Regulatory Mechanism Of L-cysteine Desulfhydrase In Tomato Fruit Ripening And Senescence

Tomato is an important economic crop in the world,and its fruit is rich in nutrients.In recent years,researches on delaying the ripening and senescence of postharvest tomato fruits and prolonging the shelf-life have been in-depth research.Hydrogen sulfide（H₂S）is a versatile signaling molecule in plants,and the ripening and aging process of postharvest fruits can be delayed by the treatment of exogenous H₂S.L-cysteine desulfhydrase（LCD）is the main enzyme that produces endogenous H₂S in tomato fruits.In this paper,bioinformatics analysis was performed on the LCD gene family in plants.The E.coli prokaryotic expression system was used to verify the enzymatic properties of the tomato LCD proteins.Using virus-induced gene silencing（VIGS）and Crispr/Cas9gene editing technology to construct Sl LCD1 silencing and mutation plants,explore the function and mechanism of Sl LCD1 in the process of tomato fruit ripening and senescence.Bioinformatics analysis identified 41 LCD genes in 17 plant species.The analysis of transcriptome data was found that the LCD expression patterns has a certain response in drought,cold and salt stress.The E.coli prokaryotic expression system verified that the tomato LCD1 and LCD2 proteins have the enzymatic function of catalyzing the L-Cys to generate H₂S.By constructing the subcellular localization vectors,it was found that the tomato LCD2 was localized in the chloroplast and the LCD1 was localized in the nucleus,and it was verified that the NLS（nuclear localization signal sequence）on the LCD1interacts with the nuclear import protein importinα3,which mediates the LCD1 nuclear localization.In order to investigate the mechanism of Sl LCD1 regulating the ripening and senescence of tomato fruits,Sl LCD1 gene silencing plants vigs-lcd1 and gene editing T2generation homozygous mutant plants lcd1-7 and lcd1-9 were constructed,and it was found that the vigs-lcd1 fruits showed a premature ripening phenotype,the plants of lcd1-7/9 were shorter and the fruits matured earlyer.During the ripening process,the fruits of the mutant strains accumulated a lot of carotenoids,and the chlorophyll content was significantly reduced,and the carotenoid synthesis related genes（PDS,PSY1 and ZDS）,the chlorophyll degradation related genes（NYC1,PAO,PPH and SGR1）,the cell wall metabolism related genes（CEL2,EXP,XTH5,PG,and TBG4）and the ethylene synthesis pathway related genes（ACO1,ACO3,ACS2,RIN,NOR,E4,and E8）were significantly up-regulated.In summary,the results showed that the subcellular localization of the tomato Sl LCD1 and Sl LCD2 proteins is the nucleus and chloroplast,respectively.LCD1 is the main enzyme catalyzing the production of H₂S in tomato.The vigs-lcd1 and lcd1-7/9 plants further proves that Sl LCD1 regulates tomato fruit ripening and senescence through pigment accumulation,cell wall degradation and ethylene synthesis and response.The results of this study provide a theoretical basis for the maturation of tomato fruit and the improvement of tomato fruit quality.