Purification And Characterization Of Two Serine Protease Correlated Leaf Senescence In Wheat Leaves

In the studies, two senescence-associated serine proteases (proteases S1 and S2) with similar biochemical properties from dark-induced senescent wheat leaves were found. These data about characters and mass spectrometry told us two proteases S1 and S2 were corresponding to the same serine protease TaSSP1. We further analysis the physiological function in the wheat with different stress and hormone treatment. The main results were showed as follows:1?Preliminary identification of two senescence-related proteases in the wheat leavesTwo senescence-associated proteases (S1 and S2) were detected by gelatin-SDS-PAGE in dark-induced senescent wheat leaves. And the activities of two proteases S1 and S2 significantly increased when leave was treated with darkness for different times, especially for 72 h. The biochemical properties of the two proteases were studied using crude enzymes and the results showed that the proteases can maintain high activity between 30-70? and the optimum temperature was about 60?. The optimum pH was at pH 8.0 around and show activities at a broad range of pH 6.0-9.0. The proteases had a good thermal stability below 70? and were completely inactive above 70?. The protease inhibitors assay indicates that protease S1 and protease S2 were completely inhibited by the general serine proteases inhibitors PMSF and AEBSF, partially inhibited by the metalloprotease inhibitors, including EDTA, EGTA and 1'10-phenanthroline, suggesting us that the two proteases belong to metal-dependent serine protease.2?Isolation, purification and identification of proteases S1 and S2Proteases S1 and S2 were purified by heat treatment, ammonium sulphate precipitation, chromatography desalination, anion exchange chromatography, concentration and gel-cut after electrophoresis using the crude enzyme of 72h dark-induced wheat leave. The crude enzyme was firstly incubated at 50? for 30 min to remove some thermal instability protein. Most of protease S1 and protease S2 could be precipitated by 60-70%(NH4)2SO4. Then active protease fractions were further pooled and loaded a pre-equilibrated Q-sephrase fast flow anion exchange chromatography, and we found protease S1 was eluted firstly and protease S2 was eluted secondly based on the gelatin-SDS-PAGE assay. The active frations were collected and concentrated, and the samples of each purification step were detected and isolated by SDS-PAGE and gelatin-SDS-PAGE, respectively. We found the activities of proteases had been improved significantly with further purification and two candidate proteins were found on SDS-PAGE corresponding to protease S1 and protease S2 according to the location of proteases on the gel of gelatin-SDS-PAGE, respectively. The mass spectrometry assay shows that protease S1 and protease S2 were corresponding to the same serine protease TaSSPl. And the molecular weights of protease S1 and protease S2 were measured as 77.9 kDa and 70.2 kDa by SDS-PAGE.3?Analysis of biochemical characters and physiological function of protease S1 and protease S2We further explored the biochemical characteristics of purified protease S1 and protease S2 including thermal stability, the optimum pH, optimum temperature. The characteristics of protease S1 were similar to those of protease S2, and also similar to those of TaSSP1. The optimum temperature was 50-60?; the optimum pH was 8.0 around; the two proteases remained high activity between 4-60?; the protease S1 and S2 were completely inhibited by serine protease inhibitors PMSF and AEBSF and partial inhibited by metalloprotease inhibitors EDTA, EGTA and 1'10-phenanthroline. We also investigated the effects of different metal ions on the protease activity and the data show that divalent metal ions (Ca2+,Mn2+,Zn2+,Co2+,Cu2+,Mg2+,Fe2+) and Al3+ could increase the activity of proteases S1 and protease S2, however K+or Na+ had no obvious effects on the activity of protease S1 or protease S2. Our results further suggested that two proteases showed the SDS-stimulating activity when the concentration of SDS were below 0.6 mM. To verify the relationship of two purified protease S1 and protease S2, we performed the western-blotting taking the anti-TaSSPl antibody, and the data showed us that two obvious immunoreactive bands with the size around 70 kDa were detected which was corresponding to sizes of protease S1 and protease S2 determined by gelatin-SDS-PAGE. We further provided the evidence to indicate the possible involvement of these two proteases in aging, stress and hormones control in plants by western-blot analysis. The expression level of two proteases was noticeable increased by dark, NaCl, PEG 6000, D-mannitol, and abscisic acid treatment. Therefore, the data imply the possible involvement of two proteases in biological control of aging and stress such as salt stress and drought stress in plants and two proteases may share the similar signaling pathway.The results above verified two senescence-associated proteases in wheat leave, and confirmed both the two proteases belong to TaSSP1. Our study provided the important biochemical basis for further understanding biological functions of TaSSP1 during leaf senescence and plant responses to environment stress.