Salicylic acid (SA) has been proposed to antagonize jasmonic acid (JA) biosynthesis and signaling. We report, however, that in salicylate hydroxylase-expressing tobacco (Nicotiana tabacum) plants, where SA levels were reduced, JA levels were not elevated during a hypersensitive response elicited by Pseudomonas syringae pv phaseolicola. The effects of cotreatment with various concentrations of SA and JA were assessed in tobacco and Arabidopsis (Arabidopsis thaliana). These suggested that there was a transient synergistic enhancement in the expression of genes associated with either JA (PDF1.2 [defensin] and Thi1.2 [thionin]) or SA (PR1 [PR1a-beta-glucuronidase in tobacco]) signaling when both signals were applied at low (typically 10-100 microm) concentrations. Antagonism was observed at more prolonged treatment times or at higher concentrations. Similar results were also observed when adding the JA precursor, alpha-linolenic acid with SA. Synergic effects on gene expression and plant stress were NPR1- and COI1-dependent, SA- and JA-signaling components, respectively. Electrolyte leakage and Evans blue staining indicated that application of higher concentrations of SA + JA induced plant stress or death and elicited the generation of apoplastic reactive oxygen species. This was indicated by enhancement of hydrogen peroxide-responsive AoPR10-beta-glucuronidase expression, suppression of plant stress/death using catalase, and direct hydrogen peroxide measurements. Our data suggests that the outcomes of JA-SA interactions could be tailored to pathogen/pest attack by the relative concentration of each hormone.

With the centenary of the first descriptions of 'hypersensitiveness' following pathogenic challenge upon us, it is appropriate to assess our current understanding of the hypersensitive response (HR) form of cell death. In recent decades our understanding of the initiation, associated signalling, and some important proteolytic events linked to the HR has dramatically increased. Genetic approaches are increasingly elucidating the function of the HR initiating resistance genes and there have been extensive analyses of death-associated signals, calcium, reactive oxygen species (ROS), nitric oxide, salicylic acid, and now sphingolipids. At the same time, attempts to draw parallels between mammalian apoptosis and the HR have been largely unsuccessful and it may be better to consider the HR to be a distinctive form of plant cell death. We will consider if the HR form of cell death may occur through metabolic dysfunction in which malfunctioning organelles may play a major role. This review will highlight that although our knowledge of parts of the HR is excellent, a comprehensive molecular model is still to be attained.

SummaryThe roles of salicylic acid (SA) and H202 in the induction of PR proteins in tobacco have been examined. Studies were conducted on wild-type tobacco and plants engineered to express a bacterial salicylate hydroxylase capable of metabolizing SA to catechol (SH-L plants). Wildtype and PR-la-GUS-transformed plants express PR-la following challenge with Pseudomonas syringae pathovar syringae, SA or 2,6-dichloro-isonicotinic acid (INA). In contrast, SH-L plants failed to respond to SA but did express PR-la following INA treatment. H202 and the irreversible catalass inhibitor 3-amino-l,2,4-triazole (3-AT) were found to be weak inducers of PR-la expression (relative to SA) in wild-type tobacco but were unable to induce PR-la in SH-L plants, suggesting that the action of these compounds depends upon the accumulation of SA. A model has been proposed suggesting that SA binds to and inhibits a catalase inducing an increase in H202 leading to PR protein expression. Catalase activity has been measured in tobacco end no significant changes in activity following infection with P. syringae pv. syringee were detected. Furthermore, inhibition of catalass activity in vitro in plant extracts requires pre-incubation and only occurs at SA concentrations above 250 I~M. Leaf disks preincubated with I mM SA do accumulate SA to these levels and PR-la is efficiently induced but there is no apparent inhibition of catalass activity. It is also shown that a SAresponsive gene, PR-le, and a H202-sensitive gene, AoPR-1, are both relatively insensitive to 3-AT suggesting that induction of these genes is unlikely to be due entirely to inhibition of an endogenous catalase.

Tobacco infected with Pseudomonas syringae pv. phaseolicola undergoes a hypersensitive response (HR). Jasmonic acid (JA) accumulated within the developing lesion 3 to 9 h after infection and this accumulation preceded protein loss, cell death, and malondialdehyde accumulation. Accumulating JA consisted largely of the (—)-JA stereoisomer and was essentially restricted to the HR lesion.

A rapid and localized programmed cell death -the hypersensitive response (HR) -is a widely utilized plant resistance mechanism against pathogens. Studies have implicated H 2 O 2 generation as a key elicitory mechanism in the HR. The causal relationship between the kinetics of the in planta oxidative burst, the HR and certain defence gene expression was examined. H 2 O 2 generation following challenge with avirulent strains of Pseudomonas syringae pv. ( P. s. pv.) syringae occurred in two phases. The effects of ROS generation were investigated using the H 2 O 2 -responsive transgene AoPR10-GUS, the dually responsive (H 2 O 2 and salicylic acid) PR1a-GUS as well as measures of cell death. Co-application of catalase with P. s. pv. syringae into tobacco leaf panels suppressed AoPR10-and PR1a-GUS expression and cell death. Conversely, varying H 2 O 2 generation with glucose: glucose oxidase influenced both defence gene expression and cell death. AoPR10-GUS proved to be primarily responsive to apoplastic not intracellular oxidative stress, suggesting that the apoplasm was a distinctive source of oxidative signals. A biphasic oxidative burst was also observed with virulent P. s. pv. tabaci , which, although delayed compared to that observed during HR, persisted at equivalent levels for a longer period. Taking all these data together we suggest that either (1) additional factors to the apoplastic oxidative burst are required to explain the rapid kinetics of defence signalling and cell death associated with the HR or (2) P. s. pv. tabaci successfully suppresses the effects of H 2 O 2 generation by an unknown mechanism.

It is reported here that salicylic acid (SA) is rapidly taken up by Arabidopsis cells, and its uptake is accompanied by media alkalization and cytosolic acidification, and it is inhibited by the ionophore nigericin, suggesting that its import is linked with that of H+ and driven by a proton gradient. Such import and accumulation declined sharply within a narrow physiological pH range (pH 5.7-6.1), corresponding to a reduction in the [H+] of the media from 1.99 micromol l(-1) to 0.79 micromol l(-1). Following the initial uptake, SA was exported back into the media as free SA against a continued [H+]-dependent import. Since the uptake and accumulation of SA declines sharply within a narrow pH range and cell wall alkalization is an early response during incompatible plant/pathogen interactions, the bacterial elicitor harpin(Pss) was used to investigate how SA transport may be modulated during defence responses. Harpin induced a rapid and sustained alkalization of the cell suspension media, reaching the critical pH (pH 5.9-6.1) at which SA import is inhibited at c. 60 min. Such media alkalization corresponded with a reduction in the SA associated with cells co-treated with harpin, and an inhibition of SA uptake in cells pretreated with harpin. Scavengers of ROS, or compounds which generate H2O2 or NO had little effect on the import or net export of SA, suggesting that media alkalization induced by harpin is sufficient to modulate the kinetics of SA transport.

Summary Using a promoter-uidA (AoPRT-L-GUS) construct, we have characterized heterologous expression controlled by an Asparagus officinalis acidic PR-5 gene promoter. The construct was found to be up-regulated following a variety of treatments with the defence signal salicylate. Similarly, AoPRT-L-GUS was induced by the SA mimic benzothiodiazole, however, unlike salicylate, this compound does not appear to be transported through the vasculature. The construct was insensitive to wounding and to the wound signal jasmonate. Pathogen challenge resulted in a restricted zone of expression at and around the infection site. High levels of NaCl or PEG 8000 failed to induce foliar expression, however, mannitol proved to be an effective inducer when applied as a root drench. The oxidants H(2)O(2) and t-butyl hydroperoxide also failed to induce AoPRT-L-GUS expression. Developmental expression of the construct appeared to be limited to leaf axils, sepal tips, a proportion of anthers and a small segment of tissue just below the stigma. Thus, the AoPRT-L promoter exhibits a limited expression profile responding principally to salicylate-related defence signals, and shows very little developmental expression. This suggests that the AoPRT-L promoter may be an ideal choice for contained gene expression.

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