Tuanyao Chai

A novel WRKY transcriptional factor from Thlaspi caerulescens negatively regulates the osmotic stress tolerance of transgenic tobacco

A novel member of the WRKY gene family, designated TcWRKY53, was isolated from a cadmium (Cd)-treated Thlaspi caerulescens cDNA library by differential screening. WRKY proteins specifically bind to W-boxes, which are found in the promoters of many genes involved in defense and response to environmental stress. TcWRKY53 contains a 975-bp open reading frame encoding a putative protein of 324 amino acids. Homology searches showed that TcWRKY53 resembles similar WRKY domain-containing proteins from rice, parsley and tobacco, especially AtWRKY53 from Arabidopsis thaliana. Semi-quantitative RT-PCR showed that the expression of TcWRKY53 was strongly induced by various environmental stresses, including an excess of NaCl, drought, cold and the signal molecule salicylic acid (SA). The expression of TcWRKY53 in response to NaCl, drought and cold suggested a possible role of TcWRKY53 in abiotic stress response. However, physiological tests indicated that the expression of TcWRKY53 in tobaccos decreases tolerance to sorbitol during seedling root development. This was consistent with PEG6000 treatment of tobacco seedlings, and together these results indicate a negative modulation of TcWRKY53 in response to osmotic stress. Furthermore, two ethylene responsive factor (ERF) family genes, NtERF5 and NtEREBP-1, were negatively induced in TcWRKY53-overexpressing transgenic plants. In contrast, a LEA family gene, NtLEA5, showed no change, suggesting that TcWRKY53 might regulate the plant osmotic stress response by interacting with an ERF-type transcription factor rather than by regulating function genes directly.

The Thlaspi caerulescens NRAMP homologue TcNRAMP3 is capable of divalent cation transport

The NRAMP gene family encodes integral membrane protein and mediates the transport of Fe, however, its function in transport of toxic metal ions is not very clear in plants. TcNRAMP3 was isolated from Thlaspi caerulescens, and encoded a metal transporter member of the NRAMP family. TcNRAMP3 was predominantly expressed in roots of T. caerulescens by semi-quantitative RT-PCR. The expression of TcNRAMP3 was induced by iron starvation and by the heavy metals Cd and Ni in roots. TcNRAMP3 was able to rescue growth of an iron uptake fet3fet4 mutant yeast strain, suggesting a possible role in iron transport. Expression of TcNRAMP3 in yeast increased Cd sensitivity and Cd content, while it enhanced the Ni resistance and reduced Ni accumulation, indicating that TcNRAMP3 could accumulate Cd and exclude Ni in yeast. Furthermore, overexpression of TcNRAMP3 in tobacco resulted in slight Cd sensitivity of root growth and did not influence Ni resistance. These results suggested that TcNRAMP3 played a role in metal cation homeostasis in plant.

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.

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.

Silicon attenuates cadmium toxicity in Solanum nigrum L. by reducing cadmium uptake and oxidative stress.

Solanum nigrum L. is considered to be a potential plant for restoring Cd-contaminated soils. Si could enhance plants tolerance to heavy metal; however, the mechanism of Si-mediated alleviation of Cd toxicity in S. nigrum was not clear. Three-week-old S. nigrum seedlings were grown in Hoagland solution containing 0 or 100 μM Cd with or without 1 mM Si for 4 days. The results showed that the Cd concentration both in roots and shoots of Si-supplied plant was significantly reduced, especially in expanding and old leaves. The relative proportion of ethanol-extractable Cd, water-extractable Cd and NaCl-extractable Cd in roots was increased by adding Si, while the root-to-shoot Cd translocation was not decreased. Furthermore, in comparison with single Cd treatment, supplying Si could reduce H₂O₂ accumulation and cell death in roots, and the electrolyte leakage and H₂O₂ concentration in functional leaves. Moreover, the activity of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7) and ascorbate peroxidase (APX, EC 1.11.1.11) in functional leaves was markedly increased by Cd exposure, while the antioxidative enzyme activities in Cd plus Si treatment seedlings were significantly lower than that in Cd treatment alone, this decrease might be attributed to the reduction of Cd concentration and Cd-induced oxidative damages. These results demonstrate that Si-enhanced Cd tolerance in S. nigrum is mainly due to the decrease of Cd uptake in roots and Cd distribution in expanding and old leaves, as well as lowering oxidative stress induced by Cd in plants.

Enhancement of Cd tolerance in transgenic tobacco plants overexpressing a Cd-induced catalase cDNA.

Catalase (CAT), an important enzyme of antioxidant system, was investigated the role in preventing the plant from Cd-induced oxidative stress caused by reactive oxygen species. A CAT gene from Brassica juncea was cloned and up-regulated in response to Cd/Zn. The CAT cDNA (BjCAT3) under the control of CaMV35S promoter was introduced into tobacco via Agrobacterium-mediated transformation. Northern blot analysis verified the BjCAT3 was expressed at high level in different transgenic lines. In morphological observation, we found that seedlings from transgenic tobacco plants grew better and showed longer root length in the presence of Cd versus wild-type (WT) seedlings. Under 100 microM Cd stress, WT plants became chlorotic and almost dead while transgenic tobacco plants still remained green and phenotypically normal. The CAT activity of transgenic T(1) generations was approximately two-fold higher than that of WT plants. In WT, endogenous CAT activity is rapidly reduced as a result of 200 microM CdCl2 exposure. Compared with WT plants, lower level of Cd-induced H2O2 accumulation and cell death were detected in roots of transgenic plants with high level of CAT activity. All our findings strongly support that overexpressing BjCAT3 in tobacco could enhance the tolerance under Cd stress.

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 SK2-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 BjDHN3Brassica 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 effects of copper, manganese and zinc on plant growth and elemental accumulation in the manganese-hyperaccumulator Phytolacca americana

Synchrotron radiation X-ray fluorescence (SRXRF) and inductively coupled plasma mass spectrometry were used to estimate major, minor and trace elements in Cu-, Zn- and Mn-treated Phytolacca americana. The effects of the addition of Cu, Zn and Mn on morphological parameters, such as root length, shoot height, and fresh and dry weights of shoots and roots, were also examined. In addition, the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidases (GPX) and catalase (CAT) and the expression of Fe-SOD, Cu/Zn-SOD, metallothionein-2 and glutathione S-transferase (GST) exposed to the highest amounts of Cu, Zn or Mn were detected. Our results confirmed the following: (1) Zn supplementation leads to chlorosis, disturbed elemental homeostasis and decreased concentrations of micro- and macroelements such as Fe, Mg, Mn, Ca and K. Cu competed with Fe, Mn and Zn uptake in plants supplemented with 25 μM Cu. However, no antagonistic interactions took place between Cu, Zn, Mn and Fe uptake in plants supplemented with 100 μM Cu. Mn supplementation at various concentrations had no negative effects on elemental deficits. Mn was co-located with high concentrations of Fe and Zn in mature leaves and the concentrations of macro elements were unchanged. (2) P. americana supplemented with increased concentrations of Zn and Cu exhibited lower biomass production and reduced plant growth. (3) When plants were supplemented with the highest Zn and Cu concentrations, symptoms of toxicity corresponded to decreased SOD or CAT activities and increased APX and GPX activities. However, Mn tolerance corresponded to increased SOD and CAT activities and decreased POD and APX activities. Our study revealed that heavy metals partially exert toxicity by disturbing the nutrient balance and modifying enzyme activities that induce damage in plants. However, P. americana has evolved hyper accumulating mechanisms to maintain elemental balance and redox homeostasis under excess Mn.

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 CdCl2 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.

Characterization of a Novel Plant Promoter Specifically Induced by Heavy Metal and Identification of the Promoter Regions Conferring Heavy Metal Responsiveness

The bean (Phaseolus vulgaris) stress-related gene number 2 (PvSR2) gene responds to heavy metals but not to other forms of environmental stresses. To elucidate its heavy metal-regulatory mechanism at the transcriptional level, we isolated and characterized the promoter region (−1623/+48) of PvSR2. Deletions from the 5′ end revealed that a sequence between −222 and −147 relative to the transcriptional start site was sufficient for heavy metal-specific induction of the promoter region of PvSR2. Detailed analysis of this 76-bp fragment indicated that heavy metal-responsive elements were localized in two regions (−222/−188 and −187/−147), each of which could separately confer heavy metal-responsive expression on the β-glucuronidase gene in the context of a minimal cauliflower mosaic virus 35S promoter. Region I (−222/−188) contains a motif (metal-regulatory element-like sequence) similar to the consensus metal-regulatory element of the animal metallothionein gene, and mutation of this motif eliminated the heavy metal-inducible function of region I. Region II (−187/−147) had no similarity to previously identified cis-acting elements involved in heavy metal induction, suggesting the presence of a novel heavy metal-responsive element. Transformed tobacco (Nicotiana tabacum) seedlings expressing β-glucuronidase under control of the PvSR2 promoter region (−687/+48) showed heavy metal-specific responsive activity that depended on the type and concentration of the heavy metal and the type of organ. These findings further our understanding of the regulation of PvSR2 expression and provide a new heavy-metal-inducible promoter system in transgenic plants.