Effect of chronic radiation on the flax (Linum usitatissimum L.) genome grown for six consecutive generations in the radioactive Chernobyl area.

The growth of plants under chronic radiation stress in the Chernobyl area may cause changes in the genome of plants. To assess the extent of genetic and epigenetic changes in nuclear DNA, seeds of the annual crop flax (Linum usitatissimum L.) of the Kyivskyi variety, sown 21 years after the accident and grown for six generations in radioactive (RAD) and remediated (REM) fields were analysed. Flaxseed used for sowing first generation, which served as a reference (REF), was also analysed. The AFLP (Amplified Fragment Length Polymorphism) revealed a higher number of specific EcoRI-MseI loci (3.4-fold) in pooled flaxseed samples harvested from the RAD field compared with the REM field, indicating a link between the mutation process in the flax genome and the ongoing adaptation process. MSAP (Methylation-Sensitive Amplified Polymorphism) detecting EcoRI-MspI and EcoRI-HpaII loci in flax nuclear DNA genome showed no significant differences in methylation level, reaching about 33% in each of the groups studied. On the other hand, significant changes in the DNA methylation pattern of flaxseed samples harvested from the RAD field compared with controls were detected. Pairwise FST comparison revealed within both, EcoRI-MspI and transformed methylation-Sensitive data sets more than a 3-fold increase of genetic divergence in the RAD field compared with both controls. These results indicate that the nuclear genome of flax exposed to chronic radiation for six generations has more mutations and uses DNA methylation as one of the adaptation mechanisms for sustainability under adverse conditions.

Basic ß-1,3-Glucanase from Drosera binata Exhibits Antifungal Potential in Transgenic Tobacco Plants.

The basic ß-1,3-glucanase of the carnivorous plant Drosera binata was tested as a purified protein, as well as under the control of a double CaMV35S promoter in transgenic tobacco for its capability to inhibit the growth of Trichoderma viride, Rhizoctonia solani, Alternaria solani, and Fusarium poae in an in-vitro assay. The purified protein inhibited tested phytopathogens but not the saprophytic fungus T. viride. Out of the analysed transgenic plants, lines 13, 16, 19, and 22 exhibited high DbGluc1 transcript abundance normalised to the actin transcript. Because of DbGluc1 transgene expression, lines 13 and 16 showed a 1.7-fold increase and lines 19 and 22 showed more than a 2-fold increase in total ß-1,3-glucanase activity compared to the non-transgenic control. In accordance with the purified ß-1,3-glucanase in-vitro antifungal assay, crude protein extracts of lines 19 and 22 significantly inhibited the growth of phytopathogens (14-34%). Further analyses revealed that the complementary action of transgenic ß-1,3-glucanase and 20% higher activity of endogenous chitinase(s) in these lines were crucial for maximising the antifungal efficiency of crude protein extracts.

Biochemical and antifungal characteristics of recombinant class I chitinase from Drosera rotundifolia.

DrChit is class I chitinase involved in the digestion of insect prey of Drosera rotundifolia plants. Herein, we cloned the DrChit-S open reading frame lacking the 5'- sequence coding signal peptide into the pET32a vector and its derivate lacking the thioredoxin tag. After DrChit-S + Trx and DrChit-S-Trx overexpression in Escherichia coli cells and purification on Ni-NTA agarose, both enzymes exhibited maximum activity at pH 6.0 and 38 °C. Surprisingly, the DrChit -S + Trx exerted double enzyme activity and improved all kinetic parameters for FITC-chitin substrate degradation resulting in catalytic efficiency three times higher (46.2 mM-1. s-1) than DrChit-S-Trx (13.63 mM-1. s-1). The 3D-structure of DrChit-S + Trx revealed different spatial arrangement of the three tyrosine residues in chitin-binding site, while their aromatic rings showed better stacking geometry for CH/π interactions with the carbohydrate substrate. In contrast, there were no significant differences between both enzymes when the effect of metal ions and their antifungal potential were tested. Quantitative in vitro assays showed growth suppression of Fusarium poae (40%), Trichoderma viride (43.8%), and Alternaria solani (52.6%) but not Rhizoctonia solani (sp.). Our study indicates that sundew chitinase has potential in biotechnology either for degradation of chitin to oligomers applicable in medicine or for plant defense fortification.

Expression of Drosera rotundifolia Chitinase in Transgenic Tobacco Plants Enhanced Their Antifungal Potential.

In this study, a chitinase gene (DrChit) that plays a role in the carnivorous processes of Drosera rotundifolia L. was isolated from genomic DNA, linked to a double CaMV35S promoter and nos terminator in a pBinPlus plant binary vector, and used for Agrobacterium-mediated transformation of tobacco. RT-qPCR revealed that within 14 transgenic lines analysed in detail, 57% had DrChit transcript abundance comparable to or lower than level of a reference actin gene transcript. In contrast, the transgenic lines 9 and 14 exhibited 72 and 152 times higher expression level than actin. The protein extracts of these two lines exhibited five and eight times higher chitinolytic activity than non-transgenic controls when measured in a fluorimetric assay with FITC-chitin. Finally, the growth of Trichoderma viride was obviously suppressed when the pathogen was exposed to 100 µg of crude protein extract isolated from line 9 and line 14, with the area of mycelium growth reaching only 56.4% and 45.2%, of non-transgenic control, respectively. This is the first time a chitinase from a carnivorous plant with substrate specificity for long chitin polymers was tested in a transgenic plant with the aim of exploring its antifungal potential.

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.

Structural and functional characterisation of a class I endochitinase of the carnivorous sundew (Drosera rotundifolia L.).

MAIN CONCLUSION: Chitinase gene from the carnivorous plant, Drosera rotundifolia , was cloned and functionally characterised. Plant chitinases are believed to play an important role in the developmental and physiological processes and in responses to biotic and abiotic stress. In addition, there is growing evidence that carnivorous plants can use them to digest insect prey. In this study, a full-length genomic clone consisting of the 1665-bp chitinase gene (gDrChit) and adjacent promoter region of the 698 bp in length were isolated from Drosera rotundifolia L. using degenerate PCR and a genome-walking approach. The corresponding coding sequence of chitinase gene (DrChit) was obtained following RNA isolation from the leaves of aseptically grown in vitro plants, cDNA synthesis with a gene-specific primer and PCR amplification. The open reading frame of cDNA clone consisted of 978 nucleotides and encoded 325 amino acid residues. Sequence analysis indicated that DrChit belongs to the class I group of plant chitinases. Phylogenetic analysis within the Caryophyllales class I chitinases demonstrated a significant evolutionary relatedness of DrChit with clade Ib, which contains the extracellular orthologues that play a role in carnivory. Comparative expression analysis revealed that the DrChit is expressed predominantly in tentacles and is up-regulated by treatment with inducers that mimick insect prey. Enzymatic activity of rDrChit protein expressed in Escherichia coli was confirmed and purified protein exhibited a long oligomer-specific endochitinase activity on glycol-chitin and FITC-chitin. The isolation and expression profile of a chitinase gene from D. rotundifolia has not been reported so far. The obtained results support the role of specific chitinases in digestive processes in carnivorous plant species.

The pollen- and embryo-specific Arabidopsis DLL promoter bears good potential for application in marker-free Cre/loxP self-excision strategy.

KEY MESSAGE: Marker-free transgenic plants can be generated with high efficiency by using the Cre/ lox P self-excision system controlled by the pollen- and embryo-specific Arabidopsis DLL promoter. In this work, we aimed to study the feasibility of using the pollen- and embryo-specific DLL promoter of the At4g16160 gene from Arabidopsis thaliana in a Cre/loxP self-excision strategy. A Cre/loxP self-excision cassette controlled by the DLL promoter was introduced into the tobacco genome via Agrobacterium-mediated transformation. No evidence for premature activation of the Cre/loxP system was observed in primary transformants. The efficiency of nptII removal during pollen and embryo development was investigated in transgenic T1 progenies derived from eight self- and four cross-pollinated T0 lines, respectively. Segregation and rooting assays were performed to select recombined T1 plants. Molecular analyses of these plants confirmed the excision event in all analysed T0 lines and marker-free transgenic T1 plants were obtained with efficiency of up to 96.2%. The Arabidopsis DLL promoter appears to be a strong candidate to drive Cre-mediated recombination not only in tobacco as a model plant, but also in other plant species.

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.

The influence of heat stress on auxin distribution in transgenic B. napus microspores and microspore-derived embryos.

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.

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.

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 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 ß-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 ß-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.

Detection of chitinolytic enzymes with different substrate specificity in tissues of intact sundew (Drosera rotundifolia L.): chitinases in sundew tissues.

The round-leaf sundew (Drosera rotundifolia L.) is a carnivorous plant expressing a wide range of chitinolytic enzymes playing role in many different processes. In this study the intact plants were analyzed for the presence of chitinase transcripts and chitinolytic activities in different organs. In situ hybridization with chitnase fragment as a probe has revealed the presence of chitinases in the mesophyll cells of leaves and vascular elements of stems of healthy, non-stressed plants. More pronounced expression was observed in cortex and stele cells of roots as well as in ovules and anthers of reproductive organs. Similarly, higher chitinase enzyme activity was typical for flowers and roots suggesting a more specific role of chitinases in these tissues. In addition to endochitinases of different substrate specificities, chitobiosidases contributed to overall chitinolytic activity of tissue extracts. The activity of chitobiosidases was again typical for flowers and roots, while their role in plant physiology remains to be elucidated.

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 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.

Heavy-metal stress induced accumulation of chitinase isoforms in plants.

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.

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.

The promiscuity of heterospecific lox sites increases dramatically in the presence of palindromic DNA.

Heterospecific lox sites are mutated lox sites that in the presence of Cre recombinase recombine with themselves but not or much less with wildtype loxP. We here show that in Escherichia coli both lox511 and lox2272 sites become highly promiscuous with respect to loxP when in the presence of Cre one of the recombination partners is present in a larger stretch of an inverted repeat of non-lox DNA. In such a palindromic DNA configuration, also the occurrence of other DNA repeat-mediated recombination events is somewhat increased in the presence of Cre. The results indicate that in recombinase mediated cassette exchange or other double lox applications based on the exclusivity of heterospecific lox sites, or in research combining Cre-lox approaches with hairpin RNA for gene silencing, the presence of duplicated DNA around lox sites has to be taken into account. It is proposed that the presence of palindromic non-lox DNA interferes with the homology search of the Cre enzyme prior to the actual recombination event.