Map-Based Cloning And Functional Analysis Of Floury Endosperm Related Gene OsNDUFA9 In Rice

Starch is the main energy reserve of seed endosperm in cereal crops,accounting for more than 80%of seed weight in rice,also is a key determinant of rice quality and yield.Starch synthesis is a complex and fine process involving a number of biosynthetic enzymes and regulators.The development of rice endosperm and endosperm is closely related.Starch in the endosperm provides energy and nutrients for embryo development and seed germination,while the abnormal development of the embryo and seedling growth limit its biological production,especially starch synthesis.A few genes have been identified for starch biosynthesis and embryo development,however,the new players and process has not well understood yet.Therefore,exploring new genes involed in starch synthesis and embryo development and studying their biological processes is of great significance for improving rice quality and increasing yield.A floury endosperm and embryo-lethal mutant named flo13 was derived from a pool of japonica rice cultivar Ningjing 3 mutants created by EMS-induced chemical mutagenesis,and then the underlying mechanism of floury mutant flo13 was investigated.Map-based gene cloning and transgenic complementation showed that the mutant gene flo13 encodes a 408aa protein of mitochondrial NADH dehydrogenase subunit,named OsNDUFA9.Previous studies have shown that NDUFA9 are mainly involved in the assembly and stability of NADH dehydrogenase.In this study,OsNDUFA9 showed to be a pleiotropic gene involed in starch synthesis and embryo development.The main findings are as follows:1.The homozygous flo13 seeds could not germinate.TTC staining revealed that the seed batches of wild type showed strongest viability as almost all seed embryos were stained into deep red,while the seed batches of flo13 exhibited maximum mortality due to its high percentage of uncolored embryos.Further freehand sections of the imbibed seeds showed that the flo13 embryos were blocked without visible counterpart structures,suggesting the embryogenesis of flo13 was arrested.Though the contents of total starch,amylose of the flo13 mutant displayed no significant differences from those of wild type,the flo13 mutant had a 30%reduction in 1000-grain weight.Both seed length and seed width of the flo13 mutant and wild type were comparable,but the seed thickness was significantly reduced in flo13.2.Scanning electron microscopy observation mature seeds of flo13 revealed that the starch granules were irregular shaped and loosely packed with large air spaces in the flo13 mutant compared with wild-type,and the size of starch granules in flo13 varies greatly.In Semi-thin section analysis of the 12 days after flowering endosperm showed that many smaller,immature and scattered SGs in the cytosol were observed both in the center and periphery of the endosperm cells in flo13.3.Because homozygous flo13(japonica)was unviable,we crossed a heterozygous flo13/+genotype to 9311(indica).Half of the F1 plants were the flo13/+genotype that could be selfed to generate an F2 segregation population.The mutant gene was finally delimited in the region of about 38kb between the markers S76-20 and S76-17 on the long arm of chromosome 2 with 2253 recessive individuals showing flo13 phenotypes.Then the genomic DNA sequencing analysis revealed that a G was changed to an A in the first exon of Os02g0816800,leading to a premature stop codon in the flo 13 mutant.4.OsNDUFA9 targets to mitochondria verified by its subcellular localization in rice protoplasts.Real-time RT-PCR and analysis showed that OsNDUFA9 was expressed constitutively in all tested organs of rice plant and endosperms during grain filling and preferred expressing in green tissues.5.Real-time RT-PCR analysis indicated that all the tested genes were obviously decreased.The expression levels of AGPS2b,AGPSL1,SS?a,SUS4,BEI,ISA2,PUL and Phol in the mutant were less than five folds than that those in the wild type.And the protein levels were also significantly reduced in the flo13 mutant.In conclusion,the flo13 mutation had important effects on the starch biosynthetic in rice endosperm.6.In flo13,transcript profiles of selected genes function in mitochondrial electron transport chain(ETC)were greately changed,genes in alternative respiratory pathway such as AOX1a and AOX1b were remarkably upregulated.In addition,the mitochondrial ultrastructure in the flo13 mutant was compromised.The mitochondria in wild-type endosperm formed distinct internal cristae surrounded with abundant matrix,while in flo13,the majority of mitochondria were aberrant and swollen,filled with very scarce matrix,lacking any obvious cristae structures formed by the inner membrane.These results indicated the mutation of OsNDUFA9 causes abnormal mitochondrial ultrastructure as well as the dysfunction of mitochondria.