Map-based Cloning And Function Analysis Of Leaf Early Senescence Gene OsPLS2 In Rice

Leaf senescence constitutes the final stage of leaf development. However, premature aging of functional leaves lead to yield reduction and quality decline. Thus, it is very important for development of novel crop germplasms with delayed leaf-senescence character through investigating the molecular mechanism of leaf senescence.In this study, a leaf early senescence mutant ospls2 (OOryza sativa premature leaf senescence 2, ospls2) from Rice Variety 7954 was obtained by EMS. Through map-based cloning technology and other related physiological, cytological and molecular methods, we successful separated target gene OsPLS2 and studied potential gene functions.Main results are as follows:1. Phenotype and main agronomic traits of mutant ospls2The early symptoms of the premature senescence mutant, started at tillering stage as rusty spots in leaf tip, which spread rapidly to leaf joint and lead to leaf decay. Compared with wild type 7954, mutant ospls2 exhibited decreased TKW (Thousand Kernel Weight), ripening rate and NFGP (number of filled grains per panicle).2. The response analysis of mutant ospls2 due to H2O2 and ABA treatmentResults suggested that growth of mutant ospls2 seedling were seriously restrained after treatment with 80 mM H2O2 and 0.25μM ABA. Plant height and root length of mutant ospls2 were decreased and were obviously shorter than wild type. After treatment with H2O2, plant height were reduced by 27.44%. When treated with ABA for 6 days, plant height and root of mutant ospls2 dropped by 19.08% and 20.08% correspondingly. We also analyzed the responses of detached mature leaves to 150 mM H2O2 and 500μM ABA solution. The assay demonstrated that the leaves of mutant ospls2 were more sensitive to both treatment than wild type leaves.3. The molecular analysis of mutant ospls2 related leaf early senescenceThe molecular analysis about ABA anabolic genes determined that those genes responsible for ABA biosynthesis, including OsNCED1, OsNCED3, OsNCED4 and OsNCED5 showed remarkably higher expression in the ospls2 mutant than in the wild-type at the heading stage; while the expression of those ABA metabolism related genes in ospls2 mutant, including ABAox2 and ABAox3 were significantly lower. These finding suggested that higher ABA concentration in ospls2 mutant, leading to earlier leaf senescence and more sensitive to exogenous ABA treatment.4. Gene mapping, sequence and function analysis of OsPLS2We fine mapped this gene into an interval of 121 Kb between RM15363 and RM15361 on rice chromosome 3. Through selection and sequence analysis of candidate genes, a gene LOC_Os03g38990 was found two one single base G substituted by A in ORF. This change leads frame shift mutation. Furthermore, the genes function was certified by genetic complement experiment. Meanwhile, OsPLS2 gene annotation and its amino acid sequence analysis indicated that the OsPLS2 protein is a UFP1 like protein. The subcellular localization of OsPLS2 showed that this gene is expressed in the cytoplasm and plasma membrane as well as UPF1.