Significance: Guard cells play a crucial role in controlling transpiration and the plant water status. Here, we show that the Arabidopsis plasma membrane aquaporin PIP2;1 is involved in stomatal closure triggered by abscisic acid (ABA) or the pathogen-associated molecular pattern flg22. The use of a genetic probe for hydrogen peroxide (H ₂ O ₂ ) revealed that PIP2;1 is also required for intracellular accumulation of H ₂ O ₂ after flg22 or ABA treatments. Our data lead to a model whereby flg22 and ABA activate PIP2;1 through phosphorylation at a conserved site to facilitate transport of both water and H ₂ O ₂ and promote stomatal closure. This study fills a gap in our understanding of stomatal regulation and suggests a general signaling role of aquaporin in contexts involving H ₂ O ₂ .

Stomatal movements in response to environmental stimuli critically control the plant water status. Although these movements are governed by osmotically driven changes in guard cell volume, the role of membrane water channels (aquaporins) has remained hypothetical. Assays in epidermal peels showed that knockout Arabidopsis thaliana plants lacking the Plasma membrane Intrinsic Protein 2;1 (PIP2;1) aquaporin have a defect in stomatal closure, specifically in response to abscisic acid (ABA). ABA induced a 2-fold increase in osmotic water permeability (P_f) of guard cell protoplasts and an accumulation of reactive oxygen species in guard cells, which were both abrogated in pip2;1 plants. Open stomata 1 (OST1)/Snf1-related protein kinase 2.6 (SnRK2.6), a protein kinase involved in guard cell ABA signaling, was able to phosphorylate a cytosolic PIP2;1 peptide at Ser-121. OST1 enhanced PIP2;1 water transport activity when coexpressed in Xenopus laevis oocytes. Upon expression in pip2;1 plants, a phosphomimetic form (Ser121Asp) but not a phosphodeficient form (Ser121Ala) of PIP2;1 constitutively enhanced the P_f of guard cell protoplasts while suppressing its ABA-dependent activation and was able to restore ABA-dependent stomatal closure in pip2;1. This work supports a model whereby ABA-triggered stomatal closure requires an increase in guard cell permeability to water and possibly hydrogen peroxide, through OST1-dependent phosphorylation of PIP2;1 at Ser-121.

Aquaporins are channel proteins that facilitate the transport of water across plant cell membranes. In this work, we used a combination of pharmacological and reverse genetic approaches to investigate the overall significance of aquaporins for tissue water conductivity in Arabidopsis (Arabidopsis thaliana). We addressed the function in roots and leaves of AtPIP1;2, one of the most abundantly expressed isoforms of the plasma membrane intrinsic protein family. At variance with the water transport phenotype previously described in AtPIP2;2 knockout mutants, disruption of AtPIP1;2 reduced by 20% to 30% the root hydrostatic hydraulic conductivity but did not modify osmotic root water transport. These results document qualitatively distinct functions of different PIP isoforms in root water uptake. The hydraulic conductivity of excised rosettes (Kros) was measured by a novel pressure chamber technique. Exposure of Arabidopsis plants to darkness increased Kros by up to 90%. Mercury and azide, two aquaporin inhibitors with distinct modes of action, were able to induce similar inhibition of Kros by approximately 13% and approximately 25% in rosettes from plants grown in the light or under prolonged (11–18 h) darkness, respectively. Prolonged darkness enhanced the transcript abundance of several PIP genes, including AtPIP1;2. Mutant analysis showed that, under prolonged darkness conditions, AtPIP1;2 can contribute to up to approximately 20% of Kros and to the osmotic water permeability of isolated mesophyll protoplasts. Therefore, AtPIP1;2 can account for a significant portion of aquaporin-mediated leaf water transport. The overall work shows that AtPIP1;2 represents a key component of whole-plant hydraulics.

Water channel proteins, AQPs (aquaporins), of the PIP (plasma membrane intrinsic protein) subfamily, provide a means for fine and quick adjustments of the plant water status. A molecular model for gating of PIPs by cytosolic protons (H⁺) and divalent cations was derived from the atomic structure of spinach SoPIP2;1 (Spinacia oleracea PIP2;1) in an open- and a closed-pore conformation. In the present study, we produced the Arabidopsis AtPIP2;1 (Arabidopsis thaliana PIP2;1) homologue in Pichia pastoris, either WT (wild-type) or mutations at residues supposedly involved in gating. Stopped-flow spectrophotometric measurements showed that, upon reconstitution in proteoliposomes, all forms function as water channels. The first functional evidence for a direct gating of PIPs by divalent (bivalent) cations was obtained. In particular, cadmium and manganese were identified, in addition to calcium (Ca²⁺) and H⁺ as potent inhibitors of WT AtPIP2;1. Our results further show that His¹⁹⁹, the previously identified site for H⁺ sensing, but also N-terminal located Glu³¹, and to a lesser extent Asp²⁸, are involved in both divalent-cation- and H⁺-mediated gating. In contrast, mutation of Arg¹²⁴ rendered AtPIP2;1 largely insensitive to Ca²⁺ while remaining fully sensitive to H⁺. The role of these residues in binding divalent cations and/or stabilizing the open or closed pore conformations is discussed.