| Chloroplast is the main organelle for photosynthesis in plants.Rice leaves are the main sites for photosynthesis and the production of organic matter.Chloroplast is mainly in the plant leaves and the development of chloroplast closely related to leaves developmental.Therefore,we can dig out more genes about chloroplast development and ready for construct chloroplast regulation network.Futher more,this can provide theoretical basis for practice.The appearance of white fine stripe in rice leaves had a great effect on the photosynthesis and dry matter accumulation.Although there are a large number of leaf color genes were cloned,but the cloning and functional study of white fine stripe leaf gene is rarely reported.Here,we cloned a gene WFSL1(White Fine Stripe Leaf 1),which encodes a HD domain-containing metal-dependent phosphohydrolase that regulates the development of rice chloroplast.A point mutation in WFSL1 leads to white fine stripe phenotype,and the wfsl1 chlorophyll content obviously decreased compared to the wild type.For the white fine stripe phenotype,we confirmed the function of WFSL1 by cell biology,biochemistry,molecular biology,transcriptome sequencing and protein expression.The results are as follows:1.The wfsl1 mutant had no white fine stripe phenotype in seedling stage,and white fine stripe phenotype was observed in the newly released leaves until tillering stage and continued to the mature stage.Leaf cross section observation and chlorophyll content determination showed that the chlorophyll content of wfsl1 mutant was obviously decreased,compared to the wild type.2.Transmission electron microscopy analysis revealed that wfsl1 leaves were composed of three types of cells: green,green and white,white.The number of chloroplast and thylakoid reduced,or even no thylakoid structure of wfsl1 white cells.The results indicated that the chloroplast were abnormal in wfsl1 mutants.3.The agronomic traits analysis revealed that the wfsl1 plant height and panicle length was shorter than that of the wild type.The number of primary and secondary branches,seed setting rate and the 1000 grain weight decreased in wfsl1 mutant.In addition,the photosynthetic rate of wfsl1 mutant was significantly lower than that of the wild type.4.The white fine stripe is controlled by recessive unclear gene,which is located in 100 kb between markers gcw56 and gcw24 on the short arm of chromosome 1.Gene predicting and sequencing revealed that encoding region of LOC_Os01g01920 had a "A" to "T" nucleotide substitution.The complementary and overexpression vector were constructed and transferred into mutant,which proved that the white fine stripe phenotype was caused by the mutation of WFSL1.5.Bioinformatics analysis showed that the WFSL1 protein had a HD-domain and the activity of phosphatase.6.Real-time PCR experiments revealed that WFSL1 was highly expressed in mature leaves and sheath.The WFSL1 protein is localized to the chloroplast.7.Transcriptome sequencing analysis revealed that the expression of genes related to chloroplast development was down regulated,and the metabolic pathway of carbon and glucose was abnormal of wfsl1 mutant.8.Western-blot assay revealed that the expression level of WFSL1 protein was significantly decreased in the wfsl1 mutant,and phosphatase activity analysis showed that the phosphatase activity significantly decreased for the mutation of WFSL1.The expression level of chloroplast encoded RNA polymerase gene was down regulated in wfsl1 mutant and the expression level of protein was also decreased,but the expression of nuclear encoded RNA polymerase gene was not affected.9.The expression of chloroplast ribosomal subunits gene was down regulated,and the expression level of protein was significantly decreased in wfsl1 mutant,indicated that mutation of WFSL1 affects the transcription or translation of chloroplast ribosomes,resulting in abnormal chloroplast development... |
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