Enhanced selenium tolerance and accumulation in transgenic Arabidopsis expressing a mouse selenocysteine lyase

Selenium (Se) toxicity is thought to be due to nonspecific incorporation of selenocysteine (Se-Cys) into proteins, replacing Cys. In an attempt to direct Se flow away from incorporation into proteins, a mouse (Mus musculus) Se-Cys lyase (SL) was expressed in the cytosol or chloroplasts of Arabidopsis. This enzyme specifically catalyzes the decomposition of Se-Cys into elemental Se and alanine. The resulting SL transgenics were shown to express the mouse enzyme in the expected intracellular location, and to have SL activities up to 2-fold (cytosolic lines) or 6-fold (chloroplastic lines) higher than wild-type plants. Se incorporation into proteins was reduced 2-fold in both types of SL transgenics, indicating that the approach successfully redirected Se flow in the plant. Both the cytosolic and chloroplastic SL plants showed enhanced shoot Se concentrations, up to 1.5-fold compared with wild type. The cytosolic SL plants showed enhanced tolerance to Se, presumably because of their reduced protein Se levels. Surprisingly, the chloroplastic SL transgenics were less tolerant to Se, indicating that (over) production of elemental Se in the chloroplast is toxic. Expression of SL in the cytosol may be a useful approach for the creation of plants with enhanced Se phytoremediation capacity.     

A family of S-methylmethionine-dependent thiol/selenol methyltransferases. Role in selenium tolerance and evolutionary relation

Several plant species can tolerate high concentrations of selenium in the environment, and they accumulate organoselenium compounds. One of these compounds is Se-methylselenocysteine, synthesized by a number of species from the genus Astragalus (Fabaceae), like A. bisulcatus. An enzyme has been previously isolated from this organism that catalyzes methyl transfer from S-adenosylmethionine to selenocysteine. To elucidate the role of the enzyme in selenium tolerance, the cDNA coding for selenocysteine methyltransferase from A. bisulcatus was cloned and sequenced. Data base searches revealed the existence of several apparent homologs of hitherto unassigned function. The gene for one of them, yagD from Escherichia coli, was cloned, and the protein was overproduced and purified. A functional analysis showed that the YagD protein catalyzes methylation of homocysteine, selenohomocysteine, and selenocysteine with S-adenosylmethionine and S-methylmethionine as methyl group donors. S-Methylmethionine was now shown to be also the physiological methyl group donor for the A. bisulcatus selenocysteine methyltransferase. A model system was set up in E. coli which demonstrated that expression of the plant and, although to a much lesser degree, of the bacterial methyltransferase gene increases selenium tolerance and strongly reduces unspecific selenium incorporation into proteins, provided that S-methylmethionine is present in the medium. It is postulated that the selenocysteine methyltransferase under selective pressure developed from an S-methylmethionine-dependent thiol/selenol methyltransferase.     

SELENIUM: TOXICITY AND TOLERANCE IN HIGHER PLANTS

1. Different plant species show considerable variation in their selenium content. Primary indicators, also termed selenium accumulators, many of which are members of the genus Astragalus, are highly tolerant of selenium; they are known to contain tissue levels of several thousand µg selenium/g. Secondary indicators, tolerant to low concentrations of the element, may absorb up to 1000 µg selenium/g. Non-accumulators are poisoned by selenium. 2. The toxicity of selenate (SeO₄^-) and selenite (SeO₃^-) to most plants can be attributed to a combination of three factors. Firstly, selenate and selenite are readily absorbed from the soil by roots and translocated to other parts of the plant. Secondly, metabolic reactions convert these anions into organic forms of selenium. Thirdly, the organic selenium metabolites, which act as analogues of essential sulphur compounds, interfere with cellular biochemical reactions. 3. Incorporation into proteins of the amino acid analogues selenocysteine and selenomethionine, in place of the equivalent sulphur amino acids, is considered to be the underlying cause of selenium toxicity. The physical and chemical differences between selenium and sulphur will result in small, but significant, changes in the biological properties of a selenium-substituted protein. 4. Selenium-tolerant accumulator plants differ in at least two respects from sensitive species. Large quantities of Se-methylselenocysteine and selenocystathionine, two non-protein selenoamino acids rarely detected in non-accumulators, have been isolated from the tissues of selenium accumulators. In addition, selenium is kept from entering proteins so that the selenium levels in proteins of accumulator plants is significantly lower than the levels in selenium-sensitive plants. 5. Exclusion of selenium from the proteins of accumulators is thought to be the basis of selenium tolerance. Discrimination against selenocysteine during protein synthesis seems to prevent incorporation of this selenoamino acid into proteins of accumulators. Furthermore, synthesis of Se-methylselenocysteine and selenocystathionine, which results in diversion of selenium away from the synthesis of selenomethionine, will restrict the amount of this compound available for protein synthesis. 6. Selenium accumulation among unrelated plant genera is a striking example of convergent evolution. The possibility that accumulation of this element is associated with a nutritional requirement for selenium, although explored in the past, is still in need of further clarification.     

Exploring the selenium phytoremediation potential of transgenic Indian mustard overexpressing ATP sulfurylase or cystathionine-gamma-synthase

Indian mustard (Brassica juncea) plants overexpressing ATP sulfurylase (APS transgenics) were previously shown to have higher shoot selenium (Se) levels and enhanced Se tolerance compared to wild type when supplied with selenate in a hydroponic system. Other transgenic Indian mustard overexpressing cystathionine-gamma-synthase (CGS) showed a higher Se volatilization rate, lower shoot Se levels, and higher Se tolerance than wild type, also in hydroponic studies. In the present study, these APS and CGS transgenics were evaluated for their capacity to accumulate Se from soil that is naturally rich in Se. Wild-type Indian mustard and the Se hyperaccumulator Stanleya pinnata were included for comparison. After 10 weeks on Se soil, the APS transgenics contained 2.5-fold higher shoot Se levels than wild type Indian mustard, similar to those of S. pinnata. The CGS transgenics contained 40% lower shoot Se levels than wild type. Shoot biomass was comparable for all Indian mustard types and higher than that of S. pinnata. These results obtained with these transgenics on soil are in agreement with those obtained earlier using hydroponics. The significance of these findings is that they are the first report on the performance of transgenic plants on Se in soil and show the potential of genetic engineering for phytoremediation.     

Overexpression of selenocysteine methyltransferase in Arabidopsis and Indian mustard increases selenium tolerance and accumulation

A major goal of phytoremediation is to transform fast-growing plants with genes from plant species that hyperaccumulate toxic trace elements. We overexpressed the gene encoding selenocysteine methyltransferase (SMT) from the selenium (Se) hyperaccumulator Astragalus bisulcatus in Arabidopsis and Indian mustard (Brassica juncea). SMT detoxifies selenocysteine by methylating it to methylselenocysteine, a nonprotein amino acid, thereby diminishing the toxic misincorporation of Se into protein. Our Indian mustard transgenic plants accumulated more Se in the form of methylselenocysteine than the wild type. SMT transgenic seedlings tolerated Se, particularly selenite, significantly better than the wild type, producing 3- to 7-fold greater biomass and 3-fold longer root lengths. Moreover, SMT plants had significantly increased Se accumulation and volatilization. This is the first study, to our knowledge, in which a fast-growing plant was genetically engineered to overexpress a gene from a hyperaccumulator in order to increase phytoremediation potential.     

Overexpression of glutathione reductase in Brassica juncea: Effects on cadmium accumulation and tolerance

To determine the importance of glutathione reductase (GR, EC 1.6.4.2) for heavy metal accumulation and tolerance, a bacterial GR was expressed in Indian mustard ( Brassica juncea L.), targeted to the cytosol or the plastids. GR activity in the cytosolic transgenics (cytGR) was about two times higher compared to wild-type plants; in the plastidic transgenics (cpGR) the activity was up to 50 times higher. When treated with 100 μ M CdSO₄, cytGR plants did not differ from wild type in cadmium tolerance or accumulation. CpGR plants, however, showed enhanced cadmium tolerance at the chloroplast level: in contrast to wild-type plants they showed no chlorosis, and their chlorophyll fluorescence parameters F_v/F_m and photochemical quenching were higher. Cadmium tolerance at the whole-plant level (plant growth) was not affected. The lower cadmium stress experienced by the cpGR chloroplasts may be the result of reduced cadmium uptake and/or translocation: cadmium levels in shoots of cpGR plants were half as high as those in wild-type shoots. These differences in cadmium tolerance and accumulation may result from increased root glutathione levels, which were up to two times higher in cpGR plants than in the wild type.     

Utilization of selenocysteine as a source of selenium for selenophosphate biosynthesis

Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate from selenide and ATP. Characterization of selenophosphate synthetase revealed the determined K(m) value for selenide is far above the optimal concentration needed for growth and approached levels which are toxic. Selenocysteine lyase enzymes, which decompose selenocysteine to elemental selenium (Se(0)) and alanine, were considered as candidates for the control of free selenium levels in vivo. The ability of a lyase protein to generate Se(0) in the proximity of SPS maybe an attractive solution to selenium toxicity as well as the high K(m) value for selenide. Recently, three E. coli NifS-like proteins, CsdB, CSD, and IscS, were characterized. All three proteins exhibit lyase activity on L-cysteine and L-selenocysteine and produce sulfane sulfur, S(0), or Se(0) respectively. Each lyase can effectively mobilize Se(0) from L-selenocysteine for selenophosphate biosynthesis.     

Ectopic expression of a cold-inducible transcription factor, CBF1/DREB1b, in transgenic rice (Oryza sativa L.)

The gene encoding C-repeat/dehydration-responsive element binding factor 1 (CBF1/DREB1b) of Arabidopsis was introduced into rice (Oryza sativa L.) under the control of the maize ubiquitin promoter. Its incorporation and expression in transgenic rice plants were confirmed by DNA and RNA gel-blot analyses. Cold tolerance in the transgenics was not significantly different from that of the wild-type plants, as determined by ion leakage, chlorophyll fluorescence, and survival rates. However, the cold-responsive genes lip5, lip9, and OsDhn1 were up-regulated in the transgenic plants, suggesting that the cold signal transduction pathway involving CBF1 is partially conserved in this cold-labile plant.     

Selenium-tolerant diamondback moth disarms hyperaccumulator plant defense

BACKGROUND: Some plants hyperaccumulate the toxic element selenium (Se) to extreme levels, up to 1% of dry weight. The function of this intriguing phenomenon is obscure. RESULTS: Here, we show that the Se in the hyperaccumulator prince's plume (Stanleya pinnata) protects it from caterpillar herbivory because of deterrence and toxicity. In its natural habitat, however, a newly discovered variety of the invasive diamondback moth (Plutella xylostella) has disarmed this elemental defense. It thrives on plants containing highly toxic Se levels and shows no oviposition or feeding deterrence, in contrast to related varieties. Interestingly, a Se-tolerant wasp (Diadegma insulare) was found to parasitize the tolerant moth. The insect's Se tolerance mechanism was revealed by X-ray absorption spectroscopy and liquid chromatography-mass spectroscopy, which showed that the Se-tolerant moth and its parasite both accumulate methylselenocysteine, the same form found in the hyperaccumulator plant, whereas related sensitive moths accumulate selenocysteine. The latter is toxic because of its nonspecific incorporation into proteins. Indeed, the Se-tolerant diamondback moth incorporated less Se into protein. Additionally, the tolerant variety sequestered Se in distinct abdominal areas, potentially involved in detoxification and larval defense to predators. CONCLUSIONS: Although Se hyperaccumulation protects plants from herbivory by some invertebrates, it can give rise to the evolution of unique Se-tolerant herbivores and thus provide a portal for Se into the local ecosystem. In a broader context, this study provides insight into the possible ecological implications of using Se-enriched crops as a source of anti-carcinogenic selenocompounds and for the remediation of Se-polluted environments.     

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.     

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

Background

Despite selenium''s 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.     

Antisense Inhibition of Threonine Synthase Leads to High Methionine Content in Transgenic Potato Plants

Methionine (Met) and threonine (Thr) are members of the aspartate family of amino acids. In plants, their biosynthetic pathways diverge at the level of O-phosphohomo-serine (Ser). The enzymes cystathionine gamma-synthase and Thr synthase (TS) compete for the common substrate O-phosphohomo-Ser with the notable feature that plant TS is activated through S-adenosyl-Met, a metabolite derived from Met. To investigate the regulation of this branch point, we engineered TS antisense potato (Solanum tuberosum cv Désirée) plants using the constitutive cauliflower mosaic virus 35S promoter. In leaf tissues, these transgenics exhibit a reduction of TS activity down to 6% of wild-type levels. Thr levels are reduced to 45% wild-type controls, whereas Met levels increase up to 239-fold depending on the transgenic line and environmental conditions. Increased levels of homo-Ser and homo-cysteine indicate increased carbon allocation into the aspartate pathway. In contrast to findings in Arabidopsis, increased Met content has no detectable effect on mRNA or protein levels or on the enzymatic activity of cystathionine gamma-synthase in potato. Tubers of TS antisense potato plants contain a Met level increased by a factor of 30 and no reduction in Thr. These plants offer a major biotechnological advance toward the development of crop plants with improved nutritional quality.     

Specific roles of alpha- and gamma-tocopherol in abiotic stress responses of transgenic tobacco

Tocopherols are lipophilic antioxidants that are synthesized exclusively in photosynthetic organisms. In most higher plants, alpha- and gamma-tocopherol are predominant with their ratio being under spatial and temporal control. While alpha-tocopherol accumulates predominantly in photosynthetic tissue, seeds are rich in gamma-tocopherol. To date, little is known about the specific roles of alpha- and gamma-tocopherol in different plant tissues. To study the impact of tocopherol composition and content on stress tolerance, transgenic tobacco (Nicotiana tabacum) plants constitutively silenced for homogentisate phytyltransferase (HPT) and gamma-tocopherol methyltransferase (gamma-TMT) activity were created. Silencing of HPT lead to an up to 98% reduction of total tocopherol accumulation compared to wild type. Knockdown of gamma-TMT resulted in an up to 95% reduction of alpha-tocopherol in leaves of the transgenics, which was almost quantitatively compensated for by an increase in gamma-tocopherol. The response of HPT and gamma-TMT transgenics to salt and sorbitol stress and methyl viologen treatments in comparison to wild type was studied. Each stress condition imposes oxidative stress along with additional challenges like perturbing ion homeostasis, desiccation, or disturbing photochemistry, respectively. Decreased total tocopherol content increased the sensitivity of HPT:RNAi transgenics toward all tested stress conditions, whereas gamma-TMT-silenced plants showed an improved performance when challenged with sorbitol or methyl viologen. However, salt tolerance of gamma-TMT transgenics was strongly decreased. Membrane damage in gamma-TMT transgenic plants was reduced after sorbitol and methyl viologen-mediated stress, as evident by less lipid peroxidation and/or electrolyte leakage. Therefore, our results suggest specific roles for alpha- and gamma-tocopherol in vivo.     

A nifS-like gene, csdB, encodes an Escherichia coli counterpart of mammalian selenocysteine lyase. Gene cloning, purification, characterization and preliminary x-ray crystallographic studies.

Selenocysteine lyase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the exclusive decomposition of L-selenocysteine to L-alanine and elemental selenium. An open reading frame, named csdB, from Escherichia coli encodes a putative protein that is similar to selenocysteine lyase of pig liver and cysteine desulfurase (NifS) of Azotobacter vinelandii. In this study, the csdB gene was cloned and expressed in E. coli cells. The gene product was a homodimer with the subunit Mr of 44,439, contained 1 mol of PLP as a cofactor per mol of subunit, and catalyzed the release of Se, SO2, and S from L-selenocysteine, L-cysteine sulfinic acid, and L-cysteine, respectively, to yield L-alanine; the reactivity of the substrates decreased in this order. Although the enzyme was not specific for L-selenocysteine, the high specific activity for L-selenocysteine (5.5 units/mg compared with 0.019 units/mg for L-cysteine) supports the view that the enzyme can be regarded as an E. coli counterpart of mammalian selenocysteine lyase. We crystallized CsdB, the csdB gene product, by the hanging drop vapor diffusion method. The crystals were of suitable quality for x-ray crystallography and belonged to the tetragonal space group P43212 with unit cell dimensions of a = b = 128.1 A and c = 137.0 A. Consideration of the Matthews parameter Vm (3.19 A3/Da) accounts for the presence of a single dimer in the crystallographic asymmetric unit. A native diffraction dataset up to 2.8 A resolution was collected. This is the first crystallographic analysis of a protein of NifS/selenocysteine lyase family.     

Overexpression of cystathionine-γ-synthase enhances selenium volatilization in<Emphasis Type="Italic"> Brassica juncea</Emphasis>

Selenium (Se) can be assimilated and volatilized via the sulfate assimilation pathway. Cystathionine--synthase (CGS) is thought to catalyze the synthesis of Se-cystathionine from Se-cysteine, the first step in the conversion of Se-cysteine to volatile dimethylselenide. Here the hypothesis was tested that CGS is a rate-limiting enzyme for Se volatilization. Cystathionine--synthase from Arabidopsis thaliana (L.) Heynh. was overexpressed in Indian mustard [Brassica juncea (L.) Czern & Coss], and five transgenic CGS lines with up to 10-fold enhanced CGS levels were compared with wild-type Indian mustard with respect to Se volatilization, tolerance and accumulation. The CGS transgenics showed 2- to 3-fold higher Se volatilization rates than wild-type plants when supplied with selenate or selenite. Transgenic CGS plants contained 20–40% lower shoot Se levels and 50–70% lower root Se levels than the wild type when supplied with selenite. Furthermore, CGS seedlings were more tolerant to selenite than the wild type. There were no differences in Se accumulation or tolerance from selenate, in agreement with the earlier finding that selenate-to-selenite reduction is rate-limiting for selenate tolerance and accumulation. In conclusion, CGS appears to be a rate-limiting enzyme for Se volatilization. Overexpression of CGS offers a promising approach for the creation of plants with enhanced capacity to remove Se from contaminated sites in the form of low-toxic volatile dimethylselenide.Abbreviations CGS cystathionine--synthase - DMSe dimethylselenide - SeCys selenocysteine - WT wild type     

A Sweetpotato Geranylgeranyl Pyrophosphate Synthase Gene,IbGGPS, Increases Carotenoid Content and Enhances Osmotic Stress Tolerance in Arabidopsis thaliana

Sweetpotato highly produces carotenoids in storage roots. In this study, a cDNA encoding geranylgeranyl phyrophosphate synthase (GGPS), named IbGGPS, was isolated from sweetpotato storage roots. Green fluorescent protein (GFP) was fused to the C-terminus of IbGGPS to obtain an IbGGPS-GFP fusion protein that was transiently expressed in both epidermal cells of onion and leaves of tobacco. Confocal microscopic analysis determined that the IbGGPS-GFP protein was localized to specific areas of the plasma membrane of onion and chloroplasts in tobacco leaves. The coding region of IbGGPS was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis thaliana to obtain transgenic plants. High performance liquid chromatography (HPLC) analysis showed a significant increase of total carotenoids in transgenic plants. The seeds of transgenic and wild-type plants were germinated on an agar medium supplemented with polyethylene glycol (PEG). Transgenic seedlings grew significantly longer roots than wild-type ones did. Further enzymatic analysis showed an increased activity of superoxide dismutase (SOD) in transgenic seedlings. In addition, the level of malondialdehyde (MDA) was reduced in transgenics. qRT-PCR analysis showed altered expressions of several genes involved in the carotenoid biosynthesis in transgenic plants. These data results indicate that IbGGPS is involved in the biosynthesis of carotenoids in sweetpotato storage roots and likely associated with tolerance to osmotic stress.     

Overexpression of phytoene synthase gene from Salicornia europaea alters response to reactive oxygen species under salt stress in transgenic Arabidopsis

A phytoene synthase gene SePSY was isolated from euhalophyte Salicornia europaea L. The 1655 bp full-length SePSY has an open reading frame of 1257 bp and encodes a 419-amino acid protein. The overexpression of SePSY enhanced the growth of transgenic Arabidopsis. When the plants were exposed to 100 mM NaCl, the photosynthesis rate and photosystem II activity (Fv/Fm) increased from 92% to 132% and from 9.3% to 16.6% in the transgenic lines than in the wild-type, respectively. The transgenics displayed higher activities of SOD and POD and lower contents of H(2)O(2) and MDA than the WT. In conclusion, the transgenic lines showed higher tolerance to salt stress than WT plants by increased photosynthesis efficiency and antioxidative capacity. This is the first report about improving the salt tolerance by genetic manipulation of carotenoid biosynthesis.