- - By Allison Marie Barbaglia A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Cell and Molecular Biology Master of Science 2015 ABSTRACT - - By Allison Marie Barbaglia To cope with adverse environments, plants rely on detection followed by signaling leading to developmental responses. While many signals are local, others require phloem - mediated long - distant transport. We have identi fied lipids and their putative protein partners in the phloem. These proteins could function by releasing the lipid from the membrane, transporting the lipid, acting as a receptor, or acting as the signal itself. I focused on three lipid - binding proteins : a putative GDSL - lipase that may release lipids into the phloem; PIG - P, a potential receptor, and PLAFP, a protein of unknown function. These proteins respond to abiotic stress and bind lipids: GDSL is downregulated by drought mimics and binds diacylgy lcerol (DAG) and phosphatidic acid (PA). PIG - P is unaffected by stress and binds phosphatidylserine (PS), PA, and phosphatidylinositol - 4 - phosphate (PIP). PLAFP is induced by drought mimics/signals and binds PA. PA is a known secondary signal in response to drought and ABA. Since both PA and PLAFP increase in response to ABA and drought , we propose that PLAFP - PA could function in an ABA - related long - distance signaling path . Changes in PLAFP levels affect root and vasculature development, plant size, seed yield, and drought tolerance. Computational modeling suggests two possible PLAFP - PA interaction models: 1) PA binds solely with the head group where the lipid likely remains inserted in the membrane or 2) PA fits into the hydrophobic groove on the prote in where PA could be mobile. Initial experiments suggest specificity of PLAFP for ligands with unsaturated acyl chains further supporting model 2 and thus long - distance signaling. Copyright by ALLISON MARIE BARBAGLIA 2015 iv ACKNOWLEDGEMENTS I would first like to thank my mentor Susanne Hoffmann - Benning for all of her support, patience, and encouragement throughout the years. My labmates have also been extremely helpful with bouncing ideas and executing experiments and I would not be where I am without them. I thank all of my friends I have acquired at MSU. They have been very great support and have been there when things were not going well. Lastly, I need to thank my family because without them I would not even be where I am today. They have watched me throughout my long journey and have always loved, encouraged, and supported me even when I doubt ed myself and my abilities. I cannot thank them enough for all they have done to help me be successful and reach my goals. v TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ................................ .. vii LIST OF FIGURES ................................ ................................ ................................ ................................ . v iii KEY TO ABBREVIATIONS ....................................................................................... ................. ............ x CHAPTER 1 Introduction: Lipid - Binding Proteins and Their Role in Long - Distance Signaling and Response to Abiotic Stress ................................ ................................ ................................ ................................ ...... 1 1.1 Significance ................................ ................................ ................................ ...................... 1 1.2 Content and Function of Plant Phloem ................................ ................................ ............ 3 1.3 Identification of Lipids in Arabidopsis Phloem Exudates ................................ ................. 8 1.4 Identification of Lipid Binding Proteins from Arabidopsis Phloem Exudates .................. 17 1.5 Lipid/Phospholipid Signaling in Plants ................................ ................................ ............. 25 1.6 Intracellular Phosphatidic Acid Signaling in Plant Response to Abiotic Stress ................ 29 1.7 Putative Lipid - binding Prote ins in the Phloem and their Potential Function in Long - Distance Signaling ................................ ................................ ................................ .................. 35 REFERENCES ................................ ................................ ................................ ........................... 39 CHAPTER 2 Functional Characterization of GDSL - lipase, PLAFP, and PIG - P and their Potential Roles in Long - Distance Signaling ................................ ................................ ................................ .............................. 5 5 2.1 Introduction ................................ ................................ ................................ ..................... 5 5 2.2 Material and Methods ................................ ................................ ................................ ..... 63 2.2.1 Plant Growth ................................ ................................ ................................ ..... 63 2.2.2 Abiotic Stress Analysis ................................ ................................ ....................... 63 2.2.3 Gene Expression Analyses ................................ ................................ ................ 64 2.2.4 GUS Reporter Gene Construct, Arabidopsi s Transformation, and GUS Assay . 64 2.2.5 Fluorescent Reporter Gene Constructs for GDSL - lipase , PIG - P and PLAFP ...... 65 2.2.6 Protein Expression and Purification ................................ ................................ .. 66 2.2.7 Protein Lipid Overlay As say ................................ ................................ ............. 67 2.2.8 Liposome Binding Ass ay ................................ ................................ .................... 68 2.3 Results and Discussion ................................ ................................ ................................ ................. 69 2.3.1 GDSL - lipase, PLAFP, and PIG - P Bind Lipids ................................ ....................... 70 2.3.2 Protein Localization to the Cell Periphery ................................ ........................ 76 2.3.3 PLAFP Promoter Activity is Associate d with the Vascular Bundles .................. 79 2.3.4 Abiotic Stresses Effect Gene Expression ................................ ........................... 79 2.4 Conclusions ................................ ................................ ................................ ................................ .. 85 APPENDIX ................................ ................................ ................................ ................................ ........... 8 7 REFERENCES ................................ ................................ ................................ ................................ ....... 91 vi CHAPTER 3 PLAFP and it s Role in Drought Tolerance and Long - Distance Lipid Signaling ................................ .... 100 3.1 Introduction ................................ ................................ ................................ ..................... 100 3.2 Material an d Methods ................................ ................................ ................................ ..... 108 3.2.1 Plant Growth ................................ ................................ ................................ ..... 108 3.2.2 Gene Expression An alyses ................................ ................................ ................ 108 3.2.3 GUS Reporter Gene Construct, Arabidopsis Transformation and GUS As say .. 108 3.2.4 Fluorescent Reporter Gene Construct for PLAFP ................................ ............. 109 3.2.5 Abiotic Stre ss Analysis ................................ ................................ ....................... 110 3.2.6 Harvest of Phloem Exudates ................................ ................................ ............. 110 3.2.7 Lipid Analysis within Phloem Exudates ................................ ............................. 111 3.2.8 Protein Expres sion and Purification ................................ ................................ .. 111 3.2.9 Protein Lipid O verlay Assay ................................ ................................ ............. 112 3.2.10 Liposome Binding Assay ................................ ................................ .................. 113 3.3 Results and Discussion ................................ ................................ ................................ ..... 114 3.3.1 PLAFP Binds a Phloem Phospholipid ................................ ................................ . 1 14 3.3.2 PLAFP is Expressed in the Vasculature ................................ ............................. 115 3.3.3 PLAFP Localizes to the Cell Periphery ................................ ............................... 120 3.3.4 Abiotic Stresses Effect PL AFP Gene Expres sion ................................ ................ 122 3.3.5 Effect of Different PLAFP Expressio n Levels on Plant Phenotype .................... 12 5 3.3.6 PLAFP Expression Effe cts Drought Tolerance ................................ ................... 129 3.3.7 PLAFP Response to Abioti c Stress in Crop Plants ................................ ............. 13 3 3.3.8 Lipid Profile of PLAFP Displays High Abundanc e of PA in O verexpression Lines ................................ ................................ ................................ ........................... 13 5 APPENDI X ................................ ................................ ................................ ............................... 141 REFERENCES ................................ ................................ ................................ ........................... 146 CHAPTER 4 Conclusions and Future Perspectives ................................ ................................ ................................ 153 4.1 Summary and Conclusions ................................ ................................ ............................... 153 4.2 Future Perspectives ................................ ................................ ................................ ......... 15 6 vii LIST OF TABLES Table 1. 1: Hydrophobic Molecules in the Phloem and their Possible Function ................... ............ 1 0 Supplementary Table 2.1: Primer Table for Cloning, RT - PCR, and qPCR ............................. ............ 88 Table 3.1: Putative Lipid - Binding Proteins Identified via Proteomics .................................. ............ 103 Supplementary Table 3.1: Primer Table for Cloning, RT - PCR, and qPCR ............................. ............ 1 42 Supplementary Table 3.2: Lipid Species Used in Protein - Lipid Binding Assays .................... ............ 145 viii LIST OF FIGURES Figure 1. 1 : Schematic of Drought Throughout the Contiguous United States Over the Past 15 Years ................................ ................................ ................................ ................................ .............. 2 2 Figure 1.2: Lipid Profile of Phloem Exudates of Arabidopsis (Chloroform Phase) Using LC - MS Positive Ion Mode ................................ ................................ ................................ .............................. 16 Figure 1.3: Cleavage Sites for Phospholipases ................................ ................................ .................. 27 Figure 1. 4. Schematic Model Depicting a Potential Role for PLAFP in the Transcriptional R egulation of ABA Signaling ................................ ................................ ................................ ............... 31 Figure 2. 1: Protein - Lipid Binding Assays for GDSL - lipase ................................ ................................ . 71 Figure 2. 2: Protein - Lipid Binding Assays for PLAFP ................................ ................................ .......... 73 Figure 2. 3: Protein - Lipid Binding Assays for PIG - P ................................ ................................ ........... 74 Figure 2. 4: Transient Localization of GDSL - lipase (A) , PLAFP (B) , a nd PIG - P (C) using Florescent Protein Tags ................................ ................................ ................................ ................................ ....... 77 Figure 2. 5 : Transient Localization of Florescently Tagged - GDSL - lipase (A) , PLAFP (B) , and PIG - P (C) with Plasma Membrane Marker in Tobacco . ................................ ................................ ............. 7 8 Figure 2. 6 : PLAFP Promoter Activity in Two Week Old Arabidopsis Seedlings Using a GUS Reporter Construct ................................ ................................ ................................ ........................... 80 Figure 2. 7 : Hydroponic System Set - up for Abiotic Stress Treatment ................................ ............... 83 Figure 2. 8 : Effect of Abiotic Stress on GDSL - lipase , PLAFP, and PIG - P ................................ ............. 84 Figure 3. 1: Model of Possible Protein - Lipid Action in Long - Distance Signaling ............................... 105 Figure 3. 2 : Model Prediction for Possible PLAFP - PA Interactions ................................ .................... 116 Figure 3. 3 : PLAFP Promoter Activity Using GUS Reporter ................................ ................................ 117 Figure 3. 4 : Transient Localization of PLAFP in Tobacco ................................ ................................ .... 121 Figure 3. 5 : Effect of Abiotic Stress on PLAFP ................................ ................................ .................... 12 4 Figure 3. 6 : Effect of Soil Drought on PLAFP in Leaves and Roots ................................ ..................... 126 ix Figure 3. 7 : Effect of PL AFP Expression on Development ................................ ................................ .. 128 Figure 3. 8 : Effect of PLAFP Phe notype under Drought Conditions ................................ .................. 130 Figure 3. 9 : Seed Yield Comparison between PLAFP Lines ................................ ................................ 132 ................................ ................................ .................. ................................ .............................. Figure 3. 1 2 : Effect of ABA on PLAFP Expression in Maize ................................ ................................ 137 Figure 3.13 : Phosphatidic Acid Quantification in PLAFP Overexpression Line ................................ . 138 Figure 3. 1 4 : Phosphatidic Acid Lipid Abundance in PLAFP ................................ ............................... 140 x KEY TO ABBREVIATIONS GDSL: Glycine, Aspartic Acid, Serine, Leucine PLAFP: Phloem Lipid Associated Family Protein PIG - P: phosphatidylinositol N - acetyglucosaminlytransferase subunit P ER: endoplasmic reticulum TAG: triacylglycerol DAG: diacylglycerol PA: phosphatidic acid PS: phosphatidylserine PE: phosphatidylethanolamine PC: phosphatidylcholine PG: phosphatidylglycerol CL: cardiolipin PI: phosphatidylinositol PIP 1 : ph osphatidylinositol - 4 - phosphate PIP 2 : phos phatidylinositol - 4,5 - phosphate PIP 3 : phospha tidylinositol - 3,4,5 - phosphate PLD: phospholipase D PLC: phospholipase C PLA: phospholipase A FT: Flowering Locus T ACBP: Acyl - CoA Binding Protein 1 Chapter 1 . Introduction : Lipid - Binding Proteins and Their Role in Long - Distance Signaling and Response to Abiotic Stress Parts of this chapter are in press at Springer Publishing ; Barbaglia, A . and Hoffmann - Benning, S. (2016 ) Lipid signaling and its role in plant development and stress response. In: Subcellular Chemistry; Eds. Nakamura, Y and Li - Beisson, Springer; Invited book chapter; in press; expected print date 201 6 . 1.1 Significance T he world population is projected to increase by 2.4 billion, from 7.2 billion in 2013 to 9.6 billion in 2050, with most of the increase occurring within less developed regions ( World, 2013 ). Thus, the need for a high and reliable agricultural yield is increasing . A similar situa tion occurred back in the 1940s Norman Borlaug. Initiatives le d by Borlaug included the development of high - yielding variet ies of cereal grains (rice and wheat), the expansion of irrigation systems, modernization of management techniques, and the distribution of hybrid seeds, synthetic fertilizers, and pesticides to farmers, which resulted in increased agricultural crop yield worldwide (Hazel, 2009). The current increase in the world population leads to an encroachment of cities on arable land. In addition, changes i n the climate negatively affect crop yield . Thus, in order to continue to be able to provide sufficient food a nd fuel for the world, we need to develop plants that are able to produce greater yield and can survive under stressful conditions. One environmental stress that has a strong impact on agricultur al yield is drought. In the past few years, the United States has been experiencing extreme to exceptional drought in over 50% of the country (droughtmonitor.unl.edu ; NDMC - UNL ; Figure 1. 1 ) . While this situation has improved recently in certain regions of the countr y , it is still an obstacle farmers are currently battling in the United States , particularly in California, and around the world. An approach that can be taken to help find a solution 2 Figure 1. 1 : Schematic of D rought T hroughout the C ontiguous United States O ver the P ast 15 Y ears. For over a decade, the effect of drought has been plaguing the United States in a fluctuating manner. Different regions have been experiencing droug ht at different magnitudes, with 2012 being one of the worst years on record. Currently in 2015, California is experiencing the worst drought conditions in the country being at the highest grade of drought: exceptional. The U.S. Drought Monitor is joint ly produced by the National Drought Mitigation Center at the University of Nebraska - Lincoln, the United States Department of Agriculture, and the National Oceanic and Atmospheric Administration. Map s courtesy of NDMC - UNL. July 18, 2000 July 19, 2005 July 24, 2012 July 21, 2015 3 to this problem is to understand the long - distance signals responsible for changes in plant development and the resulting adaptations that lead to drought resistance. Unlike animals, plants cannot escape the unpredictability of their surroundings. To cope, they have evolve d mechanisms to detect changes in their environment, communicate these changes throughout the plant, and adjust their development as needed. One of the pathways plants use to communicate is the phloem. Appreciating how signaling compounds act and are tra nsported within the phloem is vital for understanding how plants adjust to their surroundings. 1.2 Content and Function of Plant Phloem Over the years, the understanding of phloem function has changed from being the mode for simple assimilate transport to a trafficking system for abiotic/biotic responses and developmental regulators (Guelette et al., 2012). The phloem is a channel - like system that consists mainly of two cell types: the sieve elements and companion cells. The sieve element is part of the vasculature that functions in the transport of photoassimilates and other materials throughout the plant. The companion cell is a metabolically active cell that transports meta bolites and proteins into the sieve element. The phloem is essential for the t ransport of photoa ssimilates, plant viruses, virus - induced silencing, defense and resistance against pathogen infection, and signaling of environmental conditions ( Lucas et al., 2013 ). It contains a variety of compounds such as small molecules (Chen et al ., 2001; Corbesier et al., 2003) , peptides/ proteins ( Haebel and Kehr, 2001; Hoffmann - Benning et al., 2002; Giavalisco et al., 2006; Lin et al., 2009; Benning et al . 2012 . ), mRNAs ( Ruiz - Medrano et al., 1999; Pallas et al., 2013; Hannapel et al., 2013), microRNAs ( Pant et al., 2008; Buhtz et al., 2010 ; Varkonyi - Gesic et al., 2010; Rodriguez - Medina et al., 2011 ), nucleic acids ( Citovsky and Zambryski, 2 000; Haywood et al., 2005; Ding et al., 2003; Yoo et al., 2004) , and lipids ( Madey et al., 2002; Behmer et al., 2011, 2013 ; Guelette et al., 2012; Benning et al., 2012; Tetyuk et al., 2013 ). All of 4 these components have made the phloem so complex, that phloem transport has been deemed the et al., 1998). Phloem transport is likely a regulated process dependent on protein - protein or protein - RNA interactions. Movement within the phloem occurs within the sieve elements, which have evolved to enhance flow by removing organelles and increasing the permeability of the cell walls (van Bel and Knoblauch, 2000). During early development, sieve elements contain nuclei, vacuole, and organelles; however , those cell compartments degrade later in development leaving the sieve elements with only the plasma membrane , a thin cytoplasm containing ER, phloem - specific plastids, and a few mitochondria (Turgeon and Wolf, 2009 ; van Bel and Knoblauch, 2000 ). As a r esult, it is generally assumed that sieve element proteins are synthesized within the companion cell and transported to the sieve element via plasmo desmata (Lucas et al., 2013) . The phloem can be difficult to work with due to its ability to reseal itself when wounded. There are four ma in methods used to collect phloem exudates, yet they work only in select species: through cuts of the petiole for example in cucurbits ( Balachandran, et al., 1997; Haebel and Kehr, 2001; Yoo et al., 2013; Ham et al., 2014 ) , shallow cuts or punctures of the stem or petiole of lupine, Perilla , cucurbits, Brassica napus , among other species ( Hoffmann - Benning et al., 2002; Giavalisco, et al., 2006; Marentes and Grusak, 1998; Walz et al., 2004 ) , use of aphids or other phloem - fee d ing insects in plants such as Arabidopsis, lettuce , sweet orange , maize, and apple ( Kloth et al., 2015; Hijaz and Killiny, 2014; Palmer et al., 2014; Ohshima et al., 1990; Varkonyi - Gesic et al., 2010; Will and van Bel, 2006 ), or EDTA - facilitated exudation in Arabidopsis, lupine, Perilla , canola, poplar , Chinese cabbage, and tobacco ( Chen et al. 2001; Hoffmann - Benning et al., 2002; Maeda et al., 2006; Madey et al., 2002 ; Dafoe et al., 2010; Behmer et al., 2011 ) . Since Arabidopsis is more fragile than other plants, cutting of the petiole/puncturing of the stem is not sufficient to obtain a reasonable amount of 5 phloem exudate for a dependable analysis . Additionally, while utilizing aphids will suffice to harvest phloem sap, a laser is required making this method more expensive . Thus, using EDTA as a means to retrieve phloem sap from Arabidopsis leaves is most efficient ( Tetyuk et al., 2013 ). EDTA prevents the phloem from sealing itself, thus allowing for more sufficient exudate co llection for the analysis of proteins. Thus, o btaining exudates in this fashion allows for adequate analysis of proteins, small molecules, lipids, and RNAs (Guelette et al., 2012). The phloem has been known to play an important role in pathogen defense. V iruses have been shown to utilize the phloem as a means to infect the entire plant (Waigmann et al., 2004; Gopinath and Kao, 2007); however, plants employ a universal response to infection, making long - distance signaling within the plant very important. When a plant is infected by a pathogen, it first responds by local programmed cell death such as hypersensitive response and then induces systemic acquired resistance (SAR) through generation of mobile signals, accumulation of the defense hormone salicyli c acid (SA) , and secretion of the antimicrobial pathogenesis - related (PR) proteins (Fu and Dong, 2013). Thus, SAR is a mechanism of defense that allows the plant to further prepare and respond/protect itself more rapidly for future pathogen attacks. M any of the compounds involved in SAR , such as SA, a zelaic acid ( AzA ) , and a g ylcerol - 3 - phosphate* derivative ( G3P* ) have been identi fied within the phloem exudates. O ther p lant proteins such as systemin and plant hormones such as jasmonic acid (JA), both which provoke a pathogen response, have also been identified and transported within the phloem in response to pathogen infection (Schilmiller and Howe, 2005; Truman et al., 2007) further supporting the importance of long - distance signaling within the plant . Other components of the phloem, such as the protein flowering locus T (FT; Corbesier et al., 2007; Jaeger and Wigge, 2007; Mathieu et al., 2007; Turnbull, 2011), mRNAs (Bel1; Lin et al., 2013 ), and microRNAs ( reviewed in Kehr, 2012; miR399; Buhtz et al., 2010 ) have been shown to have a role 6 in long - distance transport within the phloem . FT , a universal signaling molecule for flowering plants, has been shown to act as a phloem - mobile florigen hormone. FT induces flowering in response to a photoperiodi c stimulus , but also has broader roles in seasonal developmental checkpoints including bud dormancy and tuberization, and in the reg ulation of meristem determinacy and compound leaf development ( Corbesier et al., 2007; Turnbull, 2011 ; Notaguchi et al., 200 8; Nakamura et al., 2014 ). FT encodes a 20 kDa protein that interacts with the basic region/leucine zipper ( bZIP ) transcription factor FD, which is preferentially expressed in the shoot apex and activates the transcription of meristem identity genes such as APETELA1 (AP1; Notaguchi et al., 2008; Nakamura et al., 2014). For more than 40 years, RNA has been identified in phloem exudates (Kollmann et al., 1970; Ziegler and Kluge, 1962 ), even though for a while it was believed their presence was due to contaminations brought about by sample collection. Despite this, there have been s tudies that have shown phloem samples contain a diverse set of RNAs, including viral RNAs, endogenous mRNAs, tRNAs, and a portion of small RNAs (i.e. si RNAs, miRNAs) in the size range of 20 - 26 nucleotides ( nt; reviewed in: Kragler , 2010; Kehr and Buhtz , 2008; Kehr, 2012 ; Asano et al., 2002; Vilaine et al., 2003; Omid et al., 2007; Deeken et al., 2008; Gaupels et al., 2008 ). One of these mobile RNAs is StBEL5, which encodes a BEL1 - like transcription factor that is expressed in potato ( Solanum tuberosum ; Banerjee et al., 2006). BEL1 - like transcription factors are members of the three - amino - loop - extension (TALE) superclass that interact with KNOTTED1 (KN1) - like partners to regulate numerous aspects of development (Lin et al., 2013) . These transcription factors possess very high levels of sequence conservation in the DNA - binding region, known as the homeodomain, and contain thre - helices similar to the bacterial helix - loop - helix motif (Kerstetter et al., 1994). In potato, the BEL1 - like transcription factor, StBEL5, and its Knox protein partner regulate tuber formation by targeting genes that control growth. The StBEL5 promote r is induced by light in leaves, which leads to the mRNA 7 being transported to stolons and roots under short - day conditions . This results in enhanced translation in the stolon tip or root followed by StBEL5 binding to a Knox protein partner and subsequent activation of the transcription and regulation of select genes via binding to the tandem TTGAC motif of the target promoter. This regulation leads to enhanced tuber and root growth (Banerjee et al., 2006). RT - PCR and grafting experiments demonstrated tha t StBEL5 transcripts are present with in the phloem and move across the grafting junction to the stolon tips, the site of tuber induction (Banerjee et al., 2006). The authors found that this RNA movement begins in the leaf veins and petioles, is activated under a short day photoperiod, and is regulated by the untranslated regions of the transcript. In conjunction, Lin et al. ( 2013 ) recently demonstrated the StBEL5 RNA , in addition to moving from leaves to stolon, can also move from the leaves to the roots . This movement is induced initially by light and occurs regardless of photoperiod length. Both groups found that this movement resulted in enhanced tuber production. In addition to mRNAs, micro RNAs (miRNAs) have also been identified within phloem exudates. MicroRNAs are normally around 21nt in length and function in RNA silencing and post - transcriptional regulation of gene expression by downregulating existing target mRNAs. Similarities of miRNAs identifie d within phloem samples have been observed among different plant species (Yoo et al., 2004; Buhtz et al., 2008, 2010; Varkonyi - Gasic et al., 2010). For example, miR156, miR159, miR166 and miR167 were detected in all plant species analyzed and may be membe rs of a group of miRNAs conserved within the phloem (Kehr, 2012). They regulate development of vegetative and floral organs ( miR159, miR160, miR164, miR166, miR167, and miR319 ; Mallory et al. , 2004; Kehr, 2012) or signal the nutrient status of the plant ( i.e. miR395, miR397, miR398, miR399, miR408, miR2111 ; Kehr, 2012). For instance, miR399 is a long - distance signal within the phloem induced under phosphate starvation and may act as a long - distance second messenger (Buhtz et al., 2010). 8 D espite all of the se studies exhibiting the identification of various compounds/molecules within the phloem , few studies have described lipids within the phloem. There are many lipid candidates that could have some function or role within the phloem such as JA (Truman et al., 2007) and phosphatidic acid ( PA ; Benning et al., 2012 ) . A ll have been identified within the phloem due to an induced response or stress condition. Thus, even though there is little knowledge on lipids and lipid - binding proteins within the phloem, th ey may have a substantial influence in signaling. 1.3 Identification of Lipids in Arabidopsis Phloem Exudates Within the phloem exudates of Arabidopsis , the lab has identified over 121 distinct metabo lites including 16 fatty acids/ esters using G C - MS ( Guelette et al., 2012). Similar findings have been reported previously when small amounts of lipids and large amounts of free fatty acids were identified within the phloem of canola (Madey et al., 2002). Analogous to their findings, we have encountered f atty acids of short - and medium - chain length and saturated fatty acids, including those that have odd - number carbon chains. Fatty acids of these lengths are not typically seen within membranes of plants and therefore, could either be specific to the phloe m or a result of degradation . One reason for this potential specificity could be that the fatty acids are transported into the phloem by the means of a lipid - binding protein or other transporting agent. Some studies suggest that these phloem fatty acids are a component of the plant pathogen response (Sanz et al., 1998; Hamberg et al., 1999). The hairpin gene HrpN, from the bacteria E. amylovora along with the new gene, pathogen - inducible oxygenase (piox) , which was identified from the HR analysis of tobacco leaves that had been inoculated with the HrpN protein , displayed induced e xpression of piox in response to various compounds known to act as or to generate c ellular signals mediating the plants response to p athogen infection. piox was rapidly induced by SA and JA, common pathogen defense signaling molecules, and was correlated with the production of active oxygen species (AOS) such as superoxide 9 radicals or hydrogen peroxide ( H 2 O 2 ) . T he recombinant enzyme, PIOX, from tobacco and its Arabidopsis homolog s has been suggested to work together with aldehyde dehydrogenase and NAD + to provide a pathway/mechanism for the degradation of fatty acids into shorter chain s (Sanz et al., 1998; Hamberg et al., 1999). Other lipophilic compounds previously found in the phloem are plant hormones such as auxin (IAA), abscisic acid (ABA), gibberellins (GA), c ytokinin (CK) ; SAR/plant defense signaling molecules including SA, d ehydroabietinal (DA), AzA, G3P*, and JA ; and phospho/ glycerolipids e.g. d iacylglycerols (DAGs), triacylglycerols (TAGs), phosphatidylinositol (PI), phosphatidylcholine ( PC ) , and PA ( Table 1. 1 ). All of these hormones help facilitate different aspects of plant development : Gibberellins are well kno wn for the ir role in regulating plant growth and development, including seed germination, stem elongation, leaf expansion, trichome development, pollen maturation and the induction of flowering (Achard and Genschik, 2009; reviewed in Davière and Achard, 2013 ). One of the first studies identifying gibberellin - like substances within the phloem was from phloem exudates obtained by the honeydew of aphids that fed on three different higher order plants: dandelion ( Taraxacum officinale ), broad bean ( Vicia faba ), an d willow ( Salix viminalis ). The honeydew was extracted from the aphids, chromatographed, and tested in various bioassays to confirm GA - like substances were present (Hoad and Bowen, 1968). In willow, t he aut hors found the amount of phloem sap collected wa s dependent on the day length, with more exudates collected from plant grown under long days, and less collected under short days. Due to the small volume obtained from honeydew, o ne caution to consider when interpreting data from it is the possibility fo r contamination or alteration in the metabolism of compounds during the passage of phloem sap through the gut of the aphids (Hoad et al., 1993). Thus, Hoad and his colleagues collected a larger volume of phloem sap by puncturing the stem or fruit from whi te lupine , cowpea, and castor bean and analyzed the phloem samples for 10 gibberellins using GC - MS (Hoad et al., 1993) . The results confirmed there is a wide variety of GAs within the different higher plant species. More recent studies have shown GAs to be involved in long - Table 1.1: Hydrophobic Molecules in the Phloem and their Possible Function Name Category Long - distance Function Example - Structures Indole - acetic acid (IAA) Aromatic ring with carboxylic acid Root growth; apical dominance; vascular bundle development Abscisic acid (ABA) Isoprenoid (Sesquiterpene) Abiotic stress signal in response to drought, cold, salt stress Gibberellins (GA) Isoprenoid (Pentacyclic diterpene) Germination; Plant growth Cytokinin Adenine derivatives; often isoprenylated Vascular bundle development; root development; cell division/ differentiation Salicylic acid (SA) Monohydroxybenzoic acid Pathogen defense; Systemic acquired resistance (SAR) Dehydroabietinal (DA) Isoprenoid (Abietane diterpenoid) SAR; Flowering ? Azelaic acid Dicarboxylic acid SAR Glycerol - 3 - phosphate* Unknown G - 3 - P - derivative SAR Jasmonic acid/oxylipins Oxylipins SAR; wounding; senescence; drought response 11 Diacylglycerols Diacylglycerols Precursor for PtdOH through the PLC - DGK pathway Triacylglycerols Triacylglycerols Storage lipid Phosphatidyl choline Phosphoglyerolipid Precursor for PtdOH through PLD pathway Phosphatidyl inositol Phosphoglyerolipid Abiotic stress; light - and gravitropic signaling; vesicle trafficking Phosphatidic acid Phosphoglyerolipid Drought, salt, osmotic, wounding, pathogen? 12 distance signaling in Arabidopsis. Through multiple grafting experiments between wildtype and GA - deficient mutants, the authors were able to determine that the gibberellin precursor GA12 was the transmissible signal through the graft (Regnault et al., 2015a,b). Auxins are mobile compounds that have been identified within the phloem of more than 14 species (Hoad, 1995). Auxin activity was first identified within the phloem by Hüber et al. ( 1937 ) . They collected exudates from the bark of three different tree species and determined there was auxin - like activity present using the Avena - curvature test (Hoad, 1995). More r ecent articles have shown auxin activity in phloem exudates in other plant developmental stages. For example, a uxin as well as cytokinin participates in the signaling and regulation of xylem and phloem development (Lucas et al., 2013). In addition, auxin participates in the regulation of root growth via the presence or absence of phosphate (López - Bucio et al., 2002; Chiou and Lin, 2011) and is involved in SAR (Truman et al., 2010) . Auxin has also been shown to move along two different pathways in the root apex: one phloem - facilitated and the other mediated by AUX1, a permease that promotes acropetal and basipetal auxin transport within inner and outer tissues of the root apex, res pectively after phloem unloading (Swarup et al., 2001). ABA is transported in the phloem in response to many stresses including drought. ABA was first identified by spectropolarimetric analysis of phloem sap collected from the honeydew of Tuberoluchnus sulignus aphids feeding on willow as (+) - abscisin II (+) - dormin (Hoad 1995 ). L ater studi es noted that the level of ABA within the phloem exudates increased when water was scarce as well as when the plants exposed to other stresses. ABA has been shown to move from source to sink leaves, which suggests ABA moves within the phloem and may participate in phloem loading and unloading (Hoad, 1995). A more detailed examination of ABA and its functional role in lipid signaling will be discussed in subsequent cha pters. 13 SA and JA play a role in the pathogen defense response and SAR , however, neither has been shown to be the phloem mobile signal for SAR. As stated previously, there are many compounds that contribute to SAR, such as MeSA , but which one acts as the transmittance signal is currently unknown (Truman et al., 2007 ). There has been some evidence supporting JA as a mobile signal for SAR; however it was not found in petiole exudates that had SAR - inducing ability ( Chaturvedi et a l., 2008 ). JA, but not SA, has been shown to rapidly accumulate within the phloem exudates from leaves that have been inoculated with an avirulent strain of Pseudomonas syringae . Despite this accumulation, studies support JA most likely act ing as a media tor rather than the mobile signal for long - distance signal movement. DA , a diterpenoid metabolite , aids in systemic expression of PR genes in uninfected , distant tissue in order to protect the rest of the plant from secondary infection . Additionally d uring SAR, G3P levels increase in the infected leaf, petiole exudates, and in distal, non - infected leaves, however, when radiolabeled G3P was applied to leaves, no movement was observed. Instead, an unknown compound was identified with the radiolabeled ta g in a distal leaf (Chanda et al., 2011). This suggests that rather than G3P being the mobile signal for SAR, an as - of - yet unidentified G3P derivative (G3P*) may perform that function (Fu and Dong, 2013) . AzA is a small dicarboxylic acid found within the phloem exudates of infected plants (Jung et al., 2009). It may aid in increasing levels of G3P in SAR, but when applied to plants, only a small amo u nt of the labeled compound could be recovered in more distant leaves making its ability to move long dista nces debatable. Other lipophilic signals moving through the phloem are oxylipins. Oxylipins are oxygenated metabolites of polyunsaturated fatty acids that are derived from the polyunsaturated acyl groups of galactolipids found in chloroplast membranes (Ho we and Schilmiller, 2002; Feussner and Wasternack, 2002; Yan et al., 2013). Well - known members of this group are the jasmonates. Jasmonates are 14 formed through a LOX - catalyzed peroxidation of the trienoic fatty acids to form 13 - hydroperoxide and are furth er modified through the allene oxide synthase (AOS) pathway or the hydroperoxide lyase (HPL) pathway (Chehab et al., 2008). In the AOS pathway, 12 - OPDA (cis - 12 - oxo - phytodiern ic acid) and JA are produced, while in the HPL pathway, aldehydes and their volat ile relatives (green leaf volatiles , GLV) are generated. Members of the jasmonat e family include JA, methyl - jasmonate (Me - JA), jasmonate - isoleucine (JA - Ile), and OPDA. These compounds participate in the regulation of plant growth and development as well as in response to biotic and abiotic stresses. OPDA, in addition to being the precursor for JA, is also a signaling m olecule (Dave and Graham, 2012); i n response to wounding and herbivory, JA is synthesized and travels throughout the plant to stimulate a systemic defense response (Truman et al., 2007; Thorpe et al., 2007). Lastly, JA can crosstalk with other hormones such as ethylene, SA, ABA, and GA in stress signaling pathways ( For a summary see Yan et al., 2013 ). There are many glycerolipids within the phloem . Within our phloem samples, phospholipids, di - and triacylglycerols were detected . Analogous to animal systems, lipids and lipid - binding proteins may play an important role in stress and developmental signaling in plants. Lipids, specifically ph ospholipids, are found mainly within the cell membrane where they provide structure as well as act in the regulation of plant development and environmental interactions (Wang and Chapman, 2013; Wang, 2004; Xue et al., 2009). Phospholipid signaling cascade s are characterized according to the phospholipase that generate s lipid messen gers such as PA, DAG , diacylglycerol pyrophosphate (DGPP), lysophospholipids, free fatty acids, oxylipins, PI, and inositol phosphates (IPs; Zhu, 2002; Wang, 2004 ; Wang et al., 2 007). Unlike structural lipids, signaling lipids are present in minute amou n ts , but in response to certain stimuli, their levels can drastically increase. T heir 15 accumulation/response is transient since the signal is quickly downregulated. Thus, signaling lipids have a higher rate of turnover than other structural lipids (Testerink and Munnik, 2005). More complex lipids identified within the phloem exudates were characterized using thin layer chromatog raphy and liquid chromatography mass spectrome try using multiplexed collision - induced dissociation (LC - MS - CID; Figure 1. 2 ). Lipids were detected at retention times that differed from those found within the leaf, indicating these phloem lipids are partly distinct from lipids of other parts of the plan t. To identify the types of lipids found in the phloem exudates, MS analysis using multiplexed CID displayed the presence of numerous classes of PC, PA , DAG, and TAG , as well as, PI within the exudates (Guelette et al., 2012). Lipids such as PA, PI, a nd its phosphates (PIPs) are well - known secondary messengers within plant cells (Wang, 2004; Munnik and Testerink, 2009), but very little is known regarding their role in long - distance signaling. Analysis of Arabidopsis phloem exudates led to the identifi cation of several glycerolipids in the ph l oem including PA, PC, PI, DAG, and TAG (Guelette et al., 2012; Benning et al., 2012; Tetyuk et al., 2013). Potential functions for these lipids are as follows: 1) Lipids could be transported through the phloem as energy carriers, assimilates, or building blocks; 2) Lipids could be the product of periodic membrane turnover within the sieve element; 3) Phloem lipids could serve as long - distance signals. While all three are viable options, the last possibility seems most plausible. For instance, many tissues are able to make their own lipids, thus, transport as energy carriers or building blocks would be inefficient (Ohlrogge et al., 1995). Furthermore, phloem lipids being a product of turnover seems unlikely since when lipids from the leaf and phloem samples were compared, their lipid profiles were distinct, with several lipids being unique to the phloem (Guelette et al., 2012). PA and DAGs are well - known secondary messengers within the plant and it has been sugge sted they may participate in osmotic stress and pathogen response (Testerink and Munnik, 2005, 2011). The fact 16 [M+H] + [Phospho choline] + [M+H] + : PC 34:3 Positive ion mode (Panel 1: 35V; panel 2: 80V [M+formate] - [PC 34:3+ formate] - - [C18:3] - [C16:0] - Negative ion mode (Panel 1: 35V; panel 2: 50V; panel 3: 80V) PC 36:5 PC 36:6 PC 34:2 PC 34:3 PA 34:2 TAGs N/D PI OPDA Figure 1. 2: Lipid Profile of Phloem Exudates of Arabidopsis (Chloroform Phase) Using LC - MS Positive Ion Mode. Mass spectra generated at aperture 1 voltages of 35, 50, and 80V in both positive (B) and negative (C) ion mode to show fragmentation of the lipid at a ret ention time of 16.1 min (Guelette et al., 2012) and illustrate lipid identification. PC, phosphatidylcholine; PA, phosphatidic acid; TAG, triacylglycerol; DAG, diacylglycerol; *, detergent; N/D, not determined due to multiple peaks in the spectra. Chromatograms are representatives of three biological replicates for each sample. Loss of methyl - formate; : 60 D AGs 17 that lipids are present within phloem exudates implies they are not only important in intracellular signaling, but may be involved in long - distance signaling as well. Additionally, Buseman et al. ( 2006 ) , proposed that galactolipids, which a re located in the chloroplast, are the substrate for the production of OPDA, suggesting phloem lipids have functions specific to the phloem, making t hem unlikely to be products of non - specific membrane turnover. OPDA is not only the precursor for the phloem - mobile signal JA, but can also be found in the phloem itself ( Furch et al., 2014 ). This suggests that phloem lipids not only could be transported into the phloem by proteins, but also may be synthesized within the phloem as well. The hypothesis that phloem lipids can serve as long - distance signals is further supported by observations in other biological systems, such as the bloodstream where signaling and storage lipids are transported while bound to proteins. A more detailed description of how and why lipids could act as long - distance developmen tal signals is discussed later i n this chapter. 1.4 Identification of Lip id Binding Proteins from Arabidopsis Phloem Exudates Analysis of phloem exudates has allowed for the identification of various proteins within several plant species. Some of these experiments were to study particular proteins or look at specific functions ( Chen et al., 2001; Fröhlich et al., 2012; Ishiwatari et al., 199 5 ; Vincill et al., 2013; Balachandran et al., 1997 ), while others attempted to obtain a more comprehensive protein profile ( Carvallo et al., 2011; Haebel and Kehr, 2001; Giavalisco et al., 2006; Kloth et al., 2015; Lin et al., 2009 ). Using a n EDTA - facilitated collection of phloem exudates followed by SDS - PAGE and LC - MS analysis, the lab w as able to identify 65 proteins within Arabidopsis phloem sap . Among those proteins, 14 were involved in carbohydrate metabolism, 16 identified with stress, pathogen, or hormone response, six were nucleotide - , DNA - , or RNA - binding proteins, 11 were associated with the plastid, 11 contributed to protein modification, folding, turnover and stability, and pro tein - protein 18 interaction, and 11 were characterized as putative lipid/fatty acid - binding proteins (Guelette et al., 2012). The proteins of most interest were the last group where the predicted function of the proteins detected ranged from lipid/fatty acid metabolism, binding, to signaling (Guelette et al., 2012; annexin, GRP17/oleosin, DIR1/ LTP, aspartic protease, long - chain fatty acid CoA ligase, put. GRP7, two putative lipases, hypothetical lipid - binding protein, 14 - 3 - 3 protein, major latex protein, Bet v - 1 allergen, and PLAFP). Only a few putative lipid - binding proteins found in the phloe m have been studie d further : DIR1, acyl - CoA binding protein (ACBP) , annexin, Flowering Locus T (FT) , and 14 - 3 - 3 proteins . Currently, four of these lipid binding proteins are being studied by other groups in the context o f long - distance lipid signaling : DIR1, ACBP, FT , and 14 - 3 - 3 proteins . DIR1 is a lipid transfer protein (LTP) that has been shown to play a role in long - distance signaling associated with SAR in Arabidopsis and tomato (Carella et al., 2014; Champigny et al., 2013; Shah and Zeier, 2013; La scombe et al., 2008; Maldonado et al., 2002; Mitton et al., 2009). Acyl - CoA binding protein , such as RPP10 (ACBP1 - 6 in Arabidopsis) , has been identified in rice phloem exudates (Suzui et al., 2006; Hayashi et al., 2000) and has been linked to the vascular bundles in Arabidopsis (Zheng et al., 2012). FT is a signaling molecule involved in the initiation of flowering (Turnbull, 2011; Corbesier et al., 2007). 14 - 3 - 3 proteins have been shown to have a response to drought mimic polyethylene glycol (PEG; He et al., 2015 ) and interact with the FT receptor (Taoka et al., 2011). Even though all of these proteins have been identified within the phloem, very few have been connected to phloem lipids . B elow is a brief discussion of the putative lipid - binding protein s that have been identified in the phloem , the roles they play in plant growth and development , and the lipid, if any, they have been identified to bind . On e phloem lipid binding protein is Defective in Induced Resistance 1 ( DIR1 ) , a lipid transfer protein (LTP) that plays a role in long - distance SAR signaling in Arabidopsis a nd tomato by binding 19 AzA (Fu and Dong, 2013) and transporting it to distal, non - infected t issue to help protect and prepare those tissues from potential future i nfections. Recent studies have shown DIR1 expression in the leaf vasculature (Champigny et al., 2011) and i n Plasmodesmata - Located Protein PDLP1 and PDLP5 overexpression lines, indicating DIR 1 move s between cells via plasmodesmata during SAR in Arabidopsi s (Carella et al., 2014 ) . In addition, previous studies have suggested DIR1 is required for the accumulation and/or systemic movement of a SAR - inducing factor since when pathogens were applied to the dir1 mutant, which lacks SAR activity, the distal leaves were not protected from infection (Maldonado et al., Another lipid - binding protein that has been identified within the phloem is Acy l - CoA Binding Protein (ACBP) . These proteins have been identified in the phloem exudates of rice and have been linked to the vasculature in Arabidopsis. A member of this family, ACBP6, has been shown to bind PC, and when overexpressed confers enhanced toleranc e to freezing conditions (Chen et al., 2008). This suggests that ACBP6 may play a role in medi ating freezing stress response - associated phospholipid metabolism in Arabidopsis. Furthermore, due to its ability to bind PC , it is possible ACBP6 could be involved in the intracellular transport of PC in the cytosol (Chen et al., 2008). The protein Annexin has been shown to bind phospholipids and participates in intracellular Ca 2+ signaling and callose formation (Andrawis et al., 1993; Gerke and Moss, 2002). Even though annexin has been found within phloem exudates, there are not many studies about it in the phloem pertaining to long - intracellul ar signaling ( Wang et al., 2015 ; Davies, 2014 ) . S mall, 15 - 26 kDa alkaline proteins known as GRP17/oleosins , that along with phospholipids surround oil bodies composed mostly of TAGs. Together with phospholipids, oleosins provide s tructure to oil bodies and prevent them from combining via steri c hindrance (Tzen and Huang, 20 199 2). Glycine - rich protein 17 (GRP17) is expressed predomina n tly in anther and late - stage flower development (de Oliviera et al., 1993). GRP17 is required for p ollination in Arabidopsis since the mutation, grp17 - 1, results in the removal of a single oleosin - domain protein from the Arabidopsis pollen coat (Mayfield and Preuss, 2000). Oleosins are present in oil bodies where they act in oil body biogenesis, oil bo dy stabilization , and maintenance of an appropriate size and surface/volume ratio for gradual oil body mobilization ( Bhatla et al., 2009 ). Similar to oleosins, caleosins are believed to play a role in lipid trafficking and membrane expansion (Lin et al., 2005) . They may also be involved in the docking of peroxisomes to lipid bodies and degradation of storage lipids during seed germination ( Naested et al., 2002; Poxleitner et al., 2006 ). L ipase s are the principle enzyme involved in oil body mobilization during seed germination. They catalyze the cleavage of fatty acids from the glycerol backbone. On the other hand, lipoxygenase activity can lead to oxygenation of storage TAGs to their corresponding hydroperoxy derivatives in some oilseeds (F eussner et a l., 2001) . A subfamily of endopeptidases , the aspartic proteases , has been used in various applications such as a milk coagulating enzyme. They have molecular weight s in the range of 30 - 45 kDa and contain one or two conserved aspartic acid residues at their active site. These proteins are most active and stable at acidic pH (pH 3 - 5) and are specifically inhibited by Pepstatin A (Hsiao et al., 2014). The Arabidopsis gene, Constitutive Disease Resistance 1 (CDR1) was identified via T - DNA activation tagging . This gene encodes an apoplastic aspartic protease that, when overexpressed, lead s to enhanced resistance to bacterial pathogens ( Xia et al., 2004 ). When a pathogen attacks, CDR1 accumulates in the intercellular fluid. Additionally, upon induction, CDR1 generates a small signal an endogenous peptide elicitor that stimulates SA - dependent disease resistance responses. Thus, the authors proposed CDR1 facilitates a peptide signal scheme involve d in the activation of inducible resistance mechanisms ( Xia et al., 2004). 21 A family of lipid - binding proteins that are conserved regulatory molecules expressed in all eukaryotic cells are the 14 - 3 - 3 proteins . They are acidic protein s approximately 30 kDa in size and exist primarily as homo - or heterodimers. These proteins have the ability to bind a diverse set of signaling proteins, including kinases, phosphatases, and transmembrane receptors, and thus, can be an important factor i n a wide range of regul atory processes (Yaffe, 2002). These proteins bind specifically to phoshoserine /threonine receptors, and in general, participate in the activity of bound ligands, bound ligands associated with other cellular compounds, and the lo calization of cargo bound to them (Yaffe, 2002). In addition to playing a role in regulating plasma membrane H + - ATPase activity (Palmgren, 2001) , which affects root growth and water acquisition, they also may participate in carbohydrate metabolism and transport, which also influen ces root growth management under water stress conditions (Comparot et al., 2003; Gowda et al., 2011) . In a recent study, the plant 14 - 3 - 3 protein, GRF9, displayed enhanced overall plant and root growth when overexpressed and decreased growth when mutated when subjected to water stress via PEG (He et al., 2015). They found that GRF9 is important in water stress response by regulating phloem sucrose transport in order to allow for more carbon to be transferred from the shoot to the root. Thus, it was concluded that GRF9 could be simultaneously regulated by the local and systemic response to water stress. In the shoot, GRF9 facilitates shoot carbon distribution and increases phloem sucrose transport to promote root growth (systemic). In the root, GRF9 activates the root plasma membrane H + - ATPase to release more protons (local). These two processes allow for the plant to maintain root growth under water deficient conditions (He et al., 2015). 14 - 3 - 3 proteins have also been shown to interact with the FT receptor. Taoka et al. ( 2011 ) show 14 - 3 - 3 proteins act as intracellular recep tors for Hd3a ( the rice ( Oryza sativa ) homolog to florigen), which is transported from leaves to the shoot apex. At 22 t he apex, the Hd3a - 14 - 3 - 3 complex enters the nucleus to bind OsFD1 which leads to the transcription of OsMADS15 (a homolog to AtAP1) result ing in flower initiation ( Taoka et al., 2011 ). A component of florigen , flowering locus T (FT) , is involved in the initiation of flowering. FT transmits photoperiodic flowering signals from leaf companion cells th r ough the plasmodesmata to the sieve elements where they are transported to the shoot apex and binds specifically to phosphatidylcholine (PC; Nakamura et al., 2014). FT protein that is expressed but immobilized in companion cells is unable to promote flowering unless it is transported to the phloem sieve elements by FT - INTERACTING PROTEIN 1 (FTIP1), an ER - localized protein. Once in the sieve elements, FT travels up the phloem to the shoot apex where it interacts with FD, a bZIP transcription factor, which leads to the activation of APETALA1 (AP1) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) to initiate flower development (Nakamura et al., 2014). In addition to FD, FT binds to PC at the shoot apex. The authors found that the levels of specific molecular species of PC vary depending o n the time of day, and concluded FT preferred to bind species of PC that were predominantly present during the light period (PC species containing less unsaturated fatty acids: 36:4, 36:3. 36:2, 36:1, 34:1) in order to promote flowering at the right time. The protein family , major latex proteins, was first identified in the latex of opium poppy. These proteins are only found in plants; 24 of which have been found in Arabidopsis. Other plants that contain t hese proteins include peach, strawberry, melon, cucumber, and soybean (Lytle et al., 2009). While their function is still unknown, they have been shown to be involved in fruit and flower development as well as pathogen defense responses. One of these proteins from Gossypium hirsutum , was shown to cont ain several potential cis - elements for response to biotic and abiotic stress such as a salt stress , cold stress, salicylic acid, wounding, and pathogen elicited attacks (Chen 23 and Dai, 2010). Additionally, these proteins have been characterized as members of the Bet v 1 protein superfamily on the basis of discreet similarities in sequence (Lytle et al., 2009). The Bet v 1 alle r gen proteins that belong to a ubiquitous (in angiosperms) family of pathogenesis - related (PR) proteins affiliated with birch pollen allergy, a specific cluster of food hypersensitivities where patients experience symptoms such as itching or burning sensation in the lips, mouth, ear canal, and/or pharynx (Ebner et al., 199 5 ; Karamloo e t al., 2001). In 2001, Karamloo and colleagues developed a recombinant protein, r Pyr c 1, which expresses an allergen from pear and is a new member of the Bet v 1 allergen family. Having this protein available will allow for further investigation of its allergenic properties, the determination of t he extent by which the major allergen contributes t o the IgE binding capacity of a fruit , and promote studies on the structure and cross - reactive epitopes of pollen - related food allergens (Karamloo et al., 2001). In a 2003 study by Markovi - Housley and colleagues uncovered the crystal structure of Bet v1I , a hypoallergenic isoform of the major birch pollen allergen Bet v 1 . They found that the structure displayed a stable, high affinity complex between Bet v 1l and two molecules of deoxycholate, a steroid m olecule very similar to plant brassinosteroid s (B Rs) . A mass spectrometry study revealed Bet v 1l acted more as a general, rather than specific, steroid carrier due to the specific non - covalent interaction between Bet v 1l and two BRs , brassinolide and 24 - epicastasterone. Thus, since both Bet v 1 and b rassinosteroids are highly expressed within the pollen, it was concluded that Bet v 1 proteins could fulfill a BR - carrier function - Housley et al., 2003). A putative lipase has also been identified within phloem sap. A lipase is an enzyme that fa cilitates the hydrolysis of lipids. Lipases are primarily found in tissues utilized for storage such as oilseeds. In addition, these enzymes have high substrate specificity and can also catalyze synthesis reactions (Seth et al., 2014). Similar to the mo re specific GDSL lipase below, lipases tend to respond 24 to various plant hormones such as SA in terms of pathogen attack and defense, as well as bind these hormones to stimulate their activity which could generate lipid - derived signal s ( Kumar and Klessig , 2003). A second lipase, a GDSL motif lipase (GDSL - lipase from this point forward) has also been identified within the phloem exudates. GDSL - lipase is a lipid - binding protein with lipase - like function. It is part of a new subfamily of hydrolytic/lipolyti c enzymes that has unique positioning of their active site serine. As oppose to the putative lipase described previously whose active site serine is located closer to the center of the protein, the GDSL - lipase active site Serine is positioned closer to th e N - terminus. These enzymes are also thought to play a role in the regulation of plant growth and morphogenesis and have been shown to bind lipids (DAG, PA; unpublished). GDSL - lipases from different plant species have been shown to be implicated in vario us processes such as increased expression during germination (Ling et al., 2006), response to several plant hormones such as SA, ethylene, and auxin as well as defense against pathogens (Lee and Cho, 2003; Hong et al., 2008; Oh et al., 2005; Lee et al., 20 09; Yu et al., 2010). A more elaborate description of GDSL will be discussed toward the end of the chapter. The phloem lipid - associated family protein , PLAFP , is a small protein with unknown function. It contains a PLAT/LH2 domain, which is thought to me diate interaction with lipids or membrane - bound proteins (Benning et al., 2012) . PLAFP has been shown to bind PA (Benning et al., 2012 ). A recent study on the same protein deemed PLAT1 promoted tolerance under abiotic stress conditions, particularly salt stress. In addition, they found PLAT1 expressed in the vasculature of the leaves and roots and conveyed tolerance to ER stress elicited by tunicamycin ( Hyun et al., 2014). A more detail description of PLAFP is discussed later in the chapter. The phosphat idylinositol N - acetyglucosaminyl transferase subunit P (PIG - P) - like protein is a protein with unknown function and thought to play a role in GPI - anchor synthesis. We have shown 25 that it binds lipids (see Chapter 2; Barbaglia et al., 2016, invited ma nuscript to Frontiers). Currently , not much is known about this protein in plants since there is no significant homology to other proteins. In mammals, PIG - P is referred to as phosphatidylinositol glycan class P and is required for the regulation of doli chol - phosphate mannose synthase (DPM2) and associates with the PIG - A component of the GPI - N - acetylglucosaminyl transferase (GPI - GnT) complex in GPI anchor biosynthesis ( Watanabe et al., 2000 ) . Without PIG - P, cells would not contain GPI anchors, which make it crucial for GPI - GnT activity. A homolog to the mammalian PIG - P has been identified in yeast, Gpi19, and is also involved in GPI anchor synthesis (Newman et al., 2005). In addition, PIG - P has also been implicated in insulin signaling where it has been shown to elevate IRS 1 - associated P I 3 K activity and the tyrosine phosphorylation state of IRS 1 without increasing the phosphorylation rate of the insulin receptor, as well as translocat e GLUT4 glucose transporters to the plasma membrane in order to take up glucose from the extracellular space (Jones and Varela - Nieto , 1999). PIG - P will be discussed further in 1.7. Based on this information regarding lipid - binding proteins , our goal is to investigate whether lipid - bind proteins exist in the phloem and play a role in long - distance lipid transport. 1.5 Lipid/Phospholipid Signaling in Plants Unlike their animal counterparts, plants cannot escape adverse conditions. To cope, they have evolved mechanisms that all ow them to adapt to these environmental changes. Communication is the primary way plants can survive situations they cannot avoid. Cells converse with each other through signaling which can regulate interactions between adjacent cells or processes that o ccur in other parts of the plant. One of the molecule classes within cells that contribute to signaling is lipids . Lipids are mainly found within cell membranes to which they provide both structure and act as mediators that regulate various aspects of plant development and 26 environmental interactions (Wang and Chapman, 2013; Wang, 2004; Xue et al., 2009). Signaling lipids include glycerolipids, sphingolipids, fatty acids, oxylipins, and sterols. P hospholipid signaling cascades are normally grouped according to the phospholipase that catalyze s the formation of specific phospholipids such as PA, DAG, DAG pyrophosphat e (DGPP), lysophospholipids, free fatty acids, oxylipins, phosphoinositides, and inositol ph osphates (Zhu, 2002; Wang, 2004; Wang et al., 2007). Phospholipids are generated by four main types of phospholipases : phospholipase D (PLD), phospholipase C (PLC ), phospholipase A1 (PLA 1 ), and phospholipase A2 (PLA 2 ). PLC and PLD cleave at the first and the second pho s phodiesteric bond, respectively, while the PLAs cleave the fatty acid chains from the membrane lipid (Figure 1. 3 ) . P lant phospholipases participate in different cellular and physiological processes, which can be grouped into three categories: cellular regulation, membrane lipid remodeling, and lipid degradation; in addition, some phospholipases can mediate storage lipi d biosynthesis (Wang et al., 2012). Phospholipase D catalyzes the hydrolysis of glycerophospholipids such as PC, phosphatidylethanolamine ( PE ) , or phosphatidylglycerol (PG) into PA and a free head group, e.g. choline. In Arabidopsis , there are 12 PLDs: et al., 2012). Each of these subgroups is based on molecular and biochemical properties. Additionally, PLD can be classified into two groups based on their lipid binding domains; those with both pleckstrin homology (PH) and phox homology (PX) e other class known as C2, in which the lipids contain a C2 (calcium and lipid binding) domain. Phospholipase C cleaves phospholipids to produce diacylglycerol (DAG) and a phosphorylated head group. Plants possess two different PLC families: phosphoinositide - specific PLC (PI - PLC) and nonspecific PLC (NPC). PI - PLC facilitates the hydrolysis of phosphatidylinositol 4, 5 - bisphosphate (P IP 2 ) to generate inositol 1, 4, 5 - triphosphate (IP 3 ) and DAG. The resulting DAG remains bound to the membrane, while IP 3 is released into the 27 Figure 1. 3 : Cleavage Site s for Phospholipase s . Schematic depicting the cleavage site for different phospholipase family members with X representing head groups of phospholipids. The fatty acid chain length could vary. - 28 cytosol to act as a second messenger or is phosphorylated into IP 6 to aid in Ca 2+ signaling (Wang et al., 2012 ; Munnik and Testerink, 2009 ). NPC function s in cleaving common membrane phospholipids, such as PC and PE. Phospholipase As hydrolyze phospholipids to produce lysophospholipids and free fatty acids. PLA 1 and PLA 2 cleave the fatty acid from sn - 1 and sn - 2 positions, respectively of the glycerol backbone. In plants, there are four families of PLAs: the PC - hydrolyzing PLA 1 (PLA 1 ), the PA - preferring PLA 1 ( PA - PLA 1 ), the secretory low molecular weight PLA 2 (sPLA 2 ), and the patatin - like PLA (pPLA; Wang et al., 2012). Phospholipid - derived prod ucts generated by phospho lipase A (PLA) are important in plants because they generate phospholipid - derived signal molecules that mediate a variety of cellular processes, including cell elongation, gravitropism, anther dehiscence, biosynthesis of jasmonic acid upon wounding and pathogen attack, and defense signaling through intracellular acidification ( Ryu, 2004). In phospholipid signaling, PA plays a major role acting as a signaling messenger. PA is rapidly and transiently generated in response to a variety of biotic and abiotic stresses by either the PLC - DGK pathway or directly by PLD (Munnik and Testerink, 2009; Te sterink and Munnik, 2011; Wang et al., 2014; Julkowska et al . , 2014). PLD cleaves structural phospholipids in order to generate PA and a free head group. PLC, on the other hand, hydrolyzes phosphatidylinositol - 4,5 - bisphosphate (PtdIns(4,5)P 2 or PIP 2 ) int o inositol - 1,4,5 - triphosphate (Ins(1,4,5)P 3 or IP 3 ) and DAG (Julkowska et al., 2014 ). IP 3 diffuses into the cytosol where it triggers Ca 2+ release from intracellular stores, while DAG remains within the membrane and is rapidly converted to PA by DAG kinase (DGK) via phosphorylation (Testerink and Munnik, 2005 ; 2011). PA can act as docking sites where specific proteins are recruited to the m embrane . In addition , signaling lipids affect the activity of target enzymes. PA mediate s various cellular activities through these different modes of action which include binding to its targeted proteins to increase or inhibit their activities, acting a s a membrane anchor for proteins 29 involved in signaling cascades; acting as a substrate for the production of other lipid regulators such as lysoPA , free fatty acids, DAG, and DGPP; and regulating membrane trafficking and biogenesis (Wang, 2004). PA can al so bind proteins that have undergone post - translational modifications to facilitate target regulation ( Testerink and Munnik, 2005) . 1.6 Intracellular Phosphatidic Acid Signaling in Plant Response to Abiotic Stress Unlike structural lipids, signaling lipids are present only in minute amounts, but their levels increase rapidly in response to certain stimuli. Their accumulation/response is transient since the signal is quickly downregulated. Consequently, signaling lipids usually have much faster turno ver than other structural lipids (Testerink and Munnik, 2005). Under different stresses, signaling lipids have different responses. PA, for example, is triggered in response to several biotic and abiotic stresses, such as pathogen infection, drought, sal inity, wounding, cold, cell death, and oxylipin production (Xue et al., 2009; Wang et al., 2007 ; Hong et al., 2010 ; Kim et al., 2013). PA is also an intermediate in lipid biosynthesis ( Ohlrogge and Browse, 1995 ; Testerink and Munnik, 2011) and plays a role in membrane curvature ( Kooijman et al., 2003, 2005) . On a physiological basis , PA plays a role in regulating normal plant growth and development, mainly in roots and pollen tube growth (Kim et al., 2013 ; McLoughlin and Testerink , 2013 ). PA has been shown to bind to a myriad of proteins, including transcription factors, protein kinases, lipid kinases, protein phosphatases, and proteins involved in vesicular trafficking and cytoskeletal rearrangements ( For summary see Wang et al., 20 06 ). Thus, PA can directly interact with proteins to transport signals. E xamples of effector proteins PA has interacted with in plants include abscisic acid insensitive 1 (ABI1), phosphoinositide - dependent protein kinase (PDK1), constitutive triple response 1 (CTR1), trigalactosyldiacylglycerol 2 (TGD2), and NADPH oxidase (Guo et al., 2011). When PA binds to ABI1, CTR1, and phosphoethanolamine N - methyltransferase (PEAMT), it prevents their phosphatase or kinase activity. 30 On the other hand, through direct inte raction, PA stimulates the catalytic activity of PDK1, NADPH oxidases (R ESPIRATORY BURST OXIDASE HOMOLOG D/F; RbohD/F), sphingosine kinases (SPHK1/2), mitogen activated protein kinase 6 (MPK6), and SNF1 - related kinase 2 (SnRK2; Kim et al., 2013). In addition to regulating protein activity through their active site, PA has been shown to tether some of these proteins to the membrane. A well - known example of this is its interaction with ABI1. PA binds and tethers ABI1 to the plasma membrane which prevents ABI1 from translocating to the nucleus where it would bind ATHB6, a transcription factor that negatively regulates ABA responses (Kim et al., 2013) in addition to allowing SNF1 - related kinase 2 (SnRK2) activation via phosphorylation, mediat ing downstream signaling (Guo et al., 2012 a ; Umezawa et al. , 2009 ) . mediation of stomatal closure under drought conditions ( Figure 1. 4 ) . This phospholipase, a s well as to a lesser extent stomatal closure, although they regulate different areas of the signaling pathway. is activated in response to ABA to generate PA; howe in response to ABA. On the other hand, response to H 2 O 2 , possibly via PA - mediated activation of MAPKs (Guo et al., 2012b). In addition to helping plants end et al., 2008), regulates cytoskeletal organization (Gardiner et al., 2001), and improves plant stress tolerance by reducing H 2 O 2 - induced cell death (Zha ng et al., 2003). facilitates ROS response et al., 2012; Lu et al., 2013). Stomatal aperture is tightly regulated by a variety of effector proteins and signaling molecules including ABI1, ROS, NO, cytos olic Ca 2+ , sphingosine - 1 - phosphate (S1P), phyto - S1P, and ino sitol 1,4,5 - triphosphate (IP 3 ). All of these components play a role in the regulation of water loss via the stomata under 31 Figure 1.4 : Schematic M odel D epicting a P otential R ole for PLAFP in the T ranscriptional R egulation of ABA S ignaling. Model was modified from Fujita et al., 2011 with an integration of data by Yao et al., 2013 and our findings concerning PA and PLAFP. Broken lines indicate possible routes. 32 drought condi tions. Connections have also been made between PA, sphingosine kinase (SPHK), and , in that PA is involved in the activation of SPHK (Guo et al., 2012 a ). Additionally, PA binding to SPHK results in an produce phyto - positively regulate ABA - induced stomatal closure and are involved in ABA - induced ROS and NO production. Evidence shows that constitutively expressed and slightly increa sed under drought and salt expression is induced by drought and salt stress (Uraji et al., 2012; Katagiri et al., 2001) together work under more severe environmental stress conditions (Uraji et al., 2012). Conversely , expression increases under ABA stress . For example, expression of SiPLD 1 ( Setaria italic ) in fox tail millet was upregulated under drought, ABA, and NaCl treatments. In addition, expression in SiPLD 1 in Arabidopsis displayed enhanced sensitivity to ABA, NaC l, and mannitol during post - germ i n ation growth, while expression of overexpressed SiPLD 1 unde r water - deficient conditions resulted in significant tolerance to drought stress (Peng et al., 2010). Furthermore, others studies have shown PLD 1 is involved in ABA responses such as ABA - regulated stomatal closure and when PLD 1 activity is inhibited in Arabidopsis, the plant is unable to regulate stomatal closure resulting in more water loss ( Zhang et al., 2004; Zhang et al., 2009 ). Under drought stress, ABA binds to its pyrabactin resistance/pyr1 - like protein/regulatory components of its receptor, whi ch results the inhibition of ABI1, a negative regulator, allowing SNF1 - related kinase 2 (SnRK2) to be activated, which mediates downstream signaling such as ABA - induced gene expression (Julkowska et al., ts with SPHK. Increased phyto - S1P (a phosphorylated long - chain base - 1 - 33 to a further increase in PA levels. Thus, may be due to use of different conditio ns, such as when the samples were collected after treatment PA, which functions as a lipid signaling molecule, regulates downstream proteins such as ABI1, NADPH ox idase, and ion channels to facilitate stomatal closure (Guo et al., 2012 a ). A recent study noted that under drought conditions, PA binds MYB transcription factor WEREWOLF, and may tether it to the nuclear membrane, which would allow for the movement of th e proteins from the cytosol to the nucleus (Yao et al., 2013 ). T his could further aid in the transcription al regulation of drought tolerant genes. Overall, phospholipid signaling and the role of PA in plants are very important for regulation and function not only in response to drought but also in connection with additional environmental factors. Other examples of (phospho - under cold stress/freezing tolerance as well as ABA . Plants deficient in this phospholip ase show improved tolerance to freezing through the activation of cold - responsive genes and through the increase d accumulation of osmolyte s (Xue et al., 2009). In addition , phospholipid hydrolysis and PA production is decreased in both freezing and post - lipid hydrolysis and PA production in post - freezing recovery (Xue et al., 2009). In addition, Arisz and colleagues (2013) performed transphosphatidylation assays to demonstrate t hat under cold/freezing conditions, the resultant rapid accumulation of PA is due the phosphorylation of DAG by DGK rather than by hydrolysis of structural phospholipids by PLD. In a transphosphatidylation assay, cultures are in the presence of a low concentration of a primary alcohol, such as n - ButOH, which serves as a substrate in a PLD - catalyzed reaction generating phosphatidylbutanol ( PtdBut ) , at the cost of PLD - catalyzed production of PA. Thus, the ac cumulation of PtdBut is a measure of PLD activity . In this 34 experiment, the prelabled Arabidopsis seedlings with 32 P i for 20, 60, and 180 minutes, after which they placed the seedlings at 0°C for 5 minutes. They found that the cold temperature el icited a notable increase in 32 P - PA levels in the seedlings prelabeled for 20 minutes, and when they observed the results of the other structural lipids (PC and PE) ; they found they were barely labeled which allowed for their exclusion as precursors for 32 P - PA in a PLD - mediated reaction. Therefore, since the autho rs did not observe an increase in 32 P - PtdBut under chilling conditions that stimulate d the considerable 32 P - PA response, indicated that PLD was not involved and DGK was the source for PA production. It was suggested, though, that it is possible for PLD to participate at a later phase of the cold response (Arisz et al., 2013). Lastly, it has been proposed that o xidative st ress and wounding result in the accumulation of PA , leading to the activation of the mitogen activated protein kinase ( MAPK ) signaling cascade . Zhang et al., (2003) found that H 2 O 2 hypersensitiv ity to H 2 O 2 - induced cell death, which suggests PA may play a critical role in cell death provoked by H 2 O 2 . Similarly, wounding stimulates the activation of PLD - facilitated hydrolysis of phospholipids ( Xue et al., 2009 ) . In addition, wounding also activates MAPK signaling in soybean and this MAPK signaling initiation is attenuated when PA production is prevented with n - butanol, indicating PA acts as a signaling molecule in MAPK signaling provoked by wounding. In summary , the increase in PA can grea tly influence cell function in different ways: 1) PA can act as a lipid messenger by interacting with specific target proteins, such as the tethering target proteins like ABI1, to the membrane and/or controlling their catalytic activity, or by activating downstream signaling cascades 2) PA may alter membrane structure, promoting membrane fusion and interaction of certain soluble proteins with the membranes, or 3) PA may be converted to another signaling molecule, such as DAG, lysoPA, DGPP, and free fatt y acids, or may be involved in membrane lipid metabolism ( For a 35 summary see Wang et al., 2007). Thus, phospho - /lipid signaling is essential for plant survival, communication, and response under stress conditions and play a major role in their growth and d evelopment. 1.7 Putative Lipid - binding Proteins in the Phloem and their Potential Function in Long - Distance Signaling It is well - known that animal systems use long - distance signaling as a mechanism for communication among cells of different body parts in response to different stimuli. One of these paths is the bloodstream , which is an aqueous system that transports chemicals and other compounds , cells, and signaling molecules throughout the body in response to different signals sent out by organs or regul atory systems . For example, c holesterol is either bound to low - density lipoproteins (LDL) or high - density lipoproteins (HDL). When bound to LDL, it is t ransported throughou t the bloodstream for uptake in to cells and incorporation into membranes; converse ly, when bound to HDL or within chylomicrons , it is moved to the liver for degradation (for a summary see Nelson et al. , 2008). Using this system as a comparison, it increases the likelihood that plants too can use this method to facilitate communication within the organism. More specifically, lipids and their corresponding binding proteins in the phloem may implement a similar function in plants and play an important role in stress and developmental signaling. Lipids such as PA, PI , and its phosphates (PIPs) are well - known intracellular secondary messengers in plant cells (Wang, 2004; Munnik and Testerink, 2009), but essentially nothing is known about their involvement in long - distance transport. A recent publication produced from our lab suggested that phloem (phospho - ) lipids could work as long - distance developmental signals and lipid - binding proteins within the phloem may facilitate different aspects of this signaling process (Benning et al., 2012). This was based on our finding that 11 proteins identified within the phloem of Arabidopsis have predicted lipid - binding sites 36 (Guelette et al., 2012) . T he phloem, composed of the sieve elements and companion cells , is one of the long - distance transport paths , but currently there is no proof that lipid biosynthesis occurs within the phloem. Thus, lipids need to be released into or moved into the phloem, solubiliz ed , and transport ed to its target location, or be bound by a receptor protein lead ing to a change in development . Alternatively, the lipid - binding protein act s as the signal itself, with its activity controlled by the bound lipid (Benning et al., 2012 ). This project involves three of thes e proteins, GDSL - lipase, PIG - P - like protein , and PLAFP, with the focus primarily on PLAFP. GDSL esterases/lipases are hydrolytic enzymes with multifunctional properties such as broad substrate specificity and regiospecificity (Akoh et al., 2004). They co ntain a distinct Glycine, Aspartic acid, Serine, Leucine ( GDSL ) motif and have a flexible active site that appears to change conformation with the presence and binding of different substrates. GDSL - lipases are part of a new subfamily of hydrolytic/ lipolytic enzymes where the presence of their active site serine is located closer to the N - terminus, while other lipases have their active site serine situated near the center of the protein (Akoh et al., 2004). Several members of this family have displa yed a specific lipolytic ( A. thaliana ; Arab - 1; Brick et al., 1995) or fatty acyl ester hydrolytic (sunflower : H. annuus ; Beisson et al., 1997) activity. Since members of this protein family appear to be differentially expressed it is possible t hese lipas es may play a role in the regulation of plant growth and morphogenesis (Brick et al., 1995) . For example, Enod8 is a member of the GDSL lipase/esterase family isolated from Medicago sativa root nodules (Pringle and Dickstein, 2004) , while BnLIP2 ( Brassica napus L ) encodes a GDSL lipase induced during germination and is retained in mature seedlings (Ling et al., 2006). GDSL lipases also perform important roles in plant response to abiotic and biotic stress. For instance, Br - sil1 ( Brassica rapa sal icylate - induced lipase - like 1) is induced by salicylic acid and pathogen s (Lee and Cho, 2003), GLIP1 is an Arabidopsis GDSL - lipase that has 37 antimicrobial activity and regulates pathogen resistance in relationship to ethylene signaling in Alternaria brassicicola (Hong et al., 2008; Oh et al., 2005), and GLIP2 plays a role in pathogen defense against Erwinia carotovora by negative regulation of auxin signaling (Lee et al., 2009; Yu et al., 2010 ) . The GDSL studied here is a lipase - like protein we belie ve may be involved in the release of lipids from the membrane in response to abiotic stress and aids in their t ransport through the phloem. The PIG - P - like protein is a hydrophilic protein with homology to a protein involved in glycosylphosphatidylinositol (GPI) formation. GPIs are attached to the C - terminus of many membrane proteins, acting as a membrane anchor. Since there have not been any studies done on PIG - P in plants, much of what we can deduce about its potential fun ction come from human or mammalian (yeast) studies. In humans, b iosynthesis of GPI is initiated by GPI - N - acetylglucosaminyl transferase (GPI - GnT), which transfers N - acetylglucosamine from UDP - N - acetylglucosamine to phosphatidylinositol (Watanabe et al., 2 000). The GPI - GnT complex is composed of four components, PIG - A, PIG - H, PIG - C, and GPI1. PIG - P , in humans, is required for the regulation of dolichol - phosphate mannose synthase (DPM2) and associates with the PIG - A component of the GPI - GnT complex through the major hydrophilic region on the cytoplasmic side of the protein, but the two proteins could also associate between their transmembrane domains (Watanabe et al., 2000). Without PIG - P, ce lls will be GPI - anchor negative, t hus, PIG - P is essential for GPI - GnT activity. So far, it is not possible to predict the function of PIG - P from its sequence since it does not have any significant homology to other proteins. In addition, based on membrane topology, the functional sites of PIG - P are present either with in the membrane or on the cytoplasmic side of the ER (Watanabe et al., 2009). The putative PIG - P - like protein studied in this project is a predicted lipid - binding protein we believe may act as a receptor in lipid transport within the phloem or participate in the production of a receptor . 38 The p hloem lipid - associated family protein (PLAFP) is a small lipid - binding protein with unknown function. It contains a PLAT/LH2 domain, which is thought to mediate interaction with lipids or membrane - boun d proteins (Benning et al., 2012). We have shown that PLAFP is stress induced (Chapter 2 & 3; Barbaglia et al., 2016) and bind s phosphatidic acid (PA; Benning et al., 2012) . Both PLAFP and PA have been found within the phloem exudates suggesting PA is tr ansported within or into the phloem while bound to a protein . W hether or not this protein has a role in loading/unloading the lipid into and out of the phloem, acts as a receptor or a transport molecule, and has a true signaling function remains to be det ermined. Overall , these phloem lipid - binding proteins may play a role in long - distance signaling of lipids during development and in response to abiotic stress. The response of gene expression of the lipid - binding protein in different tissues and under different stress conditions in conjunction with regulation of plant development could indicate certain lipid - binding proteins may be involved in adaptation to stress. My thesis investigates the lipid - binding properties , localization, and expression, as wel l as the stress response of the three putative lipid - binding proteins. 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Plant Biol. 53: 247 - 73. 55 Chapter 2: Functional Characterization of GDSL - lipase , PLAFP , and PIG - P and their Potential Roles in Long - Distance Signaling This chapter has been invited for publication at Frontiers in Plant Science : Allison M. Barbaglia, Veronica Greve, Banita Tamot, and Susanne Hoffmann - Benning: Phloem proteomics reveals new lipid - based signaling paths in response to environmental signals and stress. Abstract accepted/paper invited at Frontiers in Plant Science/Plant Proteomics . 2.1 Introduction The world needs food and fuel to thrive, especially when faced with an exponential increase in the population. Yet the amount of arable land that is currently available is becoming even more limited due to changes in our climate and the encroachment of humans onto arable land. Together this leads to a competition between food and fuel crops. Moreover, the global climate change has an additional and direct impact on future crop yields. Drought, heat , and cold are the most common abiotic stresses that affect crop yield. In order to continue to be able to produce and provide sufficient food and fuel for an increasing world population, we need plants that exhibit accelerated growth, higher grain yield, and/or are more resistant to stress. One approach to prevent crop loss due to abiotic stress is to better understand the signals responsible for changes in plant development and the resulting adaptation. Unlike animals, plants cannot move to escape adve rse environmental conditions. To cope, plants had to develop mechanisms to detect changes in their environment, communicate these changes to different organs, and adjust development accordingly (Thomas and Vince - Prue, 1997). As a result, plants evolved t wo long - distance transport systems: the xylem for water and mineral distribution from the roots throughout the plant, and the phloem. The phloem plays a crucial 56 role in assimilate and nutrient transport, pathogen response, and the transmission of many si gnals governing plant growth and development. The phloem consist s of two main components for transport: the sieve elements and the companion cells. To enhance transport of molecules through the sieve elements, the y optimize the longitudinal flow in the sieve elements by degrading any obstacles in the form of organelles and by increasing the porosity of the cell walls at the longitudinal ends (van Bel and Knoblauch, 2000). N uclei, vacuoles, and other organelles that are present in early sieve tubes disi ntegrate as development progresse s , leaving only the plasma membrane and a thin cytoplasm which contain s ER, phloem - specific plastids, and a few, dilated, mitochondria ( van Bel and Knoblauch, 2000; Turgeon and Wolf, 2009). The residual ER is found near t he plasmodesmata which connect the sieve elements with the companion cells (Lucas et al., 2013). Since no functional protein synthesis machinery is thought to exist within the sieve elements , molecules found within are synthesized in the adjacent companion cells for long - distance movement, and these molecules need to be transported into the sieve element via plasmodesmata . The residual ER may participate in controlling and mediating this movement (Lucas et al., 2009, 2013) . Subsequently, they mo ve along the sieve tube, and are either perceived at their target location or transported out of the phloem. Transport of photoassimilates as well as signaling molecules is thought to occur from source (photosynthetically active mature leaves) to sink (im mature leaves, roots, fruits, flowers, for a review see Froehlich et al., 2011; Lucas et al., 2013). Over the years, t he view of phloem function has progressed f rom that of a simple assimilate transport system to a trafficking pathway for stress signals and developmental regulators in the form of small molecules (Chen 57 et al., 2001; Corbesier et al., 2003), peptides/proteins (Haebel and Kehr, 2001; Hoffmann - Benning et al., 2002; Giavalisco et al., 2006; Lin et al., 2009; Benning et al. , 2012.), mRNAs (Ruiz - Medrano et al., 1999; Pallas et al., 2013; Hannapel et al., 2013), microRNAs (Pant et al., 2008; Buhtz et al., 2010; Varkonyi - Gesic et al., 2010; Rodriguez - Medina et al., 2011), nucleic acids (Citovsky, and Zambryski, 2000; Haywood et al., 2005; Ding et al., 2003; Yoo et al., 2004), and lipids (Madey et al., 2002; Behmer et al., 2011 ; Guelette et al., 2012; Benning et al., 2012; Tetyuk et al., 2013). Since the contents and the function of the phloem has become so (Jorgensen et al., 1998). Therefore, the study of signaling compounds within the phloem is essential for our under standing of the distribution of environmental cues throughout the plant. The lab has identified over 121 distinct metabolites including 16 fatty acids/esters within Ara bidopsis phloem exudates using GC - MS (Guelette et al., 2012). Similar findings have bee n reported previously in the phloem of canola when small amounts of lipids and large amounts of free fatty acids were identified ( Madey et al., 2002). To investigate the role of these lipids in the phloem exudates, we analyzed the proteins present in the phloem sap within the sieve elements. Using an EDTA - facilitated collection of phloem exudates followed by SDS - PAGE and LC - MS analysis, we were able to identify 65 proteins within Arabidopsis phloem sap. Among those proteins, 11 were characterized as lipi d/fatty acid - binding proteins (Guelette et al., 2012). The ir predicted function ranged from lipid/fatty acid metabolism, binding, to signaling (Guelette et al., 2012; annexin, GRP17/oleosin, DIR1/LTP, aspartic protease, long - chain fatty acid CoA ligase, p ut. GRP7, two putative lipases, hypothetical lipid - binding protein, 14 - 3 - 3 protein, major latex protein, Bet v - 1 allergen, and PLAFP). Since we were able to identify 58 putative proteins that may bind lipids, we used LC - MS - CID to identify the types of lipids found in the phloem exudates. The results displayed the presence of numerous classes of phosphatidylcholine (PC), phosphatidic acid (PA), di - and triacylgycerols (DAG, TAG), as well as, phosphatidylinositol (PI) within the exudates. T hese lipids could b e products of membrane turnover or degradation, transported as energy carriers, or participating in long - distance signaling. This lipid - signaling concept is not unheard of in other biological systems. For example, other aqueous environments like the human bloodstream contain a variety of lipids, many of which are very important for human health. Lipids within the bloodstream are typically bound to proteins which can serve multiple functions such as transport to other tissues for storage, use, modification, or degradation. The best - known example of a lipid found within the blood is cholesterol. Cholesterol is either bound to low - density lipoprotei ns (LDL) and transported throughout the bloodstream for uptake in to cells and incorporation into membranes; or it is bound to high - density lipoproteins (HDL) or within chylomicrons and moved to the liver for degradation (for a summary see Nelson et al., 2 008). In addition, lipids can be taken up into cells, cleaved , and the resulting fatty acids activate transcription factors regulating their own Wahli and Michalik, 2012 ). Thus, the type of protein to which a lipid binds not only det ermines its direction of transport but also its fate as well as downstream regulatory processes . In plants, we know of several lipophilic long - distance signaling molecules, yet none of them are as complex as the phospholipids we ha ve identified . For examp le, many lipid 59 hormones have been identified within the phloem such as auxin, jasmonic acid (JA), abscisic acid (ABA), and cytokinins . Auxins move via polar auxin transport relying on specific carrier proteins for passage across the membrane as well as th rough bulk flow within the phloem. JA, an oxylipin as well as lipid signaling hormone , is synthesized in response to wounding and herbivory and is involved in systemic acquired resistance (SAR) . Movement usually occurs in response to its active form JA - I le (Matsu u ra et al., 2012), but also has been shown to move as methyl - JA in the vasculature as well as the xylem (Thorpe et al., 2007; Tanogami et al., 2012). ABA and its metabolites phaseic acid and dihydrophaseic acid (DPA) have been detected within the phloem. ABA has roles in response to stomatal closure under water deficit conditions. Cytokinins have been identified in both the phloem and xylem, thus, the form of cytokinin that is present determines the mode of action and direction of its transport. Cytokinin (and auxin) has been shown to affect PIN activity and root vascular development/patterning (Bishopp et al., 2011). O ther small lipophilic molecules such as oxylipins, phytosterols, dehydroabietinal (DA), and azelaic acid (AzA) also have been identified within the phloem . Behmer et al. (2011, 2013) detected free, acylated, and glycosylated derivatives of cholesterol, sitosterol, camposterol, and stigmasterol in the phloem. Lastly, DA and AzA both participate in SAR and have be en shown to bind proteins and transport them to distal leaves to help protect the plant from future pathogen attacks (Fu and Dong, 2013). In order for long - distance lipid signaling to commence within the phloem, lipid - binding proteins may have various pote ntial functions: (i) they could mediate the release of the lipid into the sieve element either by participating in the cleavage of the lipid so it can be transported in to the sieve element or in its release from the membrane, (ii) they could bind 60 specific phloem lipids, facilitating their solubilization in the exudate as well as their targeted transport, (iii) they could be part of a receptor which senses the lipid ( - signal) and transfers it out of the sieve element, or (iv) the protein could be the signal itself, with its activity modulated by the bound lipid. For this study, we focus on the functional characterization of three of the putative lipid - binding proteins : GDSL - lipase, PIG - P, and PLAFP. GDSL esterases/lipases are part of a new subfamily of hyd rolytic/lipolytic enzymes with multifunctional properties such as broad substrate specificity and regiospecificity (Akoh et al., 2004). They contain a distinct Glycine - Aspartic acid - Serine - Leucine ( GDSL ) motif , have a flexible active site that appears to change conformation with the presence and binding of different substrates , and is located closer to the N - terminus, unlike other lipases that have their active site serine situated near the center of the protein (Akoh et al., 2004). Since members of this protein family display differential expression, it is possible these lipases may play a role in the regulation of plant growth and morphogenesis (Brick et al., 1995) . O ne example is Enod8 , a member of the GDSL - lipase/esterase family isolated from Medicago sativa root nodules (Pringle and Dickstein, 2004). BnLIP2 ( Brassica napus L ) encodes a GDSL - lipase induced during germination and is retained in mature seedlings (Ling et al., 2006). GDSL - lipases also perform important roles in plant response to abiotic and biotic stress. For instance, Br - sil1 ( Brassica rapa salicylate - induced lipase - like 1) is induced by salicylic acid and pathogens (Lee and Cho, 2003), At GLIP1 is an Arabidopsis GDSL - lipase that has antimicrobial activity and regulates pathogen resista nce in relationship to ethylene signaling in Alternaria brassicicola (Hong et al., 2008; Oh et al., 2005), and At GLIP2 plays a role in pathogen defense against Erwinia carotovora 61 by negative regulation of auxin signaling (Lee et al., 2009 ) . We are examini ng whether the GDSL - lipase found in the phloem does indeed bind lipids and may play a role in lipid signaling. The phosphatidylinositol N - acetyglucosamin y ltransferase subunit P ( PIG - P ) - like protein is a hydrophilic protein with a predicted function in glycosylphosphatidylinositol (GPI) formation. GPIs are attached to the C - terminus of many membrane proteins, acting as a membrane anchor. There have not been any studies about this in pl ants; so much of what we discover about this protein is based on what is known about it in animals/humans. In humans , b iosynthesis of GPI is initiated by GPI - N - acetylglucosaminyl transferase (GPI - GnT), which transfers N - acetylglucosamine from UDP - N - acetyl glucosamine to phosphatidylinositol (Watanabe et al., 2000). The GPI - GnT complex is composed of four components, PIG - A, PIG - H, PIG - C, and GPI1. In humans, phosphatidylinositol glycan class P ( PIG - P ) , is required for the regulation of dolichol - phosphate m annose synthase (DPM2) and associates with the PIG - A component of the GPI - GnT complex through the major hydrophilic region on the cytoplasmic side of the protein, but the two proteins could also associate between their transmembrane domains (Watanabe et al ., 2000). Without PIG - P, human cells will be GPI - anchor negative, thus, PIG - P is essential for GPI - GnT activity. So far, it is not possible to predict the function of PIG - P from its sequence since it does not have any significant homology to other protei ns. In addition, based on membrane topology, the functional sites of PIG - P are present either within the membrane or on the cytoplasmic side of the ER (Watanabe et al., 2000 ). The putative PIG - P - like protein studied in this project is a predicted lipid - b inding protein we believe may act as a receptor in lipid transport within the phloem. 62 Phloem lipid - associated family protein (PLAFP) is a small putative lipid - binding protein with unknown function. It contains a PLAT/LH2 domain, which is thought to mediat e interaction with lipids or membrane - bound proteins ( Bateman and Sanford, 1999; Benning et al., 2012). Because of the presence of this domain, it is annotated as a lipoxygenase. However, this annotation is incorrect since the protein is lacking the catal ytic domain. P rotein s containing the PLAT/LH2 domain are typically stress - induced ( Hyun et al., 2014; Bona et al., 2007; Mhaske et al., 201 3 ) . We have shown that it specifically bind s phosphatidic acid (PA) , a membrane lipid known to participate in intracellular signaling in response to several stresses ( Benning et al., 2012; Testerink and Munnik, 2011; Arisz et al., 2013; Wang, 2005, 2006 ). Both PLAFP and PA have been found within the phloem exudates, which is important because it shows that both t he lipid and its protein pa rtner are present in the phloem. In addition, this suggests PA may also function in signaling within the phloem , possibly while bound to PLAFP. Proteomic and lipidomic analyses prove that lipid - binding proteins and lipids reside within the phloem of Arabidopsis. This study provides evidence that GDSL - lipase , PLAFP , and PIG - P are localized to the phloem/vasculature, bind lipids, and respond to different abiotic stresses. Our findings show that, while all the proteins are indeed lipid - binding and act in the vasculature possibly in a function rel ated to long - distance signaling; the three proteins do not act in the same but rather in distinct signaling pathways. 63 2.2 Material and Methods 2.2.1 Plant Growth Arabidopsis seeds were sterilized (20% bleach and 0.5% Triton X - 100 for 15 min and washed 6 times with sterile, deionized water) and plated on MS, 1% sucrose, and 0.6% phyto agar with or without antibiotic depending on the experiment. Next, plates were transferred to 4°C for two days before being placed into a Percival growth chamber; 22°C /18°C , 12 - h light/12 - h dark photoperiod with 60% relative humidity, and a light intensity of 120 mol photons m - 2 s - 1 . After two weeks , seedlings were either transplanted into soil ( equal parts Bacto Soil (Michigan Pear Company, Houston), medium vermiculite, and perlite ) and grown to maturity or transferred to hydroponic culture for stress experiments . 2.2.2 Abiotic Stress Analysis Arabidopsis seeds were sterilized and plated on MS, 1% sucrose, and 0.6% phyto agar as described above . P lates were transferred to 4°C for two days before being placed into a Percival growth chamber for 14 - 16 days. Wildtype Col - 2 seedlings were then caref ully transferred to a hydroponic - like system containing water, cover ed with a clear plastic dome and left to acclimate for 24 h, at room temperature ( 22°C .) . After this period, 300 mM Mannitol, 150 mM NaCl, 100 µM of ABA, or 30% PEG were added to the syste m . S eedlings were harvested at 0, 1, 2, 5, 8, 10, 12, and 24 hours post stress (hps). For each time point 3 - 6 seedlings were pooled. Each time course was performed in triplicate. Asterisks indicate statistical differences determined by - test; P < 0. 01 ). 64 2.2.3 Gene Expression Analyses Semi - quantitative and q uantitative RT PCR analysis was performed on total RNA extracted from 2 - 3 week old Arabidopsis seedlings or a leaf from a 5 week old plant following the instructions provided by the RNEasy Plant Mini Kit (Qiagen). The first strand was synthesized by oligo dT primers using SuperScript First Strand Synthesis III system (Invitrogen). The resultant cDNA was then used for semi - quantitative RT - PCR followed by quantitative real - time PCR (qPCR) us ing SYBR Green (Affymetrix) as the detection probe. Standard conditions ( 95°C activation, gene - specific annealing temperature, 72°C elongation; repeated 40 times ) and a melting curve set at 60°C with a 20 minute run time were performed for each run. Prim ers and annealing temperatures for all the RT - PCR and qPCR are outlined in Supplementary Table 1 (See Appendix) . 2.2.4 GUS Reporter Gene Construct, Arabidopsis Transformation , and GUS Assay The 1 Kb region upstream of the transcription initiation site of GDSL - lipase , PIG - P , and PLAFP was PCR amplified using the primers indicated in Supplementary Table 1 . For GDSL - lipase and PIG - P , HindIII and BamHI sites were added and HindIII and XbaI sites were . The PCR product was cloned into pGEMT - Easy vector (Promega) and subcloned into pBI121 (Clontech) vector (from which the 35S promoter was removed by digestion using the restriction enzymes mentioned above) to generate the clones, GDSL1KbPro:GUS , PIG - P1KbPro :GUS, and P LAFP1KbPro:GUS which were then transformed into Agrobacterium tum e fac ie ns strain GV3101 and C58C1pGV2260 by electroporation , respectively . Positive transformants were selected by Kanamycin resistance , and further confirmed by colony 65 PCR using t he same set of primers mentioned above, purified, sequenced by the Research Technology Support Facility (RTSF) Genomics Core at Michigan State University, and used to transform Arabidopsis Col - 2 by floral dip method (Clough and Bent, 1998). Transgenic lin es were selected by Kanamycin resistance and the incorporation of the transgene was confirmed by PCR, using primers indicated in Supplementary Table 1 . The GUS assay was performed as described (Martí et al. , 2010) using GUS staining solution: 50 mM sodium phosphate buffer, pH 7.0, 0.5 mM potassium ferricyanide, 0.5 m M potassium ferrocyanide, 0.1% T riton X - 100 and 1 mg/ml 5 - Bromo - 4 - chloro - 3 - indoxyl - beta - D - glucuronide cyclohexylammonium salt (Gold Biotechnology). Seedlings were observed under a light microscope ( Nikon Eclipse C i ). 2.2.5 Fluorescent Reporter Gene Constructs for GDSL - lipase and PIG - P and PLAFP The coding sequence of GDSL - lipase , PLAFP, and PIG - P was PCR amplified using the primers indicated in Supplementary Table 1 , which added the att sites of the Gateway s . The PCR product was cloned into pGEMT - Easy vector (Promega) and subjected to the Gateway cloning system where the resultant DNA product was subcloned into the donor vector pDNOR 207 followed by the destination vector, pEarley Gate 103 (or pEarley Gate 102 CFP or pEarleyGate 101 YFP ) to generate the clones GDSL1KbPro:GFP (CFP), PLAFP1KbPro:YFP, and PIG - P1KbPro:GFP (CFP), which were then transformed into Agrobacterium tumefaciens strain GV3101 by electroporation. Positive transformants were selected by Kanamycin resistance, further confirmed by colony PCR using the same set of primers mentioned above, sequenced , and used to transiently transform 66 Nicotiana tabacum . Leaf samples were then observed under confocal microscopy (Olympus FV1000SP CLSM; YFP Emission wavelength: 530 - 555nm, excitation: 515nm; RFP emission wavelength: 605 - 630nm, excitation: 559nm; CFP emission wavelength: 475 - 500nm, excitation: 458nm ) to detect the subcellular localization of the proteins. 2.2.6 Protein Expression and Purification A cDNA clone s for GDSL - lipase (At1g29660), U13183 ; PLAFP (At4g39730), U21720 ; and PIG - P (At2g39435) were obtained from Arabidopsis Biological Resource Centre, Ohio State University (Columbus, OH, USA). The coding region s of GDSL - lipase , PLAFP, and PIG - P excluding the 78 and 69 nucleotide region s encoding the 26 and 23 amino - acid predicted signal peptide s for GDSL - lipase and PLAFP, respectively , was PCR amplified using the primers in dicated in Supplementary Table 1, which introduced Nde the GDSL - lipase and PIG - P PCR products and Nde I sites at both ends of the PLAFP PCR product. The PCR product s were cloned into pGEMT - Easy vector (Promeg a), and subcloned into pET15b expression vector (Novagen) using Nde I the site to generate the expression clone, pET15b - GDSL - lipase / PLAFP /PIG - P . E. coli host strain OrigamiB(DE3)pLysS (Novagen) was transformed with pET15b - GDSL - lipase / PLAFP /PIG - P and the transformants were selected by Ampicillin (Amp), Kanamycin (Kan), Chloramphenicol (Cm), and Tetracycline (Tet) resistance for PLAFP and Amp and Cm resistance for GDSL - lipase and PIG - P . Protein expression was induced by adding IPTG up to the final PLAFP p rotein was extracted and purified using , and GDSL - lipase and PIG - P proteins w ere 67 extracted and purified using the Ni - NTA resin (Qiagen) using the phosphate buffers containing . Purification steps include the clear lysate, flow through, wash fraction, a nd elution fractions. E lution fractions 1 - 2 and 2 - 3 for GDSL and PIG - P, respectively contain ing purified protein were pooled. 2 PO 4 (Lu and Benning, 2009) using a PD10 column (GE healthcare). 2.2.7 Protein Lipid Overlay Assay Lipids ; di 18:1 PE, PA, PC, PS, PG, PI ) onto a Hybond - C membrane (GE Healthcare). The protein lipid overlay assay was performed as d escribed ( Benning et al., 2012; Awai et al., 2006). Briefly, HCl, pH 8.0, buffer cont aining purified protein incubated in the blocking buffer with Anti - polyHis mouse monoclonal antibody (Sigma) at d incubated in the blocking buffer with horseradish peroxidase (HRP) - conjugated goat anti - mouse washed twice with TBST and detection was determined using a chemiluminesce nce detection system (Thermo Scientific). 68 2.2.8 Liposome Binding Assay Liposomes were prepared using di18:1 PC, di18:1 PA, or a mixture of di18:1 PC and di18:1 PA, following the method described in (Awai et al., protein a g for - cold TBS and then mixed with SDS - PAGE sample buffer. Western blot analysis was performed using anti - His and HRP - conjugated goat anti - mouse antibodies and chemiluminescence detection system describe d previously. 69 2.3 R esults and Discussion Using a proteomics approach, the lab identified several putative lipid - binding proteins in the phloem exudates (Guelette et al., 2012; Tetyuk et al., 2013). Simultaneously, we identified several phospholipid species, di - an d triacylglycerols, as well as j asmonic acid and its precursor OPDA. Our findings of lipids and lipid - binding proteins in the phloem prompted us to propose that phloem (phospho - ) lipi ds could participate in long - distance signaling possibly in response to abiotic stress, and that they are released, sensed, and moved by phloem lipid - binding proteins (Benning et al., 2012). To participate in long - distance signaling, these proteins must p erform at least one of the following tasks: cleave the lipid from the membrane, solubilize it and transport it throughout the sieve element, and/or act as a receptor for the lipid signal at its target destination. Indeed, the proteins we identified incl ude lipases, that could release the signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid - movement. This study investigates three putative lipid - binding proteins, GDSL - lipase , PLAFP, and PIG - P that may play a role in stress - related long - distance signaling : (i) GDSL - lipase may act in cleaving the lipid, thus releasing it from the membrane and into the sieve element; (ii) PLAFP may solubilize and transport the lipid; and (iii) PIG - P could be part of a receptor. To participate in any long - distance function, the respective genes need to be expressed in the vasculature of the plant and the proteins need to bind the phloem lipids. Additionally, since many long - distance signaling paths are influenced by str ess stimuli, we also tested and observed how these proteins respond to abiotic environmental factors (osmotic, ABA, salt, and water stress). This project investigates whether these lipid - binding proteins and their lipid 70 partners are needed to help the pla nt survive under different abiotic stresses via phloem - mediated long - distance signaling. 2.3.1 GDSL - lipase , PLAFP , and PIG - P Bind Lipids The protein - lipid overlay assay shows GDSL - lipase strongly binds to diacylglycerol (DAG) and PIP 3 . In addition, a weak interaction with phosphatidic acid (PA), phosphatidylserine ( PS ) and phosphatidylinositol 4,5 - bisphosphate ( PIP 2 ) can be detected ( Figure 2. 1A ). The PIP 3 - binding is biologically irrelevant since the commercial lipid membranes are pr epared with lipids found in animal systems and PIP 3 s have not been found in plants (Gillaspy, 2013 ; Munnik and Testerink, 2009 ; Wang et al. , 2007 ) . Similarly, PIP 2 s are present in plants only in small amounts. To independently confirm GDSL - lipase interac tion with the lipids , we performed a liposome - binding assay ( Figure 2. 1B ). We were able to confirm binding to different species of PA (data not shown) , however, binding to DAG, PC, and PIP 2 could not be confirmed, which is indicated by the fact that the protein could only be found in the supernatant but not in the pellet containing the liposomes. The discrepancy between these two assays is possibl y due to the difference in the acyl chain of the lipids used. The commercially - available membrane in Figure 2. 1 contains lipi ds with saturated acyl chains, which are predominant in animal membranes (Volmer et al., 2013) , while the liposome binding was per formed with lipids containing monounsaturated acyl chains, which are more likely found in plant systems ( Grison et al., 2015 ; Supplementary Table 2 ). This suggests that it is essential to test for lipids present in the experimental system. In addition, it 71 Figure 2. 1: Protein - Lipid Binding Assays for GDSL - lipase. Immunodetection of GDSL - lipase - His on a protein - lipid overlay assay demonstrates binding of phosphatidic acid and diacylglycerol (DAG; A). Assay was performed twic e. The liposome binding assay could not provide further evidence for GDSL - lipase binding lipids (B). T he assay was performed three times . Abbreviations: TAG: triacylglyceride, DAG: diacylglycerol, PA: phosphatidic acid, PS: phosphatidylserine, PE: phosphatidylethanolamine, PC: phosphatidylcholine, PG: phosphatidylglycerol, CL: cardio lipin, PI: phosphatidylinositol, PIP 1 : phosphatidylinositol - 4 - phosphate, PIP 2 : phosphatidylinositol - 4,5 - phosphate, PIP 3 : phosphatidylinositol - 3,4,5 - phosphate. A B Pellet Supernatant 72 allows for the possibility that some proteins not only distinguish their ligands by the lipid head group, but by the identity of their acyl species as well (Guo et al., 2012) . To avoid these issues we examined possible PLAFP - binding partners using a homemade lipid - membrane containing 18:1 - 18:1 PA (DOPA) . PLAFP exhibited strong and very s pecific binding to PA (Figure 2. 2A ) . The liposome assay confirmed this binding (Figure 2. 2 B ) : PLAFP can be detected in the pellet containing the liposomes. More importantly, it is not present in liposomes containing PC (negative control). Increasing the ratio of PA in the liposomes also increases the amount of protein detected, clearly confi rming PA as a ligand for PLAFP. Putative lipid - binding proteins were identified based on homology to known proteins. To confirm their lipid - binding ab ility as well as identify their lipid ligands, we performed two independent lipid binding assays, protein - lipid overlay assay and the liposome binding assay . The protein - lipid overlay assay indicates a strong interaction of PIG - P with phosphatidylserine (P S) and phosphatidylinositol - 4 - phosphate (PIP 1 ) and weakly to PA (Figure 2. 3A ) . Liposome binding confirmed this interaction with PS and PA ( Figure 2. 3B ) but not with PIP (not shown). PIG - P can be detected in the fraction containing the liposomes and not i n the supernatant. No binding to the negative control (PC) is observed as is indicated by the presence of the protein in the PC - supernatant. Our findings suggest that all three proteins have the capability to bind PA , while at least PIG - P may bind additional lipids. PA is a well - known secondary messenger involved in various signaling cascades, and may participate in osmotic stress and pathogen response (Testerink and Munnik, 2005, 2011) . Physiologically, PA regulate s normal plant growth and development, 73 Figure 2. 2: Protein - Lipid Binding Assays for PLAFP. Immunodetection of PLAFP - His on a protein - lipid overlay assay reveals PLAFP binds phosphatidic acid (PA; A). The liposome assay confirm s PLAFP binding to PA (B). PLAFP exhibits binding to any complex that contains PA and does not bind PC - only liposomes (control). Assays were performed at least three times. Abbreviations: PE: phosphatidylethanolamine, PA: phosphatidic acid, PC: pho sphatidylcholine, PS: phosphatidylserine, PG: phosphatidylglycerol, PI: phosphatidylinositol. B A 74 Figure 2. 3: Protein - Lipid Binding Assays for PIG - P. Immunodetection of PIG - P - His on a protein - lipid overlay assay displays PIG - P binding to phosphatidic acid (PA) as well as phosphatidylserine (PS) and phosphoinositol - 4 - phosphate (PIP 1 ). The liposome assay confirm s PIG - P binding to PA and PS, but not PC ( negative control) within the pellet. Both assays were performed twice. Abbreviations: TAG: triacylglyceride, DAG: diacylglycerol, PA: phosphatidic acid, PS: phosphatidylserine, PE: phosphatidylethanolamine, PC: phosphatidylcholine, PG: phosphati dylglycerol, CL: cardiolipin, PI: phosphatidylinositol, PIP 1 : phosphatidylinositol - 4 - phosphate, PIP 2 : phosphatidylinositol - 4,5 - phosphate, PIP 3 : phosphatidylinositol - 3,4,5 - phosphate. A B 75 primarily in roots and pollen tube growth (Kim et al., 2013). PA has also been shown to bind to various proteins such as transcription factors, protein kinases, lipid kinases, protein phosphatases, as well as vesicular trafficking and cytoskeletal - forming proteins (Guo et al., 2011). Thus, PA can act either directly or indirectly with other proteins in order to transmit signals. Since PA is the common lipid bound between our three proteins, this suggests o ne of two possibilities. First is that all of these proteins are anchored to the membrane via PA. Alternatively , these three proteins may function in a long - distance signaling path involving PA. This would involve GDSL - lipase releasing PA from the membrane and transfer it to PLAFP , which would then solubilize PA and transport it thro ugh the sieve element to its target destination . Here, PLAFP - PA, PLAFP, or PA would subsequently bind to PIG - P, a receptor molecule. This concept has been seen with the well - known Frizzled - W nt interaction observed in animals. Similar to what we believe is occurring with PLAFP and PA, the Frizzled (Fz) receptor must bind W nts (Wingless and Int - 1) , which are lipid - modified morphogens. This interaction is critical for proper development. Wnts help facilitate mammalian development in areas such as cell pro liferation, differentiation, and movement (Janda et al., 2012). Frizzled is a transmembrane receptor that is needed for virtually all Wnt signaling, where the N - terminal cysteine - rich domain acts as the binding site for Wnt. What is interesting about thi s interaction is the necessity of the palmitoleic acid lipid group on Wnt. This lipid is necessary for specific amino acid interactions to occur between Wnt and Frizzled as well as for full activation of each protein (Janda et al., 2012). 76 2.3.2 Protein Localization to the Cell Periphery Determination of where the three proteins localize within the cell was established by generating protein constructs containing a fluorescent tag at the C - terminus of the proteins. These constructs ( 35S - GDSL - CFP, 35S - PLAF P - YFP , and 35S - PIG - P - CFP ) were transiently expressed within tobacco ( Nicotiana tabacum ) to detect where the three proteins localize within the plant. Localization of all three proteins was at the periphery of the cell. GDSL - lipase and PLAFP show ed only p artial overlap with the ER marker (Figure 2. 4A,B ) , which implies that they are likely not retained in the ER but rather transgress through it either to the plasmodesmata or are secreted into the apoplast . This is consistent with the fact that neither protein has an ER - retention signal (HDEL/KDEL ; Montesinos et al., 2014; Xu and Liu, 2012 ). Further colocalization studies with a plasma membrane marker, shows no overlap with neither GDSL - lipase nor PLAFP (Figure 2. 5A,B ) . PLAFP is visible in a punctate p attern around the cell periphery. Similar patterns have been found for receptors, plasmodesmata, and protein s moving through plasmodesmata (knotted 1 :Kn1 ). Kn1 is a homeobox protein that has been shown to move through plasmodesmata (Duan et al., 2015; Stahl and Rüdiger , 2013; Kim et al., 2002; Jackson, 2002; Lucas et al., 1995, 1999) , suggesting that PLAFP might be mobile as well. PIG - P expresses at the cell periphery and does not overlap with the ER or plasma membrane marker s . PIG - P does not contain a signal sequences so there is no interaction between it and the ER , but rather they express adjacent to one another and sometimes the two are sandwiched with the ER marker surrounding PIG - P (Figure 2. 4C ) . Similar to expression with the ER, PIG - P expresse s adjacent to the plasma membrane marker ( Figure 2. 5C ). 77 Figure 2.4 : Transient Localization of GDSL - l ipa s e (A) , PLAFP (B) , and PIG - P (C) using Fluorescent Protein Tags. Constructs were transiently expressed in tobacco epidermis cells. Proteins are associated with the periphery of the cell but not within the ER. B C A GDSL_CFP ER_RFP Chlorophyll Autofluorescence Composite Overlay Composite Overlay Chlorophyll Autofluorescence ER_RFP PIG - P _CFP 78 Figure 2.5 : Transient Localization of Fluorescently Tagged GDSL - l ipase (A) , PLAFP (B) , and PIG - P (C) with Plasma Membrane Marker in Tobacco . The expression of GDSL appears to be in the periphery of the cell. When colocalized with the plasma membrane (PM) marker, no overlap is observed (A). PLAFP is expressed in a punctate pattern at the cell periphery. When expressed with the PM marker, there is no association, but rather PLAFP appreast to be on either side of the marker (B). Simil aer to GDSL, PIG - P is expressed around the outside of the cell and exhibits no assocition with the PM (C). A B C B B C C B C 79 2.3.3 PLAFP Promoter Activity is Associated with the Vascular Bundles To examine the promoter activity, 1 kb upstream of the transcription sta rt site was cloned in front of a GUS reporter gene ( pPLAFP - GUS ) . These experiments revealed that PLAFP is expressed within the vasculature of the young leaves and roots of 2 - 3 week old Arabidopsis seedlings (Figure 2. 6 ) . Localization of gene expression w ithin the vasculature supports our hypothesis that PLAFP is involved in long - distance signaling within the phloem , either by directly participating in signaling or by playing a role in vascular bundle development . To observe the promoter activity of GDSL - lipase and PIG - P, we designed a construct containing a 1kb promoter region of each protein tagged with GUS at the C - terminus (GDSL1KbPro:GUS; PIGP1KbPro:GUS ). Using these constructs, we transformed Arabidopsis plants via the floral dip method ( Clough and Bent, 1998 ) and then subjected the transgenic seedlings to a GUS assay in order to detect how the promoter regulates gene expression. Expression for GDSL - lipase and PIG - P was not observed in young, two - week old seedlings. From t his data we can conclude either expression does not occur in young tissue, but rather in older, more mature seedlings or the constructs are not very stable . 2.3.4 Abiotic Stresses Effect Gene Expression We have shown that all of our proteins bind PA . In plants, PA can be formed t hrough different pathways: 1) PA can be produced directly by PLD, which cleaves structural phospholipids such as PC and PE. 2) PA can be made by the dual action of phospholipase C ( PLC ) and diacylglycerol kinase ( DGK ) , where PIP 2 is hydrolyzed into IP 3 and DAG. DAG is then phosphorylated by DGK to generate PA (Testerink and Munnik, 2011). PA is a well - known 80 Figure 2. 6 : P LAFP P romoter Activity in Two W eek O ld Arabidopsis S eedlings U sing a GUS Reporter Construct. Promoter activity was identified within the leaf and root vasculature as well as in the hydathodes and shoot meristem from three different lines . 81 intracellular signal in response to various abiotic and biotic stresses . PA has been shown to have roles in pathogen resp onse and infection, drought, salinity, wounding, cold, cell death, and oxylipin production (Xue et al., 2009; Wang et al., 2007; Hong et al., 2010; Kim et al., 2013). PA together with sucrose non - fermentation 1 - related protein kinase 2 (SnRK2) and mitogen activated protein kinase (MAPK) has been shown to play a role in maintain root system architecture as well as salt tolerance (McLoughlin and Testerink, 2013). In ad dition, PA is widely known for its role in stomatal closure via the activation of the ABA pathway under drought/osmotic conditions. Here, PA binds and tethers ABI 1 , a PP2C, to the membrane which allows for ABA to bind to its receptor and SnRKs to be activ ated allowing for the stomata to close to conserve water and gene transcription to commence. Hence, it is possible that PA or the protein - PA complex is influencing how these proteins respond to the stressors in a phloem - mediated mechanism . Therefore, lon g - distance lipid signaling may be an important factor for the plant s ability to survive and adapt to its surroundings. PA mediate s cellular activities through different modes of action : These include binding to its targeted proteins , thus tethering them to the membrane which modifies their activity; alternatively PA can act as a substrate for the production of other lipid regulators such as lysoPA, free fatty acids, DAG, and DGPP , or play a role regulating membrane trafficking and biogenesis (Wang, 2004 ; Testerink and Munnik, 2005; Wang et al., 2006, 2007 ). In the context of long - distance signaling, PA could move through the phloem while bound to a phloem lipid - binding protein like PLAFP and elicit a response in distal tissues. PA is known to be induce d by a variety of stressors either through the action of Phospholipase D (PLD) or PLC/D G K. Under drought/osmotic stress, the plant stress hormone ABA is activated. When ABA is stimulated, PLD starts to produce more PA. PA 82 then is prompted to bind to abs cisic acid insensitive 1 ( ABI1 ) and tether it to the membrane. This prevents it from binding to the ABA receptor, PYR /PYL/RCAR, and inhibiting ABA from closing the stomata. With ABI1 bound, the plant can conserve the water that it has stored within it by closing the stomata and reducing CO 2 intake and transpiration. PA production by PLC/DGK occurs under hyper osmotic and salt stresses through the hydrolysis of PIP 2 to form IP 3 and DAG, followed by the phosphorylation of DAG by DAG kinase (DGK). We examin ed if any of the three PA - binding proteins are induced by the same stressors as PA . Two - week old seedlings were plated on MS plates with kanamycin sel e ction for two weeks before being transferred to a hydroponic system (Figure 2. 7 ) . After acclimation for 24 hours, seedlings were introduced to four different forms of abiotic stress : osmotic stress in the form of mannitol , salt stress via NaCl , ABA, a plant hormone stimulated by drought/osmotic stress , and PEG, a water stress mimic. Gene ex pression was monitored over 24 hours using quantitative RT - PCR ( Figure 2. 8 ). PIG - P was not affected by any of the stresses. This is not surprising since we had proposed that PIG - P may be part of a receptor and as such, should be constitutively expressed. Alternatively, its function may be unrelated to stress signaling. GDSL - lipase was downregulated by osmotic (mannitol) stress , ABA, and water stress mimic (PEG) , while PLAFP displayed almost the opposite response to these stresses by being strongly upregulated by ABA and water stress mimic (PEG). While there have been other GDSL - lipases that exhibit an increase in expression under various abiotic stresses such as ABA, drought, osmotic, salt, and SA stress (Hong et al., 2008 ), this particular lipase shows the opposite effect. Since GDSL - lipase and PLAFP have the opposite response to ABA and PEG , this indicates that they most likely do not work in the same but distinct signaling pathways. Most interestingly, 83 Figure 2.7 : Hydroponic System Set - up fo r Abiotic Stress Treatment. Wildtype seedlings were grown on MS plates for two weeks and then tra nsferred to the hydroponic culture and treated as displayed above. After 24 hours of acclimation in their new environment, the abiotic stress treatments were added: osmotic stress received 300mM Mannitol, salt stress received 150mM sodium chloride (NaCl), and drought stress signal and mimic in the form of 100µM abscisic acid (ABA) or 30% polyethylene glycol (PEG), respectively. Seedlings were collected after various time points over a 24 hour period. Method adapted from communication with Patricia Santos. 84 Figure 2.8 : Effect of Abiotic Stress on GDSL - lipase , PLAFP, and PIG - P. Two week old Arabidopsis seedlings were transferred to hydroponic culture and submitted to osmotic, salt, water stress mimic, and ABA stress signal. Values represent mean and standard error of the GDSL, PLAFP, and PIG - P gene expression levels after 0, 5, and 24 hours of 3 - 6 biological replicates as determined using qPCR (three technical replicates per biological replicate). Values were normalized to 18S mRNA. Gene expression was determined using qPCR. The asterisks indicate significance of p<0.01. 0 0.5 1 1.5 2 0 hr. 5 hr. 24 hr. Relative Gene Expression Time Effect of Abiotic Stress on GDSL Control Mannitol ABA NaCl 30% PEG 0 1 2 3 4 0hr 5hr 24hr Relative Gene Expression Time After Treatment Effect of Abiotic Stress on PLAFP Control Mannitol ABA NaCl 30% PEG 0 0.5 1 1.5 2 0 hr. 5 hr. 24 hr. Relative Gene Expression Time Effect of Abiotic Stress on PIG - P Control Mannitol ABA NaCl 30% PEG * * * * * * * * * * * * A B C 85 PLAFP is greatly upregulated by ABA and PEG. ABA is a plant hormone that signals when the plant is experiencing drought or a water deficit, and PEG is a drought/water stress mimic . This increase in PLAFP expression is parallel to the increase in the abundance of PA in response to the same stresses. Upregulation of PLAFP under ABA stress also indicates that PLAFP could contribute to the ABA signaling pathway. Under the ABA pathway, drought or osmotic stress leads to ABA to bind to its receptor, PYR/PYL/RCAR. An increase in ABA leads to the activation (ABI1), a protein phosphatase 2C, from binding to the ABA receptor by tethering it to the membrane. Th is allows ABA to regulate gene expression and to promote stomatal closure leading to a conservation of water when the plant is experiencing water - deficit conditions (Lu et al., 2013) . PA has been shown to be involved in this intracellular signaling process by regulating stomatal closure (Lu et al., 2013) and by regulating the transcription of stress tolerance genes through interaction with the MYB transcription factor, WEREWOLF. (Yao e t al., 2013). Our findings of PA as well as a PA - binding protein, PLAFP, which is induced by the same stressors as PA in the phloem suggests that both lipid and protein may be involved in the long - distance signaling. 2.4 Conclusions We have shown that th e plant phloem contains both lipids as well as putative lipid - binding proteins. We confirmed that three o f these proteins, a GDSL - lipase protei n, a PIG - P homologue, and PLAFP do indeed bind lipids . In addition, all three proteins bind phosphatidic acid, a lipid known to play an important role in intracellular signaling in plants. Expression of 86 the genes associated with these proteins is correlated with the plant vasculature, an essential prerequisite for a long - distance signaling or transport function. Ho wever, the varied responses to abiotic stress indicated that the three proteins do not act in the same but in distinct signaling paths. Nonetheless, both PA and PLAFP are induced by the same stressors and are present in the phloem, suggesting the existenc e of a novel phospholipid - associate d long - distance signaling path. 87 APPENDIX 88 Supplementary Table 2. 1: Primer Table for Cloning, RT - PCR, and qPCR. Sample Sequence Function, Sites, etc GDSL FW/Rev FW: - GCGC AAGCTT GTGTAACAAGACTTAAGGCGC - Rev: - GCGCGGATCCCGATCTCACAAAACAAAACAAAAATAC - Amplifies ~1000bp upstream of the promoter site; Contains HindIII and BamHI restriction sites; Annealing Temperature: 60 °C; Expected Size: 989bp GDSL Clone FW/Rev - ATGGAGAGTTACTTGAGGAAATGGTG - - TCAAAGCTGTGCTAATTGCGAGATATC - Amplifies the entire CDS sequence; Annealing Temperature: 52 °C; Expected Size: 1095bp GDSL_GFP FW/Rev FW: 5' - GGGGACAAGTTTGTACAAAAAAGCAGGCTACCATGGAGAGTTACTTGAGGAAAT GGTG - 3' Rev: - GGGACCACTTTGTACAAGAAAGCTGGGTCAAGCTGTGCTAATTGCGAGATATC - Cloning; Contains att sit es ; Annealing Temperature: 64 °C; Expected Size: 1150 bp GDSL F/R - CATCGATTTCGGCGGCCCCA - - GTCTTGGCTGCCCTGTGCGA - RT - PCR; Annealing Temperature: 58 °C; Expected Size: 526bp GDSLq FW/Rev - TCGGCCAACCGAATCTTCAA - - CCTTCCAAT TCCGCAACACG - qPCR; Annealing Temperature: 52 °C; Expected Size: 173bp LAFP FW/Rev At4g39730 - ATGGCTCGTCGCGATGTTCTC - - AACGACCCAAGAAAGCTTTTTCCG - Amplifies entire CDS minus the stop codon; Annealing Temperature: 53 °C; Expected Size: 543bp 89 LAFP - 1 F/R At4g39730 - GAGCAATGGCTCGCTACTGA - - ACGCCACATTACACTCACAAG - Amplifies the entire CDS sequ ence; Annealing Temperature: 52 °C; Expected Size: 210bp LAFPq FW/Rev At4g39730 - TGCTCGA CGCAGGATTTTGA - - CTCAGACCCGACCCGACTAA - qPCR; Annealing Temperature: 52 °C; Expected Size: 126bp pPLAFP FW/Rev FW: - GCCAAGCTTATTGATTATCATTGCATTGC - Rev: - GCGTCTAGATTTGTTTTTTTCCGGTGAACG - Amplifies the 1 Kb region upstream of the transcription initiation site of PLAFP ; Contains HindIII and Xba restriction sites; Annealing Temperature: 56 °C; Expected Size: 1000 bp PLAFP - GUS Confirm FW: - GCCAAGCTTATTGATTATCATTGCATTGC - Rev: - GACCGCATCGAAACGCAGCACG - Amplifies PLAFP and GUS sequence attached to the C - terminus; used to confirm PLAFP - GUS transgenic l ines; Annealing Temperature: 56 °C; Expected Size: 12 89 bp PLAFP - Protein Expression At4g39730 FW: - GCGCATATGGAAGATGATCCAGACTGTGTATACA - Rev: - GCGCATATGTTAAACGACCCAAGAAAGCTTTTTCCG - Amplifies t he coding region of PLAFP, excluding 69 nucleotide region encoding the 23 amino - acid predicted signal peptide ; Contains NdeI restriction site(s); Annealing Temperature: 59 °C; Expected Size: 495 bp PLAFP_YFP FW/Rev At4g39730 FW: - GGGGACAAGTTTGTACAAAAAAGCAGGCTACCATGGCTCGTCGCGATGTTCTC - Rev: - GGGGACCACTTTGTACAAGAAAGCTGGGTCAACGACCCAAGAAAGCTTTTTCCG - Amplifies the coding sequence of PLAFP; contains att sites; Annealing Temperature: 65 °C; Expecte d Size: 601bp 90 PIG - P FW/Rev - GCGCAAGCTTGGGTCAAATTATCTATGTGGTTC - - GCGCGGATCCTTCTTTTGAGACCTCTACTCTTC - Amplifies ~1000bp upstream of the promoter; Contains HindIII and BamHI restriction sites; Annealing Tem perature: 60°C; Expected Size: 997bp PIG - P Clone FW/Rev FW: 5' - ATG GAGTCTAAGGAAATTAGATCCTCC - 3' Rev: 5' - CTACATGAAGCTGACAACCTCAGAC - 3' Amplifies the entire CDS sequence; Contains att sites; Annealing Temperature: 52°C; Expected Size: 1395bp PIG - P_GFP FW/Rev FW: 5' - GGG GACAAGTTTGTACAAAAAAGCAGGCTACCATGGAGTCTAAGGAAATTAGATCCTCC - Rev: - GGGGACCACTTTGTACAAGAAAGCTGGGTCCATGAAGCTGACAACCTCAGAC - Cloning; Contains att s ites; Annealing Temperature: 65 °C; Expected Size: 1450 bp PIG - P F/R - TTGATGCACTA GCTAAGCCTCCTCA - - AGCGCAGTGCCTAGCCTCCTA - RT - PCR; Annealing Temperature: 56°C; Expected Size: 976bp PIG - Pq FW/Rev - GACGAATTCGGAAGATGCTC - - TCAGGGTTTCCAGCTGATTC - qPCR; Annealing Temperature: 50°C; Expected Size: 247bp At18S_F/R - TCAACTTTCGATGGTAGGATAGTG - - CCGTGTCAGGATTGGGTAATTT - Control Primers; Amplifies part of the 18S gene; Annealing Temperature: 50°C; Expected Size: 161 bp GUS - F/R - ATGTTACGTCCTGTAGAAACCCCAACCCGTG - - AGGAGTTGGCCCC AATCCAGTCCATTAA - Amplifies GUS gene; Annealing Temperature: 59°C; Expected Size: 976bp 91 REFERENCES 92 REFERENCES Akoh, C.C., Lee, G - C., Liaw, Y - C., Huang, T - H., Shaw, J - F. 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(201 3) Phosphatidic Acid Interacts with a MYB Transcription Factor and Regulates Its Nuclear Localization and Function in Arabidopsis. Plant Cell. 25: 5030 - 5042. 99 Yoo, B. C., Kragler, F., Varkonyi - Gasic, E., Haywood, V., Archer - Evans, S., Lee, Y. M., Lough, T . J. and Lucas, W. J. (2004) A systemic small RNA signaling system in plants. Plant Cell 16: 1979 - 2000 . 100 Chapter 3: PLAFP and its Role in Drought Tolerance and Long - Distance Lipid Signaling 3.1 Intro duction T he need for an increased productivity and yield in food crops is essential to feed the progressively growing world population. One factor that is impacting the production of sufficient food is climate change. Drought, heat, and cold are a few examples of environmental stresses that affect crop growth and production. Thus, in order to continue to be able to produce and provide sufficient food and fuel for an increasing world population, we need plants that exhibit accelerated growth, higher grain yield, and/or more resistance to stress. One avenue which could lead to preventing or lowering crop loss due to abiotic stress is to better understand the changes that occur in plant development, resulting in their adaptation to these conditions. Since plants are sessile, they cannot avoid harmful conditions within their surroundings. To cope, they had to evolve mechanisms to detect c hanges in their environment, communicate these changes throughout the plant , and adjust development accordingly (Thomas and Vince - Prue, 1997). As a result, plants evolved two long - distance transport systems: the xylem for water and mineral distribution f rom the roots to other organs of the plant, and the phloem. The phloem plays a crucial role in assimilate and nutrient transport, pathogen response, and plant growth and development. The phloem consists of mainly three components , the phloem parenchyma, the sieve element , and the companion cell. The latter two are essential for long - distance transport and signaling. To facilitate transport of assimilates, nutrients, and signaling molecules, the organelles within the sieve elements disintegrated early in phloem development leaving it with only a plasma membrane and a thin 101 cytoplasm which contained ER, phloem - specific plastids, and a n enlarged mitochondria (v an Bel and Knoblauch, 2000; Turgeon and Wolf, 2009). Additionally, the ER which is found near the plasmodesmata, connects the sieve elements to the companion cells (Lucas et al., 2013). Since the sieve elements do not contain the machinery to synthesize proteins and many other molecules, it relies on the companion cell for the production and transfer of these compounds. For long - distance movement, these molecules need to be transported into the sieve element via plasmodesmata, moved along the sieve tube, and are either perceived at their target location or transported out of the phloem. Over the year s, t he view of phloem function has progressed from that of a simple assimilate transport system to a trafficking pathway for stress signals and developmental regulators in the form of small molecules (Chen et al., 2001; Corbesier et al., 2003), peptides/pr oteins (Haebel and Kehr, 2001; Hoffmann - Benning et al., 200 2; Giavalisco et al., 2006 ; Lin et al., 2009; Benning et al. , 2012), mRNAs (Ruiz - Medrano et al., 1999; Pallas et al., 2013; Hannapel et al., 2013), microRNAs (Pant et al., 2008; Buhtz et al., 2010; Varkonyi - Gesic et al., 2010; Rodriguez - Medina et al., 20 11), nucleic acids (Citovsky and Zambryski, 2000; Haywood et al., 2005; Ding et al., 2003; Yoo et al., 2004), and lipids (Madey et al., 2002; Behmer et al., 2011 ; Guelette et al., 2012; Benning et al., 2012; Tetyuk et al., 2013). Thus, because the contents and the function of the phloem has become so complex, phloem et al., 1998). Therefore, the study of signaling co mpounds within the phloem is essential for the understanding of the distribution of environmental cues throughout the plant. The lab identified over 121 distinct metabolites including 16 fatty acids/esters within Ara bidopsis phloem exudates using GC - MS (Gu elette et al., 2012). In addition, further analysis 102 of the phloem lipidome using liquid chromatography mass spectrometry using collision induced dissociation (LC - MS - CID) revealed the presences of oxylipins, phospholipids, and di - and triacylglycerols within the exudates. The presence of these lipids within phloem exudates implies they are not only important in intracellular signaling, but also may be involved in long - distance signaling. To determine a potential role of lipid signaling and/or metabol ism in the phloem exudates, we analyzed the proteins present with in the phloem sap. EDTA - facilitated collection of phloem exudates followed by SDS - PAGE and LC - MS analysis, resulted in the identification of 65 proteins within Arabidopsis phloem sap. Among those proteins, 14 were involved in carbohydrate metabolism, 16 identified with stress, pathogen, or hormone response, six were nucleotide - , DNA - , or RNA - binding proteins, 11 were associated with the plastid, 11 contributed to protein modification, foldin g, turnover and stability, and protein - protein interaction, and 11 were characterized as lipid/fatty acid - binding proteins (Guelette et al., 2012). Proteins in the latter group included putative proteins involved in lipid/fatty acid metabolism, binding, a nd signaling (Table 3 . 1) . These previously identified proteins provided strong evidence that long - distance lipid signaling could occur within the phloem. This concept is not unique to plants but has been observed in other biological systems. For example, other aqueous environments like the human bloodstream contain a variety of lipids, many of which are important for human health. Lipi ds within the bloodstream typically bind to proteins which can serve multiple purposes such as transport to other tissues for storage, use, modification, or degradation. The most recognized example of a lipid found within the blood is cholesterol. Choles terol is either bound 103 Table 3. 1 : Putative Lipid - Binding Proteins Identified via Proteomics in Arabidopsis and additional plant species such as rice, canola, cucurbits, lupine, and Perilla . Genes encoding most of these proteins are expressed in companion cells (CC; Mustroph et al., 2009). Protein name Accession Number Molecular Weight Possible Function (i - iv) Expressed in CCs Lipid Identified First Report of Phloem Localization DIR1 At5g48485 ~10kDa i - iii Aza, G3P*, DA?; Shah, 2014 Maldonado et al., 2002 ACBP6 At1g31812 10kDa i X ACBP6: PC; Chen et al., 2008 Hayashi et al., 2000 Annexin At1g35720 36kDa ii X Phosphlipids (PL); Rescher and Gerke, 2004 Barnes et al., 2004 GRP17/oleosi n At5g07530 53kDa ii X PL; Tzen and Huang, 1992 Guelette et al., 2012 Put. lipase At4g16820 58kDa i Guelette et al., 2012 GDSL - lipase At1g29660 40kDa i X (DAG, PA; unpub) Guelette et al., 2012 PLAFP At4g39730 20kDa i - iv X P A ; Benning et al., 2012 Guelette et al., 2012 Aspartic protease At4g04460 56kDa i X Guelette et al., 2012 14 - 3 - 3 protein At1g22300 At2g10450 28kDa, 9kDa iii X Guelette et al., 2012 Major latex protein At1g70890 18kDa ii, iv X Giavalisco et al., 2006 Bet v1 allergen At1g23130 18kDa ii, iv X Guelette et al., 2012 PIG - P - like protein At2g39435 50kDa iii (PS, PA, PIP; unpub) Guelette et al., 2012 Flowering locus T At1g65480 22kDa ii, iv X P C ; Nakamura et al., 2014 Giavalisco et al., 2006 104 to low - density lipoproteins (LDL) and transported throughout the bloodstream for uptake in to cells and incorporation into membranes; or it is bound to high - density lipoproteins (HDL) or within chylomicrons and moved to the liver for degradation (for a su mmary see Nelson et al . , 2008). Thus, the type of protein to which a lipid binds not only determines where it will be transported but also its final outcome . In addition, the plant phloem c ontains many molecules that, though less complex than phospholipids, are still lipophilic. These contain lipophilic plant hor mones ( ABA, gibberellins, and a uxin) , oxylipins like methyl - jasmonate and its precursor OPDA, and other compounds such as azelaic acid and dehydroabietinal . These move in the phloem as either free molecules or in the form of methyl - , amino acid - or glucose esters (for a summary see Barbaglia and Hoffmann - Benning, in press; Hoffmann - Benning AOCS lipid library). In order for lon g - distance phospho lipid signaling to occur within the phloem, the plant may require lipid - binding proteins of various potential functions: (i) they could mediate the release of the lipid into sieve element by participating in either the cleavage of the li pid so it can be transported to the sieve element or in its release from the membrane, (ii) they could bind specific phloem lipids, facilitating their solubilization in the exudate as well as their targeted transport, (iii) they could be part of a receptor which senses the lipid ( - signal) and transfers it out of the sieve element, or (iv) the protein could be the signal itself, with its activity controlled by the bound lipid (Figure 3. 1 ) . In chapter 2 , we described three of the putative lipid - binding prote ins, one that could potentially participate in the first three functions: GDSL - lipase, PLAFP , and PIG - P. GDSL - lipase could aid in releasing the lipid into the phloem, PLAFP may act in the solubilization and transport of the lipid through the phloem, and PIG - P may potentially act similar to a receptor, binding the lipid once it has reached its final destination. 105 Figure 3.1: Model of Possible Protein - L ipid A ction in Long - Distance Signaling . A protein is induced by an environmental signal or during particular developmental stages. It cleaves a lipid from the membrane provoking a subsequent lipid - binding protein to bind specifically to that lipid and transports it via the phloem to its target destination where it will bind to a receptor prote in. 106 This chapt er will focus on the functional characterization of PLAFP , including its lipid - binding properties , its expression pattern throughout the plant, and its response to abiotic stress, as well as its potential role in long - distance lipid signaling . Phloem lipid - associated family protein (PLAFP) is a small lipid - binding protein with unknown function. It contains a PLAT/LH2 domain, which is thought to mediate interaction with lipids or membrane - bound proteins (Benning et al., 2012). This protein ap pears to be stress - induced and has been found, by our lab, to bind phosphatidic acid (PA; Benning et al., 2012; Refer to Figure 2. 2 ). Both PLAFP and PA have been found within the phloem exudates, which is important because it indicates that both the lipid and its protein partner are present in the phloem . In addition, this suggests PA is transported within or into the phloem while bound to PLAFP, however, whether or not PLAFP has a role in loading/unloading the lipid into and out of the phloem, acts as a receptor or a transport molecule, and has a true signaling function remains to be determined. PA has many important functions in the plant. It is a central intermediate in lipid biosynthesis ( Ohlrogge and Browse, 1995; Testerink and Munnik, 2011 ), it affects membrane curvature and can thus influence vesicle formation/fusion ( Kooijman et al., 2003, 2005 ), and it serves in intracellular signaling. For example, PA production is triggered in response to several biotic and abiotic stresses, such as pathoge n infection, drought, salinity, wounding, cold, cell death, and oxylipin production (Xue et al., 2009; Wang et al., 2007; Hong et al., 2010; Kim et al., 2013). On a developmental level , PA plays a role in regulating normal plant growth and d evelopment, ma inly in root and pollen tube growth (Kim et al., 2013). PA has been shown to 107 bind to several proteins, including transcription factors, protein kinases, lipid kinases, protein phosphatases, and proteins involved in vesicular trafficking and cytoskeletal r earrangements (Guo et al., 2011). Thus, PA can directly interact with proteins to mediate signals. The si g naling path relevant in this study is the role of PA in ABA signaling . In response to ABA, PA is produced omatal closure under drought conditions. When the plant senses water is growing scarce and PA levels are increasing, PA then binds to ABI1, a PP2C, and tethers it to the plasma me m brane to prevent the negative effect of ABI1 on ABA signaling (Lu et al., 2013). In turn, ABA lead s to activation of SNF1 - related kinase 2 (SnRK2) which mediates downstream signaling where PA binds and activates sphingosine kinase which leads to the phosphorylation of phytosphingosine to phytosphingosine 1 - phosphate (phyto - S1P for the regulation of stomatal closure (Guo et al . , 2012) . S ince PLAFP binds PA and both have been identified within the phloem exudates, we propose that PLAFP and PA, either individually or jointly play a role in long - distance lipid signaling, and PLAFP may participate or aid in stress tolerance. 108 3.2 Material and Methods 3.2.1 Plant Growth Arabidopsis seeds were sterilized (20% bleach and 0.5% Triton X - 100 ) for 15 min and wash ed 6 times with sterile, deionized water) and plated on MS, 1% sucrose, and 0.6% agar with or without antibiotic depending on the experiment. Next, plates were transferred to 4°C for two days before being placed into a Percival growth chamber; 22°C day/18 ° C night temperature in a 12 - h photoperiod with 60% relative humidity, and a light intensity of 120 mol photons m - 2 s - 1 . 3.2.2 Gene Expression Analyses Quantitative RT PCR analysis was performed on total RNA extracted from 2 - 3 week old Arabid opsis seedlings or leaves from 5 week old plant s following the instructions provided by the RNEasy Plant Mini Kit (Qiagen). The first strand was synthesized by oligo dT primers using SuperScript First Strand Synthesis III system (Invitrogen). The resulta nt cDNA was then used for quantitative real - time PCR (qPCR) using SYBR Green (Affymetrix) as the detection probe. Primers and conditions for all the RT - PCR and qPCR are outlined in Supplementary Table 1 (See Appendix) . 3.2.3 GUS Reporter Gene Construct, Arabidopsis Transformation , and GUS Assay The 1 Kb region upstream of the transcription initiation site of PLAFP was PCR a mplified using the primers indicated in Supplementary Table 1 end, respectively. The PCR product was cloned into pGEMT - E asy vector (Promega) and subcloned into pBI121 (Clontech) vector (from which 35S promoter had been removed by 109 HindIII and XbaI digestion) to generate the clone, P LAFP1KbPro:GUS which was then tran sformed into Agrobacterium tumifaceans strain C58C1pGV2260 by electroporation. Positive transformants were selected by Kanamycin resistance and further confirmed by colony PCR using the same set of primers mentioned above, and that was use d to transform Ar abidopsis Col - 2 by floral dip method (Clough and Bent, 1998). Transgenic lines were selected by Kanamycin resistance and the incorporation of the transgene was confirmed by PCR, using promoter specific primer and GUS specific primer as detailed in Suppleme ntary Table 1. The G US assay was performed as described (Martí et al. 2010) using GUS staining solution: 50 mM sodium phosphate buffer, pH 7.0, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 0.1% triton X - 100 and 1mg/ml 5 - Bromo - 4 - chloro - 3 - i ndoxyl - beta - D - glucuronide cyclohexylammonium salt (Gold Biotechnology). Seedlings were observed under a light microscope (Nikon Eclipse C i ). 3.2.4 Fluo rescent Reporter Gene Construct for PLAFP The coding sequence of PLAFP was PCR amplified using the primers indicated in Supplementary Table 1, which PLAFP, respectively. The PCR product was cloned into pGEMT - Easy vector (Promega) and subjected to the Gateway cloning system where the resultant DNA product was sub cloned into the donor vector pD O N R 207 followed by the destination vector, pEarleyGate 101 to generate the clones PLAFP1KbPro:YFP, which was then transformed into Agrobacterium tumifaceans strain GV3101 by elec troporation. Positive transformants were selected by Kanamycin resistance, further confirmed by colony PCR using the same set of primers mentioned above, 110 and used to transiently transform Nicotiana tabacum . Leaf samples were then observed under confocal microscopy (Olympus ; YFP Emission wavelength: 530 - 555nm, excitation: 515nm; RFP emission wavelength: 605 - 630nm, excitation: 559nm; CFP emission wavelength: 475 - 500nm, excitation: 458nm ) to detect the subcellular localization of the proteins. 3.2.5 Ab iotic Stress Analysis Fourteen to sixteen day old w ildtype Col - 2 seedlings were carefully transferred to a hydroponic system containing water, covered with a clear plastic dome , and left to acclimate during 24 h, under laboratory conditions ( 22°C ) . After this period, 300 mM Mannitol, 150 mM NaCl, 100 µM of ABA, or 30% PEG were added to the system. Seedlings were harvested at 0, 1, 2, 5, 8, 10, 12, and 24 hours post stress (hps). Asterisks indicate statistical differences determined by - test (* P < 0.01). 3.2.6 Harvest of Phloem Exudates Phloem exudate from 6 - 7 week old Arabidopsis plants were collected using the EDTA - facilitated method described in Guelette et al., 2012, Benning et al., 2012, and Teytuk et al., 2013. In short, leaves w ere cut at the base of petiole and placed in a petri dish containing 2 - EDTA, pH 7.0. Fifteen leaves are collected, stacked on top of one another, recut under EDTA, L of K 2 - solution was discarded, the petioles were washed thoroughly, and placed in a new autoclaved Millipore water. Exudates were collected for 6 111 3.2.7 Lipid Analysis within Phloem Exudates Phloem exudates were phase partitioned against chloroform:methanol (1:1, v/v), concentrated under N 2 and submitted to liquid chromatography mass spectrometry ( LC MS ) . Lipid analysis was performed using a Waters LCT Premier mass spectrometer (LC - TOF MS) with multiplexed CID (collision - in both positive and negative ion mode after separation on a C18 column (5 cm X 2.1 m using nitrile:isopropanol; 1:2) . I nternal standard s of di14:0 PA and di12:0 phosphatidylcholine ( PC ) w ere used at a concentration of 0.16 nmol. Data are representatives of three to four biological replicates. 3.2.8 Protein Expression and Purification A cDNA clone for PLAFP (At4g39730), U21720, was obtained from Arabidopsis Biological Resource Centre, Ohio State University (Columbus, OH, USA). Th e coding region of PLAFP, excluding the 69 nucleotide region encoding the 23 amino - acid predicted signal peptide, was PCR amplified using the primers shown in Supplementary Table 1 which introduced Nde I sites at both ends of the PCR product. The PCR product was cloned into pGEMT - Easy vector (Promega), and subcloned into pET15b expression vector (Novagen) using Nde I the site to generate the expression clone, pET15b - GDSL. E. coli host strain OrigamiB(DE3 )pLysS (Novagen) was transformed with pET15b - GDSL and the transformants were selected by Ampicillin (Amp), Kanamycin (Kan), Chloramphenicol (Cm), and Tetracycline (Tet) resistance. Protein expression was induced by adding IPTG up to the final concentratio 112 include the clear lysate, flow thro ugh, wash fraction, and elution fractions. E lution fractions 2 - 5 contain ing purified protein were pooled. KH 2 PO 4 (Lu and Benning, 2009) using a PD10 column (GE healthcare). 3.2.9 Protein Lipid Overlay Assay The phospholipid strip was purchase d from Echelon Biosciences Incorporated o r prepared by di 18:1 PE, PA, PC, PS, PG, PI; Avanti Polar Lipids) onto a Hybond - C membrane (GE Healthcare). The protein lipid overlay assay was performed as described ( Benning et al., 2012; Awai et al., 2006). Briefly, the lipid strip was blocked with the followed by overnight 4°C incubation in the blocking buffer containing purified PLAFP - His6 with Anti - polyHis mouse monoclonal antibody (Sigma) at a temperature. The membrane was washed twice with TBST and incubated in the blocking buffer with horseradish peroxidase (HRP) - conjugated goat anti - mouse antibody (BioRad) at a 1:10,000 detection was de termined using Pierce ECL Western Blotting Substrate (Thermo Scientific) and HyBlot CL Autoradiography Film (Denville Scientific Inc.) on a Mini Medical Series Imager (AFP Imaging Corp. ). 113 3.2.10 Liposome Binding Assay Liposomes were prepared using di 18:1 PC and di 18:1 PA , or a mixture of PC and PA, fol lowing the method described in Awai et al., a incuba g pellet was washed twice with ice - cold TBS and separated using SDS - PAGE. Western blot analysis was performed using anti - His and HRP - conjugated goat anti - mouse antibodi es and chemiluminescence detection system described previously. 114 3.3 Results and Discussion This study focuses on PLAFP which may play a role in stress - related long - distance signaling. To participate in long - distance signaling, PLAFP must perform at least one of the following tasks: cleave the lipid from the membrane, solubilize and transport it throughout the sieve element, a nd/or act as a receptor for the lipid signal at its target destination. To participate in long - distance phospholipid signaling, PLAFP needs to : 1) Bind a phloem phospholipid 2) Be expressed in the plant vasculature 3) Be localized in a pattern allowing for movement through plasmodesmata 4) Be induced in response to abiotic stress 5) Increase tolerance to this abiotic stress 6) Modify the phloem lipid profile 7) Is mobile itself or mediate mobility of its lipid - ligand 3.3.1 PLAFP Binds a Phloem Phospholipid Previous work from the laboratory ha s shown that PLAFP binds PA ( Refer to Fig ure 2. 2). PLAFP is annotated as a lipoxygenase , h owever, when the PLAFP structure was predicted based on known lipoxygenase structures and superimposed on those, it became clear that PLAFP co nsists of only the PLAT domain but not the catalytic domain (Figure 3 .2 A) . Thus PLAFP is not a lipoxygenase . Many lipoxygenases function as polymers, hence , the PLAT domain is essential in protein - protein interaction. Since PLAFP only consists of the PLAT domain, that could 115 indicate that it interacts with other proteins, for examples receptors. Its small size (20 kDa) should allow for movement into and throughout the sieve elements. In addition, the interaction of li poxygenases with lipids occurs in the catalytic domain, while our data show that the PLAT domain is capable of binding lipids as well. This suggests that PLAFP may either anchor to the membrane using protein - lipid interaction or transport lipids. Visual molecular dynamics modeling visualizes both options (Fig ure 3. 2 B - D ; designed ). Fig ure 3. 3 B shows a model of PLAFP - PA interaction where the negatively charged PA headgroup electrostatically interacts with a conserved arginine while the a cyl chains are embedded in a hydrophobic gro o ve. Fig ure 3. 2 D shows interaction of PA with the same arginine, however, in this case the acyl chains are extended away from the molecule. The latter is thermodynamically unfavorable in an aqueous environment and would likely suggest tethering of PLAFP to PA embedded in the membrane, while the former protein - ligand interaction suggests a phloem - mobile molecule. Future NMR analysis and crystallography will clarify the nature of the PLAFP - PA interaction. 3.3.2 PLAFP is E xpressed in the V asculature Using the PLAFP1KbPro:GUS construct, we were able monitor the activity of the PLAFP promoter , which contains several transcription factor binding sites such as TATA box and ABRE - like sequences , 1kb upstream of the transcription start site within Arabidopsis seedlings. PLAFP promoter activity is associated the vasculature of the young leaves and roots of 2 - 3 week old Arabidopsis seedlings as well as in the hydathodes (Figur e 3. 3 A,B ). Expression was also ob served in the leaf meristem, pollen grains, and vascular bundle s connected with the seeds 116 Figure 3 .2 : Model Prediction for Possible PLAFP - PA Interactions. PLAFP is consists of the PLAT domain (blue; A) which is predicted to interact with lipids or membranes suggesting PLAFP can either anchor to the membrane using protein - lipid interaction or transport lipids . Panels B displays possible PLAFP - PA interaction where the PA headgroup i nteracts with a conserved arginine with the acyl chains embedded in a hydrophobic groove . Panel D shows interaction of PA with the same arginine, but the acyl chains are e xtended away from the molecule and likely integrated in the membrane . - - MARRDVLLPF LLLLATVSAV AFAEDDPD CV YTFYLRTGSI WKAGTDSIIS ARIYDKDGDY IGIKNLQAWA GLMGPDYNYF ERGNLDIFSG RAPCLPSPIC ALNLTSDGSG DHHGWYVNYV EITTAGVHAQ CSTQDFEIEQ WLATDTSPYE LTAVRNN CPV KLRDSVSRVG SEIRKKLSWV V 117 Figure 3.3 : PLAFP Pro moter Activity Using GUS Reporter. Two - week old Arabidopsis seedlings containing the 1kb region upstream of the transcription initiation site of PLAFP were generated. GUS expression was observed in three independent lines in the root and leaf vasculature (A - C), the vascular bundles (D), the pollen (E), and the vascular bundles attached to the seeds (F). A C B F E D D 118 (Figure 3. 3 C - F ). Expression within the root vasculature as well as the leaf and silique veins supports our hypothesis that PLAFP is present within the phloem and could be involved in long - distance signaling . PLAFP expression in the early stages of seedling development (young leaf veins, roots, hydathodes, leaf meristem ), indicates PLAFP may play a role in early plant development, specifically in the development of the vasculature. Furthermore, since promoter activity in the root is enhanced in response to the signaling molecule abscisic acid (ABA ; see Figure 3. 3 above ), PLAFP might further participate in the signaling or response to ABA - mediated abiotic stresses. For example, it could signal enhanced root growth in response to drought. Several plant hormones aid in vasc ular development such as cytokinin and ABA, however, auxin is best - known for its role in this regulation. The main form of auxin in plants is indole - 3 - acetic acid (IAA), a small molecule that regulates changes in gene expression by targeting transcription al repressors for degradation (Dharmasiri et al., 2005; Spicer et al., 2013). At the cellular level, auxin acts as a signal for cell division, expansion, elongation/extension, and differentiation during the course of the plant life cycle. At the whole - pl ant level, auxin plays an important role in vascular development, lateral root initiation/formation, apical dominance, leaf growth/expansion, tropism, and senescence (Guilfoyle et al., 1998; Hagen and Guilfoyle, 2002; Gonzalez et a., 2012). Important comp onents involved in auxin signaling are the auxin response factors (ARFs), which are transcriptions factors that aid in auxin expression , and auxin response elements (AREs), which are cis - elements found within the promoter of auxin - response genes that promo te auxin expression (Hagen and Guilfoyle, 2002). These elements are usually bound by ARFs to regulate auxin response. In normal development, vascular tissues differentiate from procambial cells, which can be identified in young organ primordia . Studies 119 have shown that auxin plays a role in vascular differentiation during normal leaf development since the auxin expression patterns of Arabidopsis rosette leaf primordia precede the sites of procambial differentiation (Mattsson et al., 2003). In embryo , shoot , and root meristem development, the regulation of auxin transport is the most important aspect of the establishment and maintenance of auxin distribution patterns. Auxin transport can be easily visualized through the expression and localization of the Arabidopsis PIN - formed family of auxin efflux carrier proteins. PIN proteins have been shown to be crucial for determining the direction of auxin flow within roots. Vascular differentiation occurs at sites of maximum auxin response and that an auxin maxima is formed via polar transport of auxin to a specific location within the leaf primordia. The polar transport of auxin then lead s to the formation of secondary and tertiary leaf veins as the leaf develops (Mattsson et al., 2003). Auxin has also bee n shown to interact with other plant hormones, such as cytokinin in the regulation of root vasculature development. Auxin and cytokinin exhibit a mutually inhibitory interaction in root vascular patterning. This interaction occurs within the protoxylem a nd procambial cells. Normally in the procambium, auxin levels are high and cytokinin levels are low, while in the protoxylem, cytokinin concentrations are high and auxin concentrations are low. When auxin flows laterally between the protoxylem and the pr ocambia, a mutually inhibitory mechanism occurs where the initial amounts of auxin and cytokinin flow into the cell can spread into two well defined areas of elevated hormone response. Thus, the phenotype of the vasculature can be affected by auxin and cy tokinin concentrations . In addition to cytokinin, ABA also affects different aspects of root development. Auxin promotes lateral root and root hair growth, but inhibit s primary root elongation (Calderon - Villalobos et al., 2010 ; Overvoorde et al., 2010 ) . 120 ABA , on the other hand, inhibits lateral root growth, but promotes root growth when the plant is experiencing a water deficit (Zhang et al., 2010) . Since we see similar expression patterns from PLAFP in terms of the vasculature, it would be interesting t o see if there is any crosstalk or interaction with auxin. We could use DR5 or even the SUC2 promoter, which is specific to the phloem, to detect similar movement and expression patterns within both the root and leaves. 3.3.3 PLAFP Localizes to the Cell Periphery Determination of where PLAFP localize s within the cell was established by generating a protein construct containing a fluorescent tag at the C - terminus of the protein. Th is construct ( 35S - PLAFP - YFP ) w as transiently expressed within Nicotia na tabacum to detect where PLAFP localize d within the cell . Localization of PLAFP wa s at the periphery of the cell. Despite possessing a signal sequence, PLAFP does not exhibit very strong association with the ER, which implies PLAFP most likely transver ses the ER but does not remain associated with it. This is further substantiated by its lack of a HDEL/KDEL ER retention signal ( Refer to Figure 2.4B ; Nelson et al., 2007 ) . The fusion protein displayed a dotted pattern all around the cell periphery similar to Golgi and plasmodesmata. Thus, to make sure what we were seeing wa s not either the Golgi or plasmodesmata , we colocaliz ed PLAFP with these two cell markers and found that PLAFP does not appear overlap with the Golgi , but will need to be repeate d (data not shown) and there is partial overlap but no coexpression with the plasmodesmata l marker, a viral movement protein (Figure 3. 4 ) . Immunogold labeling will be used to confirm association with plasmodesmata. Colocalization of PLAFP with a plasma membrane marker ( Nelson et al., 2007) shows that PLAFP is present on either side of the plasma membrane and in some cases 121 Figure 3.4 : Transient Localization of PLAFP in Tobacco. PLAFP is localized in a punctuate pattern the cell periphery. No c oex pression with viral movement protein (VMP) could be observed. 122 es membrane ( Refer to Figure 2. 5 B ) . The above findings indicate that PLAFP could move through the plasmodesmata to enter the phloem and further support a possible function in long - distance signaling . P lasmodesmata are pores within the membrane that form the (symplastic) connections between cells and allow not only for intercellular movement but are also essential for the entry of proteins and signals into the sieve element . 3.3.4 Abiotic Stresses Effect PLAFP Gene Expression Long - distance , phloem - mediated signaling is one mechanism which plants communicate with themselves . This is especially true when weather conditions or other environmental changes occur. Since we know from our lipid - binding assays that PLAFP bind s PA, it is possible that PA or the PLAFP - PA complex participate in long - distance lipid signaling and thus, are very important for the plants ability to survive and adapt to their surroundings. PA is a signaling molecule that mediate s various cellular activities through different modes of action , such as binding to its targeted proteins to increase or inhibit their activities, acting as a membrane anchor during the formation of s ignaling proteins, or act as substrate for the production of other lipid regulators such as lysoPA, free fatty acids, DAG, and DGPP; and regulating membrane trafficking and biogenesis (Wang, 2004). Additionally, PA can activate proteins, directly or indir ectly, by guiding them to the location needed to perform their function, PA could act with other lipid second messengers in order for protein activation and transport, and PA can bind proteins that have undergone post - translational modifications to facilit ate target regulation (Testerink and Munnik, 2005; Wang et al., 2006, 2007). Since PLAFP was shown to bind PA, and PA is produced in response to different forms of osmotic/water stresses, such as 123 dehydration, drought, salinity, freezing, as well as with treatment with the stress hormone abscisic acid (ABA; Wang et al., 2007), we wanted to see how these treatments would affect PLAFP expression. Two - week old seedlings were plated on MS plates with kanamycin selection for two weeks before being trans ferred to a hydroponic system ( Refer to Figure 2.7 ). After 24 hours of acclimation , seedlings were introduced to four different forms of abiotic stress: osmotic in the form of mannitol, salt stress in the form of NaCl , ABA, a plant hormone stimulated by drought/osmotic stress, and PEG, a water stress mimic ( Refer to Figure 2.8A ). Samples were then collected at various time points over a 24 hour period. PLAFP displayed a slight, but insignificant increase in expression under osmotic stress (mannitol) and a decrease in expression under salt stress, in comparison to the control. On the other hand, we see a drastic increase in PLAFP expression when stressed with ABA and 30% PEG ( Figure 3. 5 ) . The increase in expression we observe could indicate that PLA FP may participate in the signaling of environmental conditions that involve a limited or depleted water supply. Thus if plants contain ed higher levels of PLAFP, then they could live longer under these conditions by conserving the water supply the current ly possess. Upregulation of PLAFP under ABA stress also indicates that PLAFP may contribute to the ABA signaling pathway. We believe this participation is in association of PLAFP with PA. Under the ABA pathway, drought or osmotic stress leads to ABA bin d ing to its receptor, PYR/PYL/RCAR. An increase in ABA leads to the , which in turn produces more PA. PA prevents abscisic acid insensitive 1 (ABI1), a protein phosphatase 2C, from binding to the ABA receptor by tet hering it to the membrane. This allows ABA to activate downstream kinases (S n RK2) to 124 Figure 3.5 : Effect of Abiotic Stress on PLAFP. Two week old Arabidopsis seedlings were transferred to hydroponic c ulture and submitted to ABA and the water stress - mimic PEG . Values represent mean and standard error of PLAFP gene expression levels after 0, 1, 2, 5, 8, 12, and 24 hours of 3 - 6 biological replicates as determined using qPCR (three technical repl icates per biological replicate ). Values were normalized to 18S mRNA. Gene expression was determined using quantitative RT - PCR. The asterisks indicate significance a p<0.01 via student t - test . 125 induce ABA - activated gene expression and to promote stomatal closure , thus , conserving water levels within the p lant. In addition, PA has recently been found to be bound by the MYB transcription factor, WEREWOLF. This interaction causes WEREWOLF to translocate in to the nucleus where it affects root hair patterning (Lu et al., 2013) . Thus, because PLAFP binds PA, we propose that PLAFP may act together with PA to help mediate or perhaps enhance the closure of the stomata allowing that plant to retain larger volumes of water during water - deficient conditions. The response PLAFP exhibits under drought - mimic and water stress conditions indicates PLAFP - overexpression could be very important for crop plant viability in a water deficit making them more drought tolerant. In the abiotic stress expression study described above, we found that PLAFP expression was significant ly upregulated under ABA and 30% PEG treatments. Since both of these stresses are water - /drought - related, we wanted to determine if actual soil drought stress had the same or a similar effect on PLAFP expression. To accomplish this, we subjected five - wee k old wildtype plants to drought for six days. Samples were collected when the plants were well - watered (Day 0) after which the bottoms of the pots were batted dry and were deprived of water for the next six days. Leaf and root samples were collected eac h day from each of the plants ( Figure 3. 6 ) . 3.3.5 Effect of D ifferent PLAFP E xpression L evels on P lant P henotype To determine if PLAFP had an effect on plant phenotype we generated ten overexpression, two complementation, and two mutant lines. The complementation line s were generated by reintroducing the PLAFP into the knock - down line. 126 Figure 3. 6 : Effect of Soil Drought on PLAFP in Leaves and Roots. PLAFP gene expression was monitored in five week old wildtype Arabidopsis seedlings subj ected to drought for six days. Samples were collected in 3 biological replicates with three technical replicates per biological replicate. Values were normalized to 18S mRNA. Gene expression was determined using qPCR. 0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 6 Relative Gene Expression Days Effect of Drought on PLAFP in Leaves Control Drought 127 plant. When the plants were grown up, one of the lines contained a phenotype similar to the wildtype and the other the other complementation line appeared to be similar to the overexpression line. When we compared the physical traits (size and root length) of the plants together, we obse rved the overexpression and complementation lines were larger than the wildtype and mutants (Figure 3.7 A - C) . The mutant line s obtained (28 and 54c) w ere knockdown line s. T he mutants were generated from a T - DNA insertion (one in the second exon and one in just upstream of the first exon of PLAFP , and h omozygous knockdown lines contain ed approximately 30% expression and displayed very low germination rate s . These seedlings had a better survival rate when grown under humid conditions. These plants are smaller in size and had shorter roots in comparison to wildtype and overexpressor plants ( Figure 3. 7 A - C ; 54c not shown). Plants that encounter drought conditions must cope by closing their stomata to conserve water stor es as well as extend their roots further into the ground in search for more water. Thus, we wanted to determine if the root length for the overexpression, complementation, wildtype, and mutant lines correlated with the levels of PLAFP they contained and t hus their susceptibility to survive drought. To do so, we measured the roots of each line . Results revealed that the roots of the overexpression lines were significantly longer than the wildtype, complementation, and mutant lines. The roots of the knock down mutant were the shortest and the complementation line displayed roots that were slightly longer than the wildtype. These observations support the phenotype exhibited by these plants and correlate with the hypothesis that PLAFP affects the plants abil ity to cope under water deficient conditions. Since the overexpression lines contain greater levels of PLAFP, the plant can survive 128 Figure 3.7 : Effect of PLAFP Expression on Development. Plant growth in dependence of PLAFP expression (28: KD - line; OX: overexpressor; Comp: complementation line) (A). Comparison of root length on day si x after seed imbibition in wildtype (WT), knock down (KD 28), co mplementation (Comp) and overexpression lines (OX); measurements were obtained from 8 - 11 seedlings per line; * and # indicate statistically significant differences compared to wild type with p < 0.05, and p < 0.07, respectively, as determined by student t - test (B). PLAFP wildtype, knockdown, and overexpression line root lengths were measured over an 11 day time course. Measurements were obtained by 4 - 6 seedlings per line (C). 0 2 4 6 8 10 12 14 16 18 20 Day 4 Day 5 Day 6 Day 8 Day 11 Root Length (mm) WT 1 28-1 OX 2-1 OX 1 A B C * * * * * * * * 129 drought more easily; their roots are longer in order to search for water deeper within the soil. The opposite is true for t he mutant line where the roots we re shorter. This makes sense since this line only contains 30% PLAFP expression, thus , the influence of PLAFP i n water scar ce conditions is less effective. From these results, w e can conclude that the amount of PLAFP affects the overall size of the plant and can influence its survival capabilities under stress. 3.3. 6 PLAFP Expression E ffects Drought Tolerance Experiments shown above indicate that PLAFP is induced in response to the drought mimic PEG, the drought signal ABA , and drought itself. To understand if PLAFP overexpression confers drought tolerance , we subjected f ive - week old wildtype, overexpression, mutant, and complementation PLAF P Arabidopsis seedlings to drought for six days. The plants were well watered before the bottoms were batted dry and water withheld for six days. Within five days of water withdrawal, PLAFP knockdown lines had died. Wildtype plants died on day six, whil e overexpression lines survived until day seven (Figure 3. 8 ) . These results suggest that PLAFP is critical in the plants tolerance to drought conditions. When PLAFP levels are very low, as is displayed in the 28 and 54 knockdown lines, the plants do not survive very long without water. When normal levels of PLAFP are expressed, like in the wildtype line, the plants last about 3 - 4 days until the effect of drought really starts to take its toll on the plant survival. Lastly, when PLAFP is overexpressed, we observe the plants surviving for 5 - 6 days and eventually succumbing to the drought after 7 - 8 days. The complementation lines died sometime between the wildtype and the overexpression lines. An interesting observation we noted in wildtype seedlings was an increase in gene expression around day four of drought (see Fig ure 3. 6 above) . 130 Figure 3.8 : Effect of PLAFP Phenotype under Drought Conditions. Five week old wildtype, overexpression, mutant, and complementation PLAFP Arabidopsis seedlings were subjected to drought for six days. - - - - - - - - - 131 What this spike in expression could mean is that at this time , the plant has utilized all of the water in the soil and signals to trigger developmental changes . Based on the phenotype exhibited by the various plant lines, the plants size also correlated with the duration of their survival. Since the 28 and 54 mutant lines have smaller roots than th e wildtype, one can infer that these plants cannot take up as much water from the roots . The complementation and overexpression lines displayed a phenotype of an overall much larger plant containing very large leaves and long, branched roots. This phenot ype is very advantageous when faced with drought conditions because the longer roots allow for the plant to dig deeper and wider in the soil in search of water in addition to the general fact that soil tends to be more saturated further down into the groun d. In conjunction with longer roots, larger leaves allow the plants to generate more assimilate products from photosynthesis and the larger leaves also can store more water by closing their stomata during water - deficit conditions. At the same time, the m ore highly branched trichomes in the overexpression lines lead to reduced transpiration and thus higher water use efficiency. Together with the larger roots that allow the plant to draw up more water and nutrients from the ground , this promotes higher drought tolerance and greater seed yield ( Figure 3. 9 ). Other studies have shown a similar pattern of survival . For instance a study in cell - specific promoter, un der hyperosmotic stress conditions including drought and high salinity, the plants displayed decreased water loss and an increase in biomass ( Lu et al., 2013) . In addition, they also generated more seeds under drought conditions. These results indicate t hat guard cell - drought tolerance and crop yield under drought conditions . This could be due to the mediated synthesis of PA which 132 Figure 3.9 : Seed Yield Comparison between PLAFP Lines. Seeds from individual plants were collected and the mass of the total number of seeds was measured. The weights represent the average of 8 plants. Student t - test was performed. Asterisk represents a p<0.01. 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 OX 3 T3 OX 1 T3 28 Comp T2 WT Seed Weight (g) Line * * 133 aids in stomata closure under water deficient conditions and allows the plant to retain more water and use that energy for generating seeds and oil production . Another study by Yao et al. , (2013) has shown plants that express a MYB transcription factor kn own as WEREWOLF (WER) exhibit better stress tolerance. This is because WER, when expressed , promotes two important factors that aid in growth and survival: 1) WER suppresses GLABRA2 (GL2), which is a negative regulator of root hair growth. Thus, WER pro motes the production of more root hairs. This allows the plant to extend deeper as well as outward into the ground to take up more water under drought conditions. 2) WER also has been shown to bind PA. This results in WER being translocated to the nucleus. These cases suggest that the PLAFP - induced drought tolerance may similarly be mediated by PA and the PA - induced control of root growth and water use efficiency. The question arises if PLAFP overexpression and the enhanced root development allow for greater uptake of water and nutrients for growth and ultimately result in improvem ent in biomass and seed yield for the plant . To test this possibility we grew plants in the environmental chamber under optimized conditions (60% humidity) but moved the m to ambient conditions (35% humidity; same light conditions and temperature) at the onset of flowering. The resulting seed yield in the overexpressi on lines was two - times that of wildtype plants ( See Figure 3. 9 above ). 3.3. 7 PLAFP Response to Abiotic Str ess in Crop Plants While drought - tolerant Arabidopsis is not required, this is an essential trait in crop plants. PLAFP has homolog s in many plant species. W e initiated PLAFP homologs in two crops with water - intensive growth conditions (Figure 3. 1 0 ) . Hydroponically grown tomato and corn 134 135 seedlings were exposed to ABA stress to see if expression of PLAFP matched the same pattern observed in Arabidopsis. The effect of ABA on the tomato homolog of PLAFP results in an upregulation in expression over a 24 hour period; the same expression pattern we obs erved in Arabidopsis (Figure 3. 1 1 ) . On the other hand, in the corn homolog, which has two alternative splice sites, both transcripts did not appear to be significantly affected by ABA stress in the leaves nor the roots (Figure 3. 1 2 ) . Nonetheless, t his shows some promise that in the future, there could be an increase in drought tolerance and in the crop yield of tomato. This will help farmers who have been struggling with drought for many years and will help reduced food costs and make food more aff ordable to everyone over the world. This could also improve the countries that are dealing with hunger by providing seeds to generate crops that survive under drought - stricken conditions. 3.3. 8 Lipid Profile of PLAFP Displays High Abundance of PA in Overe xpression Lines In previous and current studies, our lab has shown PLAFP binds PA. To determine if PLAFP affects the phloem lipid content and, specifically the PA concentration , we performed liquid chromatography mass spectrometry with collision induced dissociation (LC - MS - CID) on several PLAFP Arabidopsis lines. These include wildtype (WT), overexpression (ox), and complementation (Comp) lines , and a knockdown mutant (KD). S everal species of PA are visible in negative ion mode ( Figur e 3.13 A,B ). Other phospholipids such as different species of PC were identified in the positive ion mode genotypes (data not shown). Observing the various types of PAs within PLAFP phloem exudates cle arly indicates that PA is found within the phloem. Additionally, since PLAFP has also been 136 - 0 1 2 3 4 5 6 7 8 9 2 5 8 24 Relative Gene Expression hours Control ABA * * 137 Figure 3. 1 2 : Effect of ABA on PLAFP Expression in Maize . Eight day old maize seedlings were subjected to ABA spray treatment. Leaf samples were taken at 5 and 8 hours. Leaf and root s amples were collected in three biological replicates with three technical replicates per biological replicate. Values were normalized to 18S mRNA. Gene expression was determi ned using quantitative RT - PCR. No significant values were observed. 0 20 40 60 80 100 120 140 160 180 5 8 Relative Gene Expression Hours ZmPLAFP1 in Leaves Control ABA 0 20 40 60 80 100 120 140 160 5 8 Relative Gene Expression Hours ZmPLAFP1 in Roots Control ABA 0 0.2 0.4 0.6 0.8 1 1.2 5 8 Relative Gene Expression Hours ZmPLAFP2 in Leaves Control ABA 0 0.5 1 1.5 2 2.5 3 5 8 Relative Gene Expression Hours ZmPLAFP2 in Roots Control ABA 138 Figure 3.13 : Phosphatidic Acid Quantification in PLAFP Overexpression Line . A comparison of phloem exudates of overexpressed PLAFP and wildtype plants was monitored in negative ion mode (A, B). Abundance of the (M - H) - ion for PAs (di14:0, m/z: 591.37, RT: 17.80 min; 34:3, m/z: 669.47, RT: 18.10 min; 34:2, m/z: 671.49, RT: 18.30 min). Extracted ion chromatograms were generated in panel A and the Y - axes in panel B are set to the same scale. Slight shift in retention time is due to variations occurring in the chromatography during the LC - MS . (34:2 PA) (34:3 PA) (di14:0 PA Standard) (34:3 PA) (di14:0 PA Standard) (34:3 PA) (34:2 PA) A B (34:2 PA) 139 i dentified within the phloem, it is possible that these two molecules are involved in th e same long - distance signaling pathway. Comparison of wildtype to mutant and wildtype to overexpression line distinctly show how phenotype and lipid content correlate. As shown in Figure 3. 13 A and B , both 34:3 PA and 34:2 PA are increased in the overexpression lines and reduced in the knockdown line . This clearly shows that PLAFP expression and phloem PA content are correlated. Comparison of a PLAFP overexpression line and the wildtype with the 34:3 PA clearly shows the difference in PA abu ndance between lines ( Figure 3. 1 4 A,B ). The overexpression lines contain higher levels of PLAFP and the levels of PA follow suit. Now, whether or not PLAFP brings PA into the phloem or vice versa is still not known, and if PLAFP and PA move in a complex o r separately still needs to be elucidated. What we can conclude from this is that PLAFP modifies the levels of PA within the phloem. It also indicates that PLAFP and PA interact and provides further support that they act together in a long - distance signa ling pathway. 140 Figure 3. 14 . Phosphatidic Acid Lipid Abundance in PLAFP. The abundance of different phosphatidic acid (PA) species was analyzed in the phloem exudates of several PLAFP lines (WT, ox, Comp, mut). Panel A displays t he lipid profile of PA (34:3) . Panel B illustrates the lipid profile of PA (34:2) in PLAFP. A di14:0 PA was added as an internal standard. Each sample receive - S.E. of t w o to four samples per line. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Normalized to PA (14:0) Std Line PA (34:3) Abundance PA (34:3) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Normalized to PA (14:0) Std Line PA (34:2) Abundance PA (34:2) A B 141 APPENDI X 142 Supplementary Table 3. 1: Primer Table for Cloning, RT - PCR, and qPCR. Sample Sequence Function, Sites, etc LAFP FW/Rev At4g39730 - ATGGCTCGTCGCGATGTTCTC - - AACGACCCAAGAAAGCTTTTTCCG - Amplifies entire CDS minus the stop codon; Annealing Temperature: 53 °C; Expected Size: 543bp LAFP - 1 F/R At4g39730 - GAGCAATGGCTCGCTACTGA - - ACGCCACATTACACTCACAAG - Amplifies the entire CDS sequence; Annealing Temperature: 52 °C; Expected Size: 210bp LAFPq FW/Rev At4g39730 FW: - TGCTCGACGCAGGATTTTGA - - CTCAGACCCGACCCGACTAA - qPCR; Annealing Temperature: 52 °C; Expected Size: 126bp pPLAFP FW/Rev FW: - GCCAAGCTTATTGATTATCATTGCATTGC - Rev: - GCGTCTAGATTTGTTTTTTTCCGGTGAACG - Amplifies the 1 Kb region upstream of the transcription initiation site of PLAFP ; Contains HindIII and Xba restriction sites; Annealing Temperature: 56 °C; Expected Size: 1000 bp PLAFP - GUS Confirm FW: - GCCAAGCTTATTGATTATCATTGCATTGC - Rev: - GACCGCATCGAAACGCAGCACG - Amplifies PLAFP and GUS sequence attached to the C - terminus; used to confirm PLAFP - GUS transgenic lines; Annealing Temperature: 56 °C; Expected Size: 1289 bp 143 PLAFP - Protein Expression At4g39730 FW: - GCGCATATGGAAGATGAT CCAGACTGTGTATACA - Rev: - GCGCATATGTTAAACGACCCAAGAAAGCTTTTTCCG - Amplifies t he coding region of PLAFP, excluding 69 nucleotide region encoding the 23 amino - acid predicted signal peptide ; Contains NdeI restriction site(s); Annealing Temperature: 59 °C; Expected Size: 495 bp PLAFP_YFP FW/Rev At4g39730 FW: - GGGGACAAGTTTGTACAAAAAAGCAGGCTACCATGGCTCGTCGCGATGTTCTC - Rev: - GGGGACCACTTTGTACAAGAAAGCTGGGTCAACGACCCAAGAAAGCTTTTTCCG - Amplifies the coding sequence of PLAFP; contains att sites; Annealing Temperature: 65 °C; Expected Size: 601bp TomLAFP_F/R SOLYC04G054980 (LOC101262805) - GGGAGTTGCTGCTCACTTCA - - GTCAAGCATGGACCTCGACC - Amplifies Tomato homolog of PLAFP; Annealing Temperature: 53 °C; Expected Size: 297 bp ZmPLAFP1_FW/Rev G RMZM2G018275 .T01 - ATGAAGCTGAAGCTCCTCTCTCCC - - CTAGAGCGCGGTCACGGTGGC - Amplifies Entire CDS sequence; Annealing Temperature: 58 °C; Expected Size: 615bp ZmPLAFP2_FW/Rev GRMZM2G087245 .T01 - ATGGCCAAGCTCGCCGTCCTC - Rev: - TCACGCCGCCGAGGCGCCCTT - Amplifies Entire CDS sequence; Annealing Te mperature: 62 °C; Expected Size: 564bp ZmPLAFP1q_FW/Rev GRMZM2G018275 .T01 - CGCGCAGCAGCTCTTCACCGT C - - CTAGAGCGCGGTCACGGT GGC - qPCR; Annealing Temperature: 60 °C; Expec ted Size: 287bp 144 ZmPLAFP2q_FW/Rev GRMZM2G087245 .T01 FW: - GTGTACACGGTGTTCATCCGG - Rev: - CCACGTAGTTGCAGTACCACC - qPCR; Annealing Temperature: 53 °C; Expected Size: 274bp At18S_F/R FW: - TCAACTTTCGATGGTAGGATAGTG - - CCGTGTCAGGATTGGGTAATTT - Control Primers; Amplifies part of the 18S gene; Annealing Temperature: 50 °C; Expected Size: 161 bp GUS - F/R - ATGTTACGTCCTGTAGAAACCCCAACCCGTG - - AGGAGTTGGCCCCAATCCAGTCCATTAA - Amplifies GUS gene; Annealing Temperature: 50 °C; Expected Size: 976bp PLDa1_F/R At3g15730 FW: - GATGGGCTCATGGCTACTCATG - Rev: - CATTCCACACATCGTGGTCCTC - RT - PCR; Amplifies part of the PLDa1 protein; Annealing Temperature: 53 °C; Expected Size: 538bp PLDa1q_FW/Rev At3g15730 - CACTCC G TTCCACTC CT TGTT - - GCAATTGGACCTTCAAGACGG - qPCR; Amplifies part of PLDa1 protein; Annealing Temperature: 52 °C; Expected Size: 147bp LEA At3g62580 - ACTCAGTTCGCTACAGCTTGG - - TTCACTCCCATGACCATCGA - Amplifies part of the LEA protein; Annealing Temperature: 52 °C; Expected Size: 295bp 145 Supplementary Table 3. 2: Lipid Species Used in Protein - Lipid Binding Assays . This table depicts the different species of lipid used to test lipid binding based on acyl chain composition. Abbreviations: L: Liposome, O: protein - lipid overlay assay, lab - made membrane, O c : protein - lipid overlay assay, commercially - made membrane . Lipid Acyl Chain Assay Used DOPA 18:1, 18:1 L,O DOPC 18:1, 18:1 L,O DOPS 18:1, 18:1 L,O DG 18:1, 18:1 L DPPA 16:0, 16:0 O c DPPS 16:0, 16:0 O c DPPC 16:0, 16:0 O c PI(4)P 16:0, 16:0 O c PI(4,5)P2 16:0, 16:0 O c PI(3,4,5)P3 16:0, 16:0 O c DAG 16:0, 16:0 O c 146 REFERENCES 147 REFERENCES Awai K, Xu C, Tamot B, Benning C. A phosphatidic acid - binding protein of the chloroplast inner envelope membrane involved in lipid trafficking (2006). Proc Natl Acad Sci USA 103:10817 - 10822. Barbaglia AM and Hoffmann - Benning S. (2016) Lipid signaling and its role in plant development and stress response. In: Subcellular Chemistry; Eds. Nakamura, Y and Li - Beisson, Springer; Invited book chapter; in press; expected print date Jan 2016. 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Thus, in order to continue to be able to produce and provide sufficient food and fuel for an increasing world population, we need plants that exhibit accelerated growth, higher grain yield, and/or more tolerance to stress. One avenue which could lead to preventing or lowering crop los s due to abiotic stress is to better understand the changes that occur in plant development, resulting in their adaptation to these conditions. Thus, we set out to study the signaling compounds within th e phloem in order to understand the distribution of environmental cues throughout the plant. This project focused on three putative lipid - binding proteins GDSL - lipase , PLAFP, and PIG - P, with a major focus on PLAFP. We chose these proteins due to the roles we believe were required for the proteins to conduc t long - distance lipid signaling: 1) Release the lipid into the phloem, 2) Aid in the solubilization and transport of the lipid to its final destination, 3) Act as a receptor for the lipid at its target location, or 4) Be the signal itself. Each of th ese lipid - binding proteins fell under at least one of these categories: GDSL - lipase acting in lipid release, PLAFP acting in any of the four but primarily in the solubilzation and transport of the lipid, and PIG - P acting as a receptor. With these protein s we set out to functionally characterize their role in long - distance lipid signaling within the plant. We found they all localize to the cell periphery and bi nd lipids. An interesting observation we made was all three proteins bi nd phosphatidic 154 acid, a well - known intracellular lipid signal in plants. Expression of the genes associated with these proteins i s associated with the plant vasculature, an essential prerequisite for a long - distance signaling or transport function, but confirmation is still need ed for GDSL and PIG - P. Despite this, the varied responses to abiotic stress indicated that the three proteins do not act in the same but in distinct signaling paths. PLAFP fell under all the requirements of a putative lipid - binding protein. It binds lipi ds, and could function in any of the three roles needed for long - distance lipid transport. PLAFP binds to phosphatidic acid and LC - MS analysis proved PA and PC were present within the phloem. Since both PLAFP and PA are found in the phloem, this is the f irst exa mple of a lipid - binding protein and its lipid partner to both be present in the phloem . This provides strong evidence that PLAFP, PA or PLAFP - PA together could act in a long - distance signaling path. Gene expression for PLAFP was observed in the v asculature of the roots and leaves in addition to the - distance signaling. PLAFP is a small protein whose composition consists mostly of a PLAT/LH2 domain. This do main is known to bind to lipids and be induced by stress. Thus, we decided to determine salt, and drought - mimic, PEG as well as ABA, a drought signaling hormone. We found PLAFP was greatly induced under ABA and PEG, which suggested PLAFP may aid in drought tolerance. When tested in other crop species, tomato and corn, we found the same pattern to be true in tomato. Hence, this could prove to be beneficial for sur vival and seed yield for crop plants. 155 Since PLAFP fared well under drought signals or mimics, we also tested how wildtype PLAFP was affected by soil drought. We found the seedlings survived between 5 - 6 days and were completely dead on day 7. An interest ing observation was a peak in expression in day 3 in the roots and day 4 in the leaves, which indicates the plant sensing water deficiency first in the roots and then signaling to the leaves to conserve water and close their stomata. When PLAFP was overexpressed, an enhanced phenotype of larger leaves and longer roots was observed in comparison to the wildtype. The opposite was true for the knockdown PLAFP line, which exhibited and overall smaller stature. This supports our findings in that PLAFP a ids in drought tolerance. Since PLAFP displayed enhanced expression when subjected to drought - mimic or ABA, higher levels would result in greater tolerance and survival under these conditions, while the opposite would be true with decreased levels of expr ession. Finally, we have shown a model of how PLAF P and PA could be interacting, but further experimentation will be needed to determine the exact interaction between PLAFP and PA. Overall, we have shown the potential role of three putative lipid - binding proteins in long - distance lipid signaling. All three show lipid - binding capabilities and phloem localization. PLAFP especially is interesting with it binding phosphatidic acid, a well - known signaling lipid involved in many signaling cascades such as ABA , and its potential ability to aid in drought tolerance. Despite all three proteins binding lipids and responding to abiotic stress, they do not follow the same , but rather are involved in their own, distinct signaling path. 156 4.2 Future Perspectives This project has various novel findings from identifying both the lipid - binding protein and its binding partner within the phloem. In addition, we may have discovered a protein that aids in drought tolerance. Despite all of this, there still are questions th at need to be addressed. For PLAFP, we will need to determine its exact localization within the plant using immunogold labeling. In addition, we can perform RNA in situ hybridization to confirm our GUS/immunogold findings for further support. This will prove that PLAFP is localized and expressed within the phloem. Also, since we show PLAFP is expressed within the vasculature and have identified its presence within the phloem via GUS stain expression, we wanted to see how auxin, a plant hormone known for its influence on vascular development and patterning would effect PLAFP expression. Do they colocalize ? Does one override the other in terms of expression and activity? This w ould be interesting to find out since auxin initiates the development of the auxin activity or vice versa. We could do this by designing constructs that contain the auxin responsive promoter DR5 driving GUS expression. We could observe auxin alone and use th e DR5 promoter to drive PLAFP to see what effect that has on expression. Lastly, we need to colocalize PLAFP with a Golgi marker and lipid droplet stain. To do this, new constructs will need to be designed since the RFP construct/YFP constructs did not w ork well with the markers , respectively . This will provide evidence that PLAFP, while having a signaling sequence, is not secreted within the secretory pathway as well as if PLAFP associates with other lipids via oil bodies. 157 Since we have shown that PLAFP may be involved in drought tolerance, we wanted to further support this hypothesis by subjecting the overexpression, mutant, complementation, and wildtype lines to soil drought stress to see if the amount of PLAFP affects the plants susceptibility to drought . Based on our phenotypic observations, we expect the overexpression lines to survive the longest, followed by the complementation lines, wildtype, and lastly the knockdown mutant. We currently have preliminary data support ing t he s e finding s . Since the overex pression lines display larger leaves and longer roots due to higher levels of PLAFP, we believe these plants will survive longer under drought conditions since they have the ability to extend their roots further into the so il to obtain water that is deeper in the ground. Additionally, the leaves store/contain more water due to the larger number of stomata, thus they can close them to conserve the water they have stored. On the other hand, the knockdown mutant should die fi rst since the plants possess l ess PLAFP and as a result are physically smaller. Thus, their roots will not be able to extend as far into the ground as their overexpressing counterparts and their smaller leaves will not be able to conserve as much water du e to less stomata. The wildtype and complementation lines should survive somewhere between the overexpression and mutant lines with the wildtype succumbing first followed by the complementation lines. We observed the complementation lines to be slightly larger than the wildtype, possessing slightly longer roots and larger leaves. Overall, this experiment will confirm our suspicion that PLAFP aids in protecting the plant from drought conditions. In this project, we have shown through LC - MS analysis that P LAFP binds PA. We would like to confirm our findings that more PA is bound in overexpression lines as well as look for changes in the profile of other lipids within the phloem sap such as different species of PC, 158 OPDA/JA, and other phospholipids such as p hosphatidylinositol and its phosphates. Additionally, since the phloem contains an abundance of sugars due to assimilates, we would like to perform GC - MS to look at the different sugars and their levels within the phloem to see if one appears at higher le vels than others as well as if the same pattern holds true in the overexpression lines housing more of the sugars due to increased levels of PLAFP. F urther evidence for PLAFP binding PA will be performed via tryptophan quenching and NMR/crystallography. T ryptophan quenching will allow us to determine the orientation of PA (or any lipid) when bound by PLAFP. This will help with identification of any essential amino acid residues required for PA binding as well as provide evidence if our current model of th e PLAFP - PA interaction is correct. We would also like to look at the binding for PC, which there should not be any, and PIPs. In addition to tryptophan quenching, per forming NMR and crystallography will be vital for determining the structural properties/characteristics of PLAFP. These two techniques will allow us to obtain a high - resolution , three - dimensional structure of PLAFP with and without PA. The last and most exciting experiment that needs to be performed is determining whether or not PLAFP, PA or PLAFP - PA moves within the phloem. This will be done by grafting Arabidopsis and/or tomato wildtype and overexpression plants. Each type of plant will be used for t he rootstock and scion to show movement occurs in one or both directions. We will tag PLAFP or PA with a fluorescent marker to make movement easy to observe. This would be the final piece to prove PLAFP is involved in long - distance lipid signaling. 159 The s ame experiments described above will all need to be performed on GDSL and PIG - P. In the immediate future , we will need to confirm binding of DAG and PA to GDSL - lipase and obtain better images of PA and PS binding for PIG - P. Additionally, if we can find a method to successfully make PIP liposomes, we will test that with PIG - P (and PLAFP) as well. Determining the expression GDSL and PIG - P will be performed by GUS expression and RNA in situ hybridization. This will allow for confirmation of expression with in the vasculature. Immunogold labeling will allow for confirmation on protein localization within the plant. Lastly, colocalization with lipid droplets will be performed to determine if either GDSL or PIG - P associate with groups of lipids rather than in dividual phospholipids.