Caracterisation physiologique et fonctionnelle du transporteur anionique ATCLC-C chez Arabidopsis Thaliana

Chez les végétaux supérieurs, la régulation des mouvements stomatiques permet de contrôler les échanges de CO2 et la montée de la sève brute tout en limitant les pertes excessives d'eau par transpiration. Ce contrôle est assuré par des variations rapides de la turgescence des deux cellules de garde formant le stomate dues à l'activité de nombreux canaux et transporteurs ioniques. Nous avons identifié un nouveau membre de la famille des CLC chez A. thaliana, AtCLC-c exprimé dans la cellule de garde. L'étude de l'expression d'AtCLC-c et du phénotype de mutants invalidés ont permis de démontrer son rôle dans l'ouverture stomatique à la lumière et la fermeture en réponse à l'ABA. Les mutants clcc accumulent moins d'ions Cl- dans leurs cellules de garde par rapport aux plantes sauvages et sont hypersensibles à un stress salin. Enfin, nous avons confirmé par des études d'électrophysiologie la sélectivité d'AtCLC-c aux ions Cl-. L'ensemble de ce travail montre l'importance du transporteur vacuolaire d'ions Cl- AtCLC-c dans les mouvements stomatiques et la tolérance au stress salin.In plants, the high turgor is assured by ion transport and involves the creation and maintenance of a large vacuolar volume. In recent years, various chloride channels and transporters have been identified to be involved in specific functions such as plant nutrition, stomatal movements or metal tolerance. We have characterized a new member of the CLC family in A. thaliana, AtCLC-c, highly expressed in guard cell and up-regulated by ABA and salt treatment in the whole plant. Knock-out mutants in AtCLC-c are impaired in light-induced stomatal opening and ABA-induced stomatal closing correlated to a large decrease in guard cell Cl- content. Furthermore, clcc mutants are hypersensitive to salt stress compared to wild-type. Finally, using electrophysiological studies, we demonstrated that AtCLC-c is selective to Cl-. Altogether, this work shows that AtCLC-c is a tonoplastic Cl- transporter involved in stomatal movements and salt tolerance.AIX-MARSEILLE2-Bib.electronique (130559901) / SudocSudocFranceF

Isolation of a strong Arabidopsis guard cell promoter and its potential as a research tool

<p>Abstract</p> <p>Background</p> <p>A common limitation in guard cell signaling research is that it is difficult to obtain consistent high expression of transgenes of interest in <it>Arabidopsis </it>guard cells using known guard cell promoters or the constitutive 35S cauliflower mosaic virus promoter. An additional drawback of the 35S promoter is that ectopically expressing a gene throughout the organism could cause pleiotropic effects. To improve available methods for targeted gene expression in guard cells, we isolated strong guard cell promoter candidates based on new guard cell-specific microarray analyses of 23,000 genes that are made available together with this report.</p> <p>Results</p> <p>A promoter, <it>pGC1</it>(At1g22690), drove strong and relatively specific reporter gene expression in guard cells including GUS (beta-glucuronidase) and yellow cameleon YC3.60 (GFP-based calcium FRET reporter). Reporter gene expression was weaker in immature guard cells. The expression of YC3.60 was sufficiently strong to image intracellular Ca²⁺dynamics in guard cells of intact plants and resolved spontaneous calcium transients in guard cells. The <it>GC1 </it>promoter also mediated strong reporter expression in clustered stomata in the stomatal development mutant <it>too-many-mouths </it>(<it>tmm</it>). Furthermore, the same promoter::reporter constructs also drove guard cell specific reporter expression in tobacco, illustrating the potential of this promoter as a method for high level expression in guard cells. A serial deletion of the promoter defined a guard cell expression promoter region. In addition, anti-sense repression using <it>pGC1 </it>was powerful for reducing specific GFP gene expression in guard cells while expression in leaf epidermal cells was not repressed, demonstrating strong cell-type preferential gene repression.</p> <p>Conclusion</p> <p>The <it>pGC1 </it>promoter described here drives strong reporter expression in guard cells of <it>Arabidopsis </it>and tobacco plants. It provides a potent research tool for targeted guard cell expression or gene silencing. It is also applicable to reduce specific gene expression in guard cells, providing a method for circumvention of limitations arising from genetic redundancy and lethality. These advances could be very useful for manipulating signaling pathways in guard cells and modifying plant performance under stress conditions. In addition, new guard cell and mesophyll cell-specific 23,000 gene microarray data are made publicly available here.</p

Blue Light Induces a Distinct Starch Degradation Pathway in Guard Cells for Stomatal Opening

Stomatal pores form a crucial interface between the leaf mesophyll and the atmosphere, controlling water and carbon balance in plants [1]. Major advances have been made in understanding the regulatory networks and ion fluxes in the guard cells surrounding the stomatal pore [2]. However, our knowledge on the role of carbon metabolism in these cells is still fragmentary [3-5]. In particular, the contribution of starch in stomatal opening remains elusive [6]. Here, we used Arabidopsis thaliana as a model plant to provide the first quantitative analysis of starch turnover in guard cells of intact leaves during the diurnal cycle. Starch is present in guard cells at the end of night, unlike in the rest of the leaf, but is rapidly degraded within 30 min of light. This process is critical for the rapidity of stomatal opening and biomass production. We exploited Arabidopsis molecular genetics to define the mechanism and regulation of guard cell starch metabolism, showing it to be mediated by a previously uncharacterized pathway. This involves the synergistic action of β-amylase 1 (BAM1) and α-amylase 3 (AMY3) - enzymes that are normally not required for nighttime starch degradation in other leaf tissues. This pathway is under the control of the phototropin-dependent blue-light signaling cascade and correlated with the activity of the plasma membrane H+-ATPase. Our results show that guard cell starch degradation has an important role in plant growth by driving stomatal responses to light

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

Exploring emergent properties in cellular homeostasis using OnGuard to model K+ and other ion transport in guard cells

It is widely recognized that the nature and characteristics of transport across eukaryotic membranes are so complex as to defy intuitive understanding. In these circumstances, quantitative mathematical modeling is an essential tool, both to integrate detailed knowledge of individual transporters and to extract the properties emergent from their interactions. As the first, fully integrated and quantitative modeling environment for the study of ion transport dynamics in a plant cell, OnGuard offers a unique tool for exploring homeostatic properties emerging from the interactions of ion transport, both at the plasma membrane and tonoplast in the guard cell. OnGuard has already yielded detail sufficient to guide phenotypic and mutational studies, and it represents a key step toward ‘reverse engineering’ of stomatal guard cell physiology, based on rational design and testing in simulation, to improve water use efficiency and carbon assimilation. Its construction from the HoTSig libraries enables translation of the software to other cell types, including growing root hairs and pollen. The problems inherent to transport are nonetheless challenging, and are compounded for those unfamiliar with conceptual ‘mindset’ of the modeler. Here we set out guidelines for the use of OnGuard and outline a standardized approach that will enable users to advance quickly to its application both in the classroom and laboratory. We also highlight the uncanny and emergent property of OnGuard models to reproduce the ‘communication’ evident between the plasma membrane and tonoplast of the guard cell

Xylem K+ loading modulates K+ and Cs+ absorption and distribution in Arabidopsis under K+-limited conditions

Potassium (K+) is an essential macronutrient for plant growth. The transcriptional regulation of K+ transporter genes is one of the key mechanisms by which plants respond to K+ deficiency. Among the HAK/KUP/KT transporter family, HAK5, a high-affinity K+ transporter, is essential for root K+ uptake under low external K+ conditions. HAK5 expression in the root is highly induced by low external K+ concentration. While the molecular mechanisms of HAK5 regulation have been extensively studied, it remains unclear how plants sense and coordinates K+ uptake and translocation in response to changing environmental conditions. Using skor mutants, which have a defect in root-to-shoot K+ translocation, we have been able to determine how the internal K+ status affects the expression of HAK5. In skor mutant roots, under K+ deficiency, HAK5 expression was lower than in wild-type although the K+ concentration in roots was not significantly different. These results reveal that HAK5 is not only regulated by external K+ conditions but it is also regulated by internal K+ levels, which is in agreement with recent findings. Additionally, HAK5 plays a major role in the uptake of Cs+ in roots. Therefore, studying Cs+ in roots and having more detailed information about its uptake and translocation in the plant would be valuable. Radioactive tracing experiments revealed not only a reduction in the uptake of 137Cs+ and 42K+in skor mutants compared to wild-type but also a different distribution of 137Cs+ and 42K+ in tissues. In order to gain insight into the translocation, accumulation, and repartitioning of both K+ and Cs+ in plants, long-term treatment and split root experiments were conducted with the stable isotopes 133Cs+ and 85Rb+. Finally, our findings show that the K+ distribution in plant tissues regulates root uptake of K+ and Cs+ similarly, depending on HAK5; however, the translocation and accumulation of the two elements are different

14-3-3 Proteins in Guard Cell Signaling

International audienceGuard cells are specialized cells located at the leaf surface delimiting pores whichcontrol gas exchanges between the plant and the atmosphere. To optimize the CO$_2$uptake necessary for photosynthesis while minimizing water loss, guard cells integrateenvironmental signals to adjust stomatal aperture. The size of the stomatal pore isregulated by movements of the guard cells driven by variations in their volume andturgor. As guard cells perceive and transduce a wide array of environmental cues, theyprovide an ideal system to elucidate early events of plant signaling. Reversible proteinphosphorylation events are known to play a crucial role in the regulation of stomatalmovements. However, in some cases, phosphorylation alone is not sufficient to achievecomplete protein regulation, but is necessary to mediate the binding of interactorsthat modulate protein function. Among the phosphopeptide-binding proteins, the 14-3-3 proteins are the best characterized in plants. The 14-3-3s are found as multipleisoforms in eukaryotes and have been shown to be involved in the regulation of stomatalmovements. In this review, we describe the current knowledge about 14-3-3 roles inthe regulation of their binding partners in guard cells: receptors, ion pumps, channels,protein kinases, and some of their substrates. Regulation of these targets by 14-3-3 proteins is discussed and related to their function in guard cells during stomatalmovements in response to abiotic or biotic stresses