Map-based Cloning And Functional Analysis Of A Chlorotic Seedling Gene CS3 In Rice(Oryza Sativa L.)

As one of the vital biological process in higher plant,photosynthesis generates carbohydrate and oxygen.The organelle which responds for photosynthesis is chloroplast,and the photosynthetic pigments within it play an important role during photosynthesis.Abnormal accumulation of photosynthetic pigment and unusual chloroplast development caused by gene mutation will change the color of plant leaves,which could finally generate leaf-color mutant.And leaf-color mutants are considered as ideal materials to study the chlorophyll synthesis,chlorophyll degradation,and chloroplast development.We obtained a chlorotic seedling mutant from an ethyl methane sulfonate-?EMS?-derived mutant population in the“Jiahe212”background,which we named as cs3?chlorotic seedling 3?.Then characterization,map-based cloning,and functional verification and analysis were performed,and the main results are showed below:1.cs3 mutant seedlings displayed a severe chlorotic phenotype since germination,and persisted throughout the seedling stage.Additionally,small brown necrotic spots appeared on the leaf and leaf sheath of the cs3 mutant seedlings,and then the cs3 plants withered and died.Photosynthetic pigment content analysis showed that the content of chlorophyll a in cs3 mutant was barely detectable except at 5DAG stage,while there is no chlorophyll b in any stage.And the content of carotenoid in cs3 mutant was significantly lower compared with WT.2.The ultrastructure analysis of chloroplast showed that the development of chloroplast in cs3mutant was abnormal and there was more plastoglobuli accumulation.And the cs3 chloroplast seemed to undergo degradation.In addition,H₂O₂ accumulation could be found in leaf from cs3 mutant.3.Transcriptional level analysis in cs3 mutant revealed that the expression of genes which are involved in chlorophyll biosynthesis are changed in different manner;and the expression of genes involved in chlorophyll degradation,and photosystem and light harvest complex building are up-regulated,except Lhcb4;and except a negative regulator DOS,the accumulation of transcripts of genes associated with senescence were significantly higher in cs3 mutant;while genes associated with chloroplast development expressed significantly low in cs3 mutant.4.Through fine mapping,the CS3 gene was mapped to a 92Kb region on the long arm of chromosome 3 between marker CA40 and CA42,which contains 14 ORFs.Sequence alignment showed that in cs3 mutant there was a base substitution?C to T?in the second exon of ORF12?LOC_OsO3g21370?leading to an amino acid change from Ser to Phe.The ORF12 encodes a Ycf54 domain containing protein which has no detailed annotation.Multiple amino acid sequence alignment indicated that the Ycf54domain is conserved among many species.Furthermore,the mutant site of ORF12 protein in cs3 mutant is quite conserved.5.The complementary,over-expression,and knock-out analyses were performed.And the result suggested that the mutation of ORF12 is responsible for the phenotype of cs3 mutant.Moreover,the phenotype of RNAi lines exhibited pale-green leaf phenotype,and the phenotype was partly recovered during development.6.The subcellular localization of CS3 is chloroplast.However,the S136F mutation of CS3 protein may affect its subcellular localization.The fluorescent signal exhibited spot shape and could not gathered within the chloroplast just as the CS3-GFP.They seemed like around the chloroplast.Moreover,the S136F mutation of CS3 protein also affect its interaction with YGL8 protein in yeast system.7.The up-regulated expression of YGL8 gene was found in cs3 mutant.And the content of proto IX and Mg-proto IX are significantly higher than they in the wild type,while the accumulation of pchlide is down-regulated.It indicates that the chlorophyll biosynthesis pathway in cs3 mutant is blocked which may partly explain the chlorotic phenotype of cs3 mutant.