Map-based Cloning And Functional Analysis Of A White Stripe Leaf Gene WSL4 In Rice(Oryza Sativa L.)

Chloroplasts are not only the place of photosynthesis in plants,but also the arena for metabolites product biosynthesis and accumulation.Although the chloroplasts have their own genome,the expression and regulation of chloroplast genes are under the control of the proteins encoded by nuclear genes.PPR proteins are one of the most important proteins involved in post transcriptional RNA processing.PPR proteins are characterized by the tandem arrays of PPR motifs,which compose of 35canonical amino acids and commonly exist in the plant kingdom.The mysterious gene family has greatly expanded in higher plants,with 450 members in Arabidopsis and 477 members in rice.PPR proteins were mainly localized in chloroplast or mitochondria or both of them,and required for RNA editing,splicing,stability,and translation in plastid development.Although many of the PPR proteins were found,the regulatory mechanism of PPR involved in post transcriptional processing of chloroplast genes remains unclear.Here,we isolated and identified a rice white stripe leaf4?wsl4?mutant from a⁶⁰Co-irradiated mutant pool of japonica cultivar RX69.The wsl4 mutant develops white-striped leaves during early leaf development,characterized by decreased chlorophyll content and malformed chloroplasts.We isolated corresponding gene WSL4 using map-based cloning and further explored its biological function.Research results are summarized as follows:?1?The wsl4 mutant shows the developmental defect of chloroplasts.TEM analysis revealed the while striped sectors of the wsl4 mutant seedlings preferred to produce abnormal chloroplasts,which lacked any organized lamellar structures.The wsl4 mutant is sensitive to low temperature conditions.The degree of bleaching phenotype in wsl4 mutant was greatly influenced by low-temperature exposure,and the chlorophyll content of wsl4 mutant was undetectable under constant 20??C20?.?2?The wsl4 phenotype was inherited by a single recessive nuclear mutation.Based on 978 F₂recessive homozygous wsl4 background individuals,the WSL4 locus was ultimately narrowed down to an 86 kb genomic region.Comparison with wild-type cDNA sequences revealed a 2 bp deletion at position 1,330 and 1,331 bp from the ATG start codon in LOC_Os02g35750.The complementation analysis and RNAi showed that LOC_Os02g35750 indeed corresponds to the WSL4 gene.WSL4protein,which contains a tandem repeat of 12 PPR motifs and belongs to a member of the P subfamily,is a chloroplast nucleoid-localized protein.WSL4 was expressed in all tested tissues and significantly high in young leaves.The accumulation of WSL4 transcript was enhanced under low temperature condition.?3?WSL4 influences not only the expression levels of genes associated with photosynthesis and Chl biosynthesis but also the expression of plasmid encoding gene and proteins.qRT-PCR results show that the expression levels of several key genes associated with photosynthesis and Chl biosynthesis?cab1R,cab2R,rbcS,CHLD,CHLI,CHLH,and PORA?were significantly down-regulated in the wsl4 mutant.The expression of PEP-dependent Class I genes?e.g.psaA,psb A,and rbcL?was significantly down-regulated,while expression of NEP-dependent Class ?I genes?e.g.rpoB,rpo C1,and rpoC2?was significantly up-regulated in the wsl4 mutant.We assessed the accumulation of core subunit of photosynthetic enzyme complex in wild type and wsl4mutant,including photosystem I subunits?PsaA?,photosystem ??PsbA?,RNA polymerase subunits?RpoB?,and cytochrome b₆f?PetD?.The assayed proteins were undetectable in the wsl4 mutant compared to wild type except RpoB.?4?WSL4 functions in the splicing of chloroplast group ? introns gene atp F,ndhA,rpl2,and rps12.We detected RNA splicing events of chloroplast genes containing 17 group ? introns and one group I intron and found four chloroplast transcripts containing group ? introns were spliced with very low efficiency in the wsl4 mutant compared with wild type.The splicing defects were greatly rescued in complemented transgenic plant but were mimicked in RNAi transgenic plant.Those results were further verified by q RT-PCR and northern blot.