Characterization of Determinants for the Specificity of Arabidopsis Thioredoxins h in Yeast Complementation

International audienceThe disruption of the two thioredoxin genes in Sac-charomyces cerevisiae leads to a complex phenotype, including the inability to use methionine sulfoxide as sulfur source, modified cell cycle parameters, reduced H 2 O 2 tolerance, and inability to use sulfate as sulfur source. Expression of one of the multiple Arabidopsis thaliana thioredoxins h in this mutant complements only some aspects of the phenotype, depending on the expressed thioredoxin: AtTRX2 or AtTRX3 induce me-thionine sulfoxide assimilation and restore a normal cell cycle. In addition AtTRX2 also confers growth on sulfate but no H 2 O 2 tolerance. In contrast, AtTRX3 does not confer growth on sulfate but induces H 2 O 2 tolerance. We have constructed hybrid proteins between these two thioredoxins and show that all information necessary for sulfate assimilation is present in the C-terminal part of AtTRX2, whereas some information needed for H 2 O 2 tolerance is located in the N-terminal part of AtTRX3. In addition, mutation of the atypical redox active site WCPPC to the classical site WCGPC restores some growth on sulfate. All these data suggest that the multiple Arabidopsis thioredoxins h originate from a totipo-tent ancestor with all the determinants necessary for interaction with the different thioredoxin target proteins. After duplications each member evolved by losing or masking some of the determinants

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

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 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 signaling mechanisms which ultimately act on plant growth

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

International audienceWater channel proteins (aquaporins) of the Plasma membrane Intrinsic Protein(PIP) subfamily provide 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 in an open- and a closed-pore conformation. Here, we produced in Pichia pastoris the Arabidopsis AtPIP2;1 homolog, either wild-type (WT) or with mutations at residues supposedly involved in gating. Stopped-flow spectrophotometric measurements showed that, upon reconstitution in proteoliposomes, all forms function as water channels. First functional evidence for a direct gating of PIPs by divalent 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 data 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 cations and H+-mediated gating. By 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

Aquaporins in plants: from molecular structures to integrated functions

Aquaporins belong to a superfamily of membrane channels with members in all living organisms. In plants, aquaporins mediate a large part of the cell-to-cell and intracellular water movements. The ability of certain plant aquaporins homologues to transport nutrient such as boron or gas such as CO2 has recently been demonstrated. This present chapter specifically examines how our current understanding of aquaporin structure and function can be integrated into whole plant physiology. Expression studies coupled with physiological and genetic analyses have allowed to delineate a variety of functions for aquaporins in roots, leaves, and during plant reproduction. In addition, a large variety of molecular and cellular mechanisms have been identified that lead to fine regulation of membrane water transport, during plant development, or in response to environmental stimuli. However, central physiological questions remain, such as the role of aquaporins in carbon assimilation, or in a hydraulic control of growth and cel

GENOMIQUE ET LIPIDES Génomique et métabolisme des lipides des plantes

Il existe dans les bases de données publiques une énorme quantité de séquences d’ADN dérivées de plantes, et notamment la séquence complète du génome d’Arabidopsis thaliana, une plante modèle pour les oléagineux, proche parente du colza. Ces données constituent une ressource importante non seulement pour la compréhension de métabolisme lipidique et de sa régulation, mais aussi pour la sélection et le développement de variétés nouvelles d’oléagineux produisant davantage d’huiles ou des huiles de composition nouvelle. Cette abondance de séquences peut être exploitée, en utilisant les recherches d’homologies, pour identifier les gènes, pour obtenir des informations sur leur fonction, comme pour repérer des gènes candidats codant des fonctions nouvelles. L’analyse de ces bases de données a révélé que la majeure partie des gènes codant des enzymes impliquées dans le métabolisme lipidique appartient à des petites familles multigéniques, reflétant la diversification des fonctions des isoformes. Une analyse du catalogue des ADNc séquencés en aveugle reflète les niveaux d’expression des différents gènes et fournit un aperçu des régulations des flux au travers des voies métaboliques conduisant à la biosynthèse des lipides de réserve. La disponibilité de mutants et de lignées transgéniques d’Arabidopsis et le développement de puces à ADN qui permettent l’analyse simultanée de plusieurs milliers de gènes conduiront à une meilleure compréhension des facteurs qui régulent le métabolisme des huiles dans les graines. Une telle connaissance facilitera la manipulation de la composition des huiles et des quantités produites dans les graines

GENOMIQUE ET LIPIDES Génomique et métabolisme des lipides des plantes

Il existe dans les bases de données publiques une énorme quantité de séquences d’ADN dérivées de plantes, et notamment la séquence complète du génome d’Arabidopsis thaliana, une plante modèle pour les oléagineux, proche parente du colza. Ces données constituent une ressource importante non seulement pour la compréhension de métabolisme lipidique et de sa régulation, mais aussi pour la sélection et le développement de variétés nouvelles d’oléagineux produisant davantage d’huiles ou des huiles de composition nouvelle. Cette abondance de séquences peut être exploitée, en utilisant les recherches d’homologies, pour identifier les gènes, pour obtenir des informations sur leur fonction, comme pour repérer des gènes candidats codant des fonctions nouvelles. L’analyse de ces bases de données a révélé que la majeure partie des gènes codant des enzymes impliquées dans le métabolisme lipidique appartient à des petites familles multigéniques, reflétant la diversification des fonctions des isoformes. Une analyse du catalogue des ADNc séquencés en aveugle reflète les niveaux d’expression des différents gènes et fournit un aperçu des régulations des flux au travers des voies métaboliques conduisant à la biosynthèse des lipides de réserve. La disponibilité de mutants et de lignées transgéniques d’Arabidopsis et le développement de puces à ADN qui permettent l’analyse simultanée de plusieurs milliers de gènes conduiront à une meilleure compréhension des facteurs qui régulent le métabolisme des huiles dans les graines. Une telle connaissance facilitera la manipulation de la composition des huiles et des quantités produites dans les graines

Plant aquaporins: membrane channels with multiple integrated functions.

International audienceAquaporins 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

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