| Chloroplast is the major organelle for photosynthesis in plants,which is one of the most important organelles in higher plants.The chloroplast absorbs solar energy to assimilate carbon dioxide and water,synthesizes organic matter that stores energy,and simultaneously produces oxygen for plants.Thus,it’s worth investigating the differentiation,development and function of the chloroplast.There are more than 3,000 proteins in the chloroplast.The balance and homeostasis of these proteins are vital for the normally fundamental development of chloroplast.More than 95%of the proteins in the chloroplast are encoded by nuclear genes,they function together with those proteins encoded by the plastid to assemble a well-functional photosynthetic complex.The development of chloroplast is a complicated and precise process.The differentiation from pre-plastids to chloroplasts requires nuclear-plasma coordination.Any tiny mutation in those related genes might lead to abnormalities in chloroplast development.In this study,we isolated and identified a rice young seedling stripe（yss3）mutant,the absence of YSS3’s promoter resulted in white stripe leaves in seedling stage and the abnormal development of chloroplasts,low photosynthesis efficiency and declined agronomic traits.YSS3 encodes a chloroplast-located MORF（Multiple Organellar RNA editing Factor 9）protein,which plays an important role in the editing process of rice plastid genes and involving in chloroplast development.This study showed that YSS3 is essential for early chloroplast development in rice,providing the researching basis in the mechanisms of chloroplast development and diversity functions of MORF family protein.The main results are as follows:1.The phenotypic differences between the yss3 mutant and wild-type.The yss3 mutant exhibited white stripe leaves in seedling stage,and it turned green gradually during the tillering stage.Chlorophyll content reduced significantly and the relative expression levels of chlorophyll biosynthesis related genes were decreased in the yss3 mutant,which were consistent with the variegated phenotype.We next observed the chloroplast ultrastructure through transmission electron microscopy and found the chloroplast development was impaired in the mutant:in the white striped part,there was no typical thylakoid lamellar structure,vacuoles and eosinophilic granules were produced,and the shape and structure of the chloroplast were not normal;meanwhile,in the green striped part,the shape of the chloroplast was malformed,the thylakoid structure was obviously defected and vacuoles appeared,indicating that the chloroplast of the mutant was abnormal,and photosynthesis cannot be performed normally.2.YSS3 encodes a multiple organellar RNA editing factor.Genetic analysis indicated that the phenotype of yss3 is caused by a single gene stealth mutation.We proved that there was a large fragment deletion in the short arm of chromosome 8 in the yss3 mutant by fine-mapping,the expression level of the candidate gene Os08g0139100 was significantly reduced due to the absence of its promoter.Through transgenic complementary test we confirmed Os08g0139100 was the mutant gene YSS3 that caused the white stripe phenotype.YSS3 encodes a predicted chloroplast-located multiple organellar RNA editing factor 9 protein,which is highly homologous to MORF9 in Arabidopsis thaliana and has no annotated domains.We checked the expression pattern and sub-cellular localization of YSS3,it turned out YSS3 expressed constitutively and targeted to chloroplast.3.The impairy of YSS3 affected editing efficiency of numerous plastid editing sites,causing abnormal development of chloroplast.Based on the previous studies of MORF family protein in Arabidopsis thaliana and the chloroplast localization of YSS3,we checked the editing status of plastid genes by sequencing.The result came out that there were 10 editing sites showing significantly reduced editing efficiency in the yss3 mutant comparing to the wild-type,and 8 of them lead to animo acid change.These genes include rpoB encoding the PEP component,ndhB,ndhD and ndhG,who encode the chloroplast NDH complex subunits,and rpsl4 and rpl2 encoding two ribosomal proteins.In order to verify whether the chloroplast development was affected by the deviant editing status of these genes,we carried out the detection of mRNA and protein levels of chloroplast development-related genes,and found that the expression levels of the gene encoding PEP subunits were increased and the amount of protein was decreased.Meanwhile,the expression levels of genes dependent on PEP was down-regulated significantly,and the abundance of proteins was reduced either.Therefore,we hypothesize that due to abnormal editing status of plastid development genes,the invalid proteins were degraded by the proteasome,and the feedback regulation mechanism up-regulated the expression of genes encoded the PEP components,but it cannot always reach the wild-type level.This indicates that the chloroplast development of the mutant was arrested in the second step of chloroplast development（NEP transcribes plastid genes）,and the PEP complex may be abnormal,so the third step（PEP transcribes the nuclear genes of photosynthetic apparatus）cannot be performed normally.The deviant development of chloroplast triggered a series of downstream reactions like chlorophyll could not be synthesized normally,etc.4.YSS3 functions in a similar way to Arabidopsis MORF family.According to the study in Arabidopsis thailiana,the MORF family proteins play important but unknown roles as putative editosome components.To investigate whether the MORF protein YSS3 in rice has the same functions or not,we verified our hypothesis through yeast two-hybrid system.YSS3 interacted with other proteins of its own family and interacted with chloroplast-localized PPR proteins,which is consistent with studies in Arabidopsis.Therefore,we believe that YSS3 forms heterodimers with other proteins of the same family or functions independently,to ensure the completion of the editing process by interacting with PPR proteins. |
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