Channel characteristics of VDAC-3 from Arabidopsis thaliana.

Four different isoforms of the Voltage-Dependent Anion Channel (VDAC) have been identified in Arabidopsis plant cells. The electrophysiological characteristics of several VDAC channels from animal as well as plant cells are well documented, but those of this model plant are unknown. One isoform, AtVDAC-3 was obtained either directly by cell-free synthesis or produced in Escherichia coli, as inclusion bodies, and re-natured. An electrophysiological study of the purified proteins in planar lipid bilayers showed that both methods yielded proteins with similar channel activity. The characteristics of AtVDAC-3 are that of a bona fide VDAC-like channel.

The nitrate/proton antiporter AtCLCa mediates nitrate accumulation in plant vacuoles.

Nitrate, the major nitrogen source for most plants, is widely used as a fertilizer and as a result has become a predominant freshwater pollutant. Plants need nitrate for growth and store most of it in the central vacuole. Some members of the chloride channel (CLC) protein family, such as the torpedo-fish ClC-0 and mammalian ClC-1, are anion channels, whereas the bacterial ClC-ec1 and mammalian ClC-4 and ClC-5 have recently been characterized as Cl-/H+ exchangers with unknown cellular functions. Plant members of the CLC family are proposed to be anion channels involved in nitrate homeostasis; however, direct evidence for anion transport mediated by a plant CLC is still lacking. Here we show that Arabidopsis thaliana CLCa (AtCLCa) is localized to an intracellular membrane, the tonoplast of the plant vacuole, which is amenable to electrophysiological studies, and we provide direct evidence for its anion transport ability. We demonstrate that AtCLCa is able to accumulate specifically nitrate in the vacuole and behaves as a NO3-/H+ exchanger. For the first time, to our knowledge, the transport activity of a plant CLC is revealed, the antiporter mechanism of a CLC protein is investigated in a native membrane system, and this property is directly connected with its physiological role.

Characterization of active oxygen-producing proteins in response to hypo-osmolarity in tobacco and Arabidopsis cell suspensions: identification of a cell wall peroxidase.

The oxidative response induced by hypo-osmolarity is characterized in tobacco and Arabidopsis cells in order to identify the corresponding active oxygen-producing proteins. The pharmacological profiles of the oxidative responses were clearly different in the two plant materials, leading to the identification of distinct active oxygen producers in tobacco and Arabidopsis cells. In tobacco cells, a 100 kDa protein, localized in the plasma membrane, was demonstrated to produce active oxygen in the presence of NADPH. This production can be activated by fatty acids and is strongly depressed by diphenylene iodonium, as measured by an in vivo response. In Arabidopsis, 30 kDa and 34 kDa proteins localized in the cell wall were shown to be able to produce active oxygen in the presence of cofactors and the production is prevented by peroxidase inhibitors, as is the in vivo response. The two purified proteins were identified by mass spectrometry and both correspond to the peroxidase gene At5g64120.

Strategies to identify transport systems in plants.

Since the first molecular structures of plant transporters were discovered over a decade ago, considerable advances have been made in the study of plant membrane transport, but we still do not understand transport regulation. The genes encoding the transport systems in the various cell membranes are still to be identified, as are the physiological roles of most transport systems. A wide variety of complementary strategies are now available to study transport systems in plants, including forward and reverse genetics, proteomics, and in silico exploitation of the huge amount of information contained in the completely known genomic sequence of Arabidopsis.

Nucleotides provide a voltage-sensitive gate for the rapid anion channel of arabidopsis hypocotyl cells.

The rapid anion channel of Arabidopsis hypocotyl cells is highly voltage-dependent. At hyperpolarized potentials, the channel is closed, and membrane depolarization is required for channel activation. We have previously shown that channel gating is regulated by intracellular nucleotides. In the present study, we further analyze the channel gating, and we propose a mechanism to explain its regulation by voltage. In the absence of intracellular nucleotides, closure at hyperpolarized voltages is abolished. Structure-function studies of adenyl nucleotides show that the apparent gating charge of the current increases with the negative charge carried by nucleotides. We propose that the fast anion channel is gated by the voltage-dependent entry of free nucleotides into the pore, leading to a voltage-dependent block at hyperpolarized potentials. In agreement with this mechanism in which intracellular nucleotides need to be recruited to the channel pore, kinetic analyses of whole-cell and single-channel currents show that the rate of closure is faster when intracellular nucleotide concentration is increased, whereas the rate of channel activation is unchanged. Furthermore, decreasing the concentration of extracellular chloride enhances the intracellular nucleotide block. This result supports the hypothesis of a mechanism in which blocking nucleotides and permeant anions interact within the channel pore.

Protein kinases induced by osmotic stresses and elicitor molecules in tobacco cell suspensions: two crossroad MAP kinases and one osmoregulation-specific protein kinase.

Two protein kinases displaying mitogen-activated protein kinase (MAPK) properties are activated both by an hypoosmotic stress and by oligogalacturonides in tobacco cell suspensions [Cazalé et al. (1999) Plant J. 19, 297-307]. Using specific antibodies, they were identified as the salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK). The SIPK was also activated by an hyperosmotic stress, indicating that the same kinase may play a role both in hypo- and hyperosmotic signalling pathways, in addition to its involvement in the transduction of elicitor signals. Using immunoprecipitation followed by two-dimensional in-gel kinase assay, three molecular forms of the SIPK were observed, suggesting that additional modifications of the activated kinase may occur. In contrast to WIPK and SIPK, which are located at the crossroad of several transduction pathways initiated by elicitor or osmotic stimuli, a 44 kDa kinase, that would not belong to the MAPK family, appeared more specific to osmotic stress.

CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers.

The voltage-dependent chloride channel (CLC) family of membrane proteins has cognates in animals, yeast, bacteria and plants, and chloride-channel activity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localization of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were raised against an N-terminal polypeptide of CLC-Nt1. These antibodies were used to probe membrane proteins prepared by various cell-fractionation methods. These included aqueous two-phase partitioning (for plasma membranes), free-flow electrophoresis (for vacuolar and plasma membranes), intact vacuole isolation, Percoll-gradient centrifugation (for plastids and mitochondria) and stepped, linear, sucrose-density-gradient centrifugation (for mitochondria). Each purified membrane fraction was characterized with specific marker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma membranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In contrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and NAD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel ('IMAC') described previously in animal and plant mitochondria.

Anion channels in higher plants: functional characterization, molecular structure and physiological role.

Anion channels are well documented in various tissues, cell types and membranes of algae and higher plants, and current evidence supports their central role in cell signaling, osmoregulation, plant nutrition and metabolism. It is the aim of this review to illustrate through a few selected examples the variety of anion channels operating in plant cells and some of their regulation properties and unique physiological functions. In contrast, information on the molecular structure of plant anion channels has only recently started to emerge. Only a few genes coding for putative plant anion channels from the large chloride channel (CLC) family have been isolated, and current molecular data on these plant CLCs are presented and discussed. A major challenge remains to identify the genes encoding the various anion channels described so far in plant cells. Future prospects along this line are briefly outlined, as well as recent advances based on the use of knockout mutants in the model plant Arabidopsis thaliana to explore the physiological functions of anion channels in planta.

Disruption of putative anion channel gene AtCLC-a in Arabidopsis suggests a role in the regulation of nitrate content.

In animals and yeast, voltage-dependent chloride channels of the CLC family play a role in basic cellular functions such as epithelial transport, plasma membrane excitability, and control of pH and membrane potential in intracellular compartments. To assess the function of CLCs in plants, we searched for CLC insertion mutants in a library of Arabidopsis lines transformed by Agrobacterium tumefaciens transferred DNA (T-DNA). Using a polymerase chain reaction-based screening procedure, an Arabidopsis line that carries a T-DNA insertion within the C-terminus of the AtCLC-a coding sequence was identified. Progeny from this plant line, clca-1, showed dramatically altered transcription of the AtCLC-a gene. Plants homozygous for the clca-1 mutation exhibited normal development and a morphology indistinguishable from the wild-type. However, their capacity to accumulate nitrate under conditions of nitrate excess was reduced in roots and shoots, by approximately 50%, while chloride, sulphate and phosphate levels were similar to the wild-type. In addition, the herbicide chlorate, an analogue of nitrate, induced a faster and more pronounced chlorosis in mutant plants. Hypersensitivity to chlorate as well as decreased nitrate levels co-segregated with the T-DNA insertion. They were found at various time points of the clca-1 life cycle, supporting the idea that AtCLC-a has a general role in the control of the nitrate status in Arabidopsis. Concordant with such a function, AtCLC-a mRNA was found in roots and shoots, and its levels rapidly increased in both tissues upon addition of nitrate but not ammonium to the culture medium. The specificity of AtCLC-a function with respect to nitrate is further supported by a similar free amino acid content in wild-type and clca-1 plants. Although the cellular localization of AtCLC-a remains unclear, our results suggest that AtCLC-a plays a role in controlling the intracellular nitrate status.

Characterization of a nitrate-permeable channel able to mediate sustained anion efflux in hypocotyl cells from Arabidopsis thaliana.

We have characterized a new anionic current in Arabidopsis hypocotyl cells. This current, activated by membrane depolarization, has slow activation and deactivation kinetics in the 10 sec range. It presents many distinct properties from the rapid-type anion current already described on the same membrane. The slow-type channel is highly permeable to nitrate with a PNO3-/PCl- close to 20, but totally impermeable to sulphate. Activation of the channel requires cytosolic ATP and the slow current is partially inhibited by staurosporin, suggesting that channel regulation involves protein phosphorylation. The slow anion channel displays a unique pharmacological profile different from that of the rapid channel: the slow channel is inhibited by DIDS (4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid) with an IC50 of 26 microM. The slow and rapid anion channels are probably dedicated to specific functions: the first is able to mediate sustained anion efflux, while the second is a good candidate to be involved in fast electrical signalling.

Sulfate is both a substrate and an activator of the voltage-dependent anion channel of Arabidopsis hypocotyl cells.

On the basis of the anion content of in vitro-cultured Arabidopsis plantlets, we explored the selectivity of the voltage-dependent anion channel of the plasma membrane of hypocotyl cells. In the whole-cell configuration, substitution of cytosolic Cl(-) by different anions led to the following sequence of relative permeabilities: NO(3)(-) (2.6) >/= SO(4)(2-) (2.0) > Cl(-) (1.0) > HCO(3)(-) (0.8) >> malate(2-) (0.03). Large whole-cell currents were measured for NO(3)(-) and SO(4)(2-), about five to six times higher than the equivalent Cl(-) currents. Since SO(4)(2-) is usually considered to be a weakly permeant or non-permeant ion, the components of the large whole-cell current were explored in more detail. Aside from its permeation through the channel with a unitary conductance, about two-thirds that of Cl(-), SO(4)(2-) had a regulatory effect on channel activity by preventing the run-down of the anion current both in the whole-cell and the outside-out configuration, increasing markedly the whole-cell current. The fact that the voltage-dependent plasma membrane anion channel of hypocotyl cells can mediate large NO(3)(-) and SO(4)(2-) currents and is regulated by nucleotides favors the idea that this anion channel can contribute to the cellular homeostasis of important metabolized anions.

MAP kinase activation by hypoosmotic stress of tobacco cell suspensions: towards the oxidative burst response?

Hypoosmotic stress activates a phosphorylation-dependent oxidative burst. In-gel kinase assays were performed to characterize the protein kinases that could be implicated in osmoregulation and in the activation of the oxidative burst. Hypoosmotic stress activated several kinases among which 50 and 46 kDa proteins displayed mitogen-activated protein kinase (MAP kinase) properties. They phosphorylated myelin basic protein in the absence of calcium, were recognized by antibodies directed against human MAP kinases, and were phosphorylated on tyrosine. Immunoprecipitation with an antibody directed against the tobacco MAP kinase Ntf4 showed that at least one of the activated kinases would be Ntf4-like. Apigenin, a MAP kinase and cyclin-dependent kinase inhibitor which prevents the hypoosmotically induced oxidative burst (Cazale et al. 1998; Plant Physiol. 116, 659-669), inhibited these kinases in vitro suggesting that they may play a role in the activation of the oxidative burst. Like the oxidative response, activation of the kinases depended on extracellular calcium influx and protein kinases sensitive to staurosporine and 6-DMAP. However, kinase activation did not depend on effluxes through anion channels or on the oxidative burst. Two-dimensional in-gel kinase assays revealed the presence of three protein kinases with an apparent molecular mass of 50 kDa and one of 46 kDa, all four being activated by hypoosmotic stress. The same kinases were also activated by oligogalacturonides and salicylic acid, underlying the importance of these MAP kinases as common components of different signaling pathways triggered by different extracellular stimuli.

A novel immunological approach establishes that the auxin-binding protein, Nt-abp1, is an element involved in auxin signaling at the plasma membrane.

Interactions of a collection of monoclonal antibodies (mAbs) to the recombinant Nicotiana tabacum auxin-binding protein 1 (Nt-abp1) were extensively characterized using surface plasmon resonance. Dynamic interaction studies using combinations of Nt-abp1, synthetic peptides corresponding to conserved sequences within auxin-binding proteins, and the mAbs have shown that a number of the mAbs recognized discontinuous epitopes revealing the junction of distinct domains in the folded protein. In particular, the two putative auxin binding domains and the C terminus of the protein were shown to interact with each other in the folded protein. Using the auxin-induced electrical response of tobacco protoplasts as a functional assay, all the mAbs exhibited either auxin antagonist or hormonomimetic properties. These effects, measured for the first time in homologous conditions, confirm that Nt-abp1 is present at the plasma membrane and is involved in the activation of the auxin-dependent electrical response of tobacco protoplasts. Based on our surface plasmon resonance data, we propose that the key event leading to the activation of this auxin electrical response consists of a conformational change in Nt-abp1.

The sax1 dwarf mutant of Arabidopsis thaliana shows altered sensitivity of growth responses to abscisic acid, auxin, gibberellins and ethylene and is partially rescued by exogenous brassinosteroid.

Genetic approaches using Arabidopsis thaliana aimed at the identification of mutations affecting events involved in auxin signalling have usually led to the isolation of auxin-resistant mutants. From a selection screen specifically developed to isolate auxin-hypersensitive mutants, one mutant line was selected for its increased sensitivity to auxin (x 2 to 3) for the root elongation response. The genetic analysis of sax1 (hypersensitive to abscisic acid and auxin) indicated that the mutant phenotype segregates as a single recessive Mendelian locus, mapping to the lower arm of chromosome 1. Sax1 seedlings grown in vitro showed a short curled primary root and small, round, dark-green cotyledons. In the greenhouse, adult sax1 plants were characterized by a dwarf phenotype, delayed development and reduced fertility. Further physiological characterization of sax1 seedlings revealed that the most striking trait was a large increase (x 40) in ABA-sensitivity of root elongation and, to a lesser extent, of ABA-induced stomatal closure; in other respects, hypocotyl elongation was resistant to gibberellins and ethylene. These alterations in hormone sensitivity in sax1 plants co-segregated with the dwarf phenotype suggesting that processes involved in cell elongation are modified. Treatment of mutant seedlings with an exogenous brassinosteroid partially rescued a wild-type size, suggesting that brassinosteroid biosynthesis might be affected in sax1 plants. Wild-type sensitivities to ABA, auxin and gibberellins were also restored in sax1 plants by exogenous application of brassinosteroid, illustrating the pivotal importance of the BR-related SAX1 gene.

The sax1 mutation defines a new locus involved in the brassinosteroid biosynthesis pathway in Arabidopsis thaliana.

In this issue we described a dwarf mutant in Arabidopsis thaliana, sax1, which is affected in brassinosteroid biosynthesis. This primary defect is responsible for alterations in hormone sensitivity of sax1 plants characterized by the hypersensitivity of root elongation to abscisic acid and auxin and the insensitivity of hypocotyl growth to gibberellins and ethylene (Ephritikhine et al., 1999; Plant J. 18, 303-314). In this paper, we report the further characterization of the sax1 mutant aimed at identification of the mutated step in the brassinosteroid biosynthesis pathway. Rescue experiments with various intermediates of the pathway showed that the sax1 mutation alters a very early step catalyzing the oxidation and isomerization of 3 beta-hydroxyl, delta 5,6 precursors to 3-oxo, delta 4,5 steroids. The mapping of the mutation, the physiological properties of the mutant and the rescue experiments indicate that sax1 defines a new locus in the brassinosteroid biosynthesis pathway. The SAX1 protein is involved in brassinosteroid-dependent growth of seedlings in both light and dark conditions.

The auxin-binding protein Nt-ERabp1 alone activates an auxin-like transduction pathway.

Hyperpolarization of tobacco protoplasts is amongst the earliest auxin responses described. It has been proposed that the auxin-binding protein, ABP1, or a related protein could be involved in the first step of auxin perception at the plasma membrane. Using for the first time homologous conditions for interaction between the protein Nt-ERabp1 or a synthetic peptide corresponding to the C-terminus and tobacco protoplasts, we have demonstrated that both can induce the hyperpolarization response. The results show that Nt-ERabp1 or the C-terminal peptide alone activates the auxin pathway from the outer face of the plasma membrane.

Oxidative Burst and Hypoosmotic Stress in Tobacco Cell Suspensions

Oxidative burst constitutes an early response in plant defense reactions toward pathogens, but active oxygen production may also be induced by other stimuli. The oxidative response of suspension-cultured tobacco (Nicotiana tabacum cv Xanthi) cells to hypoosmotic and mechanical stresses was characterized. The oxidase involved in the hypoosmotic stress response showed similarities by its NADPH dependence and its inhibition by iodonium diphenyl with the neutrophil NADPH oxidase. Activation of the oxidative response by hypoosmotic stress needed protein phosphorylation and anion effluxes, as well as opening of Ca2+ channels. Inhibition of the oxidative response impaired Cl- efflux, K+ efflux, and extracellular alkalinization, suggesting that the oxidative burst may play a role in ionic flux regulation. Active oxygen species also induced the cross-linking of a cell wall protein, homologous to a soybean (Glycine max L.) extensin, that may act as part of cell volume and turgor regulation through modification of the physical properties of the cell wall.

Voltage-dependent anion channel of Arabidopsis hypocotyls: nucleotide regulation and pharmacological properties.

Plasma membrane anion channels are thought to play important roles in osmoregulation and signal transduction in higher plant cells. Knowledge of their pharmacology and regulation is of importance to unravel their physiological functions. In this study, we explore the pharmacological properties and the nucleotide regulation of the voltage-dependent anion channel of Arabidopsis hypocotyls. The pharmacological profile of this channel is characterized by a low sensitivity to most anion channel blockers. It is inhibited by niflumic acid with an IC50 of 80 microM, but poorly sensitive to IAA-94 and NPPB and insensitive to 9-AC and DIDS. Nucleotides alter the amplitude, the kinetics and the voltage-dependence of the channel. The main effect of nucleotides is a shift of the voltage-dependent gate of the channel toward depolarized potentials leading to a strong reduction of the current amplitude. This regulation does not require ATP hydrolysis as nonhydrolyzable ATP analogues-AMPPNP and ATP gamma S-also regulate the anion current. This suggests that a nucleotide binding site is involved in the regulation. The study of the properties of this putative nucleotide binding site reveals that (i) ATP regulates the channel with an EC50 of 0.7 mM, (ii) adenyl nucleotides modulate the channel with the following order of effectiveness: ATP > ADP > > AMP, and (iii) thiophosphate nucleotide analogues are the most potent agonists with EC50 in the range of 80 microM. The hypothesis that this regulation may couple the electrical properties of the membrane with the metabolic status of the cell is discussed.

Anion-channel blockers interfere with auxin responses in dark-grown Arabidopsis hypocotyls.

Anion channels are thought to participate in signal transduction and turgor regulation in higher plant cells. The regulation of hypocotyl cell elongation is a situation in which these channels could play important roles because it involves ionic fluxes that are implicated in turgor control and orchestrated by various signals. We have used a pharmacological approach to reveal the contribution of anion channels in the regulation of the development of hypocotyls by auxins. Auxins induce an inhibition of elongation, a disintegration of the cortical cell layers, and the formation of adventitious roots on Arabidopsis thaliana hypocotyls grown in the dark. Anion-channel blockers such as anthracene-9-carboxylic acid, 4,4'-diisothiocyanatostilbene-2-2'-disulfonic acid, 4-acetamido-4'-isothiocyanato-stilbene-2-2'-disulfonic acid, and R(+)-methylindazone; indanyloxyacteic acid-94, which produce little or no stimulation of hypocotyl elongation by themselves, are able to counteract the inhibition and the disintegration induced by auxins with various efficiencies. This interference appears to be specific for auxins and does not occur when hypocotyl elongation is inhibited by other growth regulators such as ethylene or cytokinins. The putative involvement of anion channels in auxin signal transduction is discussed.

Cloning and functional expression of a plant voltage-dependent chloride channel.

Plant cell membrane anion channels participate in basic physiological functions, such as cell volume regulation and signal transduction. However, nothing is known about their molecular structure. Using a polymerase chain reaction strategy, we have cloned a tobacco cDNA (CIC-Nt1) encoding a 780-amino acid protein with several putative transmembrane domains. CIC-Nt1 displays 24 to 32% amino acid identity with members of the animal voltage-dependent chloride channel (CIC) family, whose archetype is CIC-0 from the Torpedo marmorata electric organ. Injection of CIC-Nt1 complementary RNA into Xenopus oocytes elicited slowly activating inward currents upon membrane hyperpolarization more negative than -120 mV. These currents were carried mainly by anions, modulated by extracellular anions, and totally blocked by 10 mM extracellular calcium. The identification of CIC-Nt1 extends the CIC family to higher plants and provides a molecular probe for the study of voltage-dependent anion channels in plants.