Elevated CO₂ induces rapid dephosphorylation of plasma membrane H⁺‐ATPase in guard cells

Abstract

Summary Light induces stomatal opening, which is driven by plasma membrane (PM) H+‐ATPase in guard cells. The activation of guard‐cell PM H+‐ATPase is mediated by phosphorylation of the penultimate C‐terminal residue, threonine. The phosphorylation is induced by photosynthesis as well as blue light photoreceptor phototropin. Here, we investigated the effects of cessation of photosynthesis on the phosphorylation level of guard‐cell PM H+‐ATPase in Arabidopsis thaliana. Immunodetection of guard‐cell PM H+‐ATPase, time‐resolved leaf gas‐exchange analyses and stomatal aperture measurements were carried out. We found that light–dark transition of leaves induced dephosphorylation of the penultimate residue at 1 min post‐transition. Gas‐exchange analyses confirmed that the dephosphorylation is accompanied by an increase in the intercellular CO2 concentration, caused by the cessation of photosynthetic CO2 fixation. We discovered that CO2 induces guard‐cell PM H+‐ATPase dephosphorylation as well as stomatal closure. Interestingly, reverse‐genetic analyses using guard‐cell CO2 signal transduction mutants suggested that the dephosphorylation is mediated by a mechanism distinct from the established CO2 signalling pathway. Moreover, type 2C protein phosphatases D6 and D9 were required for the dephosphorylation and promoted stomatal closure upon the light–dark transition. Our results indicate that CO2‐mediated dephosphorylation of guard‐cell PM H+‐ATPase underlies stomatal closure.

Illuminating stomatal responses to red light: establishing the role ofCi-dependent vs - independent mechanisms

“…Thus, it seems that high C i -induced stomatal closure is dominant and suppresses C i -independent red-lightinduced stomatal opening. The observed competition between both pathways is consistent with recent work where high CO 2 -mediated dephosphorylation of guard cell PM H + -ATPases was delayed and required a higher C i under red light compared to in darkness (Ando et al, 2022), indicating that red light-induced phosphorylation of PM H + -ATPases is a component of the C i -independent response (Ando et al, 2022).…”

“…Indeed, the high C i -induced suppression of stomatal opening observed for Col-0 (Figure 1B & 2B) was not present in ca1ca4 (Figure 3B & 4B). Meanwhile, C i -independent mechanisms of stomatal opening remained unaffected in ca1ca4 (Hu et al, 2010;Matrosova et al, 2015;Ando et al, 2022); further supporting the idea that the red-light specific C i -independent stomatal opening response is at least partially independent of the mechanisms driven by C i .…”

“…Likewise, in the double carbonic anhydrase ca1ca4 mutant the stomatal response to CO 2, but not red light, was diminished (Ando et al, 2022;Matrosova et al, 2015). Interestingly, PM H + ATPase phosphorylation and stomatal opening under red light were both increased in ca1ca4, compared to wild type (Ando et al, 2022), which may imply that plants could compensate for the deficiency in the C i -dependent pathway by enhancing the C iindependent pathway to maintain effective carbon gain. This raises the question as to what signals are involved in sensing and communicating the red-light stimulus in the C i -independent pathway.…”

“…For example, several CALCIUM DEPENDENT PROTEIN KINASES (CPKs) appear to exclusively respond to CO 2 ; the cpk3/5/6/11/23 quintuple mutant showed impaired stomatal responses to high and low CO 2 , but stomatal opening in response to red light was unaffected (Schulze et al, 2021). Likewise, in the double carbonic anhydrase ca1ca4 mutant the stomatal response to CO 2, but not red light, was diminished (Ando et al, 2022;Matrosova et al, 2015). Interestingly, PM H + ATPase phosphorylation and stomatal opening under red light were both increased in ca1ca4, compared to wild type (Ando et al, 2022), which may imply that plants could compensate for the deficiency in the C i -dependent pathway by enhancing the C iindependent pathway to maintain effective carbon gain.…”

“…In response to elevated bicarbonate MPK4/12 directly interact with and inhibit HT1, which in turn relieves HT1-mediated repression of GC S-type anion channels and promotes stomatal closure (Takahashi et al, 2022;Yeh et al, 2023). In addition to the HT1-mediated pathway of stomatal closure, high CO 2 also promotes rapid dephosphorylation and deactivation of PM H + -ATPase channels, which suppresses stomatal opening (Edwards & Bowling, 1985;Ando et al, 2022).…”