Tomonori Kawano

Increased Anion Channel Activity Is an Unavoidable Event in Ozone-Induced Programmed Cell Death

Background Ozone is a major secondary air pollutant often reaching high concentrations in urban areas under strong daylight, high temperature and stagnant high-pressure systems. Ozone in the troposphere is a pollutant that is harmful to the plant. Principal Findings By exposing cells to a strong pulse of ozonized air, an acute cell death was observed in suspension cells of Arabidopsis thaliana used as a model. We demonstrated that O3 treatment induced the activation of a plasma membrane anion channel that is an early prerequisite of O3-induced cell death in A. thaliana. Our data further suggest interplay of anion channel activation with well known plant responses to O3, Ca2+ influx and NADPH-oxidase generated reactive oxygen species (ROS) in mediating the oxidative cell death. This interplay might be fuelled by several mechanisms in addition to the direct ROS generation by O3; namely, H2O2 generation by salicylic and abscisic acids. Anion channel activation was also shown to promote the accumulation of transcripts encoding vacuolar processing enzymes, a family of proteases previously reported to contribute to the disruption of vacuole integrity observed during programmed cell death. Significance Collectively, our data indicate that anion efflux is an early key component of morphological and biochemical events leading to O3-induced programmed cell death. Because ion channels and more specifically anion channels assume a crucial position in cells, an understanding about the underlying role(s) for ion channels in the signalling pathway leading to programmed cell death is a subject that warrants future investigation.

Post-transcriptional regulation of GORK channels by superoxide anion contributes to increases in outward-rectifying K⁺ currents.

· Ion fluxes are ubiquitous processes in the plant and animal kingdoms, controlled by fine-tuned regulations of ion channel activity. Yet the mechanism that cells employ to achieve the modification of ion homeostasis at the molecular level still remains unclear. This is especially true when it comes to the mechanisms that lead to cell death. · In this study, Arabidopsis thaliana cells were exposed to ozone (O₃). Ion flux variations were analyzed by electrophysiological measurements and their transcriptional regulation by RT-PCR. Reactive oxygen species (ROS) generation was quantified by luminescence techniques and caspase-like activities were investigated by laser confocal microscopy. · We highlighted the delayed activation of K(+) outward-rectifying currents after an O₃ -induced oxidative stress leading to programmed cell death (PCD). Caspase-like activities are detected under O₃ exposure and could be decreased by K(+) channel blocker. Molecular experiments revealed that the sustained activation of K(+) outward current could be the result of an unexpected O₂ ·⁻ post-transcriptional regulation of the guard cell outward-rectifying K(+) (GORK) channels. · This consists of a likely new mode of regulating the processing of the GORK mRNA, in a ROS-dependent manner, to allow sustained K(+) effluxes during PCD. These data provide new mechanistic insights into K(+) channel regulation during an oxidative stress response.

Crosstalk between intracellular and extracellular salicylic acid signaling events leading to long-distance spread of signals

It is well recognized that salicylic acid (SA) acts as a natural signaling molecule involved in both local and systemic plant defense responses upon attacks by pathogens. Recently, cellular SA receptors and a number of SA-related phloem-mobile signals were identified. Here, we compare the old and up-to-date concepts of plant defense signaling events involving SA. Finally, the crosstalk between intracellular and extracellular SA signaling events leading to long-distance spread of signals was outlined by focusing on the modes of both the short- and long-distance signaling events involving the actions of SA. For the above purpose, two distinct conceptual models for local SA perception and signaling mechanisms in the intracellular and extracellular paths (referred to as models i and ii, respectively) were proposed. In addition to two local SA perception models, we propose that the long-distance SA action could be attributed to three different modes, namely, (iii) local increase in SA followed by transport of SA and SA intermediates, (iv) systemic propagation of SA-derived signals with both chemical and electrical natures without direct movement of SA, and (v) integrated crosstalk allowing alternately repeated secondary signal propagation and biosynthesis of SA and/or conversion of inert SA intermediates to free SA finally contributing to the systemic spread of SA-derived signals. We review here that the long-distance SA signaling events (models iii–v), inevitably involve the mechanisms described in the local signaling models (models i and ii) as the key pieces of the crosstalk.

A role for oxalic acid generation in ozone-induced signallization in Arabidopis cells.

Ozone (O(3) ) is an air pollutant with an impact increasingly important in our industrialized world. It affects human health and productivity in various crops. We provide the evidences that treatment of Arabidopsis thaliana with O(3) results in ascorbate-derived oxalic acid production. Using cultured cells of A. thaliana as a model, here we further showed that oxalic acid induces activation of anion channels that trigger depolarization of the cell, increase in cytosolic Ca(2+) concentration, generation of reactive oxygen species and cell death. We confirmed that O(3) reacts with ascorbate in the culture, thus resulting in production of oxalic acid and this could be part of the O(3) -induced signalling pathways that trigger programmed cell death.

Hydrogen peroxide-independent generation of superoxide by plant peroxidase: hypotheses and supportive data employing ferrous ion as a model stimulus

When plants are threaten by microbial attacks or treated with elicitors, alkalization of extracellular space is often induced and thus pH-dependent extracellular peroxidase-mediated oxidative burst reportedly takes place, especially at the site of microbial challenge. However, direct stimulus involved in activation of peroxidase-catalyzed oxidative burst has not been identified to date. Here, we would like to propose a likely role for free ferrous ion in reduction of ferric native peroxidase into ferrous enzyme intermediate which readily produces superoxide anion via mechanism involving Compound III, especially under alkaline condition, thus, possibly contributing to the plant defense mechanism. Through spectroscopic and chemiluminescence (CL) analyses of reactions catalyzed by horseradish peroxidase (HRP), the present study proposed that plant peroxidase-catalyzed production of superoxide anion can be stimulated in the absence of conventional peroxidase substrates but in the presence of free ferrous ion.

Peroxyacetyl nitrate-induced oxidative and calcium signaling events leading to cell death in ozone-sensitive tobacco cell-line

It has long been concerned that some secondary air pollutants such as smog components, ozone (O3) and peroxyacetyl nitrate (PAN), are highly phytotoxic even at low concentrations. Compared with the biology of O3, we largely lack the information on the toxicity model for PAN at the cellular signaling levels. Here, we studied the cell-damaging impact of PAN using suspension culture of smog-sensitive tobacco variety (Bel-W3). The cells were exposed to freshly synthesized PAN and the induced cell death was assessed under microscope after staining with Evans blue. Involvement of reactive oxygen species (ROS) in PAN toxicity was suggested by PAN-dependently increased intracellular H2O2 and also by the cell-protective effects of ROS scavengers and related inhibitors. Calcium chelator also lowered the level of PAN-induced cell death, indicating that Ca2+ is also involved. Using a transgenic cell line expressing aequorin, an increase in cytosolic Ca2+ concentration responsive to the pulse of PAN, but sensitive to Ca2+ channel blockers, was recorded, indicating that Ca2+ channels are activated by PAN or PAN-derived signals. Above data show some similarity between the signaling mechanisms responsive to O3 and PAN.

Salicylic acid-induced superoxide generation catalyzed by plant peroxidase in hydrogen peroxide-independent manner

It has been reported that salicylic acid (SA) induces both immediate spike and long lasting phases of oxidative burst represented by the generation of reactive oxygen species (ROS) such as superoxide anion radical (O2•−). In general, in the earlier phase of oxidative burst, apoplastic peroxidase are likely involved and in the late phase of the oxidative burst, NADPH oxidase is likely involved. Key signaling events connecting the 2 phases of oxidative burst are calcium channel activation and protein phosphorylation events. To date, the known earliest signaling event in response to exogenously added SA is the cell wall peroxidase-catalyzed generation of O2•− in a hydrogen peroxide (H2O2)-dependent manner. However, this model is incomplete since the source of the initially required H2O2 could not be explained. Based on the recently proposed role for H2O2-independent mechanism for ROS production catalyzed by plant peroxidases (Kimura et al., 2014, Frontiers in Plant Science), we hereby propose a novel model for plant peroxidase-catalyzed oxidative burst fueled by SA.

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

ABSTRACT 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.

Enhanced elevations of hypo-osmotic shock-induced cytosolic and nucleic calcium concentrations in tobacco cells by pretreatment with dimethyl sulfoxide

ABSTRACT Dimethyl sulfoxide (DMSO) is a dipolar aprotic solvent widely used in biological assays. Here, we observed that DMSO enhanced the hypo-osmotically induced increases in the concentration of Ca2+ in cytosolic and nucleic compartments in the transgenic cell-lines of tobacco (BY-2) expressing aequorin.

Methanol induces cytosolic calcium variations, membrane depolarization and ethylene production in arabidopsis and tobacco

Background and Aims Methanol is a volatile organic compound released from plants through the action of pectin methylesterases (PMEs), which demethylesterify cell wall pectins. Plant PMEs play a role in developmental processes but also in responses to herbivory and infection by fungal or bacterial pathogens. However, molecular mechanisms that explain how methanol could affect plant defences remain poorly understood. Methods Using cultured cells and seedlings from Arabidopsis thaliana and tobacco BY2 expressing the apoaequorin gene, allowing quantification of cytosolic Ca2+, a reactive oxygen species (ROS) probe (CLA, Cypridina luciferin analogue) and electrophysiological techniques, we followed early plant cell responses to exogenously supplied methanol applied as a liquid or as volatile. Key Results Methanol induces cytosolic Ca2+ variations that involve Ca2+ influx through the plasma membrane and Ca2+ release from internal stores. Our data further suggest that these Ca2+ variations could interact with different ROS and support a signalling pathway leading to well known plant responses to pathogens such as plasma membrane depolarization through anion channel regulation and ethylene synthesis. Conclusions Methanol is not only a by-product of PME activities, and our data suggest that [Ca2+]cyt variations could participate in signalling processes induced by methanol upstream of plant defence responses.