Map-based Cloning Of SPP46 Gene Regulating Grain Number And Functional Characterization Of Green-revertible Albino Gene GRA78 In Rice

1.Map-based cloning of SPP46 gene regulating grain number in riceGrain number per panicle,which is one of the three major factors of rice yield,is mainly determined by the number of primary branches,secondary branches and grain density.In order to explore the regulator grain number per panicle in heavy-panicle indica sterile line G46 A,we carried out an ethyl methanesulfonate(EMS)mutagenesis in indica maintainer line G46 B,and a sparse panicle mutant 46(spp46)was isolated.In this study,phenotype identification and gene cloning of spp46 were performed,and the main results are as follows:In spp46 mutant,the number of branches,grain number per panicle and grain density were significantly reduced.Genetic analysis showed that the sparse panicle phenotype of spp46 is controlled by a single recessive nuclear gene.The gene locus responsible for the spp46 was finally localized in a 126-kb region between In Del markers I3 and I7 on the short arm of chromosome 3.In this region,only one gene was mutated,making a single nucleotide substitution(G-to-A)at position 674 of the DNA sequence(corresponding to position 340 in c DNA),and causing in an amino acid change from Glu to Lys in the encoded protein.Therefore,this gene was identified as the candidate gene of spp46 and designated tentatively as SPP46.Furthermore,we verified the candidate gene by functional complementary and CRISPR/Cas9-based knockout strategies.Transgenic lines spp46-C showed dense panicle phenotype,and numbers of allelic mutants generated by CRISPR/Cas9 in ZH11 or Nipponbare background by knocking out the candidate gene displayed sparse panicle phenotypes as spp46.These results suggested that the sparse panicle phenotype of spp46 was caused by the mutation of SPP46.The expression pattern analysis showed that SPP46 was constitutively expressed.The subcellular localization results indicated that the SPP46 protein was localized to the plasma membrane and Golgi.GUS staining assay showed that SPP46 expressed strongly in young panicles.RNA-seq assay revealed that in spp46,numbers of differentially expressed genes(DEGs)in young panicles were five folds higher than that in leaves,and these DEGs were most significantly enriched in plant hormone signal transduction pathway,especially in IAA and JA.q RT-PCR analysis confirmed expressions of these DEGs in spp46 were significantly reduced.Besides,we detected IAA and JA contents in spp46 and found significant difference compared with the wild type.In addition,the transcriptional levels of many genes that are related to grain number per panicle and panicle architecture were affected in spp46.In summary,our results suggested that SPP46 might be involved in IAA and JA signal transduction pathways,and affected the panicle formation.2.Functional characterization of green-revertible albino gene GRA78 in riceRice leaf color mutants are important materials for studying chloroplast formation and development,chlorophyll biosynthesis and degradation,and molecular mechanism of photosynthesis in rice.In this study,rice green-revertible albino mutant gra78 was isolated from japonica cultivar Nipponbare by ethyl methanesulfonate(EMS)mutagenesis.Based on the previous reseraches,we systematically identified the phenotype of gra78 and analyzed the function of candidate genes of GRA78.The main results are as follows:The second and third leaves of gra78 mutant displayed albino phenotype during the normal rice planting season,but the mutant gradually turned green from the four-leaf stage.Nevertheless,gra78 still showed pale-green leaves that could be distinguished from the wild type plant at the late development stage.Photosynthetic pigments measurement results showed that the contents of these pigments in gra78 were severely reduced than those of the wild type at the three-leaf stage,respectively.After regreening,the levels of these pigments increased in the mutant,but were still significantly lower than those of the wild type at the tillering stage and the heading stage.Transmission electron microscopy analysis results showed that in gra78,chloroplasts were hardly found in mesophyll cells,and thylakoids were completely lacked in only a few visible chloroplasts at the three-leaf stage.However,the gra78 chloroplasts had well-developed grana stacks and thylakoids similar to the wild-type chloroplasts at the tillering stage.Temperature-and light-sensitivity experiments found that gra78 displayed severe albino phenotype at the second and third leaves under 23°C condition,and could turn green from the four-leaf stage onward.However,gra78 exhibited only pale-green color at the second and third leaves under 30°C condition.On the other hand,gra78 exhibited the same albino phenotype at the early seedling stage under both short-day condition and long-day condition at 23°C.These results suggested that gra78 was a temperature-sensitive green-revertible albino mutant,but the albino phenotype of gra78 was independent of photoperiod.Complementary transgenic lines showed normal leaf color throughout the whole growth period,and whose pigment contents were recovered to the wild-type level.Therefore,the data confirmed that the single nucleotide mutation in GRA78(LOC?Os01g59920)is responsible for the albino phenotype of gra78.GRA78 is a cysteine synthase isoform gene,and the subcellular localization of GRA78 protein was localized to the chloroplast.GRA78 was constitutively expressed in all tissues detected in wild type parent,but was the highest expression in the leaf blades at booting stage.qRT-PCR analysis showed that the expression levels of the mutated gene(gra78)were dependent on both temperature and developmental stages.Besides,a negative regulation could exist between GRA78 and its homologous genes in the transcript levels.In addition,the mutation of GRA78 could reduce the expression levels of some genes associated with photosynthesis.