Cloning And Functional Analysis Of PALE-GREEN LEAF 12 In Rice

The chloroplast evolved from endosymbiotic is a place for photosynthesis of plants,providing energy for the growth and development of plants.Although spatially,the chloroplast has a genetic system independent of the nucleus,the operation of the system is controlled by the nucleus.The PPR proteins,encoded by nuclear genes,regulate the expression of organelle by participating in organelle RNA metabolism.In rice（Oryza sativa L.）,491 genes are predicted to encode proteins.However,most of their functions are still unknown by far.In our study,we identified a rice leaf colour mutant,pale-green leaf（pgl12）,with a background of ZH11 through the genetic screening of the EMS mutagenesis mutant library.At the seedling stage,the pgl12 mutants exhibited yellow-green leaves,and gradually became pale-green as the plant grew.The gene PGL12was cloned by map-based cloning after the investigation of agronomic traits and genetic analysis,and its possible molecular biological functions were analyzed.The main results are as follows:1.The chlorophyll content in leaves of the mutant pgl12 was significantly decreased,especially at the seedling stage,and the number of chloroplasts in the mesophyll cells of the leaves was less than that of the wild type ZH11.Although the thylakoids in pgl12were visible,but its arrangement was irregular and the layers were unclear.Temperature treatment experiments showed that pgl12 is a temperature-sensitive mutant with a severely yellow phenotype at 35°C,and albino at 20°C.There were only a few chloroplasts,most of which were damaged and showed an irregular arrangement with empty lamellae.These results indicated that the PGL12 protein is essential for chloroplast development.2.The photosynthetic rate of mutant pgl12 was significantly lower than that of wild type ZH11.The field agronomic traits investigation showed that several important traits related to grain yield such as the number of tillers,plant height,panicle length,number of primary branches,number of secondary branches and number of grains per panicle were significantly reduced in pgl12,which indicated that the mutation of PGL12 resulted in a significant decrease in grain yield.3.Genetic analysis of reciprocal crosses indicated that the pale-green leaf phenotype of the mutant pgl12 was controlled by a single recessive nuclear gene.F₂ obtained from a cross between NJ06,as the male parent,and pgl12 was used as the localization populations.PGL12 was located within the physical distance range of about 100Kb between the newly developed molecular markers YS22 and YS24 on rice chromosome 12.There are 14 open reading frames（ORFs）within the range.By sequencing alignment,it was found that the eighth ORF of the mutant pgl12 existed a single base mutation（C→T）at position 1681 of the LOC_Os12g10184 gene,which resulted in a premature stop codon.Transgenic complementation revealed that LOC_Os12g10184 is indeed PGL12,and that its mutation gives rise to the pgl12 phenotype.4.PGL12 encodes a PLS PPR protein composed of 17 PPR motifs,which is expressed in all tissues of rice.Among them,the expression in leaves is the highest,and the old leaves are larger than the new ones.Subcellular localization showed that PGL12targets the chloroplasts.Expression analysis of genes related to the chloroplast genetic system showed that NEP-dependent genes were significantly up-regulated in the mutant pgl12.Except the 16S rRNA gene,almost all other detected genes related to translation devices were also up-regulated.These results suggest that PGL12 protein is a new PPR protein whose mutation affects the expression of genes related to chloroplast development.5.RNA gel blot experiments revealed that a large amount of unprocessed 16S rRNA precursor accumulated in the mutant pgl12,which indicated that PGL12 protein was necessary for the processing of 16S rRNA.6.RT-PCR was used to analyze the RNA editing of 23 sites and splicing of 18introns in chloroplasts.It was found that the RNA editing of the mutant pgl12 was similar to that of wild type ZH11 and was not affected,but the splicing efficiency of ndhA gene was significantly reduced,indicating that PGL12 protein was involved in the regulation of the splicing of chloroplast gene ndhA transcripts.7.The yeast two-hybrid experiments of PGL12 protein interacting with other three proteins OsPPR4,WSL4 and WSP1 that have been reported to affect ndhA splicing in rice showed that PGL12 only interacted with WSP1,suggesting that splicing of ndhA may be mediated by a complex.