Functional Analysis Of OsPPKL1 Involved In Grain Length Regulation In Rice (Oryza Sativa L.)

Rice is one of the most important food crops worldwide and feeds half of population in China.The grain yield of rice is directly determined by four major components,namely the number of panicles,the number of grains per panicle,grain weight and the ratio of filled grains.Grain weight is mainly determined by grain size,grain size is specified by its three dimensional structures:grain length,grain width,and grain thickness.In addition,grain size is one of the most frequently selected traits during breeding.We identified an extra large-grain japonica accession N411 and through positional cloning approach,we cloned a major grain length QTL,qGL3,which encodes a putative protein phosphatase with Kelch-like repeat domains(OsPPKL1).It was found that the phosphatase activity of OsPPKL1N411 was significantly lower than OsPPKL193-11.By mRNA in situ hybridization,it was found that the OsPPKL1 predominantly expressed in young spikelet meristem of 93-11.The grain length of T-DNA insertion mutant osppkll was longer than its wild type Dongjin and the grain length of OsPPKL1-overexpression transgenic lines was obviously shorter than ZH11.In rice OsPPKL1 and OsPPKL3 fell into the same subgroup,whereas OsPPKL2 and BSU1,an Arabidopsis homolog,were in another subgroup.BSU1 and its homolog BSL directly dephosphorylate BIN2 in Arabidopsis.We supposed that OsPPKL1 might regulate rice grain length via BR signaling pathway.The mutant osppkll displayed enhanced BR signaling phenotypes,leaves with greatly increased leaf angles,while OsPKL1-overexpression transgenic lines showed decreased leaf angles.However,the leaf angle of NILggl3 was similar with 93-11.The endogenous BLs increased in osppkl1 seedlings while in NILqgl3 seedlings,the contents of BLs were not significantly changed.These results indicated that OsPPKL193-11 affected the BR synthesis pathway.D2,D11 and DLT,the essential genes in the BR pathway,were decreased in osppkll compared with Dongjin.On the contrary,expressions of these genes were increased in the OsPKL1-overexpression seedlings.These results revealed that OsPPKLl93-11 could impact the BR synthesis pathway,while OsPPKL1N411 could not affect BR synthesis pathway.Lamina inclination experiments showed that the OsPPKL1 overexpressors were insensitive to exogenous brassinolide(BL),the most active BR,while osppkll and NILqgl3 was hypersensitive to the hormone.It indicated that OsPPKL1 was involved in BR signaling pathway.OsGSKs were the BIN2 homologgenes in rice.By qRT-PCR,it was found that OsGSKs predominantly expressed in young panicle.OsPPKL1N411 and OsPPKL193-11 could interact with OsGSKs via Y2H,BiFC and GST pull-down.The grain length of T-DNA insertion mutant osgsk3 was longer than wild type Dongjin.Lamina inclination experiments showed that the osgsk3 was hypersensitive to exogenous brassinolide(BL).BRZ enhanced the cytoplasm accumulation of OsGSK3.It was indicated that OsGSK3 was also involved in BR signaling pathway.OsPPKL193-11 not OsPPKL1N411 could dephosphorylate OsGSK3 in vitro.It suggested that OsPPKL1 regulated grain length through dephosphorylating OsGSK3 in BR signaling pathway.In Arabidopsis,BIN2 regulated the subcellular location of BZR1,the dephosphorylation status of BZR1 located in nucleus.OsBZR1::GFP fusion protein was localized in the nuclear and cytoplasm,BL and LMB could enhance the nuclear accumulation of OsBZR1 while BRZ decreased the nuclear accumulation of OsBZR1,and DEX enhanced the cytoplasm accumulation of OsBZR1.OsBZR1 located in nuclear in the osppkll protoplasts and osgsk3 protoplasts.The results indicated that OsGSK3 played negative roles in BR response.Grain length,as a critical trait for rice grain size and shape,has a great effect on grain yield and appearance quality.Although several grain size/shape genes have been cloned,the genetic interaction among these genes and the molecular mechanisms of grain size/shape architecture have not yet to be explored.To investigate the genetic interaction between two major grain length loci of rice,GS3 and qGL3,we developed two near-isogenic lines(NILs),NIL-GS3(GS3/qGL3)and NIL-qgl3(gs3/qgl3),in the genetic background of 93-11(gs3/qGL3)by conventional backcrossing and marker-assisted selection(MAS).Another NIL-GS3/qgl3(GS3/qgl3)was developed by crossing NIL-GS3 with NIL-qg/3 and using MAS.By comparing the grain lengths of 93-11,NIL-GS3,NIL-qg/3 and NIL-GS3/qgl3,we investigated the effects of GS3,qGL3 and GS3× qGL3 interaction on grain length based on two-way ANOVA.We found that GS3 and qGL3 had additive effects on rice grain length regulation.Comparative analysis of primary panicle transcriptomes in the four NILs revealed that the genes affected by GS3 and qG13 partially overlapped,and both loci might be involved in brassinosteroid signaling.Our data provide new information to better understand the rice grain length regulation mechanism and help rice breeders improve rice yield and appearance quality by molecular design breeding.