 14-3-3 protein AAU93690 (At3g02520) 4 29 Regulatory processes and signal transduction 107 (R)GKIEAELSNIC*DGILK Z=2 99 (K)QAFDEAISELDTLGEESYK Z=2 * 70 (K)DSTLIM*QLLR Z=2 45(K)TVDVEELTVEERZ=260S ribosomal protein L7 At2g44120 2 28 Protein synthesis 57 (R)NHYVEGGDAGNR Z=2 49(K)NEDKLEFSKZ=214-3-3-like protein AAT06575 (At3g02520) 8 29 Regulatory processes and signal transduction; different from AAU93690 143 (K)QAFDEAISELDSLGEESYK Z=2 * 107 (R)GKIEAELSNIC*DGILK Z=2 79 (K)AAQDIALAELAPTHPIR Z=2 70 (K)DSTLIM*QLLR Z=2 64 (K)LLDSHLVPSSTAAESK Z=2 56 (R)KNEEHVNLIK Z=2 48 (K)ESAESTM*VAYK Z=2 Hypothetical protein 3 MaizeCAC21955 (At3g23560) 2 29 Unknown; low similarity to dienelactone hydrolases 88 (R)AYVSGAASSSR Z=2 36 (R)HEVPYYAK Z=2 Ribosomal protein S6 RPS6-1 maize AAB51304 (At5g10360) 5 28 Ribosome 122 (R)ISQEVSGDALGEEEFK Z=2 89 (K)KGENDLPGLTDTEKPR Z=3 48 (K)KLEIDDDQK Z=2 38 (R)LLLHR Z=2 82Table S2.2 (cont™d) 35 (K)QGVLTSGR Z=2 Putative thioredoxin peroxidase rice BAD27915 (At5g06290) 3 28 Electron transport, reduces H2O2 3.18 (K)EGVIQHSTINNLAIGR Z=2 1.95 (R)AGGVDDLPLVGNK Z=2 185(R)ASVVDDLPLVGNKZ=2Putative ascorbate peroxidase riceCB816056 (At1g07890) 2 27 H2O2 detoxification 37 (R)EDKPQPPPEGR Z=2 2.94(K)ELLSGDKEGLLQLPSDKZ=2Nucleoside diphosphate kinase 3 riceAAV59386 (At4g11010) 2 26 Cytosolic, Required for Coleoptile Elongation in Rice;similar to 18kDa sugarcane NPK1 2.87 (R)KIIGATNPLASEPGTIR Z=2 244(R)TFIAIKPDGVQRZ=2Mn superoxide dismutase maize AAA72022 (At3g10920) 2 25 Associated w/ increased mitochondrial activity during plant growth & development 2.96 (K)GDASAVVQLQGAIK Z=2 2.92(K)KLSVETTANQDPLVTKZ=240S Ribosomal protein S8 maizeAAB06330 2 25 Ribosome 64 (R)TLDPHIEEQFSSGR Z=2 63 (K)SAIVQVDAAPFK Z=2 Hypothetical protein 4 maize BU571995 (At1g09310) 3 22 Unknown ; similar to rice NP_00106738 with DUF538 3.30 (R)AKAEVYVGDAAGQEK Z=2 3.19 (R)HVTYGAEVSAVADK Z=2 2.04(K)AEVYVGDAAGQEK Z=2Formate dehydrogenase rice AAL33598 (At5g14780) 3 21 Methane metabolism; oxidoreductase activity; response to wounding 51 (R)LQIDPELEK Z=2 39 (R)LLLQR Z=2 36(R)AYDLEGKZ=2Ribosomal protein L18 NP_001054680 (At3g05590) 2 21 Cytoplasmic protein biosynthesis; ribosome 2.49 (K)NIAVIVGTVTDDKR Z=2 188(A)PLGENTVLLRZ=2Glyoxylase I CicerarientinumCAA12028 (At1g08110) 2 21 Calmodulin binding, found in etiolated hypocotyls, carbon-metabolism 3.18 (K)EAPANNPGLQTEVDPATK Z=2 2.49(R)GFGHIGVTVDDVHKZ=2Translationally controlled tumor-protein-like maize AAN40686 (At3g16640) 5 19 Ca binding, present in secretions 3.09 (K)NLTAVLEPEKADEFKK Z=2 2.27 (R)LQEQPPFDKK Z=2 2.24 (K)VVDIVDTFR Z=2 115 (-)M*LVYQDLLSGDELLSDSFTYK Z=2 Cyclophilin maize CAA48638 (At2g21130) 6 18 Cyclosporin; isomerase; rotamase; increases in germinating seedlings and growing tissue; involved in protein folding/modification; chaperone 3.75 (K)HVVFGQVVEGM*DVVK Z=2 3.19 (R)IVM*ELYANEVPK Z=2 2.96 (K)HVVFGQVVEGMDVVK Z=2 2.69 (R)GNGTGGESIYGEKFPDEK Z=2 2.60 (K)SGARGVRLEGAGRGGR Z=2 2.55 (R)IVMELYANEVPK Z=2 83Table S2.2 (cont™d) Translation initiator factor 5A maize CAA69225 (At1g13950) 4 18 Protein biosynthesis 2.80 (R)LPTDETLVAQIK Z=2 2.69 (K)DDLRLPTDETLVAQIK Z=2 92 (R)LPTDETLVAQIK Z=2 32(K)TYPQQAGTVRZ2Nucleoside diphosphate kinase 1 sugarcane AAB40609 (AT4g09320) 3 18 Synthesis of NTPs other than ATP; kinase activity towards histone H1 3.34 (R)NVIHGSDSIESANK Z=2 2.87 (K)IIGATNPLASEPGTIR Z=2 2.87(R)KIIGATNPLASEPGTIRZ=2Microtubule-associated protein AT5g44610 2 18 Epidermal cell morphogenesis 3.12 DMGQQTQGKTRVGSRVRNRKT Z=2 >2.5DMGQQTQGKTRVGSRVRNRKTLSSKZ=2Heat-shock protein 17.2 maize CAA46641 (At1g53540) 2 16 Heat shock protein for low molecular weight molecules; chaperone 2.99 (K)VEVEDGNVLLISGQR Z=2 1.95 (R)AALENGVLTVTVPK Z=1 Probable ribosomal protein S16 rice AAA33916 (At2g09990) 2 17 Protein biosynthesis; ribosome 2.60 (K)AFEPILLAGR Z=2 2.22 (K)TAVAVAYTKPGR Z=2 Ribosomal protein S14 B30097 2 16 Protein biosynthesis; ribosome 2.20 (R)IEDVTPVPTDSTR Z=2 2.13(K)ELGITALHIKZ=2Histone H2B maize CAA40565 (At3g45980) 3 16 Blocked amino end; chromosomal protein; DNA binding nucleosome core (2.66 (K)AM*SIM*NSFINDIFEK Z=2 ) 90 (K)AM*SIM*NSFINDIFEK Z=2 51 (K)QVHPDIGISSK Z=3 49(K)KPAEEEPAAEKZ=2Actin depolymerizin factor maize CAA66311 (At5g52360) 3 16 Enhancer of actin turnover 3.98 (R)YAIYDFDFVTAEDVQK Z=2 3.11 (K)EIVVDQVGDR Z=2 276(R)SGVSVDECM*LKZ=2Putative ribosomal protein L34 riceBAC99505 (At1g26880) 2 14 Protein biosynthesis; ribosome 2.77 (R)TVNRPYGGVLSGTAVR Z=2 2.24(R)CVLAGGPNDVLERR Z=2Profilin maizeAAB86960 (At2g19770) 2 14 Actin binding protein with complex effects on actin organization 81 (K)DFDEPGTLAPTGLFVGGTK Z=2 69(K)YMVIQGEPGVVIRZ=2Phospholipid transfer protein maizeAAB06443 (At2g38540) 3 12 Lipid transfer 2.98 (R)GVSGLNAGNAASIPSK Z=2 3.24(K)NAAAGVSGLNAGNAAASIPSKZ=2Lipid transfer protein(non-specific) rice BAD87070 (At5g01870) 3 12 Lipid transfer 3.46 (R)SLLQQANNTPDR Z=2 2.14 (K)NVANGANGSGTYISR Z=2 Edman AMSVSTVYSTLMPCLL/PFVQMGImmunophilin maize AAV28625 (At5g64350)2 12Peptidyl-prolyl cis-trans isomerase 91 (K)DPGQQPFSFSIGQGSVIK Z=2 84Table S2.2 (cont™d) 46 (K)VTVHCTGYGK Z=2 Histone H4 AAA33474 At1g07660 2 11 Histone 65 (R)DNIQGITKPAIR Z=2 57 (R)DAVTYTEHAR Z=2 Phospholipid transfer protein maize P19656 (At2g38540) 6 11 Lipid transfer 3.81 (K) NAAAGVSGLNAGNAASIPSK Z=2 (6B6) 3.41 (R)GVSGLNAGNAASIPSK Z=2 2.87 (K)CGVSIPYTISTSTDCSR Z=2 2.55 (K)GQGSGPSAGCCSGVR Z=2 2.11 (R)GTGSAPSASCCSGVR Z=2 Edman CGQVASAIAPCISYARGQGSGPSAGXXSG Cyclotide family protein (Umi11) NP_001105812 AY679129 1E 10 Induced during Ustilago infection and subsequent tumor formation Edman sequence ATLCYTGETCKYIGCLTPAASDNY 85Table S2.3: Statistical analysis of RT-PCR data. AverageDevAverageDevgdUDPgdUDPgpUDPgpColeoptileDay01.3865591.3001261.0844631.2570490.1555871.0871871.2106680.9427531.0802030.134094Day21.3464441.0607040.7855931.0642470.2804420.9426710.9987660.7860730.909170.110233Day41.0098650.8102790.6188630.8130020.1955150.9835270.940450.68910.8710260.159018Day50.6709280.5671890.364040.5340520.1561040.9008010.7797440.5523220.7442890.176924LeafDay01.4102641.3603291.0166971.262430.214271.3250951.091921.0569681.1579940.145764Day21.553841.5122031.1936271.419890.1970531.5167661.2024871.4477291.3889940.165167AverageDevAverageDevHP3HP3PLTPPLTPColeoptileDay01.22781.2052140.9920771.1416970.1300661.1584581.2563251.2885781.2344540.067761Day20.9700190.7886850.5664760.775060.2021161.15830.9408860.95321.0174620.122125Day40.8054190.7298090.3774020.6375430.2284391.1684370.7100210.8055220.894660.241858Day50.5786980.4324970.3306430.4472790.1246860.6171710.7227730.5327110.6242180.095227LeafDay00.9816031.0865880.808560.9589170.1403960.891770.6210160.9035120.8054330.159818Day21.2580191.2496591.1992241.2356340.0318081.3063381.5880160.9331541.2758360.32849586Table S2.3 (cont™d) AverageDevHP4HP4pValUDPgdUDPgpHP41.4083491.3438311.359931.3707030.033581day0day20.0663510.007190.1273031.4226941.2523181.2737691.316260.092796day2day40.0180210.225420.0107111.085921.0315350.8994561.0056370.095892day4day50.0057530.015780.0272370.6832250.7969330.7172440.7324670.058362day0day40.0197520.0792760.0071330.683061.0227290.7955730.8337870.173029day0day20.2011040.0723030.0201581.1507831.2873251.5970981.3450690.228692AverageDevRGPRGPpValHP3PLTPRGP1.582761.4592581.3095341.4505170.136823day0day20.0106920.0918440.1061881.5952461.0498911.0298311.2249890.320809day2day40.0375110.1122080.1193050.9775010.9390820.9175880.9447240.030352day4day50.0627110.1193010.0093960.779910.6292350.6937050.700950.075598day0day40.0207110.0633430.0097551.5385141.8782941.7809811.7325960.174981day0day20.0347970.0575690.1267721.7208952.3885682.0093792.0396140.33486287 REFERENCES 88REFERENCES 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CHAPTER 3: ARRP1 IN THE REGULATION OF CELL EXPANSION GROWTH 963.1 Abstract During growth, plants integrate cell division with cell elongation and differentiation. To better understand these processes we previously identified proteins from auxin-treated rapidly-growing corn coleoptiles (Li et al., 2013). One of these proteins, ARRP1 (Auxin rapid response protein 1), is closely associated with cell and organ expansion growth in above ground tissues. Here we show that upon auxin- treatment ARRP1 mRNA and protein increase within 30 minutes Œ prior to measurable growth and increased expression of genes encoding cell wall biosynthetic enzymes, suggesting an intracellular signaling function. This is further supported by the localization of ARRP1 in both the cytoplasm and the nucleus. ARRP1 expression is induced by red and blue light, suggesting a possible role in phytochrome and cryptochrome signaling at the interface with the auxin response. Arabidopsis plants lacking the ARRP1 homologue grew normally under standard conditions, but showed delayed growth and delayed flowering when germinated in the dark and transferred to light, a phenotype that can be reversed by expression of ZmARRP1 in the Arabidopsis mutant. In situ hybridization demonstrated that ARRP1 mRNA is enriched in the vasculature of growing corn tissues. In addition, corn ARRP1 interacts with the cytoplasmic domain of both Arabidopsis and Zea mays VH1/BRL2, a receptor-like kinase, which plays a role in the signaling of vascular development. Based on the fact that ARRP1 is involved in light- and auxin-mediated growth responses, we propose that it may act downstream of BRL2 at the intersection of several environmental and hormonal signals affecting plant development and the transition to light. 973.2 Introduction To adapt to the continuously changing environment, plants evolved a complex phytohormone network. Phytohormones are a group of small signaling molecules that are essential for plant survival, growth, development, and reproduction (Kende and Zeevaart, 1997; Wolters and Jurgens, 2009; Depuydt and Hardtke, 2011). Auxins were the first of the major phytohormones to be discovered. Auxin regulates cell division and cell elongation. It controls cell differentiation, root and shoot architecture, leaf abscission and fruit formation. In addition, it mediates plant responses to light, gravity, pathogen, and abiotic stress (Woodward and Bartel, 2005; Kazan and Manners, 2009; Vanneste and Friml, 2009; Weijers and Friml, 2009; Wang et al., 2010; Sauer et al., 2013). Auxin is mainly synthesized in the tips of shoot and young leaves. Perceiving auxin signal is the first step for an auxin triggered response. In the past decades at least three auxin receptors have been identified. The F-box protein TRANSPORT INHIBITOR RESISTANT1 (TIR1) is the best-studied auxin receptor. Binding of IAA into the ring-like structure of TIR1 promotes the interaction between TIR1 and proteins of the AUXIN/INDOLE ACETIC ACID (Aux/IAA) family (Tan et al., 2007), a group of proteins that regulates auxin-induced gene expression and development (Reed, 2001). Arabidopsis thaliana has 5 members of TIR1 (Calderon Villalobos et al., 2012), 29 members of Aux/IAA genes (Tan et al., 2007), and 23 members of ARF genes (Wilmoth et al., 2005; Korasick et al., 2014). Experimental evidence suggests that plants form different TIR/AFB-Aux/IAA co-receptor pairs with different auxin affinities, which contribute to the great variety of auxin functions (Vernoux et al., 2011; Korasick et al., 2014; Enders and Strader, 2015). 98Auxin is not only acting in a linear pathway, but also in conjunction with other hormones, such as brassinosteroid and cytokinin (Depuydt and Hardtke, 2011; Mouchel et al., 2006; Scacchi et al., 2010). In addition, there is crosstalk between auxin and light signaling. Light conveys environmental information such as local habitat conditions and seasonal change. The four major light receptors, the red and far-red light absorbing phytochromes (phy), the blue-light absorbing cryptochromes (cry), phototropins, and a ZTL-like F-box protein (Somers et al., 2004; Halliday et al., 2009) collectively control important aspects of plant development such as flowering and germination, tuberization, and bud dormancy, and also drive the plant towards an adaptive response to their local habitat. Much of this light response is achieved by linking to auxin system. Light controls auxin availability in plants by regulating both TAA1, an enhancer, and SUR2, a suppressor of IAA biosynthesis (Mikkelsen et al., 2004; Lau et al., 2008; Halliday et al., 2009). TAA1 encodes a tryptophan aminotransferase, which converts tryptophan to IPA in a parallel branch to the YUCCA metabolic pathway (Stepanova et al., 2008; Tao et al., 2008). SUR2, a cytochrome P450 monooxygenase CYP83B1, catalyzes the first step of indole glucosinolate biosynthesis. Disruption of SUR2 function in Arabidopsis mutants blocks the metabolic route from indole-3-acetaldoxime to glucosinolate, enhancing IAA production (Hoecker et al., 2004). By inducing the transcription level of SUR2 and suppressing TAA1, activated phyB controls the IAA homeostasis in plants (Halliday et al., 2009). Light also regulates auxin flow by modifying the abundance and function of PGPs and PINs, which are auxin polar transport carrier proteins. Activated phyB suppresses the transcription of PIN3, while phyB inactivation or loss leads to a dramatic increase of 99PIN3 mRNA levels (Friml et al., 2002). Within the upper portion of the hypocotyl, the PGP19 protein level is regulated by the phyA, phyB, cry1, and cry2 light receptors collectively (Nagashima et al., 2008). In addition, light signaling can modulate auxin distribution by directly modifying PIN1, PIN2 and PIN7 protein activity. The blue-light receptor pathway maintains PIN2™s plasma membrane localization, and reduces its protein turnover in the vacuole (Laxmi et al., 2008). Light not only controls auxin levels and distribution, but also directly integrates with auxin signal transduction. A number of transcription factors, PHYTOCHROME RAPIDLY REGULATED or PAR genes, play a key role in coordinating light and auxin signaling (Salter et al., 2003; Sessa et al., 2005; Roig-Villanova et al., 2006; Roig-Villanova et al., 2007). Among them, PAR1 and PAR2, members of the bHLH family of transcription factors, suppress auxin-induced expression of Small Auxin-Up RNA gene15 (SAUR15) and SAUR68. Plants with upregulated PAR1 or PAR2 activity show a diminished response to exogenous auxins (Roig-Villanova et al., 2007). Plants with reduced expression of many SAUR genes either through increased expression of PAR1/2 or artificial mircroRNA show reduced hypocotyl growth, similarly, overexpression of SAUR63, led to increased hypocotyl elongation (Chae et al., 2012). In addition, phyA may directly control auxin signaling. Recombinant oat phyA has been shown to phosphorylate recombinant SHY2/IAA3, and other AUX/IAA proteins in vitro (Colon-Carmona et al., 2000). These studies provide a model for the connection between light and auxin signaling system. However details about the integration of light and auxin signaling remain unknown. 100Maize (Zea mays L.) is an important agronomic crop. It provides food and a source of biofuel. Auxin-induced cell elongation of maize coleoptiles is one of the fastest phytohormonal responses known and has been widely used as a model system to study regulation of cell growth and tropisms (Haga and Iino, 1998). Auxin-induced cell elongation is mediated by several biochemical changes, including apoplastic space acidification, ion uptake, and de novo protein synthesis. In former study, we used a proteomics approach and identified four novel growth-related proteins in corn coleoptiles (Li et al., 2013). Here we show that a newly identified protein, Auxin-Response Protein1 (ARRP1) is induced rapidly and transiently by auxin within 30 minutes Œ and prior to known cell wall biosynthesis enzymes. In addition to auxin, its expression is also controlled by red and blue light, suggesting that it might also be involved in light signaling. However, its expression is unaffected by brassinosteroids or cytokinins. Arabidopsis mutants, deficient in ARRP1 expression, show a delayed growth phenotype when germinated in the dark and transferred to light. Overexpression of the corn gene in atarrp1-1/-2 double mutants can rescue this phenotype. Our findings suggest that ARRP1 is required during early seedling growth and during transition to light. ARRP1 interacts with BRL2, a receptor-like kinase with a function in vascular bundle development. In situ hybridization demonstrates that ARRP1 is enriched in the vasculature of growing tissues, overlapping with BRL2 expression pattern. Based on these findings, we propose that ARRP1 acts in conjunction with BRL2 at the intersection of environmental (light) and hormonal (auxin) signals affecting plant and vascular development in early plant development. 1013.3 Method and Materials 3.3.1 Plant material and growth conditions Zea mays Great Lakes 4758 hybrid seeds (Great Lakes Hybrids, Ovid, MI) were shaken in water for 0.5-1 h to remove fungicides and speed germination. Seeds were germinated in a standard soil mixture containing equal parts of Bacto Soil (Michigan Pear Company, Houston), medium vermiculite, and perlite. Seeds were germinated in the dark at 22 . For auxin treatment, coleoptiles were harvested after 4.5 days. To examine gene expression throughout the plant, four-day old seedlings were moved to a growth chamber at 22 with 16 h day/8 h night (LD). For expression studies in response to light, three-day old seedlings were moved to different light conditions as described below. Arabidopsis thaliana var. Columbia-0 seedlings were incubated for 20 min in 20% bleach/0.1% Triton X-100 for sterilization, washed several times with sterilized water, and plated on a Petri dish containing 1% sucrose and half-strength Murashige and Skoog Basal medium (Sigma-Aldrich). They were stored in darkness at 4for 2 days, then transferred to the growth chamber at 22with 16 h day/8 h night (LD) for two weeks. Arabidopsis seedlings were then planted in soil and grown in the growth chamber at same conditions. 3.3.2 Sequence analysis and bioinformatics analysis Homologues to ARRP1 and VH1/BRL2 were determined using the National Center for Biotechnology website (http://www.ncbi.nlm.nih.gov). Using ClustalW2, homologous sequence alignments were performed (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Predicted gene expression patterns were 102obtained through the Bio-Analytic Resource for Plant Biology (http://bar.utoronto.ca/). The following gene names/accession numbers were used: ZmARRP1: GRMZM2g139786; AtARRP1-1: At1g56580; AtARRP1-2: At1g09310; ZmBRL2: GRMZM2g002515; AtBRL2: At2g01950. 3.3.3 Hormone and light response analyses To investigate the ARRP1 response to auxin, brassinosteroids, and cytokinin treatment, coleoptiles were pulled off the primary leaf of maize seedlings and cut to a length of 10 mm after removal of the tips as described in Li et al., 2013. All coleoptiles were first shaken in distilled water for 1 h in the dark, transferred to fresh water as control or to 30 µM indole acetic acid, 20 µM benzyladenine, or 1 µM 24-epibrassinolide (Sigma-Aldrich) for up to five hours. The samples were collected every half hour in liquid nitrogen, and stored at -80°C. To understand the ARRP1 response to different light conditions, Zea mays seeds were germinated in the dark at room temperature for 3 days, and then transferred to growth chambers with white light, blue light (420 nm), red light (660 nm), or far red light (735 nm) at a photoperiod of 16h light and 8h dark at 22°C. The amount of light was set to 40 µmol m-2 s-1 for all chambers. Leaves were collected every 12 hours, frozen in liquid nitrogen, and stored at -80°C. RNA and protein extraction and quantification are described below. Experiments were performed with three biological replicates. 3.3.4 Total RNA isolation and quantification Equal amounts of plant tissue were harvested and ground to powder in liquid nitrogen. Using the Qiagen RNeasy Plant Mini-Kit, total RNA was extracted and treated 103with 10 units/100 mL Roche RNase-free DNase I for 1 h at 37°C. After incubation, RNA was cleaned up using the same kit. The quantification of total RNA amount was performed by a Nanodrop ND-1000 spectrophotometer (Thermo Scientific). Using Omniscript reverse transcriptase, 1µg of total RNA was reverse transcribed to cDNA (Qiagen). For quantitative PCR: Using the 2 X SYBR Green PCR core reagent (Applied Biosystems), real-time PCR analysis was performed on an ABI7000 instrument in 96-well plates (Bio-Rad).Each sample had three technical replicates and three biological replicates for a total of nine determinations.18S was used to generate the standard curve and for normalization. For semiquantitative PCR: PCR products were separated in a 1% agarose gel and quantified using ImageJ. Each sample had three biological replicates. The 18S signal for each sample was used for normalization. PCR primers and conditions are listed in table 3.1. Student™s t test was used to determine significance. 3.3.5 Recombinant ARRP1 and VH1 kinase-domain construct for protein-interaction essays, purification, and antibody production. To generate C-terminal His-tagged fusion proteins of ARRP1, AtBRL2 (Arabidopsis) or ZmBRL2 (maize), the respective cDNAs were isolated using RT-PCR from RNA extracts of five-day-old corn coleoptiles. Fragments were amplified by PCR with BamHI and NdeI sites at either end and cloned into digested pET15b expression vector using High Fidelity Taq (Roche). To create a fusion of His-tag with AtBRL2 or ZmBRL2, only the intracellular C-terminus of AtBRL2 or ZmBRL2 containing the kinase domain was used. All constructs were confirmed by sequencing. 104Protein expression was induced by 0.1 mM IPTG. Proteins were extracted from E.coli. culture and purified with Ni-NTA agarose (QIAGEN) according to the manufacturer™s instructions. Purified ARRP1 was sent to Cocalico Biologicals, Inc. for polyclonal antibody production using mice. Specificity of the antibody is shown in figure S3.1b. Purified AtBRL2 and ZmBRL2 were used to confirm the interaction between ARRP1 and these proteins. All primer sequences used are given in table S3.1a. 3.3.6 Protein extraction, and western blot analysis Protein extraction was performed as described in He et al. (1996) with some modifications: 100 mg plant tissue was ground in liquid N2, resuspended in 100 µl of 1 X tissue homogenization buffer (50 mM Tris-HCl, pH 6.8, 50 mM DTT, 0.1% Tween 20, 10% glycerol), and centrifuged at 12000 rpm for 20 min. The supernatant was transferred to a new 1.5 ml tube, boiled for 10 min, and resolved by a Bio-Rad 12% Tris-HCl SDS-PAGE gel. Extraction of nuclear proteins was performed as described by Sikorskaite et al. (2013). One gram of tissue was ground in liquid N2 into powder and resuspended in 10 ml of nuclei isolation buffer (10 mM MES-KOH (pH 5.4), 10 mM NaCl, 10 mM KCl, 2.5 mM EDTA, 250 mM sucrose, 1mM DTT). Cell debris was removed by filtration through two layers of cheesecloth twice and 10% Triton X-100 was added to the supernatant until a final concentration of 0.5%. Using Percoll/sucrose gradient centrifugation, nuclei were isolated and washed twice with NIB buffer (Sikorskaite et al., 2013). Protein gels were electroblotted (100 V, 1 h) onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad). The membranes were blocked with 2% bovine serum 105albumin (BSA) in TBST buffer for 1 h, followed by incubation with the primary antibody (diluted 1:5000) overnight at 4 , and washed with TBST buffer for 10 min each time, five times total. After the wash, membranes were incubated with secondary antibody, HRP conjugated goat anti-mouse antibody, (1:2000; Bio-RAD) for 1 h, then washed with TBST buffer five times as before. After reaction with Pierce ECL Western blotting substrate (Thermo), bands were visualized using HyBlot CL autoradiography film, and quantified with ImageJ. 3.3.7 Identification and verification of T-DNA insertion lines The SALK T-DNA database (http://signal.salk.edu/cgi-bin/tdnaexpress) was searched for insertion lines in ARRP1 Arabidopsis thaliana homologue and seeds were obtained from the Arabidopsis Biological Resource Center (Alonso et al., 2003). Using PCR genotyping, plants that were homozygous mutants for the T-DNA insertion were identified. Mutant plants were confirmed using reverse transcriptaseŒmediated PCR. 3.3.8 Transient expression and stable transgenic transformation To generate a Carboxy-terminal ARRP1-CFP fusion protein, the full-length ARRP1 ORF was amplified from five day-old corn coleoptile cDNA by PCR and introduced into the binary vector pDONR-207. Using the GATEWAY in vitro site-specific recombination, the ORF was then transferred to pEARLY-G103, according to manufacturer™s protocol (Invitrogen). Primers used are listed on table S3.1. The constitutive expression of the ARRP1:CFP was driven by a cauliflower mosaic virus 35S promoter; the construct was introduced into Agrobacterium tumefaciens strain GV3101 by electroporation. 106For transient expression of ARRP1, the cells of the transformed Agrobacterium overnight culture were pelleted by centrifugation in a 1.5 ml tube at room temperature. Using the infiltration buffer (50 mM MES, pH 5.6, 0.5% Glucose [w/v], 2 mM Na3PO4 and 100 mM acetosyringone) the pellet was washed three times and resuspended in 1 mL of the same buffer. The bacterial suspension was diluted with infiltration buffer to adjust the inoculum concentration to an optical density at 600 nm (OD600) of 0.05 and inoculated into the lower epidermis of tobacco (Nicotiana benthamiana) for transient expression. After 48 hours, localization was determined using confocal microscopy (Olympus FV1000SP CLSM). For CFP, the excitation laser wavelength is 458 nm, emission fluorescence wave length is 480-500 nm, For YFP, excitation laser wavelength is 515 nm, emission fluorescence wavelength is 525-570 nm. For stable expression, the constructs were transformed into Arabidopsis thaliana. Columbia-0 plants, using Agrobacterium-mediated floral dip, as described by Clough & Bent 1998. Kanamycin-resistant plants were selected on MS medium with 1% (w/v) sucrose, and 35 mg/L kanamycin (Sigma-Aldrich) at 21 °C and 16-h light/8-h dark photoperiod. Kanamycin-resistant 3-week-old plants were confirmed by genotyping PCR using GFP- specific primers (table S3.1). 3.3.9 In vitro co-immunoprecipitation Using 10 units of protein kinase A, one ug of purified His6::AtBRL2 or His6::ZmBRL2 was phosphorylated in the phosphorylation buffer (50 mM Hepes, pH 7.4, 10 mM MgCl2, 10 mM MnCl2, 1 mM DTT, 100 µM ATP and 5 µM cAMP). Phosphorylated and unphosphorylated protein was incubated with 2 mL of maize leaf protein extraction solution at 4 in 1 X PBS overnight in separate preparations. The 107mixture was incubated with Ni-NTA agarose for 1 h and washed three times with the 1 X PBS containing 20 mM imidazole. The Ni-NTA agarose was resuspended in 1X SDS-sample buffer and boiled for 10 min. The protein mixture (BRL2-interacting proteins) was resolved by 12% Tris-HCl ready SDS-PAGE gel and ARRP1 presence examined using western blot using the anti-ARRP1 antibody. 3.3.10 Yeast two hybrid assay The full length ORF of ZmARRP1 was inserted into the yeast two hybrid vector pGBKT7, which contains the GAL4 binding domain. The intracellular domain of AtBRL2 and ZmBRL2 PCR fragments was inserted into the pGADT7 vector, which contains the GAL4 activity domain. ZmARRP1 & AtBRL2 vectors were co-transformed into yeast AH109 Strain. After the first selection on SD/-Leu/-Trp plates, the co-transformants were tested for the GAL4 expression on SD/-Leu/-Trp/-His plates. 3.3.11 In situ hybridization Coleoptiles of 5-day-old Zea mays seedlings and two week old leaves were fixed in 4% paraformaldehyde and embedded in paraffin as described by Karlgren et al., 2009. A 150 bp fragment from DUF538 conserved domain of ARRP1 was amplified using RT-PCR, and used as the RNA probe for hybridization was generated. The PCR product was inserted into the pGEM T-easy vector (Promega) in sense direction. In vitro transcription of the probes, using digoxigenin-labeled UTP was performed by the TP7 and SP6 polymerase (NEB) according to the manufacturer™s protocol. RNA in situ hybridization was conducted according to a published protocol (Karlgren et al., 2009). The DIG-labeled RNA probe was identified using the DIG Wash and Block Buffer set (Roche). A Nikon Eclipse microscope was used for image acquisition. 1083.4 Results and Discussion 3.4.1 ARRP1 encodes a protein with a DUF538 conserved domain Using a proteomics approach, we had previously identified an auxin-induced protein, ARRP1, in rapidly growing corn coleoptiles (Chapter 2; Jie et al., 2013).The nucleotide sequence of ARRP1 revealed a single intron-less open reading frame of 675 bp that encodes a predicted protein of 224 amino acids with an estimated molecular mass of 24 kD (Fig. 3.1a). A typical TATA box sequence (TATATAA) is found upstream of the only ATG start site at -105. In addition to the TATA box, we also found an ARR1-binding element, which is known to be involved chloroplast development, leaf senescence, and vascular differentiation (table S3.2). ARRP1 belongs to a superfamily of plant proteins containing a conserved domain of unknown function 538 (DUF538) (Fig. 3.1b). A search of public databases through the National Center for Biotechnology Information (NCBI) website revealed 50 Zea mays protein sequence entries with a predicted DUF538 domain. All encode small molecular weight proteins that range from 15 kD to 25 kD. My analysis of DUF538 gene family members revealed representatives of this superfamily in 69 plant species, all within the Embryophyta. However, DUF538 proteins have not been found in algae and cyanobacteria, indicating that DUF538 proteins are not essential for photosynthesis (Fig. 3.2). Several DUF538 containing genes are induced by abiotic and biotic factors, such as mild drought stress, nutrient deficiency, crown gall, and mixed elicitors (Gholizadeh, 2011). It was suggested that DUF538 proteins may function in the regulation of various environmental signal transduction pathways (Gholizadeh and Baghban Kohnehrouz, 1092010). Based on previous evidence in other plants that DUF538-containing proteins may have a regulatory function, we examined whether ARRP1 serves as a regulatory or signal-transduction factor. 3.4.2 Sequence alignment of ZmARRP1 and its Arabidopsis homologues At1g56580 and At1g09310 Arabidopsis contains two ARRP1 homologues: At1g09310 (45% protein identity) and At1g56580 (44% protein identity), both of which contain the DUF538 conserved domain (Fig. 3.1b). Within the DUF538 domain the protein identity is 56% to At1g09310 and 57% to At1g56580. At1g56580, also called SVB (SMALLER WITH VARIABLE BRANCHES), was found highly expressed in the leaf trichomes of the glabra3Œshapeshifter (gl3Œsst) siamese (sim) double mutant that shows an arrest of trichome development, leading to smaller trichome branches of variable length (Marks et al., 2009). At1g09310 was found to be upregulated in plants exposed to biotic and abiotic stress. Its protein level was tightly controlled by ubiquitinase and metacaspase (Saracco et al., 2009; Tsiatsiani et al., 2013). More recently a large-scale protein-interaction approach showed its possible interaction with BRL2, a BRL1-like receptor kinase, suggesting that ARRP1 may play an important role in the signaling pathway downstream of the BRL2 receptor (Ceserani et al., 2009). Interestingly, this study also showed that BRL2 regulates the venation pattern in leaves, a traitthat is also controlled by auxin, providing more evidence that ARRP1 acts in the signaling path of multiple abiotic signals. 1103.4.3 ARRP1 expression is correlated with cell division and cell elongation To examine the expression of ZmARRP1, we determined the developmental profile of its expression (Fig. 3.3). Total RNA and protein from different tissues of five-day-old, dark-grown seedlings and at different times after the transfer to light were quantified using semi-quantitative RT-PCR and western blotting. ARRP1 was strongly expressed in the above ground tissue while mRNA and protein were barely detectable in the root. Once coleoptiles are exposed to light their growth ceases. ARRP1 showed reduced expression in parallel to this growth cessation: The mRNA level dropped more than 50% within five days. Similarly, a 60% decrease was observed in the protein levels (Fig. 3.3b). To further investigate the correlation between ARRP1 expression and leaf development/tissue expansion, we monitored gene expression and protein abundance in expanding corn leaves. In this case, exposure to light enhanced growth. ARRP1 expression levels increased about 30% in the developing leaf within two days of transition to light (Fig. 3.3a). Western blotting revealed a simultaneous increase in ARRP1 protein (Fig. 3.3b). Our findings suggest that both ARRP1 gene expression and protein abundance are correlated with expansion growth in above-ground tissues. In monocots, leaf differentiation proceeds basipetally (from tip to base) in a continuous manner. Young maize leaves can be divided into four distinct stages of leaf development, which include a basal zone, a transitional zone, a maturing zone, and mature zone (Li et al., 2010). Genes that encode enzymes for cell cycle regulation and chromatin structure, cell wall biosynthesis, auxin and brassinosteroid biosynthesis, and potential signaling proteins are highly expressed near the base of the leaf, corresponding to region I and II in Fig. 3.4a. Genes encoding enzymes for the Calvin 111cycle, isoprenoid biosynthesis, redox regulation and the light reactions of photosynthesis are highly expressed the maturing zone. Our analysis of the different leaf zones showed that while ARRP1 expression was seen in all tissues, both, mRNA level and protein abundance were highest in the basal zone (II; Fig. 3.4b & c), suggesting ARRP1 is correlated with the cell division and cell elongation growth. Our data suggest (1) that the ARRP1 functions predominantly in the above-ground tissues; (2) that its expression is highly correlated with plant growth; and (3) that it is highly enriched in areas of the leaf where cell division and cell elongation take place. Because the expression pattern indicates a strong connection between ARRP1 and plant growth, ARRP1 might be involved in the regulation of plant signaling processes associated with growth-promoting hormones such as auxin, brassinosteroids, cytokinin or environmental factors such as light. 3.4.4 ARRP1 expression is controlled by auxin and light Because ARRP1 was originally identified in auxin-treated corn coleoptiles (Li et al., 2013), I determined gene expression (Fig. 3.5a) and protein levels (Fig. 3.5b) at various times after addition of auxin. ARRP1 mRNA was increased immediately after the addition of auxin, reaching the highest level within 30 min. This elevated transcript level is maintained for up to two hours, then decreases slowly to a basal level within the next two hours. In comparison, the expression of genes which are involved cell wall (CW) biosynthesis (UDP-glucose dehydrogenase: UDPgd (Karkonen et al., 2005); a putative mixed-linked -glucanase; Hyp 3) is induced slowly and gradually over five hours of auxin treatment and does not display the rapid and transient auxin response of ARRP1. The ARRP1 protein level is also upregulated by auxin, but reaches the highest 112level two hours later, either due to the fact that translation is a time consuming process or due to an increased protein stability. Closer examination of the western blot revealed the presence of a doublet band suggesting possible post-translational modification. Analysis of the ARRP1 sequence indicates the presence of several putative phosphorylation sites (Fig. 3.1a). The fact that we only detect the protein doublet in some western blots is likely due to the resolution of the gel but could also be caused by selective phosphorylation only under certain conditions. The fact that the induction of ARRP1 expression precedes the expression of other CW-related genes indicates that ARRP1 is an early auxin-response gene with a possible signaling function which may regulate the expression of other genes including those related to CW biosynthesis. The fact that protein levels remain high for five hours suggests that it is necessary for the maintenance of the response as well. In addition to auxin, cytokinins and brassinosteroid are also regulators of plant growth (Depuydt and Hardtke, 2011). Hence, we examined whether ARRP1 expression was induced by application of these two hormones. Neither cytokinins nor 24- epibrassinolide induced expression of ARRP1 (Fig. S3.2). As previous data had shown that ARRP1 expression is increased in leaves when seedlings were grown under white light, I examined whether ARRP1 can be induced by specific wavelengths to understand, which photoreceptor is responsible for the induction of ARRP1 expression. Three-day old, dark-grown corn seedlings were transferred to growth chambers with white, red, blue, or far red light. Protein and mRNA levels in leaves were examined every 12 hours for the next three days (Fig. 3.6 a/b). The results showed that white light has the strongest inductive effect on ARRP1 expression and 113protein abundance; Student™s t-test showed that mRNA and protein levels are also induced significantly after 2.5 days by red (p<0.005) and blue light (p<0.01), but not by far red light. These observations that ARRP1 not only responds to auxin, but also can be induced by blue and red light, suggest that ARRP1 might integrate auxin and light signals (phytochrome and possibly cryptochrome). Light exerts influence on auxin signaling by regulating the biosynthesis and distribution of auxin (Teale et al., 2006; Halliday et al., 2009). We originally identified ARRP1 in auxin-treated dark-grown coleoptiles. Once these are exposed to light, coleoptile growth ceases while the growth of the developing leaf increases. This response is known to be regulated by phytochrome. Recent publications have shown a tight correlation between phytochrome sensing and auxin signaling: Phytochrome interacting factors (PIFs) regulate both auxin biosynthesis and signaling (Hornitschek et al., 2012). PIFs also interact with brassinosteroids (BRs) to regulate auxin biosynthesis and transport, which leads to an increase in cell expansion (de Lucas and Prat, 2014). Based on the ARRP1 response to auxin and light and its correlation with growth, I propose that it functions in this pathway and integrates auxin and light signaling. 3.4.5 AtARRP1 is required in the initiation of plant growth and dark/light transition Arabidopsis contains two close homologues to ZmARRP1. While overall sequence identity of the proteins is only 45%, it increases to over 55% within the DUF 538 domain. As with ZmARRP1 in corn, AtARRP1 and AtARRP2 are highly expressed in expanding leaves and five day old dark-grown hypocotyls but barely expressed in the root. Their expression levels were significantly decreased in fully expanded leaves further indicating a correlation with plant growth (Fig. 3.7). 114I obtained knock-out mutants of AtARRP1 in Arabidopsis from The Arabidopsis Information Resource (TAIR; Arabidopsis.org) (Fig. 3.8a and 3.8b). I used scanning electron microscopy to examine whether the trichome growth was affected in At1g09310 mutants as had been shown for At1g56580. Both At1g56580 (atarrp1-2) and At1g09310 (atarrp1-1) single mutants produced trichomes with smaller length and variable number of branches similar to those seen by Marks et al. (2009) for At1g56580 (Fig. 3.8c). Except for this trichome branching phenotype, the two single mutants do not show any visible growth phenotype compared to wild type (Fig. 3.8b). This result suggests that there might be functional redundancy among these DUF538 proteins. To examine this possibility I generated At1g56580xAt1g09310 double mutants. Since ARRP1 expression is regulated by auxin and light, we germinated seeds in the dark and examined the hypocotyl length of different mutants. While the hypocotyl length was indistinguishable from WT plants, the double mutant showed a slower growth upon transition to light after five days of growth in the dark, a difference that was especially obvious in the early development stages (Fig. 3.8d). The inflorescence emergence and flowering time are delayed by two weeks in the double mutant, but leaf number at the time of flowering and diameter of the rosette and stem are identical to the wild type control (Fig. 3.8e). These results suggest that At1g56580 and At1g09310 play a role in the early plant growth during transition into light. I expressed ZmARRP1, in Arabidopsis wild type and double mutant to examine if ZmARRP1 and AtARRP1/2 execute the same function. ZmARRP1 completely restores the wild type phenotype in the double mutant (Fig. 3.8d). Plant growth in the complementation line was restored to the wild-type level, and the delayed stem 115emergence time and flowering time were also rescued. Because At1g090310 and At1g56580 are highly expressed in the juvenile leaf (Fig. 3.7), this suggests that they may only function in the early stage of leaf development. When the leaves enter a later developmental stage, other regulatory factors may have dominant effects on plant growth and causing differences in phenotype to fade. These data suggest that ZmARRP1 and its Arabidopsis homologues At1g090310 and At1g56580 serve identical functions in both plants. This suggests that ARRP1 may be a regulator universal to all land plants. 3.4.6 ZmARRP1 interacts with BRL2 My findings imply that ARRP1 fulfills the same function in Arabidopsis and corn. As AtARRP1-2 was previously identified as an AtBRL2-interacting protein (Cesarani et al., 2013), I examined whether ZmARRP1 also interacts with AtBRL2. The intracellular portion of AtBRL2 contains a canonical kinase motif at the carboxyl end, which is proposed to mediate specific protein-protein interactions (Clay and Nelson, 2002). Using both a phosphorylated and a non-phosphorylated C-terminal 998-amino acid fragment of AtBRL2 containing a His tag as bait, I performed pull down assays to identify interacting proteins from Zea mays leaf extracts. Presence of ARRP1 in the complex was detected using western blotting with the ZmARRP1 specific antibody (Fig. 3.9b). The non-ARRP1-binding protein Hyp3 was used as a negative control. ARRP1-BRL2-interaction could be seen when BRL2 was phosphorylated but not when it was unphosphorylated (Fig. 3.9b). The result showed that ZmARRP1 only interacts with phosphorylated AtBRL2, suggesting the phosphorylation is very important for protein-protein interaction. This interaction was confirmed using Y2H (Fig. S3.4). 116 A search of public databases through the National Center for Biotechnology Information (NCBI) website revealed one predicted BRL2 protein in Zea mays (ZmBRL2) with 60% identity to AtBRL2 protein(Fig. 3.9a). An pull down assay using the 1003-amino acid C-terminal ZmBRL2 fragment fused to a His-tag showed that ZmARRP1 also interacts with the phosphorylated intracellular domain ZmBRL2 (Fig. 3.9c). This cross-reactivity between ZmARRP1 and AtBRL2 suggests that similar BRL2 signaling pathways exist in both maize and Arabidopsis. BRL2 is a receptor-like kinase. Proteins with a phosphorylated tyrosine or serine/threonine are often recognized by receptor like kinase protein interaction domains. Our western blots showed doublet bands for ZmARRP1, suggesting possible post-translational modifications. Based on a bioinformatics analysis (NetPhos 2.0) we found ARRP1 contains at least three serine and two tyrosine embedded in a phosphorylation target sequence (Fig. 3.1a), suggesting ARRP1 a good downstream candidate for the ZmBRL2 signal pathway. Despite the fact that the AtBRL2 protein sequence is closely related to Arabidopsis BRL1, a brassinosteroid-responsive receptor kinase, and shares with it the presence of an amino acid island thought to contribute to ligand specificity (Li and Chory, 1997), AtBRL2 does not respond to brassinosteroids, but can be induced 2-fold by auxin-treatment (Clay, 2002). The same study showed that BRL2 most likely is involved in vascular differentiation and leaf venation patterning, which would suggest an interaction with the auxin or cytokinin signal pathway (Clay, 2002). The fact that both BRL2 and ARRP1 are induced by auxin but not brassinosteroids or cytokinins and the 117fact that they show direct interaction, suggests that they act jointly in an auxin-mediated signaling pathway, perhaps as it relates to vascular differentiation. 3.4.7 ARRP1 and VH1/BRL2 are expressed in overlapping patterns Interacting proteins often are encoded by genes expressed in the same cells and under the same conditions. To determine whether ARRP1 is co-expressed with BRL2, we characterized the expression patterns of ARRP1, using in situ RNA hybridization. Expression of ARRP1 is broad and diffuse in the early stage of developing leaves and coleoptiles. In mature leaves, transcripts for ARRP1 decrease significantly suggesting ARRP1 might only function in the early stage of leaf development. Further examination of young leaves and the coleoptile found that ARRP1 is enhanced in the vascular bundles (Fig 3.11). Vascular bundle development is regulated by auxin (Scarpella et al., 2006; Fabregas et al., 2015). In addition, studies showed that the transcription of BRL2 was restricted to provascular and procambial cells in organ primordia. In leaves, expression of BRL2 significantly decreased in the maturing vascular elements and ends in fully differentiated mature leaf (Clay, 2002). This expression pattern overlaps with our findings for ZmARRP1. Together with the finding that ZmARRP1 interacts with BRL2, we proposed that auxin modifies the ZmARRP1-BRL2 interaction, leading to a change in gene expression and leaf expansion and venation pattern. 3.4.8 Subcellular localization of ARRP1 To investigate the subcellular localization of ARRP1, a C-terminal ARRP1-CFP fusion protein was transiently expressed in tobacco leaves co-infiltrated with ER marker and Golgi markers. As shown (Fig. 3.10), ARRP1-CFP is localized in the cytosol. 118Interestingly, it is also found in the nucleus. Colocalization with ER and Golgi markers is limited to the area surrounding the nucleus. The CaMV 35S promoter used in the ARRP1-CFP expression construct is a very strong constitutive promoter which sometimes will cause non-targeted expression. To confirm that ARRP1 is indeed localized in the nucleus, we isolated the nuclei from the leaves of Zea mays, using differential centrifugation (Fig. 3.11b). Western blot analysis showed that while ARRP1 is localized mostly in the total cell extract, it is also present in significant amounts in the nucleus (Fig. 3.11c). Histone H3, a known nuclear protein, is only found in the nuclear fraction. These data suggest that ARRP1 moves to the nucleus. Because ARRP1 does not contain any nuclear targeting signal, translocation to the nucleus is likely to require additional proteins. 1193.5 Conclusions: In this paper we showed that ARRP1, an auxin-response protein is induced by auxin as well as by red light and that its expression is associated predominantly with expanding above-ground tissues in maize and Arabidopsis. It is localized in the cytoplasm as well as in the nucleus and, thus, could regulate gene expression. ARRP1 interacts with BRL2, a receptor-like kinase that plays a role in the venation pattern of plants. These observations suggest that ARRP1 participates in auxin- and light-related signaling. We propose that ARRP1 integrates the auxin- and red-light response as they relate to the expansion of leaves and the development of the vasculature. In this function it could interact with auxin directly, with an auxin binding protein/receptor, and/or with an additional signal that is transmitted through the BRL2 kinase to ARRP1. We propose three possible mechanisms for its function: in response to a stimulus, (I) ARRP1 moves from cytosol to the nucleus where it binds additional activators and regulates gene expression; (II) it moves to the nucleus where it binds additional protein and regulates cell division; or (III) it binds and modifies other, possibly cytoplasmic proteins, thus affecting their function (Fig. 3.12). 120Figure 3.1 The ARRP1 protein belongs to a DUF538 superfamily.(a) The ARRP1 protein sequence contains a DUF538 conserved domain (amino acids 25 to 140; highlighted in gray). Predicted phosphorylation (Netphos 2.0) sites are indicated in red. (b) Alignment of the complete sequence of ZmARRP1, AtARRP1-1 and AtARRP1-2. Amino acids that are conserved among all of the compared sequences are labeled by asterisks, and those that are conserved among at least two sequences are labeled by a dot. a MTLTIPDEVRAKAEVYVGDAAGQEKTRLLLQETGLPSGLL 40 PLRDIIECGYVEETGFVWLRQRRKVDHYFAKAGRHVSYGA 80 EVSAVADKGRLKKITGVKAKEMLLWVTLHEICVDDPPTGK 120 LHCKAIGGLSRSFPVEAFEAEEPAPPGAGGVVPRDAEEEE 160 EVEKEEGEGDKNPEQEAAAEKDGDAPAAAPAPEEAESKAE 200 EKADKEVSSADPAVVHAEALAAKN 224 AtARRP1-1 MGLVT-DEVRARAEKYTGDEICREKTKEFLKEVSMPNGLLPLKDIEEVGYDRETGIVWLK 59AtARRP1-2 MGLVT-EEVRAKAEMYTGDEICREKTKCFLKEISMPNGLLPLKDIEEVGYDRESGVVWLK 59 ZmARRP1-1 MTLTIPDEVRAKAEVYVGDAAGQEKTRLLLQETGLPSGLLPLRDIIECGYVEETGFVWLR 60 * *. :****:** *.** :***: :*:* .:*.*****:** * ** .*:*.***: AtARRP1-1 QKKSITHKFEAIGKLVSYATEVIAQVEVGKIKKLTGVKAKELLIWVTLNELVLEQPTSSG 119 AtARRP1-2 QKKSITHKFTEIDKLVSYGTEVTAIVETGKIKKLTGVKAKELLIWVTINEIYTEEPPT-- 117 ZmARRP1-1 QRRKVDHYFAKAGRHVSYGAEVSAVADKGRLKKITGVKAKEMLLWVTLHEICVDDPPTG- 119 *::.: * * .: ***.:** * .: *::**:*******:*:***::*: ::*.: AtARRP1-2 KITFKTPTTLSRTFPVTAFIVPEEPAK------EEPAKEEPAKEKSSE---ATEAKEAVA 168 AtARRP1-1 KINFRTPTGLSRTFPVSAFVVPEV--------------EKPATEKNNG---TTEVKEAVA 162 ZmARRP1-1 KLHCKAIGGLSRSFPVEAFEAEEPAPPGAGGVVPRDAEEEEEVEKEEGEGDKNPEQEAAA 179 *: :: ***:*** ** . * *: **.. . :**.* AtARRP1-2 IKEAVAVKEAA---------------------------------- 179 AtARRP1-1 VTDA----------------------------------------- 166 ZmARRP1-1 EKDGDAPAAAPAPEEAESKAEEKADKEVSSADPAVVHAEALAAKN 224 b 121Figure 3.2 Illustration of the distribution of the DUF538 conserved domain in plants.Red numbers indicate the number of DUF538-containing genes identified in each individual species so far. Blue numbers indicate the number of species that possess genes that belong to the DUF538 superfamily. Only species above the blue line contain representative of DUF538-containing proteins 122Figure 3.3 ARRP1 levels throughout the corn seedlings.(a) ARRP1 expression relative to 18S RNA in different parts of the corn seedlings (modified from Li et al. 2013) Values are mean and S.E. of three biological replicates. (b) Quantification of ARRP1 protein throughout the seedling as determined by western blotting using an ARRP1- specific antibody. 00.10.20.3 0.40.50.6ARRP1/18Sa Actin ARRP1 Coleoptile0day2day5dayLeaf0day2dayRootb 12300.40.81.2 1.62IIIIIIIVVARRP1/18SFigure 3.4 ARRP1 expression in different leaf zones.(a) Illustration of a nine-day-old corn seedling. Leaf 2 was used to monitor the ARRP1 expression level. (b) Semiquantitative analysis of the ARRP1 mRNA levels in different corn leaf zones. ARRP1 was quantified relative to 18S RNA. (c) Protein levels of ARRP1 in the same leaf zones as determined using western blot. Actin was used as a control. Experiments were performed in triplicate. Zone I: predominantly cell division with initiation of cell elongation; Zone II: declining cell division, high rate of cell elongation; Zone III: transition zone; some cell division; increase of transcripts associated with photosynthetic machinery; Zone IV: maturation zone; high expression of genes involved with the photosynthetic machinery Zone V: mature, fully developed zone; initiation of apoptosis IIIIIIIVVc ARRP1 Actin I II IIIIVVa b Leaf212400.511.522.53ARRP1/18SArrp1UDPgdHyp3(Hrs) a Figure 3.5 Response of ARRP1 in five days old corn coleoptiles to Auxin treatment.(a) Semiquantitative analysis of mRNA level changes of genes encoding ARRP1, UDPgd and Hyp3 in corn coleoptiles within five hrs Auxin-treatment. UDPgd: UDP-glucose dehydrogenase; Hyp3: a putative endoglucanase. All genes were quantified relative to 18S RNA. Bars represent the mean ± S.E. of three biological replicates. (b) Protein level changes of ARRP1 within 5 hrs Auxin-treatment by western blotting analysis. Actin was used as a control. ARRP1 Actin +Auxin ½123525Control b Timeofexposure(Hrs)125Figure 3.6 ARRP1 expression in corn leaves in response to different light conditions.(a) Semiquantitative analysis of ZmARRP1 expression in corn leaves at various days after transfer of the seedlings to different light conditions. R : red light at 660 nm; FR: far red light at 735 nm; Blue: blue light at 420 nm; white: regular light condition. Values are mean and S.E. of three independent biological replicates. Asterisks indicate a significant difference (p<0.01; student™s t-test) as compared to leaves kept in the dark. (b) Protein level changes of ARRP1 response to different light conditions by western blotting analysis. Values are mean and S.E. of three independent biological replicates. 00.2 0.4 0.60.811.2 1.4 1.61.8ARRP1/18SNoLightRFRBluewhitea***12600.511.522.5ARRP1/ActinWhiteRedBlueFRNoLightFigure 3.6 (cont™d) b***12700.2 0.4 0.6 0.811.2 1.4 1.6 1.8RootDevelopingLeafMatureLeafHypocotylAtArrp11/18S00.511.522.53RootDevelopingLeafMatureLeafHypocotylAt1g09310/18SFigure 3.7 Expression levels of AtARRP1-1 (a) and AtARRP1-2 (b) in various Arabidopsis thaliana organs, as determined by RT-PCR.Material for root was harvested from 7 day old seedlings germinated on MS plate. Developing leaf corresponds to 7 day old young leaf, while mature leaf corresponds to 1 month old fully developed leaf. Hypocotyl material was harvested from 5 day old dark-grown seedling germinated on MS plate. Values are mean and S.E. of three independent biological replicates. ab128Figure 3.8 Single knock out mutants of atarrp1-1 and atarrp1-2 show trichomes with smaller branch length and variable number of branches. (a) Using RT-PCR, we confirmed that transcription of atarrp1-1 and atarrp1-2 was completely abolished in the knockout mutant. (b) Comparing to wild type, the knockout mutantsatarrp1-1 and atarrp1-2 did not show any morphological vegetative phenotype. (c) Both knockout mutants show changes in trichome development. The trichomes of mutants showed higher branch numbers and shorter branch length which is consist with a previous study (Marks et al., 2009). (d) The double mutant showed a slower growth phenotype upon transition to light after five days germination in dark, which can be complemented by overexpression of ZmARRP1 in Arabidopsis mutant. Overexpression of ZmARRP1 in WT plants did not show any significantly phenotype. (e) The inflorescence emergence time and flowering time are delayed by two weeks in the double mutant, but leaf number at the time of flowering, and diameter of the rosette and stem are identical to the wild type control dabcWTatarrp11 WTatarrp12 WTatarrp11atarrp12 WTatarrp11atarrp12 129Figure 3.8 (cont™d) e130Figure 3.9 ARRP1 interacts with BRL2 homologues from Arabidopsis and maize.(a) Alignment of the carboxyl terminal region of intracellular domain of ZmBRL2 and AtBRL2. Amino acids that are conserved between the two sequences are labeled by asterisks. (b) The carboxy-terminal region of the intracellular domain of Arabidopsis BRL2 was expressed, purified and then mixed with the protein extract of maize leaf for five hours. A Ni-Column was used to pull-down the AtBRL2 protein complex. Using an anti ZmARRP1 antibody, we confirmed the presence of ZmARRP1 in the AtBRL2 protein complex, indicating the interaction between ZmARRP1 and AtBRL2. (c) A parallel in vitro pull-down assay showed ZmARRP1-ZmBRL2 interaction. Endo-glucanase was used as a negative control. aAtBRL2 RARRRDADDAKMLHSLQAVN-SATTWKIEK-EKEPLSINVATFQRQLRKLKFSQLIEATN 58 ZmBRL2 RARRKEAREARMLSSLQDGTRTATTWKLGKAEKEALSINVATFQRQLRRLTFTQLIEATN 60 ****::* :*:** *** . :*****: * ***.*************:*.*:******* AtBRL2 GFSAASMIGHGGFGEVFKATLKDGSSVAIKKLIRLSCQGDREFMETAEMETETLGKIKHR 118 ZmBRL2 GFSAGSLVGSGGFGEVFKATLKDGSCVAIKKLIHLSYQGDREFTA----EMETLGKIKHR 116 ****.*::* ***************.*******:** ****** * ********* AtBRL2 NLVPLLGYCKIGEERLLVYEFMQYGSLEEVLHGPRTGEKRRILGWEERKKIAKGAAKGLC 178 ZmBRL2 NLVPLLGYCKIGEERLLVYEYMSNGSLEDGLHG-----RALRLPWERRKRVARGAARGLC 171 ********************:*. ****: *** : * **.**::*:***:*** AtBRL2 FLHHNCIPHIIHRDMETKSSNVLLDQDMEARVSDFGMARLISALDTHLSVSTLAGTPGYV 238 ZmBRL2 FLHHNCIPHIIHRDMK--SSNVLLDGDMEARVADFGMARLISALDTHLSVSTLAGTPGYV 229 ***************: ******* ******:*************************** AtBRL2 PPEYYQSFRCTAKGDVYSIGVVMLEILSGKRPTDKEEFGDTNLVGWSKMKAREGKHMEVI 298 ZmBRL2 PPEYYQSFRCTAKGDVYSLGVVFLELLTGRRPTDKEDFGDTNLVGWVKMKVREGTGKEVV 289 ******************:***:**:*:*:******:********* ***.***. **: AtBRL2 DEDLLKEGSSESLNEKEGFEGGVIVKEMLRYLEIALRCVDDFPSKRPNMLQVVASLRELR 358 ZmBRL2 DPELV-------IAAVDGEE-----KEMARFLELSLQCVDDFPSKRPNMLQVVATLRELD 342 * :*: : :* * *** *:**::*:*****************:**** AtBRL2 GSENNSHSHSNSL-- 371 ZmBRL2 DAPPSHQQAPASACD 362 .: . :. . * b AntiHis AntiARRP1 PhosphorylatedAtBRL2HisAtBRL2His E.coliExtract EndoGlucanase MaizeLeafExtract - - -- - + - + - - - - + + + - - + - - - - - - + - - - - - + + + + + 131AntiHis AntiZmARRP1 PhosphorylatedZmBRL2ZmBRL2 E.Coli.Extraction EndoGlucanase MaizeLeafExtract - - -- - + - + - - - - + + + - - + - - - - - - + - - - - - + + + + + Figure 3.9 (cont™d) c 132Figure 3.10 Subcellular localization of ARRP1 (a) Cyan fluorescent protein was fused to the C-terminus of ARRP1. Using confocal microscopy, ARRP1-CFP is visible mainly in the periphery of cell and in the nuclei. (b) Gradient differential centrifuge was used to purify the nuclei from the cell fraction. (c) Western blotting analysis confirmed the presence of ARRP1 in both cytosol and nuclei fraction, while the nuclear protein histone H3 was only detected in the nucleus. ARRP1CFP ERYFPMerge ARRP1CFP GolgiYFP Merge a 20µm 20µm20µm 40µm40µm40µm ZmARRP1 H3 c b 133Youngleaf Maturingleaf MatureleafFigure 3.11 In situ hybridization shows that ZmARRP1 is expressed in the young leaves and enriched in the vascular bundles. AntisenseSense 134Figure 3.12 A model for proposed function of ARRP1. ARRP1 interacts with BRL2, a receptor-like kinase. in response to a stimulus, (I) ARRP1 moves from cytosol to the nucleus where it binds additional activators and regulates gene expression; (II) it moves to the nucleus where it binds additional protein and regulates cell division; or (III) it binds and modifies other, possibly cytoplasmic proteins, thus affecting their function 135Table S3.1 Primers for the paper PrimerNamePrimerSequencepET15bArrp1FCATATGATGACGCTGACGATCCCGGAC pET15BArrp1RGGATCCTCAGTTCTTGGCGGCCAGCGC pDONR207Arrp1FGGGGACAAGTTTGTACAAAAAAGCAGGCTCACCATGACGCTGACGATCCCGGAC pDONR207Arrp1RGGGGACCACTTTGTACAAGAAAGCTGGGTCGTTCTTGGCGGCCAGCGC Arrp1RTPCRFGGGTAAGCTCCACTGCAAGG Arrp1RTPCRRGTTCTTGTCCCCCTCCTCCT At1g09310LPGGTGTTGTATGGCTGAAGCA AT1G09310RPCTTCGCTGCTCTTCTCCTTG AT1G56580LPTTGCCATTGAAGGACATTGA AT1G56580RPGCAGGCTTCTCAACTTCAGG LB3TAGCATCTGAATTTCATAACCAATCTCGATACAC pET15bVH1KZeaMaysFCATATGCGCGCGCGGCGGAAGGAGGCGCGCGAG pET15bVH1KZeaMaysRGGATCCTCAGTCACAGGCAGAGGCAGGCGCTTG pET15bVH1KArabidopsisFCATATGCGTGCGAGGAGGAGAGATGCGG pET15bVH1KArabidopsisRGGATCCTTACAAGCTGTTACTGTGACTGTG 136Table S3.2 Cis-regulatory element within 1 Kb ARRP1 promotor 137Figure S3.1 Expression and purification of ARRP1 and western blotting analysis. (a) Expression and purification of ARRP1. Different fractions were separated by SDS-PAGE gel and stained by coomassie blue. Elution fraction 2 & 3 showed accumulation of a protein with the same size of ARRP1 (b) Identity of the band as ARRP1 was confirmed using Anti-ARRP1 antibody. bARRP1 a32kD20kD15kD6kD138Figure S3.2 ARRP1 mRNA levels in corn coleoptile are unchanged by cytokinin or brassinosteroid. 00.511.5200.511.522.53Arrp1/18SControlBrassinosteroidCytokinin(Hrs)139ConstructsFigure S3.3 Y2H analysis of ZmARRP1 with AtBRL2. AtBRL2 was insert into the pGBKt7 vector, while ZmARRP1 was insert into the pGADT7 vector. 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148 CHAPTER 4: CONCLUSIONS AND OUTLOOKS 1494.1 Contribution of Proteomics in the Identification of Novel Proteins Associated with Plant Growth My first objective (chapter two) was to further understand the changes during auxin-induced expansion growth and identify associated proteins. I used auxin (IAA)-induced rapidly growing corn (Zea mays) coleoptiles as a model system for rapid growth and water treated coleoptiles as a control. By comparing the proteomics changes between auxin-treated and control tissues, and between coleoptile and epidermal tissues, I identified 82 candidates involved in this growth as well as the underlying cell wall and cuticle biosynthesis and four proteins of unknown function. Using quantitative RT-PCR, I could show that increases in the protein level are also reflected in the gene expression. In addition, expression of these genes was increased in rapidly growing coleoptiles, leaves and reduced in coleoptiles that have stopped growing, a finding which confirms their correlation with growth. I focused on two hypothetical proteins, HYP 3, which is a putative mixed-linked glucanase, and HYP 4, a DUF538-containing protein. Their gene expression is increased 2.5 and 1.7-fold, respectively. In addition, HYP4 expression is reduced by 50% in light-exposed, slow growing coleoptiles. It was also induced twofold in the developing leaves. Strong expression of HYP4 in all parts of the coleoptile suggested that it has a universal, not an epidermis-specific function. On the other hand, HYP 3 expression was higher in the epidermis than in total coleoptile tissue suggesting the epidermis as predominant locus of action. HYP3 expression levels decreased in coleoptiles and increased in leaves, suggesting that it plays a role in the growth-controlling processes within the epidermis. 1504.2 ARRP1 in the regulation of cell expansion growth The objective of the third chapter of my thesis was to elucidate the function of HYP4. HYP4, which we now call ARRP1 (Auxin rapid response protein1), is a novel protein with a DUF538 domain. Using pulldown assays and yeast two hybrid assays, I showed that like one of ARRP1™s Arabidopsis homologues, corn ARRP1 interacts with both ZmBRL2 and AtBRL2, a receptor-like kinase that plays a role in the venation pattern of plants, suggesting ARRP1 might function downstream of BRL2 signal pathway. RT-PCR and western blotting analysis, showed that ARRP1 expression is closely associated with cell and organ expansion growth, and mainly in above ground tissues. Like the receptor BRL2, with which it interacts, ARRP1 expression can be induced by the hormone auxin but shows no response to cytokinins and brassinosteroid treatment. In addition ARRP1 transcription was induced immediately after the addition of auxin, reaching the highest level within 30 min and then decreasing slowly to a basal level within the next four hours, indicating that ARRP1 is an early auxin-response gene with a possible signaling function. In addition, its expression is induced by red and blue light. These observations suggest that ARRP1 participates in auxin- and light-related signaling This argument is further supported by the finding that ARRP1 is localized in the periphery of the cell as well as in the nucleus. In addition RNA in situ hybridization demonstrated that ARRP1 is expressed in developing leaves and coleoptiles particularly in the vascular bundles. This expression pattern corresponds to the BRL2 expression pattern. 151Arabidopsis plants lacking the ARRP1 homologue produced trichomes with smaller length and variable number of branches, but no significant growth phenotype compared to wild type. However, the double mutant showed slower growth upon transition to light after five days germination in darkness, a difference that was especially obvious in the early development stages. Inflorescence emergence and flowering were delayed by two weeks in the double mutant, but leaf number at the time of flowering and diameter of the rosette and stem are identical to the wild type control. By overexpressing ZmARRP1 in the Arabidopsis double mutant, ZmARRP1 completely complements the phenotype, indicating that ZmARRP1 and AtARRP1/2 execute the same function. Taken together, these data suggest that ARRP1 acts downstream of the BRL2, a receptor-like kinase, integrates light and auxin signals, and affects plant development and the transition to light. Three pathways of ARRP1 action are possible (Figure 3.12): Ł ARRP1 moves from cytosol to the nucleus where it binds additional activators and regulates gene expression; Ł ARRP1moves to the nucleus where it binds additional protein and regulates cell division; or Ł ARRP1 binds and modifies other, possibly cytoplasmic proteins, thus affecting their function. Below I am proposing experiments to examine and distinguish between the respective ARRP1 functions 1524.3 Future perspective 4.3.1 Questions remaining about ARRP1 function In my dissertation research, I demonstrated that ARRP1 interacts with BRL2 and is expressed in overlapping patterns with BRL2. But whether ARRP1 and BRL2 are localized in the same subcellular compartment which would allow for their interaction remains unknown. To further test my hypothesis, a co-localization analysis should be performed. The YFP tag will be fused to the C-terminal BRL2, while the CFP tag would be fused to ARRP1. Both vectors will be transformed into Agrobacterium and inoculated into the epidermis of tobacco for transient expression. The fluorescence will be determined using confocal microscopy. In addition, we can also use fluorescence resonance energy transfer (FRET) technique to confirm not only BRL2 and ARRP1 co-localization but also the protein-protein interaction. As an alternative to FRET, BiFC (Bimolecular fluorescence complementation) can be used. My localization study showed that ARRP1, which doesn™t contain any signal peptide or nuclear targeting signal, is present in both cytosol and nucleus, indicating a possible translocation movement from the cytoplasm into the nucleus. To test this hypothesis, a stable Arabidopsis transformant that expresses ARRP1-CFP will be generated. Using the fluorescence recovery after photobleaching (FRAP) technique, we can photobleach the CFP signal inside the nucleus and monitor the trafficking of new unbleached protein into the nucleus and thus assess the mobility of ARRP1. If this translocation occurs, we may want to further test whether auxin can facilitate this movement. To achieve that, I will spray auxin onto the Arabidopsis leaves, use the same procedure to measure the recovery, and compared to the non-auxin treatment 153leaves as a control. Alternatively, KAEDE, a photoactivatable fluorescent protein, can be used (Patterson and Lippincott-Schwartz, 2002; Lippincott-Schwartz et al., 2003). Finally, although my data strongly suggest that ARRP1 functions downstream of the BRL2 receptor-like kinase, I still need functional data to support this conclusion. To better understand how ARRP1 and BRL2 work together, a homozygous T-DNA knockout mutant of BRL2 will be obtained, mRNA and protein level of ARRP1 should be measured in this mutant. In addition, I will cross the brl2 and atarrp1-1/1-2 to make a triple mutant. If the triple mutant shows a similar phenotype to brl2, it would suggest that BRL2 and ARRP1 are in the same pathway. Finally, I will also test whether overexpression of ARRP1 can complement the phenotype of brl2 mutant. The constitutive expression vector of ARRP1 driven by 35S promoter will be introduced into the brl2 mutant. The morphological phenotypes, leaf vein development and the secondary auxin response genes will be monitored and compared to the wild type. Together these experiments would provide insights into the ARRP1 function and further our understanding of the BRL2 signaling pathway. 4.3.2 Identification of ARRP1 interacting partners As ARRP1 is a small protein that does not contain any other known functional domains, it may require partners to trigger the plant response or to move to the nucleus. To identify these interaction partners, I performed an in vitro pull down assay, using a ARRP1 specific antibody. Proteins were extracted from young maize leaves, which have the highest ARRP1 protein level, and then incubated overnight with the antibody. Mouse serum without ARRP1 antibody was used as control. The protein complex was 154then purified using protein-A beads. The comparison revealed nine protein bands that were only showed in the pull down assay as compared to the control (Fig. 4.1a as indicated with an asterisk). In gel digestion followed by LC-ESI-MS/MS revealed 34 interaction candidates (Fig. 4.1b & Table S4.1). Based on the annotations of 28 of the 34 isolated proteins, they function in : (I) targeted protein modification/degradation; (II) regulation of cell division and cell elongation; (III) cell metabolic processes and (IV) signal transduction. I focused on candidate partners with a possible novel or not fully elucidated role in downstream signal transduction. Among them, I found two tetratricopeptide repeat domain (TPR) containing proteins, one of which is the homologue of Arabidopsis VIT protein. The TPR domain is a structural motif present in a wide range of proteins that mediate protein-protein interactions and the assembly of multiprotein complexes. TPR-containing proteins often play roles in numerous vital cell processes, such as cell cycle regulation, transcriptional control, mitochondrial and peroxisomal protein transport, and neurogenesis (Sikorski et al., 1991; Zachariae and Nasmyth, 1996; D'Andrea and Regan, 2003). Of particular interest is the ZmVIT, with 54% protein identity to AtVIT. Arabidopsis plants impaired in VIT expression results in vein pattern defects and in alterations in response to auxin. AtVIT had been shown to interact with both AtARRP1-1and AtBRL2 (Ceserani et al., 2009). Taken together, I propose that ZmVIT also interacts with both ZmBRL2 and ZmARRP1, and plays an important role in the early stage of plant growth, and integration of light- and auxin- signaling pathway. The second identified protein involved in the signal transduction is Auxin signaling F box protein 2 (AFB2). AFB2 is a member of a small subclade consisting of 155seven proteins, including TIR1, AFB1, 3, and COl1 (Gagne et al., 2002). Among them, AFB1,2,3 and TIR1 are most closely related. Like TIR1, AFB2 interacts with the Aux/IAA proteins in an auxin dependent manner and acts redundantly with the other three TIR1/AFB proteins to regulate diverse aspects of growth and development. Plants that are deficient in all four proteins are auxin insensitive and exhibit a severe embryonic phenotype (Dharmasiri et al., 2005). The quadruple mutant also forms rosettes with small, highly curled leaves and shows deficiency in leaf venation, which is similar to that of the BRL2 mutants, but more severe. In addition, AFB2 is mainly localized in the nucleus which provides a possible explanation why ARRP1 is also found there. I also identified two putative annexin proteins. Annexins are soluble proteins capable of Ca2+-dependent or independent association with membrane phospholipids (Gerke et al., 2005). They play a role in coordinating development and response to environmental changes. Plant annexins are induced by abiotic stress including salinity, cold, oxidative and mechanic stress (Mortimer et al., 2008; Laohavisit and Davies, 2011). Pathogen attack or symbiotic interaction also induced the transcription of annexins (Niebel Fde et al., 1998; Vandeputte et al., 2007). More recently, several experiments showed that the expression of a number of Arabidopsis, alfalfa, and tobacco annexins was affected by both light and auxin, as well as abiotic stress (Kovacs et al., 1998; Cantero et al., 2006; Baucher et al., 2011). The multifunctionality and the similar response to auxin and light make annexins good candidates for the downstream of BRL2-ARRP1 signal pathway. I am also interested in several novel proteins with unknown functions. Hypothetical protein 1 appears to be similar to a tomato ASR1 (abscisic acid stress 156ripening protein), now called abscisic acid stress ripening like protein (ASRL1), which is a small plant-specific protein. It is one of numerous plant gene products with unknown biological roles that become upregulated under water- and salt-stress conditions (Kalifa et al., 2004; Goldgur et al., 2007). Hypothetical protein 2 has similarity to other proteins of unknown function with a DUF250 domain which are also called triose-phosphate transporter (TPT) family. TPT Proteins mediate export of stromal triose-P into the cytosol to produce sucrose. In contrast to the aforementioned starch-storing plant species, maize uses sucrose as its major transitory storage in the leaves rather than starch. By affecting HYP2 activity, ARRP1 may exert significant influence on leaf sucrose level and the plant energy status, which then ultimately regulates plant growth. 4.3.3 Confirmation of protein-protein interaction To test the interaction between ARRP1 and proteins listed in table 1, yeast two hybrid (Y2H) analysis was performed as described in chapter 3. Interaction between ARRP1 and TPR1, Annexin1, and ASRL1 was confirmed (Fig. 4.2). Because Y2H also has several limitations including interactions involving membrane proteins, self-activating proteins, or proteins requiring post-translational modifications, it may lead to false negatives (see chapter 3). In the future, I would like to use FRET as a backup technique to check the interaction of ARRP1 candidates that do not interact in the Y2H. In addition, due to the low sensitivity of Coomassie blue staining, the list of protein interaction partners may not be complete. In the future, we can use the maize leaf cDNA library and for large scale screenings which may help us identify several important candidates that are in trace concentration. 1574.3.4 Expression study of three confirmed ARRP1- binding partners To determine whether the three confirmed candidates, TPR1, Annexin1, and ASRL1, are co-expressed with ARRP1, we characterized the expression of all three genes, using RT-PCR. My earlier study showed that ARRP1 was strongly expressed in the above ground tissue while mRNA and protein were barely detectable in the root. ARRP1 showed reduced expression in the coleoptiles when their growth ceased and significantly increased in the young leaves when their growth was enhanced by light. Along the length of maize leaf, ARRP1 expression was seen in all tissues, but both, mRNA level and protein abundance were highest in the basal zone, suggesting ARRP1 is involved with the cell division and cell elongation growth. ASRL1 exhibits a similar expression pattern as ARRP1. ASRL1 is expressed in early stage embryos, in the shoot, and in emerging and expanding leaves. The transcription level of ASRL1 increased about 50% in the developing leaf and dropped 60% in the growth ceased coleoptiles (Fig. 4.3 a/b). Like ARRP1, ASRL1 mRNA is at its highest level in the basal zone and decreased along the maize leaf. A search of the Bioanalytic Source for Plant Biology (http://bar.utoronto.ca/efp_maize/cgi-bin/efpWeb.cgi) suggests that the expression of ASRL1 is associated with the vascular cells. The expression patterns of TPR1 and Annexin1 also overlapped with ARRP1, but shows several differences. Both genes are highly expressed not only in the shoot and leaves, but also in the root. Just like ARRP1, transcript levels are correlated with plant expansion growth. In leaves, they are also enriched in the basal zone but closer to the bottom of the leaf than ARRP1. They are not predicted to be restricted in the 158vascular cells where ARRP1 was highly expressed. In the future, we can perform in situ hybridization analysis to confirm the tissue specific expression. In my dissertation research, I demonstrated that ARRP1 transcription was induced immediately after the addition of auxin, reaching the highest level within 30 min, but showed no changes upon cytokinin and brassinosteroid treatment. ARRP1 expression level increased significantly after 2.5 days in red and blue light conditions. As I proposed that these TPR1, Annexin1 and ASRL1 function in downstream of ARRP1, it would be necessary to check their response to different hormone and light conditions. Using quantitative RT-PCR, the mRNA level of each gene will be monitored and compared to the 18S. These experiments would consolidate my hypothesis of the BRL2-ARRP1 signal pathway. 4.3.5 Evaluation of the T-DNA knock-out mutants I already confirmed that TPR1, Annexin1, and ASRL1 interact with ARRP1, and that their expression pattern is similar to ARRP1. To further explore their physiological function in plant growth, homozygous T-DNA mutants defective in these genes will be obtained. Morphological phenotypes of mutants will be monitored and compared to BRL2 and ARRP1 mutant. The mRNA level of secondary auxin inducible genes will also be measured, as will be the levels of these genes in ARRP1 overexpression and knock-down lines. Together these results will further our understanding of the function of BRL2-ARRP1 signaling pathway. 159Figure 4.1a SDS-PAGE gel comparison of pull-down assay. ARRP1 specific antibody was used to pull down the interacting partners form the leaf total protein extraction. The marked bands were selected for LC-ESI-MS PulldownControlStd * * * * * * * * * 250KD 150100755037252015160CellDivisionandCellElongationSignalPathwayProteinDegradation/ModificationCellMetabolicProcesssOtherUnknownFunctionFigure 4.1b Grouping of proteins identified in the pull-down assay based on their predicted function/involvement in cellular processes. 161Figure 4.2 Yeast two hybrid analyses for confirming the protein-protein interaction of ARRP1 and its partners. TPR1: TPR domain containing protein 1; AFB2: auxin signaling F box protein; ASRL1: abscisic acid stress ripening like protein 1; HYP2: hypothetical protein 2; Annexin1: Annexin like protein1. SD/LeuTrpHis SD/LeuTrp 11:101:10011:101:100pGBKT7ZmARRP1+ pGADT7TPR1 pGBKT7ZmARRP1+ pGADT7AFB2 pGBKT7ZmARRP1+ pGADT7ASRL1 pGBKT7ZmARRP1+ pGADT7HYP2 pGBKT7ZmARRP1+ pGADT7Annexin1 Constructs16200.1 0.20.30.4 0.5 0.60.70.8ASRL1/18SFigure 4.3 Semiquantitative analyses of transcription levels of genes encoding ASRL1 (a, b), Annexin1 (c, d) and TPR1 (e, f) in different corn seedling organs and along the young leaf. fiEpidermisfl corresponds to the epidermis of coleoptiles of seedlings germinated and grown in the dark for five days; Material for coleoptiles was harvested either after five days in the dark (dark) or 1, 2, or 5 days after transition of the 5-day-old, dark-grown seedling to light. Leaf samples were taken either from the leaf within the coleoptile of the 5-day-old, dark-grown seedling (undeveloped leaf) or two days after transition to light (2 days light). Young maize leaves were taken from 2 weeks old corn seedling and divided into five zones as we described in chapter 3 (Fig 3.4a). Bars represent the mean ± S.E. of 3 biological replicates a163Figure 4.3 (cont™d) 00.20.40.60.811.21.41.61.82IIIIIIIVVASRL1/18Sb16400.20.4 0.60.811.21.41.6 1.8Annexin1/18S02468101214161820IIIIIIIVVAnnexin1/18SFigure 4.3 (cont™d) cd16500.1 0.2 0.3 0.4 0.50.60.7 0.8 0.91TPR1/18S00.20.40.60.811.21.41.61.8IIIIIIIVVTPR1/18SFigure 4.3 (cont™d) ef166Table 4.1 ARRP1-interacting proteins with a possible novel or not fully elucidated role in downstream signal transduction ProteinAccess#MWPossibleFunctionAuxinSignalingFbox2GRMZM2G09578666AuxinsignalpathwayZmVITGRMZM2G047093103HomologueofAtVIT,functionintheBRL2signalpathwayTPRdomaincontainingprotein1GRMZM2G39271081PossiblysignaltransductionmoleculeAnnexin1likeGRMZM2G06195038CoordinatedevelopmentandresponsetoenvironmentalchangesAnnexin2likeGRMZM2G06499335CoordinatedevelopmentandresponsetoenvironmentalchangesHypotheticalprotein#1(ASRL1)GRMZM2G13691016ResponsetoabioticstressHypotheticalprotein#2GRMZM2G08110541DUF250containingproteinHypotheticalprotein#3GRMZM2G38170996possibleubiquitinconjugatingenzymeE2167Table S4.1: Proteins identified in the ARRP1 pull down assay Protein Identification Access # TrpfraMW Localization/Function Sequence Charge state (Z) ATP synthase, putative, expressed GRMZM2G094497_T016 54 ATP synthase (R)TVSGVAGPLVILDK(V) Z=2 (R)GYPGYmYTDLATIYER(A) Z=2 (R)IALTTAEYLAYEcGK(H) Z=2 (K)IPLFSAAGLPHNEIAAQIcR(Q) Z=3 (R)TVSGVAGPLVILDK(V) Z=2 (R)VTLFLNLANDPTIER(I)Z=2 Os6bglu24 - beta-glucosidase homologue, GRMZM2G077015_T01 5 64 catalyzes the hydrolysis of the glycosidic bonds (K)ENGIEPYVTLFHWDTPQALVDSYGGFLDDR(I) Z=3 (K)VcFVHFGDVVK(N) Z=2 (K)NWFTFNEPQTFSSFSYGTGIcAPGR(C) Z=3 (K)LVGSYDIMGINYYTSR(F) Z=2 (K)HVDISTGYTPVLNTDDAYATQETK(G)Z3DnaK family protein, putative, expressed GRMZM2G001500_T01 2 49 Involved in chromosomal DNA replication (R)QAVVNPENTFFSVK(R) Z=2 (R)IINEPTAASLAYGFEK(K) Z=2 tetratricopeptide repeat domain containing protein, expressed ZmVIT GRMZM2G047093_T01 5 104 Signal transduction (K)ISLFEEcGMLDR(A) Z=2 (K)LLETVFGDQNVANR(W) Z=2 (K)ILQHAGNFTAAAALADEAR(S) Z=3 (K)LIQIEDPLAEATK(Y) Z=2 (K)VLLAFQAVK(Q) Z=2 annexin, putative, expressed GRMZM2G061950_T01 2 35 Coordinate development and response to environmental change (R)TPAQLFAVK(Q) Z=2 (R)LALGGMGTDEDDLTR(V) Z=2 COBW domain containing protein, putative, expressed GRMZM2G180418_T02 2 79 play a central role in zinc metabolism (K)LDGVVTLVDAK(H) Z=2 (K)GIVNEAVQQIAYADR(I) Z=2 (K)IDLVKEPEVLSLVER(I) Z=3 exo-beta-glucanase, expressed GRMZM2G076946_T02 3 64 Cell wall biosynthesis (K)ENGIEPYVTLFHWDTPQALVDSYGGFLDDR(I) Z=3 (K)LVGSYDIMGINYYTSR(F) Z=2 (K)YSPVLNTDDAYAAQETK(G) Z=2 168Table S4.1 (cont™d) 6-phosphofructokinase, putative, expressed GRMZM2G004932_T01 1 63 one of the most important regulatoryenzymesofglycolysis (K)SFGFDTAVEEAQR(A) Z=2 annexin, putative, expressed GRMZM2G064993_T01 1 38 Coordinate development and response to environmental change (R)SITDEISGDFER(A) Z=2 AUXIN SIGNALING F-BOX 2, putative, expressed GRMZM2G095786_T01 1 66 Auxin signal pathway (R)KLTRLSTSGQLTDR(A) Z=2 soluble starch synthase 3, putative, expressed GRMZM2G121612_T01 1 134 pathway starch biosynthesis (K)VGGLGDVVTSLSR(A) Z=2 abscisic stress-ripening, putative, expressed GRMZM2G136910_T01 3 15 Response to abiotic stress (K)QHLGEAGAIAAGAFALYEK(H) Z=2 (K)KDEEQPAGEYGYSETEVVTATGEGEYER(Y) Z=3 (K)ITEEVAAAAAVGAGGYVFHEHHEK(K) Z=3 ulp1 protease family, C-terminal catalytic domain containing protein, expressed GRMZM2G348480_T01 1 32 catalyzes two essential functions in the SUMO pathway (K)EIEmTYAKQTDRYK(H) Z=2 HD domain containing protein 2, putative, expressed GRMZM2G378040_T01 1 42 DNA replication (K)LVDPEQGVDAGAGR(A) Z=2 Hypothetical protein GRMZM2G381709_T01 1 96 catalyze the attachment ofubiquitinto the substrate protein (R)cPRTAIVSDVDALERTVNVK(W) Z=2 tetratricopeptide repeat domain containing protein, expressed GRMZM2G392710_T01 1 81 Signal transduction (R)NEEALEMMEK(A) Z=2 phenylalanine ammonia-lyase, putative, expressed GRMZM2G029048_T01 1 77 enzyme that catalyzes a reaction converting L-phenylalanine to ammonia and trans-cinnamic acid (R)INTLLQGYSGIR(F)Z=2phospholipase D, putative, expressed GRMZM2G054559_T01 1 92 ithydrolyzesto produce thesignalmoleculephosphatidic acid (R)LEGPIAWDVLYNFEQR(W) Z=2 peroxidase precursor, putative, expressed GRMZM2G394500_T01 3 38 biosynthesis and degradation of lignin, (K)TAPINIGLAAFEVIDEIK(A) Z=2 (R)LSAPPAQIVPAYR(N) Z=2 (R)DLPDSTFTVSELIR(N) Z=2 169Table S4.1 (cont™d) amine oxidase, flavin-containing, domain containing protein, expressed GRMZM2G034152_T01 556 catalyzes theoxidative cleavageofalkylaminesintoaldehydesandammonia (R)VIVVGAGMSGISAAK(R) Z=2 (K)RLSEAGITDLLILEATDHIGGR(M) Z=3 (R)LSEAGITDLLILEATDHIGGR(M) Z=3 (K)FDYEFAEPPR(V) Z=2 (R)AIYQFDmAVYTK(I) Z=2 phosphoenolpyruvate carboxylase, putative, expressed GRMZM2G083841_T01 4109 used forcarbon fixationinCAM/ C4 plants (K)VSEDDKLIEYDALLVDR(F) Z=3 (R)FLNILQDLHGPSLR(E) Z=3 (K)AIADGSLLDLLR(Q) Z=2 (R)QVFTFGLSLVK(L) Z=2 elongation factor Tu, putative, expressed GRMZM2G106061_T01 450 DNA replication (K)KYDEIDAAPEER(A) Z=3 (R)GITINTATVEYETETR(H) Z=2 (R)ELLSNYEYDGDEVPIVAGSALK(A) Z=2 (K)IGDTVDIVGIR(D) Z=2 Hypothetical protein GRMZM2G081105_T01 2 41 Unknown function (R)SECDDIEEA(K) Z=2 (K)TSDVFDTLLQ(K) Z=2 glyceraldehyde-3-phosphate dehydrogenase, putative, expressed GRMZM5G845611_T01 447 erves to break down glucosefor energy and carbon molecules (K)IVDDTTISVDGKPITVVSSR(D) Z=3 (K)ILDEEFGIVK(G) Z=2 (R)AAALNIVPTSTGAAK(A) Z=2 (R)VPTPNVSVVDLVINTVK(T) Z=2 lipoxygenase, putative, expressed GRMZM2G015419_T02 2102 catalyze the dioxygenation of polyunsaturated fatty acids in lipids (K)LVEDTTDHVLR(F) Z=3 (R)YTmEINALAR(E) Z=2 (K)LDPEVYGPAESAITK(E) Z=2 von Willebrand factor type A domain containing protein, putative, expressed GRMZM2G320152_T01 266 a large multimericglycoprotein (R)AGFIVLISDGLDGQSK(W) Z=2 (K)EGGDDPVLQAIAASLLQR(K) Z=3 fructose-bisphospate aldolase isozyme, putative, expressed GRMZM2G046284_T01 241 Calvin cycle is acarbonfixationpathwa (R)EAAYYQQGAR(F) Z=2 (R)ATPEQVAAYTLK(L) Z=2 170Table S4.1 (cont™d) starch synthase, putative, expressed GRMZM2G008263_T01 266 Starch biosynthesis (K)ELTSGPDKGVELDGVLR(T) Z=3 (R)AGIDDAEEIAPLAK(E) Z=2 LTPL122 - Protease inhibitor/seed storage/LTP family protein precursor, expressed GRMZM2G429000_T01 213 Protease inhibitor (K)FGVcADVLGLVK(G) Z=2 (K)LSALVNYcGK(C) Z=2 Hypothetical protein GRMZM2G095082_T01 245 unknown function (R)LLEEPDAQLVDIRPLK(D) Z=3 (K)ELIDEIKEIGQALLPLPGDAK(S) Z=3 Cupin domain containing protein, expressed GRMZM2G064096_T01 222 Signal transduction (K)SSVTANDFYFHGLAGQGK(I) Z=2 (K)VTFLDDAQVK(K) Z=2 DnaK family protein, putative, expressed GRMZM2G056039_T01 271 DNA replication (R)IINEPTAAAIAYGLDKK(A) Z=3 (K)EQVFSTYSDNQPGVLIQVYEGER(T) Z=3 aquaporin protein, putative, expressed GRMZM2G014914_T01 130 Integral membrane poreproteins (K)AFQSAYFDR(Y) Z=2 glycosyl hydrolase family 3 protein, putative, expressed AC234091.1_FGT001 283 Cell wall biosynthesis (K)VSQLGDEAAGVPR(L) Z=2 (K)NDAGILPLDR(S) Z=2 171 REFERENCES 172REFERENCES BaucherM,OukouomiLoweY,VandeputteOM,MukokoBopopiJ,MoussawiJ,VermeerschM,MolA,ElJaziriM,HombleF,PerezMorgaD(2011)Ntann12annexinexpressionisinducedbyauxinintobaccoroots.JExpBot62:40554065CanteroA,BarthakurS,BushartTJ,ChouS,MorganRO,FernandezMP,ClarkGB,RouxSJ(2006)ExpressionprofilingoftheArabidopsisannexingenefamilyduringgermination,deetiolationandabioticstress.PlantPhysiolBiochem44:1324CeseraniT,TrofkaA,GandotraN,NelsonT(2009)VH1/BRL2receptorlikekinaseinteractswithvascularspecificadaptorproteinsVITandVIKtoinfluenceleafvenation.PlantJ57:10001014D'AndreaLD,ReganL(2003)TPRproteins:theversatilehelix.TrendsBiochemSci28:655662DharmasiriN,DharmasiriS,WeijersD,LechnerE,YamadaM,HobbieL,EhrismannJS,JurgensG,EstelleM(2005)PlantdevelopmentisregulatedbyafamilyofauxinreceptorFboxproteins.DevCell9:109119GagneJM,DownesBP,ShiuSH,DurskiAM,VierstraRD(2002)TheFboxsubunitoftheSCFE3complexisencodedbyadiversesuperfamilyofgenesinArabidopsis.ProcNatlAcadSciUSA99:1151911524GerkeV,CreutzCE,MossSE(2005)Annexins:linkingCa2+signallingtomembranedynamics.NatRevMolCellBiol6:449461GoldgurY,RomS,GhirlandoR,ShkolnikD,ShadrinN,KonradZ,BarZviD(2007)Desiccationandzincbindinginducetransitionoftomatoabscisicacidstressripening1,awaterstressandsaltstressregulatedplantspecificprotein,fromunfoldedtofoldedstate.PlantPhysiol143:617628KalifaY,GiladA,KonradZ,ZaccaiM,ScolnikPA,BarZviD(2004)ThewaterandsaltstressregulatedAsr1(abscisicacidstressripening)geneencodesazincdependentDNAbindingprotein.BiochemJ381:373378KovacsI,AyaydinF,OberschallA,IpacsI,BottkaS,PongorS,DuditsD,TothEC(1998)Immunolocalizationofanovelannexinlikeproteinencodedbyastressandabscisicacidresponsivegeneinalfalfa.PlantJ15:185197LaohavisitA,DaviesJM(2011)Annexins.NewPhytol189:4053LippincottSchwartzJ,AltanBonnetN,PattersonGH(2003)Photobleachingandphotoactivation:followingproteindynamicsinlivingcells.NatCellBiolSuppl:S714MortimerJC,LaohavisitA,MacphersonN,WebbA,BrownleeC,BatteyNH,DaviesJM(2008)Annexins:multifunctionalcomponentsofgrowthandadaptation.JExpBot59:533544173NiebelFdeC,LescureN,CullimoreJV,GamasP(1998)TheMedicagotruncatulaMtAnn1geneencodinganannexinisinducedbyNodfactorsandduringthesymbioticinteractionwithRhizobiummeliloti.MolPlantMicrobeInteract11:504513PattersonGH,LippincottSchwartzJ(2002)AphotoactivatableGFPforselectivephotolabelingofproteinsandcells.Science297:18731877SikorskiRS,MichaudWA,WoottonJC,BoguskiMS,ConnellyC,HieterP(1991)TPRproteinsasessentialcomponentsoftheyeastcellcycle.ColdSpringHarbSympQuantBiol56:663673VandeputteO,LoweYO,BurssensS,DVANR,HutinD,BoniverD,GeelenD,ElJaziriM,BaucherM(2007)ThetobaccoNtann12gene,encodinganannexin,isinduceduponRhodoccocusfasciansinfectionandduringleafygalldevelopment.MolPlantPathol8:185194ZachariaeW,NasmythK(1996)TPRproteinsrequiredforanaphaseprogressionmediateubiquitinationofmitoticBtypecyclinsinyeast.MolBiolCell7:791801 174 APPENDICES 175APPENDIX A: STUDY OF A PUTATIVE ENDO-(1,3)(1,4)--GLUCANASE 1765.1 Introduction Plant cell walls not only provide mechanical support for cells, but also protect against dehydration, pathogens, and other environmental factors (Cosgrove, 1997). They consist of cellulose microfibrils embedded in a matrix of hemicelluloses, pectins, glycoproteins, and phenolics (Carpita and Gibeaut, 1993; Cosgrove, 2005). There are two types of cell wall, primary and secondary cell wall, that differ in the mobility, arrangement, and composition of matrix polymers, the higher-order organization of microfibrils, and their roles in the life of plant (Cosgrove and Jarvis, 2012). The variation of plant cell wall composition among cell types and species are significant. For instance, the walls of grass differ dramatically from dicot cell walls mainly in the relative abundance of non-cellulosic polysaccharides, their associations with cellulose, pectins, and proteins. (Carpita, 1996; Vogel, 2008).These variations contribute to the differences in mechanical properties that occur in different species and cell types (Cosgrove and Jarvis, 2012). It also influences the strength and flexibility of lumber and textiles, the pulping properties of wood fibers, and cell wall recalcitrance to deconstruction for biofuel production (Cosgrove, 2005; Kiemle et al., 2014). Primary cell walls in dicots, noncommelinoid monocts, and gymnosperms are characterized by a significant abundance of xyloglucan (XyG), pectic polysaccharides and structure proteins (Fincher, 2009). In contrast, the predominant noncellulosic polysaccharides in primary cell walls of commelinoid monocots (e.g. grasses, sedges, rushes and gingers) are glucuronoarabinoxylan (GAX) and (1,3)(1,4)--D-glucan (MLG), with relatively minor proportions of XyG and pectic polysaccharides (Buckeridge et al., 2004; Vogel, 2008). The GAX, that take the place of XyG in dicots, play an important 177structural role by crosslinking cellulose microfibrils. During the secondary cell wall formation, GAX also oxidatively cross-linked with each other and with lignin by phenolics (Ralph et al., 1995; Vogel, 2008). MLGs are considered to be unique to the grass cell wall (Carpita, 1996; Trethewey et al., 2005). Recently, they have also been found in other species like lycophyte Selaginella moellendorffii and Equisetum (Sorensen et al., 2008; Harholt et al., 2012). The grass MLGs are homopolymers of unbranched and unsubstituted chains of -glucopyranosyl linked through approximately 30% of -(1,3) bonds and 70% of -(1,4) bonds (Woodward, 1983). The unit structure was elucidated by digestion of MLGs with endo-(1,3)(1,4)--glucanase purified from Bacillus subtilis. This enzyme only cleaves a (1,4)--glucosyl linkage if it is next to a (1,3)--glucosyl linkage (Anderson and Stone, 1975). The result shows that MLGs of grasses are composed of a mixture of trisaccharide and tetrasaccharide units. Within each unit, two or three contiguous (1,4)--glucosyl residues are joined with a single (1,3)--glucosyl linkage (Woodward, 1983; Woodward, 1983). Up to 10% of the polymers contain more than three (1,4)-linked residues, however contiguous (1,3)-linked residue units have not been found. By creating an irregular shape within the linear polymer, the (1,3)--glucosyl linkages increase flexibility and solubility (Fincher, 2009). In certain grains, including barley, wheat, and oat, MLG can accumulate to large amounts in the cell walls of the endosperm and act as a storage carbohydrate (Trethewey et al., 2005; Wilson et al., 2006; Guillon et al., 2011). They are part of our natural diet and have a cholesterol-lowering effect as well as possibly immune-regulating properties (Rieder and Samuelsen, 2012). In the context of my project, MLGs 178have also been hypothesized to play a role in cell expansion (Luttenegger and Nevins, 1985; Buckeridge et al., 2004; Gibeaut et al., 2005). In the cell walls of coleoptiles and young leaves of grasses, MLG abundance peaked during the rapid elongation phases and dramatically dropped when cell expansion ceased (Carpita et al., 2001; Roulin et al., 2002). Several functional roles have been suggested for MLG, including providing a source of metabolizable energy (Wada, 1978; Roulin et al., 2002), coating cellulose microfibrils (Carpita et al., 2001), forming gel-like matrixes in the wall (Kozlova et al., 2014), and tethering cellulose microfibrils to form a load-bearing network (Kiemle et al., 2014). Grasses not only provide the majority of calories consumed by humans; their cell walls are also becoming a significant source of renewable biofuel. It is important to better understand the regulation and the processes necessary for grass growth. Auxin-induced maize coleoptile elongation is one of the best-characterized phytohormonal responses. This growth enhancement by auxin includes several cellular processes, one of which is the specific adjustment in the mechanical properties of the cell wall (Taiz, 1984). This adjustment has been correlated with qualitative and quantitative changes in certain matrix polysaccharides in the cereal grasses (Loescher and Nevins, 1972; Darvill AG, 1978). Among these changes, the effect of auxin on the composition of -glucans in cell wall appears to be the most significant one. IAA treatment of maize coleoptile segments caused a transient increase followed by a decrease in the -glucan content of the cell walls, suggesting a link between glucan metabolism and auxin responses (Luttenegger and Nevins, 1985). A higher autohydrolytic activity, mediated by a wall associated exoglucanase and an endoglucanase for degradation of MLG, was 179detected in cell wall extracts from maize coleoptiles (Inouhe and Nevins, 1991). Using the specific antisera against either of these glucanase enzymes will dramatically inhibit the coleoptile elongation growth, indicating a role of glucanases in control of plant elongation (Thomas et al., 2000). In addition, auxin also directly induces the transcription of endoglucanase, suggesting that the de novo synthesis of endoglucanase is necessary for sustained cell elongation (Kotake et al., 2000). Although it is clear that these glucanases are essential for auxin induced coleoptile elongation, how they function is still under debate. In the original theory, glucanases are thought to be involved with cell wall loosening (Inouhe and Nevins, 1991). However several recent experiments suggest that instead of increasing walls plasticity, glucanase might just provide a potential energy source, by degrading MLGs (Roulin et al., 2002; Takeda et al., 2010). Endo-(1,3)(1,4)--glucanase is a carbohydrate-acting enzyme of class GH16b . Class GH16b enzymes contain not only endo-glucanases but also xyloglucan endotransglucosylase/hydrolase (XTHs) (Strohmeier et al., 2004). The sequence similarity between XTHs and endo-glucanase suggested the possibility that endo-glucanase might be able to use XyG as an alternative substrate. In vitro experiments demonstrated that corn endo-glucanase not only hydrolyses MLG but also XyG (Hatfield and Nevins, 1987). Taken together, these results make endo-glucanase a good candidate for an ancient and universal mechanism of growth control in cereal plants. In our comparison of auxin-treated corn coleoptiles we have identified a protein of unknown function, called HYP3, which encodes a putative endo-(1,3)(1,4)--glucanase. In Chapter 2 (Jie et al., 2013) I showed that expression of HYP3 is correlated with cell 180expansion growth in coleoptiles and leaves (Fig. 2.7a) (Li et al., 2013). HYP3 cDNA was cloned from maize and the recombinant HYP3 protein expressed in E. coli and purified. However recombinant HYP3 did not exhibit any enzyme activity in vitro analysis. 1815.2 Materials and Methods 5.2.1 Protein sequence analysis and bioinformatics analysis Protein sequence and full cDNA sequence were obtained from the phytozome website (phytozome.jgi.doe.gov). Using the national center for biotechnology website, the conserved domains and two Arabidopsis homologues of HYP3 were determined and aligned. Potential signal peptide and glycosylation sites were predicted by SignalP 4.1 server and NetNGlyc 1.0 server, respectively. 5.2.2 Recombinant HYP3 expression and purification To generate C-terminal His-tagged fusion proteins of HYP3, the full length ORF was isolated from RNA extracts of five-day-old corn coleoptiles, using RT-PCR, with BamHI and NdeI sites at either end. After the digestion, the ORF was inserted into the pET42b expression vector. The construct was confirmed by sequencing. Protein expression was performed as described in chapter 3. However HYP3 was predominately enriched in the inclusion body. To obtain the protein for in vitro enzymatic analysis, the inclusion body was purified and dissolved in PBS buffer containing 8 M Urea. For protein refolding, the denatured protein was then dripped slowly into 50 volumes of PBS buffer with 10 mM DTT under constant stirring. The protein was purified by Ni-column and analyzed by SDS-PAGE. 5.2.3 In vitro enzymatic analysis The enzyme activity and substrate specificity of the purified HYP3 were determined using the dinitrosalicylic acid (DNS) method (Miller, 1959). Different substrates were dissolved in 0.1 M sodium acetate buffer at pH 5.0 and incubated with 10 µg HYP3 protein solution for at 50°C for 10 min. The reaction was then stopped and the 182absorbance of the supernatant was measured at 575 nm. Endo-glucanase (Sigma) and lichenase (megazyme) were used as control. 5.2.4 Identification and verification of T-DNA insertion lines The SALK T-DNA database (http://signal.salk.edu/cgi-bin/tdnaexpress) was searched for insertion lines in HYP3 Arabidopsis thaliana homologue and seeds were obtained from the Arabidopsis Biological Resource Center (Alonso et al., 2003). Using PCR genotyping, homozygous mutants for the T-DNA insertion were identified. Mutant plants were confirmed using reverse transcriptaseŒmediated PCR. 5.2.5 Transient expression and stable transgenic transformation Following the method described in chapter 3, a vector for expression of carboxy-terminal HYP3-GFP fusion protein was generated, transformed into Agrobacterium and inoculated into the lower epidermis of tobacco for transient expression. After 48 hours, localization was determined using confocal microscopy. To generate the HYP3 overexpression line, the constructs were transformed into Arabidopsis thaliana Columbia-0 plants, using Agrobacterium-mediated floral dip as described by Clough & Bent 1998. Overexpression plants were confirmed by genotyping PCR and RT-PCR. 1835.3 Results 5.3.1 Sequence analysis of the full-length HYP3 gene HYP3 (GRMZM2G073079) has an open reading frame of 795 bp that encodes a predicted protein of 264 amino acid residues with an estimated molecular weight of 28.6 kD and a pI of 6.3. It contains a potential 25 amino acid signal peptide sequence (SignalP 4.1) and two predicted N-linked glycosylation sites (Fig. 5.1a). Several endo-(1,3)(1,4)--glucanases in other species, including sorghum, Setaria italica, Oryza sativa, and brachypodium exhibit high protein sequence similarity, in the comparison with HYP3. HYP3 contains a dienelactone hydrolase domain (DLH domain), which can catalyze the hydrolysis of dienelactone to maleylacetate, a domain that is conserved among other endoglucanases as well (Fig. 1a). These results suggest that HYP3, a putative maize glucanase, might possess the same enzyme activity as other endo-(1,3)(1,4)--glucanase. I found two HYP3 homologues in Arabidopsis: AtHYP3-1 (At3g23570) and AtHYP3-2 (At3g23600) (Fig. 1b). Both of them contain the dienelactone hydrolase domain and are predicted to be involved with plant response to abiotic stress (Gong et al., 2001). 5.3.2 Expression of HYP3 and enzymatic analysis The full-length ORF of HYP3 was cloned into the pET42b vector and transformed into the E. coli. Protein expression was induced by 0.1mM IPTG for 3 hours. After the ultra-sonication for disrupting the cell membrane, different fractions of cell lysis were then analyzed by SDS-PAGE. Comparison between the control and induced E. coli. protein bands revealed the presence of a highly expressed protein with 29 kD molecular weight, which corresponds to HYP3. However, this recombinant HYP3 was 184predominantly located in the inclusion bodies. Low temperature, low IPTG concentration during the induction, or fusion of MBP domain to C-terminus of HYP3, could not improve the solubility of HYP3. Using the combination of a denature-renature method with the Ni-IDA affinity column (Fischer et al., 1993), I was finally able to obtain purified protein, with 90% purity. Enzymatic analysis was performed to test the hydrolytic activity of HYP3. Endo-(1,3)(1,4)--glucanase (megazyme) and -glucanase (Sigma) from Bacillus subtilis were used as positive controls. Using lichenin, one kind of MLGs, as substrate, HYP3 did not exhibit any enzyme activity while the two controls effectively hydrolyzed the substrate (Fig 2). I tested all three enzymes against various polysaccharides, including barley -glucan, sodium carboxymethyl cellulose (CMC), and XyG. As expected, endo-(1,3)(1,4)--glucanase also hydrolyzed barley -glucan, but with a lower activity compared to lichenin. -glucanase showed no substrate specificity and hydrolyzes all three polysaccharides. However HYP3 still exhibited no hydrolytic activity. This may be either due to protein misfolding during the renaturing process or missing post transcriptional modifications. 5.3.3 Phenotyping of athyp3-1 and athyp3-2 knock-out mutants, and ZmHYP3 overexpression lines. I obtained athyp3-1 and athyp3-2 knock-out mutants in Arabidopsis from the Arabidopsis Information Resource. The two single mutants do not show any growth phenotype compared to wild type (Fig. 3a). By crossing single mutants, I was able produce the double knock-out mutant, which was also indistinguishable from wild type. In addition, overexpression of ZmHYP3 in Arabidopsis also did not cause any 185morphological phenotypes (Fig 3b). The lack of an effect of ZmHYP3 is not necessarily surprising since Arabidopsis is not expected to contain MLGs. The HYP3 homologues in Arabidopsis may hence serve an entirely different function or use other glucans as substrate. 5.3.4 Subcellular localization of HYP3 To investigate the subcellular localization of HYP3, a C-terminal HYP3-green fluorescent protein (GFP) fusion protein was transiently expressed in tobacco leaves. As shown (Fig. 5.4), HYP3-GFP is localized in the periphery of the cell. 5.3.5 Conclusions and outlook In chapter 2, I had identified HYP3, a putative maize endo-(1,3)(1,4)--glucanase, from auxin induced rapidly-growing corn coleoptiles. Expression studies suggest that it plays an important role in cell elongation growth. However, recombinant HYP3 did not show any enzyme activity, which may be due to the misfolding or loss of post transcriptional modifications. To examine this possibility, I will attempt to express ZmHYP3 in wheat germ cell-free system (Olliver et al., 1998) and repeat the activity assay. An alternative method that we can try is to directly purifying the HYP3 protein from the Arabidopsis stable overexpression line, by using a GFP affinity column (ChromoTeck). MLGs are unique to monocots, which might be the reason why the Arabidopsis double mutant and overexpression lines did not show any phenotypic differences. In the future, using RNAi or CRISPR/Cas9, we can generate the HYP3 knock down mutant in maize. The morphological phenotype, auxin response and cell wall composition will be 186monitored and compared to the wildtype. Together these results will shed light on the physiological function of HYP3. 187b HYP3 MPSSAQVLLCLAAVLAAAAATTAEAHSQCLDNPPDRSIHGRQLAEAGEVVHDLPGGLRAY AtHYP3-1 -----------------------MSGHQCTENPPDLDPT------SGSGHVEKLGNLDTY AtHYP3-2 -----------------------MSGPQCCENPPTLNPV------SGSGHVEKLGGLDAY : ** :*** . :*. : * * :* HYP3 VSGAASSSRAVVLASDVFGYEAPLLRQIADKVAKAGYFVVVPDFLKGDYLD---DKKNFT AtHYP3-1 VCGSTHSKLAVLLVPHVFGYETPNLRKLADKVAEAGFYAVVPDFFHGDPYNPENQDRPFP AtHYP3-2 VSGSAESKLCVLLISDIFGFEAPNLRALADKVAASGFYVVVPDYFGGDPYNPSNQDRPIP *.*:: *. .*:* .:**:*:* ** :***** :*::.****:: ** : :.: : HYP3 EWLEAHSPVKAAEDAKPLFAALKKEGK-SVAVGGYCWGGKLSVEVGKTSDVKAVCLSHPY AtHYP3-1 IWMKDHELEKGFEESKPIVEALKNKGITSIGAAGFCWGAKVAVELAKEKLVDATVLLHPA AtHYP3-2 VWIKDHGCDKGFENTKPVLETIKNKGITAIGAAGMCWGAKVVVELSKEELIQAAVLLHPS *:: * *. *::**:. ::*::* ::...* ***.*: **:.* . :.*. * ** HYP3 SVTADDMKEVKWPIEILGAQNDTTTPPKEVYRFVHVLRERHEVPYYAKIFQGVEHGFACR AtHYP3-1 RVTVDDIKEVNLPIAVLGAEIDQVSPPELVRQFEDILASKPQVKSFVKIFPRCKHGWTVR AtHYP3-2 FVNVDDIKGGKAPIAILGAEIDQMSPPALLKQFEEILSSKPEVNSYVKIHPKVSHGWTVR *..**:* : ** :***: * :** : :* .:* .: :* :.**. .**:: * HYP3 YNTTDPFAVKTAETALAYMVSWFNKHLN AtHYP3-1 YNENDPSEVEAAMEAHKDMLAWLIDYLK AtHYP3-2 YNIDEPEAVKAAEEAHKEMLDWFVTYIK ** :* *::* * *: *: ::: MPSSAQVLLCLAAVLAAAAATTAEAHSQCLDNPPDRSIHGRQLAEAGEVV 50 ************************* HDLPGGLRAYVSGAASSSRAVVLASDVFGYEAPLLRQIADKVAKAGYFVV 100 VPDFLKGDYLDDKKNFTEWLEAHSPVKAAEDAKPLFAALKKEGKSVAVGG 150 YCWGGKLSVEVGKTSDVKAVCLSHPYSVTADDMKEVKWPIEILGAQNDTT 200 TPPKEVYRFVHVLRERHEVPYYAKIFQGVEHGFACRYNTTDPFAVKTAET 250 ALAYMVSWFNKHLN 264 a Figure 5.1 Protein sequence alignment of HYP3 (a), AtHYP3 and AtHYP3-2 (b).(a) The HYP3 protein sequence contains a DLH domain (amino acids 45 to 220; highlighted in yellow). Predicted signal peptide (SignalP 4.1) are labeled by asterisks. Two Asn amino acids, that are predicted to be N-glycosylated with high potential, are indicated in red. 18800.511.522.5012345Absorbancesubstrateconcentration(mg/ml)LicheninglucanaseEndo(1,3)(1,4)glucanaseHYP300.511.52012345absorbancesubstrateconcentration(mg/ml)barleyglucanglucanaseEndo(1,3)(1,4)glucanaseHYP3abFigure 5.2 Enzymatic analysis of HYP3 (grey), Endo-(1,3)(1,4)--glucanase (red), and -glucanase (blue) using lichenin (a), barley -glucan (b), carboxymethyl cellulose (c), and xyloglucan (d) as substrates. The absorbance were measured at 575 nm. 18900.511.5012345absorbancesubstrateconcentration(mg/ml)XyloglucanglucanaseEndo(1,3)(1,4)glucanaseHYP3dFigure 5.2 (cont™d) 00.511.52012345absorbancesubstrateconcentration(mg/ml)CMCglucanaseEndo(1,3)(1,4)glucanaseHYP3c190Figure 5.3 Phenotype of athyp3-1, athyp3-2, and ZmHYP3 overexpression lines. (a) Knock-out mutants athyp3-1 and athyp3-2 did not show any phenotype. (b) The double mutant and overexpression lines also are indistinguishable from wild type. (a) WT athyp3-1 athyp3-2 (b) WT athyp3-1 X athyp3-2 Overexpression191HYP3GFPChlorophyllBrightFieldFigure 5.4 Subcellular localization of HYP3. Green fluorescent protein was fused to the C-terminus of HYP3. 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