茉莉酸甲酯处理对绿豆下胚轴质膜H^+-ATPase水解活力及磷酸化水平的影响

Effects of Methyl Jasmonate Treatment on the Hydrolytic Activity and Phosphorylation Level of Plasma Membrane H^+-ATPase in Mung Bean (Vigna radiata L.) Hypocotyls

运用γ-32P示踪、蛋白激酶和磷酸酶抑制剂药理实验探讨茉莉酸甲酯(MeJA)对质膜H+-ATP酶水解活力及磷酸化水平的影响。结果如下:MeJA可促进H+-ATP酶水解活力30％;斑蝥素和岗田酸促进了MeJA对质膜H+-ATP酶的刺激作用;星形孢菌素和白屈菜红碱削弱了MeJA对质膜H+-ATP酶的刺激作用。H+-ATP酶活力变化同时,其上的γ-32P标记量发生变化。Ca2+对H+-ATP酶水解活力有很大的刺激作用,但对MeJA促进H+-ATP酶活力的作用没有进一步的影响。根据这些结果可以得出结论:MeJA刺激质膜H+-ATP酶水解活力的变化与H+-ATP酶磷酸化水平呈正相关,并且催化这一作用的蛋白激酶可能不依赖于Ca2+,而蛋白磷酸酶依赖于Ca2+。

Employing both protein kinase inhibitors and phosphatase inhibitors to investigate the effect of MeJA (methyl jasmonate) on the H+-ATPase hydrolytic activity of plasma membrane, and the regulations involved with phosphoryla-tion and dephosphorylation of plasma membrane H+-ATPase after treatments with MeJA. 3 d-old etiolated mung bean (Vigna radiata L.) seedlings were harvested and the hypocotyls (1-2 cm in length) under the hook were used to prepare the plasma membrane vesicles by means of aqueous two-phase partition. Hydrolytic activities of plasma membrane H+-ATPase were determined in responding to the treatment with MeJA. H+-ATPase activity stimulated by MeJA and FC was up to 30%, and the combination of MeJA and FC did not show significant additive effect. The phosphatase inhibitors, okadaic acid and cantharidin, enhanced MeJA-induced increase of the enzyme activity to 60% and 50%, respectively. Staurosporine and cheleythrine, two inhibitors of protein kinase, abolished completely the stimulative effect of MeJA on PM H+-AT-Pase activity (Fig.2). The results from γ-32P tracer experiments showed that treatment with MeJA and FC increased the level of isotope labeling on PM H+-ATPase. Okadaic acid and cantharidin could enhance the labeling of γ-32P on PMH+-ATPase induced by MeJA and FC. Both MeJA- and FC-induced increase in γ-32P level were inhibited when cheleythrine was applied (Fig.3). Ca2+ strongly stimulated the PM H+-ATPase 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 . In the presence of Ca2+, Okadaic acid could increase the MeJA stimulation slightly, but cantharidin had not any significant effect on the enzyme activity. Staurosporine and cheleythrine showed no effect on MeJA-induced increase in PM H+-ATPase, and which activity was similar to the control after treatment with either inhibitor (Figs. 5, 6). The changes in hydrolytic activity of H+-ATPase stimulating by MeJA is probably related to reversible phosphorylation, and the protein kinase is Ca2+-independent, phosphatase is Ca2+-dependent.