Changes Of Autophagy Flow And Functional Verification Of Autophagy Related Gene ATG8f During Tomato Fruit Ripening

Tomato is the main vegetable crop in facility cultivation and also an important model plant for studying fruit development.Tomato ripening is a dynamic and complex process involving cell expansion and lysis.Autophagy is a process in which eukaryotes rely on lysosome pathway for cell degradation and substance recycling,which plays an important role in regulating plant growth and development.In this study,we took‘Micro-Tom’tomato as experimental material and comprehensively used bioinformatics,genetic analysis,physiological and biochemical methods to explore the role of autophagy gene SlATG8 f in tomato fruit rippling process.The main conclusions are as follows:(1)There were 7 subtypes in tomato ATG8 gene family.Seven SlATG8 s genes were distributed in six chromosomes.The molecular weight of SlATG8 s protein ranged from 13.2 to 30.05 kDa,and the number of amino acids ranged from 116 to271.SlATG8 s gene has a fragment replication event in the process of evolution,and it is closely related to Solanaceae.SlATG8 s gene was differentially expressed during the ripening process of tomato fruits,and the expression level was mostly higher in fruit color breaking stage or late ripening stage.(2)The autophagy related gene SlATG8 f was cloned from tomato.The protein encoded by SlATG8 f is a hydrophilic protein with transmembrane structure.There is a conserved Gly residue near the C terminal of SlATG8 f gene,which corresponds to the PE receptor site of ATG8 in yeast,and SlATG8 f gene is highly conserved in Nightsolanae.Subcellular localization results showed that SlATG8 f protein was located in cytoplasm and cell membrane.(3)SlATG8 f affected tomato fruit development and autophagy activity.Subcellular localization results showed that SlATG8 f protein was located in the cytoplasm.The ventricular glial tissue of tomato fruits in SlATG8 f overexpression strains was fuller than that of the wild type,and the liquefaction rate of the ventricular glial tissue was faster,suggesting that SlATG8 f gene could positively regulate the development of tomato fruits.MDC staining and Transmission electron microscope observation showed that the number of autophagosomes in fruit of SlATG8 f overexpression strain was significantly higher than that of the wild type,and Western blot analysis showed that the expression of ATG8 protein in fruit of SlATG8 f overexpression strain was significantly increased during the color breaking stage.It was concluded that the fruit of SlATG8 f overexpression strain had relatively stronger autophagy activity,indicating that SlATG8 f expression level was positively correlated with autophagy activity.SlATG8 f overexpression can induce the formation of autophagosome in tomato fruit and increase the autophagy flow of tomato fruit cells.(4)SlATG8 f promotes tomato fruit ripening and improves fruit quality.Overexpression of SlATG8 f can promote the accumulation of ascorbic acid,soluble solids and lycopene in tomato fruits,reduce the content of titratable acid in fruit,accelerate fruit softening,and affect the expression levels of ethylene related genes,carotenoid related genes and cell wall struction-related genes.