Characterization of the gene family encoding metal tolerance proteins in Triticum urartu: Phylogenetic, transcriptional, and functional analyses.

Homeostasis of microelements in organisms is vital for normal metabolism. In plants, the cation diffusion facilitator (CDF) protein family, also known as metal tolerance proteins (MTPs), play critical roles in maintaining trace metal homeostasis. However, little is known about these proteins in wheat. In this study, we characterized the MTP family of Triticum urartu, the donor of 'A' genome of Triticum aestivum, and analysed their phylogenetic relationships, sequence signatures, spatial expression patterns in the diploid wheat, and their transport activity when heterologously expressed. Nine MTPs were identified in the T. urartu genome database, and were classified and designated based on their sequence similarity to Arabidopsis thaliana (Arabidopsis) and Oryza sativa MTPs. Phylogenetic and sequence analyses indicated that the triticum urartu metal tolerance protein (TuMTP)s comprise three Zn-CDFs, two Fe/Zn-CDFs, and four Mn-CDFs; and can be further classified into six subgroups. Among the TuMTPs, there are no MTP2-5 and MTP9-10 counterparts but two MTP1/8/11 orthologs in relation to AtMTPs. It was also shown that members of the same cluster share similar sequence characteristic, i.e. number of introns, predicted transmembrane domains, and motifs. When expressed in yeast, TuMTP1 and TuMTP1.1 conferred tolerance to Zn and Co but not to other metal ions; while TuMTP8, TuMTP8.1, TuMTP11, and TuMTP11.1 conferred tolerance to Mn. When expressed in Arabidopsis, TuMTP1 localized to the tonoplast and significantly enhanced Zn and Co tolerance. TuMTPs showed diverse tissue-specific expression patterns. Taken together, the closely clustered TuMTPs share structural features and metal specificity but play diverse roles in the homeostasis of microelements in plant cells.

Triticum urartu MTP1: its ability to maintain Zn2+ and Co2+ homeostasis and metal selectivity determinants.

KEY MESSAGE: TuMTP1 maintains Zn2+ and Co2+ homeostasis by sequestering excess Zn2+ and Co2+ into vacuoles. The mutations NSEDD/VTVTT in the His-rich loop and I119F in TMD3 of TuMTP1 restrict metal selectivity. Mineral nutrients, such as zinc (Zn) and cobalt (Co), are essential or beneficial for plants but can be toxic at elevated levels. Metal tolerance proteins (MTPs) are plant members of the cation diffusion facilitator (CDF) transporter family involved in cellular metal homeostasis. However, the determinants of substrate selectivity have not been clarified due to the diversity of MTP1 substrates in various plants. In this study, Triticum urartu MTP1 was characterized. When expressed in yeast, TuMTP1 conferred tolerance to Zn2+ and Co2+ but not Fe2+, Cu2+, Ni2+ or Cd2+ in solid and liquid culture and localized on the vacuolar membrane. Furthermore, TuMTP1-expressing yeast accumulated more Zn2+ and Co2+ when treated. TuMTP1 expression in T. urartu roots was significantly increased under Zn2+ and Co2+ stresses. Determinants of substrate selectivity were then examined through site-directed mutagenesis. The exchange of NSEDD with VTVTT in the His-rich loop of TuMTP1 restricted its metal selectivity to Zn2+, whereas the I119F mutation confined specificity to Co2+. The mutations H74, D78, H268 and D272 (in the Zn2+-binding site) and Leu322 (in the C-terminal Leu-zipper) partially or completely abolished the transport function of TuMTP1. These results show that TuMTP1 might sequester excess cytosolic Zn2+ and Co2+ into yeast vacuoles to maintain Zn2+ and Co2+ homeostasis. The NSEDD/VTVTT and I119F mutations are crucially important for restricting the substrate specificity of TuMTP1, and the Zn2+-binding site and Leu322 are essential for its ion selectivity and transport function. These results can be employed to change metal selectivity for biofortification or phytoremediation applications.

Molecular cloning and characterization of a Brassica juncea yellow stripe-like gene, BjYSL7, whose overexpression increases heavy metal tolerance of tobacco.

KEY MESSAGE: BjYSL7 encodes a plasma-localized metal-NA transporter and has transport Fe(II)-NA complexes activity. BjYSL7 is involved in the transport of Cd and Ni from roots to shoots. Heavy metal transporters play a key role in regulating metal accumulation and transport in plants. In this study, we isolated a novel member of the yellow stripe-like (YSL) gene family BjYSL7 from the hyperaccumulator Brassica juncea. BjYSL7 is composed of 688 amino acids with 12 putative transmembrane domains and is over 90 % identical to TcYSL7 and AtYSL7. Real-time PCR analysis revealed that BjYSL7 mRNA was mainly expressed in the stem under normal condition. The expression of BjYSL7 was found to be up-regulated by 127.1-, 12.7-, and 3.4-fold in roots and 6.5-, 4.3-, and 2.8-fold in shoots under FeSO4, NiCl2, and CdCl2 stresses, respectively. We have demonstrated that BjYSL7 is a Fe(II)-NA influx transporter by yeast functional complementation. Moreover, a BjYSL7::enhanced green fluorescent protein (EGFP) fusion localized to the plasma membrane of onion epidermal cells. The BjYSL7-overexpressing transgenic tobacco plants exhibited longer root lengths, lower relative inhibition rate of lengths and superior root hair development compared to that of wild-type (WT) plants in the presence of CdCl2 and NiCl2. Furthermore, the concentrations of Cd and Ni in shoots of BjYSL7-overexpressing plants are significantly higher than that of WT plants. Compared with WT plants, BjYSL7-overexpressing plants exhibited Fe concentrations that were higher in the shoots and seeds and lower in the roots. Taken together, these results suggest that BjYSL7 might be involved in the transport of Fe, Cd and Ni to the shoot and improving heavy metal resistance in plants.

[Mechanism of heavy-metal tolerance in Pseudomonas aeruginosa ZGKD2].

The cadmium-resistant bacterium Pseudomonas aeruginosa strain ZGKD2 exhibiting tolerance to various heavy-metals was isolated from gangue pile of coal area in our laboratory. This bacterium could serve as an effective metal sequestering and growth-promoting bioinoculant for plants grown in metal-contaminated soil. However, the mechanism of heavy-metal tolerance is still unclear. When the beef extract-peptone medium was supplemented with 200-3 000 micromol x L(-1) Cd2+, Cu2+, Zn2+, Ni2+, Pb2+ or Mn2+, the maximum biomass of strain ZGKD2 decreased with the increase of heavy-metal concentrations, while different concentrations of heavy-metals had no significant effect on its alkaline production. Stationary-phase cells of strain ZGKD2 were exposed to 0, 200, 600 and 1 000 micromol x L(-1) of Cd2+ , Cu2+, Zn2+ and Ni2+ or 0, 1 000, 2 000 and 3 000 micromol x L(-1) of Pb2+ and Mn2+ for 2 h, respectively. The activity of SOD and CAT increased in a heavy-metal-concentration-dependent manner, especially in the Cd2+ and Cu2+ treatments. The siderophore production of strain ZGKD2 in modified sugar-aspartic acid medium was enhanced by 200- 1 000 micromol x L(-1) of various heavy-metals. Cd2+ and Zn2+ strongly induced the siderophore production of strain ZGKD2, Ni2+ and Mn2+ had little effect, whereas Cu2+ led to significant inhibition. The siderophore production of strain ZGKD2 was positively related with its metal tolerance. These results indicated that alkaline production, siderophore production, and the increase of antioxidant enzyme activities in strain ZGKD2 might be the main mechanisms of heavy-metal tolerance.

Cd-induced changes in leaf proteome of the hyperaccumulator plant Phytolacca americana.

Cadmium (Cd) is highly toxic to all organisms. Soil contamination by Cd has become an increasing problem worldwide due to the intensive use of Cd-containing phosphate fertilizers and industrial zinc mining. Phytolacca americana L. is a Cd hyperaccumulator plant that can grow in Cd-polluted areas. However, the molecular basis for its remarkable Cd resistance is not known. In this study, the effects of Cd exposure on protein expression patterns in P.americana was investigated by 2-dimensional gel electrophoresis (2-DE). 2-DE profiles of leaf proteins from both control and Cd-treated (400µM, 48h) seedlings were compared quantitatively using ImageMaster software. In total, 32 differentially expressed protein spots were identified using MALDI-TOF/TOF mass spectrometry coupled to protein database search, corresponding to 25 unique gene products. Of those 14 were enhanced/induced while 11 reduced under Cd treatment. The alteration pattern of protein expression was verified for several key proteins involved in distinct metabolic pathways by immuno-blot analysis. Major changes were found for the proteins involved in photosynthetic pathways as well as in the sulfur- and GSH-related metabolisms. One-third of the up-regulated proteins were attributed to transcription, translation and molecular chaperones including a protein belonging to the calreticulin family. Other proteins include antioxidative enzymes such as 2-cys-peroxidase and oxidoreductases. The results of this proteomic analysis provide the first and primary information regarding the molecular basis of Cd hypertolerance in P. americana.

Phytochelatin synthase of Thlaspi caerulescens enhanced tolerance and accumulation of heavy metals when expressed in yeast and tobacco.

Phytochelatin synthase (PCS) is key enzyme for heavy metal detoxification and accumulation in plant. In this study, we isolated the PCS gene TcPCS1 from the hyperaccumulator Thlaspi caerulescens. Overexpression of TcPCS1 enhanced PC production in tobacco. Cd accumulation in the roots and shoots of TcPCS1 transgenic seedlings was increased compared to the wild type (WT), while Cd translocation from roots to shoots was not affected under Cd treatment. The root length of the TcPCS1 transgenic tobacco seedlings was significantly longer than that of the WT under Cd stress. These data indicate that TcPCS1 expression might increase Cd accumulation and tolerance in transgenic tobacco. In addition, the malondialdehyde content in TcPCS1 plants was below that of the wild type. However, the antioxidant enzyme activities of superoxide dismutase, peroxidase and catalase were found to be significantly higher than those of the WT when the transgenic plant was exposed to Cd stress. This suggests that the increase in PC production might enhance the Cd accumulation and thus increase the oxidative stress induced by the cadmium. The production of PCs could cause a transient decrease in the cytosolic glutathione (GSH) pool, and Cd and lower GSH concentration caused an increase in the oxidative response. We also determined TcPCS1 in Thlaspi caerulescens was regulated after exposure to various concentrations of CdCl(2) over different treatment times. Expression of TcPCS1 leading to increased Cd accumulation and enhanced metal tolerance, but the Cd contents were restrained by adding zinc in Saccharomyces cerevisiae transformants.

[Antioxidative response of Phytolacca americana and Nicotiana tabacum to manganese stresses].

Plant species capable of accumulating heavy metals are of considerable interest for phytoremediation and phytomining. The mechanism of Mn tolerance/hyperaccumulate in Phytolacca americana L. is less known. To elucidate the role of antioxidative enzyme in response to Mn, the 6-week-old seedling of Mn hyperaccumulator P. americana and non-accumulator-tobacco (Nicotiana tabacum) were exposed to half strength Hoagland solution with 1 mmol x L(-1) or 3 mmol x L(-1) MnCl2 for 4 days. The photosynthetic rate in P. americana decreased more slowly than that in tobacco, while the MDA content and electrolyte leakage in tobacco increased more rapidly than that in P. americana. For example, after exposure to 1 mmol x L(-1) Mn for 4 days, the photosynthetic rates of P. americana and tobacco in comparison to the control reduced by 13.3% and 75.5%, respectively. The MDA content and electrolyte leakage in tobacco increased by 347.3% and 120.1%, respectively, whereas Mn had no marked effect on both of it in P. americana, indicated that the oxidative damage in tobacco was more serious than that in P. americana. The activities of SOD and POD of both species increased rapidly with elevated Mn concentration and exposure time in both species, the increase of SOD activity in P. americana was higher than that in tobacco. CAT activity in tobacco declined rapidly, while the activity of CAT in P. americana was increased. The activities of SOD, POD and CAT in P. americana upon 1 mmol x L(-1) Mn exposure increased by 161.1%, 111.3% and 17.5%, respectively. The activities of SOD and POD in tobacco increased by 55.5% and 206.0%, respectively, while CAT activity decreased by 15.6%, indicating that the antioxidative enzymes in P. americana, particularly in CAT,could fully scavenge the reactive oxygen species generated by Mn toxicity. These results collectively indicate that the enzymatic antioxidation capacity is one of the important mechanisms responsible for Mn tolerance in hyperaccumulator plant species.

Characterization of the novel gene BjDREB1B encoding a DRE-binding transcription factor from Brassica juncea L.

A novel DREB (dehydration responsive element binding protein) gene, designated BjDREB1B, was isolated from Brassica juncea L. BjDREB1B contains a conserved EREBP/AP2 domain and was classified into the A-1 subgroup of the DREB subfamily based on phylogenetic tree analysis. RT-PCR showed that BjDREB1B was induced by abiotic stresses and exogenous phytohormones, such as drought, salt, low temperature, heavy metals, abscisic acid, and salicylic acid. Gel shift assay revealed that BjDREB1B specifically bound to the DRE element in vitro. Yeast one-hybrid assay showed that full-length BjDREB1B or its C-terminal region functioned effectively as a trans-activator. Furthermore, overexpression of BjDREB1B in tobacco up-regulated the expression of NtERD10B, and BjDREB1B transgenic plants accumulated higher levels of proline than control plants under normal and saline conditions, together showing that BjDREB1B plays important roles in improving plant tolerance to drought and salinity.

Gene manipulation of a heavy metal hyperaccumulator species Thlaspi caerulescens L. via Agrobacterium-mediated transformation.

Thlaspi caerulescens L. is well known as a Zn/Cd hyperaccumulator. The genetic manipulation of T. caerulescens through transgenic technology can modify plant features for use in phytoremediation. Here, we describe the efficient transformation of T. caerulescens using Agrobacterium tumefaciens strain EHA105 harboring a binary vector pBI121 with the nptII gene as a selectable marker, the gus gene as a reporter and a foreign catalase gene. Based on the optimal concentration of growth regulators, the shoot cluster regeneration system via callus phase provided the basis of the genetic transformation in T. caerulescens. The key variables in transformation were examined, such as co-cultivation period and bacterial suspension density. Optimizing factors for T-DNA delivery resulted in kanamycin-resistant transgenic shoots with transformation efficiency more than 20%, proven by histochemical GUS assay and PCR analysis. Southern analysis of nptII and RT-PCR of catalase gene demonstrated that the foreign genes were integrated in the genome of transformed plantlets. Moreover, the activity of catalase enzyme in transgenic plants was obviously higher than in wild-type plants. This method offers new prospects for the genetic engineering of this important hyperaccumulator species.

BjDHNs confer heavy-metal tolerance in plants.

Dehydrin gene transcript could be induced by heavy metals, and some dehydrins possess the ability to bind metals. However, the correlation between dehydrins and heavy-metal stress is unknown. In order to elucidate the contribution of dehydrins to heavy-metal stress tolerance in plants, we cloned two SK(2)-type dehydrin genes from heavy-metal hyperaccumulator Brassica juncea, and investigated their Cd/Zn tolerance in transgenic plants. Semi-quantitative RT-PCR analysis revealed that BjDHN2/BjDHN3 expressed in the leaves, stems and roots at a low level and were up-regulated by heavy metals. Antisense BjDHN3 Brassica juncea plants showed more electrolyte leakage and higher malondialdehyde production than the control plants when exposed to heavy metals, and the total amount of metals accumulated in the whole plant was reduced. Transgenic tobacco plants overexpressing BjDHN2/BjDHN3 showed lower electrolyte leakage and malondialdehyde production than the control plants when exposed to Cd/Zn. These results indicated that BjDHN2/BjDHN3 enhanced the tolerance for heavy metals by reducing lipid peroxidation and maintaining membrane stability in the plants.

[Research advances in drought resistance and heavy metals tolerance of transgenic plant].

Drought and heavy metals contamination greatly affect plant growth, while the cloning and function analysis of stress-inducible genes provide an effective approach to improve the stress tolerance and yield of crops by genetic engineering. The expression of LEA (late embryogenesis abundant protein) gene could be induced by various stresses such as drought, high salinity, cold, and heavy metals. The study on transgenic plants showed that LEA could increase plant tolerance to water stress, had ion-binding activity, and acted as an antioxidant under abiotic stresses. Aquaporins largely presented in plasma membrane and vacuolar membrane, and played a key role in root water uptake and transportation both at cellular and at whole plant level. The expression of aquaporins was up-regulated in response to drought and salinity, and conferred the water stress tolerance in plant. Cation-efflux transport proteins were involved in the absorption, transportation, and accumulation of heavy metals in plants. All of the proteins mentioned above could have potential applied profits on improving the biological water-saving, drought resistance, and heavy metals tolerance of lawny grass.

The bean PvSR2 gene produces two transcripts by alternative promoter usage.

The bean (Phaseolus vulgaris) stress-related gene number 2 (PvSR2) is heavy metal-inducible. Here, the intron of PvSR2 (I-PvSR) within the coding sequence was isolated and characterized. I-PvSR exhibited a weak and constitutive promoter activity and enhanced the PvSR2 promoter activity in transiently transformed tobacco protoplasts. The transcription start site of I-PvSR promoter was mapped 72 bp upstream of the 3'-splice site. The shorter PvSR2 transcript (768nt) in bean is generated from this intronic promoter and lacks the last 56 bases of 3'-end sequence of longer PvSR2 transcript (829nt) by utilizing an alternative polyadenylation site. Quantitative competitive PCR analysis further revealed that two transcripts were differently accumulated in response to Hg(2+)-exposure and the longer transcript was more abundant than the shorter one. These results demonstrate an additional non-metal inducible transcription of PvSR2 via alternative intronic promoter usage and provide new insights into expression mechanism of metal inducible gene.

[A new method for EMSA by modifying DIG high prime DNA labeling and detection starter Kit II].

Gel retardation, also named electrophoretic mobility shift assay (EMSA), is a useful tool for identifying protein-DNA interactions. Typically, 32P-labeled DNA probes used in EMSA are sensitive. However, it relies on the handling of hazardous radioisotopes, and is not easily quantified. Recently, some successful cases have been reported using non-radio labelled probes instead of radiolabelled probes in EMSA. The method is rapid, convenient, and safe, but it depends on a very expensive kit. In this study, we offered a new method performing EMSA by modifying DIG High Prime DNA Labeling and Detection Starter Kit II (Rohe). Firstly, the prepared labeled probe was introduced the EcoR I stick in the end of probe for 3'-end labeling, and then was performed the probe labeling and detecting the signals of EMSA with the relatively cheap DIG High Prime DNA Labeling and Detection Starter Kit II Rohe. By adjusting the experiment parameters, the successful result was obtained. The present study provides a successful example and method for modifying DIG High Prime DNA Labeling and Detection Starter Kit II.

[Advances in the research of genetic engineering of heavy metal resistance and accumulation in plants].

Using plants to remove or inactivate heavy metal pollutants from soils and surface waters provide a cheap and sustainable approach of Phytoremediation. However, field trials suggested that the efficiency of contaminant removal using natural hyperaccumulators is insufficient, due to that many of these species are slow growing and produce little shoot biomass. These factors severely constrain their potential for large-scale decontamination of polluted soils. Moreover, both the micronutrient and toxic metal content accumulated in crops determine the quality and safety of our food-chain. By a transgenic approach, the introduction of novel genes responsible for hyperaccumulating phenotype into high biomass plants and/or stable crops uptaking minerals as food is a promising strategy for the development of effective techniques of phytoremediation and improvement of nutritional value of stable food through a viable commercialization. Recently, the progress at molecular level for heavy metal uptaking, detoxification and hyperaccumulation in plants, and also the clarification of some functional genes in bacteria, yeasts, plants and animals, have advanced the research on genetic engineering plants of heavy metal resistance and accumulation, and on the functional genes (e . g. gsh1, MerA and ArsC) and their genetic transformated plants. These studies demonstrated commercialization potentials of phytoremediation. In this paper, the molecular approach, effects and problems in gene transformation were discussed in details, and also the strategy and emphases were probed into the future research.