Plant chitinases belong to so-called pathogenesis related proteins and have mostly been detected in plants exposed to phytopathogenic viruses, bacteria or fungi. A few studies revealed that they might also be involved in plant defence against heavy metals. This work was undertaken to monitor the accumulation of chitinases in a set of heavy-metal stressed plants and bring evidence on their involvement during this kind of stress. Roots of different plant species including Vicia faba cvs. Astar and Piestanský, Pisum sativum, Hordeum vulgare, Zea mays and Glycine max were exposed to different concentrations of lead (300 and 500 mg l(-1) Pb(2+)), cadmium (100 and 300 mg l(-1) Cd(2+)) and arsenic (50 and 100 mg l(-1) As(3+)). In each case, the toxicity effects were reflected in root growth retardation to 80-10% of control values. The most tolerant were beans, most sensitive was barley. Extracts from the most stressed roots were further assayed for chitinase activity upon separation on polyacrylamide gels. Our data showed that in each combination of genotype and metal ion there were 2-5 different chitinase isoforms significantly responsive to toxic environment when compared with water-treated controls. This confirms that chitinases are components of plant defence against higher concentrations of heavy metals. In addition, accumulation of some isoforms in response to one but not to other metal ions suggests that these enzymes might also be involved in a more (metal) specific mechanism in affected plants and their biological role is more complex than expected.

Induction of plant-derived chitinases in the leaves of a carnivorous plant was demonstrated using aseptically grown round-leaf sundew (Drosera rotundifolia L.). The presence of insect prey was mimicked by placing the chemical inducers gelatine, salicylic acid and crustacean chitin on leaves. In addition, mechanical stirring of tentacles was performed. Chitinase activity was markedly increased in leaf exudates upon application of notably chitin. Application of gelatine increased the proteolytic activity of leaf exudates, indicating that the reaction of sundew leaves depends on the molecular nature of the inducer applied. In situ hybridization of sundew leaves with a Drosera chitinase probe showed chitinase gene expression in different cell types of nontreated leaves, but not in the secretory cells of the glandular heads. Upon induction, chitinase mRNA was also present in the secretory cells of the sundew leaf. The combined results indicate that chitinase is likely to be involved in the decomposition of insect prey by carnivorous plants. This adds a novel role to the already broad function of chitinases in the plant kingdom and may contribute to our understanding of the molecular mechanisms behind the ecological success of carnivorous plants in nutritionally poor environments.

Defense responses against cadmium, arsenic and lead were compared in two crop plants such as the monocotyledonous maize (Zea mays cv. Quintal) and dicotyledonous soybean (Glycine max cv. Korada). The applied metals caused root growth retardation, membrane damage and subsequent loss of cell viability, while enhanced H(2)O(2) generation, lipid peroxidation and lignification were detected with respect to corresponding controls. The measured data suggest that soybean was in general more tolerant to tested doses of metals and showed more pronounced defense responses than maize. Concurrently, the total activity of β-1,3-glucanases, a subgroup of so called pathogenesis-related defense proteins, was comparable in root extracts of both plant types. Though in a view of previous comparative genome approaches the β-1,3-glucanases do not mirror the differences in the cell wall structure and architecture between the monocots and dicots, we show that in both plant types they clearly respond to metal stress. Accumulation patterns of different glucanase isoforms upon exposure to tested metals indicate that they do contribute to plant defense mechanisms during exposure to heavy metals and their biological role is more complex than expected.

Plant embryogenesis is regulated by differential distribution of the plant hormone auxin. However, the cells establishing these gradients during microspore embryogenesis remain to be identified. For the first time, we describe, using the DR5 or DR5rev reporter gene systems, the GFP- and GUS-based auxin biosensors to monitor auxin during Brassica napus androgenesis at cellular resolution in the initial stages. Our study provides evidence that the distribution of auxin changes during embryo development and depends on the temperature-inducible in vitro culture conditions. For this, microspores (mcs) were induced to embryogenesis by heat treatment and then subjected to genetic modification via Agrobacterium tumefaciens. The duration of high temperature treatment had a significant influence on auxin distribution in isolated and in vitro-cultured microspores and on microspore-derived embryo development. In the “mild” heat-treated (1 day at 32 °C) mcs, auxin localized in a polar way already at the uni-nucleate microspore, which was critical for the initiation of embryos with suspensor-like structure. Assuming a mean mcs radius of 20 μm, endogenous auxin content in a single cell corresponded to concentration of 1.01 μM. In mcs subjected to a prolonged heat (5 days at 32 °C), although auxin concentration increased dozen times, auxin polarization was set up at a few-celled pro-embryos without suspensor. Those embryos were enclosed in the outer wall called the exine. The exine rupture was accompanied by the auxin gradient polarization. Relative quantitative estimation of auxin, using time-lapse imaging, revealed that primordia possess up to 1.3-fold higher amounts than those found in the root apices of transgenic MDEs in the presence of exogenous auxin. Our results show, for the first time, which concentration of endogenous auxin coincides with the first cell division and how the high temperature interplays with auxin, by what affects delay early establishing microspore polarity. Moreover, we present how the local auxin accumulation demonstrates the apical–basal axis formation of the androgenic embryo and directs the axiality of the adult haploid plant.

Oilseed rape is considered relatively recalcitrant for genetic modification. This work was performed to establish conditions for effective transformation and regeneration of commercially used cultivars Campino, Haydn, Heros, Hunter and Topas (Brassica napus L.). Cotyledonary petioles and hypocotyls (obtained from the seedlings grown under dark conditions) as a source of explants were used. Our experiments revealed that a lower selection pressure in combination with postponing of selection by 14 days after co-cultivation resulted in recovery of transgenic plants from all cultivars. Transformants were obtained with efficiency from 1 to 5.5%. Histochemically GUS-positive plants were analysed by PCR using primers corresponding to the internal fragments of gus and nptII genes. The transgenic nature was confirmed by Southern blot analyses using a specific nptII probe. This work enriches the list of oilseed rape cultivars available for genetic modification.

This work is focused on the generation of selectable marker-free transgenic tobacco plants using the self excision Cre/loxP system that is controlled by a strong seed specific Arabidopsis cruciferin C (CRUC) promoter. It involves Agrobacterium-mediated transformation using a binary vector containing the gus reporter gene and one pair of the loxP sites flanking the cre recombinase and selectable nptII marker genes (floxed DNA). Surprisingly, an ectopic activation of CRUC resulting in partial excision of floxed DNA was observed during regeneration of transformed cells already in calli. The regenerated T(0) plants were chimeric, but no ongoing ectopic expression was observed in these one-year-long invitro maintained plants. The process of the nptII removal was expected in the seeds; however, none of the analysed T(0) transgenic lines generated whole progeny sensitive to kanamycin. Detailed analyses of progeny of selected T(0)-30 line showed that 10.2% GUS positive plants had completely removed nptII gene while the remaining 86.4% were still chimeras. Repeated activation of the cre gene in T(2) seeds resulted in increased rate of marker-free plants, whereas four out of ten analysed chimeric T(1) plants generated completely marker-free progenies. This work points out the feasibility as well as limits of the CRUC promoter in the Cre/loxP strategy.

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.

Callose plays important roles in a variety of processes of plant development, and/or in a response to a range of biotic and abiotic stresses. In the current work we have studied and compared the effect of lead, cadmium and arsenic on accumulation of newly formed callose deposits in the roots of maize and soybean. We observed formation of characteristic callose deposits in the root cell walls, probably associated with plasmodesmata, depending on the type of metal and the plant species investigated. Further, the callose turnover was analysed by measuring of total callose content as well as activities of total β-(1,3)-glucanases in roots. The latter enzymes are responsible for callose depletion, and their possible role during metal stress has previously been proposed. However, neither of these biochemical values appeared to be sufficiently reliable for scoring the altered callose turnover (including local deposits) in plant tissue. The microscopical observations are discussed in light of the biochemical data obtained.