Dephosphorylate to close: type 2C protein phosphatases regulate stomatal aperture

Stomata are minute epidermal pores that regulate the balance between carbon uptake and water loss in plants. Stomatal pores are created by a pair of guard cells (GCs) whose swelling and shrinkage due to water influx and efflux, respectively, define the pore size. Swollen or turgid GCs lead to opened stomata, allowing the uptake of carbon dioxide for photosynthesis and loss of water through transpiration. The mechanisms of stomatal opening and closure have gathered much attention among researchers due to their critical roles in carbon fixation and drought tolerance (Lawson and Matthews, 2020).

Light acts as the most important regulator of stomatal opening in terrestrial plants with photoactive stomata. Both blue and red wavelengths of the light spectrum can induce stomatal opening (Yang et al., 2020) by controlling the activities of ion transporter proteins localized in the plasma membrane (PM) and tonoplast (the membrane surrounding vacuoles). The PM localized proton (H⁺) pump (PM H⁺-ATPase) removes H⁺ out of GCs, causing the influx of potassium (K⁺) and other ions through the inwardly rectifying channels. Accumulation of K⁺ decreases the water potential within GCs, promoting water uptake and turgidity, leading to stomatal opening (Jezek and Blatt, 2017). Within 30 s of blue light exposure, the phototropin photoreceptors (PHOT1 and PHOT2) promote the phosphorylation of a penultimate threonine residue (Thr-947) in the C-terminal auto-inhibitory domain of PM H⁺-ATPase, leading to its activation (Hayashi et al., 2011). Thr-947 phosphorylation creates binding sites for the 14-3-3 regulatory proteins that stabilize the active conformation of PM H⁺-ATPase (Figure  1; Falhof et al., 2016). As demonstrated recently, red light-mediated stomatal opening also involves the phosphorylation of PM H⁺-ATPase (Ando and Kinoshita, 2018). In contrast, the divalent magnesium- or manganese-dependent activity of type 2C protein phosphatases (PP2Cs) dephosphorylates Thr-947, thereby deactivating the PM H⁺-ATPase (Hayashi et al., 2010). Specifically, the isoforms of PP2Cs belonging to the D-clade (PP2C.Ds) dephosphorylate the PM H⁺-ATPase during auxin-mediated hypocotyl elongation (Ren and Gray, 2015). In addition, PP2C.D2, D5, and D6 suppress stomatal opening (Wong et al., 2021), suggesting a potential role of PP2C.Ds in regulating the PM H⁺-ATPase dephosphorylation in GCs.

Figure 1.

A simple model of blue light-mediated stomatal movements regulated by PP2C.Ds in Arabidopsis thaliana. The photoreceptors PHOT1 and PHOT2 are activated upon blue light exposure in the GCs (closed stomata; left) and mediate the phosphorylation of the Thr-947 residue in the PM H⁺-ATPase. Simultaneously, the light-activated PHOTs mediate the suppression of PP2C.D activity via unknown mechanisms. The phosphorylated Thr-947 facilitates the binding of regulatory 14-3-3 proteins that maintain the active state of PM H⁺-ATPase. The active H⁺-ATPase pumps out H⁺ ions, creating hyperpolarization of GC membranes that drives the uptake of K⁺ ions via internally rectifying channels. The increase in K⁺ and other ions within the GCs decreases the water potential allowing the influx of water, increase in GC turgidity, and stomatal opening (opened stomata; right). In contrast, the lack of illumination relieves the repression of PP2C.Ds, which dephosphorylate Thr-947, subsequently reducing PM H⁺-ATPase activity. The resulting depolarization facilitates the movement of K⁺ and other ions out of GCs, shifting the water potential and efflux of water leading to stomatal closure (closed stomata; left). The red dotted arrows or lines indicate efflux or inhibitory effects. Green dashed arrows show activation or influx. Question mark (?) represents unknown mechanisms. PHOT, phototropin; PM H⁺-ATPase, plasma membrane-localized ATP proton pump; Thr-947, penultimate threonine residue in the C-terminal auto-inhibitory region of PM H⁺-ATPase.

In this issue of Plant Physiology, Akiyama et al. (2021) demonstrate the redundant roles of PP2C.D6 and D9 in dephosphorylating the PM H⁺-ATPase in GCs. As all nine members of the PP2C.D clade suppressed Thr-947 phosphorylation of endogenous H⁺-ATPase in vivo, the authors searched publicly available transcriptome data to check the specificity of PP2C.D expression in GCs. Among the D-clade isoforms, PP2C.D6 and D9 represented more than half of the PP2C.D transcripts in GCs, suggesting their potential role in stomatal dynamics. Consistent with this finding, the native promoters of PP2C.D6 and D9 clearly expressed the β-glucuronidase reporter in GCs. In addition, fluorophore-tagged transiently expressed PP2C.D6 and D9 localized in the cytoplasm and the GC peripheral regions. Further assays using tagged PP2C.D9 driven by a GC-expressed promoter revealed that PP2C.D9 localizes to the PM of GCs (Akiyama et al., 2021).

To investigate the potential role of PP2C.D6 and D9 in light-mediated stomatal opening, the authors isolated and analyzed the respective T-DNA insertion null mutants pp2c.d6 and pp2c.d9 (Akiyama et al., 2021). While the single mutants did not differ significantly from wild-type (WT) plants (Col-0) in terms of stomatal responses, the double mutant pp2c.d6/9 exhibited more widely opened stomata in response to light, suggesting negative and redundant roles of PP2C.D6 and D9 in regulating stomatal aperture. In line with this observation, levels of Thr-947 phosphorylation in PM H⁺-ATPase in the GCs, detected via immunohistochemical assays, were significantly higher than WT in pp2c.d6/9 double mutants exposed briefly to blue light. Moreover, the dephosphorylation kinetics in pp2c.d6/9 showed persistent phosphorylation of H⁺-ATPase compared with WT, even 40 min after the exposure to a blue light pulse (Akiyama et al., 2021). Interestingly, the widened stomatal aperture in the pp2c.d6/9 double mutant was also observed in darkness. Furthermore, transgenic plants expressing PP2C.D9 in GCs failed to open stomata even upon illumination, presumably due to the enhanced dephosphorylation of PM H⁺-ATPase.

In summary, the work by Akiyama et al. (2021) provides in vivo and in vitro evidence for PP2C.D6- and D9-mediated dephosphorylation of PM H⁺-ATPase in GCs (Figure  1). Although delayed in pp2c.d6/9 double mutants, dephosphorylation was not completely abolished, suggesting additional factors such as other isoforms of PP2C.D acting in the same pathway. Future research may address this question by testing the possibility of the remaining PP2C.D members directly dephosphorylating the PM H⁺-ATPase, specifically in GCs. Another outstanding question that remains to be answered: How does blue light control the phosphorylation status of Thr-947 in PM H⁺-ATPase? Despite many efforts in the past, the protein kinase/s responsible for blue light-dependent direct phosphorylation of Thr-947 in PM H⁺-ATPase has not been identified (Yang et al., 2020). Alternatively, since Thr-947 phosphorylation can be induced without blue light illumination (Akiyama et al., 2021; Wong et al., 2021), the blue light signals may predominantly act by suppressing the dephosphorylation function of PP2C.Ds. Hence, future efforts may be directed toward deciphering the mechanisms underlying blue light-dependent regulation of PP2C.Ds in GCs. Auxin-induced SMALL AUXIN UPREGULATED (SAUR) proteins inhibit PP2C.D activity during hypocotyl growth and stomatal opening via direct protein–protein interactions (Inoue and Kinoshita, 2017; Wong et al., 2021). Furthermore, the SAUR proteins and PP2C.Ds antagonistically regulate stomatal movements by targeting PM H⁺-ATPases and K⁺ channels in GCs (Wong et al., 2021). However, in preliminary experiments, auxin failed to induce PM H⁺-ATPase phosphorylation in GCs (Akiyama et al., 2021), suggesting the involvement of additional unknown factors. Understanding the stomatal kinetics in response to light signals may facilitate better ways of improving water-use efficiency without compromising carbon fixation.

Conflict of interest statement. The author declares no conflict of interest.