The involvement of calmodulin and protein kinases in the upstream of cytosolic and nucleic calcium signaling induced by hypoosmotic shock in tobacco cells.

Changes in Ca2+ concentrations in cytosol ([Ca2+]C) or nucleus ([Ca2+]N) may play some vital roles in plants under hypoosmotic shock (Hypo-OS). Here, we observed that Hypo-OS induces biphasic increases in [Ca2+]C and [Ca2+]N in two tobacco cell lines (BY-2) expressing apoaequorin either in the cytosol or in the nucleus. Both [Ca2+]C and [Ca2+]N were sensitively modulated by the inhibitors of calmodulin and protein kinases, supporting the view that calmodulin suppresses the 1st peaks and and protein kinases enhance 2nd peaks in [Ca2+]C and [Ca2+]N. Data also suggested that the 1st and 2nd events depend on the internal and extracellular Ca2+ sources, respectively.

Calcium- and ROS-mediated defence responses in BY2 tobacco cells by nonpathogenic Streptomyces sp.

AIMS: The early molecular events underlying the elicitation of plant defence reactions by Gram-positive bacteria are relatively unknown. In plants, calcium and reactive oxygen species are commonly involved as cellular messengers of a wide range of biotic stimuli from pathogenic to symbiotic bacteria. In the present work, we checked whether nonpathogenic Streptomyces sp. strains could induce early signalling events leading to defence responses in BY2 tobacco cell suspensions. METHODS AND RESULTS: We have demonstrated that nonpathogenic Streptomyces sp. OE7 strain induced a cytosolic Ca(2+) increase and a biphasic oxidative burst in the upstream signalling events, leading to defence responses in BY2 tobacco cell suspensions. Streptomyces sp. OE7 also elicited delayed intracellular free scopoletin production and programmed cell death. In agreement with scopoletin production, OE7 induced accumulation of PAL transcripts and increased accumulation of transcripts of EREBP1 and AOX genes that are known to be regulated by the jasmonate/ethylene pathway. Transcript levels of PR1b and NIMIN2α, both salicylic acid pathway-linked genes, were not modified. Moreover, Streptomyces sp. OE7 culture filtrates could reduce Pectobacterium carotovorum- and Pectobacterium atrosepticum-induced death of BY2 cells and soft rot on potato slices. CONCLUSIONS: New insights are thus provided into the interaction mechanisms between Streptomyces sp. and plants; Streptomyces sp. could be sensed by plant cells, and through cytosolic Ca(2+) changes and the generation of reactive oxygen species, defence responses were induced. SIGNIFICANCE AND IMPACT OF THE STUDY: These induced defence responses appeared to participate in attenuating Pectobacterium-induced diseases in plants. Thus, Streptomyces sp. OE7 could be a biocontrol agent against Pectobacterium sp.

HrpZ harpins from different Pseudomonas syringae pathovars differ in molecular interactions and in induction of anion channel responses in Arabidopsis thaliana suspension cells.

HrpZ, a type three secretion system helper protein from the plant-pathogen Pseudomonas syringae, can be recognized by many plants as a defence elicitor. Responses of Arabidopsis thaliana suspension cells to different HrpZ variants were studied by electrophysiological methods and cell death assay. Purified HrpZ originating from a compatible pathogen P. syringae pv. tomato DC3000 (HrpZ(Pto)) and incompatible P. syringae pv. phaseolicola (HrpZ(Pph)) both promoted Arabidopsis cell death. As an early response, both HrpZ variants induced an increase in time dependent K(+) outward rectifying current. In contrast, the effects of HrpZ proteins on anion currents were different: HrpZ(Pph) had no effect, and HrpZ(Pto) induced an anion current increase. This suggests that the observed responses of the K(+) channels and anion channels resulted from different and separable interactions and that the interaction implied in anion current modulation is host-specific. HrpZ(Pto) and HrpZ(Pph) also had a different sequence preference in phage display screen for peptide-binding. These peptides presumably represent a part of a putative target protein in the host, and HrpZ proteins of different P. syringae pathovars might have different binding specificities to match the allelic variation between plant species. Supporting the idea that the peptide-binding region of HrpZ is important for interactions with host cell components, we found that a mutation in that region changed the anion channel response of Arabidopsis cells.

Occurrence OF Pectobacterium carotovorum strains isolated from potato soft rot in Morocco.

Pectobacterium carotovorum subsp. carotovorum, Pectobacterium astrosepticum and Pectobacterium chrysanthemi are the soft rot tuber of potatoes pathogens (Solanum tuberosum). The aim of this study was to determine the occurrence of these pathogens in Moroccan regions producing potatoes. Fifty three isolates of Pectobacterium were isolated on medium Crystal Violet Pectate. The comparison of their bacteriological characteristics with standard strains allowed us to conclude that all the isolates belonged to the Pectobacterium. With regard to phenotype characteristics, the variability that was found included 32 typical Pectobacetrium carotovorum subsp. carotovorum, 3 typical Pectobacterium atrosepticum, and 18 atypical Pectobacterium carotovorum subsp. carotovorum. Three strains of the atypical group; showed that the biochemical properties overlap among the Pectobacterium carotovorum and Pectobacterium chrysanthemi. These data were needed molecular characterization. However, the PCR amplification of total genomic DNA of 53 isolates with the two primers Y1/Y2 and P143/P145 yielded an amplified fragment of the expected size (434 bp) only with Y1/Y2, indicated that all the isolates collected and tested belonged to the Pectobacterium carotovorum species. On the basis the pathogenicity tests, these strains revealed that they were pectinolytic, and showed differences in aggressiveness against potato and leaves of tobacco.

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.

Early physiological responses of Arabidopsis thaliana cells to fusaric acid: toxic and signalling effects.

Fusaric acid (FA) is a toxin produced by Fusarium species. Most studies on FA have reported toxic effects (for example, alteration of cell growth, mitochondrial activity and membrane permeability) at concentrations greater than 10(-5) m. FA participates in fungal pathogenicity by decreasing plant cell viability. However, FA is also produced by nonpathogenic Fusarii, potential biocontrol agents of vascular wilt fusaria. The aim of this study was to determine whether FA, at nontoxic concentrations, could induce plant defence responses. Nontoxic concentrations of FA were determined from cell-growth and O2-uptake measurements on suspensions of Arabidopsis thaliana cells. Ion flux variations were analysed from electrophysiological and pH measurements. H2O2 and cytosolic calcium were quantified by luminescence techniques. FA at nontoxic concentrations (i.e. below 10(-6) m) was able to induce the synthesis of phytoalexin, a classic delayed plant response to pathogen. FA could also induce rapid responses putatively involved in signal transduction, such as the production of reactive oxygen species, and an increase in cytosolic calcium and ion channel current modulations. FA can thus act as an elicitor at nanomolar concentrations.

Harpin, a hypersensitive response elicitor from Erwinia amylovora, regulates ion channel activities in Arabidopsis thaliana suspension cells.

HrpN, the hypersensitive response elicitor from Erwinia amylovora, stimulated K(+) outward rectifying currents in Arabidopsis thaliana suspension cells. It also decreased anion currents. These data demonstrate the ability of harpin to regulate different plasma membrane ion channels, putative components of signal transduction chains leading to defense responses and programmed cell death.

Ion currents involved in early Nod factor response in Medicago sativa root hairs: a discontinuous single-electrode voltage-clamp study.

Nod factor [NodRm-IV(Ac,S)], isolated from the bacterium Rhizobium meliloti, induces a well-known depolarization in Medicago sativa (cv Sitel) root hairs. Analysis of this membrane response using the discontinuous single-electrode voltage-clamp technique (dSEVC) shows that anion channel, K+ channel and H+-ATPase pump currents are involved in young growing root hairs. The early Nod-factor-induced depolarization is due to increase of the inward ion current and inhibition of the H+ pump. It involved an instantaneous inward anion current (IIAC) and/or a time-dependent inward K+ current (IRKC). These two ion currents are then down-regulated while the H+ pump is stimulated, allowing long-term rectification of the membrane potential (Em). Our results support the idea that the regulation of inward current plays a primary role in the Nod-factor-induced electrical response, the nature of the ions carried by these currents depending on the activated anion and/or K+ channels at the plasma membrane.

Transient outward K+ currents across the plasma membrane of laticifer from Hevea brasiliensis.

Non-inactivating outward rectifying K+ channel currents have been identified in a variety of plant cell types and species. The present study of laticifer protoplasts from Hevea brasiliensis, cells which are specialized for stress response, has revealed, through a switch-clamp method, an outward rectifying current displaying rapid inactivation. The inactivation depended on the external K+ concentration and on the voltage. This current inactivation appeared clearly different from all those previously described in plant cells and it shared homology with current kinetics of animal Shaker family channels. These results, given the recent cloning of plant K+ channel beta-subunits, shed new light on possible plant K+ channel regulation.

Evidence of multiple sugar uptake across the plasma membrane of laticifer protoplasts from Hevea.

In Hevea, rubber synthesis is confined to the cytosol of the highly differentiated laticifer cells. Agronomic and biochemical studies showed that this process uses high amounts of sugars that are efficiently imported into the laticifer. A H(+)-sugar symport system located in the plasma membrane is involved in sugar uptake into laticifers. Laticifer protoplasts were prepared and used in electrophysiological and labeling experiments to test the capacity of this system to transport a variety of sugars such as oligosaccharides from the raffinose family, trace compounds in rubber. Translocation of sugars known to be transported with different efficiency across the plasma membrane of plant cells was also tested. A 1 mM sucrose affinity was found for the symport. All the sugars tested, except palatinose induce membrane depolarization indicating that they were actively absorbed by the laticifer network. This reveals the wide capacity of this peculiar sink for the uptake of sugars.

HIV-1 gp160 decreases the K+ voltage-gated current from Jurkat E6.1 T cells by up-phosphorylation.

HIV-1 gp120/gp160 is known to disturb the activity of p56lck, protein kinase C (PKC) and Ca2+ homeostasis in T lymphocytes. We found that gp160 decreases the Kv1.3 current of Jurkat E6.1 cells probably by increasing the PKC-dependent phosphorylation of Kv channel protein after 5 days. This decrease is dose-dependent. In contrast, gp160 did not decrease the Kv1.3 current of the JCaM1.6 cell line, a p56(lck)-defective Jurkat cell line. This shows that p56lck was at the beginning of the events which induced the Kv1.3 current decrease. As a consequence of this decrease, Jurkat E6.1 cells were depolarized and exhibited a volume increase.

Pharmacological properties of slow anion currents in intact guard cells of Arabidopsis. Application of the discontinuous single-electrode voltage-clamp to different species.

More electrophysiological studies have been carried out on guard cells than on any other cell type of vascular plants. The characterization of their ion channels has been achieved using mainly the whole-cell patch-clamp technique applied to guard-cell protoplasts. The aim of this study was to obtain recordings of ion channel currents in intact guard cells and especially of slow anion channels of Arabidopsis thaliana, a species of fundamental genetic interest. Application of the discontinuous single-electrode voltage-clamp technique enabled the first characterization of K+ currents in Commelina communis and of slow anion currents in C. communis and A. thaliana in intact guard cells to be made. Inward K+ channels from A. thaliana were inhibited by external application of tetraethylammonium (TEA) or Ca2+. In the presence of K+ channel blockers, slow anion channel currents were elicited in almost all guard cells tested and were confirmed by the application of anion channel blockers. In A. thaliana, only anthracene-9 carboxylic acid was able to inhibit slow anion currents, to promote stomatal opening in the dark and to reverse the effect of 25 microM abscisic acid under light. Use of a single microelectrode and preservation of cell integrity make this technique well suited for the study of ion channel regulation in species that have guard cell protoplasts with which it is difficult to form good seals.