Plant chitinases (EC 3.2.1.14) are considered as typical defense components under various environmental stresses, including heavy metals. In addition, some of them play crucial role in normal plant growth and development. In this work the profile and activities of these enzymes were analyzed to study the variability of defense within soybean plants. For this, two cultivars with contrasting tolerance to metals were exposed to ecologically relevant doses of arsenic and cadmium. Enzyme profiles revealed a spatial distribution of chitinase activities throughout the individual plants, tending to decrease upwards to the top of the plants. Under metal stress, there was a single responsive isoform detected in roots that behaved opposingly in the studied soybean cultivars. In contrast, several isoforms were activated in aboveground tissue, predominantly in mature (older) leaves. Of these, two were identified (21 and 42 kDa) as more specifically involved in defense against metal stress in soybean. The 21 kDa isoform was concluded as possibly contributing to metal tolerance and deserves further investigations at molecular level. Nevertheless, no sound interaction was detected between leaf developmental stage and responsiveness to metals for either of the chitinase isoforms. Further studying the distribution of induced defense within plants is important in understanding the defense strategy of plants against environmental cues including metals.

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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.

Plants have a potential for the uptake and accumulation of essential and non-essential trace elements. The ability to take up and tolerate metals varies between and within species as well as between metals. For most metals, the mechanisms involved in plant tolerance, uptake and accumulation are still not fully known and it is not known to what extent the plant response is metal-specific rather than a general stress response. In the present study, the growth response of soybean to Cd, As, Al and NaCl was compared and contrasted to simple sequence repeat (SSR) marker analysis results for Cda1, a dominant gene located in a major quantitative trait locus that regulates Cd accumulation in soybean, to evaluate the hypothesis that general effect patterns are induced by the individual metals. Principal component analysis revealed that the root growth response was most diverse for Al exposure and decreased in the order of Al > As > Cd > NaCl. NaCl did not exert a differentiating effect, indicating response mechanisms similar, at least partially, to metal exposure. The applied stressors yielded a distinguishable pattern of root responses, indicating the potential of such screens to identify agents acting similarly or differently. The SSR marker analysis also facilitated characterization of the Cd accumulation potential of the 22 soybean cultivars studied, and thereby identification of cultivars with potential health risk under cultivation in Cd-contaminated soils.

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 β-1,3-glucanases have previously been identified in digestive fluid of some carnivorous species. Here we show that a single acidic endo-β-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 β-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 β-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 β-1,3-glucanases in the digestive fluid of carnivorous sundew are also discussed.

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.

Heavy metals perturbate water balance in plants and hence impact stomatal aperture. After longer exposure, stomatal development also is affected, and stomatal density and size can change. Two soybean (Glycine max) varieties were experimentally selected for contrasting tolerance to arsenic and cadmium. For these cultivars, natural variability in leaf epidermal cell parameters was detected. Pot plants at first assimilating leaf stage were exposed for 10 days to 5 mg kg-1 soil As 3+ and 50 mg kg-1 soil Cd 2+ (respectively). Metals accumulated primarily in roots and exerted relatively low impact on biomass. Despite this, we observed diverse adjustments of stomata and pavement cells. In cv. Bólyi 44 the stomatal size decreased upon stress treatment, possibly to avoid further water loss. In contrast, the other cultivar Cordoba uses larger stomata that might be advantageous in gaining further resources. The observed responses varied depending on leaf type. In addition, dorsoventral stomatal responses in width, yet undescribed under metal stress, were observed. Our data show that leaf epidermal cell adjustments are flexible components of plant defense even at low metal doses, and possibly help to compromise the structural and functional needs of plant (tissue) under metal stress.

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.