Functional Analyis Of Two Genes,RGG2 And OsPP2C09,in Regulating Grain Size In Rice

Rice(Oryza sativa)is one of the most important crops,feeding more than half of the people in the world.Rice grain yield is determined by three components:grain weight,panicle number and grain number.Grain weight is controlled by grain size and filling.Although many genes responsible for rice grain size have been characterized,the molecular mechanism remains unclear.More genes involved in the regulation of grain size in rice should be identified.In this study,two genes,RGG2 and OsPP2C09,were found to play essential roles in regulating grain size in rice.The main results are as follows.Part ? Mutation of RGG2,which encodes a type B heterotrimeric G protein y subunit,increases grain size and yield production in rice.Heterotrimeric G proteins,which consist of G?,G? and G? subunits,function as molecular switches to regulate a wide range of developmental processes in plants.However,the biological function of the RGG2 gene,which encodes a type B Gy subunit in rice,has not been characterized.In this study,we investigate the function of rice RGG2 in grain size and yield production.The main results obtained are as follows.1.Using yeast two-hybrid system and co-immunoprecipitation assays,we confirmed that RGG2 interacts with RGB 1.2.qPCR and GUS staining analyses revealed that RGG2 was constitutively expressed in all plant tissues,including the leaf,sheath,stem,panicle,node,and root tissues.Additionally,the transcript accumulation of RGG2 decreased as the inflorescence and grain development progressed.3.Subcellular localization analysis showed that rice RGG2 was localized to the nucleus,cytoplasm,and plasma membrane.4.RGG2 overexpression in Nipponbare(NIP)leads to reduced plant height and decreased grain size.By contrast,two mutants generated by the clustered,regularly interspaced,short palindromic repeat(CRISPR)/CRISPR-associated 9(Cas9)system in the Zhenshan 97(ZS97)background,zrgg2-1 and zrgg2-2,exhibit enhanced growth,including elongated internodes,increased 1000-grain weight and plant biomass,and enhanced grain yield per plant(+11.8%and 16.0%,respectively).These results demonstrate that RGG2 acts as a negative regulator of plant growth and organ size in rice.5.The leaf sheaths of overexpression lines were less sensitive to GA3 than those of NIP,whereas the leaf sheaths of zrgg2-1 and zrgg2-2 were more sensitive to GA3 than those of ZS97.This result was confirmed by the GA-induced ?-amylase activity of the seeds.These findings suggest that RGG2 is involved in the rice GA response.6.The haplotype(Hap)analysis based on the genomic sequence in 132 rice germplasms yielded a total of 6 SNPs and one indel representing six haplotypes.Hapl and Hap2 were mostly distributed in japonica and indica rice,respectively.NIP carries Hapl,while ZS97carries Hap2.However,they have the same RGG2 protein sequence.One rare haplotype containing Indell(Hap6)was found in Suyunuo and Dalijing.These two germplasms produce extremely large grains,which imply that Hap6 has beneficial effects on grain size and weight.However,the genetic effect and breeding value of the rare RGG2 allele needs to be further investigated.In summary,we propose that RGG2 may regulate grain and organ size via the GA pathway and that manipulation of RGG2 provides a novel strategy for rice grain yield enhancement.Part II OsPP2C09 negatively regulates abscisic acid signaling and drought resistance,but promotes plant growth and grain yield in riceType 2C protein phosphatases(PP2Cs)belonging to clade A have been extensively studied and are known to negatively regulate ABA signaling in Arabidopsis thaliana.However,very few clade A PP2Cs in rice have been functionally characterized.In this study,we investigated the functions of OsPP2C09(a rice clade A PP2C)using a CRISPR/Cas9 knockout mutant(ospp2c09)and two OsPP2C09-overexpressing lines in a NIP background.The main results are as follows.1.A phylogenetic analysis revealed that OsPP2C09 belongs to the clade A PP2C family in rice,and is most closely related to HAI2(AT1G07430)in A.thaliana.2.OsPP2C09 was widely expressed in various tissues,and at a relatively high level in the leaf and sheath.The OsPP2C09 transcript abundance decreased as the panicle and spikelet elongated,suggesting that OsPP2C09 may influence panicle and spikelet development.3.To determine the subcellular location of OsPP2C09,we transiently expressed GFP alone and the OsPP2C09-GFP fusion protein under the control of the CaMV 35S promoter in N.benthamiana leaf epidermal cells.Fluorescence from the OsPP2C09-GFP fusion protein was predominantly localized in the nucleus.4.OsPP2C09 interacted with most of the tested OsPYLs and OsSAPKs in the presence of ABA,suggesting that OsPP2C09 is involved in ABA signaling in rice.The results of ABA and drought stress response indicated that OsPP2C09 negatively regulated ABA signaling,stomatal closure and drought tolerance.5.OsPP2C09 also affects plant growth in addition to ABA signaling in rice.The ospp2c09 mutant was shorter than normal and produced small grains.In contrast,the overexpression of OsPP2C09 markedly increased plant height,grain size,and grain yield per plant.A significant grain yield improvement was also observed in a high-yielding variety(Suken118)that ubiquitously overexpressed OsPP2C09.These findings reveal that OsPP2C09 is a component of the ABA signaling pathway and negatively influences ABA-mediated drought responses.Additionally,OsPP2C09 affects plant growth and grain yield.Thus,OsPP2C09 represents a novel candidate gene potentially useful for breeding new high-yielding rice varietiesin water resource-rich areas.