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Persulfidation of Nitrate Reductase 2 Is Involved in l-Cysteine Desulfhydrase-Regulated Rice Drought Tolerance.

ABSTRACT: Hydrogen sulfide (H₂S) is an important signaling molecule that regulates diverse cellular signaling pathways through persulfidation. Our previous study revealed that H₂S is involved in the improvement of rice drought tolerance. However, the corresponding enzymatic sources of H₂S and its regulatory mechanism in response to drought stress are not clear. Here, we cloned and characterized a putative l-cysteine desulfhydrase (LCD) gene in rice, which encodes a protein possessing H₂S-producing activity and was named OsLCD1. Overexpression of OsLCD1 results in enhanced H₂S production, persulfidation of total soluble protein, and confers rice drought tolerance. Further, we found that nitrate reductase (NR) activity was decreased under drought stress, and the inhibition of NR activity was controlled by endogenous H₂S production. Persulfidation of NIA2, an NR isoform responsible for the main NR activity, led to a decrease in total NR activity in rice. Furthermore, drought stress-triggered inhibition of NR activity and persulfidation of NIA2 was intensified in the OsLCD1 overexpression line. Phenotypical and molecular analysis revealed that mutation of NIA2 enhanced rice drought tolerance by activating the expression of genes encoding antioxidant enzymes and ABA-responsive genes. Taken together, our results showed the role of OsLCD1 in modulating H₂S production and provided insight into H₂S-regulated persulfidation of NIA2 in the control of rice drought stress.

SUBMITTER: Zhou H

PROVIDER: S-EPMC8624084 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Aims: Pepper fruit is a horticultural product worldwide consumed that has great nutritional and economic relevance. Besides the phenotypical changes that undergo pepper fruit during ripening, there are many associated modifications at transcriptomic, proteomic, biochemical, and metabolic levels. Nitric oxide (NO) and hydrogen sulfide (H2S) are recognized signal molecules that can exert regulatory functions in diverse plant processes. This study aims at analyzing the interrelationship between NO and H2S during fruit ripening. Results: Our data indicate that the H2S-generating cytosolic L-cysteine desulfhydrase (LCD) and the mitochondrial D-cysteine desulfhydrase (DCD) activities are downregulated during ripening but this effect was reverted after NO treatment of fruits. Innovation and Conclusion: Using as a model the non-climacteric pepper fruits at different ripening stages and under an NO-enriched atmosphere, the activity of the H2S-generating LCD and DCD was analyzed. LCD and DCD activities were downregulated during ripening, but this effect was reverted after NO treatment of fruits. The analysis of LCD activity by non-denaturing polyacrylamide gel electrophoresis (PAGE) allowed identifying three isozymes designated CaLCD I to CaLCD III, which were differentially modulated by NO and strictly dependent on pyridoxal 5'-phosphate (PLP). In vitro analyses of green fruit samples in the presence of different compounds including NO donors, peroxynitrite (ONOO-), and reducing agents such as reduced glutathione (GSH) and L-cysteine (L-Cys) triggered an almost 100% inhibition of CaLCD II and CaLCD III. This redox adaptation process of both enzymes could be cataloged as a hormesis phenomenon. The protein tyrosine (Tyr) nitration (an NO-promoted post-translational modification) of the recombinant LCD was corroborated by immunoblot and by mass spectrometry (MS) analyses. Among the 11 Tyr residues present in this enzyme, MS of the recombinant LCD enabled us to identify that Tyr82 and Tyr254 were nitrated by ONOO-, this occurring near the active center on the enzyme, where His237 and Lys260 together with the cofactor PLP are involved. These data support the relationship between NO and H2S during pepper fruit ripening, since LCD and DCD are regulated by NO during this physiological event, and this could also be extrapolated to other plant species.

Project description:BackgroundPlant stress responses and mechanisms determining tolerance are controlled by diverse sets of genes. Transcription factors (TFs) have been implicated in conferring drought tolerance under drought stress conditions, and the identification of their target genes can elucidate molecular regulatory networks that orchestrate tolerance mechanisms.ResultsWe generated transgenic rice plants overexpressing the 4 rice TFs, OsNAC5, 6, 9, and 10, under the control of the root-specific RCc3 promoter. We showed that they were tolerant to drought stress with reduced loss of grain yield under drought conditions compared with wild type plants. To understand the molecular mechanisms underlying this tolerance, we here performed chromatin immunoprecipitation (ChIP)-Seq and RNA-Seq analyses to identify the direct target genes of the OsNAC proteins using the RCc3:6MYC-OsNAC expressing roots. A total of 475 binding loci for the 4 OsNAC proteins were identified by cross-referencing their binding to promoter regions and the expression levels of the corresponding genes. The binding loci were distributed among the promoter regions of 391 target genes that were directly up-regulated by one of the OsNAC proteins in four RCc3:6MYC-OsNAC transgenic lines. Based on gene ontology (GO) analysis, the direct target genes were related to transmembrane/transporter activity, vesicle, plant hormones, carbohydrate metabolism, and TFs. The direct targets of each OsNAC range from 4.0-8.7% of the total number of up-regulated genes found in the RNA-Seq data sets. Thus, each OsNAC up-regulates a set of direct target genes that alter root system architecture in the RCc3:OsNAC plants to confer drought tolerance. Our results provide a valuable resource for functional dissection of the molecular mechanisms of drought tolerance.ConclusionsMany of the target genes, including transmembrane/transporter, vesicle related, auxin/hormone related, carbohydrate metabolic processes, and transcription factor genes, that are up-regulated by OsNACs act as the cellular components which would alter the root architectures of RCc3:OsNACs for drought tolerance.

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Persulfidation of Nitrate Reductase 2 Is Involved in l-Cysteine Desulfhydrase-Regulated Rice Drought Tolerance.

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