质膜Ca2+-ATPase及H+-ATPase与细胞信号转导研究

Jasmonates (JAs) are "stress hormone" involved in signaling of defense responses. Our present knowledge of JAs signaling pathway is still limited. In the present study we investigated the effect of MeJA on H+-ATPase hydrolysis activity of the plasma membrane (PM ) isolated and purified from mung bean hypocotyls by aqueous two-phase partition and on the the phosphorylation and dephosphorylation of the enzyme comparing to fusicosin (FC) which is an activator of H+-ATPase. Moreover, the role of the calcium ion in MeJA- and FC-induced increases of the plasma membrane H+-ATPase was studied. .Application of MeJA (50μmol/L) to 3-d-old seedlings resulted in an increase (about 30%) in PM H+-ATPase activity in vivo. The enzyme activity after 10μmol/L MeJA treatment in vitro also showed about 30% increase 2hr after incubation. FC stimulated PM H+-ATPase activity and the maximal increase reached to 72% in vivo, while it had the same stimulation (about 30%) as MeJA had in vitro. The combination of MeJA and FC did not show significant additive effect on enzyme activity. In vitro, phosphatase inhibitors, okadaic acid and cantharidin, enhanced MeJA- induced increase of the enzyme activity. Staurosporine and cheleythrine, two inhibitors of protein kinase, inhibited the stimulation of MeJA on PM H+-ATPase activity completely. Both protein kinase and phosphatase inhibitors showed the same effect on FC-induced increases of enzyme activity. The results from γ-32p tracing experiments showed that the level of isotope labeling on PM H+-ATPase increased after treatment with MeJA and FC, respectively, demonstrating that the phosphorylation took place in the enzyme after MeJA treatment. Ca2+ strongly stimulated the PM H+-ATPase in vitro, and the increase of the enzyme activity was two times higher than that of the control. But Ca2+ had no enhancement of the enzyme activity induced by FC and MeJA .Trypsin digestion experiment was conducted to investigate the MeJA effect. After digesting and removing of a 7-10 kD segment of PM H+-ATPase C-terminus, the PM H+-ATPase activity increased by 30%. Same increasing of the enzyme activity was observed after MeJA and FC treatments, indicating the regulation of C-terminus of PM H+-ATPase might be involved in MeJA stimulation.in mung bean.[Ca2+]c changes were directly measured in Arabidopsis leaf from 10 d seedlings by using confocal laser scanning microscopy in conjunction with a calcium dye of Fluo-3AM. We found that [Ca2+]c increased rapidly after JA (50-150 mm) treatment. This increasing was inhibited by the pretreatment of the leaf with nifedipine, a Ca2+ channel blocker. But verlapamil, another Ca2+ channel blocker had no significant effect on JA induced [Ca2+]c changes. Moreover, the expression of two JA induced genes, vsp and jr1 were also inhibited when nifedipine was applied. And verlapamil could not affect two gene expression..Our data reported here demonstrated firstly that MeJA activated PM H+-ATPase hydrolysis activity and the the phosphorylation and dephosphorylation of the enzyme were involved in MeJA stimulation in mung bean; Ca2+/CaM were required in the JA signaling and nifedipine-sensitive channel was involved in [Ca2+]c changes induced by JA in Arabidopsis.

在以往用原生质体实验系统，比较研究钙信使在光和激素信号转导中作用的基础上，分离纯化原生质膜，测定光和激素刺激后，质膜Ca2+-ATPase和H+-ATPase的变化，研究钙、钙调素的作用，并探讨光和激素刺激对酶的磷酸化、去磷酸化的影响。拟在同一实验体系比较光和激素信号转导的异同之处。此项课题对深入细致地研究植物信号转导的机理有重要理论意义。