Jean-Pierre Rona

Plasma Membrane Depolarization Induced by Abscisic Acid in Arabidopsis Suspension Cells Involves Reduction of Proton Pumping in Addition to Anion Channel Activation, Which Are Both Ca2+ Dependent

In Arabidopsis suspension cells a rapid plasma membrane depolarization is triggered by abscisic acid (ABA). Activation of anion channels was shown to be a component leading to this ABA-induced plasma membrane depolarization. Using experiments employing combined voltage clamping, continuous measurement of extracellular pH, we examined whether plasma membrane H+-ATPases could also be involved in the depolarization. We found that ABA causes simultaneously cell depolarization and medium alkalinization, the second effect being abolished when ABA is added in the presence of H+ pump inhibitors. Inhibition of the proton pump by ABA is thus a second component leading to the plasma membrane depolarization. The ABA-induced depolarization is therefore the result of two different processes: activation of anion channels and inhibition of H+-ATPases. These two processes are independent because impairing one did not suppress the depolarization. Both processes are however dependent on the [Ca2+]cyt increase induced by ABA since increase in [Ca2+]cyt enhanced anion channels and impaired H+-ATPases.

Electrochemical data on compartmentation into cell wall, cytoplasm, and vacuole of leaf cells in the CAM genusKalanchoë

SummaryTranscellular electrical profiles ofKalanchoë leaf cells were obtained by pushing a glass micro-saltbridge through cells with the tip consecutively in the cell wall, cytoplasm, and vacuole. The electrical resistance of the cell wall was too small to be detectable, that of the plasmalemma and tonoplast was about 0.18–0.21 and 0.16–0.18 Ωm2, respectively. The electrical potential difference between the cytoplasm and the external medium,ψco, was ≈−180 mV, the potential difference between the vacuole and the medium,ψvo, was ≈−155mV, and thus the mean potential difference at the tonoplast,ψvc, was about +25 mV. Potential difference,ψvo, was independent of proton concentration in the external medium between pH 9 and 5.5, and behaved like an H+-electrode between pH 5 and 3. Depolarizations and hyperpolarizations ofψvo obtained by increasing and decreasing, respectively, the Na+-concentrations in the medium were smaller than with changing K+-concentrations, suggesting that permeabilities arePNa+/PK+≈-0.23. Assessment of K+-compartmentation by flux analysis gave K+-concentrations in the cytoplasm including chloroplasts (cc) and vacuole (cv) ascc between 200 and 400 mmol kg−1 FrWt andcv ≈-15 mmol kg−1 FrWt. The Nernst criterion suggests that metabolically regulated K+ transport out of the vacuoles concentrates K+ in the cytoplasm. Fusicoccin (10−5m) hyperpolarizedψco by about 100 mV and depolarized the positiveψvc by about 10 mV, the latter presumably being an insignificant effect. The evidence for the existence of proton pumps exchanging H+ and K+ at the plasmalemma and at the tonoplast is discussed.

The indolic compound hypaphorine produced by ectomycorrhizal fungus interferes with auxin action and evokes early responses in nonhost Arabidopsis thaliana.

Signals leading to mycorrhizal differentiation are largely unknown. We have studied the sensitivity of the root system from plant model Arabidopsis thaliana to hypaphorine, the major indolic compound isolated from the basidiomycetous fungus Pisolithus tinctorius. This fungi establishes ectomycorrhizas with Eucalyptus globulus. Hypaphorine controls root hair elongation and counteracts the activity of indole-3-acetic acid on root elongation on A. thaliana, as previously reported for the host plant. In addition, we show that hypaphorine counteracts the rapid upregulation by indole-3-acetic acid and 1-naphthalenic-acetic acid of the primary auxin-responsive gene IAA1 and induces a rapid, transient membrane depolarization in root hairs and suspension cells, due to the modulation of anion and K+ currents. These early responses indicate that components necessary for symbiosis-related differentiation events are present in the nonhost plant A. thaliana and provide tools for the dissection of the hypaphorine-auxin interaction.

Abscissic acid specific expression of RAB18 involves activation of anion channels in Arabidopsis thaliana suspension cells

The abscissic acid (ABA) transduction cascade following the plasmalemma perception was analyzed in intact Arabidopsis thaliana suspension cells. In response to impermeant ABA, anion currents were activated and K+ inward rectifying currents were inhibited. Anion current activation was required for the ABA specific expression of RAB18. By contrast, specific inhibition of K+ channels by tetraethylammonium or Ba2+ did not affect RAB18 expression. Thus, outer plasmalemma ABA perception triggered two separated signaling pathways.

An early Ca2+ influx is a prerequisite to thaxtomin A-induced cell death in Arabidopsis thaliana cells

The pathogenicity of various Streptomyces scabies isolates involved in potato scab disease was correlated with the production of thaxtomin A. Since calcium is known as an essential second messenger associated with pathogen-induced plant responses and cell death, it was investigated whether thaxtomin A could induce a Ca2+ influx related to cell death and to other putative plant responses using Arabidopsis thaliana suspension cells, which is a convenient model to study plant-microbe interactions. A. thaliana cells were treated with micromolar concentrations of thaxtomin A. Cell death was quantified and ion flux variations were analysed from electrophysiological measurements with the apoaequorin Ca2+ reporter protein and by external pH measurement. Involvement of anion and calcium channels in signal transduction leading to programmed cell death was determined by using specific inhibitors. These data suggest that this toxin induces a rapid Ca2+ influx and cell death in A. thaliana cell suspensions. Moreover, these data provide strong evidence that the Ca2+ influx induced by thaxtomin A is necessary to achieve this cell death and is a prerequisite to early thaxtomin A-induced responses: anion current increase, alkalization of the external medium, and the expression of PAL1 coding for a key enzyme of the phenylpropanoid pathway.

Anion channel activity is necessary to induce ethylene synthesis and programmed cell death in response to oxalic acid

Oxalic acid is thought to be a key factor of the early pathogenicity stage in a wide range of necrotrophic fungi. Studies were conducted to determine whether oxalate could induce programmed cell death (PCD) in Arabidopsis thaliana suspension cells and to detail the transduction of the signalling pathway induced by oxalate. Arabidopsis thaliana cells were treated with millimolar concentrations of oxalate. Cell death was quantified and ion flux variations were analysed from electrophysiological measurements. Involvement of the anion channel and ethylene in the signal transduction leading to PCD was determined by using specific inhibitors. Oxalic acid induced a PCD displaying cell shrinkage and fragmentation of DNA into internucleosomal fragments with a requirement for active gene expression and de novo protein synthesis, characteristic hallmarks of PCD. Other responses generally associated with plant cell death, such as anion effluxes leading to plasma membrane depolarization, mitochondrial depolarization, and ethylene synthesis, were also observed following addition of oxalate. The results show that oxalic acid activates an early anionic efflux which is a necessary prerequisite for the synthesis of ethylene and for the PCD in A. thaliana cells.

Plasmalemma Abscisic Acid Perception Leads to RAB18 Expression via Phospholipase D Activation in Arabidopsis Suspension Cells

Abscisic acid (ABA) plays a key role in the control of stomatal aperture by regulating ion channel activities and water exchanges across the plasma membrane of guard cells. Changes in cytoplasmic calcium content and activation of anion and outward-rectifying K+ channels are among the earliest cellular responses to ABA in guard cells. In Arabidopsis suspension cells, we have demonstrated that outer plasmalemma perception of ABA triggered similar early events. Furthermore, a Ca2+influx and the activation of anion channels are part of the ABA-signaling pathway leading to the specific expression ofRAB18. Here, we determine whether phospholipases are involved in ABA-induced RAB18 expression. Phospholipase C is not implicated in this ABA pathway. Using a transphosphatidylation reaction, we show that ABA plasmalemma perception results in a transient stimulation of phospholipase D (PLD) activity, which is necessary for RAB18 expression. Further experiments showed that PLD activation was unlikely to be regulated by heterotrimeric G proteins. We also observed that ABA-dependent stimulation of PLD was necessary for the activation of plasma anion current. However, when ABA activation of plasma anion channels was inhibited, the ABA-dependent activation of PLD was unchanged. Thus, we conclude that in Arabidopsis suspension cells, ABA stimulation of PLD acts upstream from anion channels in the transduction pathway leading to RAB18 expression.

The HrpN ea Harpin from Erwinia amylovora Triggers Differential Responses on the Nonhost Arabidopsis thaliana Cells and on the Host Apple Cells

Erwinia amylovora is a gram-negative necrogenic bacterium causing fire blight of the Maloideae subfamily of Rosaceae such as apple and pear. It provokes progressive necrosis in aerial parts of susceptible host plants (compatible interaction) and a hypersensitive reaction (HR) when infiltrated in nonhost plants (incompatible interaction). The HrpN(ea) harpin is a type three secretion system effector secreted by E. amylovora. This protein is involved in pathogenicity and HR-eliciting capacity of E. amylovora. In the present study, we showed that, in nonhost Arabidopsis thaliana cells, purified HrpN(ea) induces cell death and H2O2 production, two nonhost resistance responses, but failed to induce such responses in host MM106 apple cells. Moreover, HrpN(ea) induced an increase in anion current in host MM106 apple cells, at the opposite of the decrease of anion current previously shown to be necessary to induce cell death in nonhost A. thaliana cells. These results suggest that HrpN(ea) induced different signaling pathways, which could account for early induced compatible or incompatible interaction development.

Anion Channel Activation and Proton Pumping Inhibition Involved in the Plasma Membrane Depolarization Induced by ABA in Arabidopsis thaliana Suspension Cells are Both ROS Dependent

In Arabidopsis thaliana suspension cells, ABA was previously shown to promote the activation of anion channels and the reduction of proton pumping that both contribute to the plasma membrane depolarization. These two ABA responses were shown to induce two successive [Ca(2+)](cyt) spikes. As reactive oxygen species (ROS) have emerged as components of ABA signaling pathways especially by promoting [Ca(2+)](cyt) variations, we studied whether ROS were involved in the regulation of anion channels and proton pumps activities. Here we demonstrated that ABA induced ROS production which triggered the second of the two [Ca(2+)](cyt) increases observed in response to ABA. Blocking ROS generation using diphenyleneiodonium (DPI) impaired the proton pumping reduction, the anion channel activation and the RD29A gene expression in response to ABA. Furthermore, H(2)O(2) was shown to activate anion channels and to inhibit plasma membrane proton pumping, as did ABA. However, ROS partially mimicked ABAs effects since H(2)O(2) treatment elicited anion channel activation but not the subsequent expression of the RD29A gene as did ABA. This suggests that expression of the RD29A gene in response to ABA results from the activation of multiple concomitant signaling pathways: blocking of one of them would impair gene expression whereas stimulating only one would not. We conclude that ROS are a central messenger of ABA in the signaling pathways leading to the plasma membrane depolarization induced by ABA.

Induction of abscisic acid-regulated gene expression by diacylglycerol pyrophosphate involves Ca2+ and anion currents in arabidopsis suspension cells

Diacylglycerol pyrophosphate (DGPP) was recently shown to be a possible intermediate in abscisic acid (ABA) signaling. In this study, reverse transcription-PCR of ABA up-regulated genes was used to evaluate the ability of DGPP to trigger gene expression in Arabidopsis (Arabidopsis thaliana) suspension cells. At5g06760, LTI30, RD29A, and RAB18 were stimulated by ABA and also specifically expressed in DGPP-treated cells. Use of the Ca2+ channel blockers fluspirilene and pimozide and the Ca2+ chelator EGTA showed that Ca2+ was required for ABA induction of DGPP formation. In addition, Ca2+ participated in DGPP induction of gene expression via stimulation of anion currents. Hence, a sequence of Ca2+, DGPP, and anion currents, constituting a core of early ABA-signaling events necessary for gene expression, is proposed.