Resistance of cotton towards Xanthomonas campestris pv. malvacearum.

Interactions between Gossypium spp. and the bacterial pathogen Xanthomonas campestris pv. malvacearum are understood in the context of the gene-for-gene concept. Reviewed here are the genetic basis for cotton resistance, with reference to resistance genes, resistance gene analogs, and bacterial avirulence genes, together with the physiological mechanisms involved in the hypersensitive response to the pathogen, including production of signaling hormones, synthesis of antimicrobial molecules and alteration of host cell structures. This host-pathogen interaction represents the most complex resistance gene/avr gene system yet known and is one of the few in which phytoalexins are known to be specifically localized in HR cells at anti-microbial concentrations.

Identification of a Cd2+- and Zn2+-binding site in cytochrome c using FTIR coupled to an ATR microdialysis setup and NMR spectroscopy.

Fourier transform infrared (FTIR) difference spectroscopy allows the study of molecular changes occurring at active sites in proteins with high sensitivity. Reactions are triggered by light, potential, or temperature steps and more recently by the diffusion of buffers containing effectors above membrane proteins deposited as films on ATR crystals. We have adapted a microdialysis system to an ATR, to study metal sites in soluble proteins. In this study, we identified a Cd(2+)- or Zn(2+)-binding site in cytochrome c with dissociation constants of 17 and 42 microM, respectively, which affects the oxidation rate of ferrocytochrome c by hydrogen peroxide. Using the microdialysis ATR-FTIR setup, we determined that a histidine and the carboxylate group of a glutamate are involved in Zn(2+) binding. The implication of His 33 and Glu 104 in the binding site was deduced from the comparison of FTIR data recorded with horse heart and the variant tuna cytochrome c lacking these two amino acids. A two-dimensional NMR analysis of the Zn(2+)-binding site in horse heart cytochrome c confirmed that His 33 and residues close to the C terminus are sensitive to Zn(2+) binding. This study demonstrates that the microdialysis ATR-FTIR setup is promising for the analysis of metal sites in proteins. From H(2)O/(2)H(2)O exchange experiments, we concluded that the impact of Zn(2+) and Cd(2+) binding on the oxidation kinetics of ferrocytochrome c by H(2)O(2) is associated to the perturbation of a hydrogen-bonding network involving His 33 that is sensitive to the redox state of cytochrome c.

Lipid peroxidation in cotton: Xanthomonas interactions and the role of lipoxygenases during the hypersensitive reaction.

Lipid peroxidation, often associated with hypersensitive cell death, may be initiated either by active oxygen species (AOS) or lipoxygenases (LOX). Here we report a detailed analysis of this oxidative process in both incompatible and compatible interactions between the cotton cultivar Reba B50 and Xanthomonas campestris pv. malvacearum (Xcm). The hypersensitive reaction (HR) was characterized by a massive production of polyunsaturated fatty acid (PUFA) hydroperoxides together with typical tissue dehydration. Among these, isomers peroxidized on carbon 9, largely predominant, were chiral, showing an excess in the S enantiomer. The HR process was accompanied by an increase in 9S-LOX activity and preceded by transcription of a LOX gene (GhKLox1). These results showed that: (i) AOS produced during the oxidative burst were not involved in PUFA peroxidation during HR; and (ii) as previously described in elicited leaves of tobacco, the massive enzymatic lipid peroxidation was closely associated with hypersensitive cell death. During disease development in this cotton cultivar, the 9-lipoxygenation of PUFAs was late, weak, preceded by a faint accumulation of GhKLox1 transcripts, and associated with chlorosis but not with necrosis. Consequently, the main difference between incompatible and compatible interactions was in the precocity and intensity of the oxidative process, rather than in its nature. These data provide the evidence for a correlation between lipid peroxidation and hypersensitive cell death induced by pathogens.

Salicylic acid mediated by the oxidative burst is a key molecule in local and systemic responses of cotton challenged by an avirulent race of Xanthomonas campestris pv malvacearum.

We analyzed the production of reactive oxygen species, the accumulation of salicylic acid (SA), and peroxidase activity during the incompatible interaction between cotyledons of the cotton (Gossypium hirsutum) cv Reba B50/Xanthomonas campestris pv malvacearum (Xcm) race 18. SA was detected in petioles of cotyledons 6 h after infection and 24 h post inoculation in cotyledons and untreated leaves. The first peak of SA occurred 3 h after generation of superoxide (O(2)(.-)), and was inhibited by infiltration of catalase. Peroxidase activity and accumulation of SA increased in petioles of cotyledons and leaves following H(2)O(2) infiltration of cotyledons from 0.85 to 1 mM. Infiltration of 2 mM SA increased peroxidase activity in treated cotyledons and in the first leaves, but most of the infiltrated SA was rapidly conjugated within the cotyledons. When increasing concentrations of SA were infiltrated 2. 5 h post inoculation at the beginning of the oxidative burst, the activity of the apoplastic cationic O(2)(.-)-generating peroxidase decreased in a dose-dependent manner. We have shown that during the cotton hypersensitive response to Xcm, H(2)O(2) is required for local and systemic accumulation of SA, which may locally control the generation of O(2)(.-). Detaching cotyledons at intervals after inoculation demonstrated that the signal leading to systemic accumulation of SA was emitted around 3 h post inoculation, and was associated with the oxidative burst. SA produced 6 h post infection at HR sites was not the primary mobile signal diffusing systemically from infected cotyledons.

Evaluation of lipid peroxidation as a toxicity bioassay for plants exposed to copper.

Agricultural soils may contain toxic levels of copper (Cu) due to sewage sludge spreading or industrial pollution but chemical analyses may not be representative of Cu bioavailability, defined as the soil Cu fraction that plants can actually absorb (i.e. Cu fraction which is not strongly adsorbed to soil components). Lipid peroxidation caused by Cu in plants was investigated as a relevant bioassay of toxicity. Seven-day-old rapeseed plantlets were grown on Cu-supplemented medium in controlled conditions. Lipid-peroxidation was assessed by measuring: (1) the 2-thiobarbituric acid (TBA)-reactive substances; (2) the hydroperoxy acids by HPLC analysis; and (3) the alkane outputs by gas chromatography. We first verified the correlation between the results obtained by each method and then discussed their advantages and disadvantages within the context of a bioassay, showing that the volatile alkane output measurement is the most precise and easy to perform method for this purpose.

Involvement of lipoxygenase-dependent production of fatty acid hydroperoxides in the development of the hypersensitive cell death induced by cryptogein on tobacco leaves.

Lipid peroxidation was investigated in relation with the hypersensitive reaction in cryptogein-elicited tobacco leaves. A massive production of free polyunsaturated fatty acid (PUFA) hydroperoxides dependent on a 9-lipoxygenase (LOX) activity was characterized during the development of leaf necrosis. The process occurred after a lag phase of 12 h, was accompanied by the concomitant increase of 9-LOX activity, and preceded by a transient accumulation of LOX transcripts. Free radical-mediated lipid peroxidation represented 10% of the process. Inhibition and activation of the LOX pathway was shown to inhibit or to activate cell death, and evidence was provided that fatty acid hydroperoxides are able to mimic leaf necrotic symptoms. Within 24 h, about 50% of leaf PUFAs were consumed, chloroplast lipids being the major source of PUFAs. The results minimize the direct participation of active oxygen species from the oxidative burst in membrane lipid peroxidation. They suggest, furthermore, the involvement of lipase activity to provide the free PUFA substrates for LOX. The LOX-dependent peroxidative pathway, responsible for tissue necrosis, appears as being one of the features of hypersensitive programmed cell death.

The extensin multigene family responds differentially to superoxide or hydrogen peroxide in tomato cell cultures.

Changes in extensin gene expression were examined in cultured tomato cells following treatments leading to the production of activated oxygen species. Digitonin, a steroid glycoalkaloid compound, has been shown to trigger a rapid and transient production of superoxide anion, O2-*. 6 h after application of 50 or 100 microM of digitonin, the accumulation of four extensin transcripts (1.5, 2.6, 4.0 and 6.1 kb) was observed. Superoxide dismutase strongly inhibited the digitonin-mediated response, suggesting a key role of O2-* in the signalling cascade. Furthermore, cells treated with enzymatically produced O2-* generated by xanthine oxidase (0.015 U/ml) gave a similar extensin response and again, SOD exerted a strong inhibitory effect on the response. On the other hand, H2O2 (2 mM) or the enzymatic H2O2 generator, glucose oxidase (0.34 U/ml), elicited the accumulation of only three of the four transcripts (1.5, 2.6 and 4.0 kb), indicating that the corresponding genes could be regulated either by H2O2 or O2-* but that the gene encoding the 6.1 kb transcript was exclusively expressed in response to O2-*. Finally, it was shown that lipid peroxidation, which was only induced when cells were exposed to H2O2, did not participate in the AOS-mediated gene expression for extensin. It can be concluded from these results that tomato cells are able to discriminate H2O2 from O2-* and they probably sense the latter by the specific oxidation of an extracellular component.

Are elicitins cryptograms in plant-Oomycete communications?

Stimulation of plant natural defenses is an important challenge in phytoprotection prospects. In that context, elicitins, which are small proteins secreted by Phytophthora and Pythium species, have been shown to induce a hypersensitive-like reaction in tobacco plants. Moreover, these plants become resistant to their pathogens, and thus this interaction constitutes an excellent model to investigate the signaling pathways leading to plant resistance. However, most plants are not reactive to elicitins, although they possess the functional signaling pathways involved in tobacco responses to elicitin. The understanding of factors involved in this reactivity is needed to develop agronomic applications. In this review, it is proposed that elicitins could interact with regulating cell wall proteins before they reach the plasma membrane. Consequently, the plant reactivity or nonreactivity status could result from the equilibrium reached during this interaction. The possibility of overexpressing the elicitins directly from genomic DNA in Pichia pastoris allows site-directed mutagenesis experiments and structure/function studies. The recent discovery of the sterol carrier activity of elicitins brings a new insight on their molecular activity. This constitutes a crucial property, since the formation of a sterol-elicitin complex is required to trigger the biological responses of tobacco cells and plants. Only the elicitins loaded with a sterol are able to bind to their plasmalemma receptor, which is assumed to be an allosteric calcium channel. Moreover, Phytophthora and Pythium do not synthesize the sterols required for their growth and their fructification, and elicitins may act as shuttles trapping the sterols from the host plants. Sequence analysis of elicitin genes from several Phytophthora species sheds unexpected light on the phylogenetic relationships among the genus, and suggests that the expression of elicitins is under tight regulatory control. Finally, general involvement of these lipid transfer proteins in the biology of Pythiaceae, and in plant defense responses, is discussed. A possible scheme for the coevolution between Phytophthora and tobacco plants is approached.

Measurement of thermally produced volatile alkanes: an assay for plant hydroperoxy fatty acid evaluation.

A new method designed to monitor lipid peroxidation in plants has been set up with soybean hypocotyl/radicles. The hydroperoxy fatty acids present in situ are converted by rapid thermal treatment (80 s and 210 J g-1) of the biological sample into ethane and n-pentane, which are analyzed by gas chromatography. The method has been directly calibrated by quantification of the hydroperoxy fatty acids by silica-phase HPLC analysis of their reduced hydroxy derivatives. Hypocotyl/radicles from the two soybean cultivars Argenta and Soriano were submitted to various chemical oxidative treatments and were analyzed for both thermally produced volatile alkanes and hydroperoxy fatty acid levels. Our results showed that ethane and n-pentane production are in both cases closely correlated with linolenic as well as linoleic acid hydroperoxide levels (P < 0.001). Within a given plant material, thermal conversion of both hydroperoxides into alkanes occurred with yields which were not dependent on the oxidative treatment. These yields are however functions of the biological material since in Soriano and Argenta cultivars they were around 6 and 25%, respectively. Taking into account the last point, the alkane test cannot be used to directly quantify the absolute lipid hydroperoxide levels of plant tissues but it is convenient to monitor the peroxidative phenomenon as it occurs. The assay is easy and rapid to perform (analysis of 50 samples per day) since no sample preparation is needed, and the low detection limit (20 pmol of alkane g-1) permits the analysis of small samples.

The protective function of the xanthophyll cycle in photosynthesis.

The rapid conversion of the carotenoid violaxanthin to zeaxanthin via antheraxanthin (xanthophyll cycle) in potato leaves exposed at 23 degrees C to a strong white light of 2000 microE.m-2.s-1 was associated with a slight inhibition of photosynthetic electron transport (as estimated from chlorophyll fluorescence measurements) and a low lipid peroxidation (as estimated from ethane measurements). When the xanthophyll cycle was blocked by dithiothreitol (3 mM) or low temperature (3 degrees C), photoinhibition of electron transport was exacerbated and pronounced lipid peroxidation occurred concomitantly. Accumulation of zeaxanthin and antheraxanthin in potato leaves by a non-photoinhibitory light treatment at 23 degrees C (900 microE.m-2.s-1 for 1 h) considerably reduced the level of lipid peroxidation during subsequent light stress at 3 degrees C. The presented results indicate that one of the functions of the xanthophyll cycle could be the protection of thylakoid membranes against lipid peroxidation, suggesting that zeaxanthin and antheraxanthin synthesized in strong light are present as free pigments in the membrane lipid bilayer.

Involvement of Oxidative Processes in the Signaling Mechanisms Leading to the Activation of Glyceollin Synthesis in Soybean (Glycine max).

The efficiency of hydroperoxides (tert-butyl hydroperoxide, hydrogen peroxide) and sulfhydryl reagents (iodoacetamide, p-chloromercuribenzene sulfonic acid) as glyceollin elicitors was examined in relation to sulfhydryl oxidation, or alteration, and to lipid peroxidation, in 3-d-old soybean hypocotyl/radicle, Glycine max. These oxidative events were investigated as possible early steps in the transduction mechanisms leading to phytoalexin synthesis. Free protein sulfhydryl groups were not modified after any of the eliciting treatments, thus indicating that immediate massive protein oxidation or modification cannot be considered a signal transduction step. Unlike sulfhydryl reagents, which led to a decrease of the free nonprotein sulfhydryl group (free np-SH) pool under all of the eliciting conditions, the results obtained with hydroperoxides indicated that immediate oxidation of the np-SH is not required for the signal transduction. Moreover, elicitation with 10 mM tertbutyl hydroperoxide did not lead to further oxidation or to changes in np-SH level during the critical phase of phenylalanine ammonialyase activation (the first 20 h), suggesting that np-SH modifications are probably not involved in hydroperoxide-induced elicitation. On the other hand, all treatments leading to significant glyceollin accumulation were able to trigger a rapid (within 2 h) lipid peroxidation process, whereas noneliciting treatments did not. In addition, transition metals, such as Fe2+ and Cu+, were shown to stimulate both hydrogen peroxide-induced lipid peroxidation and glyceollin accumulation, again emphasizing that the two processes are at least closely linked in soybean. Among the oxidative processes triggered by activated oxygen species, oxidation of sulfhydryl compounds, or lipid peroxidation, our results suggest that lipid peroxidation is sufficient to initiate glyceollin accumulation in soybean. This further supports the hypothesis that lipid peroxidation could be involved as a step in the signal cascade that leads to induction of plant defenses.

Radioimmunoassay of the phytotoxic compound zinniol.

The phytotoxic compound zinniol, produced by phytopathogenic fungi such as Alternaria spp. and Phoma macdonaldii was conjugated to bovine serum albumine by the mixed anhydride method. Antiserum against zinniol was obtained by injection at multiple intradermal sites of rabbits. Sensitivity of the R.I.A. was 0.14 ng/tube, the within and between-assay coefficient of variation were less than 10 and 14% respectively. Negligible binding occurred when analogs of zinniol were tested for cross reactivity. The excellent accuracy of this R.I.A., applied to ethanolic extracts of plants, might allow to determine the production of toxin by the parasite during the infection process.