Vadim Demidchik

Reactive oxygen species produced by NADPH oxidase regulate plant cell growth.

Cell expansion is a central process in plant morphogenesis, and the elongation of roots and root hairs is essential for uptake of minerals and water from the soil. Ca2+ influx from the extracellular store is required for (and sets the rates of) cell elongation in roots. Arabidopsis thaliana rhd2 mutants are defective in Ca2+ uptake and consequently cell expansion is compromised—rhd2 mutants have short root hairs and stunted roots. To determine the regulation of Ca2+ acquisition in growing root cells we show here that RHD2 is an NADPH oxidase, a protein that transfers electrons from NADPH to an electron acceptor leading to the formation of reactive oxygen species (ROS). We show that ROS accumulate in growing wild-type (WT) root hairs but their levels are markedly decreased in rhd2 mutants. Blocking the activity of the NADPH oxidase with diphenylene iodonium (DPI) inhibits ROS formation and phenocopies Rhd2-. Treatment of rhd2 roots with ROS partly suppresses the mutant phenotype and stimulates the activity of plasma membrane hyperpolarization-activated Ca2+ channels, the predominant root Ca2+ acquisition system. This indicates that NADPH oxidases control development by making ROS that regulate plant cell expansion through the activation of Ca2+ channels.

Extracellular Ca2+ ameliorates NaCl-induced K+ loss from Arabidopsis root and leaf cells by controlling plasma membrane K+ -permeable channels.

Calcium can ameliorate Na+ toxicity in plants by decreasing Na+ influx through nonselective cation channels. Here, we show that elevated external [Ca2+] also inhibits Na+-induced K+ efflux through outwardly directed, K+-permeable channels. Noninvasive ion flux measuring and patch-clamp techniques were used to characterize K+ fluxes from Arabidopsis (Arabidopsis thaliana) root mature epidermis and leaf mesophyll under various Ca2+ to Na+ ratios. NaCl-induced K+ efflux was not related to the osmotic component of the salt stress, was inhibited by the K+ channel blocker TEA+, was not mediated by inwardly directed K+ channels (tested in the akt1 mutant), and resulted in a significant decrease in cytosolic K+ content. NaCl-induced K+ efflux was partially inhibited by 1 mm Ca2+ and fully prevented by 10 mm Ca2+. This ameliorative effect was at least partially attributed to a less dramatic NaCl-induced membrane depolarization under high Ca2+ conditions. Patch-clamp experiments (whole-cell mode) have demonstrated that two populations of Ca2+-sensitive K+ efflux channels exist in protoplasts isolated from the mature epidermis of Arabidopsis root and leaf mesophyll cells. The instantaneously activating K+ efflux channels showed weak voltage dependence and insensitivity to external and internal Na+. Another population of K+ efflux channels was slowly activating, steeply rectifying, and highly sensitive to Na+. K+ efflux channels in roots and leaves showed different Ca2+ and Na+ sensitivities, suggesting that these organs may employ different strategies to withstand salinity. Our results suggest an additional mechanism of Ca2+ action on salt toxicity in plants: the amelioration of K+ loss from the cell by regulating (both directly and indirectly) K+ efflux channels.

Free oxygen radicals regulate plasma membrane Ca2+- and K+-permeable channels in plant root cells

Free oxygen radicals are an irrefutable component of life, underlying important biochemical and physiological phenomena in animals. Here it is shown that free oxygen radicals activate plasma membrane Ca2+- and K+-permeable conductances in Arabidopsis root cell protoplasts, mediating Ca2+ influx and K+ efflux, respectively. Free oxygen radicals generate increases in cytosolic Ca2+ mediated by a novel population of nonselective cation channels that differ in selectivity and pharmacology from those involved in toxic Na+ influx. Analysis of the free oxygen radical-activated K+ conductance showed its similarity to the Arabidopsis root K+ outward rectifier. Significantly larger channel activation was found in cells responsible for perceiving environmental signals and undergoing elongation. Quenching root free oxygen radicals inhibited root elongation, confirming the role of radical-activated Ca2+ influx in cell growth. Net free oxygen radical-stimulated Ca2+ influx and K+ efflux were observed in root cells of monocots, dicots, C3 and C4 plants, suggesting conserved mechanisms and functions. In conclusion, two functions for free oxygen radical cation channel activation are proposed: initialization/amplification of stress signals and control of cell elongation in root growth.

Arabidopsis root K+-efflux conductance activated by hydroxyl radicals: single-channel properties, genetic basis and involvement in stress-induced cell death

Reactive oxygen species (ROS) are central to plant stress response, signalling, development and a multitude of other processes. In this study, the plasma-membrane hydroxyl radical (HR)-activated K+ channel responsible for K+ efflux from root cells during stress accompanied by ROS generation is characterised. The channel showed 16-pS unitary conductance and was sensitive to Ca2+, tetraethylammonium, Ba2+, Cs+ and free-radical scavengers. The channel was not found in the gork1-1 mutant, which lacks a major plasma-membrane outwardly rectifying K+ channel. In intact Arabidopsis roots, both HRs and stress induced a dramatic K+ efflux that was much smaller in gork1-1 plants. Tests with electron paramagnetic resonance spectroscopy showed that NaCl can stimulate HR generation in roots and this might lead to K+-channel activation. In animals, activation of K+-efflux channels by HRs can trigger programmed cell death (PCD). PCD symptoms in Arabidopsis roots developed much more slowly in gork1-1 and wild-type plants treated with K+-channel blockers or HR scavengers. Therefore, similar to animal counterparts, plant HR-activated K+ channels are also involved in PCD. Overall, this study provides new insight into the regulation of plant cation transport by ROS and demonstrates possible physiological properties of plant HR-activated K+ channels.

Sodium Fluxes through Nonselective Cation Channels in the Plasma Membrane of Protoplasts from Arabidopsis Roots

The aim of the present work was to characterize Na+currents through nonselective cation channels (NSCCs) in protoplasts derived from root cells of Arabidopsis. The procedure of the protoplast isolation was modified to increase the stability of Arabidopsis root protoplasts in low external Ca2+ by digesting tissue in elevated Ca2+. Experiments in whole-cell and outside-out modes were carried out. We found that Na+ currents in Arabidopsis root protoplasts were mediated by cation channels that were insensitive to externally applied tetraethylammonium+ and verapamil, had no time-dependent activation (permanently opened or completely activated within 1–2 ms), were voltage independent, and were weakly selective for monovalent cations. The selectivity sequence was as follows: K+ (1.49) > NH4 + (1.24) > Rb+ (1.15) ≈ Cs+ (1.10) ≈ Na+ (1.00) > Li+ (0.73) > tetraethylammonium+ (0.47). Arabidopsis root NSCCs were blocked by H+ (pK ≈ 6.0), Ca2+(K1/2 ≈ 0.1 mm), Ba2+, Zn2+, La3+, Gd3+, quinine, and the His modifier diethylpyrocarbonate. They were insensitive to most organic blockers (nifedipine, verapamil, flufenamate, and amiloride) and to the SH-group modifier p-chloromercuriphenyl sulfonic acid. Voltage-insensitive, Ca2+-sensitive single channels were also resolved. Properties of Arabidopsis root NSCCs are discussed and compared with characteristics of similar conductances studied previously in plants and animals. It is suggested that NSCCs present a distinct group of plant ion channels, mediating toxic Na+ influx to the cell and probably having other important roles in physiological processes of plants.

Genes for calcium-permeable channels in the plasma membrane of plant root cells

In plant cells, Ca(2+) is required for both structural and biophysical roles. In addition, changes in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) orchestrate responses to developmental and environmental signals. In many instances, [Ca(2+)](cyt) is increased by Ca(2+) influx across the plasma membrane through ion channels. Although the electrophysiological and biochemical characteristics of Ca(2+)-permeable channels in the plasma membrane of plant cells are well known, genes encoding putative Ca(2+)-permeable channels have only recently been identified. By comparing the tissue expression patterns and electrophysiology of Ca(2+)-permeable channels in the plasma membrane of root cells with those of genes encoding candidate plasma membrane Ca(2+) channels, the genetic counterparts of specific Ca(2+)-permeable channels can be deduced. Sequence homologies and the physiology of transgenic antisense plants suggest that the Arabidopsis AtTPC1 gene encodes a depolarisation-activated Ca(2+) channel. Members of the annexin gene family are likely to encode hyperpolarisation-activated Ca(2+) channels, based on their corresponding occurrence in secretory or elongating root cells, their inhibition by La(3+) and nifedipine, and their increased activity as [Ca(2+)](cyt) is raised. Based on their electrophysiology and tissue expression patterns, AtSKOR encodes a depolarisation-activated outward-rectifying (Ca(2+)-permeable) K(+) channel (KORC) in stelar cells and AtGORK is likely to encode a KORC in the plasma membrane of other Arabidopsis root cells. Two candidate gene families, of cyclic-nucleotide gated channels (CNGC) and ionotropic glutamate receptor (GLR) homologues, are proposed as the genetic correlates of voltage-independent cation (VIC) channels.

Is ATP a Signaling Agent in Plants

Physiological processes in plant cells are regulated by intrinsic and extrinsic signals. Numerous signaling molecules have been identified, including hormones, elicitors, and secondary metabolites. Cognate receptors and receptor genes have been reported in some cases ([Hua and Meyerowitz, 1998][1

Plant extracellular ATP signalling by plasma membrane NADPH oxidase and Ca2+ channels

Extracellular ATP regulates higher plant growth and adaptation. The signalling events may be unique to higher plants, as they lack animal purinoceptor homologues. Although it is known that plant cytosolic free Ca2+ can be elevated by extracellular ATP, the mechanism is unknown. Here, we have studied roots of Arabidopsis thaliana to determine the events that lead to the transcriptional stress response evoked by extracellular ATP. Root cell protoplasts were used to demonstrate that signalling to elevate cytosolic free Ca2+ is determined by ATP perception at the plasma membrane, and not at the cell wall. Imaging revealed that extracellular ATP causes the production of reactive oxygen species in intact roots, with the plasma membrane NADPH oxidase AtRBOHC being the major contributor. This resulted in the stimulation of plasma membrane Ca2+-permeable channels (determined using patch-clamp electrophysiology), which contribute to the elevation of cytosolic free Ca2+. Disruption of this pathway in the AtrbohC mutant impaired the extracellular ATP-induced increase in reactive oxygen species (ROS), the activation of Ca2+ channels, and the transcription of the MAP kinase3 gene that is known to be involved in stress responses. This study shows that higher plants, although bereft of purinoceptor homologues, could have evolved a distinct mechanism to transduce the ATP signal at the plasma membrane.

Zea mays Annexins Modulate Cytosolic Free Ca2+ and Generate a Ca2+-Permeable Conductance

Regulation of reactive oxygen species and cytosolic free calcium ([Ca2+]cyt) is central to plant function. Annexins are small proteins capable of Ca2+-dependent membrane binding or membrane insertion. They possess structural motifs that could support both peroxidase activity and calcium transport. Here, a Zea mays annexin preparation caused increases in [Ca2+]cyt when added to protoplasts of Arabidopsis thaliana roots expressing aequorin. The pharmacological profile was consistent with annexin activation (at the extracellular plasma membrane face) of Arabidopsis Ca2+-permeable nonselective cation channels. Secreted annexins could therefore modulate Ca2+ influx. As maize annexins occur in the cytosol and plasma membrane, they were incorporated at the intracellular face of lipid bilayers designed to mimic the plasma membrane. Here, they generated an instantaneously activating Ca2+-permeable conductance at mildly acidic pH that was sensitive to verapamil and Gd3+ and had a Ca2+-to-K+ permeability ratio of 0.36. These results suggest that cytosolic annexins create a Ca2+ influx pathway directly, particularly during stress responses involving acidosis. A maize annexin preparation also demonstrated in vitro peroxidase activity that appeared independent of heme association. In conclusion, this study has demonstrated that plant annexins create Ca2+-permeable transport pathways, regulate [Ca2+]cyt, and may function as peroxidases in vitro.

The Effect of Cu2+ on Ion Transport Systems of the Plant Cell Plasmalemma

Changes in plasmalemma permeability caused by excessive Cu2+ levels were examined in cells of a freshwater alga (Nitella flexilis) using a conventional microelectrode voltage-clamp technique. A rapid Cu2+-induced increase of plasmalemma conductance starting from 5 [mu]M Cu2+ was shown. Cu2+-induced plasmalemma conductance (ClGm) was nonselective and potential-independent, resembling the conductance of nonselective ionic leakage of the plasmalemma. The K+ channel conductance was shown to be unaltered by Cu2+, and a decrease in plasmalemma Cl- channel conductance at Cu2+ concentrations above 5 [mu]M was found. The depression of Cl- channels and ClGm were time-, dosage-, and Ca2+-dependent processes, revealing a great similarity in all parameters, with Ca2+ causing the preventive effect by shifting the effective Cu2+ concentrations to higher levels. This phenomenon may be explained by the same Cu2+-modified target on the plasmalemma both for ClGm and Cl- channel depression. In addition, a reversible, inhibitory effect of Cu2+ (>10 [mu]M) on the light-stimulated H+-ATPase electrogenic pump in the plasmalemma was demonstrated. This effect was Ca2+- independent, which made it possible to distinguish it from ClGm. Therefore, the Cu2+-induced dramatic alterations in plant cell plasmalemma permeability are caused mainly by nonselective conductance increases and electrogenic pump inhibition.