<p dir="ltr">Exocytosis is crucial for delivering proteins, lipids, and cell wall polysaccharides to the plasma membrane and extracellular spaces, playing a vital role in normal plant development as well as responses to biotic and abiotic stresses. One key molecular player, the exocyst, is an octameric protein complex that tethers secretory vesicles to the plasma membrane (PM). Chapter 1 is a literature survey that introduces the function of the exocyst, as well as the characterization of Endosidin2 (ES2), a synthetic molecule that targets the EXO70 subunit of exocyst. This chapter also defines existing knowledge gaps in the profiling of cargo proteins trafficked by the exocyst and the identification of novel modulators of exocytosis. Chapter 2 employs a comparative proteomics approach to examine the changes of PM proteome of root cells following ES2-treatment. Proteins with decreased abundance at the PM were considered candidate cargo proteins of ES2-targeted trafficking and several were validated with quantitative live-cell imaging. Chapter 3 describes the use of ES2 as a tunable and reversible chemical genetics tool as demonstrated by the development and deployment of a large-scale mutant screen in Arabidopsis that identified 70 <u>ES2</u>-hyper<u>s</u>ensitive mutants (<i>es2s</i>). Among these, candidate mutations for 14 non-allelic lines were mapped and reported. T-DNA insertion lines were subsequently screened as alternative alleles to identify causal mutations. In Chapter 4, the causal mutation of <i>es2s-15-12</i> was confirmed as <i>ArgJ</i> with a second T-DNA insertion mutant allele as well as genetic and chemical complementation. <i>ArgJ</i> encodes an enzyme in the arginine biosynthesis pathway. It was demonstrated that arginine biosynthesis deficiency synergizes with ES2 to inhibit root growth in Arabidopsis. Root growth in <i>argj</i> mutants was not hypersensitive to other inhibitors with different modes of action, such as LatB, ES9-17, and BFA. Additionally, roots of <i>argj-1</i> displayed a reduced abundance of PIN2 at the apical PM in epidermal cells; however, PIN2 polar distribution was not further reduced by ES2 treatment. Our findings point to a functional connection between arginine metabolism and exocytosis. Chapter 5 discusses potential future directions and experiments, including technological advances and the testing of new hypotheses. Overall, this study presents a detailed application of chemical genetics to dissect the exocytosis process in Arabidopsis and uncovers novel modulators of exocytosis in plants.</p>

<p><i>Agrobacterium tumefaciens </i>transfers virulence effector proteins to infected host plants to facilitate the transfer and trafficking of a piece of its tumor inducing (Ti) plasmid, (T-[transfer] DNA), into and through plant cells.T-DNA integrates into the host genome where it uses the host’s gene expression machinery to express transgenes. Scientists have used this process to insert beneficial genes into plants by replacing native T-DNA in the bacteria with engineered T-DNA, making <i>Agrobacterium</i>-mediated transformation the preferred method for crop genetic engineering. In spite of its wide-spread use in research and agriculture, we still do not have a complete understanding of the transformation process. Consequently, many important crop species remain highly resistant to transformation. One of my lab’s major goals is to define the molecular interactions between <i>Agrobacterium</i> and its host plants which mediate transformation. I study the role of the <i>Agrobacterium</i> effector protein, VirE2, which is important for plant transformation. VirE2 likely coats the transferred DNA (T-DNA) after it enters the plant cell and protects it from degradation. VIP1 is a host transcription factor that interacts with VirE2 and is involved in activating plant defense responses. VIP1 localizes to both the cytoplasm and the nucleus.Under stress, VIP1 localizes to the nucleus where it activates expression of defense response genes.This observation led to the model that T-DNA-bound VirE2 binds VIP1 and uses VIP1 nuclear localization to deliver T-DNA into the nucleus (the “Trojan Horse” model). In contrast to this model, our lab has obtained data showing that VirE2 holds at least a portion of the VIP1 pool outside the nucleus. We also showed that VIP1 and its homologs are not necessary for transformation. VirE2 interacts with several host proteins in addition to VIP1, and these interactions could lead to changes in host gene expression and protein levels, possibly facilitating transformation. We investigated this model by placing VirE2 under the control of an inducible promoter in <i>Arabidopsis</i> and performing RNA-seq and proteomics under non-induced and induced conditions, and in the presence of <i>Agrobacterium</i> to determine its individual effect on plant RNA and protein levels during infection. Some genes differentially expressed after VirE2 induction are known to be important for transformation. Knockout mutant lines of some VirE2 differentially expressed genes showed altered transformation phenotypes. Protein levels of genes known to be important for transformation were also increased in response to VirE2 induction, and overexpression of some of these genes resulted in increased transformation susceptibility. We therefore conclude that VirE2 modulates both plant RNA and protein levels to facilitate transformation.</p>

<p>Plants are used as a primary food source by humans. Some plants produce edible roots or leaves, but most crops used today are grown to harvest their nutrient-rich seeds which are a product of double fertilization in flowering plants. </p> <p>Cell-cell recognition, adhesion, and fusion are widespread phenomena in many biological processes, where fertilization is an exemplary process. Many players have been identified to mediate sperm-egg fusion in both animals and plants. Interestingly several of these components were shown to be structurally and functionally conserved across kingdoms. In animals Tetraspanins act as facilitators of sperm-egg fusion. Tetraspanins are known to associate in clusters in the plasma membrane of cells, where they recruit diverse signaling proteins, forming the so called Tetraspanin-enriched microdomains (TEMs). TEMs are therefore recognized as major signaling platforms mediating specific cellular processes in the plasma membrane of cells. Two <i>Arabidopsis</i>-expressed tetraspanins, <i>TET11</i> and <i>TET12</i>, are highly expressed in the sperm cells (SCs), however their function in fertilization are unknown. Using fluorescence microscopy, we quantified the expression of TETs in SCs and found evidence for the existence of a Tetraspanin-enriched microdomain (TEM) at the SC-SC adhesion interface. Sperm cell factors which are necessary for fertilization were found to accumulate at the TEM, suggesting that plant SC TEMs may function as protective platforms for fertilization factors. Sperm-expressed TETs directly interact with members of a novel, plant-specific family of unknown proteins, <i>DMP8/9</i>. DMP8/9 function as negative regulators of SC-SC adhesion and are required for double fertilization. Structural and functional analysis suggest that these two proteins may perform unique functions as membrane remodelers in SCs. In addition, we provide evidence of a new GEX2 function as a SC-SC adhesion factor and potential partner of TET-DMP complexes at the SC-SC interface.</p>

<p> </p> <p>Phosphorus (P) is an essential macronutrient for plant growth and development. P deficiency is becoming one of the most limiting factors for crop productivity. It has been discovered that vascular tissue-mediated systemic signaling plays important roles in plant responses to P deficient growth conditions. In order to understand vascular tissue-specific molecular alterations in response to P deficiency, I used <em>Plantago major </em>as a model species to study the transcriptomic alterations in vascular tissues because it is fast and easy to dissect pure vascular tissues from this plant. I identified 237 differentially expressed genes involved in various roles to P deficiency, such as “phosphate metabolism and remobilization”, “sucrose metabolism, loading and synthesis” and “plant hormone metabolism and signal transduction”. In addition, translating ribosome affinity purification (TRAP) was used to identify 547 differentially expressed genes from the Arabidopsis vascular tissues. <em>AtERF</em>, one of the downregulated genes, was chosen for further functional characterization. My results demonstrated that <em>AtERF </em>is specifically expressed in vascular tissues and it encodes a transcription factor. Over-expression of <em>AtERF </em>led to a purple vein phenotype, decreased growth of shoots and roots, and reduced Pi concentrations in shoots and roots. The <em>erf </em>mutant plants displayed larger shoots and roots, and increased Pi concentration in shoots and roots. Molecular analysis in the over-expression and mutant plants showed that genes related to hormone metabolisms and root architecture establishment might be the major players enabling plants to cope with low P. The discoveries from this study may be used to implement strategies for the production of crops with increased P uptake efficiency. </p>

<p dir="ltr">Plants are capable of perceiving and responding to biotic and abiotic stress. They have evolved a variety of mechanisms to help them recognize and trigger rapid responses to both chemical and mechanical stimuli. These signals coordinate plant growth, development, and innate immune responses. However, we have limited knowledge about how mechanical signals are perceived and transduced during the plant immune response. In this study, we investigated the potential role of PIEZO, a mechanosensitive ion channel that is responsible for cellular mechanotransduction in both the plant and animal kingdoms, during the plant immune responses. Publicly- available RNAseq data revealed that <i>PIEZO</i> expression remained constant and unaltered in response to a variety of phytopathogens or elicitors. We, then, conducted infectious growth assays on <i>piezo</i> mutants in <i>Arabidopsis thaliana</i> plants. Our results indicated that <i>piezo</i> mutants, <i>pzo1-1 </i>and<i> pzo1-5,</i> were more susceptible to <i>Pseudomonas syringae </i>pv. tomato<i> </i>(Pst) DC3000 and to the <i>P. syringae hrcC</i>^<em>-</em> mutant confirming PIEZO’s role in plant defense and PTI. We further explored disease progression with necrotrophic fungi, <i>Alternaria brassisicola</i> and <i>Botrytis cinerea, </i>on <i>piezo</i> mutant plants and found enhanced fungal growth compared to the wild type (Col-0) with <i>Botrytis</i>. Building upon these findings, we probed the role of PIEZO in the growth-defense tradeoff using a root growth inhibition assay with flg22 as the MAMP elicitor. <i>pzo1-1</i> was less sensitive to flg22 treatment with less reduction in root growth compared to wild type whereas <i>pzo1-5</i> shows no difference in reduction compared to Col-0. In addition, we investigated whether PIEZO operates upstream of the main NADPH-oxidase, RBOHD, and the associated oxidative burst that occurs in early defense. There was no significant difference in Reactive Oxygen Species (ROS) production between <i>piezo</i> mutants and the wild type in an apoplastic ROS assay with a MAMP elicitor (flg22) and also with Ca²⁺ flux leaf disk assay. In conclusion, we demonstrated a potential role for PIEZO in plant immune defense responses and the growth-defense tradeoff.</p>

<p>A thesis concerning the effects of water deficit on stomatal traits in plants. The relationships between different traits and their influence on overall stomatal anatomy is discussed. Genetic work to investigate molecular regulation of stomatal development is also presented and discussed.</p>

<p dir="ltr">Plants emit an amazing diversity of volatile organic compounds (VOCs) that in addition to being utilized by humans for a multitude of applications, allow plants to communicate with their environment, and play numerous roles in plant growth and development. Plants must be able to perceive and distinguish between VOC cues mediating plant-plant, plant-insect, and plant-microbe interactions to appropriately respond to stimuli. Due to the plethora of biological processes dependent on VOCs, significant progress has been made towards understanding the biosynthesis of plant VOCs and their regulation, and, in recent years, the molecular mechanisms involved in VOC emission. However, to date, little is known about how VOCs are perceived by plants and trigger cellular response(s). In animals, VOCs are recognized by odorant receptors known as G-protein-coupled receptor (GPCR) proteins. However, the few GPCR genes identified in plants appear to have different functions and the lack of a reliable marker for VOC perception has hampered research in this field.</p><p dir="ltr">The discovery of natural fumigation of terpenoids in petunias provides a means of studying VOC perception and the downstream signaling pathways by providing a visual indicator of perception. Transcriptomic analysis of wild-type and transgenic petunias deficient in terpenoid synthesis revealed a link between terpene perception in pistils with the karrikin-like signaling pathway. By utilizing biochemical, computational, and in planta experiments, we demonstrate that of the four petunia karrikin-insensitive receptors (PhKAI2), one of the Lamiid-specific KAI2 intermediate clade receptors, PhKAI2ia, can stereo-specifically perceive the (−)-germacrene D signal emitted from the floral tubes, triggering a KAI2-mediated signaling cascade and affecting plant fitness. Downregulation of PhKAI2ia results in significantly smaller stigmas compared to wild-type, and the phenotype cannot be complemented by the treatment of pistils with (−)-germacrene D, indicating that PhKAI2ia transgenic plants are acting as deaf receptors. We also show that the binding of (−)-germacrene D to PhKAI2ia is sufficient to induce complex formation with more axillary growth 2 (PhMAX2) and the subsequent degradation of suppressor of MAX2 (PhSMAX1a).</p><p dir="ltr">Altogether, our research uncovers the role(s) of the intermediate clade of KAI2 receptors, illuminates the involvement of a KAI2ia-dependent signaling pathway in volatile communication, and provides new insights into plant olfaction and the long-standing question about the nature of potential endogenous KAI2 ligand(s).</p>

<p dir="ltr">Tomato, being a tropical species, is sensitive to temperatures below 10°C, thus limiting its growth to warmer regions and greenhouses. Understanding the cold response pathways in tomato will help improve its climate resiliency through breeding and biotechnology. Reportedly, plant genes undergo alternative splicing (AS) in response to various environmental stresses, however, the scope and dynamics of alternative splicing events in response to cold are unknown in tomato. To fill this knowledge gap, a fine-scale time-series cold (4°C) experiment was performed followed by RNA-sequencing of shoot and root tissues in tomato. Computational analysis revealed that various AS events occur within the first 20 minutes of temperature reduction and later on. Many AS genes were common between shoots and roots, however, the majority of the changes were organ-specific. Circadian rhythm and photosynthesis were the most significant among the various impacted biological processes, highlighting their importance in cold stress response. This study will help us gain insights into cold response pathways of tomato and other commercially important, closely related Solanaceae species.</p>

<p> The plant cytoskeleton is commonly considered a vital component of cell growth and development; however, it also plays a critical role in plant immunity. During plant immunity, the cytoskeleton orchestrates rapid and precise immune-associated processes. For instance, the cytoskeleton mobilizes and orients the movement of organelles, proteins, and chemical signaling. To counter plant immunity, bacterial pathogens deliver virulence proteins, known as T3Es (type III effectors), into plant cells through a needle-like apparatus called the type III secretion system (T3SS). A novel T3E, called RipU, interacts with the cytoskeleton. Data has shown that RipU co-localizes with cytoskeletal markers in tobacco leaves. Ectopic expression of RipU can suppress PTI responses like ROS bursts or seedling growth inhibition. Tomato plants inoculated with <em>Rs</em> K60 lacking RipU showed less wilting and root colonization, suggesting that RipU plays a role in pathogenesis and virulence. Furthermore, inducible expression of RipU in Arabidopsis dramatically alters plant development. These plants have wavy roots, branching root hairs, and underdeveloped true leaves. Our results suggest that by targeting the cytoskeleton, RipU contributes to <em>Rs</em> K60s pathogenicity and virulence. </p>

<p>Nitrogen and phosphorus are the two macronutrients which play important roles in the plant, both structurally and functionally, e.g., starting from being constituents of cellular integrity to being signal molecules in signal transduction. Since they are required by plants in higher concentrations, it becomes indispensable to replenish their pools in soils by the application of chemical fertilizers. However, this practice is not only costly, the sources of Phosphorus and Nitrogen are not renewable and the excessive application in the form of fertilizers is not environmentally sustainable. Therefore, it warrants a better understanding of the plant responses during the nutrient deficiency because such knowledge will help implement strategies for breeding crops with more efficient use of minerals.</p><p>Most prior efforts in studying the molecular and physiological responses to low minerals were focused on roots. However, recently it has been found that shoot-to-root long distance signaling plays an important role in the adaptation of roots to low nitrogen or phosphorus. Here, we measured different physiological and morphological parameters and used RNA-Seq to elucidate the physiological and molecular responses in the vascular tissues of <i>Plantago major</i>, a new model species established in our laboratory, to low nitrogen, low phosphate or combined nitrogen and phosphate starvation<i>. </i>In this study, <i>P major </i>showed reduced photosynthesis and Fv/Fm, increased catalase and ascorbate peroxidase activity, reduced phosphate and nitrate contents in respective treatments. In addition, assessment of root morphological parameters revealed that nutrient deficiencies could lead to higher root densities and increased root to shoot ratios.</p><p>For molecular analysis of transcriptome changes, 24 hours of nutrient starvation exhibited an alteration of 33, 221, and 329 genes for the deficiencies of phosphorus, nitrogen and combined nitrogen and phosphorus, respectively. Our study helped to dissect several novel pathways associated with the vascular system in response to the deficiencies of major macronutrients. </p>