Roles of a Cysteine Desulfhydrase LCD1 in Regulating Leaf Senescence in Tomato

Hydrogen sulfide (H2S), a novel gasotransmitter in both mammals and plants, plays important roles in plant development and stress responses. Leaf senescence represents the final stage of leaf development. The role of H2S-producing enzyme L-cysteine desulfhydrase in regulating tomato leaf senescence is still unknown. In the present study, the effect of an L-cysteine desulfhydrase LCD1 on leaf senescence in tomato was explored by physiological analysis. LCD1 mutation caused earlier leaf senescence, whereas LCD1 overexpression significantly delayed leaf senescence compared with the wild type in 10-week tomato seedlings. Moreover, LCD1 overexpression was found to delay dark-induced senescence in detached tomato leaves, and the lcd1 mutant showed accelerated senescence. An increasing trend of H2S production was observed in leaves during storage in darkness, while LCD1 deletion reduced H2S production and LCD1 overexpression produced more H2S compared with the wild-type control. Further investigations showed that LCD1 overexpression delayed dark-triggered chlorophyll degradation and reactive oxygen species (ROS) accumulation in detached tomato leaves, and the increase in the expression of chlorophyll degradation genes NYC1, PAO, PPH, SGR1, and senescence-associated genes (SAGs) during senescence was attenuated by LCD1 overexpression, whereas lcd1 mutants showed enhanced senescence-related parameters. Moreover, a correlation analysis indicated that chlorophyll content was negatively correlated with H2O2 and malondialdehyde (MDA) content, and also negatively correlated with the expression of chlorophyll degradation-related genes and SAGs. Therefore, these findings increase our understanding of the physiological functions of the H2S-generating enzyme LCD1 in regulating leaf senescence in tomato.

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

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.

Delaying Broccoli Floret Yellowing by Phytosulfokine α Application During Cold Storage

During postharvest life, broccoli suffers from floret yellowing confining its economic and nutritional value. The objective of the present study was to explore the mechanisms employed by phytosulfokine α (PSKα) at 150 nM for delaying floret yellowing in broccoli during storage at 4°C for 28 days. Our results showed that the higher endogenous accumulation of hydrogen sulfide (H2S) resulting from the higher gene expression and activities of l-cysteine desulfhydrase (LCD) and d-cysteine desulfhydrase (DCD) in broccoli floret treated with 150 nM PSKα may serve as an endogenous signaling molecule for delaying senescence. Moreover, the suppressed ethylene biosynthesis in broccoli floret treated with 150 nM PSKα might be ascribed to lower gene expression and activities of ACC synthase (ACS) and ACC oxidase (ACO). Furthermore, lower gene expression and activities of Mg2+ dechelatase (MDC), pheophytinase (PPH), and pheophorbide a oxygenase (PaO) might be the reasons for the higher accumulation of chlorophyll in broccoli floret treated with 150 nM PSKα. Based on our findings, exogenous PSKα application could be employed as signaling bioactive hormone for retarding floret yellowing of broccoli during storage at 4°C for 28 days.

A nuclear-localized cysteine desulfhydrase plays a role in fruit ripening in tomato

Hydrogen sulfide (H2S) is a gaseous signaling molecule that plays multiple roles in plant development. However, whether endogenous H2S plays a role in fruit ripening in tomato is still unknown. In this study, we show that the H2S-producing enzyme l-cysteine desulfhydrase SlLCD1 localizes to the nucleus. By constructing mutated forms of SlLCD1, we show that the amino acid residue K24 of SlLCD1 is the key amino acid that determines nuclear localization. Silencing of SlLCD1 by TRV-SlLCD1 accelerated fruit ripening and reduced H2S production compared with the control. A SlLCD1 gene-edited mutant obtained through CRISPR/Cas9 modification displayed a slightly dwarfed phenotype and accelerated fruit ripening. This mutant also showed increased cysteine content and produced less H2S, suggesting a role of SlLCD1 in H2S generation. Chlorophyll degradation and carotenoid accumulation were enhanced in the SlLCD1 mutant. Other ripening-related genes that play roles in chlorophyll degradation, carotenoid biosynthesis, cell wall degradation, ethylene biosynthesis, and the ethylene signaling pathway were enhanced at the transcriptional level in the lcd1 mutant. Total RNA was sequenced from unripe tomato fruit treated with exogenous H2S, and transcriptome analysis showed that ripening-related gene expression was suppressed. Based on the results for a SlLCD1 gene-edited mutant and exogenous H2S application, we propose that the nuclear-localized cysteine desulfhydrase SlLCD1 is required for endogenous H2S generation and participates in the regulation of tomato fruit ripening.

Impairment in O-acetylserine-(thiol) lyase A and B, but not C, confers higher selenate sensitivity and uncovers role for A, B and C as L-Cys and L-SeCys desulfhydrases in Arabidopsis

ABSTRACTThe role of the cytosolic O-acetylserine-(thiol) lyase A (OASTLA), chloroplastic OASTLB and mitochondrion OASTLC in plant resistance/sensitivity to selenate was studied in Arabidopsis plants. Impairment in OASTLA and B resulted in reduced biomass, chlorophyll and soluble protein levels compared with impaired OASTL C and Wild-Type treated with selenate. The lower organic-Se and protein-Se levels followed by decreased organic-S, S in proteins and total glutathione in oastlA and oastlB compared to Wild-Type and oastlC are indicative that Se accumulation is not the main cause for the stress symptoms, but rather the interference of Se with the S-reduction pathway. The increase in sulfite oxidase, adenosine 5′-phosphosulfate reductase, sulfite reductase and OASTL activity levels, followed by enhanced sulfite and sulfide, indicate a futile anabolic S-starvation response to selenate-induced organic-S catabolism in oastlA and oastlB compared to Wild-Type and oastlC.Additionally, the catabolic pathway of L-cysteine degradation was enhanced by selenate, and similar to L-cysteine producing activity, oastlA and B exhibited a significant decrease in L-cysteine desulfhydrase (DES) activity, compared with WT, indicating a major role of OASTLs in L-cysteine degradation. This notion was further evidenced by sulfide dependent DES in-gel activity, immunoblotting, immunoprecipitation with specific antibodies and identification of unique peptides in activity bands generated by OASTLA, B and C. Similar responses of the OASTLs in Seleno-Cysteine degradation was demonstrated in selenate stressed plants. Notably, no L-cysteine and L-Seleno-Cysteine DES activity bands but those related to OASTLs were evident. These results indicate the significance of OASTLs in degrading L-cysteine and L-SelenoCysteine in Arabidopsis.SummaryThe cytosolic OASTLA and chloroplastic OASTLB have significantly higher desulfhydrase activity rates than the cytosolic DES1 and are able to degrade L-Cys and L-SeCys to sulfide and selenide, respectively in Arabidopsis.

Involvement of l-Cysteine Desulfhydrase and Hydrogen Sulfide in Glutathione-Induced Tolerance to Salinity by Accelerating Ascorbate-Glutathione Cycle and Glyoxalase System in Capsicum

The aim of this study is to assess the role of l-cysteine desulfhydrase (l-DES) and endogenous hydrogen sulfide (H2S) in glutathione (GSH)-induced tolerance to salinity stress (SS) in sweet pepper (Capsicum annuum L.). Two weeks after germination, before initiating SS, half of the pepper seedlings were retained for 12 h in a liquid solution containing H2S scavenger, hypotaurine (HT), or the l-DES inhibitor dl-propargylglycine (PAG). The seedlings were then exposed for three weeks to control or SS (100 mmol L−1 NaCl) and supplemented with or without GSH or GSH+NaHS (sodium hydrosulfide, H2S donor). Salinity suppressed dry biomass, leaf water potential, chlorophyll contents, maximum quantum efficiency, ascorbate, and the activities of dehydroascorbate reductase, monodehydroascorbate reductase, and glyoxalase II in plants. Contrarily, it enhanced the accumulation of hydrogen peroxide, malondialdehyde, methylglyoxal, electrolyte leakage, proline, GSH, the activities of glutathione reductase, peroxidase, catalase, superoxide dismutase, ascorbate peroxidase, glyoxalase I, and l-DES, as well as endogenous H2S content. Salinity enhanced leaf Na+ but reduced K+; however, the reverse was true with GSH application. Overall, the treatments, GSH and GSH+NaHS, effectively reversed the oxidative stress and upregulated salt tolerance in pepper plants by controlling the activities of the AsA-GSH and glyoxalase-system-related enzymes as well as the levels of osmolytes.