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Plasma membrane H+-ATPase provides the driving force for light-induced stomatal opening. However, the mechanisms underlying the regulation of its activity remain unclear. Here, we show that the phosphorylation of two Thr residues in the C-terminal autoinhibitory domain is crucial for H+-ATPase activation and stomatal opening in Arabidopsis thaliana. Using phosphoproteome analysis, we show that blue light induces the phosphorylation of Thr-881 within the C-terminal region I, in addition to penultimate Thr-948 in AUTOINHIBITED H+-ATPASE 1 (AHA1). Based on site-directed mutagenesis experiments, phosphorylation of both Thr residues is essential for H+ pumping and stomatal opening in response to blue light. Thr-948 phosphorylation is a prerequisite for Thr-881 phosphorylation by blue light. Additionally, red light-driven guard cell photosynthesis induces Thr-881 phosphorylation, possibly contributing to red light-dependent stomatal opening. Our findings provide mechanistic insights into H+-ATPase activation that exploits the ion transport across the plasma membrane and light signalling network in guard cells. Light-induced stomatal opening is crucial for photosynthesis. Here the authors show that blue light triggers phosphorylation of two Thr residues in the C-terminal autoinhibitory domain of plasma membrane H+-ATPase, thereby promoting stomatal opening. [ABSTRACT FROM AUTHOR]

Blue light (BL)-dependent H+ pumping by guard cells, which drives stomatal opening, is inhibited by abscisic acid (ABA). We investigated this response with respect to the activity of plasma membrane H+-ATPase using Vicia guard cell protoplasts. ATP hydrolysis by the plasma membrane H+-ATPase, phosphorylation of the H+-ATPase, and the binding of 14-3-3 protein to the H+-ATPase stimulated by BL were inhibited by ABA at 10 µM. All of these responses were similarly inhibited by hydrogen peroxide (H202) at 1 mM. The ABA-induced inhibitions of BL-dependent H+ pumping and phosphorylation of the H+-ATPase were partially restored by ascorbate, an intracellular H2O2 scavenger. A single-cell analysis of the cytosolic H2O2 using 2',7'-dichlorofluorescin revealed that H2O2 was generated by ABA in guard cell protoplasts. We also indicated that H+ pumping induced by fusicoccin and the binding of 14-3-3 protein to the H+-ATPase were inhibited slightly (approximately 20%) by both ABA and H2O2. By contrast, H2O2 at 1 mM did not affect H+ pumping by the H+-ATPase in microsomal membranes. From these results, we concluded that inhibition of BL-dependent H+ pumping by ABA was due to a decrease in the phosphorylation levels of H+-ATPase and that H2O2 might be involved in this response. Moreover, there are at least two inhibition sites by ABA in the BL signaling pathway of guard cells. [ABSTRACT FROM AUTHOR]

Phototropins (phot1 and phot2) are plant blue light receptor kinases that function to mediate phototropism, chloroplast movement, leaf flattening, and stomatal opening in Arabidopsis. Considerable progress has been made in understanding the mechanisms associated with phototropin receptor activation by light. However, the identities of phototropin signaling components are less well understood by comparison. In this study, we specifically searched for protein kinases that interact with phototropins by using an in vitro screening method (AlphaScreen) to profile interactions against an Arabidopsis protein kinase library. We found that CBL-interacting protein kinase 23 (CIPK23) interacts with both phot1 and phot2. Although these interactions were verified by in vitro pull-down and in vivo bimolecular fluorescence complementation assays, CIPK23 was not phosphorylated by phot1, as least in vitro. Mutants lacking CIPK23 were found to exhibit impaired stomatal opening in response to blue light but no deficits in other phototropin-mediated responses. We further found that blue light activation of inward-rectifying K + (K + in ) channels was impaired in the guard cells of cipk23 mutants, whereas activation of the plasma membrane H + -ATPase was not. The blue light activation of K + in channels was also impaired in the mutant of BLUS1, which is one of the phototropin substrates in guard cells. We therefore conclude that CIPK23 promotes stomatal opening through activation of K + in channels most likely in concert with BLUS1, but through a mechanism other than activation of the H + -ATPase. The role of CIPK23 as a newly identified component of phototropin signaling in stomatal guard cells is discussed., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Stomata regulate gas exchange between plants and atmosphere by integrating opening and closing signals. Stomata open in response to low CO 2 concentrations to maximize photosynthesis in the light; however, the mechanisms that coordinate photosynthesis and stomatal conductance have yet to be identified. Here we identify and characterize CBC1/2 (CONVERGENCE OF BLUE LIGHT (BL) AND CO 2 1/2), two kinases that link BL, a major component of photosynthetically active radiation (PAR), and the signals from low concentrations of CO 2 in guard cells. CBC1/CBC2 redundantly stimulate stomatal opening by inhibition of S-type anion channels in response to both BL and low concentrations of CO 2 . CBC1/CBC2 function in the signaling pathways of phototropins and HT1 (HIGH LEAF TEMPERATURE 1). CBC1/CBC2 interact with and are phosphorylated by HT1. We propose that CBCs regulate stomatal aperture by integrating signals from BL and CO 2 and act as the convergence site for signals from BL and low CO 2.

Phototropin (phot1) is a blue light-activated plasma membrane-associated kinase that acts as the principal photoreceptor for shoot phototropism in Arabidopsis in conjunction with the signalling component Non-Phototropic Hypocotyl 3 (NPH3). PHOT1 is uniformly expressed throughout the Arabidopsis hypocotyl, yet decapitation experiments have localized the site of light perception to the upper hypocotyl. This prompted us to investigate in more detail the functional role of the hypocotyl apex, and the regions surrounding it, in establishing phototropism. We used a non-invasive approach where PHOT1-GFP (P1-GFP) expression was targeted to the hypocotyl apex of the phot-deficient mutant using the promoters of CUP-SHAPED COTYLEDON 3 (CUC3) and AINTEGUMENTA (ANT). Expression of CUC3::P1-GFP was clearly visible at the hypocotyl apex, with weaker expression in the cotyledons, whereas ANT::P1-GFP was specifically targeted to the developing leaves. Both lines showed impaired curvature to 0.005 μmol m -2  sec -1 unilateral blue light, indicating that regions below the apical meristem are necessary for phototropism. Curvature was however apparent at higher fluence rates. Moreover, CUC3::P1-GFP partially or fully complemented petiole positioning, leaf flattening and chloroplast accumulation, but not stomatal opening. Yet, tissue analysis of NPH3 de-phosphorylation showed that CUC3::P1-GFP and ANT::P1-GFP mis-express very low levels of phot1 that likely account for this responsiveness. Our spatial targeting approach therefore excludes the hypocotyl apex as the site for light perception for phototropism and shows that phot1-mediated NPH3 de-phosphorylation is tissue autonomous and occurs more prominently in the basal hypocotyl., (© 2016 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Phototropins are light-activated receptor kinases that mediate a wide range of blue light responses responsible for the optimization of photosynthesis. Despite the physiological importance of phototropins, it is still unclear how they transduce light signals into physiological responses. Here, we succeeded in reproducing a primary step of phototropin signaling in vitro using a physiological substrate of phototropin, the BLUS1 (BLUE LIGHT SIGNALING1) kinase of guard cells. When PHOT1 and BLUS1 were expressed in Escherichia coli and the resulting recombinant proteins were incubated with ATP, white and blue light induced phosphorylation of BLUS1 but red light and darkness did not. Site-directed mutagenesis of PHOT1 and BLUS1 revealed that the phosphorylation was catalyzed by phot1 kinase. Similar to stomatal blue light responses, the BLUS1 phosphorylation depended on the fluence rate of blue light and was inhibited by protein kinase inhibitors, K-252a and staurosporine. In contrast to the result in vivo, BLUS1 was not dephosphorylated in vitro, suggesting the involvement of a protein phosphatase in the response in vivo. phot1 with a C-terminal kinase domain but devoid of the N-terminal domain, constitutively phosphorylated BLUS1 without blue light, indicating that the N-terminal domain has an autoinhibitory action and prevents substrate phosphorylation. The results provide the first reconstitution of a primary step of phototropin signaling and a clue for understanding the molecular nature of this process., (© The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

PP1 (protein phosphatase 1) is among the most conserved enzymes known, with one or more isoforms present in all sequenced eukaryotic genomes. PP1 dephosphorylates specific serine/threonine phosphoproteins as defined by associated regulatory or targeting subunits. In the present study we performed a PP1-binding screen to find putative PP1 interactors in Arabidopsis thaliana and uncovered a homologue of the ancient PP1 interactor, I-2 (inhibitor-2). Bioinformatic analysis revealed remarkable conservation of three regions of plant I-2 that play key roles in binding to PP1 and regulating its function. The sequence-related properties of plant I-2 were compared across eukaryotes, indicating a lack of I-2 in some species and the emergence points from key motifs during the evolution of this ancient regulator. Biochemical characterization of AtI-2 (Arabidopsis I-2) revealed its ability to inhibit all plant PP1 isoforms and inhibitory dependence requiring the primary interaction motif known as RVXF. Arabidopsis I-2 was shown to be a phosphoprotein in vivo that was enriched in the nucleus. TAP (tandem affinity purification)-tag experiments with plant I-2 showed in vivo association with several Arabidopsis PP1 isoforms and identified other potential I-2 binding proteins.