Map-based Cloning And Functional Study Of Rice Mutant Gene Wlpl Controlling White Leaf And Panicle

Rice is the most important food crops in the world. High yield is the most effective safeguard forfood safty and agricultural sustainable development. The photosynthesis is one of the predominantinfluences for rice yield determination,and the photosynthetic efficiency is decided largely by leaf color.Leaf coloration mutants are very important materials for photosynthesis research. At present, lots of leafcolor mutants were obtained from rice, the cause of leaf color mutant is largely due to loss-function ofrelated protein or abnormal expression of related genes, but the mutation on the translation system itselfis rarely reported. In this study, a mutant charaterized by white leaf and white panicle under lowtemperature was recovered among a population of regenerants from in vitro cultured tissue of thejaponica rice cv. Asominori, and some studies were done in physiology, cell morphological, map-basedcloning and functional analysis.1. The wlp1displayed low temperature sensitive chlorophyll deficiency. When grown underrestrictive temperature23℃，the wlp1shows albino phenotype at2nd leaf stage and3th leaf stage, butturned green from the4th leaf stage and appeared normal green like the leaves of parental Asominoriplants before the panicle appeared albino at heading stage. The contents of chlorophyll a, chlorophyll band total chlorophyll were drastically lower than those in the parental Asominori plants at L3andheading stage. Ultrastructural changes in the chloroplasts were also compared between the wlp1mutantand wild-type plants L3leaves under C23and C30using TEM. The result shows that leaf chloroplastsof wlp1plants were fewer in number and smaller in size and more detail the chloroplasts appeared lesswell developed with no well structured lamellar structure and more OG. When grown under30℃condition, no great differences were found in phenotype, chlorophyll content and chloroplastultrastructure. In field condition, the mutant flowered slightly later and was taller than the wild type.2. The phenotype of white leaf and panicle under low temperature in the wlp1mutant is controlledby a single recessive nuclear locus by genetic analysis. To map the wlp1locus, a F2mapping populationderived from a cross between wlp1and the cultivar Nanjing11was generated and wlp1was mapped toan interval between marker RM1095and RM8097on Chromosome1. To narrow down the region for acandidate gene affected in wlp1, the new developing Indel markers and a larger F2mapping populationconsisting of1100segregants showing the wlp1mutant phenotype were used for fine-mapping. Thewlp1locus was finally located in a17.5-kb DNA region between the Indel markers In15and In12,P0481E12. Within this region, three opening reading frames (ORFs) were predicted and encode50Sribosome protein L13, heat shock factors and trehalose synthase, respectively. To define the molecularlesions of wlp1mutant, the three ORFs were sequenced and only the1st ORF carries a single nucleotidemutation(C to T), resulting in an amino acid change from Thr-80to Ile. Therefore, we tentativelydesignated the1st ORF as the WLP1gene, which was subsequently verified by Complement test andRNA interference experiment. 3. A comparison between the WLP1gDNA and cDNA sequences showed that the gene comprisedfour exons and three introns. The predicted WLP1protein was a228residue polypeptide of molecularmass~26kDa. Its C-terminal region showed some structural similarity to the50S E. coli ribosome L13protein large subunit, and its N-terminal56residues were characteristic of a chloroplast transit peptide.The WLP1sequence as a whole also has homologs among rice, zea may, Arabidopsis thaliana, Spinaciaoleracea and so on, but particularly with sequences encoded by monocotyledonous species such asmaize and sorghum.4. Real-time PCR analysis showed that WLP1most strongly transcribed in leaves and less in leafsheaths, young panicles,culms and root at the booting stage, with a low level of expression detected inroot, corresponding to the results revealed by histochemical staining of the transgenic plants.Subcellular localization of WLP1protein was localized in the chloroplast. WLP1transcript abundancein L3seedlings was higher under C23than under C30conditions. In the latter environment, WLP1inthe wild type (and similarly wlp1in the mutant) was most strongly transcribed in the newly emergingthird leaf and much less with leaf development. The same profile applied to WLP1in wild type plantsexposed to C23, but in the mutant, the peak abundance of wlp1transcript was delayed. The chlorophyllsynthesis genes such as HEMA1,CAO1, DVR, YGL1and chloroplast development genes such as V1, V2and OsPPR1were all up-regulated under C23conditions, while the expression levels ofphotosynthesis-related genes such as cab1R, cab2R, psaA, psbA and RbcL were significantly reduced inthe wlp1mutant. However, The transcription abundance of all these genes, except for cab2and psaA,was the same in the mutant and the wild type plants grown under C30conditions. The rates of Chlaccumulation was slowed in the wlp1mutant. The expressions levels of RNA polymerase-related genes,rpoA, rpoB and RpoTp, were markedly up-regulated in the wlp1mutant (especially under C23conditions). These results suggest that WLP1play a role in the early stages of leaf development. Thetranslation in chloroplast was damage caused by the mutation under low temperature conditions. Theupstream gene was overexpressed by feedback regulation and downstream genes were down-regulatedby the damage of transcription/translation system.