© 2003 American Association for the Advancement of ScienceHugh H. Harris, Ingrid J. Pickering and Graham N. Georg

The biological activity of selenium is dependent upon its speciation. We aim to integrate selenium speciation and metabolism into a discussion of the mechanisms by which selenium exerts its biological activity. First, we present the current status of selenium in the prevention of cancer, cardiovascular and neurodegenerative diseases with particular attention paid to the results of major chemoprevention trials involving selenium supplementation. A comprehensive review of the current understanding of the metabolism of common dietary selenium compounds – selenite, selenomethionine, methylselenocysteine and selenocystine – is presented, with discussion of the evidence for the various metabolic pathways and their products. The antioxidant, prooxidant and other mechanisms of the dietary selenium compounds have been linked to their disease prevention and treatment properties. The evidence for these various mechanisms – in vitro, in cells and in vivo – is evaluated with emphasis on the selenium metabolites involved. We conclude that dietary selenium compounds should be considered prodrugs, whose biological activity will depend on the activity of the various metabolic pathways in, and the redox status of, cells and tissues. These factors should be considered in future laboratory research and in selecting selenium compounds for trials of disease prevention and treatment by selenium supplementation.

It has become increasingly evident that dietary Se plays a significant role in reducing the incidence of lung, colorectal and prostate cancer in humans. Different forms of Se vary in their chemopreventative efficacy, with Se-methylselenocysteine being one of the most potent. Interestingly, the Se accumulating plant Astragalus bisulcatus (Two-grooved poison vetch) contains up to 0.6% of its shoot dry weight as Se-methylselenocysteine. The ability of this Se accumulator to biosynthesize Se-methylselenocysteine provides a critical metabolic shunt that prevents selenocysteine and selenomethionine from entering the protein biosynthetic machinery. Such a metabolic shunt has been proposed to be vital for Se tolerance in A. bisulcatus. Utilization of this mechanism in other plants may provide a possible avenue for the genetic engineering of Se tolerance in plants ideally suited for the phytoremediation of Se contaminated land. Here, we describe the overexpression of a selenocysteine methyltransferase from A. bisulcatus to engineer Se-methylselenocysteine metabolism in the Se non-accumulator Arabidopsis thaliana (Thale cress). By over producing the A. bisulcatus enzyme selenocysteine methyltransferase in A. thaliana, we have introduced a novel biosynthetic ability that allows the non-accumulator to accumulate Se-methylselenocysteine and γ-glutamylmethylselenocysteine in shoots. The biosynthesis of Se-methylselenocysteine in A. thaliana also confers significantly increased selenite tolerance and foliar Se accumulation. These results demonstrate the feasibility of developing transgenic plant-based production of Se-methylselenocysteine, as well as bioengineering selenite resistance in plants. Selenite resistance is the first step in engineering plants that are resistant to selenate, the predominant form of Se in the environment.

Summary: The fractionation of stable copper (Cu) isotopes during uptake into plant roots and translocation to shoots can provide information on Cu acquisition mechanisms. Isotope fractionation (⁶⁵Cu/⁶³Cu) and intact tissue speciation techniques (X‐ray absorption spectroscopy, XAS) were used to examine the uptake, translocation and speciation of Cu in strategy I (tomato–Solanum lycopersicum) and strategy II (oat–Avena sativa) plant species. Plants were grown in controlled solution cultures, under varied iron (Fe) conditions, to test whether the stimulation of Fe‐acquiring mechanisms can affect Cu uptake in plants. Isotopically light Cu was preferentially incorporated into tomatoes (Δ⁶⁵Cu_{whole plant‐solution} = c. −1‰), whereas oats showed minimal isotopic fractionation, with no effect of Fe supply in either species. The heavier isotope was preferentially translocated to shoots in tomato, whereas oat plants showed no significant fractionation during translocation. The majority of Cu in the roots and leaves of both species existed as sulfur‐coordinated Cu(I) species resembling glutathione/cysteine‐rich proteins. The presence of isotopically light Cu in tomatoes is attributed to a reductive uptake mechanism, and the isotopic shifts within various tissues are attributed to redox cycling during translocation. The lack of isotopic discrimination in oat plants suggests that Cu uptake and translocation are not redox selective.

Contents Summary 432 I. Introduction 433 II. Preparation of plant samples for X‐ray micro‐analysis 433 III. X‐ray elemental mapping techniques 438 IV. X‐ray data analysis 442 V. Case studies 443 VI. Conclusions 446 Acknowledgements 449 Author contributions 449 References 449 Summary: Hyperaccumulators are attractive models for studying metal(loid) homeostasis, and probing the spatial distribution and coordination chemistry of metal(loid)s in their tissues is important for advancing our understanding of their ecophysiology. X‐ray elemental mapping techniques are unique in providing in situ information, and with appropriate sample preparation offer results true to biological conditions of the living plant. The common platform of these techniques is a reliance on characteristic X‐rays of elements present in a sample, excited either by electrons (scanning/transmission electron microscopy), protons (proton‐induced X‐ray emission) or X‐rays (X‐ray fluorescence microscopy). Elucidating the cellular and tissue‐level distribution of metal(loid)s is inherently challenging and accurate X‐ray analysis places strict demands on sample collection, preparation and analytical conditions, to avoid elemental redistribution, chemical modification or ultrastructural alterations. We compare the merits and limitations of the individual techniques, and focus on the optimal field of applications for inferring ecophysiological processes in hyperaccumulator plants. X‐ray elemental mapping techniques can play a key role in answering questions at every level of metal(loid) homeostasis in plants, from the rhizosphere interface, to uptake pathways in the roots and shoots. Further improvements in technological capabilities offer exciting perspectives for the study of hyperaccumulator plants into the future.