Exploration of PM H+-ATPase phosphorylation mechanism using stomatal opening inhibitors and their derivatives.

Shogo Kuwayama, Koji Takahashi, Maki Hayashi, Yuki Hayashi, Kohei Fukatsu, Yusuke Aihara, Ayato Sato, Toshinori Kinoshita
Graduate School of Science, Nagoya University, Chikusa, Nagoya, 464-8602, Japan

The phosphorylation of plasma membrane (PM) H+-ATPase, including Thr948 and Thr881, is a key process in light-induced stomatal opening. However, the phosphorylation mechanism of PM H+-ATPase in stomatal opening is still largely unknown. To reveal this, we performed screening of a mammalian kinase inhibitor chemical library and identified AG126, as an inhibitor of blue light-induced Thr948 phosphorylation of PM H+-ATPase and stomatal opening. Interestingly, AG126 also suppressed fungal toxin fusicoccin-induced Thr948 phosphorylation of PM H+-ATPase and stomatal opening. Furthermore, we analyzed the structure-activity relationship using synthesized AG126 derivatives. As a result, we identified AGD-1, which has lower 50% inhibitory concentration (IC50) at around 2.0 µM than that of AG126 at around 7.7 µM in light-induced stomatal opening. This IC50 is almost the same as that of ABA in our experimental conditions. In addition, we synthesized acetylated AGD-1 (AcAGD-1) to improve the permeability of AGD-1. Further investigations revealed that, AcAGD-1 specifically inhibited only Thr948 phosphorylation but not Thr881, suggesting that AGD1 suppresses a protein kinase, which phosphorylates Thr948 of PM H+-ATPase. Moreover, we also synthesized linker-elongated compounds from AGD-1 for target identification. We will report the results of further characterization using AGD-1 and AGD-1 related compounds.