CNGC2 Negatively Regulates Stomatal Closure and Is Not Required for flg22- and H2O2-Induced Guard Cell [Ca2+]cyt Elevation in Arabidopsis thaliana.

Abstract

In guard cells, cytosolic Ca²⁺ acts as a second messenger that mediates abscisic acid (ABA)- and pathogen-associated molecular pattern (PAMP)-induced stomatal closure. It was reported that Arabidopsis cyclic nucleotide-gated ion channel 2 (CNGC2) functions as hydrogen peroxide (H₂O₂)- and PAMP-activated Ca²⁺-permeable channels at the plasma membrane of mesophyll cells and mediates Ca²⁺-dependent PAMP-triggered immunity. In this study, we examined the role of CNGC2 in the regulation of stomatal movement because CNGC2 is also expressed in guard cells. We found that stomata of the CNGC2 disruption mutant cngc2-3 are constitutively closed even in the absence of ABA or the flagellar-derived PAMP, flg22. Consistently, leaf temperatures of the cngc2-3 mutant were higher than those of wild-type (WT) plants. The stomatal phenotype of the cngc2-3 mutant was restored by complementation with wild-type CNGC2 under the control of the guard cell preferential promoter, pGC1. Elevation of cytosolic free Ca²⁺ concentration in guard cells induced by flg22 and H₂O₂ remained intact in the cngc2-3 mutant. The introduction of the ost1-3 mutation into the cngc2-3 background did not alter the stomatal phenotype. However, the stomatal phenotype of the cngc2-3 mutant was successfully rescued in the double disruption mutant cngc2-3aba2-2. Taken together, these results suggest that CNGC2 negatively regulates stomatal closure response and does not function as flg22- and H₂O₂-activated Ca²⁺ channels in guard cells. Though CNGC2 is responsive for H₂O₂- and flg22-induced [Ca²⁺]_cyt elevation in mesophyll cells, the involvement of CNGC2 in the response to H₂O₂ and flg22 in guard cells is questionable.