Jana Libantová

Tentacles of in vitro-grown round-leaf sundew (Drosera rotundifolia L.) show induction of chitinase activity upon mimicking the presence of prey

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 non-treated 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.

Biochemical and physiological comparison of heavy metal-triggered defense responses in the monocot maize and dicot soybean roots

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 H2O2 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.

Study on metal-triggered callose deposition in roots of maize and soybean

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.

Feasibility of the seed specific cruciferin C promoter in the self excision Cre/loxP strategy focused on generation of marker-free transgenic plants

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 T0 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 T0 transgenic lines generated whole progeny sensitive to kanamycin. Detailed analyses of progeny of selected T0-30 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 T2 seeds resulted in increased rate of marker-free plants, whereas four out of ten analysed chimeric T1 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.

Expression of a cucumber class III chitinase and Nicotiana plumbaginifolia class I glucanase genes in transgenic potato plants

The genes encoding for a cucumber class III chitinase and Nicotianaplumbaginifolia class I glucanase were co-introduced into Slovak potato (Solanum tuberosum L.) breeding line 116/86 using Agrobacterium tumefaciens. For both transgenes the number of integrated copies and level of RNA expression were determined. These analyses demonstrated low variation and significant correlation in expression of the introduced transgenes. The effect of transgene expression on fungal susceptibility of transformants was evaluated in vitro. Hyphal extension assays revealed no obvious differences in the ability of extracts from transformants to inhibit growth of Rhizoctonia solani comparing to non-transformed potato.

Stress-induced expression of cucumber chitinase and Nicotiana plumbaginifoliaβ-1,3-glucanase genes in transgenic potato plants

The genes encoding for a cucumber class III chitinase and Nicotianaplumbaginifolia class I glucanase were co-introduced into Slovak potato (Solanum tuberosum L.) cultivar ETA using Agrobacterium tumefaciens. Expression of both genes was driven by wound-inducible polyubiquitin promoter isolated from Slovak potato breeding line 116/86. Analyses showed inducible, peel-specific expression of both transgenes under stress conditions. The effect of transgene expression on fungal susceptibility of transformants was evaluated in vitro and in vivo. Experiments with crude protein extracts isolated from transgenic microtubers showed growth inhibition of Rhizoctonia solani hyphae in the range from 7.3 to 14.2%. In contrast, experiments performed in growth chamber conditions revealed that the polyubiquitin promoter driven transgene expression did not ensure any obvious increase of transgenic potato resistance against Rhizoctonia solani.

β-1,3-glucanase and chitinase activities in winter triticales during cold hardening and subsequent infection by Microdochium nivale

The accumulation of pathogenesis-related proteins such as β-1,3-glucanases and chitinases was studied in cold induced snow mould resistance in two Polish cultivars of winter triticale, cv. Hewo and cv. Magnat that substantially differ in resistance to Microdochium nivale. The plants were pre-hardened at 12°C for 10 days and hardened at 4°C for 28 days. Subsequently, cold hardened plants were inoculated with fungal mycelium (M. nivale) and incubated at 4°C for 7 days in dark. Cold acclimatisation resulted in suppression of the total glucanase and chitinases activities in the resistant Hewo as well as sensitive Magnat cultivars that possibly coincides with altered metabolism. However, upon infection with M. nivale the chitinases were markedly induced in the cv. Hewo. At the same time, total β-1,3 glucanases activities did not seem to be affected by fungus in any of the tested triticale cultivars. The pattern and/or the activity of chitinases in plants might be indicative for the resistance/susceptibility against M. nivale.

Plant tissue-specific promoters can drive gene expression in Escherichia coli

Plant transgenesis often requires the use of tissue-specific promoters to drive the transgene expression exclusively in targeted tissues. Although the eukaryotic promoters are expected to stay silent in Escherichia coli, when the promoter-transgene units within the plant transformation vectors are constructed and propagated, some eukaryotic promoters have been reported to be active in prokaryotes. The potential activity of plant promoter in E. coli cells should be considered in cases of expression of proteins that are toxic for host cells, environmental risk assessment or the stability in E. coli of plant vectors for specific Cre/loxP applications. In this study, DNA fragments harbouring four embryo- and/or pollen-specific Arabidopsis thaliana promoters were investigated for their ability to drive heterologous gene expression in E. coli cells. For this, they were fused to gfp:gus reporter genes in the pCAMBIA1304 vector. Although BPROM, bacterial sigma70 promoter recognition program identified several sequences with characteristics similar to bacterial promoters including -10 and -35 sequences in each of tested fragments, the experimental approach showed that only one promoter fragment was able to drive relatively strong- and one promoter fragment relatively weak-GUS expression in E. coli cells. Remaining two tested promoters did not drive any transgene expression in bacteria. Our results also showed that cloning of a shorter plant promoter sequence into vectors containing lacZ α-complementation system can increase the probability of gene expression driven by upstream located lac promoter. This should be considered when cloning of plant expression units, the expression of which is unwanted in E. coli.

Plant chitinase responses to different metal-type stresses reveal specificity

Key messageChitinases inGlycine maxroots specifically respond to different metal types and reveal a polymorphism that coincides with sensitivity to metal toxicity.AbstractPlants 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.

A modified low copy number binary vector pUN for Agrobacterium-mediated plant transformation

A modified low copy number plant binary vector pUN has been constructed and successfully used to clone unstable DNA sequences. The vector pUN comprises of low copy number, broad host range RK2 replicon from pBin19 and of multiple cloning site (MCS) and T-DNA region, both from a pBINPLUS-derived pLV06 vector. The absence of the ColE1 replicon in the backbone of the binary vector significantly contributed to stability of hardly clonable DNA sequences and enabled their transfer into the tobacco plants through Agrobacterium-mediated transformation.