Maria E. Donkin

Comparison of ultraviolet-induced genotoxicity detected by random amplified polymorphic DNA with chlorophyll fluorescence and growth in a marine macroalgae, Palmaria palmata.

The random amplified polymorphic DNA (RAPD) technique was used to detect DNA damage in the sublittoral macroalgae Palmaria palmata (Rhodophyta) exposed to both ambient and elevated irradiances of UV-B (280-315 nm). To investigate the potential of this method in ecotoxicological assessments, the qualitative and quantitative modifications in RAPD profiles were compared with changes in a number of physiological and fitness parameters. RAPD detectable modifications in DNA profiles were observed in all UV exposed individuals compared with controls. Changes in chlorophyll fluorescence (F(v)/F(m) ratio), in vivo pigment absorptance, thallus growth and RAPD profiles, examined simultaneously, provided a sensitive measure of UV-induced toxicity. In conclusion, the application of the RAPD method in conjunction with other suitable physiological and fitness measurements, may prove to be a valuable tool for investigating the specific effects of genotoxic agents upon marine algal populations. Ultimately, this methodology may allow the ecotoxicological examination of the link between molecular alterations and measurable adverse effects at higher levels of biological organisation.

Hsp70 expression in Enteromorpha intestinalis (Chlorophyta) exposed to environmental stressors

Numerous studies have indicated that stress proteins have potential as biomarkers of exposure to environmental contaminants. Analysis of stress proteins in animals, especially Hsp70, has dominated this research area despite increasing interest in the use of plants as pollution monitors. In the present study Hsp70 expression in Enteromorpha intestinalis has been investigated under exposure to a variety of stressors, to evaluate the potential use of Hsp70 as a biomarker of contaminant exposure in this seaweed. E. intestinalis was exposed to thermal stress, copper under differing conditions of nutrient availability and the triazine herbicide Irgarol 1051. Measurements were also taken to compare Hsp70 with conventional sublethal endpoints of toxicity such as growth and fluorescence induction parameters such as Fv/Fm. E. intestinalis exhibited a typical heat shock response. Hsp70 expression was increased with copper exposure, but proved to be a relatively insensitive biomarker of copper exposure compared to growth measurements. Nutrient limitation enhanced copper toxicity and significantly impaired growth, Fv/Fm and Hsp70 production. Fv/Fm and growth were strongly affected by Irgarol 1051 exposure, but Hsp70 levels were unaltered following exposure to the herbicide. The implications of using Hsp70 expression in E. intestinalis as a biomarker are discussed.

Risk posed by the antifouling agent Irgarol 1051 to the seagrass Zostera marina

Irgarol 1051 (2-(tert-butylamino)-4-cyclopropylamino)-6-(methylthio)-1,3,5-triazine) is a triazine herbicide that is increasingly being used to boost the effectiveness of antifouling paints. Estuarine plants, such as the marine angiosperm Zostera marina L. (eelgrass) may accumulate, and be affected by, Irgarol 1051, in locations with high boat densities. Bioconcentration of Irgarol 1051 within Zostera tissue was determined in field plants and laboratory semi-static exposure experiments. Effects of Irgarol 1051 upon the growth rate and photosystem II photosynthetic efficiency of Zostera were examined over a concentration range of 0 to 25 μg dm−3. Growth rate was assessed by comparison of leaf specific biomass ratios, and was found to be reduced at and above an Irgarol 1051 concentration of 10 μg dm−3. Photosynthetic efficiency was assessed using fluorescence induction kinetics: efficiency was significantly reduced at 0.18 μg dm−3 (0.4 μg g−1 dry weight leaf tissue) and a 10-day EC50 value of 2.5 μg dm−3 (1.1 μg g−1) calculated. Longer-term exposure revealed a 36-day EC50 value of 0.2 μg dm−3. Uptake of Irgarol 1051 was rapid within the Zostera leaves: tissue concentrations (dry weight basis) in excess of 300 times the water concentration were found within 2 days of exposure. Leaf concentrations in excess of 14 times root tissue concentration were found. Estuaries sampled in S.W. England had low aqueous Irgarol 1051 contamination, typically <0.003 μg dm−3, but Zostera leaf tissue concentrations (dry weight basis) were up to 25 000 times the aqueous values; this was only 15 times below the 10-day EC50 value. The reported results will enable the level of risk to isolated Zostera meadows from Irgarol 1051 to be assessed based on leaf tissue concentration and also have implications for the siting of marinas.

The influence of UV-B radiation on the reproductive cells of the intertidal macroalga, Enteromorpha intestinalis

The relative sensitivity of the reproductive cells of the seaweed Enteromorpha intestinalis to UV-B was assessed by measuring in vivo chlorophyll fluorescence (F(v) variable fluorescence), germination success and growth rates. Zoospores (the asexual reproductive cells) exhibit up to a 6 fold higher sensitivity to UV-B exposure than the mature thalli (measured as chlorophyll fluorescence, F(v)), and differences in growth rates were also found. Consistent patterns emerged throughout these experiments in variable fluorescence, germination success and growth rates, indicating a greater sensitivity in the sexual reproductive phase of the life cycle compared with the asexual phase. Inhibition of germination success (up to 50%) and growth rates (up to 16.4%) of settled gametes and zoospores after 1-h exposures to elevated levels of UV-B (equivalent to 27 and 31% ozone depletion) showed that damage to the reproductive cells was irreversible. In conclusion, the ecological significance of elevated UV-B exposure in the marine environment may be seriously under-estimated if effects on the early lifestages of algae are not considered.

Teaching botanical identification to adults: experiences of the UK participatory science project ‘Open Air Laboratories’

Taxonomic education and botany are increasingly neglected in schools and universities, leading to a ‘missed generation’ of adults that cannot identify organisms, especially plants. This study pilots three methods for teaching identification of native plant species to forty-three adults engaged in the participatory science project ‘Open Air Laboratories’ (OPAL). The three teaching methods (dichotomous key, word association exercise based on a mnemonic approach and pictorial card game) proved equally effective in teaching plant identification to participants for the groups of plants used. The dichotomous key is an established method for teaching transferrable identification skills, whilst the other two methods could be useful tools for stimulating initial interest and awareness in novices. The reasons for the decline in botanical knowledge are discussed, alongside the importance of using appealing identification resources and making botany relevant to people’s lives.

The influence of copper and heatshock on the physiology and cellular stress response of Enteromorpha intestinalis

Abstract Enteromorpha intestinalis was exposed to a range of copper concentrations (0-500 μg−1) for 5 days, to assess the effect of copper exposure on stress proteins (Stress-70 levels) and physiology of the seaweed. A second experiment investigated the combined effects of copper and heatshock on Stress-70. Stress-70 was induced by copper exposure, but is no better an indicator of copper exposure, in E. intestinalis, than measurement of growth. Copper and heatshock were additive inducers of Stress-70 expression. Both copper exposed and control groups exhibited the same relative increase in Stress-70 when heatshocked, indicating that copper has no effect on the ability of E. intestinalis to raise a heatshock response.

Evaluation of in vivo thallus absorptance and chlorophyll fluorescence as biomarkers of UV-B exposure and effects in marine macroalgae from different tidal levels

Abstract A field survey and an outdoor experiment were conducted to evaluate the potential of chlorophyll fluorescence and in vivo absorptance spectra as biomarkers of short-term experimental and long-term solar UV radiation exposure in macroalgae. The eulittoral macroalga Porphyra umbilicalis (L.) (Rhodophyta) and the sublittoral macroalga Palmaria palmata (L.) (Rhodophyta) were collected at three different sites along a vertical transect on the beach and in the water column. Significant decreases in the absorptance spectra at 497 and 569 nm (consistent with the presence of R-phycoerythrin and/or carotenoids and phycoerythrobilin, respectively) occurred in P. palmata after exposure to elevated UV-B (2.7 Wm−2). Furthermore, depth-dependent decreases in thallus absorptance at specific wavelengths were found after all exposures, indicating that in vivo thallus absorptance may be a useful general indicator of UV exposure in conjunction with other biomarkers. Also, a depth-dependent decrease in F v F m was detected in P. palmata. A 60% decrease in thallus absorptance and lack of recovery in chlorophyll fluorescence F v F m ratio indicated irreversible damage to accessory pigments, chlorophyll a and photosystem II after exposure to elevated UV-B. Moreover, a depth-dependent increase in thallus absorptance between 290 and 325 nm was observed in P. palmata in response to short-term experimental elevated UV-B exposure and between 290 and 380 nm in response to long-term solar UV-B. In contrast, the eulittoral alga Porphyra umbilicalis exhibited a greater degree of tolerance of UV-B exposure. The results highlight the potentially damaging effects of high irradiances of photosynthetically active radiation and UV and the necessity of utilising several biomarkers when assessing the biological effects of UV-B irradiation associated with ozone depletion.

Calmodulin Binding Drugs Trifluoperazine and Compound 48/80 Modify Stomatal Responses of Commelina communis L.

Summary Trifluoperazine and Compound 48/80 are drugs which bind to the calcium activated form of the calcium binding protein calmodulin. In this study these drugs were applied to epidermal strips of Commelina communis L. to see whether stomatal apertures under a range of incubation conditions could be affected by their presence. Significant effects of both calmodulin binding drugs on the normal stomatal responses of C. communis were found. Furthermore the drugs effects were observed to be independent of changes in stomatal aperture resulting from the application of calcium ions in millimolar quantities. It is concluded that the gross effects of high exogenous Ca ** ion concentrations on stomatal responses do not involve the calcium binding protein calmodulin. A role in stomatal control is postulated for Ca ** ions in the µMolar range based on the effects observed with the two calmodulin binding drugs. The reported effects of the calmodulin binding drugs uncovered here are in agreement with others reported in the literature.

A comparison of the kinetic properties of phosphoenolpyruvate carboxylase from guard-cell and mesophyll-cell protoplasts of Commelina communis

Some kinetic properties of partially purified phosphoenolpyruvate carboxylase (PEPCase) from guard-cell and mesophyll-cell protoplasts of Commelina communis are described. The PEPCase activity inherent to each cell type was determined and the apparent Km (phosphoenolpyruvate) and Ki (malate) were compared. Malate sensitivity was much higher (Ki malate 0.4 mol m−3) in the extract of guard-cell protoplasts than in that of mesophyllcell protoplasts (Ki malate 4.2 mol m−3). The stimulation of activity by glucose-6-phosphate in the presence of malate (‘deinhibition’) was also investigated in extracts from both cell types and was found to be similar to previously reported results with epidermal tissue. The effect of contamination of an extract of guard-cell protoplasts with mesophyll-cell protoplasts was measured in the presence and absence of malate. It was found that a small amount to mesophyll-cell contaminant appears to desensitize the malate inhibition of PEPCase from guard-cell protoplasts. It is concluded that experiments which use epidermal tissue to study guardcell PEPCase may give misleading information as a consequence of mesophyll contamination.

The development and evaluation of Freeze-Fracture/Cytoplasmic maceration for the SEM to investigate algal ultrastructure

Freeze-fracture/cytoplasmic maceration (FF/CM) to expose chloroplast ultrastructure for the SEM was developed for the green alga Enteromorpha intestinalis (Linnaeus) Link, and the results were compared with conventional TEM preparation. Preliminary investigations showed that the method used for higher plants was inadequate to expose algal chloroplast membrane detail, but, by decreasing the molarity of the buffer during maceration to make it more hypotonic relative to the tissue, sufficient cytoplasmic extraction was accomplished in 7 days using osmium tetroxide at a lower concentration than suggested in previous work. Post-maceration in tannic acid was omitted from the processing after the preliminary study to assess its effects on the final images, and consequently preparation time was shortened drammatically and now compares favourably with the time take to prepare material for the TEM. Ultrastructural integrity is preserved and is consistent with normal TEM preparations.