The Physiological Mechanism Research Of H₂S And D/L-CD Regulating Fruit Ripening And Senescence Of Tomato

As an important global crop,tomato has high yield and rich nutritional value,which is deeply loved by people.Tomato is also a typical respiratory climacteric fruit.The peak of ethylene appears during fruit ripening,which is very easy to promote fruit ripening,and it is not resistant to storage,affecting its postharvest nutritional and economic benefits.After the fruit is ripe,it is easily rotten and deteriorated,causing a great deal of loss.It is of great economic and social significance to study the mechanism of ripening and senescence oftomato fruit.Ethylene plays a key role in the process of plant growth and development,and plays a key role in the ripening and senescence of fruit,especially in the respiratory climacteric fruit.Recent studies have shown that hydrogen sulfide(H2S)is the third endogenous gaseous signaling molecule following NO and CO.In plants,H2 S is involved in regulating physiological processes such as stomatal closure,seed germination,root development,photosynthesis,biological and abiotic stress.Recent research indicates that H2 S can delay the ripening and senescence process of many fruits,but whether H2 S can antagonize ethylene toalleviatethe ripening and senescence process of tomato has not been reported yet.In this paper,green mature tomato(Solanum lycopersicum)was used as experimental material,the effects of exogenous H2 S and ethylene on the ripening and senescence process of postharvest tomato fruits were investigated by hydrosulfide(Na HS)and ethephon aqueous solutions as the donors of H2 S and ethylene respectively.The results showed that compared with the control group,ethylene treatment promoted the ripening and senescence process of tomato fruits,while H2 S treatment could delay the process.Treated with H2 S and ethylene,compared with the ethylene treatment group,H2 S could inhibit the effect of ethylene,prolong the storage time and delay the riping and senescenceof of tomato fruit.Hardness is an important indicator of fruit softening.Further study found that H2 S could delay the decrease of tomato fruit hardness and maintain the quality of fruit.In the later stage of fruit ripening,H2 S treatment group decreased the activities of fruit softening enzymes.Compared with the C2H4 treatment,H2S and C2H4 treatment group could inhibit the increase of PG,cellulase and pectin lyase activities.Ethylene is closely related to the process of fruit ripening and senescence.Therefore,we investigated the effects of H2 S and ethylene from the gene level inethylene synthesis and signal transduction and fruit softening resistance-relatedgenesexpression.The results showed that,comparison with the results of C2H4 treatment,H2 S and C2H4 treatment could down-regulatedthe expression of ACS2,ACS3,ACS4,ACO1,ACO3 and ACO4 genes in the middle and late stage of tomato fruit ripening.The expression of ETR3 and ETR4 genes was induced by H2 S and C2H4 treatmentin the mid-term of fruit ripening,and the expression of CRF2 and ERFD2 genes was alsoinduced in the late stage of fruit maturation.It is suggested that exogenous H2 S signaling can involvein the regulation of the physiological processes induced by ethylene,such as ethylene synthesis,signal transduction and fruit softening,to inhibit endogenousethylene synthesisanddelay fruit softening,and then delay the ripening and senescence process of postharvest tomato fruits.Exogenous H2 S can retard the ripening and senescence process of postharvest tomato fruits,and whether endogenous H2 S has the same function.DCD and LCD are two main genes encoding endogenous H2 S synthesis in plants.The subcellular localization and tissue expression of endogenous H2 S generation genes,DCD and LCD,were investigated.The results showed that DCD was located in the cytoplasm and LCD was located in the nucleus.DCD and LCD genes are not only species specific,but also tissue-specific,and express the highest in roots,and their expression is low or almost not expressed in stems,leaves,flowers and fruits.In addition,in order to study the function ofendogenous H2 S gene,CRISPR/Cas9 vector technology was used to construct the Cas9 vector of tomato cysteine dehydrase gene DCD and LCD.Meanwhile,overexpression vector of tomato DCD and LCD was constructed.The Cas9 positive plants of DCD and LCD genes were successfully screened by Agrobacterium mediated plant tissue culture,and the deletion of DCD and LCD genes reduced their own gene expression.