一株龙葵内生细菌SDE06去除Cd2+的实验

植物内生菌广泛存在于各种植物中, 对宿主的生命活动产生了各种影响。本研究通过对重金属镉(Cd)超累积植物龙葵(Solanum nigrum L.)内生菌优势种进行分离纯化, 并用含Cd2+培养基初步筛选, 得到7株有抗性的菌株, 分别命名为SDE01-07,其中SDE06在Cd2+浓度为80 mg/L的条件下仍能生长。经鉴定此株菌属芽孢杆菌属(Bacillus sp.)。对SDE06在不同条件下去除Cd2+的情况进行研究, 结果表明：正交实验得最佳实验条件为培养时间36 h, pH 6.0, 温度37°C     

Organic Acids Accumulation and Antioxidant Enzyme Activities in Thlaspi caerulescens under Zn and Cd Stress

Growth, organic acid and phytochelatin accumulation, as well as the activity of several antioxidative enzymes, i.e. superoxide dismutase (SOD), ascorbate peroxidase (APX) guaiacol peroxidase (POX) and catalase (CAT) were investigated under Zn and Cd stress in hydroponically growing plants of Thlaspi caerulescens population from Plombières, Belgium. Tissue Zn and Cd concentration increased (the highest concentration of both was in roots) as the concentration of these metals increased in the nutrient solution. Increasing Zn concentration enhanced plant growth, while with Cd it declined compared to the control. Both metals stimulated malate accumulation in shoots, Zn also caused citrate to increase. Zn did not induce phytochelatin (PC) accumulation. In plants exposed to Cd, PC concentration increased with increasing Cd concentration, but decreased with time of exposure. Under Zn stress SOD activity increased, but APX activity was higher at 500 and 1000 μM Zn and CAT activity only at 500 μM Zn in comparison with the control. CAT activity decreased in Cd- and Zn-stressed plants. The results suggest that relative to other populations, a T. caerulescens population from Plombières, when grown in hydroponics, was characterized by low Zn and Cd uptake and their translocation to shoots and tolerance to both metals. The accumulation of malate and citrate, but not PC accumulation was responsible for Zn tolerance. Cd tolerance seems to be due to neither PC production nor accumulation of organic acids.     

Antioxidative defense and proline/phytochelatin accumulation in a newly discovered Cd-hyperaccumulator, Solanum nigrum L.

Changes in the activity of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and the contents of malondialdehyde (MDA), chlorophyll, free proline and phytochelatins (PCs) in Solanum nigrum, the newly discovered Cd-hyperaccumulator were examined and compared with a non-hyperaccumulator Solanum melongena. It was indicated that leaf SOD and POD activity of S. nigrum was significantly higher than that of S. melongena. The Cd treatments significantly increased root SOD activity, leaf POD activity, and CAT activity and free proline content in the leaves and roots of S. nigrum. On the contrary, the Cd treatments decreased SOD activity, and did not change CAT activity in the leaves and roots of S. melongena. Moreover, there were no significant differences in free proline levels in the roots of S. melongena. These results validated that S. nigrum had a greater capacity than S. melongena to adapt to oxidative stress caused by Cd and free proline accumulation might be responsible for the tolerance of S. nigrum to Cd. Treated with 10 μg Cd g⁻¹, growth of S. nigrum and its contents of chlorophyll and MDA were basically unaffected. In contrast, there were a decrease in the growth and chlorophyll content, and an increase in MDA in the roots of S. melongena. Although lipid peroxidation was promoted in both the hyperaccumulator and non-hyperaccumulator by high Cd stress, the greater increase took place in the tissues of S. melongena. The PCs level in roots of S. nigrum was significantly lower than that of S. melongena. On the contrary, the content of leaf PCs was much higher in S. nigrum than that in S. melongena. These results further suggested that antioxidative defense in the Cd-hyperaccumulator might play an important role in Cd tolerance, and PCs synthesis is not the primary reason for Cd tolerance although PCs in S. nigrum increased significantly by Cd.     

Metal accumulation and arbuscular mycorrhizal status in metallicolous and nonmetallicolous populations of <Emphasis Type="Italic">Pteris vittata</Emphasis> L. and <Emphasis Type="Italic">Sedum alfredii</Emphasis> Hance

Although Pteris vittata L. and Sedum alfredii Hance have been identified as an As hyperaccumulator and a Zn/Cd hyperaccumulator, respectively, for a few years, variations in metal accumulation among populations and their arbuscular mycorrhizal (AM) status have not been fully explored. Six populations of P. vittata and four populations of S. alfredii from southeast China were investigated. Up to 1,373 As, 680 Pb, 376 Zn, 4.8 Cd, 169 Cu mg kg⁻¹ in fronds of P. vittata and 358 As, 2,290 Pb, 23,403 Zn, 708 Cd, 342 Cu mg kg⁻¹ in shoots of S. alfredii were detected. Constitutive properties of As and Zn hyperaccumulation in metallicolous populations of P. vittata and S. alfredii, respectively, were confirmed. However, Cd hyperaccumulation in S. alfredii varied among populations. The two hyperaccumulators varied in efficiency in taking up other heavy metals. Different metal tolerance strategies adopted by the two hyperaccumulators varied among plant species and metal species. Low to moderate levels of AM colonization in P. vittata (4.2–12.8%) and S. alfredii (8.5–45.8%) were observed at uncontaminated and metal-contaminated sites. The relationship between metal concentrations and AM colonization in the two hyperacumulators was also examined. The abundance of AM fungal spores ranged from 16 to 190 spores per 25 g soil. Glomus microaggregatum, Glomus mosseae, Glomus brohultii and Glomus geosporum were the most common species associated with both P. vittata and S. alfredii. To our knowledge, this is the first report of AM fungal status in rhizosphere of P. vittata and S. alfredii.     

LC-MS and GC-MS metabolite profiling of nickel(II) complexes in the latex of the nickel-hyperaccumulating tree Sebertia acuminata and identification of methylated aldaric acid as a new nickel(II) ligand

Targeted liquid chromatography-mass spectrometry (LC-MS) technology using size exclusion chromatography and metabolite profiling based on gas chromatography-mass spectrometry (GC-MS) were used to study the nickel-rich latex of the hyperaccumulating tree Sebertia acuminata. More than 120 compounds were detected, 57 of these were subsequently identified. A methylated aldaric acid (2,4,5-trihydroxy-3-methoxy-1,6-hexan-dioic acid) was identified for the first time in biological extracts and its structure was confirmed by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. After citric acid, it appears to be one of the most abundant small organic molecules present in the latex studied. Nickel(II) complexes of stoichiometry NiII:acid=1:2 were detected for these two acids as well as for malic, itaconic, erythronic, galacturonic, tartaric, aconitic and saccharic acids. These results provide further evidence that organic acids may play an important role in the transport and possibly in the storage of metal ions in hyperaccumulating plants.     

Plant regeneration of the arsenic hyperaccumulator <Emphasis Type="Italic">Pteris vittata</Emphasis> L<Emphasis Type="Italic">.</Emphasis> from spores and identification of its tolerance and accumulation of arsenic and copper

An in vitro plant regeneration system was established from the spores of Pteris vittata and identification of its tolerance, and accumulation of gametophytes and callous, to arsenic (As) and copper (Cu) was investigated. The highest frequency (100%) of callus formation was achieved from gametophyte explants treated with 0.5 mg l^?1 6-benzylaminopurine (6-BA) + 0.5 mg l^?1 gibberellin acid (GA). Furthermore, sporophytes were differentiated from the callus tissue derived from gametophyte explants on MS medium supplemented with 0.5 mg l^?1 6-BA, 0.5–1.0 mg l^?1 GA and additional 300 mg l^?1 lactalbumin hydrolysate (LH) for 4 weeks. The optimum combination of ½ MS + 1.0 mg l^?1 GA + 0.5 mg l^?1 6-BA + 300 mg l^?1 LH promoted sporophyte formation on 75 ± 10% of the callus. Every callus derived from gametophyte explants could achieve 3–4 sporophytes. The in vitro growth of gametophyte and callus was accelerated in the medium containing Na₃AsO₄ lower than 0.5 mM, but this growth was inhibited with 2 mM Na₃AsO₄. And with the increase of Na₃AsO₄ in the culture medium from 0 to 2 mM, the As accumulation in gametophytes and callus increased and achieved a level of 763.3 and 315.4 mg kg^?1, respectively. Gametophytes and calluses transplanted to culture medium, supplemented with different concentrations of CuSO₄, are similar to those in Na₃AsO₄, and the Cu accumulation in gametophytes could achieve 7,940 mg kg^?1 when gametophytes were subcultured in medium containing 3 mM CuSO₄. These results suggested that the high efficiency propagation system could be a useful and rapid means to identify other heavy metal tolerance and accumulation. Further, the regeneration ability of callus made it possible for genetic transformation of this fern.     

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.     

Pb hyperaccumulation and tolerance in common buckwheat (Fagopyrum esculentum Moench)

Common buckwheat grown in Pb-contaminated soil was found to accumulate a large amount of Pb in its leaves (8,000 mg/kg DW), stem (2,000 mg/kg DW), and roots (3,300 mg/kg DW), without significant damage. This indicates that buckwheat is a newly recognized Pb hyperaccumulator, which is defined as a plant containing over 1,000 mg/kg of Pb in its shoots on a dry-weight basis. Moreover, it was shown that application of the biodegradable chelator methylglycinediacetic acid trisodium salt at concentrations of up to 20 mmol/kg resulted in a more than five times higher concentration of Pb in the shoot without notable growth inhibitation at up to 10 mmol/kg. These results indicate that buckwheat is a potential phytoremediator of Pb-contaminated soils.     

Diversity and heavy metal tolerance of endophytic fungi from Dysphania ambrosioides,a hyperaccumulator from Pb–Zn contaminated soils

The diversity and heavy metal (HM) tolerance of endophytic fungi (EF) associated with Dysphania ambrosioides, a hyperaccumulator from two Pb–Zn contaminated sites were investigated. A total of 237 culturable EF were isolated and identified to 43 taxa based on morphological characteristics and rDNA internal transcribed spacer analysis, of which 13 occurred as endophytes of both sites, while other taxa were only found in either site. The colonization rate, dominant genera, community structure of EF as well as the HM content in the plant from two sites were significantly different. We suggest that these differences may result from the difference in the soil HM content: lower HM content in the soil, more EF in the plant, which may enhance the plant HM accumulation and thus result higher HM in it. HM tolerance tests indicated that 50% of the isolates exhibited HM tolerance. Among them, two isolates exhibited better HM tolerance, of which FT2G59 could tolerate Pb, Zn, and Cd, and the minimum inhibitory concentration (MIC) of them were 30–50, >?680, 20–30?mmol/l, respectively. While, the isolate FT2G7 could tolerate Cd, and the MIC was 30–50?mmol/l. These isolates may have potential application in phytoremediation.     

Hyperaccumulators, arbuscular mycorrhizal fungi and stress of heavy metals

Use of plants, with hyperaccumulating ability or in association with soil microbes including the symbiotic fungi, arbuscular mycorrhiza (AM), are among the most common biological methods of treating heavy metals in soil. Both hyperaccumulating plants and AM fungi have some unique abilities, which make them suitable to treat heavy metals. Hyperaccumulator plants have some genes, being expressed at the time of heavy metal pollution, and can accordingly localize high concentration of heavy metals to their tissues, without showing the toxicity symptoms. A key solution to the issue of heavy metal pollution may be the proper integration of hyperaccumulator plants and AM fungi. The interactions between the soil microbes and the host plant can also be important for the treatment of soils polluted with heavy metals.