Map-based Cloning And Functional Analysis Of Albino To Lethal Leaf Gene OsPPR6 In Rice(Oryza Sativa L.)

Chloroplast is one of the most critical components of higher plants,who is the only that can absorb solar energy into chemical energy for the body to maintain organelles life.Thus chloroplast differentiation,development and function are fundamental importance to all life forms on the earth.Differentiation from pro-plastids into chloroplasts is an elaborate process which needs the coordinated expression of chloroplast-and nuclear-encoded genes because most chloroplast proteins(more than 95%)are encoded by the nuclear genome and imported into the chloroplast,where they are assembled together with the chloroplast-encoded proteins to form functional photosynthetic and metabolic complexes.Thus any related gene mutation may lead to poor chloroplast development resulting in varying types of leaf color variation.In this study,we identified and cloned a new PPR protein OsPPR6,the protein plays a crucial role in early for chloroplast development in rice.A single nucleotide substitution in OsPPR6 resulted in albino leaves in seedling stage,accompanied by abnormal chloroplast development,low photosynthesis efficiency and reduced chlorophyll content.The osppr6 mutant seedlings are lacking of power,leading to death.OsPPR6 is a protein located on the chloroplast,involving in chloroplast genes editing ndhB and splicing ycf3.This study showed that OsPPR6 is essential for early chloroplast development in rice,providing the researching basis in the mechanisms of chloroplast development and diversity functions of PPR family protein.The main findings are as follows:1)The phenotype difference between the osppr6 mutant and the wild-type is that the mutant seedlings emergence albino leaves in early seedling stage,and wither to dead in the two-leaf stage(about 15 days).3%sucrose treatment found that the osppr6 mutant seedlings changed to yellow at the tip of leaf,gradually turn green,indicating that the mutant is invalid of photosynthesis,then the energy supply shortage causes death.Observing chloroplast by TEM:The osppr6 mutants have no typical chloroplast thylakoid lamellae,but form many bubbles,or a huge vacuole and swollen thylakoids in it,or osmiphilic bodies.No photosynthesis could take place in abnormal of chloroplasts shape and structure.Analysis of genes expression indicated that chlorophyll biosynthesis-related genes were significantly decreased,and the plastid biosynthesis-related genes expression levels were abnormal.In osppr6 mutants,PEP-dependent plastid gene expression decreased obviously,and NEP-dependent plastid genes increased significantly.Western blot analysis showed the consistent results with the gene expression.This shows that chloroplast development is stuck in the second period.DAB and NBT staining found that the osppr6 mutants accumulate excess ROS.2)Genetic analysis showed that osppr6 is a single recessive gene mutation.By map-based cloning we identified a gene who a single base substitution produced mutant at a conservative amino acid position.The target gene is one of PPR protein family member,which is exist in major higher plants and has a relatively conserved amino acid sequence in homologous genes.OsPPR6 share homology with Arabidopsis protein AtECB2 encoded by AT1G15510,which is essential for early chloroplast biogenesis across their entire lengths(50%amino acid identity).Phylogenetic analysis was performed using NCBI BLAST for OsPPR6 and the homologous amino acids sequences to determine their evolutionary relatedness.We found that this type of PPR proteins were very conservative in dicotyledons and monocotyledons,and could be obviously divided into two categories.OsPPR6 belongs to PLS subfamily,containing 14 PPR motifs DYW subpopulation protein.By complementation and RNAi transgenic,we indeed verified OsPPR6 mutation resulted in lethal albino phenotypes.3)OsPPR6 functional analysis:Lessons basis of previous studies,through directly sequencing and RT-PCR found that only ndhB was different in WT and osppr6 mutant.The 737-bp of ndhB changed C to T,which implied that the editing site event did not occur in the osppr6 mutant.We carried out direct RT-PCR and compared the length of the amplified products between the WT and osppr6 mutant.We found that chloroplast transcript ycf3(photosystem I assembly protein)was spliced with very low efficiency in osppr6 mutant seedlings as compared to the WT.OsPPR6 may affect ycf3 splicing efficiency.