Molecular Mechanism And Evolutionary Analysis Of Grain Size Gene GS3 In Rice

Improving grain yield and quality are the most important goals of scientific research.In rice,one of the most important food crops in the word,grain yield is determined by three components: number of effective panicles,grain number per panicle and grain weight.Grain weight,restricted by grain size and grain filling,is the most heritable trait.Therefore,grain size determined by grain length,grain width and grain thickness is an important yield trait.In addition,grain size is also an important quality trait according to the national standard requirements,such that the ratio of length to width higher that 2.8 is required by high quality indica rice.Therefore,revealing the genetic basis and molecular mechanism of grain size control is beneficial to improve grain yield and quality,which provide more strategies and genetic resources for breeding.GS3,located near the centromere of the third chromosome,is a major gene responsible for the negative regulation of grain length,and it can be detected in many studies.Polymorphic markers developed by GS3 have been widely used in molecular marker assisted breeding.GS3 encodes an atypical Gγ subunit,which contains an organ size regulation domain（OSR）at the N terminal and a cysteine-rich domain（cys-rich）at the C terminal.There are 4 major alleles in natural populations: short grain varieties contain GS3-1 and GS3-2 alleles encoding full-length GS3 protein,and this is the wild type allele;long grain varieties contain a loss of function allele GS3-3 encoding a protein lack of OSR and cys-rich;super short grain variety Chuan 7 contains a GS3-4 allele,encoding a protein with OSR only,which shows enhanced function relative to GS3-1.DEP1 is another atypical Gγ subunit in rice,its truncated allele（dep1）without cys-rich domain promotes the proliferation of meristematic cells and increases the number of grains per panicle.In addition to the atypical Gγ subunit GS3 and DEP1,the rice G proteins also include one Gβ subunit RGB1,one Gα subunit RGA1 and two typical Gγ subunits RGG1 and RGG2.Based on above understandings,this study further explores the molecular mechanism of GS3 and the regulatory network of G protein signaling in grain size control.The main results are as follows:1.Transgenic plants with overexpression of Flag fused GS3-1 and GS3-4 in zhonghua 11（ZH11）background reduced ¹2% and ³0% of the grain size,respectively.Protein accumulation of GS3-4-Flag was found in the ubi::GS3-4::Flag transgenic plants comparing with GS3-1-Flag.Protein level of GS3-1-Flag increased in ubi:GS3-1:Flag transgenic plants under the treatment of proteasome inhibitor MG132,suggesting that the cys-rich domain may mediate the ubiquitination and degradation of GS3.2.Genetic transformation showed that transgenic plants with overexpression of GS3-1（GS3-1OE）or suppression of DEP1（DEP1Ri）exhibited the same phenotype,such as dwarfed plant,dense and erect panicles and small seeds,where as transgenic plants with overexpression of DEP1（DEP1OE）or suppression of GS3-1（GS3-1Ri）increased grain size,suggesting that GS3 and DEP1 function completely opposite in seed size regulation.In addition,the transgenic plants overexpressing dep1 decreased the grain size,which was consistent with the GS3-1OE plants,suggesting that DEP1 and GS3 may work in the same regulatory network.3.Genetic analysis showed that DEP1 OE or knock out DEP1(DEP1^ko)could not reduced the grain size in GS3-1OE background,where as DEP1 OE in GS3 Ri background further increased the grain size.Meanwhile,genetic evidence also showed that GS3 played the same function as dep1,because overexpressing dep1 can restored the phenotype of GS3 Ri,thus further proved by the evidence that dep1 under native promoter recovered the phenotype of gs3 knock out mutant.4.Knock out mutants of GS3(GS3^ko),DEP1(DEP1^ko)and their double mutants(GS3^koDEP1^ko)were constructed using CRISPR/Cas9 technology.GS3 ^ko mutant showed increased grain size,whereas DEP1 ^ko mutant decreased the grain size.However,the GS3^koDEP1 ^ko double mutants showed additive grain size between GS3 ^ko and DEP1 ^ko,suggesting that there may be another Gγ gene in the pathway,which play the same role as DEP1.5.The third atypical Gγ subunit GGC2 was found by sequence blast in rice.Overexpression of GGC2 increased the grain size,whereas knock out GGC2 reduced the grain size,indicating that GGC2 was a positive regulator of grain size regulation.The grain length of GGC2^koDEP1 ^ko double mutant was ³0% smaller than that of GGC2 ^ko or DEP1 ^ko single mutants,indicating that GGC2 and DEP1 have additive effects in grain size control.The grain length of GGC2^koDEP1^koGS3 ^ko triple mutant did not show significant increase compared with GGC2^koDEP1 ^ko double mutant,suggesting that the negative function of GS3 was dependent on GGC2 and DEP1.6.RNAi or knock out mutants of Gα gene RGA1 and Gβ gene RGB1 in ZH11 background were generated in order to validated their genetic interactions with Gγ genes GS3,DEP1 and GGC2.The RGB1 Ri and RGA1 ^ko mutants reduced the grain size,and even overexpressing DEP1 and GGC2 or suppressing GS3 could not increased the grain size in RGB1 Ri and RGA1 ^ko background,suggesting that RGB1 and RGA1 are necessary for the function of DEP1 and GGC2.Yeast two hybrid and in vivo Bi FC and luciferase complementation experiments showed that GS3,DEP1 and GGC2 directly interacted with RGB1 through the OSR domain.In addition,yeast three hybrid experiments showed that GS3,DEP1,and GGC2 competed in interaction with RGB1.Furthermore,in vivo experiments indicated that the interaction between RGB1 and DEP1 or GGC2 was moderately reduced in GS3-1 overexpressing background,whereas in GS3-4 overexpressing background,a more severe reduction of interaction was observed,which was due to the accumulation of GS3-4 protein relative to GS3-1.7.Sequence alignment of GS3,DEP1,and GGC2 revealed that the OSR domain,which mediate the interaction with RGB1,was very conserved,but the cys-rich domain shows large variation.Transgenic plants overexpressed the chimeric genes which replaced the cys-rich domain of GS3（116-232）by the cys-rich domain of DEP1（121-426）or GGC2（113-335）increased the grain size,showing opposite function to GS3,whereas transgenic plants overexpressed the chimeric genes which replaced the cys-rich domain of DEP1 or GGC2 by the cys-rich domain of GS3 reduced the grain size,showing opposite function to DEP1 and GGC2.These results showed that the variations of the cys-rich domain determine the functional differentiation of GS3,DEP1 and GGC2.Further sequence analysis showed that the cys-rich domain of DEP1 and GGC2 contained conserved repeated elements,which did not exist in GS3 with a short cys-rich domain,suggesting that the evolution of repeated elements triggered the function diversification.8.Sequence analysis also revealed that the Arabidopsis atypical Gγ subunit AGG3,like GS3,did not contain the conserved repeat elements.Transgenic evidences showed that AGG3 played the same role as GS3 in rice,which negatively regulated the grain size dependent on their OSR domains.But in Arabidopsis,all of the three Gγ subunits,GS3,DEP1 and GGC2,restored the phenotype of agg3 mutant,indicating that the function differentiation caused by cys-rich variation occured only in rice,but not in Arabidopsis.In summary,this study systematically analyzed the genetic relationship between subunits of rice G proteins in grain size regulation.The Gγ subunits exhibited functional differentiation through cys-rich domain variation,in which DEP1 and GGC2 mediated the RGA1 and RGB1 dependent signal transduction,whereas the GS3 protein was responsible for signal termination by competing interaction with RGB1.Meanwhile,the cys-rich domain of GS3-1 mediated the degradation of GS3 protein,which in turn achieved fine regulation of the G protein signaling in rice.