Map-based Cloning And Functional Analysis Of SHMT4,A Key Gene Regulating Endosperm Development In Rice

Rice(Oryza sativa L.)is one of the most important crops in the world and the largest ration crop in China.Starch and protein are the two most important nutrients in rice endosperm,and their content and composition directly affect rice various quality indicators and nutritional value.With the improvement of people’s living standards,improving the quality of rice has become an urgent demand and challenge for our country.Therefore,an intensive analysis of the molecular mechanism of rice endosperm development and elucidation of the accumulation regulation of storage substance such as starch and protein are conducive to improve rice quality by modern molecular breeding.Although a number of key factors regulating rice endosperm development have been cloned,our understanding of endosperm development is still limited due to the complexity of storage substance synthesis,transport,and accumulation.Through abnormal endosperm mutants,excavating key regulatory genes is an effective strategy to comprehensively analyze the molecular regulatory network of endosperm development.In this study,seven alleles endosperm dysplasia mutants were isolated and identified: flo20-1 to flo20-7.We chose flo20-1 for in-depth studies and systematically analyzed its phenotype and the biological function of the target gene by means of cytology,genetics and biochemistry.The main research results are as follows:1.The flo20-1 mutant was defective in endosperm development,with a floury endosperm.We found that starch granules were shape-irregular and loosely arranged,and the structure of protein bodies also changed greatly in flo20-1 by cytological observation.Among them,PBI(type I protein body)lacked the typical concentric ring structure,PBII(type II protein body)were more twisted than wild type.In addition to the endosperm phenotype,the flo20-1 mutant exhibited seedling lethality.2.Through map-based cloning,we mapped the FLO20 to a 40 kb region of chromosome 1 using the segregated population of flo20-1 and Dular,which contains 5 open reading frames.Sequencing analysis revealed a missense mutation in the first exon of the gene LOC＿Os01g65410,which encodes a serine hydroxymethyltransferase SHMT4 containing an SHMT domain.Complementation experiments showed that the genome sequence of SHMT4 could complement the phenotype of flo20-1,indicating that SHMT4 was the target gene responsible for flo20-1 phenotype.3.SHMT4 protein fused with GFP can complement the phenotype of flo20-1 mutant,suggesting that SHMT4-GFP was biological-functional protein.Confocal fluorescence microscopy observation combined with colocalization analysis showed that SHMT4 was localized in the nucleus.Detection with anti-SHMT4 antibody confirmed that the protein was constitutively accumulated in various tissues of rice.4.As a serine hydroxymethyltransferase,SHMT4 had no catalytic activity in vitro.SHMT4 could interact with itself and its homologous proteins SHMT3 & SHMT5.We speculated that they may function in vivo in the form of a complex.5.Co-immunoprecipitation confirmed that SHMT4 can interact with S-adenosylmethionine(SAM)synthase SAMS2.The methyl-donor SAM content was significantly elevated in the flo20-1 mutant.Sequencing revealed that the methylation level of the whole genome of flo20-1 endosperm was significantly increased.In addition,we found that the expression of genes related to the synthesis,transport and accumulation of storage substance such as starch and protein were significantly altered using q RT-PCR analysis.Taken together,we screened and identified seven allelic SHMT4 mutants with defective endosperm development,all of which exhibited abnormal starch and storage protein accumulation.The nuclearlocalized SHMT4 may interact with homologous proteins SHMT3 and SHMT5 to form a protein complex to function in vivo by biochemical analysis.In addition,SHMT4 physically interacted with SAMS2,and its mutation affected the content of SAM.Based on the above data,we speculated that SHMT4 may form a functional protein complex with SHMT3,SHMT5 and SAMS2 to participate in rice endosperm development by regulating the epigenetic modification of essential regulators for starch and storage protein synthesis.This study established a functional link between SHMT proteins and endosperm storage products accumulation.