Francisca F. del Campo

Differential alterations of antioxidant defenses as bioindicators of mercury and cadmium toxicity in alfalfa.

Several physiological parameters related to oxidative stress, which is a characteristic of plants exposed to toxic metals, were studied in 3-week-old alfalfa plants treated with cadmium (Cd) or mercury (Hg) at doses of 0, 3, 10 and 30 microM for 7d. The concentrations of biothiols, glutathione (GSH), homoglutathione (hGSH) and phytochelatins (PCs) increased dramatically in metals-treated plants, in particular in the presence of Cd. This was accompanied by a remarkable up-regulation of gamma-glutamyl cysteine synthetase gene, probably in response to the higher demand for GSH|hGSH needed for PC synthesis. The presence of metals enhanced lipid peroxidation in shoots, while chlorophyll content declined in a concentration dependent manner. Ascorbate peroxidase (APX) activity increased moderately in roots of Cd-exposed plants, and a new basic root peroxidase isoform was found in both Cd- and Hg-treated plants. Glutathione reductase (GR) activity was enhanced in shoots of plants exposed to Cd and Hg. However, this enzymatic activity showed a metal dependent response in roots, and was enhanced in Cd-treated plants but was severely inhibited in roots of plants treated with Hg. Inhibition of GR by Hg was confirmed in vitro by incubating a commercially available GR and control shoot extracts with several doses of Hg and Cd. Ascorbate concentrations were elevated with treatments of 3 microM Hg, 10 microM Cd and 30 microM Cd, indicating that this compound is necessary for redox cellular homeostasis. The different responses observed with Cd and Hg treatments might be the basis for specific stress bioindicators.

Global warming and hepatotoxin production by cyanobacteria: what can we learn from experiments?

Global temperature is expected to rise throughout this century, and blooms of cyanobacteria in lakes and estuaries are predicted to increase with the current level of global warming. The potential environmental, economic and sanitation repercussions of these blooms have attracted considerable attention among the worlds scientific communities, water management agencies and general public. Of particular concern is the worldwide occurrence of hepatotoxic cyanobacteria posing a serious threat to global public health. Here, we highlight plausible effects of global warming on physiological and molecular changes in these cyanobacteria and resulting effects on hepatotoxin production. We also emphasize the importance of understanding the natural biological function(s) of hepatotoxins, various mechanisms governing their synthesis, and climate-driven changes in food-web interactions, if we are to predict consequences of the current and projected levels of global warming for production and accumulation of hepatotoxins in aquatic ecosystems.

Physiological changes in Triticum durum, Zea mays, Pisum sativum and Lens esculenta cultivars, caused by irrigation with water contaminated with microcystins: a laboratory experimental approach.

The aim of the present study was to investigate the effect of exposure to a microcystin (MC)-containing extract from a cyanobacteria bloom on growth, development, mineral nutrient accumulation, and photosynthetic activity of Triticum durum, Zea mays, Pisum sativum and Lens esculenta cultivars. The MCs in the extract, identified by HPLC and/or mass spectrometry (MS) were: MC-RR, -LR, -YR, -(H4)YR, -WR, and -FR. Plant growth and development was tested along 30 exposure days. After this period, MC-extract caused a clear reduction in plant growth and productivity, as well as deleterious effects on development and Photosystem II activity, measured by Fv/Fm fluorescence. However, the chlorophyll (a + b) content hardly varied, and the accumulation of Na+, K+, Ca2+, P and N was enhanced. All the effects observed were plant species, MC concentration, and exposure-time dependent. Relative accumulation of each MC variant greatly varied among plant species and plant organ. The data obtained supports the idea that the use of surface water containing MCs for crop irrigation can affect both plant yield and quality, and secondly, that MC accumulation in edible plants might pose a potential risk for human and animal health, if the MC intake exceeded the recommended tolerable limits.

Identification of microcystins from three collection strains of Microcystis aeruginosa

Microcystins (MCs) are toxic cyclic heptapeptides produced by various cyanobacteria genera, especially Microcystis. We identified 10 out of 12 MCs produced by three Microcystis aeruginosa strains from cyanobacteria collections, UTEX 2666, UTEX 2670 and UAM 1303, by using two analytical methods: Matrix-assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF/MS) and HPLC Photodiode Array Detector coupled to a hybrid Quadrupole Time of Flight Mass Spectrometry (HPLC-PDA-QTOF/MS). MALDI-TOF/MS failed to detect non-polar MCs, such as MC-LY and MC-LW. HPLC-QTOF/MS permitted the accurate identification of most MCs present in methanolic extracts. Besides, three new MCs, namely: [D-Glu(OCH3)6, D-Asp3] MC-LAba, MC-YL and MC-YM were detected by HPLC-QTOF/MS.

Isolation of the LEMMI9 gene and promoter analysis during a compatible plant-nematode interaction

Plant-endoparasitic root-knot nematodes feed on specialized giant cells that they induce in the vascular cylinder of susceptible plants. Although it has been established that a number of plant genes change their expression pattern during giant cell differentiation, virtually no data are available about the mechanisms involved in that change. One possibility is differential promoter recognition by the transcription factor(s) responsible for the expression of specific genes. We have isolated and characterized a genomic clone from tomato containing the promoter region of LEMMI9, one of the few plant genes that have been reported to be highly expressed in galls (predominantly in giant cells). The analysis of transgenic potato plants carrying a LEMMI9 promoter-beta glucuronidase (GUS) fusion has demonstrated that the tomato promoter was activated in Meloidogyne incognita-induced galls in a heterologous system. We have located putative regulatory sequences in the promoter and have found that nuclear proteins from the galls formed specific DNA-protein complexes with the proximal region of the LEMMI9 promoter. The nuclear protein-binding sequence mapped to a region of 111 bp immediately upstream from the TATA box. This region contains a 12-bp repeat possibly involved in the formation of DNA-protein complexes, which might be related to the LEMMI9 transcriptional activation in the giant cells.

Effect of different microcystin profiles on toxin bioaccumulation in common carp (Cyprinus carpio) larvae via Artemia nauplii

In this study, a 12-day growth trial was conducted to compare the effect of the variation in microcystin (MC) composition in two Microcystis aeruginosa bloom samples on the growth performance and MC accumulation/transfer in the common carp (Cyprinus carpio L.) larvae. Fish were fed Artemia salina nauplii that had been preexposed to extracts from two M. aeruginosa natural blooms with different microcystins (MCs) profiles. Bloom A had MC-LR as major toxin (74.05%) while bloom B had a diversity of MC (MC-RR; MC-(H4)YR; MC-YR; MC-LR; MC-FR; MC-WR) with no dominance of MC-LR. Newly-hatched Artemia nauplii were exposed separately to the two M. aeruginosa extracts A and B (100 microg L(-1)EqMC-LR) for 2h. The MC concentration in the nauplii was 73.60+/-7.88ngEqMC-LRg(-1)FW (n=4, mean+/-SE) for bloom A and 87.04+/-10.31ngEqMC-LRg(-1)FW for bloom B. These contaminated nauplii were given at the same ration to different groups (A and B) of fish larvae. Larval weight and length from day 9 were significantly different between groups A and B, and in both cases lower than that of a control group fed non-exposed nauplii. MCs accumulation by larvae, inversely correlated with the growth performance, was also significantly different between groups A and B (37.43+/-2.61 and 54.55+/-3.01ngEqMC-LRg(-1) FW, respectively, at the end of the experimental period). These results indicate that MC profile of a bloom may have differential effects on toxin accumulation/transfer and toxicity.

Detection of potentially producing cylindrospermopsin and microcystin strains in mixed populations of cyanobacteria by simultaneous amplification of cylindrospermopsin and microcystin gene regions.

Cyanobacterial blooms are frequently formed by heterogeneous populations of toxin-producing and non-producing strains. Microcystins (MC) and cylindrospermopsin (CYN) are the most representative cyanobacterial toxins. We have developed a multiplex PCR assay that allows simultaneous detection of MC(+) and/or CYN(+) strains in mixed populations of cyanobacteria. Various primer sets were designed using mcy and aoa gene sequences related with MC and CYN synthesis respectively, to amplify at the same time aoa and mcy sequences. Purified DNA, cultured cell mixtures and field samples with MC and CYN producing strains were used as DNA template. The results show: (i) the expected amplicons were only observed with toxic strains; (ii) cells were suitable as a source of purified DNA for the multiplex PCR; (iii) the assay could detect simultaneously 3 aoa and 3 mcy gene regions with mixed CYN(+) and MC(+) cyanobacteria cells. The method could be applied to environmental samples, allowing in a rapid, economical and easy way to detect simultaneously the presence of CYN(+) and MC(+) cyanobacteria in sestonic fractions of water samples.

Regulation of Gene Expression in Feeding Sites

The profound structural and physiological transformations that initial root cells experience to become nematode feeding sites (NFSs) must be paralleled by molecular changes. Although it seemed obvious that these molecular changes should occur mostly through modifications in gene expression, experimental support for this idea has only been obtained in the last few years. In this Chapter we review the different approaches that have led to the identification of genes whose expression patterns are modified during the differentiation of NFSs. These approaches involve comparisons between infected and non-infected roots, including protein analysis by two-dimensional gel electrophoresis, construction and screening of cDNA libraries, differential display of transcribed sequences and the analysis of reporter genes in transgenic plants, either fused to known promoters or as part of promoter-trap constructions. The still limited information gathered through these means is clustered around two main questions: i) the role in feeding sites of those genes that respond to nematodes, and ii) the mechanisms underlying their responsiveness to the parasites, particularly at the level of transcriptional regulation. The identification of promoter elements in nematode-responsive genes is finally being addressed, thanks to the application to the study of plant-nematode interactions of techniques for the functional and structural analysis of promoters. The information is still too limited to allow the formulation of a comprehensive model, although some connections with differentiation processes in plants are starting to be unveiled.

Compensatory Growth Induced in Zebrafish Larvae after Pre-Exposure to a Microcystis aeruginosa Natural Bloom Extract Containing Microcystins

Early life stage tests with zebrafish (Danio rerio) were used to detect toxic effects of compounds from a Microcystis aeruginosa natural bloom extract on their embryolarval development. We carried out the exposure of developing stages of fish to complex cyanobacterial blooms containing hepatotoxic molecules - microcystins. Fish embryo tests performed with the bloom extract containing 3 mg·L−1 Eq microcystin-LR showed that after 24 h of exposure all fish embryos died. The same tests performed with other diluted extracts (containing 0.3, 0.1 and 0.03 mg·L−1 Eq microcystin-LR) were shown to have an influence on zebrafish development and a large number of embryos showed malformation signs (edema, bent and curving tail). After hatching the larvae were transferred to a medium without toxins to follow the larval development under the new conditions. The specific growth of the pre-exposed larvae was significantly more important than that of the control larvae. This may represent a compensatory growth used to reduce the difference in size with the control fish noted after hatching.

Typing of toxinogenic Microcystis from environmental samples by multiplex PCR

Microcystin (MC)-producing Microcystis strains from environmental samples were assessed by the simultaneous amplification of up to five DNA sequences, corresponding to specific regions of six mcy genes (mcyA, mcyB, mcyC, mcyD, mcyE and mcyG), codifying for key motifs of the non-ribosomal peptide synthetase and polyketide synthase of the microcystin synthetase complex. Six primer pairs with the same melting temperature, one of them of new design, were used. A crucial point for the good performance of the new multiplex PCR test was the concentration of each primer pair. In the test, cell suspensions from laboratory cultures, field colonies and blooms were directly used as DNA source. The results of the multiplex PCR were consistent with the toxinogenic character of the samples, as checked by high performance liquid chromatography and/or matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. As a whole, the newly developed test could be used for a reliable, rapid and low-cost screening of potential MC-producing Microcystis in field samples, even scattered colonies.