The Study On The Mechanisms Of Ethylene-Regulated Early Ripening Apple Fruit Softening And Dehiscence

Apple is one of the most important fruit crop in China. It is important to investigate themechanism of texture quality including firmness and fragility which influences the eatingand shipping quality.‘Taishanzaoxia’ is one of the early-ripening apple cultivars and hasexcellent quality, but it is easy softening and even dehiscence. This greatly reduce itscommercial value. Therefore, studying the mechanism of fruit texture quality is very importfor improving the fruit quality. In this study, the ethylene production, PG activity and fruitfirmness during fruit development and storage stages were analysed. The treatment with1-MCP(1.0μL·L-1) was also used during storage stage. Five ethylene signal transcriptionfactors (ZMdEIL1, ZMdEIL2, ZMdEIL3, ZMdERF1and ZMdERF2) and one functional geneZMdPG1which encodes polygalacturonase were identified. The expression patterns ofZMdEIL1, ZMdEIL2, ZMdEIL3, ZMdERF1, ZMdERF2and ZMdPG1were studied.Transcription factors and promoter interaction and functional transgenic works were alsoconducted in the study. The main results are as follows:1. PG activity and fruit softening of ‘Taishanzaoxia’ fruit rapidly increased followed byethylene burst. With treatment of1-MCP, ethylene production was sharply reduced.Furthermore, the PG activity, fruit softening and dehiscence were significantly inhibited. Itsuggested that endogenous ethylene could play important role in the regulation of fruit softingand dehiscence.2. ZMdERF1and ZMdEIL2transcription were more abundant in ‘Taishanzaoxia’softening fruit and dehiscent fruit and their expression were inhibited by an ethylene inhibitor1-MCP. Therefore, ZMdERF1and ZMdEIL2expression were response to endogenousethylene and associated with fruit softening and dehiscence. ZMdPG1expression was inducedduring fruit softening and dehiscence, but it can be blocked by1-MCP, indicating thatZMdPG1was essential for fruit softening and dehiscence and its expression was mediated bythe endogenously occurred ethylene. The expression of ZMdERF2，ZMdEIL1and ZMdEIL3maintained low level during fruit development, and there was little increase in ZMdERF2butit was inhibited by1-MCP in softening fruit. These results indicated that the transcription ofZMdERF1，ZMdERF2，ZMdEIL2and ZMdPG1were closely connected in ethylene during fruit softening and dehiscence stages.3. ZMdERF1, ZMdERF2, ZMdEIL1, ZMdEIL2, ZMdEIL3and ZMdPG1were clonedfrom ‘Taishanzaoxia’ fruit. ZMdERF1and ZMdERF2belong to ERFs family including YRGand RAYD conserved domains. ZMdEIL1, ZMdEIL2and ZMdEIL3belong to EIN3/EILsfamily shared characteristic factors AD, BDI, BDII, BDIII, BDIV, BDV and PR. TheZMdPG1promoter was cloned which contains A-box, AT-rich sequence, BoxI, G-Box,BoxIII, CCGTCC-box and CGTCA-motif regulatory factors. ZMdEIL1, ZMdEIL2andZMdEIL3proteins were located in cell nucleus, while ZMdPG1was located in cell wall andcytoplasmic membrane.4. ZMdPG1overexpression in Arabidopsis led to seed silique early dehiscence whilesuppressing ZMdPG1expression by antisense ZMdPG1prevented seed silique naturallyopening. The ectopic expression of ZMdPG1was involved in cell separation and contributedto fruit dehiscence. ZMdPG1overexpression in tomato resulted in flower and fruit abscission.5. There were multiple regulatory factors in ZMdPG1promoter including bindingprotein factor BOXIII, however, ZMdERF1didn’t bound to ZMdPG1promoter. Therefore,ZMdERF1did not directly regulate the functional gene ZMdPG1.6. BiFC(Bimolecular fluorescence complementation) experiment evidenced thatZMdERF1and ZMdERF2interacted physically with ZMdEIL2.