Role Of OsU496A In The Regulation Of Grain Filling By Auxin And Cytokinin In Rice

Grain filling is a key stage that contributes greatly to the grain yield formation and quality construction of rice,and its physiological and molecular mechanism exploration has always been the focus and difficulty.In fact,the grain filling is the importing and accumulating of assimilates to grain,which is regulated by phytohormones,especially of auxin and cytokinin.The research on the molecular mechanism of plant hormones regulating the grain filling of rice is being carried out gradually.OsU496A is an uncharacterized membrane protein mainly localized to the endosperm of rice.In this study,OsU496A RNA interference transgenic rice(RNAi-37),overexpressed transgenic rice(OX-43)and wild type rice(Nipponbare,WT)were used as materials.The role of OsU496A in the grain filling regulated by auxin and cytokinin was researched by the analysis of endogenous auxin and cytokinin concentration,exogenous IAA and 6-BA treatments,two phytohormone-and grain filling-related genes expression,and OsU496A-interacting proteins.The main results are as follows:The content of IAA and Z+ZR in the grain of RNAi-37 was significantly lower than that in the wild type during the whole grain filling,while the peak value of IAA and Z+ZR content in the grain of OX-43 was slightly higher than that in the wild type,but there was no significant difference between the OX-43 and wild type.Results of the expression of genes related to auxin and cytokinin metabolism and regulation in the grains of RNAi-37 and OX-43 showed that OsU496A was involved in the synthesis and transportation of IAA and the decomposition of cytokinin,resulting in changes of IAA and CTK content in grains,and participated in the regulation of IAA and CTK-mediated signal transduction during grain filling.The expression of OsU496A in grains was significantly increased by IAA and 6-BA treatments.Compared with the wild type,exogenous IAA and 6-BA treatments significantly increased the grain weight of RNAi-37,but did not significantly increase the grain weight of OX-43.The dynamic analysis of rice grain filling showed that exogenous hormone treatment significantly increased the maximum grain filling rate(Gmax)of RNAi-37 and WT,while significantly reducing the active grain filling period(D)of RNAi-37 grains and the time to reach the maximum grain filling rate(Tmax),thus significantly improving the grain filling efficiency.Further analysis of grain sucrose and starch metabolism,key enzyme activities and gene expression showed that OsU496A might promote the conversion of sucrose and starch synthesis through the regulation of auxin and cytokinin on the activities of sucrose-starch conversion key enzymes(SuS,AGPase,SSS,DBE),especially SuS and SSS,thereby affecting grain weight,which positively regulates auxin and cytokinin-mediated grain filling of rice.54 positive yeast clones were screened from rice cDNA library by using yeast two hybrid technique.Among them,26 positive clones were the same gene,E3 ubiquitin ligase encoding ARI2 genes.In summary,it is speculated that OsU496A may interact with E3 ubiquitin ligase to promote the degradation of IAA transcriptional inhibitors,thus activating the auxin transcription factor,causing down-regulation of auxin-regulated gene expression,and increasing the activity of grain-filling related enzymes in grains,thus positively regulating the grain filling of rice;On the other hand,OsU496A may increase the cytokinin content in grains by inhibiting the expression of cytokinin oxidase gene,which in turn positively regulates the cytokinin signaling and promotes grain filling of rice.The results preliminarily explored the role of OsU496A in the regulation of grain filling by auxin and cytokinin in rice,which could lay a foundation for further research on the function of OsU496A and the improving rice yield.