Gene Cloning And Functional Analysis Of Rice White-Core Endosperm Mutant W59

Starch,the most important storage reserve,accounting for about 90%in rice endosperm,is the main determinant of rice processing,eating and cooking qualities.Starch synthesis is a fine and complex process.A large number of starch synthesis genes and regulatory factors have been identified,but the synthesis and regulation of starch is not well understood.Therefore,in-depth understanding of starch biosynthesis and exploring new genes involved in starch synthesis is of great significance in improving rice yield and quality.In the chemically induced mutant population of japonica cv Koshihikari,a stably inherited white-core endosperm mutant w59 was obtained.The mutant gene w59,which encodes a plastidic pyruvate kinase(PKp),was cloned and validated by map-based cloning and transgenic complementation.Previous studies have shown that PKps are mainly involved in the de novo synthesis of fatty acids in plastids.However,the role of PKps in starch-storing tissues has not been investigated.In the w59 endosperm,both the fatty acid and the starch synthesis are significantly blocked,these may reveal a functional connection between lipid and starch synthesis in rice endosperm.The fact that OsPKpas interact with FL06,a starch-binding protein involved in starch synthesis and compound starch grain formation provides further supporting evidence for this connection.Moreover,the isoforms of PKps in rice were analyzed.The main findings are as follows:1.Compared with the wild type,the w59 mutant showed the remarkably lower grain filling rate and an about 25%decrease in 1,000-grain weight.The grain size of w59 mature seeds was reduced significantly.The total starch,amylose,protein and lipid contents of w59 seeds decreased;meanwhile,the viscosity characteristics of starch and the chain length distribution of amylopectin were changed obviously.In all,the mutation had pleiotropic effects on the accumulation of storage substances in the endosperm.2.The mature seeds of w59 exhibited a white-core endosperm.Scanning electron microscopy observation indicated that the starch granules were small and round,loosely packed in the mutant.Semi-thin sections showed that the center of the w59 endosperm had large empty spaces,with fewer and smaller amyloplasts.The amyloplasts were scattered in the w59 endosperm,including abnormal amyloplasts strikingly amorphous and stained weakly with iodine.Besides,fewer and smaller oil bodies appeared in the aleurone layer of w59 endosperm.At the same time,the contents of different fatty acid species and galactosyldiacylglycerol(the major components in plastid membranes)were decreased in w59.Therefore,the w59 mutation affected the development of amyloplasts and oil bodies.3.Using an F2 population derived from a cross between w59(japonica)and Nanjing 11(indica),the mutant gene was finally delimited in the region of about 170 kb between the markers W59-40 and W59-45 on the short arm of chromosome 7.Then the cDNA sequencing analysis revealed that a G was changed to an A in the third exon of Os07g0181000,resulting in premature termination of protein translation.Overexpression and interference experiments confirmed that the Os07g0181000 gene was responsible for the w59 white-core phenotype.The W59 locus encodes a PKp containing 578 amino acids.4.The phylogenetic tree analysis announced that four PKps in the rice genome,enconding two ? and two ? subunits.The W59 locus encoded OsPKpal,and the other three were OsPKpa2,OsPKp?1 and OsPKp?2,all constitutively expressed.The four subunits were confirmed to localize in plastids.OsPKpal can be detected in amyloplast stroma.5.The activity of PKp in mutant w59 was decreased and cytosolic PK(PKc)activity was increased significantly.In addition,the content of pyruvate(Pyr)in w59 was decreased and phosphoenolpyruvate(PEP)was increased.The expression levels of phosphopyruvate carboxylases(PEPC)PEPC1 and PEPC2 were significantly increased in w59 seedlings or developing seeds,as did that of chloroplastic NADP-dependent malic enzyme(ME)ME6,while the expression of pyruvate dehydrogenase(PDH)was decreased.6.Recombination experiments showed that the active PKp complexs could be formed between inactive a subunit and ? subunit in vitro.The mutant OsPKpal activity was only 20%of the wild type in vitro.Yeast two-hybrid,pull-down,bimolecular fluorescence complementation assays indicated that at least two hetetomeric ?1?1,?1?2 existed in rice endosperm.The mature seeds of OsPKp?2 RNA interference lines also showed white core endosperm.In addition,the expression of OsPKp?2 was similar to that of OsPKpal,both were high expressed during seed development,suggesting that ?1?2 may be the major PKp complex in rice ednosperm.7.Western blot analysis indicated that the protein contents of AGPL2,AGPS2b,AGPS1,GBSSI,Pho?,SS?a,SBE?,SBE?b decreased in w59 seeds.In addition,compared with the wild type,the AGPase activity was significantly reduced in w59.In conclusion,the w59 mutation had important effects on the starch biosynthetic pathway in rice seed.8.Yeast two-hybrid assay showed that FL06 was able to interact with OsPKpal and OsPKpa2.BiFC experiments confirmed the interaction of FL06 with OsPKpal and OsPKpa2 in plastids.The direct interaction between FL06 and OsPKpa2 was demonstrated by the pull-down experiment.In conclusion,OsPKpa interacted with the starch-binding protein FL06.The fatty acid synthesis was blocked by the mutation of OsPKpal,resulting in defects in the development of amyloplasts and oil bodies and starch synthesis was impaired.OsPKpal could interact with FL06,which may affect starch synthesis and the formation of compound starch granules.