Luisa Ederli

Interaction between Nitric Oxide and Ethylene in the Induction of Alternative Oxidase in Ozone-Treated Tobacco Plants

The higher plant mitochondrial electron transport chain contains, in addition to the cytochrome chain, an alternative pathway that terminates with a single homodimeric protein, the alternative oxidase (AOX). We recorded temporary inhibition of cytochrome capacity respiration and activation of AOX pathway capacity in tobacco plants (Nicotiana tabacum L. cv BelW3) fumigated with ozone (O3). The AOX1a gene was used as a molecular probe to investigate its regulation by signal molecules such as hydrogen peroxide, nitric oxide (NO), ethylene (ET), salicylic acid, and jasmonic acid (JA), all of them reported to be involved in the O3 response. Fumigation leads to accumulation of hydrogen peroxide in mitochondria and early accumulation of NO in leaf tissues. Although ET accumulation was high in leaf tissues 5 h after the start of O3 fumigation, it declined during the recovery period. There were no differences in the JA and 12-oxo-phytodienoic acid levels of treated and untreated plants. NO, JA, and ET induced AOX1a mRNA accumulation. Using pharmacological inhibition of ET and NO, we demonstrate that both NO- and ET-dependent pathways are required for O3-induced up-regulation of AOX1a. However, only NO is indispensable for the activation of AOX1a gene expression.

Ozone-Induced Cell Death in Tobacco Cultivar Bel W3 Plants. The Role of Programmed Cell Death in Lesion Formation

Treatment of the ozone-sensitive tobacco (Nicotiana tabacum L. cv Bel W3) with an ozone pulse (150 nL L–1 for 5 h) induced visible injury, which manifested 48 to 72 h from onset of ozone fumigation. The “classical” ozone symptoms in tobacco cv Bel W3 plants occur as sharply defined, dot-like lesions on the adaxial side of the leaf and result from the death of groups of palisade cells. We investigated whether this reaction had the features of a hypersensitive response like that which results from the incompatible plant-pathogen interaction. We detected an oxidative burst, the result of H2O2 accumulation at 12 h from the starting of fumigation. Ozone treatment induced deposition of autofluorescent compounds and callose 24 h from the start of treatment. Total phenolic content was also strongly stimulated at the 10th and 72nd h from starting fumigation, concomitant with an enhancement in phenylalanine ammonia-lyase a and phenylalanine ammonia-lyase b expression, as evaluated by reverse transcriptase-polymerase chain reaction. There was also a marked, but transient, increase in the mRNA level of pathogenesis-related-1a, a typical hypersensitive response marker. Overall, these results are evidence that ozone triggers a hypersensitive response in tobacco cv Bel W3 plants. We adopted four criteria for detecting programmed cell death in ozonated tobacco cv Bel W3 leaves: (a) early release of cytochrome c from mitochondria; (b) activation of protease; (c) DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling of DNA 3′-OH groups; and (d) ultrastructural changes characteristic of programmed cell death, including chromatin condensation and blebbing of plasma membrane. We, therefore, provide evidence that ozone-induced oxidative stress triggers a cell death program in tobacco cv Bel W3.

Ozone and nitric oxide induce cGMP‐dependent and ‐independent transcription of defence genes in tobacco

Here, we analyse the temporal signatures of ozone (O3)-induced hydrogen peroxide(H2O2) and nitric oxide (NO) and the role of the second messenger guanosine3′,5′-cyclic monophosphate (cGMP) in transcriptional changes of genes diagnostic for biotic and abiotic stress responses. Within 90 min O3 induced H2O2 and NO peaks and we demonstrate that NO donors cause rapid H2O2 accumulation in tobacco (Nicotiana tabacum) leaf. Ozone also causes highly significant, late (> 2 h) and sustained cGMP increases, suggesting that the second messenger may not be required in all early (< 2 h) responses to O3,but is essential and sufficient for the induction of some O3-dependent pathways.This hypothesis was tested resolving the time course of O3-induced transcript accumulation of alternative oxidase (AOX1a), glutathione peroxidase (GPX),aminocyclopropancarboxylic acid synthase (ACS2) that is critical for the synthesis of ethylene, phenylalanine ammonia lyase (PALa) and the pathogenesis-related protein PR1a.The data show that early O3 and NO caused transcriptional activation of the scavenger encoding proteins AOX1a, GPX and the induction of ethylene production through ACS2 are cGMP independent. By contrast, the early response of PALa and the late response of PR1a show critical dependence on cGMP.

NO release by nitric oxide donors in vitro and in planta

Artificial nitric oxide (NO) donors are widely used as tools to study the role of NO in plants. However, reliable and reproducible characterisation of metabolic responses induced by different NO donors is complicated by the variability of their NO release characteristics. The latter are affected by different physical and biological factors including temperature and light. Here we critically evaluate NO release characteristics of the donors sodium nitroprusside (SNP), S-nitrosoglutathione (GSNO) and nitric oxide synthase (NOS), both in vitro and in planta (Nicotiana tabacum L. cv. BelW3) and assess their effects on NO dependent processes such as the transcriptional regulation of the mitochondrial alternative oxidase gene (AOX1a), accumulation of H(2)O(2) and induction of cell death. We demonstrate that, contrary to NOS and SNP, GSNO is not an efficient NO generator in leaf tissue. Furthermore, spectrophotometric measurement of NO with a haemoglobin assay, rather than diaminofluorescein (DAF-FM) based detection, is best suited for the quantification of tissue NO. In spite of the different NO release signatures by SNP and NOS in tissue, the NO dependent responses examined were similar, suggesting that there is a critical threshold for the NO response.

Photoinhibition and oxidative stress : effects on xanthophyll cycle, scavenger enzymes and abscisic acid content in tobacco plants

Summary The effects of short-term ozone pollution at high doses (300 ppb for 30 min) on the xanthophyll cycle under photoinhibitory conditions (1800 μmol m − 2 s − 1) were studied. The experiments were conducted on two different tobacco ( Nicotiana tabacum L.) cultivars, one O 3 -tolerant (Havana) and the other O 3 -sensi-tive (Bel-W3). Given that the phytotoxicity of O 3 is due to the formation of active oxygen species, the plants were also subjected to direct treatment with H 2 O 2 , · O 2 − , · OH and paraquat, a herbicide that, under certain conditions, produces the above-mentioned toxic forms. Our results show that in both cultivars oxidative stress inhibits the function of the xanthophyll cycle with a consequent decrease of anthera-xanthin and zeaxanthin synthesis, particularly during exposure to the superoxide ion and the · OH radical. In addition, we determined the activities of the scavenger enzymes, i.e. ascorbate peroxidase, dehydroas-corbate reductase, glutathione reductase, and glutathione-S-transferase involved in the ascorbate metabolism, the basic substrate for the de-epoxidation reaction of violaxanthin. The quantity of abscisic acid was also determined after ozone exposure. The results showed a greater presence of this hormone in the leaves of the O 3 -treated plants, a simultaneous degradation of violaxanthin was observed, which was not compensated by the transformation into antheraxanthin + zeaxanthin. It is hypothesized that, under photoinhibitory conditions combined with strong oxidative stress, violaxanthin is used in large part not for the xanthophyll cycle reaction but for the synthesis of growth inhibitory substances such as abscisic acid.

Deciphering cGMP signatures and cGMP-dependent pathways in plant defence.

The second messenger, 3’, 5’-cyclic monophosphate (cGMP), is a critical component of many different processes in plants while guanylyl cyclases that catalyse the formation of cGMP from GTP have remained somewhat elusive in higher plants. Consequently, two major aims are the discovery of novel GCs and the identification of cGMP mediated processes. Recently, we have reported temporal signatures of ozone (O3)-induced hydrogen peroxide (H2O2) and nitric oxide (NO) generation, their effect on cGMP generation, and consequent transcriptional changes of genes diagnostic for stress responses in tobacco. We demonstrated that O3 and NO induced early transcriptional activation of the scavenger encoding proteins, alternative oxidase (AOX1a), glutathione peroxidase (GPX) and the induction of ethylene production through aminocyclopropancarboxylic acid synthase (ACS2) are cGMP-independent. By contrast, the early response of the phenylalanine ammonia lyase gene (PALa) and the late response of the gene encoding the pathogenesis-related protein (PR1a) show critical dependence on cGMP. Here we show differential cGMP responses to virulent and avirulent Pseudomonas syringae strains and propose that host-pathogen recognition and/or down-stream processes are transduced by complex cGMP signatures. This is in accordance with the identification of a growing number of multi-domain molecules in Arabidopsis that are reported to contain putative functional GC catalytic centres.

Ozone affects pollen viability and NAD(P)H oxidase release from Ambrosia artemisiifolia pollen

Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O3) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O3 fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O3 fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O3, determined from the mRNA levels of the major allergens. We conclude that O3 can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase.

Morphological and cytological development and starch accumulation in hermaphrodite and staminate flowers of olive (Olea europaea L.)

In olive (Olea europaea L.), the formation of functionally staminate flowers rather than fully functional hermaphrodites is one of the major factors limiting fruit set, as flowers with aborted pistils are incapable of producing fruit. Studies conducted on various angiosperm species have shown a correlation between flower abortion and starch content. Thus, it is important to know if starch content plays a role in regulating pistil development in olive and if so, what mechanism regulates starch distribution. Cyto-histological observations of staminate and hermaphrodite olive flowers show that pistil development in staminate flowers is interrupted after the differentiation of the megaspore mother cell. At that stage, starch grains were only detected in the ovary, style and stigma of the hermaphrodite flowers. No starch was observed in the pistils of the staminate flowers. This finding suggests a tight correlation between starch content and pistil development. The secondary origin of starch within the flower is indicated by low chlorophyll content in the gynoecium, undetectable Rubisco activity in the pistils of these two kinds of flowers and by the ultrastructure of the plastids observed by transmission electron microscope analysis. The plastids have few thylakoid membranes and grana and in the staminate flowers appeared very similar to proplastids. Considering differences in starch content between staminate and hermaphrodite flowers and the secondary origin of the starch, differences in pistil development in the staminate and hermaphrodite flowers could be related to differences in the sink strength of these two types of flowers.

The Arabidopsis thaliana cysteine-rich receptor-like kinase CRK20 modulates host responses to Pseudomonas syringae pv. tomato DC3000 infection

In plants, the cysteine-rich repeat kinases (CRKs) are a sub-family of receptor-like protein kinases that contain the DUF26 motif in their extracellular domains. It has been shown that in Arabidopsis thaliana, CRK20 is transcriptionally induced by pathogens, salicylic acid and ozone (O(3)). However, its role in responses to biotic and abiotic stress remains to be elucidated. To determine the function of CRK20 in such responses, two CRK20 loss-of-function mutants, crk20-1 and crk20-2, were isolated from public collections of Arabidopsis T-DNA tagged lines and examined for responses to O(3) and Pseudomonas syringae pv. tomato (Pst) DC3000. crk20-1 and crk20-2 showed similar O(3) sensitivities and no differences in the expression of defense genes when compared with the wild-type. However, pathogen growth was significantly reduced, while there were no differences in the induction of salicylic acid related defense genes or salicylic acid accumulation. Furthermore, correlation analysis of CRK20 gene expression suggests that it has a role in the control of H(2)O and/or nutrient transport. We therefore propose that CRK20 promotes conditions that are favorable for Pst DC3000 growth in Arabidopsis, possibly through the regulation of apoplastic homeostasis, and consequently, of the environment of this biotrophic pathogen.

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.