The Effects Of Nitric Oxide On Anti-oxidative Enzymes During Leaf Senescence And Leaf Proteome In Cotton

To study the mechanism of early senescence and the effect of NO on leaf proteome in short season cotton, 2 cultivars with non-premature senescence (CCRI16 and Liao4086) trait and 1 clutivar with premature senescence (CCRI10) trait were selected as the experiment samples. Under field condition, various physiological parameters,activity of anti-oxidative enzymes and their corresponding genes were investigated at different stage of leaf senescence; under indoor condition, the effect of NO on anti-oxidative enzymes were also studied. Meanwhile, the effect of NO on cotton leaf proteome was also analyzed by using label-free method. The results are as follows:Under natural condition, the 35 day can be regarded as a watershed for the onset of leaf senescence in the two genotypes. The content of H2O2 and MDA was higher in the premature senescence (PS) genotype than in the non-premature senescence (NPS) plants; the content of NO decreased sharply and was significantly lower in PS than that of NPS cultivar at the late stage of leaf senescence, hence NO might be important factor delaying leaf senescence.The AsA concentration was lower in the PS genotype when compared with NPS genotype, and no significant distinction of GSH in both genotypes was observed throughout the whole lifespan of leaf development and among different materials. It is suggested AsA plays a more crucial role than GSH in terms of ROS scavenging during cotton leaf senescence. The activity of CAT was significantly higher in the NPS plant than in the PS plant, and change in its relative mRNA expression of CAT was consistent with enzyme activity. The activity of APX differed significantly between the NPS and PS from 35- to 50-days and the relative mRNA expression level of APX exhibited the similar pattern with its coresponding enzyme activity. No significant change in the relative expression level of Mn-SOD during leaf aging was observed; throughout the whole leaf development, there was also no significant difference of the relative expression level of Fe-SOD among the three cotton genotypes. The cCu/Zn-SOD and eCu/Zn-SOD play more pronounced roles than chloroplastic Cu/Zn-SODs in terms of ROS scavenging and delaying senescence. Overall POD activity in leaf increased strikingly when undergoing natural senescence, therefore it is suggested that POD did not act to eliminate H2O2 but involved in H2O2 generation.Under indoor condition, after exogenous application of SNP, the NO content was significantly higher in the treatment group than that of control group during the whole cotyledon development stage. At the same stage, the activity of CAT and APX in treatment group was significantly higher than that of control group, especially at the late stage of cotyledons. The activity of POD and its gene expression substantially declined in the subject cultivar by exogenous spray of SNP. Although exogenous NO could inhibit the activity of SOD at early stage of cotyledon development, the treatment group demonstrated greater SOD activity than that of control group with the advancement of leaf senescence. The responses to NO varied among different types of SOD, and the Cu/Zn SOD was the most sensitive isoforms among which cCu/Zn SOD's genes played a more potent role. The physiological and molecular mechanism underlying the delaying effect of NO on leaf senescence is thus revealed by fine coordination of the activity of oxidation and anti-oxidation systems (CAT, APX, POD and SOD) in plant.To date, it is the first time that the effect of different concentrations of NO on the leaf proteome in cotton was investigated using the label-free approach. In this study, 121 differentially expressed proteins were obtained which were functionally divided into 13 groups and spread accross chloroplast, Gogi apparatus, cytoplasm and mitochondria. The pathway analysis demonstrated that NO involved in various physiological activities and had a pronounced impact on photosynthesis, oxidative phosphorylation and protein processing. High concentrations of NO could constitute a toxic threat on cells and the toxic effect may be induced by inhibiting expression of photosynthetic components, decreasing the capability of ATP synthesis and leading to the misfold and assembly of proteins.