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.

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.

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.

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.