Cloning And Functional Analysis Of Rice Floury Endosperm Gene OsPORR1

Rice is an important food crop,which feeds half people of all the world.With the popularization of semi-dwarf varieties and hybrid rice,rice yield has increased greatly.After defeating hunger,people had higher request for rice quality than ever.Rice belongs monocot and its endosperm is edible part.Starch,the main component of endosperm,determines rice quality.Thus,studies on endosperm development and starch synthesis have great significance for improvement of rice quality.To study underlying mechanisms of endosperm development and starch synthesis in rice,an N-methyl-N-nitrosourea（MNU）-induced mutant pool of japonica strain W017 was generated.From the pool,a lethal mutant b438 with floury shrunken endosperm was screened.The mutant gene of the b438 mutant was located on the long arm of chromosome9 by map-based cloning.It was conformed that LOC＿Os09g29760 is the target gene by results of sequencing and transferring gene.As annotation,LOC＿Os09g29760 encodes a protein containing a plant organelle RNA recognition（PORR）domain.Because the gene is the first PORR gene cloned in rice,it is named as Os PORR1 in this study.Studies in Arabidopsis and maize showed that PORR genes were involved in intron splicing of mitochondria and chloroplasts.However,their impacts on rice endosperm development and starch synthesis retain unknown.The results of this study are as following:1.Compared with those of the WT W017 seeds,both the 1000-grain weight and grain thickness of mature b438 seeds are significantly reduced.The contents of starch and amylose of mature b438 seeds are reduced,while the contents of protein and lipid are increased.Homozygous b438 seeds（osporr1/-）were only harvested from heterozygous plants（Os PORR1/-）.The progenies of heterozygous plants exhibited a 3:1 segregation ratio of transparent grains to opaque ones,suggesting the b438 mutant phenotypes were controlled by a recessive nuclear gene.2.Compound starch grains（CSGs）developed slowly and assemble loosely in b438developing endosperm cells.After riping,CSGs were smooth and loosely assembled in b438 seeds while sharp-edged and tightly assembled in WT seeds.The accumulations of granule-bound starch synthase GBSSI,starch synthase SSIIa,starch branching enzyme SBEI and SBEIIb were reduced in b438 developing endosperm.3.Mature b438 seeds with hulls can not germinate.Although b438 dehulled seeds can germinate,the germination percentage is much lower than that of WT seeds.The growth of b438 seedlings was delayed and they all died within 30 days after sowing（DAS）.After observing longitudinal sections of mature seeds,it was found that embryo differentiation of b438 was not obvious.The TTC（2,3,5-Triphenyltetrazolium Chloride）staining experiment showed most mature b438 seeds had little vigor.4.To identify the locus on chromosome of the target gene,an F₂population derived from a cross between heterozygous plants（Os PORR1/-）and Nanjing 11（NJ11,indica）was generated.Nine hundred and seventy-eight floury individuals were adopted and finally the target gene was located in a 54 kb region between wi-10 and wi-11,two molecular markers on the long arm of chromosome 9.Sequencing analysis revealed a 408 bp fragment was deleted in the allele of LOC＿Os09g29760 in the b438 mutant.Transgenic complementation and knockout of LOC＿Os09g29760 further confirmed the results of map-based cloning.Therefore,LOC＿Os09g29760 is the target gene.5.LOC＿Os09g29760 encodes a PORR protein named Os PORR1 that contains 399amino acids.Zm WTF1 and At WTF9 were two reported PORR proteins those had RNA-binding activity and the sequences of the PORR domain in them were 49.24%and38.23%similar,respectively,to those of Os PORR1.There are totally 17 proteins containing PORR domain in rice.Their sequence alignment showed that there are 6 amino acids highly conserved and all the 6 amino acids are within the PORR domain.6.q RT-PCR（Quantitative Real-time Polymerase Chain Reaction）analysis andβ-glucuronidase（GUS）staining showed that Os PORR1 expressed in young seedlings and roots,stems,leaves,sheaths,panicles,developing seeds of adult plants.The subcellular localization of Os PORR protein infused green fluorescent protein（GFP）showed that Os PORR1 is a mitochondrion-targeted protein.7.The transcript analysis of 9 mitochondrion-encoded genes with introns showed that the nad4 transcript in the b438 mutant had a higher molecular weight than that in WT.q RT-PCR analysis of all the rice mitochondrial introns and amplication results of three nad4 introns indicated that the splicing of nad4 intron 1 was abolished in the b438 mutant.These data suggest that Os PORR1 affects the splicing of nad4 intron 1.8.Blue native polyacrylamide gel electrophoresis（BN-PAGE）showed that the accumulation of mitochondrial complex I in the b438 mutant was much lower than that in the WT,and NADH dehydrogenase activity was almost completely lost.The inner envelope cristae of mitochondria were well organized and surrounded by a dense stroma in the WT,whereas cristae development was poor and the stroma was thin in the b438 mutant.The ATP content and respiration rate were reduced in the b438 mutant compared with the WT.The expression of alternative oxidase（AOX）gene Os AOX1a and NADH dehydrogenase gene Os NDB2 and Os NDB3 was higher in the b438 mutant than in the WT.These data show that defective splicing of nad4 intron 1 affects the mitochondrial structure and function.9.Sequence alignment of nucleic acids showed that sequences of nad4 intron 1among rice,maize and Arabidopsis were conserved.Studies in Arabidopsis and maize showed that DEAD-box RNA helicase At ABO6（ABA Overly Sensitive 6）and PPR proteins Zm DEK35（DEFECTIVE KERNEL 35）,Zm DEK43,Zm EMP8（EMPTY PERICARP 8）and At MISF68（Mitochondrial Intron Splicing Factor 68）also affected the splicing of nad4 intron 1.The results of yeast two-hybrid（Y2H）assays showed that Os MISF68 interacted together with Os ABO6,Os DEK35,Os DEK43,Os EMP8,Os PORR1and itself.Os PORR1 is involved in the splicing of nad4 intron 1,which is indispensable for normal mitochondrial structure and function.Abnormal mitochondrial cristae and a reduced respiration rate in the b438 mutant led to a lower ATP content compared with that in the WT and ultimately affected seed development and viability.These results provide valuable clues for revealing the roles of PORR proteins in rice seed development and seedling growth.