Variations in Mn(II) speciation among organisms: what makes D. radiodurans different.

The manganese(II) speciation in intact cells of D. radiodurans, E. coli, S. cerevisiae and Arabidopsis thaliana seeds was measured using high-field electron paramagnetic resonance techniques. The majority of the Mn(II) ions in these organisms were six-coordinate, bound predominately by water, phosphates and nitrogen-based molecules. The relative distribution of the different phosphates in bacteria and S. cerevisiae was the same and dominated by monophosphate monoesters. Mn(II) was also found bound to the phosphate backbone of nucleic acids in these organisms. Phosphate ligation in Arabidopsis seeds was dominated by phytate. The extent of nitrogen ligation in the four organisms was also determined. On average, the Mn(II) in D. radiodurans had the most nitrogen ligands followed by E. coli. This was attributed to higher concentrations of Mn(II) bound to proteins in these species. Although constitutively expressed in all four organisms, MnSOD was only detected in D. radiodurans. As previously reported, D. radiodurans also accumulates a second abundant Mn containing protein species. The high concentration of proteinaceous Mn(II) is a unique feature of D. radiodurans.

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.

Phylogenetic relationships within cation transporter families of Arabidopsis.

Uptake and translocation of cationic nutrients play essential roles in physiological processes including plant growth, nutrition, signal transduction, and development. Approximately 5% of the Arabidopsis genome appears to encode membrane transport proteins. These proteins are classified in 46 unique families containing approximately 880 members. In addition, several hundred putative transporters have not yet been assigned to families. In this paper, we have analyzed the phylogenetic relationships of over 150 cation transport proteins. This analysis has focused on cation transporter gene families for which initial characterizations have been achieved for individual members, including potassium transporters and channels, sodium transporters, calcium antiporters, cyclic nucleotide-gated channels, cation diffusion facilitator proteins, natural resistance-associated macrophage proteins (NRAMP), and Zn-regulated transporter Fe-regulated transporter-like proteins. Phylogenetic trees of each family define the evolutionary relationships of the members to each other. These families contain numerous members, indicating diverse functions in vivo. Closely related isoforms and separate subfamilies exist within many of these gene families, indicating possible redundancies and specialized functions. To facilitate their further study, the PlantsT database (http://plantst.sdsc.edu) has been created that includes alignments of the analyzed cation transporters and their chromosomal locations.

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.

Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells.

Drought is a major threat to agricultural production. Plants synthesize the hormone abscisic acid (ABA) in response to drought, triggering a signalling cascade in guard cells that results in stomatal closure, thus reducing water loss. ABA triggers an increase in cytosolic calcium in guard cells ([Ca2+]cyt) that has been proposed to include Ca2+ influx across the plasma membrane. However, direct recordings of Ca2+ currents have been limited and the upstream activation mechanisms of plasma membrane Ca2+ channels remain unknown. Here we report activation of Ca2+-permeable channels in the plasma membrane of Arabidopsis guard cells by hydrogen peroxide. The H2O2-activated Ca2+ channels mediate both influx of Ca2+ in protoplasts and increases in [Ca2+]cyt in intact guard cells. ABA induces the production of H2O2 in guard cells. If H2O2 production is blocked, ABA-induced closure of stomata is inhibited. Moreover, activation of Ca2+ channels by H2O2 and ABA- and H2O2-induced stomatal closing are disrupted in the recessive ABA-insensitive mutant gca2. These data indicate that ABA-induced H2O2 production and the H2O2-activated Ca2+ channels are important mechanisms for ABA-induced stomatal closing.

Cadmium and iron transport by members of a plant metal transporter family in Arabidopsis with homology to Nramp genes.

Metal cation homeostasis is essential for plant nutrition and resistance to toxic heavy metals. Many plant metal transporters remain to be identified at the molecular level. In the present study, we have isolated AtNramp cDNAs from Arabidopsis and show that these genes complement the phenotype of a metal uptake deficient yeast strain, smf1. AtNramps show homology to the Nramp gene family in bacteria, yeast, plants, and animals. Expression of AtNramp cDNAs increases Cd(2+) sensitivity and Cd(2+) accumulation in yeast. Furthermore, AtNramp3 and AtNramp4 complement an iron uptake mutant in yeast. This suggests possible roles in iron transport in plants and reveals heterogeneity in the functional properties of Nramp transporters. In Arabidopsis, AtNramps are expressed in both roots and aerial parts under metal replete conditions. Interestingly, AtNramp3 and AtNramp4 are induced by iron starvation. Disruption of the AtNramp3 gene leads to slightly enhanced cadmium resistance of root growth. Furthermore, overexpression of AtNramp3 results in cadmium hypersensitivity of Arabidopsis root growth and increased accumulation of Fe, on Cd(2+) treatment. Our results show that Nramp genes in plants encode metal transporters and that AtNramps transport both the metal nutrient Fe and the toxic metal cadmium.

Differences in expression of acetylcholinesterase and collagen Q control the distribution and oligomerization of the collagen-tailed forms in fast and slow muscles.

The collagen-tailed forms of acetylcholinesterase (AChE) are accumulated at mammalian neuromuscular junctions. The A(4), A(8), and A(12) forms are expressed differently in the rat fast and slow muscles; the sternomastoid muscle contains essentially the A(12) form at end plates, whereas the soleus muscle also contains extrajunctional A(4) and A(8) forms. We show that collagen Q (ColQ) transcripts become exclusively junctional in the adult sternomastoid but remain uniformly expressed in the soleus. By coinjecting Xenopus oocytes with AChE(T) and ColQ mRNAs, we reproduced the muscle patterns of collagen-tailed forms. The soleus contains transcripts ColQ1 and ColQ1a, whereas the sternomastoid only contains ColQ1a. Collagen-tailed AChE represents the first evidence that synaptic components involved in cholinergic transmission may be differently regulated in fast and slow muscles.

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.

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.

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.

The mammalian gene of acetylcholinesterase-associated collagen.

The collagen-tailed or asymmetric forms (A) represent a major component of acetylcholinesterase (AChE) in the neuromuscular junction of higher vertebrates. They are hetero-oligomeric molecules, in which tetramers of catalytic subunits of type T (AChET) are attached to the subunits of a triple-stranded collagen "tail." We report the cloning of a rat AChE-associated collagen subunit, Q. We show that collagen tails are encoded by a single gene, COLQ. The ColQ subunits form homotrimers and readily form collagen-tailed AChE, when coexpressed with rat AChET. We found that the same ColQ subunits are incorporated, in vivo, in asymmetric forms of both AChE and butyrylcholinesterase. A splice variant from the COLQ gene encodes a proline- rich AChE attachment domain without the collagen domain but does not represent the membrane anchor of the brain tetramer. The COLQ gene is expressed in cholinergic tissues, brain, muscle, and heart, and also in noncholinergic tissues such as lung and testis.

ATP-Dependent Regulation of an Anion Channel at the Plasma Membrane of Protoplasts from Epidermal Cells of Arabidopsis Hypocotyls.

Although Arabidopsis is the object of many genetic and molecular biology investigations, relatively few studies deal with regulation of its transmembrane ion exchanges. To clarify the role of ion transport in plant development, organ-and tissue-specific ion channels must be studied. We identified a voltage-dependent anion channel in epidermal cells of Arabidopsis hypocotyls, thus providing a new example of the occurrence of voltage-dependent anion channels in a specific plant cell type distinct from the stomatal guard cell. The Arabidopsis hypocotyl anion channel is able to function under two modes characterized by different voltage dependences and different kinetic behaviors. This switch between a fast and a slow mode is controlled by ATP. In the presence of intracellular ATP (fast mode), the channels are closed at resting potentials, and whole-cell currents activate upon depolarization. After activation, the anion current deactivates rapidly and more and more completely at potentials negative to the peak. In the absence of ATP, the current switches from this fast mode to a mode characterized by a slow and incomplete deactivation at resting potentials. In addition, the whole-cell currents can be correlated with the activity of single channels. In the outside-out configuration, the presence of ATP modulates the mean lifetimes of the open and closed states of the channel at hyperpolarized potentials, thus controlling its open probability. The fact that ATP-dependent voltage regulation was observed in both whole-cell and outside-out configurations suggests that a single type of anion channel can switch between two modes with distinct functional properties.

Calcium channel antagonists induce direct inhibition of the outward rectifying potassium channel in tobacco protoplasts.

Verapamil, nifedipine and bepridil, three antagonists of L-type calcium channels in animal cells, were shown to induce direct inhibition of outward rectifying potassium current in Nicotiana tabacum cv. Xanthi protoplasts with IC50 of 5 microM, 5 microM and 1 microM, respectively. In the outside-out configuration, verapamil reduced the open probability of the ion channel responsible for the outward rectifying potassium conductance. Verapamil also blocked the outward rectifying potassium conductance in protoplasts from the N. tabacum cv. Bright Yellow cell line. Thus, studies using these molecules to demonstrate the involvement of calcium channels in plant physiological responses should be regarded with caution.

Cytosolic protons as secondary messengers in elicitor-induced defence responses.

A variety of early elicitor-induced membrane responses have been described, and their possible role in the generation of second messengers involved in the cascades of events leading to the activation of defence genes is actively investigated. Treatment of tobacco cells with a crude elicitor preparation from Phytophthora megasperma, purified oligouronides and a commercial pectate lyase, induce a common set of membrane reactions similar to those described in a variety of plant material, i.e. efflux of K+, extracellular alkalinization, net Ca2+ uptake and membrane depolarization. In the same conditions the three elicitors stimulate the activity of phenylalanine ammonia-lyase (PAL) and O-diphenol methyltransferase (OMT), two enzymes of the phenylpropanoid pathway. A good correlation between the intensity of the membrane response and the extent of enzyme stimulation has been observed. Cytosolic acidifications have also been measured as a rapid response to the different elicitor preparations used. These results show that plant cells (which usually succeed in counteracting pH-perturbing processes associated with their metabolism, with the transport of solutes or with the effect of various factors from the environment) display significant variation in the concentration of cytosolic protons in specific physiological circumstances, such as the perception of signals inducing defence reactions. Direct evidence that these cytosolic pH changes could be interpreted by plant cells as messages involved in triggering defence responses is provided by experiments showing that artificial acidifications of the cytoplasm lead to a co-ordinated stimulation of PAL and OMT. These results stress the need to explore in more detail the role played by cytoplasmic mechanisms underlying those pH changes.