Biotic stresses such as infection by bacteria negatively affect plant growth and pose a severe threat to human food production. Improving our understanding of the immune systems of plants should help ensure food supplies in the years ahead. Bacterial infections induce Pattern-Triggered Immunity (PTI), a process in which plants perceive bacterial molecules and trigger an immune response. Mitogen- Activated Protein Kinase (MAPK) cascades are key players in this immunity process. Since the MAP Kinases (MPKs) 3/4/6 are mainly responsible for flg22- dependent phosphorylation events, we sought to find out how oxidation of MPK4 affects its ability to respond to stresses. Previous studies have shown varying kinase activity of MPK4 upon oxidation. Therefore, this project aims to provide an insight into the oxidative defense signaling mechanism of A. thaliana by investigating the role of MPK4 Cysteine181 in vitro and in vivo. Analysis of oxidation-mimicking as well as oxidation-dead mutants gave first hints that Cysteine181, which is located in the MPK4 substrate binding pocket, is a highly important regulatory residue of oxidative stress signaling by affecting MPK4 kinase activity and the activation of MPK3 and MPK6. Binding studies revealed that those events are due to sterical hindrance within the binding pocket of MPK4 and the blockage of upstream activator binding. The second part of this study characterizes compositional and post-translational changes of plant ribosomes during pathogen infection. Ribosomal proteins selectively participate in the formation of polysomes under different environmental and developmental conditions. However, the function of these changes still remains elusive. The current research project attempts to understand the plant ribosomal changes that occur upon exposure to bacterial pathogens. To observe ribosomal changes, A. thaliana plants were treated with a pathogen associated molecular pattern (PAMP), flg22. Mass spectrometric analysis identified quantitative changes of PAMP-induced ribosomal proteins in polysomes as well as changes in post-translational modifications. Spatial simulations of ribosomes revealed specific regions within the ribosomes to be PTI specific. This study demonstrates that MPK6 contributes to modification of P-stalk composition and phosphorylation status. The MPK6 mediated modifications may affect translation and in combination indicate a mechanism of PTI-related translational control.

Light and temperature are two of the most important environmental factors regulating plant development. Although heat stress has been well studied, little is known about the interaction between light and temperature. In this study, we performed phenotypic assays comparing seedling responses to heat under light and dark conditions. Seedlings exposed to heat in the dark show lower survival rates than seedlings stressed in the light. To identify transcriptional changes underlying light-dependent heat tolerance, we used RNA-sequencing. The light-dependent heat stress responses involved a plethora of genes which could be potential candidate genes for light-induced heat tolerance, including transcription factors (bHLH) and genes commonly associated with biotic stress. By using the latest high-throughput phenotyping facility, we found that the light-dependent heat tolerance is reflected more on the maintenance of photosynthetic capacity, rather than leaf temperature. These results provide insights into how light increases heat stress tolerance in Arabidopsis seedlings and suggest its underlying mechanisms.

Although the cell wall is an essential plant cell structure influencing several important aspects of plant development, little is known about the genes and proteins that regulate its structure and function. In this dissertation, we first examined the regulation of the nuclear proteome of rice (Oryza sativa) in response to cell wall removal. Using labelree comparative proteome analysis we found that, upon removal of the cell wall, 142 nuclear proteins were up regulated and 112 nuclear proteins were down regulated. The differentially expressed proteins included transcription factors, histones, histone domain containing proteins, and histone modification enzymes. This study led to a novel discovery that removal of the cell wall results in dynamic changes in the nuclear proteome affecting the regulation of proteins involved in various molecular processes such as chromatin and nucleosome assembly, protein-DNA complex assembly, and DNA packaging. To further study cell wall development, we utilized the unique features of cotton fiber and performed a proteomic study using four stages during cotton fiber cell wall development including 10 days post anthesis (dpa), 15 dpa, 25 dpa, and 35 dpa. In addition, we aimed to improve protein extraction for recalcitrant fiber stages using pressure cycling technology (PCT). To our knowledge, this study identified the largest number of proteins and differentially expressed proteins in the G. hirsutum cotton species including the 35 dpa fiber proteome which has not been examined in prior reports. Additionally, in order to identify key genes regulating cell wall cellulose content, a mutant with a substantial reduction in cellulose was characterized in Arabidopsis. It was found that the mutated gene was VHA-E1. We found that the VHA-E1 protein formed a distinct plate in the boundary of two fusion-destined vacuoles to tether vacuoles together. The eventual vacuole fusion was achieved by pinching off the vacuole-boundary plate producing a stable membrane-bound intravacuolar globoid. These observations demonstrate that VHA-E1 may be involved in a novel cellular process regulating fusion of vacuoles by forming a cellular structure referred to as the vacuole boundary plate. Altogether, these findings suggest plant vacuole fusion and central vacuole biogenesis involve an unprecedented mechanism in Arabidopsis.

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The effector-triggered immunity (ETI) is activated at the site of pathogen infection and results in a state of enhanced immunity called systemic acquired resistance (SAR) in distal, uninfected plant organs. SAR relays on mobile signals transported from infected cells to distal organs, and on signal amplification which supports transcriptional re-programming associated with priming and execution of SAR. Previous research in our lab has identified the chloroplastic TOP1 and cytosolic TOP2 as salicylic acid (SA)–binding oligopeptidases, non-competitively inhibited by SA. We demonstrate that SAR triggered with P. syringae DC3000 AvrRpt2 is abolished in top2 whereas top1 top2 exhibits a SAR slightly but consistently stronger than wild type (WT) controls, indicating that top1 is epistatic to top2. In agreement with the observed SAR phenotypes, top2 is defective in the induction of SAR markers including SA and Pip synthesis and SA signaling genes, whereas top1 top2 shows significantly higher induction of these markers. SAR- phenotype of top2 is rescued by exogenous SA, H2O2 and Pip applications. Interestingly, neither top1 nor top 1top2 are unable to mount SAR in response to Pip and H2O2 treatments. Analysis of ROS-responsive transcription factors and antioxidant gene induction in infected and distal tissues reveal significantly dysregulated patterns in all mutants, with top2 and top1 top2 most affected, indicating that TOP1 and TOP2 function together to support a pattern of successive waves of oxidation and reduction during SAR. The local and systemic oscillations are anti-corelated in Wt. The local vs. systemic anti-correlation is lost in the mutant genotypes. The amplitude of the mRNA oscillations is significantly lower in top2 plants, and significantly increased in top1top2 plants. top1 and top1top2 lost the oscillation compared to WT but they are still able to keep the expression up in time. top2 is unable to support the expression of some of the genes and oscillations and continued the expression of these genes in time. Overall, our results argue for a defining role of TOP chloroplastic and cytosolic proteolytic pathways in maintaining redox signaling necessary for the induction of SAR transcriptional re-programming and execution.

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When exposed to either abiotic or biotic stressors, plants release chemical compounds that can serve as defense mechanisms. For example, plants of the mustard and cabbage family produce a class of anti-herbivory compounds called glucosinolates. In the model mustard plant Arabidopsis thaliana, some glucosinolates are produced from the amino acid tryptophan and are called indole glucosinolates (IGs). Expression of IG synthesis genes is positively regulated by the partially redundant transcription factors, MYB34 and MYB51. Recent studies have shown that these two transcription factors have distinct roles in regulating IG production in different tissues and in mediating responses to different environmental cues. To understand the distinct roles of these transcription factors at a more detailed temporal and spatial level, reporters for CYP79B2, a transcriptional target of both MYB34 and MYB51, were used. CYP79B2-GFP and CYP79B2-GUS reporter expression was analyzed in wild-type and MYB34 and MYB51 mutant plants in response to increased ambient temperature, increased light intensity, ATP exposure, and chitin exposure. Reverse-phase HPLC quantification of IGs was also performed to determine how these transcription factors are mediating the synthesis of IGs in response stressors. Overall, it was found that MYB51 is responsible for the temperature induction of IG production, while increased light intensity has no impact on IG synthesis. Furthermore, ATP appears to induce IG production independently of both MYB34 and MYB51, while chitin does not increase IG synthesis. Taken together, these studies allow us to better understand how plants respond to and defend themselves from different abiotic and biotic stress / 2025-02-26T00:00:00Z

<p> Age-Related Resistance (ARR) has been observed in numerous plant species, resulting in increased disease resistance as the plant matures. The ARR defective mutant, iap1-1, (important in the ARR Pathway,) was discovered in an ARR mutant screen and EDS1, (enhanced disease susceptibility,) which is involved in other disease resistance pathways, was shown to be required for ARR. lntercellular accumulation of salicylic acid (SA) is required for ARR suggesting that SA may act as an anti-microbial agent. Mature (6 wpg) iap1-1 does not accumulate intercellular or intracellular SA in response to Pst inoculation. lntercellular and intracellular SA accumulation is also partially reduced in young (4 wpg) plants during R gene-mediated resistance to Pst(AvrRpt2) which is partially compromised suggesting that the two pathways share common elements. The novel discovery of the presence of intercellular SA during R gene-mediated resistance suggests that it may act as an antimicrobial agent during R gene-mediated resistance as it is hypothesized to during ARR. The iap1-1 mutation maps to chromosome four between 17,938,268bp and 18,133,423b. The semi-dominant, loss of function nature of the iap1-1 mutation suggests that IAP1 is a positive regulator in the ARR pathway.</p> / Thesis / Doctor of Philosophy (PhD)

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