Dataset Information

Discovery of a Nitric Oxide-Responsive Protein in Arabidopsis thaliana.

ABSTRACT: In plants, much like in animals, nitric oxide (NO) has been established as an important gaseous signaling molecule. However, contrary to animal systems, NO-sensitive or NO-responsive proteins that bind NO in the form of a sensor or participating in redox reactions have remained elusive. Here, we applied a search term constructed based on conserved and functionally annotated amino acids at the centers of Heme Nitric Oxide/Oxygen (H-NOX) domains in annotated and experimentally-tested gas-binding proteins from lower and higher eukaryotes, in order to identify candidate NO-binding proteins in Arabidopsis thaliana. The selection of candidate NO-binding proteins identified from the motif search was supported by structural modeling. This approach identified AtLRB3 (At4g01160), a member of the Light Response Bric-a-Brac/Tramtrack/Broad Complex (BTB) family, as a candidate NO-binding protein. AtLRB3 was heterologously expressed and purified, and then tested for NO-response. Spectroscopic data confirmed that AtLRB3 contains a histidine-ligated heme cofactor and importantly, the addition of NO to AtLRB3 yielded absorption characteristics reminiscent of canonical H-NOX proteins. Furthermore, substitution of the heme iron-coordinating histidine at the H-NOX center with a leucine strongly impaired the NO-response. Our finding therefore established AtLRB3 as a NO-interacting protein and future characterizations will focus on resolving the nature of this response.

SUBMITTER: Zarban R

PROVIDER: S-EPMC6696476 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Discovery of a Nitric Oxide-Responsive Protein in <i>Arabidopsis thaliana</i>.

Zarban Randa R Vogler Malvina M Wong Aloysius A Eppinger Joerg J Al-Babili Salim S Gehring Chris C

Molecules (Basel, Switzerland) 20190724 15

In plants, much like in animals, nitric oxide (NO) has been established as an important gaseous signaling molecule. However, contrary to animal systems, NO-sensitive or NO-responsive proteins that bind NO in the form of a sensor or participating in redox reactions have remained elusive. Here, we applied a search term constructed based on conserved and functionally annotated amino acids at the centers of Heme Nitric Oxide/Oxygen (H-NOX) domains in annotated and experimentally-tested gas-binding p ...[more]

PMID: 31344907

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Project description: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.

Project description: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 donor S-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 several cis-regulatory elements were activated under biotic and/or abiotic stress conditions. The ABA biosynthetic gene AtAO3 was selected for validation using functional genomics. The loss of function mutant atao3 was found to differentially regulate oxidative and nitrosative stress. Further investigations for determining the role of AtAO3 in plant defense suggested a negative regulation of plant basal defense and R-gene-mediated resistance. The atao3 plants showed resistance to virulent Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) with gradual increase in PR1 gene expression. Similarly, atao3 plants showed increased hypersensitive response (HR) when challenged with Pst DC3000 (avrB). The atgsnor1-3 and atsid2 mutants showed a susceptible phenotype with reduced PR1 transcript accumulation. Drought tolerance assay indicated that atao3 and atnced3 ABA-deficient mutants showed early wilting, followed by plant death. The study of stomatal structure showed that atao3 and atnced3 were 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 in atao3 than in WT. CONCLUSIONS:AtAO3 negatively regulates pathogen-induced salicylic acid pathway, although it is required for drought tolerance, despite the fact that ABA production is not totally dependent on AtAO3, and that drought-related genes like DREB2 and ABI2 show response to drought irrespective of ABA content.

Dataset Information

Discovery of a Nitric Oxide-Responsive Protein in Arabidopsis thaliana.

Publications

Discovery of a Nitric Oxide-Responsive Protein in <i>Arabidopsis thaliana</i>.

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