Gurrieri Libero, Capuzzi Anna Clara, Müller-Schüssele Stefanie J, Trost Paolo, Sparla Francesca
Department of Pharmacy and Biotechnology, University of Bologna, 40126 Bologna, Italy.
Department of Biology, University of Kaiserslautern-Landau, 63663 Kaiserslautern, Germany.
Plant Physiol. 2025 Aug 4;198(4). doi: 10.1093/plphys/kiaf344.
Guard cells control the opening and closure of stomatal pores in response to internal and external stimuli, ensuring gas exchange in plants. In Arabidopsis (Arabidopsis thaliana), β-AMYLASE1 (BAM1), assisted by α-AMYLASE3, begins degrading starch at dawn in guard cells to promote stomatal opening. Both enzymes are controlled by reversible disulfide bond formation, which decreases their activity. In the present study, we investigated the sensitivity of BAM1 to other redox-dependent post-translational modifications (PTM) both in vitro and in vivo. In vitro, H2O2 reversibly inactivates BAM1 and, in the presence of glutathione (GSH), induces S-glutathionylation of BAM1. Glutathionylated BAM1 is active and transiently protected from H2O2 inhibition. However, the glutathionylated state of BAM1 has limited stability and can be slowly resolved by a second cysteine with the formation of the intramolecular disulfide bond that inhibits BAM1 activity. Thioredoxin f can fully revert the inhibition by reducing the disulfide to a dithiol. In vivo, Arabidopsis mutants with lower plastidial GSH reductase activity, and consequently modified GSH homeostasis, showed higher BAM1 activity, lower starch levels in guard cells, and altered stomata aperture, indicating that GSH redox potential impacts stomatal physiology, possibly through BAM1. Moreover, plastidial BAM1 presents a prime example for the role of glutathionylation functioning as a transiently protective PTM, interfering with the formation of inhibitory disulfide bonds. This example illustrates how transitions between protein cysteinyl thiol PTMs can orchestrate dynamic responses involving several redox systems.
保卫细胞响应内部和外部刺激控制气孔的开闭，确保植物中的气体交换。在拟南芥中，β-淀粉酶1（BAM1）在α-淀粉酶3的协助下，于黎明时分开始在保卫细胞中降解淀粉以促进气孔开放。这两种酶都受可逆二硫键形成的控制，这种形成会降低它们的活性。在本研究中，我们在体外和体内研究了BAM1对其他氧化还原依赖性翻译后修饰（PTM）的敏感性。在体外，H2O2可逆地使BAM1失活，并且在谷胱甘肽（GSH）存在的情况下，诱导BAM1的S-谷胱甘肽化。谷胱甘肽化的BAM1具有活性，并能暂时免受H2O2抑制。然而，BAM1的谷胱甘肽化状态稳定性有限，可被第二个半胱氨酸缓慢分解，形成抑制BAM1活性的分子内二硫键。硫氧还蛋白f可以通过将二硫键还原为二硫醇来完全恢复抑制作用。在体内，质体谷胱甘肽还原酶活性较低，因此谷胱甘肽稳态发生改变的拟南芥突变体表现出较高的BAM1活性、保卫细胞中较低的淀粉水平以及气孔孔径改变，这表明谷胱甘肽氧化还原电位可能通过BAM1影响气孔生理。此外，质体BAM1是谷胱甘肽化作为一种暂时保护性PTM发挥作用的主要例子，它干扰抑制性二硫键的形成。这个例子说明了蛋白质半胱氨酰硫醇PTM之间的转变如何协调涉及多个氧化还原系统的动态反应。
