Alexis De Angeli

Vacuolar transporters in their physiological context.

Vacuoles in vegetative tissues allow the plant surface to expand by accumulating energetically cheap inorganic osmolytes, and thereby optimize the plant for absorption of sunlight and production of energy by photosynthesis. Some specialized cells, such as guard cells and pulvini motor cells, exhibit rapid volume changes. These changes require the rapid release and uptake of ions and water by the vacuole and are a prerequisite for plant survival. Furthermore, seed vacuoles are important storage units for the nutrients required for early plant development. All of these fundamental processes rely on numerous vacuolar transporters. During the past 15 years, the transporters implicated in most aspects of vacuolar function have been identified and characterized. Vacuolar transporters appear to be integrated into a regulatory network that controls plant metabolism. However, little is known about the mode of action of these fundamental processes, and deciphering the underlying mechanisms remains a challenge for the future.

Anion channels/transporters in plants: from molecular bases to regulatory networks.

Anion channels/transporters are key to a wide spectrum of physiological functions in plants, such as osmoregulation, cell signaling, plant nutrition and compartmentalization of metabolites, and metal tolerance. The recent identification of gene families encoding some of these transport systems opened the way for gene expression studies, structure-function analyses of the corresponding proteins, and functional genomics approaches toward further understanding of their integrated roles in planta. This review, based on a few selected examples, illustrates that the members of a given gene family exhibit a diversity of substrate specificity, regulation, and intracellular localization, and are involved in a wide range of physiological functions. It also shows that post-translational modifications of transport proteins play a key role in the regulation of anion transport activity. Key questions arising from the increasing complexity of networks controlling anion transport in plant cells (the existence of redundancy, cross talk, and coordination between various pathways and compartments) are also addressed.

AtALMT9 is a malate-activated vacuolar chloride channel required for stomatal opening in Arabidopsis

Water deficit strongly affects crop productivity. Plants control water loss and CO2 uptake by regulating the aperture of the stomatal pores within the leaf epidermis. Stomata aperture is regulated by the two guard cells forming the pore and changing their size in response to ion uptake and release. While our knowledge about potassium and chloride fluxes across the plasma membrane of guard cells is advanced, little is known about fluxes across the vacuolar membrane. Here we present the molecular identification of the long-sought-after vacuolar chloride channel. AtALMT9 is a chloride channel activated by physiological concentrations of cytosolic malate. Single-channel measurements demonstrate that this activation is due to a malate-dependent increase in the channel open probability. Arabidopsis thaliana atalmt9 knockout mutants exhibited impaired stomatal opening and wilt more slowly than the wild type. Our findings show that AtALMT9 is a vacuolar chloride channel having a major role in controlling stomata aperture.

Malate transport by the vacuolar AtALMT6 channel in guard cells is subject to multiple regulation

Gas exchange in plants is controlled by guard cells, specialized cells acting as turgor pressure-driven valves. Malate is one of the major anions accumulated inside the vacuole during stomatal opening counteracting the positive charge of potassium. AtALMT6, a member of the aluminum-activated malate transporter family, is expressed in guard cells of leaves and stems as well as in flower organs of Arabidopsis thaliana. An AtALMT6-GFP fusion protein was targeted to the vacuolar membrane both in transient and stable expression systems. Patch-clamp experiments on vacuoles isolated from AtALMT6-GFP over-expressing Arabidopsis plants revealed large inward-rectifying malate currents only in the presence of micromolar cytosolic calcium concentrations. Further analyses showed that vacuolar pH and cytosolic malate regulate the threshold of activation of AtALMT6-mediated currents. The interplay of these two factors determines the AtALMT6 function as a malate influx or efflux channel depending on the tonoplast potential. Guard cell vacuoles isolated from Atalmt6 knock-out plants displayed reduced malate currents compared with wild-type vacuoles. This reduction, however, was not accompanied by phenotypic differences in the stomatal movements in knock-out plants, probably because of functional redundancy of malate transporters in guard cell vacuoles.

CLC-mediated anion transport in plant cells

Plants need nitrate for growth and store the major part of it in the central vacuole of cells from root and shoot tissues. Based on few studies on the two model plants Arabidopsis thaliana and rice, members of the large ChLoride Channel (CLC) family have been proposed to encode anion channels/transporters involved in nitrate homeostasis. Proteins from the Arabidopsis CLC family (AtClC, comprising seven members) are present in various membrane compartments including the vacuolar membrane (AtClCa), Golgi vesicles (AtClCd and AtClCf) or chloroplast membranes (AtClCe). Through a combination of electrophysiological and genetic approaches, AtClCa was shown to function as a 2NO3−/1H+ exchanger that is able to accumulate specifically nitrate into the vacuole, in agreement with the main phenotypic trait of knockout mutant plants that accumulate 50 per cent less nitrate than their wild-type counterparts. The set-up of a functional complementation assay relying on transient expression of AtClCa cDNA in the mutant background opens the way for studies on structure–function relationships of the AtClCa nitrate transporter. Such studies will reveal whether important structural determinants identified in bacterial or mammalian CLCs are also crucial for AtClCa transport activity and regulation.

ATP Binding to the C Terminus of the Arabidopsis thaliana Nitrate/Proton Antiporter, AtCLCa, Regulates Nitrate Transport into Plant Vacuoles

Nitrate, one of the major nitrogen sources for plants, is stored in the vacuole. Nitrate accumulation within the vacuole is primarily mediated by the NO3−/H+ exchanger AtCLCa, which belongs to the chloride channel (CLC) family. Crystallography analysis of hCLC5 suggested that the C-terminal domain, composed by two cystathionine β-synthetase motifs in all eukaryotic members of the CLC family is able to interact with ATP. However, interaction of nucleotides with a functional CLC protein has not been unambiguously demonstrated. Here we show that ATP reversibly inhibits AtCLCa by interacting with the C-terminal domain. Applying the patch clamp technique to isolated Arabidopsis thaliana vacuoles, we demonstrate that ATP reduces AtCLCa activity with a maximum inhibition of 60%. ATP inhibition of nitrate influx into the vacuole at cytosolic physiological nitrate concentrations suggests that ATP modulation is physiologically relevant. ADP and AMP do not decrease the AtCLCa transport activity; nonetheless, AMP (but not ADP) competes with ATP, preventing inhibition. A molecular model of the C terminus of AtCLCa was built by homology to hCLC5 C terminus. The model predicted the effects of mutations of the ATP binding site on the interaction energy between ATP and AtCLCa that were further confirmed by functional expression of site-directed mutated AtCLCa.

Intra- and extra-cellular excretion of carboxylates

Carboxylates, such as malate and citrate, are widely acknowledged to have a central role in plant metabolism. They are involved in the production of energy and its storage as well as contributing to the cellular osmolyte pool and participating in the regulation of cellular pH. As we discuss here, recent research has demonstrated the functional importance of carboxylate excretion into the soil, apoplast and vacuole, particular with respect to the regulation of stomatal and root function.

The proline 160 in the selectivity filter of the Arabidopsis NO3-/H+ exchanger AtCLCa is essential for nitrate accumulation in planta.

Nitrate, the major nitrogen source for plants, can be accumulated in the vacuole. Its transport across the vacuolar membrane is mediated by AtCLCa, an antiporter of the chloride channel (CLC) protein family. In contrast to other CLC family members, AtCLCa transports nitrate coupled to protons. Recently, the different behaviour towards nitrate of CLC proteins has been linked to the presence of a serine or proline in the selectivity filter motif GXGIP. By monitoring AtCLCa activity in its native environment, we show that if proline 160 in AtCLCa is changed to a serine (AtCLCa(P160S) ), the transporter loses its nitrate selectivity, but the anion proton exchange mechanism is unaffected. We also performed in vivo analyses in yeast and Arabidopsis. In contrast to native AtCLCa, expression of AtCLCa(P160S) does not complement either the ΔScCLC yeast mutant grown on nitrate or the nitrate under-accumulation phenotype of clca knockout plants. Our results confirm the significance of this amino acid in the conserved selectivity filter of CLC proteins and highlight the importance of the proline in AtCLCa for nitrate metabolism in Arabidopsis.

Anion channels and transporters in plant cell membranes

Anion channels/transporters appear as key players in signaling pathways leading to the adaptation of plant cells to abiotic and biotic environmental stresses, in the control of metabolism and in the maintenance of electrochemical gradients. Focusing on the most recent advances, this review aims at providing a description of the role of these channels in various physiological functions such as control of stomatal movements, plant–pathogen interaction, xylem loading, compartmentalization of metabolites and coupling with proton gradients. These functions have been demonstrated by a combination of electrophysiology, pharmacology and genetics approaches, the key issue being to identify the corresponding proteins and genes.

Phosphorylation of the vacuolar anion exchanger AtCLCa is required for the stomatal response to abscisic acid.

The Arabidopsis vacuolar anion/proton exchanger AtCLCa contributes to both opening and closing of stomata. One Exchanger to Open and Close the Pores Turgor pressure in the guard cells of plant leaves controls pores called stomata, which enable gas exchange and photosynthesis when open and limit water loss when closed. Light opens these pores, whereas the hormone abscisic acid (ABA) keeps them closed. Ions and water imported into or released from the vacuole control the turgor pressure of guard cells. Wege et al. found that Arabidopsis plants lacking the vacuolar anion/proton exchanger AtCLCa did not effectively open or close their stomata. In light, AtCLCa mediated the uptake of anions into the vacuole to aid in opening the pores, and ABA stimulated AtCLCa phosphorylation and enhanced its efflux activity, which helped to close the pores. Thus, a single exchanger can move ions bidirectionally depending on the plant’s photosynthetic and water conservation needs. Eukaryotic anion/proton exchangers of the CLC (chloride channel) family mediate anion fluxes across intracellular membranes. The Arabidopsis thaliana anion/proton exchanger AtCLCa is involved in vacuolar accumulation of nitrate. We investigated the role of AtCLCa in leaf guard cells, a specialized plant epidermal cell that controls gas exchange and water loss through pores called stomata. We showed that AtCLCa not only fulfilled the expected role of accumulating anions in the vacuole during stomatal opening but also mediated anion release during stomatal closure in response to the stress hormone abscisic acid (ABA). We found that this dual role resulted from a phosphorylation-dependent change in the activity of AtCLCa. The protein kinase OST1 (also known as SnRK2.6) is a key signaling player and central regulator in guard cells in response to ABA. Phosphorylation of Thr38 in the amino-terminal cytoplasmic domain of AtCLCa by OST1 increased the outward anion fluxes across the vacuolar membrane, which are essential for stomatal closure. We provide evidence that bidirectional activities of an intracellular CLC exchanger are physiologically relevant and that phosphorylation regulates the transport mode of this exchanger.