Doug Van Hoewyk

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.

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.

A tale of two toxicities: malformed selenoproteins and oxidative stress both contribute to selenium stress in plants.

BACKGROUND Despite seleniums toxicity in plants at higher levels, crops supply most of the essential dietary selenium in humans. In plants, inorganic selenium can be assimilated into selenocysteine, which can replace cysteine in proteins. Selenium toxicity in plants has been attributed to the formation of non-specific selenoproteins. However, this paradigm can be challenged now that there is increasingly abundant evidence suggesting that selenium-induced oxidative stress also contributes to toxicity in plants. SCOPE This Botanical Briefing summarizes the evidence indicating that selenium toxicity in plants is attributable to both the accumulation of non-specific selenoproteins and selenium-induced oxidative stress. Evidence is also presented to substantiate the claim that inadvertent selenocysteine replacement probably impairs or misfolds proteins, which supports the malformed selenoprotein hypothesis. The possible physiological ramifications of selenoproteins and selenium-induced oxidative stress are discussed. CONCLUSIONS Malformed selenoproteins and oxidative stress are two distinct types of stress that drive selenium toxicity in plants and could impact cellular processes in plants that have yet to be thoroughly explored. Although challenging, deciphering whether the extent of selenium toxicity in plants is imparted by selenoproteins or oxidative stress could be helpful in the development of crops with fortified levels of selenium.

Malformed Selenoproteins Are Removed By The Ubiquitin-Proteasome Pathway In Stanleya pinnata

Despite the widely accepted belief that selenium toxicity in plants is manifested by the misincorporation of selenocysteine into selenoproteins, there is a lack of data suggesting that selenoproteins are malformed or misfolded. Plant mechanisms to prevent the formation of selenoproteins are associated with increased selenium tolerance, yet there is no evidence to suggest that selenoproteins are malformed or potentially misfolded. We reasoned that if selenoproteins are malformed, then they might be degraded by the ubiquitin-proteasome pathway. The data demonstrate that selenate treatment induced the accumulation of both oxidized and ubiquitinated proteins, thus implicating both the 20S and 26S proteasome of Stanleya pinnata, a selenium-hyperaccumulating plant, in a selenate response. Inhibition of the proteasome increases the amount of selenium incorporated into protein, but not other elements. Furthermore, a higher percentage of selenium was found in a ubiquitinated protein fraction compared with other elements, suggesting that malformed selenoproteins are preferentially ubiquitinated and removed by the proteasome. Additionally, levels of the 20S and 26S proteasome and two heat shock proteins increase upon selenate treatment. Arabidopsis mutants with defects in the 26S proteasome have decreased selenium tolerance, which further supports the hypothesis that the 26S proteasome probably prevents selenium toxicity by removing selenoproteins.

Selenite activates the alternative oxidase pathway and alters primary metabolism in Brassica napus roots: evidence of a mitochondrial stress response

BackgroundHuman requirements for dietary selenium are met mainly by crops. However, excessive uptake of selenium in plants can restrict growth, and its toxicity has been postulated to target roots. Selenite toxicity can be attributed to its assimilation into selenocysteine, which can replace cysteine to yield malformed selenoproteins. Additionally, selenite has pro-oxidant properties. In this study, the effects of selenite on root tissue in Brassica napus (canola) were investigated to better understand its mode of toxicity and the metabolic adjustments needed to mediate a selenite-response.ResultsSelenite induced the rapid formation of mitochondrial superoxide, which led to decreased aconitase activity and involvement of the alternative oxidase pathway. Although selenite altered primary metabolism, as observed by the increased amino acids and decreased TCA cycle metabolites, increased glucose presumably supported higher respiratory rates and ATP levels reported in this study. Additionally, evidence is presented indicating that selenite suppressed the ubiquitin-proteasome pathway, and induced the pentose phosphate pathway needed to maintain antioxidant metabolism. Selenite treatment also elevated glutathione concentration and coincided with increased levels of γ-glutamyl cyclotransferase, which may possibly degrade selenium metabolites conjugated to glutathione.ConclusionCollectively, the data indicate that selenite necessitates the reconfiguration of metabolic pathways to overcome the consequences of mitochondrial oxidative stress in root tissue. Efforts to mitigate the detrimental effects of selenite-induced oxidative stress may ultimately improve selenium tolerance and accumulation in crops.

The ubiquitin–proteasome pathway protects Chlamydomonas reinhardtii against selenite toxicity, but is impaired as reactive oxygen species accumulate

Plants subjected to stress imposed by their environment often accumulate misfolded proteins, that would be cytotoxic if not properly removed by the ubiquitin-proteasome pathway (UPP). During severe stress, the UPP becomes impaired, but the mechanisms that damage it are not well understood in plants. In this study, the effects of mild and severe stress selenium on the UPP were analyzed using the unicellular green algae Chlamydomonas. Mild selenium stress increased proteasome activity. However, inhibition of the UPP caused by severe selenium stress was associated with reactive oxygen species, including mitochondrial superoxide. Additionally, this is the first time that proteasome activity has been reported in lower plants.

Stuck between a ROS and a hard place: Analysis of the ubiquitin proteasome pathway in selenocysteine treated Brassica napus reveals different toxicities during selenium assimilation.

During the selenium assimilation pathway, inorganic selenate and selenite are reduced to form selenocysteine (Sec). Tolerance to selenium in plants has long been attributable to minimizing the replacement of cysteine with selenocysteine, which can result in nonspecific selenoproteins that are potentially misfolded. Despite this widely accepted assumption, there is no evidence in higher plants demonstrating that selenocysteine induces toxicity by resulting in malformed proteins. In this study, we use Brassica napus to analyze the ubiquitin-proteasome pathway, which is capable of removing misfolded proteins. Sec rapidly increased proteasome activity and levels of ubiquitinated proteins, strongly indicating that selenocysteine induces protein misfolding. Proteasome inhibition increased the amount of selenium in protein in Sec-treated plants. Collectively, these data provide a mechanism that accounts for Sec toxicity. Additionally, Sec did not cause oxidative stress as judged by examining levels of superoxide using fluorescent microscopy. Therefore, the cellular response to Sec is different compared to selenite, which was recently shown to increase antioxidant metabolism in response to elevated mitochondrial superoxide that ultimately impaired proteasome activity. Therefore, plants must contend with two divergent modes of cytotoxicity during selenium assimilation. Selenite can result in oxidative stress, but increased flux of selenite reduction can yield Sec that in turn can cause protein misfolding.

Defects in endoplasmic reticulum-associated degradation (ERAD) increase selenate sensitivity in Arabidopsis

Stress can impair protein folding in the endoplasmic reticulum (ER). Minimizing the accumulation of misfolded proteins in the ER is achieved by ER-associated degradation (ERAD), which involves the retrograde transport and proteasomal removal of aberrant proteins. Recently, the proteasome has been implicated in a selenium stress response. However, it remains unknown if selenium causes ER stress in plants similar to animals, and if ERAD is associated with optimal selenium tolerance. This deficiency was addressed by monitoring selenate-treated Arabidopsis plants with mutations in HRD1 and SeL1L, participants of ERAD. hrd1a/hrd1b and sel1l mutants treated with selenate demonstrate decreased tolerance and ER stress, as judged by BiP2 accumulation. The data indicate that optimal plant growth during selenate stress requires ERAD.ABSTRACT Stress can impair protein folding in the endoplasmic reticulum (ER). Minimizing the accumulation of misfolded proteins in the ER is achieved by ER-associated degradation (ERAD), which involves the retrograde transport and proteasomal removal of aberrant proteins. Recently, the proteasome has been implicated in a selenium stress response. However, it remains unknown if selenium causes ER stress in plants similar to animals, and if ERAD is associated with optimal selenium tolerance. This deficiency was addressed by monitoring selenate-treated Arabidopsis plants with mutations in HRD1 and SeL1L, participants of ERAD. hrd1a/hrd1b and sel1l mutants treated with selenate demonstrate decreased tolerance and ER stress, as judged by BiP2 accumulation. The data indicate that optimal plant growth during selenate stress requires ERAD.

Manipulating Selenium Metabolism in Plants: A Simple Twist of Metabolic Fate Can Alter Selenium Tolerance and Accumulation

Selenium (Se) is a micronutrient for many organisms including humans. But like many trace elements, Se can be toxic at high concentrations and become a public health concern if it accumulates in soils or groundwater. Although higher plants don’t require Se, plants can still accumulate and metabolize Se via the sulfur assimilatory pathway. Genetic manipulation of plant selenium metabolism primarily stems from two areas of interest: it has the potential to improve the phytoremediation of Se in contaminated areas, and it may aid the development of Se-containing phytochemical compounds that possess health benefits. This review highlights studies that have successfully altered Se metabolism in plants, and concludes by focusing on novel genes and pathways that might be targeted to manipulate Se metabolic processes.

Bringing the Dead Compartment of a Plant Cell to Life: A Novel Imaging Technique Resurrects the Dynamic Nature of the Apoplast

In vivo imaging has made significant contributions to plant biology over the past decade. Notable advances in bioimaging have enabled a more comprehensive understanding of many plant-related topics, including photosynthetic electron transport (Ehlert and Hincha, 2008), viral pathogenesis (Tilsner and Oparka, 2010), and the role of reactive oxygen species (Swanson et al., 2011). Now it appears that the benefits of in planta imaging have been extended to the realms of the non-living. Geilfus and Mühling (2011) report in Frontiers in Plant Nutrition a novel non-invasive technique that allows for the direct quantification of ion and pH dynamics in the apoplast. Perhaps most noteworthy, the authors demonstrate the ability to quantify pH fluctuation in different apoplastic compartments in the leaves less than 3 h after roots are subjected to salt stress. The newly developed method reliably measures apoplastic pH both temporally and spatially in a living plant. Briefly, the technique uses a diffusible and fluorescent dye (Oregon Green 488) that is loaded into the apoplast of intact leaves in the common bean (Vicia faba). The pH sensitive, photostable dye is conjugated to a dextran molecule that prevents its movement into the symplast. Fluorescence microscopy is used to estimate apoplastic pH using the fluorescence ratio of F495/F444. Such ratio imaging is possible, because while the fluorescence of the dye at F495 is pH dependent, the concentration of Oregon Green is corrected by the fluorescence at F444, which is not sensitive to H+ concentration. Therefore, the in situ fluorescence ratio of the two wavelengths only provides a direct estimation of apoplastic pH, and is not affected by the dye concentration. The functions of the apoplast in plant physiology and development varies enormously (Sattelmacher, 2001). Similarly, the potential application of this new method could extend far beyond the studies pertaining to crop stress physiology; the newly described technique to measure apoplastic pH both temporally and spatially could be transformative to a variety of disciplines in plant biology. First, the new approach to study changes in apoplastic pH could improve plant nutrient acquisition. For example, although the apoplast provides a continuum for the bulk flow of water and essential plant nutrients, membrane-bound proton pumps can greatly affect the acidity in localized regions of the apoplast. H+ ATPases can therefore create a polarized microenvironment in the apoplast that can (i) drive cations down their electrochemical gradient into the symplast or (ii) facilitate the movement of anions across the cell membrane via co-transporters. Therefore, a better conceptual framework of apoplastic pH dynamics may potentially allow the screening and development of new crop varieties with enhanced nutritional content. Secondly, because accumulation of cations is dependent upon apoplastic pH, advances in the phytoremediation of soils or groundwater containing excessive amounts of Al, Cu, Fe, and Zn may also be possible due to knowledge gleaned by utilizing the new technique developed by Geilfus and Mühling (2011). Lastly, a decrease in apoplastic pH is associated with cell-wall relaxation and cell elongation. In view of the acid growth theory, enhancing our understanding of apoplastic pH dynamics is relevant to plant growth and biomass. In this context, knowledge of apoplastic pH fluctuations could be far-reaching and applied to research in agriculture or cellulosic ethanol. A reliable method to measure apoplastic pH in intact leaves has long been awaited. A review and assessment of techniques to quantify apoplastic pH notes the limitations of previously employed methods (Yu et al., 2000). The new technique by Geilfus and Mühling (2011) overcomes difficulties encountered with previous methods to estimate apoplastic pH, and comes with the added bonus that pH can be discriminated both spatially and temporally. The new protocol presented by Geilfus and Mühling (2011) ushers in the coming of age for the in planta imaging of apoplastic pH dynamics, and perhaps with it a better understanding of the extracellular space in plants.