Two Poplar Hybrid Clones Differ in Phenolic Antioxidant Levels and Polyphenol Oxidase Activity in Response to High Salt and Boron Irrigation.

Poplar hybrids can be used for selenium (Se) and boron (B) phytoremediation under saline conditions. The phenolic antioxidant stress response of two salt and B tolerant poplar hybrids of parentage Populus trichocarpa × nigra × deltoides was studied using high-performance liquid chromatography (HPLC) and absorption-based assays to determine the antioxidant capacity, total phenolic content, hydroxycinnamic acid levels, and the enzyme activity of l-phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), phenol peroxidase (POD), and laccase. Most remarkable was the contrasting response of the two poplar clones for PPO activity and phenolic levels to irrigation with high salt/B water. To cope with stressful growing conditions, only one clone increased its phenolic antioxidant level, and each clone displayed different PPO isoform patterns. Our study shows that poplar hybrids of the same parentage can differ in their salt/B stress coping mechanism.

Complexation of Hg with phytochelatins is important for plant Hg tolerance.

Three-week-old alfalfa (Medicago sativa), barley (Hordeum vulgare) and maize (Zea mays) were exposed for 7 d to 30 µm of mercury (HgCl(2) ) to characterize the Hg speciation in root, with no symptoms of being poisoned. The largest pool (99%) was associated with the particulate fraction, whereas the soluble fraction (SF) accounted for a minor proportion (<1%). Liquid chromatography coupled with electro-spray/time of flight mass spectrometry showed that Hg was bound to an array of phytochelatins (PCs) in root SF, which was particularly varied in alfalfa (eight ligands and five stoichiometries), a species that also accumulated homophytochelatins. Spatial localization of Hg in alfalfa roots by microprobe synchrotron X-ray fluorescence spectroscopy showed that most of the Hg co-localized with sulphur in the vascular cylinder. Extended X-ray Absorption Fine Structure (EXAFS) fingerprint fitting revealed that Hg was bound in vivo to organic-S compounds, i.e. biomolecules containing cysteine. Albeit a minor proportion of total Hg, Hg-PCs complexes in the SF might be important for tolerance to Hg, as was found with Arabidopsis thaliana mutants cad2-1 (with low glutathione content) and cad1-3 (unable to synthesize PCs) in comparison with wild type plants. Interestingly, high-performance liquid chromatography-electrospray ionization-time of flight analysis showed that none of these mutants accumulated Hg-biothiol complexes.

Study of phytochelatins and other related thiols as complexing biomolecules of As and Cd in wild type and genetically modified Brassica juncea plants.

The accumulation of As and Cd in Brassica juncea plants and the formation of complexes of these elements with bioligands such as glutathione and/or phytochelatins (PCs) is studied. The genetic manipulation of these plants to induce higher As and Cd accumulation has been achieved by overexpressing the genes encoding for gamma-glutamyl cysteine synthetase (gamma-ECS) and glutathione synthetase (GS). These two enzymes are responsible for glutathione (GSH) formation in plants, which is the first step in the production of PCs. The biomass produced in both the wild type and the genetically modified plants, has been evaluated. Additionally, the total Cd and As concentration accumulated in the plant tissues was measured by inductively coupled plasma mass spectrometry (ICP-MS) after extraction. Speciation studies on the extracts were conducted using size exclusion liquid chromatography (SEC) coupled online with ICP-MS to monitor As, Cd and S. For further purification of the As fractions, reversed phase high performance liquid chromatography (RP-HPLC) was used. Structural elucidation of the PCs and other thiols, as well as their complexes with As and Cd, was performed by electrospray-quadrupole-time-of-flight (ESI-Q-TOF). In both the Cd and As exposed plants it was possible to observe the presence of oxidized PC2 ([M + H]+, m/z 538), GS-PC2(-Glu) ([M + H]+, m/z 716) as well as reduced GSH ([M + H]+, m/z 308) and oxidized glutathione (GSSG) ([M + H]+, m/z 613). However, only the GS plants exhibited the presence of As(GS)3 complex ([M + H]+, m/z 994) that was further confirmed by MS/MS. This species is reported for the first time in B. juncea plant tissues.

Field trial of transgenic Indian mustard plants shows enhanced phytoremediation of selenium-contaminated sediment.

Three transgenic Indian mustard [Brassica juncea (L.) Czern.] lines were tested under field conditions for their ability to remove selenium (Se) from Se- and boron-contaminated saline sediment. The transgenic lines overexpressed genes encoding the enzymes adenosine triphosphate sulfurylase (APS), gamma-glutamyl-cysteine synthetase (ECS), and glutathione synthetase (GS), respectively. The APS, ECS, and GS transgenic plants accumulated 4.3, 2.8, and 2.3-fold more Se in their leaves than wild type, respectively (P < 0.05). GS plants significantly tolerated the contaminated soil better than wild type, attaining an aboveground biomass/area almost 80% of that of GS plants grown on clean soil, compared to 50% for wild type plants. This is the first report showing that plants genetically engineered for phytoremediation can perform successfully under field conditions.

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.

Speciation of volatile selenium species in plants using gas chromatography/inductively coupled plasma mass spectrometry.

Gas chromatography/inductively coupled plasma mass spectrometry (GC/ICP-MS) coupled with solid phase micro-extraction can provide a simple, extremely selective and sensitive technique for the analysis of volatile sulfur and selenium compounds in the headspace of growing plants. In this work, the technique was used to evaluate the volatilization of selenium in wild-type and genetically-modified Brassica juncea seedlings. By converting toxic inorganic selenium in the soil to less toxic, volatile organic selenium, B. juncea might be useful in bioremediation of selenium contaminated soil.

Overexpression of cystathionine-gamma-synthase enhances selenium volatilization in Brassica juncea.

Selenium (Se) can be assimilated and volatilized via the sulfate assimilation pathway. Cystathionine-gamma-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-gamma-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.