P450 Protein ES2 Is Involved In Leaf Senescence And Heat Stress In Rice

Rice(Oryza sativa)is one of the most important cereal crops in the world and increasing rice yields and cultivating high-quality varieties are of great significance to social stability and economic development.As one of the six major organs of plants,leaves sustain the dorminant role to produce carbohydrate by photosynthesis.During the phase transition of plant from vegetative to reproductive stage,leaf senescence are naturally happened.Leaf senescence involves a coordinated action at the cellular,tissue,organ,and organism levels and has a significant impact on crop yields and grain quality.Therefore,it is important to explore the molecular mechanism of leaf senescence.Several previous studies had made a forward in understanding of leaf senescence,however,the molecular mechanism of leaf senescence is still not clear so far.In this study,we isolated an early senescence mutant,called as early senescence2(short to es2),by EMS mutagenesis and cloned ES2 gene by Map-based cloning approach.We analyzed the physiological characteristics of the es2 mutant and explored the basic role of ES2 to affect rice leaf senescence and stress tolerance.The results are as follows:1.The es2 mutant gradually appeared spotted leaves after three-week-old seeding with the entire growth cycle.It showed a phenomenon that the es2 mutant displayed premature senescence in the later stages of vegetative growth.The results showed that the spike length of the mutant was shorter than wild type and the numbers of primary branch and secondary branch decreased.The chlorophyll content of the mutant was significantly lower than that of the wild type and death cells appeared in the leaves of the es2 mutant.2.We obtained the ES2 by Map-based cloning.We found there were 135 bp bases deletion in the second exon of ES2 in the mutant resulting in the deletion of 45 amino acids.3.Bioinformatics analysis showed that ES2 encodes a protein of the cytochrome P450 family,and the promoter region contains several stress-related elements,suggesting that ES2 may be associated with stress reaction.Tissue expression profiling analysis showed that ES2 was expressed in all tested tissues,and with the highest expression level in leaf sheath.4.The activities analysis of antioxidation enzyme,including SOD,POD,CAT and APX,revealed that SOD and POD showed obvious decrease in es2 mutant.In addition,ROS analysis by DAB staining showed that the mutant leaves were stained with brown spots.Meanwhile MDA(malondialdehyde)content analysis revealed that MDA in the es2 mutant is more than wild type.In summary,the accumulation of reactive oxygen species in the es2 mutant may be one of the reason of leaf senescence.5.The expression analysis of chlorophyll degradation-related genes(CDGs)and senescence-associated genes(SAGs)showed that the expression of several SAGs changed significantly in es2,which indicated that ES2 delay leaf senescence through regulating the expression of SAGs.6.Under high temperature treatment,the es2 mutant displayed more strong phenotype than wild type,including curling leaf and decreased survival rate.Additionally,the ES2 expression level increased obviously under high temprature and the expression of several heat shock proteins was upregulated earlier in the mutant.Therefore,ES2 is involved in high temperature tolerance.