Map-Based Cloning And Functional Analysis Of GW9 Gene Controlling Grain Width In Rice

Rice is one of the most important food crops in the world,and feeds more than half of the global’s population.Given the ongoing environmental deterioration and the decrease in cultivable land,it is of great significance to further improve rice yield to ensure the food security in China.Rice yield is mainly determined by the number of panicles,number of grains per panicle and grain weight.The grain weight is the comprehensive embodiment of grain shape(including the grain width,grain length and grain thickness),and grain filling degree.In recent years,although numerous genes related to grain shape have been identified,the molecular mechanism of grain shape regulatory network remain limited.Therefore,it is necessary to screen new grain shape regulatory genes to enriching the genetic regulatory network of grain shape in rice.In this research,the grain width 9(gw9)mutant was obtained from an EMS-treated library of W7,and the molecular mechanism of gw9 in grain width regulation was studied.The main results are showed as follows:Compared with W7,the plant height,grain width,grain length and 1,000-grain weight of gw9 mutant were increased strikingly.In addition,the grain filling rate was improved and the flowering time was advanced in gw9.Cytological observations showed that GW9 mainly regulated grain width and grain length by regulating the glume cells proliferation and expansion.Map based cloning found that GW9 was located on chromosome 9,and the gentic sequencing result revealed that a single base substitution on the11 th intron of LOC＿Os09g13630,resulting in the wrong splicing of the 11 th exon.Genetic complementation and overexpression demonstrated that LOC＿Os09g13630 is GW9,which responsible for the phenotypes of gw9.GW9 encoding a PRC2 complex protein EMF2b,which can negatively regulate rice grain width.Subcellular localization analysis showed that GW9 was localized in the nucleus.RT-qPCR and GUS staining assay showed that GW9 was widely expressed in roots,stems,leaves,panicles,and the highest expression were found in young panicles.RNA-seq revelaed that GW9 may regulate plant height by participating in gibberellin pathway.The expression of gibberellin synthesis genes and metabolic genes in gw9 mutant were significantly changed,and the gibberellin contents were significantly increased in gw9 mutant.GW9 protein contains a C2H2 zinc finger domain and a VEFS domain,which can regulate rice grain width and flower organ development,respectively.The results of gene knockout at different domain showed that plant height and grain width remarkably increased,and the flowering time anvanced when the zinc finger domain was impaired but VEFS domain was normal.Knockout of the VEFS domain results in dwarfing,delayed flowering,malformation of flower organs,and reduced pollen fertility.Multiple molecular experiments have demonstrated that GW9 physically interacts with GW2,a negative regulator of grain width,and the zinc finger domain of GW9 plays an important role in this interaction.Further studies revealed that the E3 ubiquitin ligase GW2 could ubiquitinate GW9,and the loss function of zinc finger domain of GW9 reduced the ubiquitination degree.Genetic analysis showed that GW2 and GW9 were not completely independent in genetic relationship,and they could jointly regulate grain shape.Taken together,the GW9 can not only functions in flowering transition and flower organ identity,but also regulate grain width through GW2 pathway.These results not only expand the function of PRC2 complex,but also provid new sights into the genetic regulation mechanism underlying grain shape in rice.