Functional Study Of ISP Overexpression In Delaying Senescence Of Rice Leaves

Senescence,a highly programmed process,largely affects the yield and quality of crops.The associated studies of rice leaf senescence indicated that rice leaf can generate a lot of changes in morphology and physiology during this process which was under the regulation of genes,including production of a large number of hydrolase,reduction of the amount of nucleic acid,production and accumulation of harmful substances(including reactive oxygen),decomposition of many kinds of protein and the chlorophyll,and the damage of leaf tissue.Studies have shown that leaf senescence is regulated by various senescence-associated genes,such as STAY-GREEN,RED CHLOROPHYLL CATABOLITE REDUCTASE 1,and NON-YELLOW COLORING 1 and 3.In our previous study,we identified a special protein ISP(Indica Special Protein)by analyzing the differential expression of proteomics between indica and japonica rice,but its function is unclear.Thus,transgenic techniques,transcriptome analyses and real-time quantitative PCR were used to investigate the function of ISP.The main contents and results were as follows:(1)Throughout the phenotypic observation between wild type and ISP overexpression(ISP-OE)plants in the whole growth cycle,we found the phenotypes of ISP-OE plants were similar to wild type in the growth period.In the mature period,the leaves of ISP-OE plants could keep green,but the leaves of the wild type became yellow.(2)In this study,transcriptomic method was used to analyze the gene expression differences between wild type and ISP overexpressing rice.Throughout comparing ISP-OE plants with wild type,889 differentially expressed genes(FDR ? 0.05 and the absolute value of log2.0 Fold_change ? 1)were obtained,including 543 up-regulated genes and 346 down-regulated genes.Based on the result of transcriptome sequencing,we screened some senescence related genes and chlorophyll degradation related genes.The expression levels of these genes in ISP-OE were significantly lower than that in wild-type plants.These results indicated that overexpression of ISP gene could inhibit the expression of some senescence related genes.In addition,we found that there were significant differences in the key genes of ABA synthesis pathway between wild-type and ISP-OE plants.The expression levels of ABA synthesis pathway related genes in overexpressing plants were significantly lower than that in wild-type plants.(3)The expression levels of senescence related genes and chlorophyll degradation related genes in wild-type and ISP-OE plants were detected by real-time quantitative PCR.We also measured the content of chlorophyll in leaves of wild-type and ISP-OE plants.The results showed that ISP can inhibit the age-dependent senescence of rice leaf.(4)With the dark treatment of wild type and ISP-OE plant leaves in vitro,the detached leaves of wild type began to become yellow while the leaves of ISP overexpression still remained green.By real-time quantitative PCR we measured the expression levels of senescence associated genes and chlorophyll degradation associated genes in ISP-OE plants and wild-type plants under the dark-induced senescence condition.Meanwhile,the chlorophyll content of leaves was measured.In conclusion,the overexpression of ISP can inhibit dark-induced senescence of rice leaf.(5)In this study,we also measured the expression levels of ABA biosynthesis pathway key genes in wild-type and ISP-OE plants by real-time quantitative PCR.The results showed that the expression levels of ABA biosynthetic pathway genes in overexpressing plants were significantly lower than that in wild-type plants.The results also verified the accuracy of transcriptome sequencing results.Furthermore,we used 50 ?M ABA hormone to deal with wild-type and ISP overexpressing germinated seeds,ISP-OE plants were found to be sensitive to ABA after 10 d growth.