The Function Of WRKY Transcription Factors In Fruit Ripening Of Tomato

Tomato(Solanum lycopersicum) is a typical climacteric fruit. Physiological changes during the fruit ripening of tomato include a burst of ethylene production, degradation of chlorophyll and formation of lycopene, which make the fruit colour turn from green to red, and fruit softening, which isrelated to the action of hydrolytic enzymes.Transcription regulation is a vitalprocess inplants.WRKYtranscription factors are plant specific and key regulators of many processes, including plant morphogenesis, metabolism, responses to biotic and abiotic stresses, etc. Recent researches on model plants, Arabidopsis(Arabidopsis thaniana) and rice(Oryza sativa), provide evidence that WRKYsregulate leaf senescence, but there is little research related to the regulation of postharvest physiology and fruit ripening. In this study, several Sl WRKYs which responded to ethylene treatmentwere identified based on the former research of our team, then we analyzedtheirexpression pattern in tomato leaves and fruits, identified probable target genes, and investigated their interacting proteins.The main results were as follows:1. Using quantitative real-time PCR, the expression pattern of nine Sl WRKY genes in tomato leaves and fruit in plants was detected.(1)Sl WRKY16, Sl WRKY17,Sl WRKY22,Sl WRKY23,Sl WRKY25,Sl WRKY31,Sl WRKY33,Sl WRKY53, Sl WRKY54 genes expressed highest at the onset of leaf senescense,(2)In the ripening process of tomato fruits, expression level of Sl WRKY16, Sl WRKY17, Sl WRKY22, Sl WRKY23, Sl WRKY25, Sl WRKY31, Sl WRKY33, Sl WRKY53, Sl WRKY54 genes were up-regulated differently.(3) The expression of Sl WRKY16, Sl WRKY17, Sl WRKY23, Sl WRKY25 reached a peak in pink fruits, and the peak of expression level of Sl WRKY31、Sl WRKY33、Sl WRKY53、Sl WRKY54 appeared in red ripe fruits. The expression level of Sl WRKY22 increaced gradually during the process of fruit ripening.2. YFP recombinant vector was constructed and YFP signals were observed with confocal fluorescence microscopy. Sl WRKY17, Sl WRKY22, Sl WRKY23, Sl WRKY25, Sl WRKY31, Sl WRKY33, Sl WRKY53 and Sl WRKY54 are all located in the nucleus.3.Sl WRKYs have no influence on the promoter activity of Sl ACO1, Sl ACO3 and Sl PAO. Sl WRKY16, Sl WRKY23, Sl WRKY33, Sl WRKY53 increased the promoteractivity of Sl PPH to 2-3 folds, Sl WRKY25 increased promoter activity of Sl PDS to 6 folds.The combined action of Sl WRKY16 and Sl WRKY17 largely increased the promoter activity of Sl PSY1.4. The results of bimolecular fluorescence complementation assays informed the interaction between Sl WRKYs and transcription fators like Sl ERF2 b, Sl ERF7 and Sl RIN. Sl ERF2 b interacts with Sl WRKY17 and Sl WRKY54, Sl ERF7 interactswith Sl WRKY17 and Sl WRKY33,Sl RIN interacted with Sl WRKY17 and Sl WRKY53,Sl WRKY17 interacted with Sl WRKY16 and Sl WRKY54.5.Sl WRKY23 and Sl WRKY53 proteins were obtained by prokaryotic expression and protein purification. Electrophoretic mobility shift assays(EMSAs) were done with the promoter fragment containing W-box.Results revealed that Sl WRKY23 could conbine with the promoter fragment of Sl ACO1, Sl ACO2, Sl ACO3, Sl PAO, Sl PDS, Sl PG, Sl PPH, Sl PSY1, Sl SGR, and Sl WRKY53 could not bind with any promoter fragment above.