Positional Cloning And Function Analysis Of White Stripe Leaf 8(WSL8)in Rice

Chloroplasts are semi-autonomous organelles that contain their own transcription and translation systems.Plants mainly perform photosynthesis in chloroplasts,and converting light energy into chemical energy and providing energy-material ATP for organism survival.Therefore,the normal differentiation and development of chloroplasts is essential for plant growth.As one of the apparent traits of rice,leaf color affects photosynthesis of rice and affects yield and quality.The leaf color mutant can be used as an ideal material for studying plant physiological processes such as photomorphogenesis and photosynthesis,as well as for plant hormone physiology and plant pathology research and high-efficiency breeding,and also as a marker trait for cross breeding.Therefore,it is of great theoretical significance and actual production value to continuously discover new rice leaf color mutants and carry out gene cloning and functional research.In this study,a longitudinal white stripe leaf mutnat wsl8 was isolated from indica rice cultivar(cv.)9311 afer Ethyl methanesulfonate mutagenesis.A new deoxyribonucleoside kinase gene was identified by map-based cloning and the phenotypic characterization and functional analysis were carried out.The main results are as follows:(1)The wsl8 mutant showed a white stripe phenotype before four-leaf stage,and the phenotype in three-leaf stage was the most obvious.Newly developed leaves starting from the four-leaf stage displayed normal green color.Transmission electron microscopy(TEM)analysis showed that the chloroplasts in the wild type leaves were normal.There were two types of chloroplasts in the wsl8 mutant:one was similar to the wild type,but the thylakoids were loosely arranged;the other could not form a lamellar structure.qRT-PCR analysis indicated that the expression of chlorophyll synthesis and chloroplast development related genes were significantly down-regulated in the wsl8.In addition,the plant height,panicle length,1000-grain weight and seed setting rate in wsl8 decreased significantly compared with wild type.(2)Genetic analysis indicated that the wsl8 mutation was controlled by a single recessive nuclear gene.The WSL8 locus was mapped to a 22.9 kb interval between the markers vf2 and dl on the long arm of chromosome 5,which contains three open reading frames(ORF).A single base mutation from C→G occurred in LOC_Os05g35520 by whole genome sequencing analysis,resulting in the replacement of leucine(Leu)by valine(Val).LOC_Os05g35520 encodes a deoxyribonucleoside kinase protein family and is a key enzyme in the nucleotide salvage synthesis pathway.Phylogenetic analysis showed that the WSL8 protein was highly conserved in most monocotyledons.It was confirmed by complementation,CRISPR knockout and overexpression experiments that the LOC_Os05g35520 was the WSL8 gene.(3)wsl8 is a temperature-sensitive type and exhibited more serious phenotype at low temperature.WSL8 is localized to chloroplast and is constitutively expressed in various tissues.It is highly expressed in young leaf and panicle,and with a low expression in mature leaf,stem,root,and sheath.The GUS staining results were consistant with qRT-PCR results.Tissue expression analysis showed that the recovery of phenotype of wsl8 mutant at late stage is due to a significant down-regulation of OsdNK1(WSL8)and a significant up-regulation of OsdNK2(LOC_Os01g65350).(4)WSL8 encodes a deoxyribonucleoside kinase that plays an important role in the salvage pathway of nucleotides.Exogenous addition experiments showed that the dNK mainly performs the function of thymidine kinase and catalyzes the formation of thymidine monophosphate from thymidine.Enzyme activity assay showed that the enzyme activity in the mutant decreased significantly,resulting in insufficient synthesis of dTTP,which ultimately disturb the balance of the dNTP pools.Therefore,WSL8 plays an important role in the normal development of chloroplasts and the normal growth of rice plant.