Hydrogen Sulfide/Cysteine Synthase In Regulating Physiological Mechanism Storage Quality Of Tomato

China is a big country of fruits production and consumption,due to the imperfection of the conditions and technology in storage and transportation,picked fruits gradually lost its value accompanied by the process of aging,death and rot,consequently causing huge waste in agricultural production.Investigating the mechanism on ripening and senescence of fruits,so as to provide effective storage technology,has always been an urgent problem for people to solve.Tomato is one of the most widely cultivated and most consumed crops in China,with extremely high nutritional value.It is not resistant to storage and prone to rot,thus resulting in huge lost.How to extend its postharvest storage period and keep the nutritional value of tomato,is a subject that many scholars pay close attention to.In recent years,studies indicated that hydrogen sulfide?H₂S?significantly inhibit the aging and senescence process of postharvest fruits and extend the postharvest storage period of fruits.However,it remains to be explored whether the fruit is still kept its nutritional quality during storage.Ethylene?C₂H₄?,a plant endogenous hormone,plays a key role in the storage of postharvest fruits and which is an important regulatory factor of fruits ripening and aging.It is also a hot topic for people to research.The effect of H₂S and C₂H₄ on the quality of fruits during storage period is not very clear.In this paper,the effects of exogenous H₂S and C₂H₄ on the quality of postharvest tomato were studied by using sodium thiohydride?NaHS?and ethylene-derived solutions as the donor of H₂S and C₂H₄,respectively.The results showed that the H₂S treatment group compared with the control group,or the C₂H₄ treatment group compared with H₂S and C₂H₄,can be seen that C₂H₄ significantly accelerate the speed of tomato reddening,while H₂S slowed down the speed and maintained a better color of tomatoes.The H₂S treatment inhibited the decomposition of chlorophyll and the accumulation of carotenoids and anthocyanin,increased the content of antioxidant substances such as flavonoids and total phenols,and made the fruits appear fresh and plump.At the same time H₂S and C₂H₄ treatment,H₂S can delay the degradation of ascorbic acid,titratable acid,reducing sugar,soluble protein and starch,but also significantly inhibit the activity of proteolytic enzymes and amylase.These show that C₂H₄ treatment can accelerate the ripening and aging process and the consumption of nutrients of tomato fruit,reduce the storage quality of tomato.While H₂S can slow down the degradation of nutrients,and better maintain the commodity value and nutritive value of tomato fruit,so as to ensure the quality during storage.In addition,H₂S can inhibit the role of ethylene to some extent,thus prolonging the storage period of postharvest tomatoes.We found that compared to the control group,the presence of exogenous H₂S inhibited the activity of cysteine synthetase?OASTL?and decreased the relative expression of cysteine synthetase-related genes.During the storage,the indexes related to tomato quality had been measured with H₂S and C₂H₄ treatment?chlorophyll a,chlorophyll b,total chlorophyll,carotenoid,flavonoids,total phenol,anthocyanin,ascorbic acid,titration acid,reducing sugar,soluble protein,starch,proteolytic enzyme and amylase activity?which have a high correlation with the expression levels of cysteine synthase family gene.Cysteine synthase is a key enzyme in the synthesis of cysteine.The production of cysteine is a precursor substance of many metabolic products with important biological functions in plants.The biosynthesis ability of cysteine is closely related to the resistance,tolerance and fruit quality of plants,which are all regulated by cysteine synthase.Therefore,the bioinformatics analysis of cysteine synthetase family genes OASTL1 and OASTL2 was carried out in this paper.It is included the construction of the evolutionary tree,the prediction of protein structure and motif analysis.The OASTL1 and OASTL2 of tomato have high homology with the corresponding genes of Arabidopsis,tobacco,rice and corn,and the function of the gene is more conservative by the analysis of protein domain and Motif.The homology of tomato OASTL1 and tobacco was up to 99%and the homology between tomato OASTL2 gene and Arabidopsis reached 97%.The function of the gene was conserved by the analysis of protein structure domain and Motif.By constructing GFP-OASTL1/OASTL2 vectors and transfection of tobacco leaves for subcellular localization,the results showed that the OASTL1 and OASTL2 genes were mainly located in the chloroplast and cytoplasm of plant cells.In addition,quantitative PCR was performed on the expression of OASTL1 and OASTL2 genes in different tissues of tomato.The expression in different tissues was different,and the expression in leaves and fruits were relatively high,which was consistent with the prediction results of the website.The Crispr/cas9 knockout vector and PBI121 overexpression vector of OASTL1 and OASTL2 were constructed,then transferred to Agrobacterium tumefaciens,and infected tomato stems and cotyledons.The gene deletion plants and overexpression plants of tomato OASTL1/OASTL2 were screened and obtained by the plant tissue culture.The expression of OASTL in tomato overexpression plant was analyzed.RT-PCR results showed that expression of OASTL1-1 was increased about twice times relative to wild type plant,OASTL1-2 increased by about 4 times times,OASTL1-3increased by about 7 times times.The relative expression of OASTL2-1 plant was about 25 times that of wild type tomato.The expression of OASTL gene of tomato overexpression plant was increased in different degree,which showed that the carrier played a corresponding role in the plant.Finally,the callus was induced by the stem and leaf of wild tomato plant and overexpression plants of OASTL1 and OASTL2.It was found that the browning degree of overexpression plant callus was significantly lower than that of wild type in the experiment under the heavy metal cadmium stress.This shows that the callus of tomato plants overexpression OASTL had stronger resistance to the heavy metal stress,the overexpression of OASTL enhances the tolerance of plants to heavy metals,and preliminary explores the function of OASTL.