| One-year-old plants of 70 days after budding and berries of 30days after fruit setting of grape (Vtis vinifera L. cv. Jingxiu) were used as experiment materials. After they were treated by heat stress (40℃) and salicylic acid (SA) with concentration of 150 umol/L in different times, respectively, the effects of heat stress and SA on the induction and activities of protein kinases in grape leaves and pulp cells were studied. The signaling transduction of SA in cells stressed by heat was demonstrated from molecular level. The following results were obtained:Protein kinase was activated by heat stress and exogenous SA in cell tissues of grape leaves, which could phosphorylated MBP as artificial substrate in vitro. The activity of this protein kinase reached a peak value as the concentration of MBP was 0.5 mg/mL, and then, its activity declined as the concentration of MBP increased continuously. However, this protein kinase could not phosphorylate histone-III as substrate in vitro. The best concentration of ATP in reaction system was 50 umol/L. When the concentration of ATP in reaction solution exceeded 50 umol/L, the activity of protein represented a decreasing trend. As concentration of Mg2+ in solution was 5 mmol/L, the activity of protein kinase ascended a maximum value. The stimulation of Ca2+ to this protein kinase was extremely weak. Mn2+ had no effect of stimulation to this protein kinase, and it could not replace the role of Mg2+ in reaction system. The result of autoradiography of substrate phosphorylation in gel showed that the molecular weight of this protein kinase activated by heat stress 10-60 min and the treatment of SA 30-180 min was about 52 kD. This protein kinase could phosphorylate MBP as substrate embedded in gel, and the phosphorylating products labeled by 32P revealed a very high radiation activity in gel. But histone-III embedded in gel was not phosphorylated by this protein kinase and did not display any radiation activity in gel. The high phosphorylation activity of this protein kinase was found with MBP substrate in solution reaction system. After 60 min heat stress, the activity of this protein kinase reached its maximum value, and then declined rapidly. Compared with control, the activity of protein kinase had no difference when histone-III was used as substrate in reaction system. Ca2+ could not enhance the activity of this protein kinase, showing that this protein kinase was Ca2+-independent. The activity of protein kinase reached a peak value as pH was 7.5 in the reaction system with MBP substrate. The tyrosine-specific protein phosphatase (Yersinia enterocolitica protein, YOP) could significantly inactivate the phosphorylation activity of protein kinase. All results demonstrate that the 52 kD protein kinase activated by heat stress belong to the MAPK family.After the discs of grape berries were treated in the incubation solution with heat stress and SA, the types and activities of protein kinases in crud extracts of pulp cells of the berry discs were mensurated through the methods of in-gel protein activity assay and protein acitivity assay in solution. The results showed that a 52kD and a 42kD protein kinase were stimulated by heat stress 5-20 min and SA treatment 5-60 min. The phosphorylated MBP and histone-III were displayed by autoradiography at theposition of 52kD and 42kD in gels, respectively. The phosphorylation activity of 52kD protein kinase was very strong as artificial substrate MBP was added in reaction solution with Mg +, but Ca + in solution had no stimulation to 52kD protein kinase. Tyrosine-specific protein phosphatase Yersinia enterocolitica protein (YOP) could clearly inactivate 52kD protein kinase. The phosphorylation activity of 42kD protein kinase was strongly represented when histone-III was used as substrate in reaction solution with Ca2+, and 42kD protein kinase characterized Ca2+ dependent, and CaM independent. CaM antagonists Calmidazolium and W7 could significantly inhibit the phosphorylation activity of 42kD protein kinase to histone-I...
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