Mechanistic insights into phosphoactivation of SLAC1 in guard cell signaling

Li Qina, Ya-nan Denga, Xiang-yun Zhang a, Ling-hui Tanga, Chun-rui Zhanga, Shi-min Xua, Ke Wanga, Mei-hua Wanga, Xian-hui Zhanga, Min Sua, Qi Xieb, Wayne A. Hendricksone, & Yu-hang Chen,
State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

Stomata in leaves regulate gases (CO₂ and water vapor) exchange and water transpiration between plants and the atmosphere. SLAC1 mediates anion efflux from guard cells and plays a crucial role in controlling stomatal aperture. It serves as a central hub for multiple signaling pathways in response to environmental stimuli, with its activity regulated through phosphorylation via various plant protein kinases. However, the molecular mechanism underlying SLAC1 phosphor-activation has remained elusive. Through a combination of protein sequence analyses and AlphaFold-based modeling, we unveiled that the highly conserved motifs on the N- and C-terminal segments of SLAC1 form a cytosolic regulatory domain (CRD) that interacts with the transmembrane domain (TMD), thereby maintaining the channel in an autoinhibited state. Mutations in these conserved motifs destabilize the CRD, releasing autoinhibition in SLAC1 and enabling its transition into an activated state. Our further electrophysiological studies demonstrated that the SLAC1 activation undergoes an autoinhibition-release process and subsequent structural changes in the pore-helices. These findings provide mechanistic insights into the activation mechanism of SLAC1 and shed light on understanding how SLAC1 controls stomatal closure in response to environmental stimuli.