José M. Becerril

A rapid high‐performance liquid chromatography method to measure lipophilic antioxidants in stressed plants: simultaneous determination of carotenoids and tocopherols

A new reversed-phase HPLC method has been developed for the simultaneous determination of all major photosynthetic pigments in higher plants (antheraxanthin, α- and β-carotene, chlorophyll a and b, lactucaxanthin, lutein, lutein-epoxide, neoxanthin, pheophytin a, violaxanthin and zeaxanthin) and tocopherols (α- and γ-tocopherol) in the same extract. The method is also suitable for the determination of other carotenoids and their intermediates, for example, γ- and ζ-carotene, lycopene, phytoene and phytofluene. The quantitative analysis of such compounds is of paramount importance in studies dealing with pigment biosynthesis and degradation, as well as in studies on plant ecophysiology, since many of these compounds seem to be important in enabling a plant to withstand environmental stresses. The method uses a C-18 column and a simple solvent gradient, resolving in 16 min all carotenoids and tocopherols. Chromatograms are generated by a photodiode array detector at three wavelengths (295, 410 and 445 nm). The use of a fluorescence detector significantly improves the sensitivity of detection of tocopherols and also allows the determination of δ-tocopherol. The HPLC method provides a sensitive, accurate and reproducible procedure for the quantitative analysis of pigments and tocopherols and, since these compounds are usually analysed by separate analytical procedures in plant stress physiology studies, also represents a reduction in the requirements of equipment, the amounts of solvents used and the analysis time compared with the methods currently employed. Copyright

Chelate-Enhanced Phytoremediation of Soils Polluted with Heavy Metals

In general, hyperaccumulators are low biomass, slow-growing plants. High biomass non-hyperaccumulator plants by themselves are not a valid alternative for phytoextraction as they also have many limitations, such as small root uptake and little root-to-shoot translocation. In this context, chemically-induced phytoextraction (based on the fact that the application of certain chemicals, mostly chelating agents, to the soil significantly enhances metal accumulation by plants) has been proposed as an alternative for the cleaning up of metal polluted soils. But chelate-induced phytoextraction increases the risk of adverse environmental effects due to metal mobilization during extended periods of time. In order to minimize the phytotoxicity and environmental problems associated with the use of chelating agents, nowadays, research is being carried out on the gradual application of small doses of the chelating agent during the growth period. However, EDTA utilization in the future will most likely be limited to ex situconditions where control of the leachates can be achieved. There are other mobilizing agents which are much less harmful to the environment such as citric acid, NTA, and particularly EDDS. Research should also be aimed towards more innovative agronomic practices. Environmentally safe methods of chelate-induced phytoextraction must be developed before steps towards further development and commercialization of this remediation technology are taken. Most importantly, more applied projects in this field are needed to clarify the real potential and risks of this technology.

Seasonal changes in photosynthetic pigments and antioxidants in beech (Fagus sylvatica) in a Mediterranean climate: implications for tree decline diagnosis

Seasonal changes in all major and minor photosynthetic pigments and antioxidants were studied in sun and shade in beech (Fagus sylvatica L.) leaves and buds under Mediterranean conditions. Both sun and shade leaves differed progressively in their biochemical and morphological characteristics during the course of leaf ageing. Sun leaves were 3-fold thicker, pigment content was highly reduced and the leaves were protected by a much more powerful antioxidative system. Pigment content decreased progressively during summer in sun leaves and remained stable in shade leaves. From July to September, there was no chlorophyll (Chl) biosynthesis, so photodegradation of Chl was not compensated by production of new Chl. This reduction in Chl occurred in parallel with an increment in tocopherols. Natural leaf senescence occurred first in sun leaves. During this process, Chl was completely degraded, xanthophylls were esterified with fatty acids, and violaxanthin was converted into zeaxanthin, while carotenes remained stable. One of the main indicators of tree decline and air pollution is accelerated leaf ageing, and this process is characterised in beech leaves by tocopherol and pigment destruction, so we have used these criteria to verify whether these parameters can be used as early indicators of tree decline. All pigments and antioxidants (including α -tocopherol) decreased with beech decline, except xanthophyll cycle pigments. As reduction of these compounds is concomitant with a similar level of Chl degradation, Chl determination is proposed as a simple method sufficient to predict the early stages of beech decline at a biochemical level.

Effects of chelates on plants and soil microbial community: comparison of EDTA and EDDS for lead phytoextraction.

Most studies on chelate-induced phytoextraction have focused on EDTA-mediated Pb phytoextraction. But EDTA and the formed EDTA-Pb complexes have low biodegradability and high solubility in soil, resulting in an elevated risk of adverse environmental effects. EDDS is an easily biodegradable chelating agent that has recently been proposed as an environmentally sound alternative to EDTA. Consequently, a greenhouse experiment, using a completely randomized factorial design with four replications, was carried out to compare the potential of EDTA and EDDS for chelate-induced Pb phytoextraction with Cynara cardunculus, as well as to investigate the toxicity of these two chelates to both cardoon plants and soil microorganisms. The effects of chelate addition on soil microbial communities were studied through the determination of a variety of biological indicators of soil quality such as soil enzyme activities, basal and substrate-induced respiration, potentially mineralizable nitrogen, and community level physiological profiles. EDTA was much more efficient than EDDS for the enhancement of root Pb uptake and root-to-shoot Pb translocation. In a soil polluted with 5000 mg Pb kg(-1), as a result of the addition of 1 g EDTA kg(-1) soil, a value of 1332 mg Pb kg(-1) DW shoot was obtained. EDDS application resulted in a shoot Pb accumulation of only 310 mg kg(-1)DW. Plants treated with EDDS showed lower values of biomass than those treated with EDTA. EDDS proved to be rapidly degraded, and less toxic to the soil microbial community in control non-polluted soils. Pb-polluted EDDS-treated soils showed significantly higher values of basal and substrate-induced respiration than those treated with EDTA. Although EDDS had a lower capacity to enhance Pb phytoextraction than EDTA, it has the advantage of rapid biodegradation.

Diurnal changes in antioxidant and carotenoid composition in the Mediterranean schlerophyll tree Quercus ilex (L) during winter

Abstract Seasonal changes of pigment composition and antioxidant content were characterized in the Mediterranean evergreen holm oak ( Quercus ilex L.). Higher contents of antioxidants and carotenoids, with a photoprotective role during winter, indicated that this period was highly stressful, so a study of diurnal changes in photosynthesis, pigments and carotenoids was conducted during January in sun and shade leaves. Sun and shade leaves were used to compare the effects due only to low temperature separate from those resulting from the interaction of light. During winter, a relatively high rate of CO 2 fixation on sun leaves represented an important sink for photosynthetic electrons contributing to the annual carbon balance of the plant. This high rate contrasted with a reduced F v / F m , even at predawn. This reduction was correlated with the accumulation of zeaxanthin at the expense of violaxanthin by de-epoxidation. Sun leaves were also protected by a higher concentration of antioxidants (ascorbate, glutathione and tocopherol) and carotenoids (except lutein epoxide). Ascorbate was 10–50-fold greater than the other antioxidants, indicating a central role in protection against photooxidative stress. Nevertheless those mechanisms were unable to avoid a loss of hydrophilic antioxidants (glutathione and ascorbate) and xanthophylls during the initial morning hours after dawn, indicating that the first target of photooxidative damage was these molecules.

Effects of drought on photoprotective mechanisms in European beech (Fagus sylvatica L.) seedlings from different provenances

The effects of drought on photoprotection mechanisms of three beech provenances from three contrasting climatic origins have been studied. Morphology differed among provenances, with a lower leaf area/fine roots ratio in the xeric populations (Moncayo). This characteristic allowed this provenance to respond later than the others to a drought-stress treatment. However, in desiccating leaves the stability of PSII was higher in the plants from the stressful climate of the Pyrenees (Belagoa). Drought resulted in a reduction in pigment content (except in Belagoa) with simultaneous increases in antheraxanthin, zeaxanthin and tocopherol pools. These molecules play an important photoprotective role and reached similar values in stressed leaves from the three provenances. These antioxidants also protected the leaves from paraquat-induced oxidation. It is concluded that drought tolerance in the studied xeric ecotype is based to a greater extent on morphological characteristics than on a better photoprotective system.

The operation of the lutein epoxide cycle correlates with energy dissipation

A new xanthophyll cycle involving de-epoxidation of lutein epoxide (Lx) into lutein in the light and epoxidation back in the dark has been recently described in parasitic plants and in trees from the genus Quercus. To explore the role of the Lx cycle in photoprotection, shade leaves of red oak (Q. rubra), with a relatively high Lx pool, were exposed to different light intensities. Both violaxanthin and Lx were de-epoxidised to the same extent, although the initial kinetics differed, with a rate proportional to the light intensity. De-epoxidation of violaxanthin and Lx was inhibited by dithiothreitol, suggesting that the same enzyme, violaxanthin de-epoxidase (VDE), catalyses both reactions. Dark recovery lagged in the case of Lx, and after 5 h in darkness, the Lx cycle was much more de-epoxidised than the violaxanthin cycle. The different rates of epoxidation of the violaxanthin and Lx cycles were used to study the role of the Lx cycle in photoprotection. Statistical approaches (partial correlation and multiple regression) indicate that in these leaves, maximal photochemical efficiency of PSII (Fv/Fm) and non-photochemical quenching are correlated with the level of Lx de-epoxidation. The potential implications of this finding for the understanding of the photosynthetic process in shaded and understorey leaves are discussed.

Microbial Monitoring of the Recovery of Soil Quality During Heavy Metal Phytoremediation

Soil pollution with heavy metals is a worldwide environmental problem. Phytoremediation through phytoextraction and phytostabilization appears to be a promising technology for the remediation of polluted soils. It is important to strongly emphasize that the ultimate goal of a heavy metal remediation process must be not only to remove the heavy metals from the soil (or instead to reduce their bioavailability and mobility) but also to restore soil quality. Soil quality is defined as the capacity of a given soil to perform its functions. Soil microbial properties are increasingly being used as biological indicators of soil quality due to their quick response, high sensitivity, and, above all, capacity to provide information that integrates many environmental factors. Indeed, microbial properties are among the most ecologically relevant indicators of soil quality. Consequently, microbial monitoring of the recovery of soil quality is often carried out during heavy metal phytoremediation processes. However, soil microbial properties are highly context dependent and difficult to interpret. For a better interpretation of microbial properties as indicators of soil quality, they may be grouped within categories of higher ecological relevance, such as soil functions, ecosystem health attributes, and ecosystem services.

Interactions between plant and rhizosphere microbial communities in a metalliferous soil.

In the present work, the relationships between plant consortia, consisting of 1-4 metallicolous pseudometallophytes with different metal-tolerance strategies (Thlaspi caerulescens: hyperaccumulator; Jasione montana: accumulator; Rumex acetosa: indicator; Festuca rubra: excluder), and their rhizosphere microbial communities were studied in a mine soil polluted with high levels of Cd, Pb and Zn. Physiological response and phytoremediation potential of the studied pseudometallophytes were also investigated. The studied metallicolous populations are tolerant to metal pollution and offer potential for the development of phytoextraction and phytostabilization technologies. T. caerulescens appears very tolerant to metal stress and most suitable for metal phytoextraction; the other three species enhance soil functionality. Soil microbial properties had a stronger effect on plant biomass rather than the other way around (35.2% versus 14.9%). An ecological understanding of how contaminants, ecosystem functions and biological communities interact in the long-term is needed for proper management of these fragile metalliferous ecosystems.

Differential responses of three fungal species to environmental factors and their role in the mycorrhization of Pinus radiata D. Don

Three ectomycorrhizal (ECM) isolates of Rhizopogon luteolus, R. roseolus and Scleroderma citrinum were found to differ markedly in their in vitro tolerance to adverse conditions limiting fungal growth, i.e. water availability, pH and heavy metal pollution. S. citrinum was the most sensitive, R. luteolus intermediate and R. roseolus the most tolerant species. Pinus radiata D. Don seedlings were inoculated in the laboratory and in a containerised seedling nursery with spore suspensions of the three ECM species. Colonisation percentage was considerably lower under nursery conditions, probably due to competition by native fungi. The effects of nursery ECM inoculation on seedling growth depended on the fungal species. Only R. roseolus-colonised plants showed a significantly higher shoot growth than non-mycorrhizal plants. All three fungi induced significantly higher root dry weights relative to control plants. Despite the low mycorrhizal colonisation, mycorrhization with all three species improved the physiological status of nursery-grown seedlings, e.g. enhanced root enzyme activity, shoot nutrient and pigment content, net photosynthesis rate and water use efficiency. Of the three fungal species, R. roseolus was the most effective; this species was also the most adaptable and showed the greatest range of tolerance to adverse environmental conditions in pure culture. It is, therefore, proposed as a promising fungal species for ECM inoculation of P. radiata in the nursery.