The Cell-Wall ß-d-Glucan in Leaves of Oat (Avena sativa L.) Affected by Fungal Pathogen Blumeria graminis f. sp. avenae.

In addition to the structural and storage functions of the (1,3; 1,4)-ß-d-glucans (ß-d-glucan), the possible protective role of this polymer under biotic stresses is still debated. The aim of this study was to contribute to this hypothesis by analyzing the ß-d-glucans content, expression of related cellulose synthase-like (Csl) Cs1F6, CslF9, CslF3 genes, content of chlorophylls, and ß-1,3-glucanase content in oat (Avena sativa L.) leaves infected with the commonly occurring oat fungal pathogen, Blumeria graminis f. sp. avenae (B. graminis). Its presence influenced all measured parameters. The content of ß-d-glucans in infected leaves decreased in all used varieties, compared to the non-infected plants, but not significantly. Oats reacted differently, with Aragon and Vaclav responding with overexpression, and Bay Yan 2, Ivory, and Racoon responding with the underexpression of these genes. Pathogens changed the relative ratios regarding the expression of CslF6, CslF9, and CslF3 genes from neutral to negative correlations. However, changes in the expression of these genes did not statistically significantly affect the content of ß-d-glucans. A very slight indication of positive correlation, but statistically insignificant, was observed between the contents of ß-d-glucans and chlorophylls. Some isoforms of ß-1,3-glucanases accumulated to a several-times higher level in the infected leaves of all varieties. New isoforms of ß-1,3-glucanases were also detected in infected leaves after fungal infection.

Molecular characterization and evolution of carnivorous sundew (Drosera rotundifolia L.) class V ß-1,3-glucanase.

MAIN CONCLUSION: A gene for ß-1,3-glucanase was isolated from carnivorous sundew. It is active in leaves and roots, but not in digestive glands. Analyses in transgenic tobacco suggest its function in germination. Ancestral plant ß-1,3-glucanases (EC 3.2.1.39) played a role in cell division and cell wall remodelling, but divergent evolution has extended their roles in plant defense against stresses to decomposition of prey in carnivorous plants. As available gene sequences from carnivorous plants are rare, we isolated a glucanase gene from roundleaf sundew (Drosera rotundifolia L.) by a genome walking approach. Computational predictions recognized typical gene features and protein motifs described for other plant ß-1,3-glucanases. Phylogenetic reconstructions suggest strong support for evolutionary relatedness to class V ß-1,3-glucanases, including homologs that are active in the traps of related carnivorous species. The gene is expressed in sundew vegetative tissues but not in flowers and digestive glands, and encodes for a functional enzyme when expressed in transgenic tobacco. Detailed analyses of the supposed promoter both in silico and in transgenic tobacco suggest that this glucanase plays a role in development. Specific spatiotemporal activity was observed during transgenic seed germination. Later during growth, the sundew promoter was active in marginal and sub-marginal areas of apical true leaf meristems of young tobacco plants. These results suggest that the isolated glucanase gene is regulated endogenously, possibly by auxin. This is the first report on a nuclear gene study from sundew.

Plant chitinase responses to different metal-type stresses reveal specificity.

KEY MESSAGE: Chitinases in Glycine max roots specifically respond to different metal types and reveal a polymorphism that coincides with sensitivity to metal toxicity. Plants evolved various defense mechanisms to cope with metal toxicity. Chitinases (EC 3.2.1.14), belonging to so-called pathogenesis-related proteins, act as possible second line defense compounds in plants exposed to metals. In this work their activity was studied and compared in two selected soybean (Glycine max L.) cultivars, the metal-tolerant cv. Chernyatka and the sensitive cv. Kyivska 98. Roots were exposed to different metal(loid)s such as cadmium, arsenic and aluminum that are expected to cause toxicity in different ways. For comparison, a non-metal, NaCl, was applied as well. The results showed that the sensitivity of roots to different stressors coincides with the responsiveness of chitinases in total protein extracts. Moreover, detailed analyses of acidic and neutral proteins identified one polymorphic chitinase isoform that distinguishes between the two cultivars studied. This isoform was stress responsive and thus could reflect the evolutionary adaptation of soybean to environmental cues. Activities of the individual chitinases were dependent on metal type as well as the cultivar pointing to their more complex role in plant defense during this type of stress.

Glucan-rich diet is digested and taken up by the carnivorous sundew (Drosera rotundifolia L.): implication for a novel role of plant ß-1,3-glucanases.

Carnivory in plants evolved as an adaptation strategy to nutrient-poor environments. Thanks to specialized traps, carnivorous plants can gain nutrients from various heterotrophic sources such as small insects. Digestion in traps requires a coordinated action of several hydrolytic enzymes that break down complex substances into simple absorbable nutrients. Among these, several pathogenesis-related proteins including ß-1,3-glucanases have previously been identified in digestive fluid of some carnivorous species. Here we show that a single acidic endo-ß-1,3-glucanase of ~50 kDa is present in the digestive fluid of the flypaper-trapped sundew (Drosera rotundifolia L.). The enzyme is inducible with a complex plant ß-glucan laminarin from which it releases simple saccharides when supplied to leaves as a substrate. Moreover, thin-layer chromatography of digestive exudates showed that the simplest degradation products (especially glucose) are taken up by the leaves. These results for the first time point on involvement of ß-1,3-glucanases in digestion of carnivorous plants and demonstrate the uptake of saccharide-based compounds by traps. Such a strategy could enable the plant to utilize other types of nutritional sources e.g., pollen grains, fungal spores or detritus from environment. Possible multiple roles of ß-1,3-glucanases in the digestive fluid of carnivorous sundew are also discussed.

Cultivar-specific kinetics of chitinase induction in soybean roots during exposure to arsenic.

The kinetics of defense responses was studied in soybean exposed to ecologically relevant concentrations of arsenic for 96 h. In the roots of two soybean cultivars with contrasting tolerance to this metalloid there were observed differences in basal levels of membrane lipid peroxidation as well as a significantly different course of peroxidation upon exposure to As. The different course of stress was reflected in the accumulation of defense components. The responses of individual chitinase isoforms were studied since these enzymes had previously been shown to be stable components of defense against metals. The kinetics and magnitude of accumulation of the three isoforms during exposure to As significantly differed within as well as between the studied cultivars. Furthermore, accumulation of these isoforms appeared to be related to oxidative status in the root tissue. The timing of induced responses is likely to be important for efficient defense against metal(oid) pollution in environment.

Defense responses of soybean roots during exposure to cadmium, excess of nitrogen supply and combinations of these stressors.

Heavy metal pollution is a serious environmental problem in agricultural soils since the uptake of heavy metals by plants represents an entry point into the food chain and is influenced by the form and amount of nitrogen (N) fertilization. Here we studied the defense responses in soybean roots exposed to ions of cadmium (applied as 50 mg l(-1) Cd(2+)) when combined with an excessive dose of N in form of NH(4)NO(3). Our data indicate that despite of stunted root growth, several stress symptoms typically observed upon cadmium treatment, e.g. peroxidation of lipid membranes or activation of chitinase isoforms, become suppressed at highly excessive N. At the same time, other defense mechanisms such as catalases and proline accumulation were elevated. Most importantly, the interplay of ongoing responses resulted in a decreased uptake of the metal into the root tissue. This report points to the complexity of plant defense responses under conditions of heavy metal pollution combined with intensive fertilization in agriculture.