Function Analysis Of Precocious Leaf Senescence Gene OsPLS1 In Rice

Leaf senescence is a normal and inevitable stage of plants during the process of growth and development, and it is affected and controlled by kinds of internal and external factors, such as, endogenous hormone levels, Leaf age, temperature, light, moisture, mineral nutrition and so on. Especially, SA plays an important role on regulating leaf senescence and disease response. By 60Co radiation treatment of indica cultivar Zixuan 1, an precocious leaf senescence mutant osplsl (oryza sativa precocious leaf senescence) was identified. Though physiological, cytological and molecular methods, we studied potential functions of OsPLS1 gene. The main results are the following.1. The phenotype of the osplsl mutantCompared to its wild-type, the ospls1 mutant did not display noticeable phenotypic abnormalities including leaf appearance at the early developmental stage (prior to the 4-5-leaf seedlings). However, the osplsl mutant exhibited lesion-mimics and early senescence after tillering, initially from the tips of the lower leaves, followed by exacerbated red-brown lesions rapidly spreading downward to cover the whole leaf blade except the upper 1-2 fully expanded leaves and heart leaves. At the jointing and early heading stage, all of the leaves except the flag ones of the osplsl mutant manifested early senescence. Relative to the wild-type, the osplsl mutant leaves were significantly withered at both the flowering and grain filling stages.2. Cytological observation of aspls 1 mutant leafSEM showed stomatal density was greater on the adaxial and abaxial surfaces of osplsl leaf; in addition, TEM revealed that chloroplasts had disintegrated in ospls/mutant leaf during the heading stage.3. ROS accumulation in ospls 1 mutantCompared with the wild type, ROS was seriously accumulated in the cells of mutant. NBT and DAB staining showed that there were more O2- and H2O2 in the cells of mutant.4. SA accumulation in ospls 1 mutantThe ospls1 mutant exhibited increased levels of the endogenous SA; exogenous SA inhibited the roots and shoots length of mutant; it also accelerated the senescence of detached leaves of mutant.5. SA pathwaysThe result from qRT-PCR analysis indicated that relative to the wild-type, mRNA expression of OsSGT1 encoding enzyme for SA metabolism was significantly lower, while mRNA levels of SA biosynthetic genes (OsPAL2 and OsPAL6) were significantly higher in the mutant of the seedling stage; most of the genes responsible for SA biosynthesis, including OsPAL1, OsPAL2, OsPAL6, OsPAL1 and OsPAL8 showed remarkably higher expression in the ospls 1 mutant than in the wild-type at the heading stage; what’s more, WRKY gene expressed more in osplsl mutant; these results indicated that OsPLS1 gene involved the SA pathways.6. Seed dormancy examination of osplsl mutant seedsThe results of seed germination showed that the germination rate of mutant was extremely low than wild type when they were completely submerged in the water; but both seeds of mutant and wild type could germination normally when they were decorticated or on the wetted paper or submerged in the water with oxygen supplying. The dormancy of mutant seeds may relate to the change of the micropyles.7. Gene complementation testTransformation of OsPLS1 gene into osplsl mutant rescued the precocious leaf senescence phenotype.