Do chromium-resistant bacterial symbionts of hyperaccumulator Callitriche cophocarpa support their host in phytobial remediation of water?

Callitriche cophocarpa Sendtn. is a macrophyte widely distributed in aquatic systems of the temperate climate zone and a known hyperaccumulator of chromium. Ten pure symbiotic bacterial isolates of C. cophocarpa were obtained and identified. Three of the isolates showed the highest resistance to Cr(VI): Microbacterium sp. (Ct1), Aeromonas sp. (Ct3) and Acinetobacter sp. (Ct6). Acinetobacter sp. (Ct6) was able to survive up to a concentration of 104 mg/L (2 mM). The isolates were also able to effectively detoxify Cr(VI) by reducing it to Cr(III). We tested whether inoculation of plants with a consortium consisting of Ct1, Ct3 and Ct6 affects: (1) the phytoextraction of chromium from leachates, (2) the physiological state of plants after Cr(VI) treatment. The solutions were landfill leachates and contained 10.7 mg/L of Cr(VI) - an amount 530 times exceeding the legal limits. We influenced the plants with Cr in two steps, each lasting for 10 days, first using mature shoots and then apical ones. The highest Cr content concomitant with the highest bioconcentration factor (BCF) were found in the inoculated plants: 1274 and 119 mg/kg dry mass (d.m.), respectively. The physiological status of the plants was assessed by biometric tests and advanced chlorophyll fluorescence analyses. The photosynthetic activity of mature shoots was influenced by Cr(VI) more negatively than that of young apical shoots. The inoculation with the bacterial consortium significantly reduced the negative effect of Cr(VI) on mature organs. In some cases the inoculated mature plants exhibited photosynthetic activity that was even higher than in the control plants. The results unequivocally show a beneficial effect of C. cophocarpa inoculation with the tested isolates resulting in a significant improvement of the phytoremediation properties of this aquatic chromium hyperaccumulator.

Reproductive Biology of Dry Grassland Specialist Ranunculus illyricus L. and Its Implications for Conservation.

Ranunculus illyricus, a component of xerothermic grasslands, is a declining species and deserves active conservation treatments in many countries preceded by studies on the biology of its reproduction. So far, our knowledge of R. illyricus, a species with two modes of reproduction, has been fragmentary. The purpose of the studies presented here was to describe the annual development cycle of R. illyricus with particular emphasis on the production of underground tuber clusters that serve as vegetative propagation. Based on three-year-long observations in an ex situ collection, the efficiency of vegetative propagation was estimated and compared with the efficiency of generative propagation. It was found that in 3 years the best clones could produce up to 57 progeny clusters followed by flowering specimens in the first season. Meanwhile, the high potential for generative reproduction was suppressed by many limitations including fruit setting, the germination capacity of seeds, seedling survival rate, and additionally, the first flowering plant was observed only in the third year. It seems that the efficiency of vegetative propagation of this species can be higher than the efficiency of generative propagation. Moreover, vegets bloomed in the first year after emergence, whereas the first plant of generative origin was observed to bloom only after 3 years. A large proportion of individuals of vegetative origin can negatively affect the genetic diversity of the population but their survival rate against competing plants is higher. To enhance the existing populations or to create new ones, it would be best to use plants derived from clonal propagation of genets carried out in ex situ conditions.

The Effectiveness of the Sexual Reproduction in Selected Clonal and Nonclonal Species of the Genus Ranunculus.

Generative processes have been evaluated in six European buttercup species in order to verify the hypothesis that the reproduction efficiency of clonal species is lower than that of nonclonal ones. The study covered common species (Ficaria verna, Ranunculus auricomus, R. bulbosus, R. cassubicus, R. lanuginosus) and the endangered R. illyricus. The following properties have been assessed: pollen viability (staining method), pollen grain germination and the pollen-tube elongation in pistil tissues (fluorescence microscopy), seed formation efficiency, seed viability (tetrazolium test) and germination ability by introducing factors interrupting dormancy (low temperature and gibberellin application). Additionally, the pistil morphology was documented for R. bulbosus, R. illyricus and R. cassubicus using SEM techniques. It was demonstrated that the reproductive efficiency, expressed as the production of viable seeds able to germinate, was significantly higher in the species reproducing sexually (especially in R. lanuginosus) compared to the clonal ones. However, the complexity observed leads to separation of an additional group (cluster) of apomictic species: R. auricomus and R. cassubicus, distinguished by the lowest pollen viability and a low ability of the seeds to germinate. In the vegetatively reproducing R. illyricus, the seed formation efficiency was just 13.2% despite the having highest number of pistils in its flowers. The developed seeds of this species observed in our experiment were viable, but in general effective methods to stimulate their germination have not been proposed yet. Here, the first comparative study concerning the biology of sexual reproduction of R. illyricus is presented in the context of its decreasing distribution in natural habitats.

Response of physiological parameters in Dionaea muscipula J. Ellis teratomas transformed with rolB oncogene.

BACKGROUND: Plant transformation with rol oncogenes derived from wild strains of Rhizobium rhizogenes is a popular biotechnology tool. Transformation effects depend on the type of rol gene, expression level, and the number of gene copies incorporated into the plant's genomic DNA. Although rol oncogenes are known as inducers of plant secondary metabolism, little is known about the physiological response of plants subjected to transformation. RESULTS: In this study, the physiological consequences of rolB oncogene incorporation into the DNA of Dionaea muscipula J. Ellis was evaluated at the level of primary and secondary metabolism. Examination of the teratoma (transformed shoots) cultures of two different clones (K and L) showed two different strategies for dealing with the presence of the rolB gene. Clone K showed an increased ratio of free fatty acids to lipids, superoxide dismutase activity, synthesis of the oxidised form of glutathione, and total pool of glutathione and carotenoids, in comparison to non-transformed plants (control). Clone L was characterised by increased accumulation of malondialdehyde, proline, activity of superoxide dismutase and catalase, total pool of glutathione, ratio of reduced form of glutathione to oxidised form, and accumulation of selected phenolic acids. Moreover, clone L had an enhanced ratio of total triglycerides to lipids and accumulated saccharose, fructose, glucose, and tyrosine. CONCLUSIONS: This study showed that plant transformation with the rolB oncogene derived from R. rhizogenes induces a pleiotropic effect in plant tissue after transformation. Examination of D. muscipula plant in the context of transformation with wild strains of R. rhizogenes can be a new source of knowledge about primary and secondary metabolites in transgenic organisms.

Methylation of protein phosphatase 2A-Influence of regulators and environmental stress factors.

Protein phosphatase 2A catalytic subunit (PP2A-C) has a terminal leucine subjected to methylation, a regulatory mechanism conserved from yeast to mammals and plants. Two enzymes, LCMT1 and PME1, methylate and demethylate PP2A-C, respectively. The physiological importance of these posttranslational modifications is still enigmatic. We investigated these processes in Arabidopsis thaliana by mutant phenotyping, by global expression analysis, and by monitoring methylation status of PP2A-C under different environmental conditions. The lcmt1 mutant, possessing essentially only unmethylated PP2A-C, had less dense rosettes, and earlier flowering than wild type (WT). The pme1 mutant, with 30% reduction in unmethylated PP2A-C, was phenotypically comparable with WT. Approximately 200 overlapping genes were twofold upregulated, and 200 overlapping genes were twofold downregulated in both lcmt1 and pme1 relative to WT. Differences between the 2 mutants were also striking; 97 genes were twofold upregulated in pme1 compared with lcmt1, indicating that PME1 acts as a negative regulator for these genes. Analysis of enriched GO terms revealed categories of both abiotic and biotic stress genes. Furthermore, methylation status of PP2A-C was influenced by environmental stress, especially by hypoxia and salt stress, which led to increased levels of unmethylated PP2A-C, and highlights the importance of PP2A-C methylation/demethylation in environmental responses.

Accumulation patterns of Cr in Callitriche organs--qualitative and quantitative analysis.

The aims of this study were both the qualitative and quantitative analysis of chromium accumulation in the shoots of Callitriche cophocarpa. This globally distributed, submersed macrophyte exhibits outstanding Cr phytoremediation capacity in an aquatic environment. Cr was applied separately for 7 days at two stable forms as Cr(VI) and Cr(III), known from their diverse physicochemical properties and toxicities. The maps of Cr depositions in young leaves, mature leaves, and stems were obtained by micro X-ray fluorescence spectroscopy (µXRF). The detailed analysis of XRF maps was done based on Image-Pro PLUS (Media Cybernetics) software. Cr was accumulated either in trichomes or vascular bundles in respect to the element speciation and the plant organ. The concentration of Cr significantly increased in the following order: Cr(VI) mature leaves < Cr(VI) young leaves = Cr(VI) stems < Cr(III) young leaves ≤ Cr(III) mature leaves ≤ Cr(III) stems. The observed differences in distribution and accumulation of Cr were correlated with the different reduction potential of Cr(VI) by particular plant organs. The reduction of Cr(VI) is considered the main detoxification mechanism of the highly toxic Cr(VI) form. The unique L-band electron resonance spectrometer (L-band EPR) was applied to follow the reduction of Cr(VI) to Cr(III) in the studied material.