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Nitric oxide (NO) is a very active molecule involved in many and diverse biological pathways where it has proved to be protective against damages provoked by oxidative stress conditions. In this work, we studied the effect of two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine SNP-treated on the response of wheat (Triticum aestivum) to water stress conditions. After 2 and 3 h of drought, detached wheat leaves pretreated with 150 microM SNP retained up to 15% more water than those pretreated with water or NO(2)(-)/NO(3)(-). The effect of SNP treatment on water retention was also found in wheat seedlings after 7 d of drought. These results were consistent with a 20% decrease in the transpiration rate of SNP-treated detached wheat leaves for the same analyzed time. In parallel experiments, NO was also able to induce a 35%, 30%, and 65% of stomatal closure in three different species, Tradescantia sp. (monocotyledonous) and two dicotyledonous, Salpichroa organifolia and fava bean (Vicia faba), respectively. In SNP-treated leaves of Tradescantia sp., the stomatal closure was correlated with a 10% increase on RWC. Ion leakage, a cell injury index, was 25% lower in SNP-treated wheat leaves compared with control ones after the recovery period. Carboxy-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide), a specific NO scavenger, reverted SNP action by restoring the transpiration rate, stomatal aperture, and the ion leakage to the level found in untreated leaves. Northern-blot analysis showed that SNP-treated wheat leaves display a 2-fold accumulation of a group three late embryogenesis abundant transcript with respect to control leaves both after 2 and 4 h of drought periods. All together, these results suggest that the exogenous application of NO donors might confer an increased tolerance to severe drought stress conditions in plants.

The proteolytic activity present in the extracellular preparation (ECP) from suspension media of infective structures of the late blight pathogen Phytophthora infestans was partially characterized. The proteolytic activity was analyzed in gelatin-containing SDS-PAGE. A discrete band of digested gelatin was visualized at approximately 45 kDa in ECPs from zoospores and germinating cysts media. Treatment of ECP with the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) or incubation at 100°C for 5 min completely abolished the proteolytic activity in the zymograph assay. When microinjected in potato leaves, ECP induce localized necrosis within 24 h post inoculation. This necrosis appeared in potato and was not visible in two non-host plants. Moreover, the necrosis seems to be dependent on active host metabolism. Treatments of ECP with Proteinase K, PMSF and boiling inhibited their ability to induce the necrotic response. These results suggest a correlation between, proteolytic and necrosis-inducing activities in ECP. A preliminary characterization with protease inhibitors suggests that the ECPs contain serine protease(s).

Nitric oxide (NO) is a bioactive molecule involved in many physiological processes. Among its biological function, NO has been proved to be cytotoxic against microorganisms in cells of the immune response, thus preventing infection. We have specifically studied the effect of a NO donor, sodium nitroprusside (SNP), on the chlorophyll content in potato leaves infected with the pathogenic fungus Phytophthora infestans (Pi). Fifteen days after infection, chlorophyll content strongly decayed in water-treated potato leaf sections. SNP was able to partially revert that loss in a dose-dependent manner, being the effective SNP concentrations between 10 µM and 100 µM. NaNO2and NaNO3, the SNP-derived residual products, were unable to prevent the chlorophyll loss. Treatments with SNP did not affect the survival of Pi and the fungus was able to grow in a V8-agar medium containing 100 µM SNP. Both the amount and the extent of germination of Pi sporangia resulted similar in the absence and in the presence of SNP. Respiratory inhibitors of the cyanide-sensitive and cyanide-resistant pathways, 2,4-dinitrophenol and salicylhydroxamic acid respectively, did not change the chlorophyll levels in infected potato leaves, suggesting that NO effect should not be on mitochondrial respiration. These results indicate that NO could be a protective molecule, either preserving the chloroplast membrane of infected leaf sections against the toxicity of reactive oxygen species or being directly involved in any step of the chlorophyll metabolic pathway.

The initial infection process involving adhesion/recognition events between plants and fungal pathogens is essential for the establishment of pathogenesis. The basis of the specificity betweenPhytophthora infestansand potato is not yet known. It could lie in the recognition between extracellular matrix and plasma membrane components of the penetrating structures of the fungus and host cells during the earliest stages of infection processes. We have used polyclonal antibodies (AZandAC) obtained after immunizing rabbits with either 107zoospores (Z) or germinating cysts (C) ofP. infestansto detect putative fungal components located on the surface of invasive structures.AZrecognized several bands, whileACrecognized three polypeptides of approximately 87, 73 and 45 kDa in western blots. Zoospore suspensions preincubated either withAZorACwere unable to infect potato leaves. Since it was already known that some anti-idiotypic (anti-IT)antibodies may resemble the antigen, thus mimicking its structure,anti-ITantibodies (a-AZanda-AC)were generated in mice and used as inducers on potato tuber discs. In northern blotsa-AZtreatments increased 1.5-fold the glyceraldehyde-3-P dehydrogenase (GAPDH) and 1.8-fold the phenylalanine ammonia-lyase (PAL) transcript levels, while an increase of 1.8 and 1.7-fold for GAPDH and PAL, respectively, was observed when the inducer wasa-AC.Both PAL and GAPDH transcript levels have previously been shown to be modulated by fungal and elicitor treatments. These results show that the anti-idiotypic antibodies are able to mimic the pathogen components, leading to the accumulation of transcripts related to defence responses in potato–P. infestansinteraction.

Wheat leaves(Triticum aestivumL. cv. San Agustin INTA) were detached at the moment they had reached maximum expansion, put in tubes containing water and left in darkness. Under these conditions, leaf protein content decreased mainly as consequence of an increased rate of breakdown. In the range of 0 to 72 h after detachment, western blot analysis of leaf protein extracts displayed both similar proportions of total protein and quality of ubiquitin conjugates. Northern blot analysis of leaf RNA extracts revealed a 1.6 kb ubiquitin mRNA transcript which increased 3.5-fold after 48 h of treatment. Thus wheat leaves maintain both their ability for the ubiquitination of proteins and the transcription of ubiquitin mRNA at stages of senescence in which rates of protein breakdown are increased. The results suggest that the ubiquitin-dependent proteolytic pathway contributes to leaf protein breakdown during senescence

The changes in ubiquitin mRNA level in intact plants of both susceptible (cv. Spunta) and partially resistant (cv. Pampeana-INTA) potato cultivars after inoculation with low doses (l03sporangia cm−3) ofPhytophthora infestonswere studied after 72 h of treatment. Inoculation leads to 5-fold accumulation of potato ubiquitin transcripts in both cultivars. This result supports the connection between ubiquitin expression and defense reaction in plants.