The Function Of D-cysteine Desulfhydrase In Regulating Tomato Fruit Ripening And Senescence And Disease Resistance Response

Solanum lycopersicum is a model plant for studying fruit development and an important experimental material for studying plant disease resistance.Hydrogen sulfide（H₂S）is a novel gas signaling molecule,which plays an important role in regulating fruit ripening/senescence and disease resistance.D-cysteine desulfhydrase（DCD）is a key enzyme in plants that produces H₂S with D-Cys as a specific substrate.Studies have shown that DCD is involved in regulating plant stomatal development and mediating plant responses to drought stress and heavy metal stress,but whether DCD is involved in regulating fruit ripening/senescence and disease resistance response has not been reported.In this paper,DCD1/2 silenced plants and transient high expression plants constructed by virus induced gene silencing（VIGS）and transient high expression technology were used to preliminarily analyze the function of DCD1/2 in the process of tomato fruit ripening and senescence.By exploring the pathways of pigment metabolism and ethylene metabolism during the ripening and senescence process of DCD1/2 silenced and transient high expression fruits,it was found that after DCD1/2silencing,the fruit entered the color breaker stage 5 days in advance,the accumulation of carotenoids and the degradation of chlorophyll accelerated,which led to an acceleration of the ripening and senescence process of the fruits.When infected for 7d,the DCD1/2 transient high expression fruits were in the white ripening stage,and the control group fruits had entered the color breaker stage,indicating that the DCD1/2 transient high expression delayed the ripening and senescence of the fruit.By q RT-PCR technology,it was found that in the DCD1/2 silenced fruits,the transcription levels of the ethylene synthesis and signaling genes ACO1,ACS2 and E4,cell wall metabolism genes CEL2,EXP,carotenoid synthesis genes PSY1,PDS and ZDS,chlorophyll degradation genes NYC1,PAO and SGR1 were significantly upregulated compared with the control group tomatoes,and their transcriptional levels in DCD1/2 transient high-expression fruits were significantly downregulated,indicating that DCD1/2 were the negative regulators of fruit ripening and senescence.By using dcd2 gene edited plant constructed by CRISPR/Cas9 gene editing technology,combined with previously constructed dcd1 in my laboratory,the function of DCD1/2 in the process of tomato fruit ripening and senescence was further explored.It was found that the dcd1 fruit entered the color breaker stage 8 d earlier than the WT fruit,and the dcd2 fruit entered the color breaker stage 6 d earlier than the WT fruit.At 43 days post flowering,the chlorophyll content in dcd2 fruits was about 50%of WT,and the carotenoid content was about 4 times that of WT.The deletion of DCD2 also led to an increase in the expression of ripening/senescence-related genes in the fruit,further verifying that DCD1/2 are negative regulators of fruit ripening and senescence,and the negative regulation of DCD1 is stronger.In order to investigate whether DCD1 is involved in the disease resistance response of tomatoes,this paper investigates the effect of DCD1 on the defense mechanism of tomato response to the invasion of the pathogenic bacteria Pseudomonas syringae and pathogenic fungus Botrytis cinerea with WT and dcd1 as infection objects.After infection by Pseudomonas syringae for 5 days,the death cell percentage in dcd1 leaves was significantly higher than that of WT leaves by trypan blue staining,indicating that DCD1 deletion led to an increase in cell mortality when tomato leaves were infected with Pseudomonas syringae.The distribution of H₂O₂in the leaves of dcd1 was found to account for 50%of the total leaf area,while the H₂O₂in wild-type tomato leaves was only concentrated near the infection wound,indicating that DCD1 deletion exacerbated the accumulation of H₂O₂when tomato leaves were infected with Pseudomonas syringae.In addition,the deletion of DCD1 also decreased antioxidant enzyme activities such as ascorbate peroxidase（APX）,catalase（CAT）,superoxide dismutase（SOD）,and peroxidase（POD）,O^2-production rate,H₂O₂content and malondialdehyde content increased in tomato leaves.The transcription levels of defense-related genes POD,SOD and PAL,and disease resistance-related genes PR1,NPR1 and GOX1 were inhibited in the leaves of dcd1.In addition,in order to investigate whether DCD1 is involved in tomato responses to the pathogenic fungus Botrytis cinerea,the effect of infection with Botrytis cinerea on dcd1 was similar to that of Pseudomonas syringae.Based on the above results,it can be concluded that the deletion of DCD1 reduces the disease resistance of tomatoes,thus DCD1 is a positive regulator of tomato response to pathogenic microorganisms.In order to explore the molecular mechanism of DCD in exerting the above functions,in this paper,the p Cold-DCD1/2 prokaryotic expression vectors were constructed,the DCD1/2 crude proteins were obtained by the E.coli prokaryotic expression system,and the proteins were further purified by nickel column affinity chromatography.Thereafter,the DCD1/2 enzyme activities were determined after purification,and the enzyme activities of DCD1 and DCD2 proteins were found to be70.9 nmol·mg^-1（protein）·min^-1and 31.2 nmol·mg^-1（protein）·min^-1,respectively.Studies have shown that H₂S-mediated sulfhydration modification can modulate the activity of enzymes.DCD1/2 proteins were treated by exogenous H₂S,and it was found that DCD1 protein could be modified by sulfhydration and the enzyme activity was increased by 20%after modification,while DCD2 protein did not have sulfhydration signal.The DCD1 protein sequence was analyzed by GPS-SNO software,and the 4th,135th and 266th Cys sites were found to be the key sites for the sulfhydration modification of DCD1,and then the above key sites were verified by the site-directed mutation technique,and the results showed that the 266th Cys site was the key site of the sulfhydration modification of DCD1.The upstream transcription factors WRKYs and MYB48,which may regulate the DCD1 promoter,were screened by correlation analysis,and whether the upstream transcription factors could activate the DCD1 promoter region were explored by double-luciferase assay.It was found that MYB48 had a strong activation effect on the DCD1 promoter,and WRKY6 has a weak activation effect on the DCD1 promoter,but has a strong activation effect on the DCD1 promoter when injected together with MYB48,indicating that WRKY6 and MYB48 have a synergistic activation effect on the DCD1promoter.Then Y1H is used to verify that MYB48 can bind to the DCD1 promoter fragment,and the interaction between WRKY6 and MYB48 is verified by Y2H.The results suggest that WRKY6 and MYB48 could form a complex to synergistically activate the expression of DCD1.In summary,DCD1/2 is involved in regulating the ripening/senescence of tomato fruits and the disease resistance response of tomatoes.At the same time,H₂S-mediated sulfhydration modifications can improve DCD1 enzyme activity,and WRKY6 and MYB48 can form complexes to synergistically activate DCD1expression.This study is of great significance in analyzing the regulatory mechanisms of plant fruit ripening/senescence and disease resistance,prolonging the storage period of fruits,and reducing agricultural losses.