Imbalance between the accumulation and removal of nitric oxide and its derivatives is a challenge faced by all plants at the cellular level, and is especially important under stress conditions. Exposure of plants to various biotic and abiotic stresses causes rapid changes in cellular redox tone potentiated by the rise in reactive nitrogen species that serve as signaling molecules in mediating defensive responses. To understand mechanisms mediated by these signaling molecules, we performed a large-scale analysis of the Arabidopsis transcriptome induced by nitrosative stress. We generated an average of 84 and 91 million reads from three replicates each of control and 1 mM S-nitrosocysteine (CysNO)-infiltrated Arabidopsis leaf samples, respectively. After alignment, more than 95% of all reads successfully mapped to the reference and 32,535 genes and 55,682 transcripts were obtained. CysNO infiltration caused differential expression of 6,436 genes (3,448 up-regulated and 2,988 down-regulated) and 6,214 transcripts (3,335 up-regulated and 2,879 down-regulated) 6 h post-infiltration. These differentially expressed genes were found to be involved in key physiological processes, including plant defense against various biotic and abiotic stresses, hormone signaling, and other developmental processes. After quantile normalization of the FPKM values followed by student’s T-test (P<0.05) we identified 1,165 DEGs (463 up-regulated and 702 down-regulated) with at least 2-folds change in expression after CysNO treatment. Expression patterns of selected genes involved in various biological pathways were verified using quantitative real-time PCR. This study provides comprehensive information about plant responses to nitrosative stress at transcript level and would prove helpful in understanding and incorporating mechanisms associated with nitrosative stress responses in plants.

Background: Exposure of plants to different environmental insults instigates significant changes in the cellular redox tone driven in part by promoting the production of reactive nitrogen species. The key player, nitric oxide (NO) is a small gaseous diatomic molecule, well-known for its signaling role during stress. In this study, we focused on abscisic acid (ABA) metabolism-related genes that showed differential expression in response to the NO donorS-nitroso-l-cysteine (CySNO) by conducting RNA-seq-based transcriptomic analysis.Results: CySNO-induced ABA-related genes were identified and further characterized. Gene ontology terms for biological processes showed most of the genes were associated with protein phosphorylation. Promoter analysis suggested that severalcis-regulatory elements were activated under biotic and/or abiotic stress conditions. The ABA biosynthetic geneAtAO3was selected for validation using functional genomics. The loss of function mutantatao3was found to differentially regulate oxidative and nitrosative stress. Further investigations for determining the role ofAtAO3in plant defense suggested a negative regulation of plant basal defense andR-gene-mediated resistance. Theatao3plants showed resistance to virulentPseudomonas syringaepv.tomatostrain DC3000 (PstDC3000) with gradual increase inPR1gene expression. Similarly,atao3plants showed increased hypersensitive response (HR) when challenged withPstDC3000 (avrB). Theatgsnor1–3andatsid2mutants showed a susceptible phenotype with reducedPR1transcript accumulation. Drought tolerance assay indicated thatatao3andatnced3ABA-deficient mutants showed early wilting, followed by plant death. The study of stomatal structure showed thatatao3andatnced3were unable to close stomata even at 7 days after drought stress. Further, they showed reduced ABA content and increased electrolyte leakage than the wild-type (WT) plants. The quantitative polymerase chain reaction analysis suggested that ABA biosynthesis genes were down-regulated, whereas expression of most of the drought-related genes were up-regulated inatao3than in WT.Conclusions: AtAO3negatively regulates pathogen-induced salicylic acid pathway, although it is required for drought tolerance, despite the fact that ABA production is not totally dependent onAtAO3, and that drought-related genes likeDREB2andABI2show response to drought irrespective of ABA content.

Exposure of plants to different biotic and abiotic stress condition instigates significant change in the cellular redox status; resulting in the elevation of reactive nitrogen species that play signaling role in mediating defense responses. Heavy metal associated (HMA) domain containing genes are required for spatio-temporal transportation of metal ions that bind with various enzymes and co-factors within the cell. To uncover the underlying mechanisms mediated by AtHMA genes, we identified 14 Arabidopsis HMA genes that were differentially expressed in response to nitrosative stress through RNA-seq analysis. Of those 14 genes, the expression of eight HMA genes was significantly increased, whereas that of six genes was significantly reduced. We further validated the RNA-seq results through quantitative real-time PCR analysis. Gene ontology analysis revealed the involvement of these genes in biological processes such as hemostasis and transport. The majority of these nitric oxide (NO)-responsive AtHMA gene products are carrier/transport proteins. AtHMAD1 (At1g51090) showed the highest fold change to S-nitrosocystein. We therefore, further investigated its role in oxidative and nitrosative mediated stress conditions and found that AtHMAD1 has antagonistic role in shoot and root growth. Characterization of AtHMAD1 through functional genomics showed that the knock out mutant athmad1 plants were resistant to virulent Pseudomonas syringae (DC3000) and showed early induction and high transcript accumulation of pathogenesis related gene. Furthermore, inoculation of athamd1 with avirulent strain of the same bacteria showed negative regulation of R-gene mediated resistance. These results were supported by hypersensitive cell death response and cell death induced electrolyte leakage. AtHMAD1 was also observed to negatively regulate systemic acquired resistance SAR as the KO mutant showed induction of SAR marker genes. Overall, these results imply that NO-responsive AtHMA domain containing genes may play an important role in plant development and immunity.

TFs are important proteins regulating plant responses during environmental stresses. These insults typically induce changes in cellular redox tone driven in part by promoting the production of reactive nitrogen species (RNS). The main source of these RNS is nitric oxide (NO), which serves as a signalling molecule, eliciting defence and resistance responses. To understand how these signalling molecules regulate key biological processes, we performed a large scale S-nitrosocysteine (CySNO)-mediated RNA-seq analysis. The DEGs were analysed to identify potential regulatory TFs. We found a total of 673 (up- and down-regulated) TFs representing a broad range of TF families. GO-enrichment and MapMan analysis suggests that more than 98% of TFs were mapped to the Arabidopsis thaliana genome and classified into pathways like hormone signalling, protein degradation, development, biotic and abiotic stress, etc. A functional analysis of three randomly selected TFs, DDF1, RAP2.6, and AtMYB48 identified a regulatory role in plant growth and immunity. Loss-of-function mutations within DDF1 and RAP2.6 showed compromised basal defence and effector triggered immunity, suggesting their positive role in two major plant defence systems. Together, these results imply an important data representing NO-responsive TFs that will help in exploring the core mechanisms involved in biological processes in plants.