Protein kinase SnRK2.4 is a key regulator of aquaporins and root hydraulics in Arabidopsis.

Soil water uptake by roots is a key component of plant water homeostasis contributing to plant growth and survival under ever-changing environmental conditions. The water transport capacity of roots (root hydraulic conductivity; Lpr ) is mostly contributed by finely regulated Plasma membrane Intrinsic Protein (PIP) aquaporins. In this study, we used natural variation of Arabidopsis for the identification of quantitative trait loci (QTLs) contributing to Lpr . Using recombinant lines from a biparental cross (Cvi-0 x Col-0), we show that the gene encoding class 2 Sucrose-Non-Fermenting Protein kinase 2.4 (SnRK2.4) in Col-0 contributes to >30% of Lpr by enhancing aquaporin-dependent water transport. At variance with the inactive and possibly unstable Cvi-0 SnRK2.4 form, the Col-0 form interacts with and phosphorylates the prototypal PIP2;1 aquaporin at Ser121 and stimulates its water transport activity upon coexpression in Xenopus oocytes and yeast cells. Activation of PIP2;1 by Col-0 SnRK2.4 in yeast also requires its protein kinase activity and can be counteracted by clade A Protein Phosphatases 2C. SnRK2.4 shows all hallmarks to be part of core abscisic acid (ABA) signaling modules. Yet, long-term (>3 h) inhibition of Lpr by ABA possibly involves a SnRK2.4-independent inhibition of PIP2;1. SnRK2.4 also promotes stomatal aperture and ABA-induced inhibition of primary root growth. The study identifies a key component of Lpr and sheds new light on the functional overlap and specificity of SnRK2.4 with respect to other ABA-dependent or independent SnRK2s.

Aquaporins in Plants.

Aquaporins are membrane channels that facilitate the transport of water and small neutral molecules across biological membranes of most living organisms. In plants, aquaporins occur as multiple isoforms reflecting a high diversity of cellular localizations, transport selectivity, and regulation properties. Plant aquaporins are localized in the plasma membrane, endoplasmic reticulum, vacuoles, plastids and, in some species, in membrane compartments interacting with symbiotic organisms. Plant aquaporins can transport various physiological substrates in addition to water. Of particular relevance for plants is the transport of dissolved gases such as carbon dioxide and ammonia or metalloids such as boron and silicon. Structure-function studies are developed to address the molecular and cellular mechanisms of plant aquaporin gating and subcellular trafficking. Phosphorylation plays a central role in these two processes. These mechanisms allow aquaporin regulation in response to signaling intermediates such as cytosolic pH and calcium, and reactive oxygen species. Combined genetic and physiological approaches are now integrating this knowledge, showing that aquaporins play key roles in hydraulic regulation in roots and leaves, during drought but also in response to stimuli as diverse as flooding, nutrient availability, temperature, or light. A general hydraulic control of plant tissue expansion by aquaporins is emerging, and their role in key developmental processes (seed germination, emergence of lateral roots) has been established. Plants with genetically altered aquaporin functions are now tested for their ability to improve plant tolerance to stresses. In conclusion, research on aquaporins delineates ever expanding fields in plant integrative biology thereby establishing their crucial role in plants.

Hormonal and environmental signaling pathways target membrane water transport.

Plant water transport and its molecular components including aquaporins are responsive, across diverse time scales, to an extremely wide array of environmental and hormonal signals. These include water deficit and abscisic acid (ABA) but also more recently identified stimuli such as peptide hormones or bacterial elicitors. The present review makes an inventory of corresponding signalling pathways. It identifies some main principles, such as the central signalling role of ROS, with a dual function of aquaporins in water and hydrogen peroxide transport, the importance of aquaporin phosphorylation that is targeted by multiple classes of protein kinases, and the emerging role of lipid signalling. More studies including systems biology approaches are now needed to comprehend how plant water transport can be adjusted in response to combined stresses.

Root Membrane Ubiquitinome under Short-Term Osmotic Stress.

Osmotic stress can be detrimental to plants, whose survival relies heavily on proteomic plasticity. Protein ubiquitination is a central post-translational modification in osmotic-mediated stress. In this study, we used the K-Ɛ-GG antibody enrichment method integrated with high-resolution mass spectrometry to compile a list of 719 ubiquitinated lysine (K-Ub) residues from 450 Arabidopsis root membrane proteins (58% of which are transmembrane proteins), thereby adding to the database of ubiquitinated substrates in plants. Although no ubiquitin (Ub) motifs could be identified, the presence of acidic residues close to K-Ub was revealed. Our ubiquitinome analysis pointed to a broad role of ubiquitination in the internalization and sorting of cargo proteins. Moreover, the simultaneous proteome and ubiquitinome quantification showed that ubiquitination is mostly not involved in membrane protein degradation in response to short osmotic treatment but that it is putatively involved in protein internalization, as described for the aquaporin PIP2;1. Our in silico analysis of ubiquitinated proteins shows that two E2 Ub-conjugating enzymes, UBC32 and UBC34, putatively target membrane proteins under osmotic stress. Finally, we revealed a positive role for UBC32 and UBC34 in primary root growth under osmotic stress.

Aquaporins facilitate hydrogen peroxide entry into guard cells to mediate ABA- and pathogen-triggered stomatal closure.

Stomatal movements are crucial for the control of plant water status and protection against pathogens. Assays on epidermal peels revealed that, similar to abscisic acid (ABA), pathogen-associated molecular pattern (PAMP) flg22 requires the AtPIP2;1 aquaporin to induce stomatal closure. Flg22 also induced an increase in osmotic water permeability (Pf) of guard cell protoplasts through activation of AtPIP2;1. The use of HyPer, a genetic probe for intracellular hydrogen peroxide (H2O2), revealed that both ABA and flg22 triggered an accumulation of H2O2 in wild-type but not pip2;1 guard cells. Pretreatment of guard cells with flg22 or ABA facilitated the influx of exogenous H2O2 Brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1) and open stomata 1 (OST1)/Snf1-related protein kinase 2.6 (SnRK2.6) were both necessary to flg22-induced Pf and both phosphorylated AtPIP2;1 on Ser121 in vitro. Accumulation of H2O2 and stomatal closure as induced by flg22 was restored in pip2;1 guard cells by a phosphomimetic form (Ser121Asp) but not by a phosphodeficient form (Ser121Ala) of AtPIP2;1. We propose a mechanism whereby phosphorylation of AtPIP2;1 Ser121 by BAK1 and/or OST1 is triggered in response to flg22 to activate its water and H2O2 transport activities. This work establishes a signaling role of plasma membrane aquaporins in guard cells and potentially in other cellular context involving H2O2 signaling.

Aquaporins Contribute to ABA-Triggered Stomatal Closure through OST1-Mediated Phosphorylation.

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 (Pf) 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 Pf 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 and plant transpiration.

Although transpiration and aquaporins have long been identified as two key components influencing plant water status, it is only recently that their relations have been investigated in detail. The present review first examines the various facets of aquaporin function in stomatal guard cells and shows that it involves transport of water but also of other molecules such as carbon dioxide and hydrogen peroxide. At the whole plant level, changes in tissue hydraulics mediated by root and shoot aquaporins can indirectly impact plant transpiration. Recent studies also point to a feedback effect of transpiration on aquaporin function. These mechanisms may contribute to the difference between isohydric and anisohydric stomatal regulation of leaf water status. The contribution of aquaporins to transpiration control goes far beyond the issue of water transport during stomatal movements and involves emerging cellular and long-distance signalling mechanisms which ultimately act on plant growth.

Plant aquaporins: membrane channels with multiple integrated functions.

Aquaporins are channel proteins present in the plasma and intracellular membranes of plant cells, where they facilitate the transport of water and/or small neutral solutes (urea, boric acid, silicic acid) or gases (ammonia, carbon dioxide). Recent progress was made in understanding the molecular bases of aquaporin transport selectivity and gating. The present review examines how a wide range of selectivity profiles and regulation properties allows aquaporins to be integrated in numerous functions, throughout plant development, and during adaptations to variable living conditions. Although they play a central role in water relations of roots, leaves, seeds, and flowers, aquaporins have also been linked to plant mineral nutrition and carbon and nitrogen fixation.

Structure-function analysis of plant aquaporin AtPIP2;1 gating by divalent cations and protons.

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 (Ca(2+)) and H(+) as potent inhibitors of WT AtPIP2;1. Our results further show that His(199), the previously identified site for H(+) sensing, but also N-terminal located Glu(31), and to a lesser extent Asp(28), are involved in both divalent-cation- and H(+)-mediated gating. In contrast, mutation of Arg(124) rendered AtPIP2;1 largely insensitive to Ca(2+) 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.

Methylation of aquaporins in plant plasma membrane.

A thorough analysis, using MS, of aquaporins expressed in plant root PM (plasma membrane) was performed, with the objective of revealing novel post-translational regulations. Here we show that the N-terminal tail of PIP (PM intrinsic protein) aquaporins can exhibit multiple modifications and is differentially processed between members of the PIP1 and PIP2 subclasses. Thus the initiating methionine was acetylated or cleaved in native PIP1 and PIP2 isoforms respectively. In addition, several residues were detected to be methylated in PIP2 aquaporins. Lys3 and Glu6 of PIP2;1, one of the most abundant aquaporins in the PM, occurred as di- and mono-methylated residues respectively. Ectopic expression in Arabidopsis suspension cells of PIP2;1, either wild-type or with altered methylation sites, revealed an interplay between methylation at the two sites. Measurements of water transport in PM vesicles purified from these cells suggested that PIP2;1 methylation does not interfere with the aquaporin intrinsic water permeability. In conclusion, the present study identifies methylation as a novel post-translational modification of aquaporins, and even plant membrane proteins, and may represent a critical advance towards the identification of new regulatory mechanisms of membrane transport.

Plant aquaporins on the move: reversible phosphorylation, lateral motion and cycling.

Aquaporins are channel proteins present in the plasma membrane and most of intracellular compartments of plant cells. This review focuses on recent insights into the cellular function of plant aquaporins, with an emphasis on the subfamily of Plasma membrane Intrinsic Proteins (PIPs). Whereas PIPs mostly serve as water channels, novel functions associated with their ability to transport carbon dioxide and hydrogen peroxide are emerging. Phosphorylation of PIPs was found to play a central role in the mechanisms that determine their gating and subcellular dynamics. Dynamic tracking of single aquaporin molecules in native plant membranes and the search for cell signaling intermediates acting upstream of aquaporins are now used to dissect their cellular regulation by hormonal and environmental stimuli.

The cellular dynamics of plant aquaporin expression and functions.

Aquaporins are channel proteins that facilitate the transport of water and small neutral molecules, including gases, across cell membranes of most of the living organisms. Integrative studies have stressed the role of aquaporins in maintaining the whole plant water and nutrient status. Cellular aspects of plant aquaporin functions and regulations are also extensively investigated. The present review provides a glance at recent progresses in this area. One first direction concerns the mechanisms that determine aquaporin targeting to specific subcellular membranes and a dynamic and stimulus-dependent control of their density in these membranes. The regulation of aquaporin opening and closing and its links to cell signalling cascades are also discussed. Multiple cellular functions are now attributed to plant aquaporins. They include the dynamic equilibration and subcellular partitioning of their various substrates and a contribution to cell expansion and possibly cell division.

Mutational and structural analysis of aglycone specificity in maize and sorghum beta-glucosidases.

Plant beta-glucosidases display varying substrate specificities. The maize beta-glucosidase isozyme Glu1 (ZmGlu1) hydrolyzes a broad spectrum of substrates in addition to its natural substrate DIMBOA-Glc (2-O-beta-d-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxaxin-3-one), whereas the sorghum beta-glucosidase isozyme Dhr1 (SbDhr1) hydrolyzes exclusively its natural substrate dhurrin (p-hydroxy-(S)-mandelonitrile-beta-d-glucoside). Structural data from cocrystals of enzyme-substrate and enzyme-aglycone complexes have shown that five amino acid residues (Phe198, Phe205, Trp378, Phe466, and Ala467) are located in the aglycone-binding site of ZmGlu1 and form the basis of aglycone recognition and binding, hence substrate specificity. To study the mechanism of substrate specificity further, mutant beta-glucosidases were generated by replacing Phe198, Phe205, Asp261, Met263, Phe377, Phe466, Ala467, and Phe473 of Glu1 by Dhr1 counterparts. The effects of mutations on enzyme activity and substrate specificity were studied using both natural and artificial substrates. The simple mutant replacing Phe198 by a valine had the most drastic effect on activity, because the capacity of this enzyme to hydrolyze beta-glucosides was almost completely abolished. The analysis of this mutation was completed by a structural study of the double mutant ZmGlu1-E191D,F198V in complex with the natural substrate. The structure reveals that the single mutation F198V causes a cascade of conformational changes, which are unpredictable by standard molecular modeling techniques. Some other mutations led to drastic effects: replacing Asp261 by an asparagine decreases the catalytic efficiency of this simple mutant by 75% although replacing Tyr473 by a phenylalanine increase its efficiency by 300% and also provides a new substrate specificity by hydrolyzing dhurrin.

Structural determinants of substrate specificity in family 1 beta-glucosidases: novel insights from the crystal structure of sorghum dhurrinase-1, a plant beta-glucosidase with strict specificity, in complex with its natural substrate.

Plant beta-glucosidases play a crucial role in defense against pests. They cleave, with variable specificity, beta-glucosides to release toxic aglycone moieties. The Sorghum bicolor beta-glucosidase isoenzyme Dhr1 has a strict specificity for its natural substrate dhurrin (p-hydroxy-(S)-mandelonitrile-beta-D-glucoside), whereas its close homolog, the maize beta-glucosidase isoenzyme Glu1, which shares 72% sequence identity, hydrolyzes a broad spectrum of substrates in addition to its natural substrate 2-O-beta-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxaxin-3-one. Structural data from enzyme.substrate complexes of Dhr1 show that the mode of aglycone binding differs from that previously observed in the homologous maize enzyme. Specifically, the data suggest that Asn(259), Phe(261), and Ser(462), located in the aglycone-binding site of S. bicolor Dhr1, are crucial for aglycone recognition and binding. The tight binding of the aglycone moiety of dhurrin promotes the stabilization of the reaction intermediate in which the glycone moiety is in a deformed (1)S(3) conformation within the glycone-binding site, ready for nucleophilic attack to occur. Compared with the broad specificity maize beta-glucosidase, this different binding mode explains the narrow specificity of sorghum dhurrinase-1.

Characterization of the yeast peroxiredoxin Ahp1 in its reduced active and overoxidized inactive forms using NMR.

Peroxiredoxins (Prx's) are a superfamily of thiol-specific antioxidant proteins present in all organisms and involved in the hydroperoxide detoxification of the cell. The catalytic cysteine of Prx's reduces hydroperoxides and is transformed into a transient sulfenic acid (Cys-SOH). At high hydroperoxide concentration, the sulfenic acid can be overoxidized into a sulfinate, or even a sulfonate. We present here the first peroxiredoxin characterization by solution NMR of the Saccharomyces cerevisiae alkylhydroperoxide reductase (Ahp1) in its reduced and in vitro overoxidized forms. NMR (15)N relaxation data and ultracentrifugation experiments indicate that the protein behaves principally as a homodimer (2 x 19 kDa) in solution, regardless of the redox state. In vitro treatment of Ahp1 by a large excess of tBuOOH leads to an inactive form, with the catalytic cysteine overoxidized into sulfonate, as demonstrated by (13)C NMR. Depending on the amino acid sequence of their active site, Prx's are classified into five different families. In this classification, Ahp1 is a member of the scarcely studied D-type Prx's. Ahp1 is unique among the D-type Prx's in its ability to form an intermolecular disulfide. The peptidic sequence of Ahp1 was analyzed and compared to other D-type Prx sequences.