The Function Of Phosphorylation Of Soybean Pm18 In Stress Resistance

Protein phosphorylation is an important posttranslational modification and widely exists in both eukaryotes and prokaryotes. It is almost involved in all kinds of life events. The growth of plants is influenced by various environmental factors like temperature, water and salinity, and a range of plant proteins are induced to resist the abiotic stress. Late embryogenesis abundant protein(LEA), which accumulate in late embryogenesis stage and arereported to participate in a variety of plant stress responses. The members of LEA protein family can be divided into 7 groups according to the characteristics of sequence and conserved domains. To date, the studies of phosphorylation of the LEA proteins were restricted to the members in group 2. In this study, we chose PM18(LEA3) to investigate the function of phosphorylation of PM18.In this study, we separated soybean root R0 stage proteins by 2DE and identified three phosphorylation sites(S90, Y136 and T266) of PM18 by LC-MS/MS. In order to study the correlation between the phosphorylation and the protective activity of PM18 protein under salt stress, a set of mutations were introduced to these three phosphorylation sites. We constructed the gain-of-phosphorylation and loss-of-phosphorylation types of PM18. The mutational and wild-type PM18 s were later transformed into salt sensitive yeast(?nha1 and ?nhx1) and hypertonic sensitive yeast(?hog1) respectively to observe their growth under different stress conditions. The results showed that the gain-of-phosphorylation and the wild-type PM18 s improvedthe salt tolerance capacity of the salt sensitive yeast strains better than dephosphorylated PM18. The phosphorylation of the S90 and Y136 sites of PM18 played an important role in salt tolerance reaction, while there was no obvious effect of the phosphorylation of T266. Further investigation revealed that there might be a synergistic effect of the phosphorylation of the S90 and Y136 sites in salt tolerance.In vitro phosphorylation of PM18 by casein kinase II(CKII) and the structural analysis by circular dichroism showed that PM18 was a disordered protein in water. While the ?-helix could be induced more easily in phosphorylated PM18 in the presence of TFE and SDS. In addition, PM18 effectively reduced the activity loss of the lactate dehydrogenase under freeze and thaw stress, and the phosphorylated PM18 had better protective activity in contrast to the dephosphorylated one.Taken together, our results indicated that the phosphorylation of PM18(LEA3) played a certain role in response to stress, which provided a novel insight to the investigation on the phosphorylation modification of LEA proteins.