Przemysław Malec

Physiological responses of Lemna trisulca L. (duckweed) to cadmium and copper bioaccumulation

Abstract Aquatic plants are known to accumulate and bioconcentrate heavy metals. In this study, several physiological responses of aquatic vascular plant Lemna trisulca L. to elevated concentrations of cadmium (up to 10 mM) and copper (up to 50 μM) were investigated. It was found that Lemna fronds were able to accumulate both cadmium and copper, but Cu-treated material showed pronounced toxic symptoms at concentrations 1000-fold lower in comparison to Cd. Lemna trisulca could tolerate elevated levels of Cd, i.e. up to 10 mM, without significant changes in photosynthetic pigments concentration. On the contrary, Cu in concentrations 25 and 50 μM promoted significant pigment degradation. The main processes affected by Cd in Lemna fronds were total gas exchange and net photosynthesis. On the contrary, the inhibition of total gas exchange and net photosynthesis caused by Cu (2–50 μM) correlated with Chl a and carotenoid concentrations decrease as well as with the decay of fluorescence from PS II. Also, an increasing impact of respiration in total oxygen exchange was observed after treatment of Lemna with increasing Cd concentrations (up to 5 mM) and with Cu in concentration range between 2 and 50 μM. In Cd-treated fronds, a dose-dependent accumulation of two polypeptides with apparent molecular weights 18 and 10 kDa, respectively as well as the appearance of two smaller polypeptides (apparent molecular weights 8 and 7 kDa) was observed in SDS-PAGE. The nature of these polypeptides remains to be determined. On the contrary, in Cu-treated fronds neither accumulation of existing proteins nor appearance of any extra protein was observed.

Phosphatidylglycerol Is Essential for Oligomerization of Photosystem I Reaction Center

Our earlier studies with the pgsA mutant of Synechocystis PCC6803 demonstrated the important role of phosphatidylglycerol (PG) in PSII dimer formation and in electron transport between the primary and secondary electron-accepting plastoquinones of PSII. Using a long-term depletion of PG from pgsA mutant cells, we could induce a decrease not only in PSII but also in PSI activity. Simultaneously with the decrease in PSI activity, dramatic structural changes of the PSI complex were detected. A 21-d PG depletion resulted in the degradation of PSI trimers and concomitant accumulation of monomer PSI. The analyses of PSI particles isolated by MonoQ chromatography showed that, following the 21-d depletion, PSI trimers were no longer detectable in the thylakoid membranes. Immunoblot analyses revealed that the PSI monomers accumulating in the PG-depleted mutant cells do not contain PsaL, the protein subunit thought to be responsible for the trimer formation. Nevertheless, the trimeric structure of PSI reaction center could be restored by readdition of PG, even in the presence of the protein synthesis inhibitor lincomycin, indicating that free PsaL was present in thylakoid membranes following the 21-d PG depletion. Our data suggest an indispensable role for PG in the PsaL-mediated assembly of the PSI reaction center.

Arabidopsis FUSCA5 encodes a novel phosphoprotein that is a component of the COP9 complex.

The COP9 complex is a regulator essential for repression of light-mediated development in Arabidopsis. Using partial amino acid sequence data generated from purified COP9 complexes, we cloned the Arabidopsis cDNA encoding the 27-kD subunit of the COP9 complex and showed that it is encoded by the previously identified FUSCA5 (FUS5) locus. fus5 mutants exhibit constitutive photomorphogenic phenotypes similar to those of cop9 and fus6. Point mutations in FUS5 that led to a loss of FUS5 protein were detected in four fus5 allelic strains. FUS5 contains the PCI/PINT and mitogen-activated protein kinase kinase activation loop motifs and is highly conserved with the mammalian COP9 complex subunit 7 and the Aspergillus nidulans AcoB proteins. FUS5 is present in both complex and monomeric forms. In the COP9 complex, FUS5 may interact directly with FUS6 and COP9. Mutations in FUS6 and COP9 result in a shift in the electrophoretic mobility of FUS5. This shift can be mimicked by in vitro phosphorylation of FUS5 by plant extracts. These findings further support the hypothesis that the COP9 complex is a central and common regulator that may interact with multiple signaling pathways.

Zinc protects Ceratophyllum demersum L. (free-floating hydrophyte) against reactive oxygen species induced by cadmium.

Evidence for Zn protection against Cd-induced reactive oxygen species in the free-floating hydrophyte Ceratophyllum demersum L. is presented in this paper. Metal treatments of 10 micromol/L Cd, 10 Cd micromol/L supplemented with Zn (10, 50, 100 and 200 micromol/L) and Zn-alone treatments of the same concentrations were used. Using 5,5 dimethyl pyrroline-N-oxide as the spin-probe, electron spin resonance spectra indicated a drastic increase in hydroxyl radicals (OH()) in Cd-10 micromol/L treatments, which was closely correlating with the enhanced formation of hydrogen peroxide (H(2)O(2)) and generation of superoxide radical (O(2)(-)) triggered by the oxidation of NADPH. The supplementation of adding Zn (10-200 micromol/L) to the Cd-10 micromol/L treatments significantly decreased the production of free radicals especially by eliminating the precursors of OH() through inhibition of NADPH oxidation. Cd-enhanced ROS production which substantially increased the oxidative products of proteins measured as carbonyls was effectively inhibited by Zn supplementation.

Increased genetic diversity of Viola tricolor L. (Violaceae) in metal-polluted environments

Changes in DNA sequences affecting cryptic intraspecific variability are very important mechanisms of plant microevolutionary processes, initiating species diversification. In polluted environments, intra- and interpopulation changes at the molecular level proceed rapidly and lead to the formation of new ecotypes in a relatively short time. We used ISSR PCR fingerprinting data to analyze the genetic diversity and genetic structure of seven populations of Viola tricolor: four growing on soil contaminated with heavy metals (Zn, Pb, Cd; waste heaps) and three from control soil. The populations from the polluted sites showed higher genetic polymorphism (%(poly)=84%) and gene diversity (H(T)=0.1709) than the control populations (%(poly)=75% and H(T)=0.1448). The number of private markers we detected within metallicolous (MET) populations was more than double that found within non-metallicolous (NON) populations (15 vs. 7). The STRUCTURE and UPGMA analyses showed clear genetic differences between the NON and MET populations. Based on broad analyses of the genetic parameters, we conclude that the effect of these polluted environments on the genetic diversity of the MET populations, separating them from the NON populations, is evidence of microevolutionary processes at species level, leading to species divergence and the emergence of local ecotypes better adapted to their different environments.

Light-induced chloroplast movements in Lemna trisulca. Identification of the motile system

Abstract In mesophyll cells of the water plant Lemna trisulca L. chloroplasts redistribute in response to blue light. In the present study it is shown that an actin depolymerizing agent cytochalasin D, a crosslinker of actin subunits in F-actin m-maleimidobenzoic acid N -hydroxysuccinimide ester (MBS) as well as N -ethylmaleimide (NEM)—a sulfhydryl group reagent, are potent inhibitors of these blue light-induced chloroplast movements in Lemna . Extraction with cold, buffered glycerol solution preserves light-induced chloroplast arrangements within cells producing permeabilized cell models. ‘Reactivation’ of these cell models by Mg-ATP results in remarkable movements which can be inhibited by treatment with NEM and cytochalasin D. Immunofluorescence microscopy demonstrates that a component which is associated with isolated Lemna chloroplasts cross-reacts with antibodies directed against bovine myosin. These results indicate that a contractile actomyosin system is involved in blue light-induced chloroplast movements in Lemna and a putative motor protein, similar to myosin, is associated with the surface of Lemna chloroplasts.

Involvement of Carotenoids in the Synthesis and Assembly of Protein Subunits of Photosynthetic Reaction Centers of Synechocystis sp. PCC 6803

The crtB gene of Synechocystis sp. PCC 6803, encoding phytoene synthase, was inactivated in the Delta crtH mutant to generate a carotenoidless Delta crtH/B double mutant. Delta crtH mutant cells were used because they had better transformability than wild-type cells, most probably due to their adaptation to partial carotenoid deficiency. Cells of the Delta crtH/B mutant were light sensitive and could grow only under light-activated heterotrophic growth conditions in the presence of glucose. Carotenoid deficiency did not significantly affect the cellular content of phycobiliproteins while the chlorophyll content of the mutant cells decreased. The mutant cells exhibited no oxygen-evolving activity, suggesting the absence of photochemically active PSII complexes. This was confirmed by 2D electrophoresis of photosynthetic membrane complexes. Analyses identified only a small amount of a non-functional PSII core complex lacking CP43, while the monomeric and dimeric PSII core complexes were absent. On the other hand, carotenoid deficiency did not prevent formation of the cytochrome b(6)f complex and PSI, which predominantly accumulated in the monomeric form. Radioactive labeling revealed very limited synthesis of inner PSII antennae, CP47 and especially CP43. Thus, carotenoids are indispensable constituents of the photosynthetic apparatus, being essential not only for antioxidative protection but also for the efficient synthesis and accumulation of photosynthetic proteins and especially that of PSII antenna subunits.

Ecophysiological tolerance of Elodea canadensis to nickel exposure

Biological accumulation of nickel and concomitant ecophysiological responses were studied in the leaves of Elodea canadensis treated with different concentrations of Ni (1-50 microM) for 5d. In low concentrations nickel was accumulated mainly in the soluble protein fraction, which correlated with its highest observed accumulation coefficient. In higher concentrations, Ni binding in the non-protein soluble fraction was observed. The effects of increasing nickel concentrations on the accumulation of photosynthetic pigments, gas exchange rates, lipid peroxidation, biosynthesis of thiol-containing compounds and the activity of selected enzymes--markers of oxidative stress were investigated. The appearance of several new polypeptides with apparent molecular weights below 20 kDa, was found by SDS-PAGE in Ni-treated Elodea leaves. Our results indicate that Ni, in concentrations up to 10 microM could induce sub-lethal oxidative stress in Elodea leaves. In response, plants developed detoxification mechanisms including an enhanced biosynthesis of thiol-containing compounds which facilitated Ni accumulation and sequestration in plant tissues effectively. Hence, E. canadensis could be used in the biological removal of Ni from polluted water up to 10 microM concentration.

Formaldehyde and methanol biodegradation with the methylotrophic yeast Hansenula polymorpha. An application to real wastewater treatment

The application of methylotrophic yeast Hansenula polymorpha to the treatment of methanol and formaldehyde-containing wastewater was experimentally verified. Avariety of real wastewater samples originating from chemical industry effluent were examined. The yeast cell culture could grow in the wastewater environment, revealing low trophic requirements and a very high adaptation potential to poor cultivation conditions.The proliferation of cells was accompanied by a concomitant xenobiotic biodegradation. Grown, preadapted cellular suspension at a density of about 1 × 107 cells/ml proved to be able to utilize formaldehyde present in wastewater at concentrations up to1750 mg/l, levels toxic to most microorganisms. The biological waste treatment method presented shows the enhanced potential by means of specific enzymatic activities of monocarbonic compound oxidations through methylotrophic pathway reactions. The need to obtain mutants highly resistant to formaldehyde has also been rationalized.

Identification and characterization of Cd-induced peptides in Egeria densa (water weed): Putative role in Cd detoxification.

Egeria densa has ability to grow in heavy metal contaminated and polluted bodies of water. Shoots exposed to Cd at concentrations up to 300microM for 7 days showed a pronounced decrease in chlorophyll a and in total protein concentration. Thiol-containing compounds and low-molecular-weight polypeptides were detected in Cd-treated plant extracts by gel filtration chromatography. Two Cd-binding fractions, a thiol-enriched fraction and a non-thiol fraction with a lower molecular weight were identified in extracts by gel filtration. The main fraction of thiol-containing polypeptide, purified by gel filtration and anion-exchange chromatography had a molecular weight of approximately 10kDa. This peptide was characterized by a broad absorption band specific to mercaptide bonds and Cd-sensitive fluorescence emission of aromatic amino acid residues. Our results indicate that cadmium exposure of plants resulted in both a formation of thiol-enriched cadmium complexing peptides and a synthesis of low-molecular-weight metal chelators. The putative role of these compounds in Cd detoxification is discussed.