Nitric oxide (NO), a diffusible gas molecule, is a major signal molecule in both plants and animals and regulates a plethora of biological processes. S-nitrosylation, a post-translation modification, is conducted by NO, which covalently attaches protein cysteine thiols and forms an S-nitroso thiol. S-nitrosylation plays an important role in plant development and plant immune systems. In Arabidopsis thaliana, S-nitrosoglutathione (GSNO) is the major NO donor for S-nitrosylation, and GSNO reductase (GSNOR) indirectly controls the S-nitrosylation level by turning over the GSNO. An A. thaliana T-DNA insertion mutant gsnor1-3 shows the loss of GSNOR activity and increases the S-nitrosylation level, resulting in loss of apical dominance, reduction of SA accumulation, increased hypersensitive response (HR) cell death and reduced disease resistance against virulence, avirulence and non-host pathogens. Interestingly, loss of GSNOR in Drosophila melanogaster, an animal model system, reduces the resistance against gram-positive and fungal pathogens. Catalase is an antioxidant enzyme and regulates the redox environment through scavenging the hydrogen peroxide (H2O2) to oxygen and water. Previous work in our lab had discovered two gsnor1-3 suppressor mutants, gsnor1-3 spl7 and gsnor1-3 spl8, which restore the loss of apical dominance and partially restore disease resistance. These two suppressor mutants were then identified as the point mutation in CAT3. CAT3, one of the three CAT genes in Arabidopsis, expresses catalase specifically in vascular tissues. To further extend the suppression of cat3 in gsnor1-3, the mutations in CAT3 and its paralogs CAT2 and CAT1, as well as other redox-related genes in gsnor1-3 background, were generated. In the developmental phenotype, only the gsnor1-3 cat3 showed significant changes compared with gsnor1-3. The disease susceptibility and HR cell death in gsnor1-3 cat3 were less than gsnor1- 3 and similar to wild-type. Moreover, the redox-related genes and CAT3 paralog mutations in gsnor1-3 background showed no significant changes in disease resistance against virulence pathogen compared with gsnor1-3 plant. Meanwhile, an SA-dependent (salicylic acid) defence-related gene (PR1, pathogenesis-related gene 1) showed the early expression in gsnor1-3 cat3 plant compared with gsnor1-3 plant. Results of developmental and disease-related phenotypes suggest the suppression effects which turn-over the malfunction in gsnor1- 3 are highly specific to CAT3. The previous report demonstrates that the hydroxyl radical, a reactive oxygen species by-product from H2O2, decomposes GSNO to oxidised glutathione in vitro. The interaction of GSNO and hydroxyl radical may be the possible mechanism of how cat3 suppresses gsnor1- 3. Therefore, we speculated less amount of GSNO in gsnor1-3 cat3 plant than in gsnor1-3 plant and lower level of hydroxyl radicals in gsnor1-3 cat3 plant than in cat3 plant. To evaluate our hypothesis, the content hydroxyl and GSNO were analysed in wild-type, gsnor1-3, cat3 and gsnor1-3 cat3 plants. The total S-nitrosylated protein, which indicates the GSNO content in vivo, was less in gsnor1-3 cat3 than in gsnor1-3. Furthermore, the level of hydroxyl radical in gsnor1-3 cat3 was lower than cat3. Accordingly, the reduction of hydroxyl radical in gsnor1- 3 cat3 may occur due to the reaction with GSNO and vice versa. Similar to what has been found in Arabidopsis, D. melanogaster also reported partial restoration of the immunodeficiency phenotypes of gsnor knock-out flies with an additional mutation in CAT gene. Interestingly, the content of hydroxyl radical in gsnor-/- cat-/- line was less than cat+/-. Collectively, our results suggest an interaction of hydroxyl radical and GSNO may happen both in Arabidopsis and Drosophila. Further research is needed to clarify the interaction between hydroxyl radical and GSNO in Arabidopsis as well as in Drosophila.

The plant hormone salicylic acid (SA) is an essential activator of plant immune responses directed against biotrophic pathogens. The transcription cofactor NPR1 (Nonexpressor of pathogenesis- related (PR) genes 1) functions to transduce the SA signal into an operational response directed to limited pathogen damage. In the absence of pathogen, NPR1 protein resides in the cytoplasm as a large molecular weight oligomer held together by disulphide bonding. Initiation of defence signalling leads to changes in intracellular redox conditions that promote NPR1 momomer release. Translocation of monomeric NPR1 to the nucleus results in the activation of over 2200 immune-related genes in Arabidopsis. NPR1 lacks a canonical DNA-binding domain but is known to perform part of its regulatory function through engagement of TGA factors (bZIP transcription factor). Induction of SA-dependent signalling is invariably associated with PR-1 gene expression and accumulation of mRNA for this gene serves as a useful marker of defence activation. However, both functional redundancy and stochastic factors limit the effectiveness of standard genetic approaches used in plant research, and thus much of the hierarchal processes surrounding NPR1-dependent gene activation are not fully understood. Using a synthetic biology approach we aim to complete exploratory work and set the foundations for the development of a yeast tool that can be used to manipulate and subsequently understand NPR1 function in relation to interacting partners and gene activation. Accordingly, using this tool we sought to create a conceptual protein circuit based on theoretical plant immunity. In completing this work we have developed a Saccharomyces cerevisiae strain that exhibits a highly oxidising intracellular redox environment. This was achieved by knocking out genes encoding S-nitrosoglutathione reductase (SFA1), flavohemoglobin (YHB1) and YAP1 (bZIP transcription factor), all important components in regulating cellular redox homeostasis and protein S-nitrosylation state in S. cerevisiae. Characterisation of this cell (designated Δsfa1yap1yhb1) reveals a high tolerance to such redox perturbations. Importantly, NPR1 is by default, assembled predominantly in the oligomeric form in this biological chassis. By activating two inducible inputs in the form of Arabidopsis S-nitrosoglutathione reductase (AtGSNOR) and Thioredoxin (AtTRXh5) which both function to promote NPR1 monomerisation, we have created a switch to selectively control NPR1 oligomer-monomer equilibrium. To complete the synthetic circuit, TGA3 was included, along with a modified yeast MEL1 promoter that has been customised to contain the TGA-responsive upstream activation sequence (termed the as-1 element) present in the promoter region of the PR-1 gene. Using FRET tools we were able to confirm nuclear interaction between monomeric NPR1 and TGA3, with this association appearing to induce as-1 element binding. However this process is not sufficient to activate a Luciferase (LUC) reporter gene, even when the GAL4 activation domain (GAL4 AD) is fused to NPR1. Ordinarily, a CUL3-dependent proteolysis-coupled transcription cycle is necessary to maintain efficient NPR1-dependent gene transcription in Arabidopsis. Although S. cerevisiae encodes an evolutionarily related CUL3 ortholog, examination by western blot demonstrates that NPR1 protein is stable in this cell, indicating an endogenous mechanism to degrade NPR1 is either not present or not functional in yeast. As such, this synthetic yeast tool represents a completely novel approach to identify missing components functioning in NPR1-mediated transcriptional regulation. Furthermore, in collaboration with a skilled bioinformatician, and using a rule-based stochastic modeling tool known as Kappa, we have been able to develop, for the first time, a preliminary mathematical simulation representative of NPR1-dependent gene activation that can be used as a foundation for future works.

Plants have evolved a plethora of effective mechanisms to protect themselves from biotic stresses. Jasmonates (JAs) are employed as vital defence signals against both insect and pathogen attack. Jasmonic acid (JA) signalling plays a central role in plant defence and development. S-nitrosylation, a redox-based post-translational modification plays an important role in plant disease resistance. S-nitrosoglutathione (GSNO) is formed by the reaction of antioxidant glutathione (GSH) and nitric oxide (NO) and acts as a mobile reservoir of NO bioactivity. The Arabidopsis thaliana S-NITROSOGLUTATHIONE REDUCTASE (AtGSNOR1) controls multiple modes of disease resistance via S-nitrosylation. In this context, the Arabidopsis lossof- function mutant atgsnor1-3 exhibits higher susceptibility to Botrytis cinerea a necrotrophic pathogens and Pieris rapae insect attack. Accumulation of JA was reduced in atgsnor1-3 after mechanical wounding. JA marker genes were also downregulated in atgsnor1-3 compared to Col-0 after Methyl Jasmonate (Me-JA) treatment. The relative gene expression of Vegetative Storage Protein (VSP) was reduced in atgsnor1-3 compared to wild type. Further, protein-protein interaction experiments in yeast two hybrid assays revealed an inhibition of Coronatine-insensitive 1 (COI1) and Jasmonate ZIM domain (JAZ1) interactions upon NO donor application. Interestingly it was also shown that Nitric oxide donor may inhibited the degradation of JAZ1-β-glucoronidase (GUS) fusion protein driven by a CaMV35s:: JAZ1-GUS transgene in GUS histochemical analysis but not in flurometric assay. A biotin switch assay of recombinant JAZ1-Maltose-binding protein (MBP) has shown that JAZ1-MBP was S-nitrosylated and mass spectrometry suggested Cysteine229 (Cys229) was the site of this modification. Further, CaMV35S::JAZ1-Flag transgene expressed in either a wild-type or atgsnor1-3 genetic background, suggested that JAZ1 was S-nitrosylated in vivo. Collectively, our data imply that JA-signalling engaged in response to either insect predation or attempted B. cinerea infection is under redox control as high SNO in atgsnor1-3 has disrupted the JA signalling pathway. Furthermore, our data suggest that S-nitrosylation of Cys-229 of JAZ1 may control JA-mediated signalling by blocking the interaction of this protein with COI1, thus reducing the turnover of JAZ1 by the 26S proteasome and consequently enabling continued JAZ1-mediated repression of JA-dependent gene expression in the presence of Me-JA. Thus our findings highlight the importance of NO and associated S-nitrosylation in JA signalling during plant immune response.

Following pathogen recognition, nitric oxide (NO) is rapidly produced in plants, this small molecule has emerged as a key signal in plant defence responses. S-nitrosylation is the major route of NO signal transduction in plants, a redox-based modification by addition of an NO moiety on cysteine thiol to form an S-nitrosothiol (SNO). S-nitrosoglutathione reductase (GSNOR) regulates cellular levels of S-nitrosylation and displays a key role in regulating the plant defence response. In this context, NO is important to orchestrate both defence gene expression and the hypersensitive response (HR) during attempted microbial infection. However, how the plant immune system recognizes NO and how NO level could elicit plant defence responses are poorly understood. The Arabidopsis thaliana (Arabidopsis) mutant NO overproducing 1 (nox1) was employed to characterize how NO level elicits defence dynamics. In response to microbial infection, resistance (R) gene-mediated defence and basal resistance were found to be compromised in the nox1 mutant relative to wild type Col-0 plants. Interestingly, nox1 mutant exhibit similar levels of HR and pathogen susceptibility to the GSNOR loss-of-function mutant atgsnor1-3. This phenomenon suggests that NO might regulate defence responses via GSNOR-mediated S-nitrosylation. Therefore, the nox1 atgsnor1-3 double mutant was generated and characterized to clarify this hypothesis. Accelerated HR and increased pathogen susceptibility are shown in the double mutant, which implies that increased NO mediated by nox1 and elevated SNOs resulting from atgsnor1-3, are additive with respect to the plant defence response. To identify genes responsible for NO perception, forward genetic screens were developed to identify Arabidopsis mutants with abnormal NO recognition. NO marker genes for genetic screens were identified from both lab and open source microarray data. Two genes, At3g28740 and At1g76600 were selected and experimentally confirmed to be strongly induced by NO. Transgenic Arabidopsis plants were generated carrying a NO reporter cassette, which consist of a luciferase reporter gene (LUC) driven by the promoter of NO marker gene. This forward genetic approach might be a powerful tool to identify genes integral to NO signal transduction. Three C2H2 zinc finger transcription factors (ZnTFs) ZAT7, ZAT8 and ZAT12 were identified as being rapidly and strongly induced by NO donors, which could be modulators of redox/NO-dependent signalling pathway. T-DNA insertion mutants within these ZnTFs have been identified. Basal resistance against Pseudomonas syringae pv tomato (Pst) DC3000 is compromised in all single knockout lines. Therefore, the full characterisation of defence phenotype of these mutants would be necessary to explore the role of these TFs in the plant defence. Furthermore, zat8 mutant is more sensitive to nitrosative stress when compared to wild type Col-0. This suggests that ZAT8 may be involved in protecting plants against nitrosative stress. However, the molecular mechanisms that underpin this function remain to be determined. In conclusion, NO and SNOs might regulate plant disease resistance via distinct pathways. Our work has also established NO-reporter lines to identify genes responsible for NO perception. In addition, three NO-induced ZnTFs have been identified that participate in regulation of basal resistance, which might unveil aspects of NO signalling related to the regulation of transcription.

Salinity stress is one of the main challenges for crop growth and production. The estimated loss of crop yield due to salinity stress is up to 20% worldwide each year. Plants have evolved an array of mechanisms to defend themselves against salinity stress. A key aspect of plant responses to salinity stress is the engagement of a nitrosative burst that results in nitric oxide (NO) accumulation. A major mechanism for the transfer of NO bioactivity is S-nitrosylation which is a modification of the reactive thiol group of a rare but highly active cysteine residue within a protein through the addition of a NO moiety to generate an S-nitrosothiol (SNO). S-nitrosylation can result in altered structure, function and cellular localisation of a protein. Our findings suggest that S-nitrosylation is a key regulator of plant responses to salinity stress. Glutathione (GSH), a tripeptide cellular antioxidant, is S-nitrosylated to form S-nitrosoglutathione (GSNO), which functions as a stable store of NO bioactivity. Cellular GSNO levels are directly controlled by S-nitrosoglutathione reductase (GSNOR), thereby, regulating global SNO levels indirectly. The absence of this gene results in high levels of SNOs. In Arabidopsis, previous research has shown that loss-of-function mutation in GSNOR1 results in pathogen susceptibility (Feechan et al., 2005). In our study, we investigated salt tolerance in gsnor1-3 plants. We have found that this line is salt sensitive at various stages of their life cycle. Interestingly, classical salt stress signalling pathways are fully functional in gsnor1-3 plants. We have also explored non-classical pathways involved in salt tolerance. Autophagy is a cellular catabolic process which is involved in the recycling and degradation of unwanted cellular materials under stressed and non-stressed conditions. We have demonstrated that gsnor1-3 plants have impaired autophagy during salt stress. An accumulation of the autophagy marker NBR1 supports the lack of autophagosome formation. We hypothesised that S-nitrosylation might regulate upstream nodes of autophagosome formation. Our study demonstrated that at least one key player involved in autophagosome biogenesis is regulated by S-nitrosylation. ATG7, an E1-like activating enzyme, which regulates ATG8-PE and ATG12-ATG5 ubiquitin like conjugation systems, is S-nitrosylated in vitro and in vivo. S-nitrosylation of ATG7 impairs its function in vitro. We showed that S-nitrosylation of ATG7 is mediated by GSNO. Interestingly, ATG7 is also transnitrosylated by thioredoxin (TRX), another important redox regulatory enzyme. We suggest that similar mechanisms might exist in planta. Finally, work in this study revealed that S-nitrosylation of Cys558 and Cys637 cause the inhibition of ATG7 function. In aggregate, this study revealed a novel mechanism for the redox-based regulation of autophagy during salt stress.

Nitric oxide (NO) as well as reactive oxygen species (ROS) play an important role in defence signalling in plants. After successful recognition of an invading pathogen, an increase in ROS occurs, the ’oxidative burst’; and a ’nitrosative burst’ is also observed. This leads to the induction of defence responses, including the ’hypersensitive response’ (HR), a form of programmed cell death. A balanced production of hydrogen peroxide and NO is crucial for HR induction. In a process called S-nitrosylation, NO can react with cysteine thiols to form S-nitrosothiols, or react with glutathione to form S-nitrosoglutathione (GSNO). The enzyme GNSO reductase (GSNOR) indirectly regulates SNO levels by turning over GNSO. The Arabidopsis thaliana T-DNA insertion mutant atgsnor1-3 shows a complete loss of GNSOR activity and has drastically increased SNO levels, resulting in stunted growth, loss of apical dominance, increased HR, loss of salicylic acid (SA) accumulation and increased susceptibility to avirulent, virulent and non-host pathogens. Two recessive and allelic EMS suppressor mutants in the atgsnor1-3 background were isolated, which showed mostly wild type growth. The mutations were identified by map-based cloning as two different point mutations in At1g20620 or CAT3, one of three catalase genes in Arabidopsis. Catalases break down hydrogen peroxide, with CAT2 being the major catalase in Arabidopsis. All three catalases are structurally very similar, but show temporal and spatial differences in their expression patterns. The suppressor mutants recovered apical dominance, and partially recovered disease resistance to avirulent pathogens, but were still susceptible to virulent pathogens and showed decreased SA levels. The suppressor mutants showed wild type HR in response to different avirulent bacteria. Interestingly, loss-of-function of the other catalase genes as well as loss-of-function of other redox-related genes did not restore apical dominance of atgnsor1-3 plants. This effect seems to be highly specific to CAT3, possibly because of its expression pattern or its expression levels. Further research is needed to fully understand the mechanisms at work here, but these results certainly seem to show a direct connection between redox signalling and S-nitrosylation.

S-nitrosylation (or S-nitrosation) has emerged as an important pathway of non-classical nitric oxide signaling. This post-translational modification involves the transfer of a nitroso group onto a cysteine residue and has been shown to regulate protein function. However, very little is known about the mechanism and structure-dependent factors of the modification. Understanding of S-nitrosothiol chemistry has lagged behind that for the classical nitric oxide signaling pathway due to challenges and limitations of current detection methods of S-nitrosothiols. The S-N bond is typically labile and indirect detection by traditional biotin switch method has low sensitivity and is prone to false positives. In this work, I have explored phosphine derivatization as a new direct approach to labeling protein S-nitrosothiols. Syntheses of aza-ylide derivatives of small organic S-nitrosothiols were successful and the termolecularity of the reaction was overcome by using a bisphosphine. Similarly, S-nitrosated cysteines of thioredoxin were successfully derivatized with the phosphine TCEP and identified by tandem mass spectrometry of the digested protein. Surprisingly, derivatization of S-nitrosoglutathione was found to be unsuccessful and ¹⁸O-labeling of the reaction indicated hydrolysis of the aza-ylide product. We hypothesize that solvent effects are the source of this discrepancy. In addition, x-ray crystallography studies were undertaken to investigate structural rearrangement of a thioredoxin helix to expose residue Cys 62 to S-nitrosation. A new structure of thioredoxin Q63A/C69S/C73S mutant was found to exhibit a highly dynamic N-terminal loop surrounding the pocket of Cys 62 which could have an effect on S-nitrosation of this residue.

Oxidative stress is one of the major characteristics in Alzheimer’s disease, and converging evidence indicates that cysteine S-nitrosylation might be related in AD pathology. My results demonstrated exogenous S-nitrosoglutathione was able to S-nitrosylate vAChT, vMAT2, vGluT1 and vGluT2. S-nitrosylation of these vesicular transporters inhibited the uptake of [3H]acetylcholine, [3H]dopamine and [3H]glutamate respectively. APP/PS1 transgenic mice were used to investigate neurotransmission dysfunctions of Alzheimer’s disease. Global protein S-nitrosylation was increased in the 9 and 12 month APP/PS1 mice. Further investigation demonstrated an increase of vAChT and vGluT1 S-nitrosylation in frontal cortex of 6, 9 and 12 month APP/PS1 mice and an increased vAChT and vGluT1 S-nitrosylation was found in hippocampus of 3 month APP/PS1 mice. These findings together suggest that S-nitrosylation of vesicular transporters inhibits the uptake of neurotransmitters, and S-nitrosylation of vAChT and might be associated with the neurotransmission dysfunction of acetylcholine and glutamate in Alzheimer’s disease.

<p>S-nitrosylation, the prototypic mechanism of redox-based signal transduction, involves the covalent attachment of a nitrogen monoxide group to a Cys-thiol side chain. S-nitrosylation of proteins has been demonstrated to affect a broad range of functional parameters including enzymatic activity, subcellular localization, protein-protein interactions and protein stability. The primary focus of my dissertation was to solve a problem of great importance in the field of S-nitrosylation, which is, to identify denitrosylase(s) i.e., enzymes that remove NO groups from S-nitrosothiols. Recent progress in elucidating the cellular regulation of S-nitrosylation has led to the identification of two physiologically relevant denitrosylating activities that remove the NO group from S-nitrosylated substrates. Thioredoxin/thioredoxin reductase (Trx system) functions as an NADPH-dependent denitrosylase across a broad range of S-nitrosylated proteins (SNO-proteins). S-nitroso-glutathione reductase (GSNOR), which is highly conserved across phylogeny, metabolizes GSNO utilizing NADH as a reducing coenzyme, thereby shifting equilibria between GSNO and SNO-proteins. This dissertation describes the discovery of two novel denitrosylases: one from yeast and the other from mammals. Using technique of column chromatography we have purified these novel denitrosylases to homogeneity and have demonstrated a principal contribution of these enzymes towards S-nitrosothiol metabolism.</p> / Dissertation

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