Somatic hybridization between the zinc accumulator Thlaspi caerulescens and Brassica napus

Somatic hybrids between the zinc hyperaccumulator Thlaspi caerulescens and Brassica napus were produced by electrofusion of protoplasts isolated from each species. Optimization of electrofusion parameters yielded interspecies heteroplasmic fusion rates of up to 13%. Hybrids were selected by screening the growing calli for Zn tolerance. In addition, a second novel selection technique was developed based on the observation that a high proportion of hybrid microcalli grown in liquid media did not adhere to the wall of the culture vessel, while microcalli derived from parental cells did. Seventeen from a total of 64 regenerated plants were conclusively verified as hybrids by AFLP DNA analysis. The hybrid plants were grown in soil for up to 4 months, and at least five flowered. Several of these hybrids survived when grown on high-zinc media.These hybridsaccumulated levels of zinc and cadmium that would have been toxic for B. napus. The data indicate that transfer of the trait for metal hyperaccumulation in plants is possible through somatic hybridization. Received: 1 December 1998 / Accepted: 30 January 1999     

Distribution of Cd, Pb, Zn, Mo, and S in juvenile and mature Brassica napus L. var. napus

The study was conducted at three locations in the Savinjska region of Slovenia, where soil is contaminated with heavy metals due to the zinc industry (Cinkarna Celje). In Ponikva the soil to a depth of 30 cm contains 0.8 mg kg(-1) Cd, 32.2 mg kg(-1) Pb, and 86 mg Zn kg(-1), in Medlog 1.4 mg kg(-1) Cd, 37.4 mg kg(-1) Pb, and 115 mg kg(-1) Zn and in Skofja vas 10.9 mg kg(-1) Cd, 239.7 mg kg(-1) Pb, and 1356 mg kg(-1) Zn. The pH at the selected sites was between 7.3 and 7.6. In the beginning of September 2006 two hybrids of Brassica napus L. var. napus, PR45 D01 and PR46 W31 suitable for production of biodiesel obtained from Pioneer Seeds Holding GmbH, were sown. After 96 days juvenile and after 277 days mature plants were collected. Parts of plants (root, shoot and seed) were separated and Cd, Pb, Zn, Mo, and S determined by ultra-trace ICP-MS. We compared the uptake of Cd, Pb, Zn, Mo and S in different parts of juvenile and mature plants of the two different hybrids, TF (translocation factor), BAF (bioaccumulation factor), and PP (phytoextraction potential) were calculated. The mature hybrid PR46 W31 had higher shoot/root ratio and higher PP for metals (Cd, Pb, and Zn) and lower PP for the micronutrient (Mo) and macronutrient (S) on the polluted site. The study demonstrated the potential use of oilseed rape on multiply polluted soils for production of 1st and 2nd generation biofuels. The potential restoration of degraded land could also disburden the use of agricultural land.     

重金属对油菜种子萌发和胚根生长的影响

分析了Hg2 、Cd2 、Ni2 、Co2 、Zn2 5种重金属离子对油菜种子萌发和胚根伸长的影响,以及金属离子K 、Mg2 和Ca2 与重金属的交互作用。结果表明:(1)重金属对油菜种子萌发的抑制作用依次为Hg2 >Cd2 和Co2 >Ni2 >Zn2 ,而对胚根生长的毒害作用依次为Hg2 >Cd2 >Co2 >Ni2 >Zn2 。(2)萌发率为40%以上时,K 和Ca2 可以提高Ni2 、Zn2 和Co2 胁迫下油菜种子的萌发率,却进一步降低了Hg2 、Cd2 胁迫下种子的萌发;Mg2 可以提高Ni2 、Zn2 、Cd2 和Co2 胁迫下种子的萌发率,但对Hg2 毒害却没有缓解。(3)胚根伸长率达到60%以上时,K 和Mg2 增强了Ni2 、Hg2 、Cd2 和Co2 对胚根生长的抑制,而Ca2 则缓解了Zn2 、Ni2 和Co2 对胚根生长的抑制作用。研究结果对于重金属复合污染土壤中植物种子的萌发和定植具有理论和实践意义。     

Construction of the cybrid transplastomic Brassica napus plants containing Lesquerella fendleri chloroplasts

Transferring of Lesquerella fendleri genetically transformed plastids to Brassica napus plants has been performed with the somatic hybridization method. The plastome of the previously engineered transplastomic L. fendleri plants contained the aadA16gfp selective marker gene conferring spectinomycin/streptomycin resistance and green fluorescence under UV light. The protoplasts of B. napus chlorophyll-deficient plants were fused with gamma-irradiated protoplasts of L. fendleri transplastomic plants. A total of 59 green hybrid colonies have been isolated followed by spectinomycin/streptomycin selection. Shoot regeneration has been observed for two cell lines. Morphologically normal plants have been regenerated for one of them. PCR and isozyme analyses showed that the plants were transplastomic cybrids containing B. napus nuclei and L. fendleri transformed chloroplasts.     

Phytoextraction of zinc, copper, nickel and lead from a contaminated soil by different species of Brassica

In a pot culture experiment, five different species of Brassica (Brassica juncea, Brassica campestris, Brassica carinata, Brassica napus, and Brassica nigra) were grown for screening possible accumulators of heavy metals, viz. Zn, Cu, Ni, and Pb. The plants were grown to maturity in a soil irrigated with sewage effluents for more than two decades in West Delhi, India. The soil analysis showed enhanced accumulation of Zn, Cu, Ni, and Pb in this sewage-irrigated soil. Among all species, B. carinata showed the highest concentration (mg kg(-1)) as well as uptake (microg pot(-1)) of Ni and Pb at maturity. Although B. campestris showed a higher concentration of Zn in its shoots (stem plus leaf), B. carinata extracted the largest amount of this metal due to greater biomass production. However, B. juncea phytoextracted the largest amount of Cu from the soil. In general, the highest concentration and uptake of metal was observed in shoots compared to roots or seeds of the different species. Among the Brassica spp., B. carinata cv. DLSC1 emerged as the most promising, showing greater uptake of Zn, Ni, and Pb, while B. juncea cv. Pusa Bold showed the highest uptake of Cu. The B. napus also showed promise, as it ranked second with respect to total uptake of Pb, Zn, and Ni, and third for Cu. Total uptake of metals by Brassica spp. correlated negatively with available as well as the total soil metal concentrations. Among the root parameters, root length emerged as the powerful parameter to dictate the uptake of metals by Brassica spp. Probably for the first time, B. carinata was reported as a promising phytoextractor for Zn, Ni, and Pb, which performed better than B. juncea.     

In vitro breeding of Brassica juncea L. to enhance metal accumulation and extraction properties

In vitro breeding and somaclonal variation were used as tools to improve the potential of Indian mustard (Brassica juncea L.) to extract and accumulate toxic metals. Calli from B. juncea were cultivated on a modified MS medium supplemented with 10–200 μM Cd or Pb. Afterwards, new B. juncea somaclones were regenerated from metal-tolerant callus cells. Three different phenotypes with improved tolerance of Cd, Zn and Pb were observed under hydroponic conditions: enhanced metal accumulation in both shoots and roots; limited metal translocation from roots to shoots; reduced accumulation in shoots and roots. Seven out of thirty individual variants showed a significantly higher metal extraction than the control plants. The improvement of metal shoot accumulation of the best regenerant (3× Cd, 1.6× Zn, 1.8× Pb) and metal extraction (6.2× Cd, 3.2× Zn, 3.8× Pb) indicated a successful breeding and selection of B. juncea, which could be used for phytoremediation purpose.     

Heavy Metal Tolerance and Accumulation in Indian Mustard (Brassica Juncea L.) Expressing Bacterial γ-Glutamylcysteine Synthetase or Glutathione Synthetase

The overexpression of either γ-glutamylcysteine synthetase (γ-ECS) or glutathione synthetase (GS) in Brassica juncea transgenics was shown previously to result in higher accumulation of glutathione (GSH) and phytochelatins (PCs), as well as enhanced Cd tolerance and accumulation. The present study was aimed at analyzing the effects of γ-ECS or GS overexpression on tolerance to and accumulation of other metal/loids supplied individually in agar medium (seedlings) or in hydroponics (mature plants). Also, as pollution in nature generally consists of mixtures of metals, glutamylcysteine synthetase (ECS) and GS seedlings were tested on combinations of metals. Compared to wild-type plants, ECS and GS transgenics exhibited a significantly higher capacity to tolerate and accumulate a variety of metal/loids (particularly As, Cd, and Cr) as well as mixed-metal combinations (As, Cd, Zn/As, Pb, and Zn). This enhanced metal tolerance and accumulation of the ECS and GS transgenics may be attributable to enhanced production of PCs, sustained by a greater availability of GSH as substrate, as suggested by their higher concentrations of GSH, PC2, PC3, and PC4 as compared to wild-type plants. Overexpression of GS and γ-ECS may represent a promising strategy for the development of plants with an enhanced phytoremediation capacity for mixtures of metals.     

Dietary and water-borne Zn exposures affect energy reserves and subsequent Zn tolerance of Daphnia magna

Enhanced tolerance of aquatic organisms to metal toxicity is one of the important issues of environmental monitoring programs. Determination of dominant uptake route(s) of metals may help to better predict the toxic effects posed by metals. This study aimed to investigate the importance of Zn uptake routes on tolerance and energy reserves of Daphnia magna. Neonates of D. magna were exposed to water-borne zinc, dietary zinc (algae Pseudokichneriella subcapitata loaded with Zn) or to combination of both for 4 days. LC50 (48 h) values of Zn were considerably different from different zinc pre-exposures. Four-day pre-exposure of D. magna neonates produced enhanced tolerance to Zn toxicity. The lowest LC50 values were found in controls (48.2 microM) (no Zn added to their exposure medium and food) and after water-borne Zn pre-exposures (46.2 microM). The level of tolerance increased when dietary Zn was included in pre-exposures, reaching the highest level of LC50 value (70.8 microM) in the highest pre-exposure concentration of diet and water-borne combination experiment. The energy reserves of D. magna also varied significantly under different pre-exposure routes of zinc. In all cases, control animals contained lowest levels of protein, sugar and lipid. Likewise, they represented the lowest energy reserves. Protein levels were highest in the highest dietary Zn exposure, and lowest in the water-borne exposures. Highest and lowest sugar levels were measured in the lowest and highest water-borne Zn exposures, respectively. In contrast, lipid levels were higher in the higher Zn exposure of all exposure routes, the combination exposure resulting in highest lipid levels. The highest total energy reserve was measured in animals that lived in the highest Zn exposure of diet and water-borne combination experiment, mainly due to greater lipid reserves in algae reared in Zn containing media. Results suggest that the dietary exposure route should be considered carefully in natural monitoring studies, and be considered in regulatory assessments of zinc and population dynamics of cladocerans.     

Effects of zinc and influence of Acremonium lolii on growth parameters, chlorophyll a fluorescence and antioxidant enzyme activities of ryegrass (Lolium perenne L. cv Apollo)

The effects of zinc on growth, mineral content, chlorophyll a fluorescence, and detoxifying enzyme activity (ascorbate peroxidase (APX), EC 1.11.1.11; superoxide dismutase (SOD), EC 1.15.1.1) of ryegrass infected or not by Acremonium lolii, and treated with nutrient solution containing 0-50 mM ZnSO(4) were studied. The introduction of zinc induces stress with a decrease in growth at 1, 5 and 10 mM ZnSO(4) and a cessation of growth at 50 mM ZnSO(4), in ryegrass plants infected by A. lolii or not. This decrease in growth may be due to an accumulation of zinc in leaves. Nevertheless, symbiotic plants showed higher values in tiller number, an advantage conferred by the fungus. After 24 d of Zn exposure, leaf fresh weights and leaf water content were lower in plants growing with Zn in the culture medium and no advantage was conferred by the fungus to its host. An increase in Zn supply resulted in a decrease of the Ca, K, Mg, and Cu content of the leaves, a reduction in the quantum yield of electron flow throughout photosystem II (DeltaF/F(1)(m))and a lowering of the efficiency of photosynthetic energy conversion (F(v)/F(m)), compared to control plants. To counter this zinc stress, detoxifying enzymes APX and SOD increased (100%) when Zn reached the value of 50 mM in the nutrient solution. At 10 mM ZnSO(4), the presence of the fungus in the plant led to an increase in the threshold toxicity of plants to zinc by a diminution of APX activity.     

一种快速生长的甘蓝型油菜DH系的获得和鉴定

从快速生长的甘蓝型油菜的小孢子培养中共获得23个再生植株。经倍性鉴定,其中自发加倍成二倍体的有10株,单倍体13株。单倍体再用秋水仙碱处理后获得DH系,所得材料对油菜功能基因组学的研究可能有一定的价值。     

The role of intracellular zinc in chromium(VI)-induced oxidative stress, DNA damage and apoptosis

Several studies have demonstrated that zinc is required for the optimal functioning of the skin. Changes in intracellular zinc concentrations have been associated with both improved protection of skin cells against various noxious factors as well as with increased susceptibility to external stress. Still, little is known about the role of intracellular zinc in hexavalent chromium (Cr(VI))-induced skin injury. To address this question, the effects of zinc deficiency or supplementation on Cr(VI)-induced cytotoxicity, oxidative stress, DNA injury and cell death were investigated in human diploid dermal fibroblasts during 48 h. Zinc levels in fibroblasts were manipulated by pretreatment of cells with 100 microM ZnSO4 and 4 or 25 microM zinc chelator TPEN. Cr(VI) (50, 10 and 1 microM) was found to produce time- and dose-dependent cytotoxicity resulting in oxidative stress, suppression of antioxidant systems and activation of p53-dependent apoptosis which is reported for the first time in this model in relation to environmental Cr(VI). Increased intracellular zinc partially attenuated Cr(VI)-induced cytotoxicity, oxidative stress and apoptosis by enhancing cellular antioxidant systems while inhibiting Cr(VI)-dependent apoptosis by preventing the activation of caspase-3. Decreased intracellular zinc enhanced cytotoxic effects of all the tested Cr(VI) concentrations, leading to rapid loss of cell membrane integrity and nuclear dispersion--hallmarks of necrosis. These new findings suggest that Cr(VI) as a model environmental toxin may damage in deeper regions residing skin fibroblasts whose susceptibility to such toxin depends among others on their intracellular Zn levels. Further investigation of the impact of Zn status on skin cells as well as any other cell populations exposed to Cr(VI) or other heavy metals is warranted.     

The isochorismate pathway is negatively regulated by salicylic acid signaling in O<Subscript>3</Subscript>-exposed <Emphasis Type="Italic">Arabidopsis</Emphasis>

Phytochelatins (PCs) are heavy metal binding peptides that play an important role in sequestration and detoxification of heavy metals in plants. In this study, our goal was to develop transgenic plants with increased tolerance for and accumulation of heavy metals from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A 35S promoter fused to a FLAG–tagged AtPCS1 cDNA was expressed in Indian mustard, and transgenic lines, designated pc lines, were evaluated for tolerance to and accumulation of Cd and Zn. Transgenic plants with moderate AtPCS1 expression levels showed significantly higher tolerance to Cd and Zn stress, but accumulated significantly less Cd and Zn than wild type plants in both shoot and root tissues. However, transgenic plants with highest expression of the transgene did not exhibit enhanced Cd and Zn tolerance. Shoots of Cd-treated pc plants had significantly higher levels of phytochelatins and thiols than wild-type plants. Significantly lower concentrations of gluthatione in Cd-treated shoot and root tissues of transgenic plants were observed. Moderate expression levels of phytochelatin synthase improved the ability of Indian mustard to tolerate certain levels of heavy metals, but at the same time did not increase the accumulation potential for Cd and Zn.     

Production of asymmetric hybrids between Arabidopsis thaliana and Brassica napus utilizing an efficient protoplast culture system

Application of the protoplast culture method developed for Brassica protoplasts to protoplasts of Arabidopsis thaliana has increased the opportunities for interspecific hybridizations involving Arabidopsis. A more-efficient and much-simpler method was established compared to the earlier-reported protocol developed for A. thaliana protoplasts in which alginate beads were utilized. Mesophyll protoplasts of A. thaliana (ecotypes 'Landsberg erecta' and 'Wassilewskija') were cultured in the modified 8p liquid medium, which had been developed for Brassica protoplasts. For comparison, protoplasts were cultured in sodium alginate beads supplied with B5 medium according to the protocol for A. thaliana. The protoplasts divided with high frequencies in the 8p medium, and calli proliferated more rapidly than in the sodium alginate beads. High frequencies of shoot differentiation and regeneration were observed in calli of both ecotypes, from about 30% in the ecotype 'Wassilewskija' to about 60% for 'Landsberg erecta'. The more-rapidly the calli developed, the higher the regeneration frequencies were. Asymmetric hybrids between A. thaliana and Brassica napus were obtained by treating the protoplasts of A. thaliana with iodoacetamide (IOA) and B. napus protoplasts with UV-irradiation before fusion with polyethylene glycol (PEG). By using the culture procedure developed for Brassica protoplasts, calli developed and plants were regenerated. Although most of the plants regenerated after cell fusion were A. thaliana-like and were judged to be escapes from IOA treatment, more than ten plants showed hybrid features of both morphological and molecular characters. Among the hybrids that have flowered so far, both male-fertile and male-sterile plants have been obtained. Back-crossings to A. thaliana are now in progress as is morphological and molecular characterization of the plants.     

Uptake and distribution of zinc,cadmium, lead and copper in Brassica napus var. oleífera and Helianthus annus grown in contaminated soils

Brassica napus var. oleifera and Helianthus annus were grown in artificially contaminated soils. Accumulation and translocation of the environmental pollutants zinc, cadmium, lead, and copper, was evaluated in different portions of the plants at two harvesting times. The distribution into the plants of these metal ions, as well as their capacity for contaminant phytoextraction and accumulation was assessed. For this purpose, an analytical method utilizing focused ultrasound employed for extraction and stripping voltammetry for measurement has been optimized and validated for the simultaneous measurement of Zn, Cd, Pb, and Cu in plant extracts.     

Root endophytes enhance stress‐tolerance of Cicuta virosa L. growing in a mining pond of eastern Japan

Cicuta virosa L. plants can grow in a pond subjected to heavy‐metal inputs at the Hitachi mine, eastern Japan. They accumulate heavy‐metal elements, especially high concentrations of zinc (Zn), in their roots. We focused on the role that root bacterial endophytes play in the heavy‐metal uptake of plants and the provision of heavy‐metal tolerance within plants. Our purpose was to clarify the effects of endophytes on: (i) Zn accumulation in C. virosa roots; (ii) growth of C. virosa seedlings; and (iii) heavy‐metal tolerance of C. virosa plants. Root endophytic Pseudomonas putida and Rhodopseudomonas sp., which induced the high production of Zn‐chelating compounds, were selected for the seedling inoculation test. The results of the inoculation test demonstrated that both strains of endophytes increased Zn accumulation in C. virosa roots by solubilizing Zn in the sediment. Both strains also increased the growth of seedlings by possible production of indole‐3‐acetic acid in the plant. The heavy‐metal tolerance of C. virosa seedlings was likely promoted by producing metal‐chelating compounds that detoxify the metals in the plant tissues, and by decreasing the heavy‐metal contents in the tissues via rapid seedling growth. Thus, such mutualistic interactions between plants and bacteria contribute to the persistence of C. virosa in this severe environment.