Du Z, Aghoram K, Outlaw W H
Department of Biological Science, Florida State University, Tallahassee 32306-3050, USA.
Arch Biochem Biophys. 1997 Jan 15;337(2):345-50. doi: 10.1006/abbi.1996.9790.
Plants regulate water loss and CO2 gain by modulating the aperture sizes of stomata that penetrate the epidermis. Aperture size itself is increased by osmolyte accumulation and consequent turgor increase in the pair of guard cells that flank each stoma. Guard cell phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), which catalyzes the regulated step leading to malate synthesis, is crucial for charge and pH maintenance during osmolyte accumulation. Regulation of this cytosolic enzyme by effectors is well documented, but additional regulation by posttranslational modification is predicted by the alteration of PEPC kinetics during stomatal opening (FEBS Lett. 352, 45-48). In this study, we have investigated whether this alteration is associated with the phosphorylation status of this enzyme. Using sonicated epidermal peels ("isolated" guard cells) preloaded with 32PO4, we induced stomatal opening and guard cell malate accumulation by incubation with 5 microM fusicoccin (FC). In corroboratory experiments, guard cells were incubated with the FC antagonist, 10 microM abscisic acid (ABA). The phosphorylation status of PEPC was assessed by immunoprecipitation, electrophoresis, immunoblotting, and autoradiography. PEPC was phosphorylated when stomata were stimulated to open, and phosphorylation was lessened by incubation with ABA. Thus, we conclude that regulation of guard cell PEPC in vivo is multifaceted; the effects of regulatory metabolites and the activation status of the enzyme are integrated to control malate synthesis. These results, together with the coincident alteration in the kinetics of the enzyme (FEBS Lett. 352, 45-48), constitute the first unequivocal demonstration of regulatory posttranslational modification of a guard cell protein that is specifically implicated in stomatal movements.
植物通过调节穿透表皮的气孔孔径大小来控制水分流失和二氧化碳吸收。气孔孔径大小本身会因渗透溶质积累以及随之而来的围绕每个气孔的一对保卫细胞膨压增加而增大。保卫细胞磷酸烯醇式丙酮酸羧化酶（PEPC，EC 4.1.1.31）催化导致苹果酸合成的调控步骤，在渗透溶质积累过程中对电荷和pH维持至关重要。效应物对这种胞质酶的调节已有充分记录，但气孔开放过程中PEPC动力学的改变预示着存在翻译后修饰的额外调节（《欧洲生物化学学会联合会快报》352卷，45 - 48页）。在本研究中，我们调查了这种改变是否与该酶的磷酸化状态相关。我们使用预先加载了32PO4的超声处理表皮条（“分离的”保卫细胞），通过与5微摩尔的壳梭孢菌素（FC）孵育来诱导气孔开放和保卫细胞苹果酸积累。在确证实验中，保卫细胞与FC拮抗剂10微摩尔脱落酸（ABA）一起孵育。通过免疫沉淀、电泳、免疫印迹和放射自显影来评估PEPC的磷酸化状态。当气孔被刺激开放时，PEPC发生磷酸化，而与ABA孵育会减少磷酸化。因此，我们得出结论，保卫细胞PEPC在体内的调节是多方面的；调节代谢物的作用和酶的激活状态相互整合以控制苹果酸合成。这些结果，连同酶动力学的同步改变（《欧洲生物化学学会联合会快报》352卷，45 - 48页），首次明确证明了一种专门参与气孔运动的保卫细胞蛋白存在调节性翻译后修饰。
