在真核细胞中，细胞自噬是一个将细胞物质吞噬到自噬体降解的保守的代谢过程。自噬体起始于自噬前体结构(PAS) 并由其逐渐扩展和延伸形成其最后的双膜结构，最后和溶酶体（lysosome）或液泡（vacuole）融合得以降解。在酵母和动物细胞中，研究已发现一系列自噬体相关基因（ATG）蛋白参与调控自噬体的形成。自噬体形成的相关研究存在两个主要的未解决的问题，它们包括自噬体的膜来源和膜变形机制。而在植物中，相当一部分关键的自噬体同源蛋白的缺失导致其分子机制研究仍处于初步阶段。在本研究中，我主要通过利用SH3P2，一个N 端含有BAR (Bin-Amphiphysin-Rvs) 结构域及C 端含有SH3（Src homology 3）结构域的蛋白作为探针, 在拟南芥中研究自噬体的形成. 在进一步的研究中，借助了免疫细胞化学技术（抗体），分子技术（萤光蛋白标记），基因技术（RNAi 干扰）以及蛋白作用（酵母双杂交和免疫共沉淀）等不同的生化以及细胞生物学手段，我发现在植物细胞中也存在保守的自噬体的形成模式，而在其过程中, SH3P2 起着重要的调控作用。通过研究我发现：1）在拟南芥植物中，绿色荧光蛋白标记的SH3P2-GFP 蛋白具有对自噬诱导的应答反应；；2）在拟南芥转基因植物和PSBD 悬浮细胞中，SH3P2-GFP 蛋白与自噬体标记蛋白共定位； 3） 在自噬途径中，SH3P2-GFP 活跃地参与在自噬体膜变形过程中并且定位在自噬前体结构包括其扩展结构的膜上； 4）基因敲低SH3P2 在拟南芥植物中是致死的并且抑制自噬体的形成过程；5) SH3P2 能通过它的BAR 结构域互相聚合; 6）SH3P2 可以结合磷脂酰肌醇-3-磷酸（PI3P）并且与磷脂酰肌醇-3-激酶复合体存在联系；7）SH3P2 通过它的SH3 结构域直接与ATG8 结合。综上所述，此项研究发掘了一个新型的膜相关蛋白SH3P2 参与在拟南芥植物自噬途径中，而其与ATG8 的直接相互结合同时也揭示了一个新的自噬形成调控机制。 / In eukaryotic cells, autophagy is a conserved catabolic mechanism by engulfing the cytoplasmic cargoes into a structure termed autophagosome. In general, autophagosome is initiated from a site named PAS (phagophore assembly site preautophagosome structure), which then expands and elongates to form a double membrane structure. Ultimately, the outer membrane of autophagosome will fuse with the lysosome or vacuole membrane and deliver the cargoes for degradation or recycling. In yeast and animal cells, a number of ATGs (autophagy related genes) have been identified to regulate the autophagosome formation. Studies of the autophagosome formation involve two main unsolved questions: the membrane origin and the membrane deformation mechanism. In plants, several key players responsible for autophagosome biogenesis are missing and the molecular mechanisms for the autophagosome formation remain elusive. In this study, I have used SH3P2, which contains a N-terminus BAR (Bin-Amphiphysin-Rvs) domain and C-terminus SH3 (Src homology 3) domain, as a probe, to study the autophagosome formation in plants. Using a combination of immunocytochemical (antibodies), molecular (GFP fusions), genetic (RNAi) and interaction (Yeast two-hybrid and Co-IP) approaches, I have shown that a conserved autophagosome formation model exists in plant cells and SH3P2 plays an essential role in the autophagy pathway in Arabidopsis thaliana. I have found that 1) SH3P2-GFP fusion proteins response to autophagic induction in transgenic Arabidopsis plants; 2) SH3P2-GFP colocalize with the known autophagosome markers in both transgenic Arabidopsis plant and PSBD cells; 3) SH3P2-GFP localizes on the PAS membrane and actively participates in membrane deformation events during autophagosome formation throughout its expansion process via the dynamic and ultra structural analysis; 4) Knock-down of SH3P2 is developmental lethal and suppresses the autophagosome formation and autophagic flux; 5) SH3P2 has a self-interaction via its BAR domain; 6) SH3P2 binds to PI3P (Phosphatidylinositol-3-Phosphate) and associates with the PI3K (Phosphatidylinositol-3-Phosphate Kinase) complex; 7) SH3P2 directly interacts with ATG8 via its SH3 domain. Taken together, this thesis research has identified a novel membrane-associated protein and demonstrated its essential role in autophagy in plant. The demonstration for the direct association between SH3P2 and the ATG8 complex may provide an insightful mechanism for autophagosome regulation in Arabidopsis thaliana. / Detailed summary in vernacular field only. / Zhuang, Xiaohong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 97-104). / Abstracts also in Chinese. / Statement --- p.I / Abstract --- p.II / 摘要 --- p.IV / Acknowledgements --- p.VI / Table of Contents --- p.VIII / List of Tables --- p.X / List of Figures --- p.XI / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Introduction of Autophagy --- p.2 / Chapter 1.2 --- Molecular Machinery for Autophagy --- p.5 / Chapter 1.3 --- Membrane Origins of Autophagosome --- p.8 / Chapter 1.4 --- Membrane Sensors for Autophagosome Formation --- p.10 / Chapter 1.4.1 --- ATG14 --- p.10 / Chapter 1.4.2 --- Bif1 (Bax-interacting factor 1) --- p.11 / Chapter 1.5 --- Autophagy in Plants --- p.12 / Chapter 1.6 --- Research Objectives --- p.13 / Chapter Chapter 2 --- SH3P2 Defines a Conserved Autohagosome Formation Process in Arabidopsis --- p.15 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.2 --- Materials and Methods --- p.19 / Chapter 2.2.1 --- Plasmid Construction --- p.19 / Chapter 2.2.2 --- Plant Materials, Growth and Treatment Conditions --- p.24 / Chapter 2.2.3 --- Transient Expression in Protoplasts and Confocal Imaging --- p.24 / Chapter 2.2.4 --- Antibody Generation, Protein Extraction and Western Blot Analysis --- p.25 / Chapter 2.2.5 --- Immunofluorescence Confocal Study --- p.26 / Chapter 2.2.6 --- Electron Microscopy (EM) Study --- p.26 / Chapter 2.2.7 --- Accession Numbers --- p.27 / Chapter 2.3. --- Results --- p.28 / Chapter 2.3.1. --- SH3P2-GFP Fusion Proteins Response to Autophagic Induction in Arabidopsis --- p.28 / Chapter 2.3.2 --- The SH3P2-GFP Positive Compartments are Overlapped with Autophagosome Markers --- p.36 / Chapter 2.3.3 --- Dynamic Analysis of SH3P2-GFP Positive Compartments in Arabidopsis Transgenic Plants upon Autophagic Induction --- p.42 / Chapter 2.3.4 --- EM Analysis of the subcellular localization of SH3P2 after autophagic induction --- p.44 / Chapter 2.4 --- Discussion --- p.52 / Chapter Chapter 3 --- SH3P2 is Essential for Plant Development and Autophagic Pathway in Arabidopsis --- p.54 / Chapter 3.1 --- Introduction. --- p.55 / Chapter 3.2.1 --- Plasmid Construction --- p.57 / Chapter 3.2.2 --- Plant Materials, Growth and Treatment Conditions --- p.57 / Chapter 3.2.3 --- Transient Expression in Protoplasts and Confocal Imaging --- p.58 / Chapter 3.2.4 --- Protein Extraction and Immunoblot Analysis --- p.58 / Chapter 3.2.5 --- RT-PCR --- p.59 / Chapter 3.3 --- Results --- p.60 / Chapter 3.3.1 --- RNAi Knockdown of SH3P2 is Developmental Lethal --- p.60 / Chapter 3.3.2 --- RNAi Knockdown of SH3P2 Suppresses the Autophagosome Formation and Autophagic Flux --- p.63 / Chapter 3.4 --- Discussion --- p.71 / Chapter Chapter 4 --- SH3P2 is Associated with the ATG Machinery --- p.73 / Chapter 4.1 --- Introduction --- p.74 / Chapter 4.2 --- Materials and Methods --- p.76 / Chapter 4.2.1 --- Plasmid Construction --- p.76 / Chapter 4.2.2 --- Plant Materials, Growth and Treatment Conditions --- p.76 / Chapter 4.2.3 --- Recombinant Protein Expression --- p.77 / Chapter 4.2.4 --- In Vitro Lipid Binding Assay --- p.77 / Chapter 4.2.5 --- Yeast-two Hybrid Analysis --- p.78 / Chapter 4.2.5 --- Immunoprecipitation Analysis --- p.78 / Chapter 4.3 --- Results --- p.80 / Chapter 4.3.1 --- SH3P2 Binds to PI3P --- p.80 / Chapter 4.3.2 --- SH3P2 Has a Strong Self-interaction via the BAR Domain --- p.82 / Chapter 4.3.3 --- SH3P2 is Associated with the PI3K Complex and Interacts with ATG8 --- p.84 / Chapter 4.4 --- Discussion --- p.86 / Chapter Chapter 5 --- Discussions and Perspectives --- p.87 / Chapter 5.1 --- Discussions --- p.88 / Chapter 5.1.1 --- Autophagosome Formation is Conserved in Arabidopsis thaliana --- p.88 / Chapter 5.1.2 --- SH3P2 Interacts with the ATG8 Complex and is Required for the Autophagic Pathway in Arabidopsis thaliana --- p.90 / Chapter 5.1.3 --- A Novel Membrane-associated Regulator for Autophagosome formaiton in Arabidopsis thaliana --- p.92 / Chapter 5.2 --- Working Model of SH3P2 during Autophagosome Formation in Arabidopsis --- p.93 / Chapter 5.3 --- Future Perspectives --- p.96 / References --- p.97 / List of Publications --- p.104

胞吐作用定義為囊運小泡將物質運輸到質膜或細胞外空間的轉運過程。其中關鍵的一步發生在同源SNARE 蛋白介導的膜融合之前，即將胞吐囊泡瞄向並靶定在適當的質膜位點。先前在酵母和哺乳動物中的研究表明，一個名為exocyst 的蛋白質複合體在這一關鍵步驟發揮作用。exocyst 蛋白複合體最早在酵母發現，之後這個複合體也在哺乳動物中被發現。這個複合體包含8 個不同的亞基：SEC3，SEC5，SEC6，SEC8，Sec10，Sec15，Exo70 和Exo84。Exocyst 同源蛋白也已在植物中發現。相比酵母和動物，exocyst 在植物體內的功能還鮮為人知，尤其是在胞吐運輸過程中的作用 。通過瞬時表達熒光蛋白標記的擬南芥同源的exocyst 蛋白Exo70：AtExo70E2 以及使用這個同源物的特異抗體，我們在擬南芥和煙草BY－2 懸浮培養細胞中發現了一種新的細胞器，並命名為exocyst 陽性細胞器（EXPO）。這種細胞器分別位於質膜或是細胞質中。由於它未能與任何傳統的細胞器標記物重合，或是被布雷菲爾德菌素A，渥曼青黴素和刀豆素A 影響，以及不能與FM4-64 重合，我們判斷這些細胞器不定位於常規的分泌或胞吞途徑中。對於快速冷凍樣本進行的免疫電子顯微鏡顯示EXPO 的雙膜性質，同時也發現了陽性標記的位於質膜外的單膜囊泡的存在。與此同時，在野生型細胞中也發現了同樣結構的細胞器。EXPO和自噬體非常相似， 都有兩層膜。然而，EXPO 不能被的自噬標記物（AtAtg8e）所標記。同時，在營養脅迫條件下，EXPO 的數量也沒有增加。因此，EXPO 代表著植物所特有的一種非常規分泌形式。 / 此外，通過在擬南芥原生質體內進行瞬時表達，我進一步證實在AtExo70E2 存在的條件下， 一些exocyst 成員可以被招募到EXPO 。AtExo70E2 的旁系同源物AtExo70A1 是在這方面物法取代AtExo70E2 的作用。蛋白蛋白相互作用分析證實了AtSec10 或AtSec6 與AtExo70E2 之間的相互作用。 AtExo70E2，而不是它在酵母或是動物中的同源蛋白，可以誘導EXPO 在動物細胞中的形成。反之，人或是酵母Exo70 同源蛋白都不能誘導EXPO 在植物細胞中的形成。這些結果表明AtExo70E2 在EXPO 形成過程中的特定的以及至關重要的作用。 / Exocytosis defines the process in which vesicles transport substances to the plasma membrane (PM)/extracellular space of the cell. One key step of exocytosis is the targeting and docking of the exocytic vesicles to the appropriate PM sites, which is prior to membrane fusion mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE). Previously studies have demonstrated that a protein complex called exocyst complex is involved in this key step in yeast and mammals. The exocyst complex, containing eight different subunits: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84, was first identified in yeast and subsequently in mammals. Exocyst homologs have also been found in plants. In comparison to its yeast and animal counterparts, little is known about the function of exocyst proteins in plants especially in the process of exocytosis. By using both antibodies specific for one of the orthlogs of exocyst protein: AtExo70E2 as well as transiently-expressed fluorescently-tagged constructs for this exocyst subunit, a novel organelle termed exocyst-positive organelle (EXPO) was identified in suspension cultured Arabidopsis and tobacco BY-2 cells. These organelles were located to both the plasma membrane and cytosol. Based on their failure to overlap with any conventional organelle markers or response to brefeldin A (BFA), wortmannin or concanamycin A (ConcA) treatments, as well as their inability to take up the endocytic dye FM4-64, these organelles were thus not lie on the conventional secretory or endocytic pathways of plant cells. Immunogold electron microscopy (EM) of cryofixed samples revealed the double membrane nature of EXPO and also produced labeling of large single-membrane bound vesicles outside of the PM. These structures were also identified in wild type cells. EXPO and autophagosomes are similar in that both have two boundary membranes. However, EXPO did not label positively with YFP-AtAtg8e, a standard marker for autophagosomes, nor did the number of EXPO increase when the cells were subjected to nutrient stress. Therefore, EXPO represents a form of unconventional secretion unique to plants. / Further studies demonstrated that a number of exocyst subunits can be positively recruited to EXPO in the presence of AtExo70E2 by performing transient expression in Arabidopsis protoplasts. The paralog AtExo70A1 is unable to substitute for AtExo70E2 in this regard. Protein-protein interaction assay have confirmed the interaction between AtExo70E2 and AtSec6 and AtSec10. AtExo70E2, but not its yeast counterpart, is also capable of inducing EXPO formation in animal cells. Inversely, neither human nor yeast Exo70 homologs are able to cause the formation of EXPO in Arabidopsis protoplasts. These results point to a specific and crucial role for AtExo70E2 in EXPO formation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ding, Yu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 101-118). / Abstracts also in Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vii / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xiv / Chapter CHAPTER 1 --- p.1 / General Introduction --- p.1 / Chapter 1.1 --- The secretory system in eukaryotic cells --- p.2 / Chapter 1.2 --- Exocytosis and exocyst complex --- p.6 / Chapter 1.3 --- Project Objectives --- p.7 / Chapter CHAPTER 2 --- p.9 / Exocyst-positive organelles (EXPOs) mediate unconventional protein secretion in plant cells --- p.9 / Chapter 2.1 --- Abstract --- p.10 / Chapter 2.2 --- Introduction --- p.11 / Chapter 2.3 --- Materials and Methods --- p.12 / Chapter 2.4 --- Results --- p.20 / Chapter 2.4.1 --- Expression pattern of different AtExo70 paralogs with fluorescent tag in Arabidopsis protoplasts --- p.20 / Chapter 2.4.2 --- The organelles labeled by AtExo70E2 are distinct from well known endomembrane markers --- p.23 / Chapter 2.4.3 --- The AtExo70E2 positive organelles do not lie on the secretory or endocytic pathways --- p.27 / Chapter 2.4.4 --- Arabidopsis Exo70E2-specific antibodies confirm identity of AtExo70E2-positive organelles --- p.31 / Chapter 2.4.5 --- AtExo70E2 positive organelles are true and novel double membrane organelles --- p.33 / Chapter 2.4.6 --- EXPO are not autophagosomes but sequester cytosolic proteins to release them into the apoplast --- p.41 / Chapter 2.5 --- Discussion --- p.53 / Chapter 2.5.1 --- EXPO: novel organelles labeled by exocyst --- p.53 / Chapter 2.5.2 --- EXPO and autophagosome: same or not? --- p.55 / Chapter 2.5.3 --- EXPO: the evidence of unconventional secretion in plant cells --- p.56 / Chapter 2.6 --- Perspectives --- p.56 / Chapter CHATER 3 --- p.58 / AtExo70E2 is essential for exocyst subunit recruitment and for EXPO formation in both plants and animals --- p.58 / Chapter 3.1 --- Abstract --- p.59 / Chapter 3.2 --- Introduction --- p.60 / Chapter 3.3 --- Materials and Methods --- p.62 / Chapter 3.4 --- Results --- p.70 / Chapter 3.4.1 --- AtExo70E2 is required for the membrane recruitment of a number of exocyst subunits --- p.70 / Chapter 3.4.2 --- AtExo70E2 is required for the recruitment of some other, but not all, AtExo70 subunits --- p.74 / Chapter 3.4.3 --- AtExo70A1 is unable to recruit other exocyst subunits --- p.74 / Chapter 3.4.4 --- FRET and BiFC confirm interactions between AtExo70E2 and other exocyst subunits --- p.80 / Chapter 3.4.5 --- Arabidopsis Exo70E2 can also induce EXPO formation in animal cells --- p.84 / Chapter 3.4.6 --- Neither human nor yeast Exo70 can induce EXPO in plant protoplasts --- p.84 / Chapter 3.4.7 --- EXPO induced by AtExo70-GFP expression in HEK cells do not colocalize with standard organelle markers --- p.87 / Chapter 3.4.8 --- Electron microscopy confirms the presence of EXPO-like, double membrane structures in HEK cells after expression of AtExo70E2-GFP --- p.87 / Chapter 3.5 --- Discussion --- p.91 / Chapter 3.5.1 --- Plant exocyst and the discovery of EXPO --- p.91 / Chapter 3.5.2 --- AtExo70E2 is a key player in exocyst recruitment onto EXPO --- p.93 / Chapter 3.5.3 --- AtExo70E2 expression as a signal for EXPO formation --- p.96 / Chapter 3.6 --- Perspectives --- p.100 / References: --- p.101 / Chapter List of publications derived from this Ph.D. thesis research --- p.119

San, Wan Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 75-78). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.i / Statement --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Acknowledgements --- p.vi / Table of Contents --- p.viii / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- The Plant Secretory Pathway --- p.1 / Chapter 1.2 --- AtEMP70 As a Potential Candidate in PVC Proteomics Analysis --- p.4 / Chapter 1.3 --- EMP70 Protein Family --- p.6 / Chapter 1.3.1 --- Arabidopsis EMP70 Protein Family --- p.6 / Chapter 1.3.2 --- EMP70 Homologs Among Different Species --- p.9 / Chapter 1.4 --- Aims of This Study --- p.10 / Chapter Chapter 2 --- Materials and Methods --- p.12 / Chapter 2.1 --- Generation of Arabidopsis cDNA --- p.12 / Chapter 2.2 --- Plasmid Construction --- p.13 / Chapter 2.3 --- Transformation of Tobacco BY-2 Cells --- p.14 / Chapter 2.4 --- Confocal Immunofluorescence Studies --- p.15 / Chapter 2.5 --- Drug Treatments --- p.16 / Chapter 2.6 --- Transient Expression in Protoplasts --- p.16 / Chapter 2.7 --- Generation of Antibodies --- p.18 / Chapter 2.8 --- SDS-PAGE and Western Blot Analysis --- p.19 / Chapter 2.9 --- Microsomal Protein Extraction --- p.21 / Chapter 2.10 --- Subcellular Fractionation --- p.21 / Chapter 2.11 --- Membrane Strip-off --- p.23 / Chapter Chapter 3 --- Results --- p.24 / Chapter 3.1 --- Subcellular Localization Study of GFP-tagged AtEMP2 Fusions via Transient Expression --- p.24 / Chapter 3.1.1 --- AtEMP2-GFP Localized to TGN in BY-2 Protoplasts --- p.24 / Chapter 3.1.2 --- AtEMP2-GFP Localized to TGN in Arabidopsis Protoplasts --- p.30 / Chapter 3.1.3 --- N-terminal GFP-tagged AtEMP2 Fusions Localized to the Golgi Apparatus in Arabidopsis Protoplasts --- p.33 / Chapter 3.2 --- Generation and Characterization of Transgenic Tobacco BY-2 Cells and Arabidopsis PSB-L Cells Expressing AtEMP2-GFP Fusion --- p.36 / Chapter 3.2.1 --- Subcellular Localization of AtEMP2-GFP Fusion in Transgenic BY-2 Cell Lines --- p.36 / Chapter 3.2.2 --- Subcellular Localization of AtEMP2-GFP Fusion in Transgenic Arabidopsis PSB-D Cell Lines --- p.39 / Chapter 3.3 --- Immunofluorescent Labeling Study --- p.41 / Chapter 3.3.1 --- ManI Antibodies Did Not Label the Punctate Organelles --- p.41 / Chapter 3.3.2 --- AtEMP2 Antibodies Labeled the Golgi Apparatus --- p.43 / Chapter 3.4 --- Generation of AtEMP70 Antibodies --- p.46 / Chapter 3.5 --- Western Blot Analysis --- p.50 / Chapter 3.5.1 --- Heat Treatment Caused Aggregation of AtEMP2-GFP Fusion Proteins --- p.51 / Chapter 3.5.2 --- Size Change of AtEMP2-GFP Fusion Proteins in Response to Heat Treatment --- p.52 / Chapter 3.5.3 --- Aggregation Formation of AtEMP2-T7 Fusion Proteins in 95°C --- p.56 / Chapter 3.5.4 --- Distribution of Endogenous AtEMP70 in Arabidopsis Wild Type Cells --- p.58 / Chapter 3.6 --- Subcellular Fractionation --- p.61 / Chapter 3.6.1 --- C-terminal GFP- or T7-tagged Fusion Affected the Subcellular Localization of AtEMP2 --- p.61 / Chapter 3.6.2 --- Endogenous AtEMP70 Localized to the Golgi Apparatus --- p.64 / Chapter Chapter 4 --- Discussion and Future Perspectives --- p.67 / Chapter 4.1 --- Discussion --- p.67 / Chapter 4.1.1 --- ER Export Signal in the Cytosolic Tail of AtEMP70 --- p.71 / Chapter 4.1.2 --- Potential Golgi Retention Signal in the Cytosolic Tail of AtEMP70 --- p.73 / Chapter 4.2 --- Future Perspectives --- p.74 / References --- p.75

Indiana University-Purdue University Indianapolis (IUPUI) / The phototropin photoreceptors are important regulators of plant growth and development and can therefore affect the photosynthetic activity of plants. Phototropin1 and Phototropin2 are versatile protein kinases that become activated when exposed to blue light. Their photobiological actions are best understood in the model plant Arabidopsis thaliana, where they are known to trigger several responses to blue light, one of which is phototropism, the bending of plant organs towards light. Additionally, phot1 and phot2 drive stomatal opening, chloroplast arrangement in leaf cells, leaf expansion, and leaf orientation. The phot1-specific response is rapid inhibition of hypocotyl growth, leaf positioning and mRNA stability whereas phot2 mediates the chloroplast avoidance response to high light. These responses impact a plant’s ability to capture light for photosynthesis, therefore the phototropins play important roles in optimizing a plant’s photosynthetic activity. Soybean (Glycine max) is a very important crop plant in Indiana known for its nutritional versatility and is also utilized for biodiesel production.In spite of soybean being a key crop, there is currently no information about the functionality of soybean phototropins. Also, being a legume, soybean has many structural and functional features that are not present in Arabidopsis. Interestingly, PsPHOT1A (a photoreceptor from garden pea) was found to be a functional phototropin as it was able to complement the phot1 mutation in Arabidopsis. The roles of these proteins in soybean will be elucidated based on the hypothesis that soybean phototropins play essential roles in regulating photosynthetic activity as do the Arabidopsis phototropins. To date, five soybean phototropins, 3 PHOT1s and 2 PHOT2s, are believed to exist. These GmPHOT protein coding regions were amplified by RT-PCR and cloned into pCR8/TOPO or pENTR-D/TOPO vectors via TOPO cloning to utilize Gateway cloning technology to create plant transformation constructs subsequently. The cloned GmPHOT cDNAs from each of the 5 GmPHOTs were sequenced and compared to the GmPHOT sequences from the Phytozome database to assess the accuracy of the gene models. The gene models of all the GmPHOTs were found to be accurate except that of GmPHOT1B-2. The high level of sequence identity between the GmPHOTs and AtPHOTs and the conservation of LOV domains and catalytic domains indicate structural resemblance between them. This suggests that soybean phototropins should encode active photoreceptors. The cloned protein coding regions from soybean were then recombined into a plant expression vector via Gateway technology,which were then used for transformation of Agrobacterium tumefaciens. These plant expression constructs will be utilized in the future to determine the functionality of soybean phototropins in Arabidopsis.