Ethylene Antagonizes Jasmonate Acid Signaling Pathway During Tomato Ripening And The Manipulation Of Fruit Color By CRISPR/Cas9 System

Tomato is not only a worldwide cultivated cash crop,but also a model plant for studying fruit ripening and postharvest.Therefore,it plays an important role in production and scientific research.During the process of tomato fruit ripening,the resistance of tomato fruit to necrotrophic pathogen decreased gradually,which significantly affected the number of commercial fruit and postharvest storage or transportation.Jasmonic acid is a key defense hormone that regulates the resistance of plants to necrotrophic pathogens.The interaction mechanism between ethylene-mediated fruit ripening and jasmonic acidmediated defense remains elusive.On the other hand,there is amount of different fruit color varieties,but most of the tomatoes sold in the market are red.In agricultural production,tomato is mainly produced by hybrids because of the super-parent phenotype caused by heterosis.However,most of the fruit color control genes that have been identified are recessive genes,thus makes an extremely difficult to cultivate rare fruit color hybrids,expecially for the phenotype caused by multilocus variations.How to quickly polymerize target genes to produce homozygous inbred lines is always a problem for breeders.In this paper,two studies were carried out,and the main results are as follows:I.The mechanism of ethylene antagonizing jasmonic acid signal during fruit ripening.1.During fruit ripening,the resistance of fruit to necrotrophic pathogen Botrytis cinerea decreased gradually.Ethylene receptor mutant Nr and jasmonic acid synthetic mutant spr8 showed increased and decreased resistance to B.cinerea,respectively.These results showed that the two hormones regulated the resistance to necrotrophic pathogen together in tomato fruit.2.The determination of the two hormones showed that the synthesis of ethylene increased during fruit ripening,and the content of bioactive form of jasmonic acid,jasmonic acid isoleucine(JA-Ile)decreased after ripening.Correspondingly,the expression of jasmonic acid biosynthesis and responsive genes decreased gradually,while the expression of catabolic genes increased during fruit ripening.When the core transcription factor EILs of ethylene signaling pathway was silenced,the fruit displayed a non-ripening phenotype and the decrease of JA-Ile content was delayed.The expression of jasmonic acid synthesis and responsive genes in EILs-RNAi was higher than that of wild type,and the expression of catabolic genes was supressed.These results indicated that ethylene had an antagonistic effect on jasmonic acid signal during fruit ripening.3.The expression of JA-Ile catabolic gene JC1 depends on the activation of EILs by Real-time fluorescent quantitative PCR analysis and tobacco promoter activation assay.Chromatin immunoprecipitation(Ch IP-q PCR)and gel migration(EMSA)assays showed that EIL1 could directly bind to EBS motif in JC1 promoter region in vivo and in vitro.The chromatin conformation capture experiment(3C)showed that the distal end of the JC1 promoter and the proximal EBS motif formed a ripen-specific chromatin loop.4.In vitro enzyme activity assay showed that JC1 could hydrolyze the bioactive JAIle into inactive 12-COOH-JA-Ile.It was found that knockout of JC1 could improve the resistance to B.cinerea without affecting other agronomic traits in ripen tomato fruits.The above experiments suggest that EILs,the core transcription factor of ethylene signaling pathway,directly binding to JC1 promoter EBS rigon and activates its expression when fruit ripening.The bioactive JA-Ile was hydrolyzed into inactive 12-COOH-JA-Ile by JC1.In addition,knockout of JC1 has a certain application value to improve resistance to B.cinerea in ripen fruits.II.Generating multiple-color tomato fruit inbred lines and hybrids by CRISPR/Cas9 gene editing system.1.In this study,we used a six-target CRISPR/Cas9 system to knock out three fruit color genes PSY1,MYB12 and SGR1 simultaneously.After two generations of hybridization and isolation,seven kinds(i.e.yellow,light yellow,brown pink,yellow green,pink,brown red and green fruit)of homozygous inbred lines without transgenic T-DNA insertions under the same background within one year,which successfully shortened the breeding period.Moreover,this breeding strategy avoid the influence of linkage encumbrance as well as the problem of foreign gene insertion faced by transgenic.2.The pink-fruit inbred parental lines obtained through myb12-gene editing were crossed to generat three hybrid lines.Compared with corresponding wild-type red fruit hybrids,the myb12-edited hybrid tomatoes showed no difference in agronomic characters except fruit color.These results showed that it was very feasible to quickly generate a variety of rare fruit color inbred lines by gene editing and apply them to the production of hybrids.