Elizabeth A. H. Pilon-Smits

Physiological functions of beneficial elements.

Aluminum (Al), cobalt (Co), sodium (Na), selenium (Se), and silicon (Si) are considered beneficial elements for plants: they are not required by all plants but can promote plant growth and may be essential for particular taxa. These beneficial elements have been reported to enhance resistance to biotic stresses such as pathogens and herbivory, and to abiotic stresses such as drought, salinity, and nutrient toxicity or deficiency. The beneficial effects of low doses of Al, Co, Na and Se have received little attention compared to toxic effects that typically occur at higher concentrations. Better understanding of the effects of beneficial elements is important to improve crop productivity and enhance plant nutritional value for a growing world population.

Selenium in higher plants: understanding mechanisms for biofortification and phytoremediation

Selenium (Se) is an essential micronutrient for many organisms, including plants, animals and humans. As plants are the main source of dietary Se, plant Se metabolism is therefore important for Se nutrition of humans and other animals. However, the concentration of Se in plant foods varies between areas, and too much Se can lead to toxicity. As we discuss here, plant Se uptake and metabolism can be exploited for the purposes of developing high-Se crop cultivars and for plant-mediated removal of excess Se from soil or water. Here, we review key developments in the current understanding of Se in higher plants. We also discuss recent advances in the genetic engineering of Se metabolism, particularly for biofortification and phytoremediation of Se-contaminated environments.

Phytoremediation of metals using transgenic plants

An ideal plant for environmental cleanup can be envisioned as one with high biomass production, combined with superior capacity for pollutant tolerance, accumulation, and/or degradation, depending on the type of pollutant and the phytoremediation technology of choice. With the use of genetic engineering, it is feasible to manipulate a plants capacity to tolerate, accumulate, and/or metabolize pollutants, and thus to create the ideal plant for environmental cleanup. In this review, we focus on the design and creation of transgenic plants for phytoremediation of metals. Plant properties important for metal phytoremediation are metal tolerance and accumulation, which are determined by metal uptake, root-shoot translocation, intracellular sequestration, chemical modification, and general stress resistance. If we know which molecular mechanisms are involved in these tolerance and accumulation processes, and which genes control these mechanisms, we can manipulate them to our advantage. This review aims to give a succinct overview of plant metal tolerance and accumulation mechanisms, and to identify possible strategies for genetic engineering of plants for metal phytoremediation. An overview is presented of what has been achieved so far regarding the manipulation of plant metal metabolism. In fact, both enhanced metal tolerance and accumulation have been achieved by overproducing metal chelating molecules (citrate, phytochelatins, metallothioneins, phytosiderophores, ferritin) or by the overexpression of metal transporter proteins. Mercury volatilization and tolerance was achieved by introduction of a bacterial pathway. The typical increase in metal accumulation as the result of these genetic engineering approaches is 2-to 3-fold more metal per plant, which could potentially enhance phytoremediation efficiency by the same factor. As for the applicability of these transgenics for environmental cleanup, results from lab and greenhouse studies look promising for several of these transgenics, but field studies will be the ultimate test to establish their phytoremediation potential, their competitiveness, and risks associated with their use.

Trehalose-producing transgenic tobacco plants show improved growth performance under drought stress

Summary Trehalose plays a role in drought stress resistance in a variety of organisms, including the extremely drought-tolerant «resurrection plants». Transgenic tobacco plants that produce trehalose were engineered by introduction of the Escherichia coli ots A and ots B genes, encoding trehalose-6-P synthase and trehalose-6-P phosphatase, respectively. The introduction of these genes had a pronounced effect on plant morphology and growth performance under drought stress. The transgenic Ots plants had larger leaves and altered stem growth. When grown under drought stress imposed by limiting water supply, the two transgenic tobacco lines Ots2 and Ots 5 yielded total dry weights that were 28 % and 39 % higher than those of wild-type tobacco. These increases in dry weight were due mainly to increased leaf production: leaf dry weights were up to 85 % higher for the best trehalose accumulator, Ots 5. No significant differences were observed under well-watered conditions. Chlorophyll fluorescence analysis of drought-stressed plants showed a higher photochemical quenching (qQ) and a higher ratio of variable fluorescence over maximal fluorescence (Fv/Fm), indicating a more efficient photosynthesis. The Ots 5 plants showed more negative leaf osmotic potentials than wild-type plants, particularly under drought stress, as well as higher levels of nonstructural carbohydrates; Ots2 plants showed intermediate values. Detached leaves from young, well-watered Ots plants had a better capacity than wild-type leaves to retain water when air-dried. They had lower osmotic potentials than wild-type leaves, and higher levels of glucose, fructose and sucrose.

Environmental cleanup using plants: biotechnological advances and ecological considerations

Plants and their associated microbes can be used in the cleanup and prevention of environmental pollution. This relatively new and growing technology uses natural processes to break down, stabilize, or accumulate pollutants. Knowledge of the biochemical processes involved may lead to the development of more efficient plants and better management practices. One approach for improving the efficiency of phytoremediation includes developing transgenic plants. Here, we give an overview of phytoremediation methods and their associated biological processes, and discuss approaches that have been used successfully to breed transgenic plants with advanced phytoremediation properties. Much is still unknown about the ecological implications of phytoremediation, especially when using transgenic plants. Phytoremediation-related processes can change the location or chemical makeup of contaminants; the question is how those processes will affect the interactions among organisms in the ecosystem, and how transgenic plants...

Molecular Mechanisms of Selenium Tolerance and Hyperaccumulation in Stanleya pinnata

The molecular mechanisms responsible for selenium (Se) tolerance and hyperaccumulation were studied in the Se hyperaccumulator Stanleya pinnata (Brassicaceae) by comparing it with the related secondary Se accumulator Stanleya albescens using a combination of physiological, structural, genomic, and biochemical approaches. S. pinnata accumulated 3.6-fold more Se and was tolerant to 20 μm selenate, while S. albescens suffered reduced growth, chlorosis and necrosis, impaired photosynthesis, and high levels of reactive oxygen species. Levels of ascorbic acid, glutathione, total sulfur, and nonprotein thiols were higher in S. pinnata, suggesting that Se tolerance may in part be due to increased antioxidants and up-regulated sulfur assimilation. S. pinnata had higher selenocysteine methyltransferase protein levels and, judged from liquid chromatography-mass spectrometry, mainly accumulated the free amino acid methylselenocysteine, while S. albescens accumulated mainly the free amino acid selenocystathionine. S. albescens leaf x-ray absorption near-edge structure scans mainly detected a carbon-Se-carbon compound (presumably selenocystathionine) in addition to some selenocysteine and selenate. Thus, S. albescens may accumulate more toxic forms of Se in its leaves than S. pinnata. The species also showed different leaf Se sequestration patterns: while S. albescens showed a diffuse pattern, S. pinnata sequestered Se in localized epidermal cell clusters along leaf margins and tips, concentrated inside of epidermal cells. Transcript analyses of S. pinnata showed a constitutively higher expression of genes involved in sulfur assimilation, antioxidant activities, defense, and response to (methyl)jasmonic acid, salicylic acid, or ethylene. The levels of some of these hormones were constitutively elevated in S. pinnata compared with S. albescens, and leaf Se accumulation was slightly enhanced in both species when these hormones were supplied. Thus, defense-related phytohormones may play an important signaling role in the Se hyperaccumulation of S. pinnata, perhaps by constitutively up-regulating sulfur/Se assimilation followed by methylation of selenocysteine and the targeted sequestration of methylselenocysteine.

Metallophytes—a view from the rhizosphere

Some plants hyperaccumulate metals or metalloids to levels several orders of magnitude higher than other species. This intriguing phenomenon has received considerable attention in the past decade. While research has mostly focused on the above-ground organs, roots are the sole access point to below-ground trace elements and as such they play a vital role in hyperaccumulation. Here we highlight the role of the root as an effective trace element scavenger through interactions in the rhizosphere. We found that less than 10% of the known hyperaccumulator species have had their rhizospheres examined. When studied, researchers have focused on root physical characteristics, rhizosphere chemistry, and rhizosphere microbiology as central themes to understand plant hyperaccumulation. One physical characteristic often assumed about hyperaccumulators is that their roots are small, but this is not true for all species and many species remain unexamined. Transporters in root membranes provide avenues for root uptake, while root growth and morphology influence plant access to trace elements in the rhizosphere. Some hyperaccumulators exhibit unique root scavenging and direct their growth toward elements in soil. Studies on hyperaccumulator rhizosphere chemistry have examined the role of the root in altering elemental solubility through exudation and pH changes. Different interpretations have been reported for mobilization of non-labile trace element pools by hyperaccumulators. However, there is a lack of evidence for a novel role for rhizosphere acidification in hyperaccumulation. As for microbiological studies, researchers have shown that bacteria and fungi in the hyperaccumulator rhizosphere may exhibit increased metal tolerance, act as plant growth promoting microorganisms, alter elemental solubility, and have significant effects on plant trace element concentrations. New evidence suggests that symbiosis with arbuscular mycorrhizae may not be rare in hyperaccumulator taxa, even in some members of the Brassicaceae. Although there are several reports on the presence of mycorrhizae, a cohesive interpretation of their role in hyperaccumulation remains elusive. In summary, we present the current state of knowledge about how roots hyperaccumulate and we suggest ways in which this knowledge can be applied and improved.

Transcriptome analyses give insights into selenium-stress responses and selenium tolerance mechanisms in Arabidopsis.

Selenate is chemically similar to sulfate and can be taken up and assimilated by plants via the same transporters and enzymes. In contrast to many other organisms, selenium (Se) has not been shown to be essential for higher plants. In excess, Se is toxic and restricts development. Both Se deficiency and toxicity pose problems worldwide. To obtain better insights into the effects of Se on plant metabolism and into plant mechanisms involved in Se tolerance, the transcriptome of Arabidopsis plants grown with or without selenate was studied and Se-responsive genes identified. Roots and shoots exhibited different Se-related changes in gene regulation and metabolism. Many genes involved in sulfur (S) uptake and assimilation were upregulated. Accordingly, Se treatment enhanced sulfate levels in plants, but the quantity of organic S metabolites decreased. Transcripts regulating the synthesis and signaling of ethylene and jasmonic acid were also upregulated by Se. Arabidopsis mutants defective in ethylene or jasmonate response pathways exhibited reduced tolerance to Se, suggesting an important role for these two stress hormones in Se tolerance. Selenate upregulated a variety of transcripts that were also reportedly induced by salt and osmotic stress. Selenate appeared to repress plant development, as suggested by the downregulation of genes involved in cell wall synthesis and auxin-regulated proteins. The Se-responsive genes discovered in this study may help create plants that can better tolerate and accumulate Se, which may enhance the effectiveness of Se phytoremediation or serve as Se-fortified food.

Characterization of a NifS-Like Chloroplast Protein from Arabidopsis. Implications for Its Role in Sulfur and Selenium Metabolism

NifS-like proteins catalyze the formation of elemental sulfur (S) and alanine from cysteine (Cys) or of elemental selenium (Se) and alanine from seleno-Cys. Cys desulfurase activity is required to produce the S of iron (Fe)-S clusters, whereas seleno-Cys lyase activity is needed for the incorporation of Se in selenoproteins. In plants, the chloroplast is the location of (seleno) Cys formation and a location of Fe-S cluster formation. The goal of these studies was to identify and characterize chloroplast NifS-like proteins. Using seleno-Cys as a substrate, it was found that 25% to 30% of the NifS activity in green tissue in Arabidopsis is present in chloroplasts. A cDNA encoding a putative chloroplast NifS-like protein, AtCpNifS, was cloned, and its chloroplast localization was confirmed using immunoblot analysis and in vitro import. AtCpNIFS is expressed in all major tissue types. The protein was expressed in Escherichia coli and purified. The enzyme contains a pyridoxal 5′ phosphate cofactor and is a dimer. It is a type II NifS-like protein, more similar to bacterial seleno-Cys lyases than to Cys desulfurases. The enzyme is active on both seleno-Cys and Cys but has a much higher activity toward the Se substrate. The possible role of AtCpNifS in plastidic Fe-S cluster formation or in Se metabolism is discussed.

Cooperative Ethylene and Jasmonic Acid Signaling Regulates Selenite Resistance in Arabidopsis

Selenium (Se) is an essential element for many organisms, but excess Se is toxic. To better understand plant Se toxicity and resistance mechanisms, we compared the physiological and molecular responses of two Arabidopsis (Arabidopsis thaliana) accessions, Columbia (Col)-0 and Wassilewskija (Ws)-2, to selenite treatment. Measurement of root length Se tolerance index demonstrated a clear difference between selenite-resistant Col-0 and selenite-sensitive Ws-2. Macroarray analysis showed more pronounced selenite-induced increases in mRNA levels of ethylene- or jasmonic acid (JA)-biosynthesis and -inducible genes in Col-0 than in Ws-2. Indeed, Col-0 exhibited higher levels of ethylene and JA. The selenite-sensitive phenotype of Ws-2 was attenuated by treatment with ethylene precursor or methyl jasmonate (MeJA). Conversely, the selenite resistance of Col-0 was reduced in mutants impaired in ethylene or JA biosynthesis or signaling. Genes encoding sulfur (S) transporters and S assimilation enzymes were up-regulated by selenite in Col-0 but not Ws-2. Accordingly, Col-0 contained higher levels of total S and Se and of nonprotein thiols than Ws-2. Glutathione redox status was reduced by selenite in Ws-2 but not in Col-0. Furthermore, the generation of reactive oxygen species by selenite was higher in Col-0 than in Ws-2. Together, these results indicate that JA and ethylene play important roles in Se resistance in Arabidopsis. Reactive oxygen species may also have a signaling role, and the resistance mechanism appears to involve enhanced S uptake and reduction.