Involvement of protein kinases and calcium in the NO-signalling cascade for defence-gene induction in ozonated tobacco plants.

This study analyses the signalling pathways triggered by nitric oxide (NO) in response to ozone (O(3)) fumigation of tobacco plants, with particular attention to protein kinase cascades and free cytosolic Ca(2+) in defence-gene activation. NO was visualized with the NO probe DAF-FM. Using a pharmacological approach, the effects of different inhibitors on the expression profiles of NO-dependent defence genes were monitored using RT-PCR. The assay of the kinase activity of the immunoprecipitates complexes shows that O(3) stimulates a 48 kDa salicylic acid (SA)-induced protein kinase (SIPK) in an NO-dependent manner. The O(3)-induced alternative oxidase 1a (AOX1a) and phenylalanine ammonia lyase a (PALa) genes are modulated by phosphorylation by protein kinases, and SIPK might have a role in this up-regulation. By contrast, protein dephosphorylation mediates pathogenesis-related protein 1a (PR1a) expression in O(3)-treated tobacco plants. Ca(2+) is essential, but not sufficient, to promote NO accumulation in ozonated tobacco plants. Intracellular Ca(2+) transients are also essential for PALa up-regulation and cGMP-induced PR1a expression. Partial dependence on intracellular Ca(2+) suggests two different pathways of SA accumulation and PR1a induction. A model summarizing the signalling networks involving NO, SA, and the cellular messengers in this O(3)-induced defence gene activation is proposed.

Effect of zinc imbalance and salicylic acid co-supply on Arabidopsis response to fungal pathogens with different lifestyles.

In higher plants, Zn nutritional imbalance can affect growth, physiology and response to stress, with effect variable depending on host-pathogen interaction. Mechanisms through which Zn operates are not yet well known. The hormone salicylic acid (SA) can affect plant ion uptake, transport and defence responses. Thus, in this study the impact of Zn imbalance and SA co-supply on severity of infection with the necrotrophic fungal pathogen B. cinerea or the biotroph G. cichoracearum was assessed in A. thaliana Col-0. Spectrophotometric assays for pigments and malondialdehyde (MDA) content as a marker of lipid peroxidation, plant defensin 1.2 gene expression by semi-quantitative PCR, callose visualization by fluorescence microscopy and diseases evaluation by macro- and microscopic observations were carried out. Zinc plant concentration varied with the supplied dose. In comparison with the control, Zn-deficit or Zn-excess led to reduced chlorophyll content and PDF 1.2 transcripts induction. In Zn-deficient plants, where MDA increased, also the susceptibility to B. cinerea increased, whereas MDA decreased in G. cichoracearum. Zinc excess increased susceptibility to both pathogens. Co-administration of SA positively affected MDA level, callose deposition, PDF 1.2 transcripts and plant response to the two pathogens. The increased susceptibility to B. cinerea in both Zn-deficient and Zn-excess plants could be related to lack of induction of PDF 1.2 transcripts; oxidative stress could explain higher susceptibility to the necrotroph and lower susceptibility to the biotroph in Zn-deficient plants. This research shows that an appropriate evaluation of Zn supply according to the prevalent stress factor is desirable for plants.

Effects of different routes of application on ethylenediurea persistence in tobacco leaves.

Ethylenediurea (EDU) is a common research tool for investigating ozone impacts on vegetation, although the role of different application routes (foliar spray vs soil drench) on EDU persistence in the leaves is unknown. We quantified EDU concentrations in leaves of the O3-sensitive Bel-W3 cultivar of tobacco treated with EDU as either foliar spray or soil drench. Foliar EDU concentrations were measured by Q-TOF LC/MS. When EDU was applied as foliar spray, 1 h was enough for reaching a measurable concentration within the leaf. EDU concentration increased over the 21-day period when the leaf was not washed after the application (treatment #1), while it decreased when the leaf was washed after the application (treatment #2). These results suggest that: a) dry deposition of EDU onto the leaf surface was gradually absorbed into the unwashed leaf, although the mechanisms of such uptake were unclear; b) concentration of EDU was decreased quickly (-35%) during the first 24 h from application and more slowly during the following three days (-20%) in the washed leaves. Degradation did not involve enzymatic reactions and was not affected by the presence of ROS. When EDU was applied as soil drench, foliar concentrations increased over time, likely due to adsorption onto soil organic matter and gradual re-solubilization by irrigation water. An analysis of EDU concentration in protoplast and intercellular washing fluid showed that EDU did not enter the cells, but was retained in the apoplast only. Possible implications of EDU in the apoplast and recommendations for EDU application are discussed.

Responses of the xanthophyll cycle pool and ascorbate-glutathione cycle to ozone stress in two tobacco cultivars.

Plants of Nicotiana tabacum (O3-tolerant cv Bel-B and O3-sensitive cv Bel-W3) were exposed to 150 ppb of ozone for 5 h; the fumigation produced visual injury in mature leaves, particularly in Bel-W3. After O3-treatment the pigments of the xanthophyll cycle pool decreased in both cvs, with a strong reduction in violaxanthin content, while antheraxanthin and zeaxanthin increased slightly. Under these conditions the content of leaf abscisic acid (ABA) markedly increased, particularly in O3-sensitive cv, indicating that the violaxanthin may have been partially converted into ABA. The control plants of Bel-B showed an ascorbic acid content four times greater than Bel-W3 and the ozone treatment did not produce significant differences in the ascorbic acid content and in the redox state. The two tobacco cvs were found to have similar total glutathione content, however the redox state was lower in O3-sensitive cv and decreased after ozone exposure. Ozone fumigation caused an increase in oxidized glutathione, particularly in Bel-W3, associated with a reduced glutathione reductase (GR) activity and a reduced GR protein content.

Gene expression in snapbeans exposed to ozone and protected by ethylenediurea.

Ethylenediurea (EDU) is the most common chemical used to prevent ozone (O3) injury on vegetation. Despite considerable research, its mode of action remains elusive and gene expression has not been studied. Transcripts of major antioxidant enzymes (catalase, glutathione reductase, glutathione peroxidase) were measured for the first time in a model plant (Phaseolus vulgaris cv S156) after short-term O3 exposure (0 or 90 ppb, 5 h/d, 4 days) and a single spray with EDU (0 or 300 ppm). Visible, physiological and biochemical parameters were assessed as indices of O3-induced stress. In O3-exposed EDU-protected plants, levels of transcript, enzyme activity, H2O2 accumulation, gas exchange and foliar visible injury were similar to those in control plants. These results suggest that EDU may halt the O3-induced ROS generation within 24 h from the exposure, and thus the downstream cascade mechanisms leading to increased H2O2 production, impaired gas exchange, and occurrence of leaf lesions.

Assessing the genotoxicity of urban air pollutants using two in situ plant bioassays.

Genotoxicity of urban air has been analysed almost exclusively in airborne particulates. We monitored the genotoxic effects of airborne pollutants in the urban air of Perugia (Central Italy). Two plant bioindicators with different genetic endpoints were used: micronuclei in meiotic pollen mother cells using Tradescantia-micronucleus bioassay (Trad-MCN) and DNA damage in nuclei of Nicotiana tabacum leaves using comet assay (Nicotiana-comet). Buds of Tradescantia clone # 4430 and young N. tabacum cv. Xanthi plants were exposed for 24 h at three sites with different pollution levels. One control site (indoor control) was also used. The two bioassays showed different sensitivities toward urban pollutants: Trad-MCN assay was the most sensitive, but DNA damage in N. tabacum showed a better correlation with the pollutant concentrations. In situ biomonitoring of airborne genotoxins using higher plants combined with chemical analysis is thus recommended for characterizing genotoxicity of urban air.