Relative Research Of H₂S And CAS In Regulating Postharvest Antioxidant Signaling Mechanism And CAS Function Of Tomato Fruit

China has a large area of fruit and vegetable cultivation,and it is a big country for fruit and vegetable production and consumption.At present,the loss rate of fruit picking in China is as high as 20%to 25%.Therefore,it is of great significance to study the mechanism of ripening and aging of fruits and vegetables.In the post-harvest ripening and aging process of fruits and vegetables,there will be a lot of reactive oxygen species?ROS?bursts.At this point,it is necessary to remove the ROS,start the antioxidant system in the plant,and protect the plant tissue from ROS.The plant hormone phytohormone ethylene?C₂H₄?also plays an important regulatory role in the process of fruit ripening and aging,and can promote fruit ripening,aging and ROS production.Hydrogen sulfide?H₂S?has been considered as an important gas signal molecule in recent years.It participates in the plant's sulfur metabolic pathway,it can delay ripening and aging of fruits and vegetables,inhibit the production of ROS and enhance the antioxidant capacity of fruits.At the later stage of C₂H₄ metabolism,cyanide is produced.Cyanoalanine synthase?CAS?generates H₂S at the same time of cyanide decomposition.In addition,when the plant is subjected to external stress,CAS also plays a role in protecting the plant from injury.At present,there are many researches on delaying plant aging by H₂S.However,whether it is antagonistic to ethylene and delays post-harvest ripening and aging of tomato fruit has not been reported.At the same time,CAS,as a key enzyme for cyanide decomposition,is also a key enzyme for endogenous H₂S production and its role in fruit ripening and aging is not yet clear.In this dissertation,the fruit of small tomatoes in the broken color period was used as the experimental material,and the effects of exogenous H₂S and C₂H₄ on the ripening and aging of the post-harvest tomato were studied using sodium hydrogen sulfide?NaHS?which was H₂S donor and ethephon which was C₂H₄ gas donor.And through screening tomato plants of CAS gene editing and overexpression,the function of CAS was explored.In this paper,we studied the relationship between H₂S and C₂H₄ on antioxidative metabolism in the repening process of postharvest tomato fruits.The results showed that compared with the H₂S+C₂H₄ group,H₂S could inhibit the accumulation of superoxide anion(·O^2-),hydrogen peroxide?H₂O₂?and malondialdehyde?MDA?content,improve antioxidant enzyme including catalase?CAT?,ascorbate peroxidase?APX?,guaiacol peroxidase?POD?and superoxide dismutase?SOD?,inhibit lipoxygenase?LOX?and polyphenol oxidase?PPO?activity,enhance the activity of phenylalanine ammonia-lyase?PAL?during the late storage period.In contrast,C₂H₄accelerated the production of ROS and inhibited the activity of antioxidant enzymes.These results show that H₂S can reduce the accumulation of free radicals in fruits,maintain the balance of ROS in cells,enhance the antioxidant capacity of tomato fruits and delay the ripening and senescence of postharvest fruits.In this paper,the relationship between H₂S,C₂H₄ and the genes of antioxidant enzymes and CAS family related enzymes in the process of post-harvest ripening and aging of tomatoes was also studied from genetic level.The results showed that compared with H₂S+C₂H₄,H₂S could up-regulate the expression of APX3,CAT1,CAT2,POD3,FeSOD and Cu/ZnSOD in tomato during the whole storage period,up-regulate APX2 and CAT3 in early storage,SOD1 and SOD2,and promote the expression of CAS1,CAS2 and NIT4 B,suggenting that H₂S treatment alleviated the harm of HCN during fruit ripening,and an extremely close relationship existed between CAS,H₂S and antioxidant.In this experiment,the bioinformatics analysis of CAS showed that the subcellular localization of CAS1 and CAS2 genes might be located in the chloroplasts and mitochondria,and then the results were observed and verified by the subcellular localization experiment.The homology of tomato CAS1 and CAS2 gene was 82.1%.The homology of tomato CAS1 and potato was up to 99%and the homology between tomato CAS2 gene and Nicotiana tobaccos reached 86.2%.CAS1 and CAS2 all had 1 domain and no transmembrane domain existed.The Microtom tomato was used as the material to construct the CAS gene editing mutant and overexpression vector.The T0 generation tomato plants were obtained by screening and identification,and the response mechanism of CAS family genes in the stress resistance was analyzed by the callus tissue.The results showed that CAS1 and CAS2 gene expression patterns were different,and CAS1 gene was induced more strongly when dealing with drought and high salt stress.Under drought stress,compared with WT,browning was accelerated in the callus of CAS1 gene knockout plant,while the water content was reduced,the permeability of cell membrane was increased,and the integrity of cell membrane was destroyed.The expression of Sl SGN-213276,SlAREB1,Sl DREB and SlDREB1 was decreased in callus of CAS1 knockdown plants,while the expression of SlSGN-213276,Sl AREB1,SlDREB,SlDREB1 and SlUSP genes was increased in callus of OECAS1 plants.So,CAS1 might resist drought stress through the regulation of drought-related gene expression.Under the stress of high salinity,compared with WT,sensitivity to stress in appearance was not observed in the callus of CAS1 gene knockout plants,but under stress,the water content and cell membrane permeability were similar to that of CAS1,indicating that the CAS1 gene caused the tomato callus to have a certain resistance to high salt stress in physiological and biochemical level.In conclusion,exogenous H₂S can antagonize ethylene,reduce ROS accumulation,increase antioxidant enzyme activity and participate in antioxidative metabolism,postponing ripening and senescence of postharvest fruits.Meanwhile,CAS has a close relationship with the antioxidant activity of tomato.The CAS1 deficient callus of tomato is more sensitive to high salt and drought stress.CAS can protect tissue from drought stress by regulating the expression of drought related genes,and it is resistant to drought and high salt stress.