Dissecting blue light signal transduction pathway in leaf epidermis using a pharmacological approach.

MAIN CONCLUSION: Blue light signalling pathway in broad bean leaf epidermal cells includes key membrane transporters: plasma- and endomembrane channels and pumps of H (+) , Ca (2+) and K (+) ions, and plasma membrane redox system. Blue light signalling pathway in epidermal tissue isolated from the abaxial side of fully developed Vicia faba leaves was dissected by measuring the effect of inhibitors of second messengers on net K(+), Ca(2+) and H(+) fluxes using non-invasive ion-selective microelectrodes (the MIFE system). Switching the blue light on-off caused transient changes of the ion fluxes. The effects of seven groups of inhibitors were tested in this study: CaM antagonists, ATPase inhibitors, Ca(2+) anatagonists or chelators, agents affecting IP3 formation, redox system inhibitors, inhibitors of endomembrane Ca(2+) transport systems and an inhibitor of plasma membrane Ca(2+)-permeable channels. Most of the inhibitors had a significant effect on steady-state (basal) net fluxes, as well as on the magnitude of the transient ion flux responses to blue light fluctuations. The data presented in this study suggest that redox signalling and, specifically, plasma membrane NADPH oxidase and coupled Ca(2+) and K(+) fluxes play an essential role in blue light signal transduction.

Enhanced resistance to the cellulose biosynthetic inhibitors, thaxtomin A and isoxaben in Arabidopsis thaliana mutants, also provides specific co-resistance to the auxin transport inhibitor, 1-NPA.

BACKGROUND: Thaxtomin A (TA) is a phytotoxin produced by plant pathogenic Streptomyces spp. responsible for potato common scab. TA inhibits cellulose biosynthesis in expanding plant tissues and is essential for disease induction. Auxin treatment of various plant tissues has been repeatedly demonstrated to inhibit TA toxicity and to reduce common scab. This work utilises Arabidopsis thaliana mutants with resistance to cellulose biosynthesis inhibitors (CBIs) to investigate the interaction between TA, other CBIs and auxins. RESULTS: Three CBI resistant A. thaliana mutants; txr1-1 (tolerance to TA), ixr1-1 (tolerance to isoxaben - IXB) and KOR1 (cellulose deficiency), showed no altered root growth response to treatment with natural or synthetic auxins, nor with the auxin efflux transport inhibitor 2,3,5-Triiodobenzoic acid (TIBA). However, all mutants had significantly enhanced tolerance to 1-napthylphthalamic acid (NPA), another auxin efflux transport inhibitor, which blocks polar auxin transport at a site distinct from TIBA. NPA tolerance of txr1-1 and ixr1-1 was further supported by electrophysiological analysis of net H+ fluxes in the mature, but not elongation zone of roots. All three mutants showed increased tolerance to IXB, but only txr1-1 showed tolerance to TA. No mutant showed enhanced tolerance to a third CBI, dichlobenil (DCB). CONCLUSIONS: We have demonstrated that plant tolerance to TA and IXB, as well as cell wall synthesis modifications in roots, have resulted in specific co-resistance to NPA but not TIBA. This suggests that CBI resistance has an impact on polar auxin efflux transport processes associated with the NPA binding protein. We also show that NPA inhibitory response in roots occurs in the mature root zone but not the elongation zone. Responses of mutants to CBIs indicate a similar, but not identical mode of action of TA and IXB, in contrast to DCB.

The waterlogging/salinity interaction in higher plants revisited - focusing on the hypoxia-induced disturbance to K+ homeostasis.

Salinity and waterlogging (root-zone hypoxia) are abiotic stresses that often occur together on saltland. It is widely recognised that these two factors interact to increase Na+ and/or Cl- concentrations in shoots, which can have adverse effects on plant growth and survival. This review expands on this understanding, providing evidence that the adverse effects of the interaction are also associated with a disturbance to plant K+ homeostasis. This conclusion is based on a comparative analysis of changes in ion concentrations and growth reported in the literature between species (glycophytes vs halophytes) and within a single species (Hordeum marinum L.). Comparisons between species show that hypoxia under saline conditions causes simultaneous increases in Na+ and Cl- concentrations and decreases in K+ concentrations in shoots and that these changes can all be related to changes in shoot dry mass. Comparisons between accessions of a single species (Hordeum maritima L.) strengthen the argument, with increases in Na+ and decreases in K+ being related to decreases in shoot relative growth rate.

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.

Methyl-beta-cyclodextrin reversibly alters the gating of lipid rafts-associated Kv1.3 channels in Jurkat T lymphocytes.

The voltage-dependent Kv1.3 potassium channels mediate a variety of physiological functions in human T lymphocytes. These channels, along with their multiple regulatory components, are localized in cholesterol-enriched microdomains of plasma membrane (lipid rafts). In this study, patch-clamp technique was applied to explore the impact of the lipid-raft integrity on the Kv1.3 channel functional characteristics. T lymphoma Jurkat cells were treated for 1 h with cholesterol-binding oligosaccharide methyl-beta-cyclodextrin (MbetaCD) in 1 or 2 mM concentration, resulting in depletion of cholesterol by 63 +/- 5 or 75 +/- 4%, respectively. Treatment with 2 mM MbetaCD did not affect the cells viability but retarded the cell proliferation. The latter treatment caused a depolarizing shift of the Kv1.3 channel activation and inactivation by 11 and 6 mV, respectively, and more than twofold decrease in the steady-state activity at depolarizing potentials. Altogether, these changes underlie the depolarization of membrane potential, recorded in a current-clamp mode. The effects of MbetaCD were concentration- and time-dependent and reversible. Both development and recovery of the MbetaCD effects were completed within 1-2 h. Therefore, cholesterol depletion causes significant alterations in the Kv1.3 channel function, whereas T cells possess a potential to reverse these changes.

Spatial variation in H2O2 response of Arabidopsis thaliana root epidermal Ca2+ flux and plasma membrane Ca2+ channels.

Hydrogen peroxide is an important regulatory agent in plants. This study demonstrates that exogenous H2O2 application to Arabidopsis thaliana root epidermis results in dose-dependent transient increases in net Ca2+ influx. The magnitude and duration of the transients were greater in the elongation zone than in the mature epidermis. In both regions, treatment with the cation channel blocker Gd3+ prevented H2O2-induced net Ca2+ influx, consistent with application of exogenous H2O2 resulting in the activation of plasma membrane Gd3+-sensitive Ca2+-influx pathways. Application of 10 mm H2O2 to the external plasma membrane face of elongation zone epidermal protoplasts resulted in the appearance of a hyperpolarization-activated Ca2+-permeable conductance. This conductance differed from that previously characterized as being responsive to extracellular hydroxyl radicals. In contrast, in mature epidermal protoplasts a plasma membrane hyperpolarization-activated Ca2+-permeable channel was activated only when H2O2 was present at the intracellular membrane face. Channel open probability increased with intracellular [H2O2] and at hyperpolarized voltages. Unitary conductance decreased thus: Ba2+ > Ca2+ (14.5 pS) > Mg2+ > Zn2+ (20 mM external cation, 1 mM H2O2). Lanthanides and Zn2+ (but not TEA+) suppressed the open probability without affecting current amplitude. The results suggest spatial heterogeneity and differential sensitivity of Ca2+ channel activation by reactive oxygen species in the root that could underpin signalling.

Role of a mitogen-activated protein kinase cascade in ion flux-mediated turgor regulation in fungi.

Fungi normally maintain a high internal hydrostatic pressure (turgor) of about 500 kPa. In response to hyperosmotic shock, there are immediate electrical changes: a transient depolarization (1 to 2 min) followed by a sustained hyperpolarization (5 to 10 min) prior to turgor recovery (10 to 60 min). Using ion-selective vibrating probes, we established that the transient depolarization is due to Ca(2+) influx and the sustained hyperpolarization is due to H(+) efflux by activation of the plasma membrane H(+)-ATPase. Protein synthesis is not required for H(+)-ATPase activation. Net K(+) and Cl(-) uptake occurs at the same time as turgor recovery. The magnitude of the ion uptake is more than sufficient to account for the osmotic gradients required for turgor to return to its original level. Two osmotic mutants, os-1 and os-2, homologs of a two-component histidine kinase sensor and the yeast high osmotic glycerol mitogen-activated protein (MAP) kinase, respectively, have lower turgor than the wild type and do not exhibit the sustained hyperpolarization after hyperosmotic treatment. The os-1 mutant does not exhibit all of the wild-type turgor-adaptive ion fluxes (Cl(-) uptake increases, but net K(+) flux barely changes and net H(+) efflux declines) (os-2 was not examined). Both os mutants are able to regulate turgor but at a lower level than the wild type. Our results demonstrate that a MAP kinase cascade regulates ion transport, activation of the H(+)-ATPase, and net K(+) and Cl(-) uptake during turgor regulation. Other pathways regulating turgor must also exist.

A novel mutant with modified tropic responses in Pisum sativum L.

A single-gene recessive mutant which displays increased phototropic and gravitropic responses has been isolated in Pisum sativum L. cv. Torsdag and is provisionally named mtr-1, for its modified tropic response. Mutant plants attain a greater degree of bending during both phototropic and gravitropic induction due to an extension of the curvature phase. In addition to their increase in tropic curvature, mutant plants have longer and narrower leaves as mature plants, attenuated blue-light-induced ion flux responses, and lower levels of PsPK5 mRNA (a PHOT1 orthologue). Possible causes of these effects are discussed.

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 Ca(2+)- and K(+)-permeable conductances in Arabidopsis root cell protoplasts, mediating Ca(2+) influx and K(+) efflux, respectively. Free oxygen radicals generate increases in cytosolic Ca(2+) 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 Ca(2+) influx in cell growth. Net free oxygen radical-stimulated Ca(2+) 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.

Kinetics of ammonium and nitrate uptake by eucalypt roots and associated proton fluxes measured using ion selective microelectrodes.

Ion-selective microelectrodes were used non-invasively to measure the concentration dependence of NH4+ and NO3- fluxes around the roots of intact solution-cultured Eucalyptus nitens (Deane & Maiden) Maiden. In addition, NH4+ and H+ fluxes were measured simultaneously at a range of NH4+ concentrations, and NO3- and H+ fluxes were measured simultaneously at a range of NO3- concentrations. Nitrogen concentrations ranged from 10-250 µM, i.e. in the range corresponding to the high affinity transport system (HATS). Both NH4+ and NO3- fluxes exhibited saturating Michaelis-Menten-style kinetics. The Km was 16 µM for NH4+ and 18 µM for NO3-. Values of Vmax were 53 nmol m-2 s-1 for NH4+ and 37 nmol m-2 s-1 for NO3-. Proton fluxes were highly correlated with NH4+ and NO3- fluxes, but the relationships were different. Proton efflux increased with increasing NH4+ concentration and mirrored the changing NH4+ fluxes. The ratio between NH4+ and H+ fluxes was 1 : -1.6. Proton influx was evident with initial exposure to NO3-, with the flux stoichiometry for NO3- : H+ being 1 : 1.4. Subsequent increases in NO3- concentration caused a gradual increase in H+ efflux such that the flux stoichiometry for NO3- : H+ became 1 : -0.8. The presence of 100 µM NH4+ greatly reduced NO3- fluxes and caused a large and constant H+ efflux. These results are evidence that E. nitens has a preference for NH4+ as a source of N, and that the fluxes of NH4+ and NO3- are quantitatively linked to H+ flux.

Arabidopsis thaliana root non-selective cation channels mediate calcium uptake and are involved in growth.

Calcium is a critical structural and regulatory nutrient in plants. However, mechanisms of its uptake by root cells are poorly understood. We have found that Ca2+ influx in Arabidopsis root epidermal protoplasts is mediated by voltage-independent rapidly activating Ca2+-permeable non-selective cation channels (NSCCs). NSCCs showed the following permeability (P) sequence: PCa (1.00) = PBa (0.93) > PZn (0.51), PCa/PNa = 0.19, PCa/PK = 0.14. They were inhibited by quinine, Gd3+, La3+ and the His modifier diethylpyrocarbonate, but not by the Ca2+ or K+ channel antagonists, verapamil and tetraethylammonium (TEA+). Single channel conductance measured in 20 mm external Ca2+ was 5.9 pS. Calcium-permeable NSCCs co-existed with hyperpolarisation-activated Ca2+ channels (HACCs), which activated 40-60 min after forming the whole-cell configuration. HACCs activated at voltages <-130 to -150 mV, showed slow activation kinetics and were regulated by cytosolic Ca2+ ([Ca2+]cyt). Using aequorin-expressing plants, a linear relationship between membrane potential (Vm) and resting [Ca2+]cyt was observed, indicating the involvement of NSCCs. Intact root 45Ca2+ influx was reduced by Gd3+ (NSCC blocker) but was verapamil and TEA+ insensitive. In the root elongation zone, both root net Ca2+ influx (measured by Ca2+-selective vibrating microelectrode) and NSCC activity were increased compared to the mature epidermis, suggesting the involvement of NSCC in growth. A Ca2+ acquisition system based on NSCC and HACC co-existence is proposed. In mature epidermal cells, NSCC-mediated Ca2+ influx dominates whereas in specialised root cells (root hairs and elongation zone cells) where elevated [Ca2+]cyt activates HACCs, HACC-mediated Ca2+ influx predominates.

Turgor regulation in osmotically stressed Arabidopsis epidermal root cells. Direct support for the role of inorganic ion uptake as revealed by concurrent flux and cell turgor measurements.

Hyperosmotic stress is known to significantly enhance net uptake of inorganic ions into plant cells. Direct evidence for cell turgor recovery via such a mechanism, however, is still lacking. In the present study, we performed concurrent measurements of net ion fluxes (with the noninvasive microelectrode ion flux estimation technique) and cell turgor changes (with the pressure-probe technique) to provide direct evidence that inorganic ion uptake regulates turgor in osmotically stressed Arabidopsis epidermal root cells. Immediately after onset of hyperosmotic stress (100/100 mM mannitol/sorbitol treatment), the cell turgor dropped from 0.65 to about 0.25 MPa. Turgor recovery started within 2 to 10 min after the treatment and was accompanied by a significant (30-80 nmol m-2 s-1) increase in uptake of K+, Cl-, and Na+ by root cells. In most cells, almost complete (>90% of initial values) recovery of the cell turgor was observed within 40 to 50 min after stress onset. In another set of experiments, we combined the voltage-clamp and the microelectrode ion flux estimation techniques to show that this process is, in part, mediated by voltage-gated K+ transporters at the cell plasma membrane. The possible physiological significance of these findings is discussed.