Hyperaccumulation of cadmium by hairy roots of Thlaspi caerulescens

Hairy roots were used to investigate cadmium uptake by Thlaspi caerulescens, a metal hyperaccumulator plant with potential applications in phytoremediation and phytomining. Experiments were carried out in nutrient media under conditions supporting root growth. Accumulation of Cd in short-term (9-h) experiments varied with initial medium pH and increased after treating the roots with H(+)-ATPase inhibitor. The highest equilibrium Cd content measured in T. caerulescens roots was 62,800 microg g(-1) dry weight, or 6.3% dry weight, at a liquid Cd concentration of 3710 ppm. Cd levels in live T. caerulescens roots were 1.5- to 1.7-fold those in hairy roots of nonhyperaccumulator species exposed to the same Cd concentration, but similar to the Cd content of autoclaved T. caerulescens roots. The ability to grow at Cd concentrations of up to 100 ppm clearly distinguished T. caerulescens hairy roots from the nonhyperaccumulators. The specific growth rate of T. caerulescens roots was essentially unaffected by 20 to 50 ppm Cd in the culture medium; in contrast, N. tabacum roots turned dark brown at 20 ppm and growth was negligible. Up to 10,600 microg g(-1) dry weight Cd was accumulated by growing T. caerulescens hairy roots. Measurement of Cd levels in whole roots and in the cell wall fraction revealed significant differences in the responses of T. caerulescens and N. tabacum roots to 20 ppm Cd. Most metal was transported directly into the symplasm of N. tabacum roots within 3 days of exposure; in contrast, T. caerulescens roots stored virtually all of their Cd in the wall fraction for the first 7 to 10 days. This delay in transmembrane uptake may represent an important defensive strategy against Cd poisoning in T. caerulescens, allowing time for activation of intracellular mechanisms for heavy metal detoxification.     

Investigation of heavy metal hyperaccumulation at the cellular level: development and characterization of Thlaspi caerulescens suspension cell lines

The ability of Thlaspi caerulescens, a zinc (Zn)/cadmium (Cd) hyperaccumulator, to accumulate extremely high foliar concentrations of toxic heavy metals requires coordination of uptake, transport, and sequestration to avoid damage to the photosynthetic machinery. The study of these metal hyperaccumulation processes at the cellular level in T. caerulescens has been hampered by the lack of a cellular system that mimics the whole plant, is easily transformable, and competent for longer term studies. Therefore, to better understand the contribution of the cellular physiology and molecular biology to Zn/Cd hyperaccumulation in the intact plant, T. caerulescens suspension cell lines were developed. Differences in cellular metal tolerance and accumulation between the cell lines of T. caerulescens and the related nonhyperaccumulator, Arabidopsis (Arabidopsis thaliana), were examined. A number of Zn/Cd transport-related differences between T. caerulescens and Arabidopsis cell lines were identified that also are seen in the whole plant. T. caerulescens suspension cell lines exhibited: (1) higher growth requirements for Zn; (2) much greater Zn and Cd tolerance; (3) enhanced expression of specific metal transport-related genes; and (4) significant differences in metal fluxes compared with Arabidopsis. One interesting feature exhibited by the T. caerulescens cell lines was that they accumulated less Zn and Cd than the Arabidopsis cell lines, most likely due to a greater metal efflux. This finding suggests that the T. caerulescens suspension cells represent cells of the Zn/Cd transport pathway between the root epidermis and leaf. We also show it is possible to stably transform T. caerulescens suspension cells, which will allow us to alter the expression of candidate hyperaccumulation genes and thus dissect the molecular and physiological processes underlying metal hyperaccumulation in T. caerulescens.     

Cadmium tolerance and antioxidative defenses in hairy roots of the cadmium hyperaccumulator,Thlaspi caerulescens

Plant species capable of hyperaccumulating heavy metals are of considerable interest for phytoremediation and phytomining. This work aims to identify the role of antioxidative metabolism in heavy metal tolerance in the Cd hyperaccumulator, Thlaspi caerulescens. Hairy roots of T. caerulescens and the non-hyperaccumulator, Nicotiana tabacum (tobacco), were used to test the effects of high Cd environments. In the absence of Cd, endogenous activities of catalase were two to three orders of magnitude higher in T. caerulescens than in N. tabacum. T. caerulescens roots also contained significantly higher endogenous superoxide dismutase activity and glutathione concentrations. Exposure to 20 ppm (178 microM) Cd prevented growth of N. tabacum roots and increased hydrogen peroxide (H(2)O(2)) levels by a factor of five relative to cultures without Cd. In contrast, growth was maintained in T. caerulescens, and H(2)O(2) concentrations were controlled to low, nontoxic levels in association with a strong catalase induction response. Treatment of roots with the glutathione synthesis inhibitor, buthionine sulfoximine (BSO), exacerbated H(2)O(2) accumulation in Cd-treated N. tabacum, but had a relatively minor effect on H(2)O(2) levels and did not reduce Cd tolerance in T. caerulescens. Lipid peroxidation was increased by Cd treatment in both the hyperaccumulator and non-hyperaccumulator roots. This work demonstrates that metal-induced oxidative stress occurs in hyperaccumulator tissues even though growth is unaffected by the presence of heavy metals. It also suggests that superior antioxidative defenses, particularly catalase activity, may play an important role in the hyperaccumulator phenotype of T. caerulescens.     

Organic acid complexation,heavy metal distribution and the effect of ATPase inhibition in hairy roots of hyperaccumulator plant species

Heavy metal uptake and distribution were investigated in hairy roots of the Cd hyperaccumulator, Thlaspi caerulescens, and the Ni hyperaccumulator, Alyssum bertolonii. Hairy roots of both species contained high constitutive levels of citric, malic and malonic acids. After treatment with 20 ppm Cd or 25 ppm Ni, about 13% of the total Cd in T. caerulescens roots and 28% of the total Ni in A. bertolonii were associated with organic acids. T. caerulescens and A. bertolonii hairy roots remained healthy and grew well at high concentrations of Cd and Ni, respectively, whereas hairy roots of the non-hyperaccumulator, Nicotiana tabacum, did not. Most of the Cd in T. caerulescens and N. tabacum roots was localised in the cell walls. In contrast, 85-95% of the Ni in A. bertolonii and N. tabacum was associated with the symplasm. Growth of T. caerulescens and A. bertolonii hairy roots was severely reduced in the presence of diethylstilbestrol (DES), an inhibitor of plasma membrane H(+)-ATPase. Treatment with DES increased the concentration of Cd in the symplasm of T. caerulescens about 6-fold with retention of root viability, whereas viability and Ni transport across the plasma membrane were both reduced in A. bertolonii. These results suggest that the mechanisms of Cd tolerance and hyperaccumulation in T. caerulescens hairy roots are capable of withstanding the effects of plasma membrane depolarisation, whereas Ni tolerance and hyperaccumulation in A. bertolonii hairy roots are not.     

Zinc and cadmium uptake by the hyperaccumulator Thlaspi caerulescens in contaminated soils and its effects on the concentration and chemical speciation of metals in soil solution

The hyperaccumulator Thlaspi caerulescens J & C Presl. was grown in seven different soils collected from around Europe that had been contaminated with heavy metals by industrial activity or the disposal of sewage sludge to land. Zinc accumulation factors (shoot concentration/initial soil solution concentration) ranged from 3500–85 000 with a mean value of around 36 000. This compares with mean accumulation factors of 636, 66 and 122 for Cd, Ca and Mg, respectively. The concentration of Zn in the shoots was much greater than in the roots. The total removal of Zn and Cd ranged from 8 to 30 and from 0.02 to 0.5 mg kg^-1 soil, respectively. The Zn concentration in shoots of T. caerulescens correlated, using a curvilinear relationship, with the initial Zn concentration in soil solution (R² = total Zn 0.78; Zn²+ 0.80). There was no relationship between the uptake of Zn and the total Zn concentration in the soil. In most soils, solution pH increased only slightly after growth of T. caerulescens, indicating that acidification was not the mechanism used to mobilise Zn in the soil. Dissolved organic carbon concentrations generally increased but characterisation of the component organic compounds was not attempted. The concentrations of Zn and Cd in soil solution decreased considerably after growth of T. caerulescens. The percentages of Zn and Cd in soil solution present as free ions also decreased. However, the decrease of Zn in soil solution after growth accounted for only about 1% of the total Zn uptake by T. caerulescens. This was much lower than for Cd, Ca and Mg. The results suggest that either T. caerulescens was highly efficient at mobilising Zn which was not soluble initially, or the soils used had large buffering capacities to replenish soil solution Zn within a short time. This work highlights the need to investigate the role of root exudates on the mobilisation of Zn and Cd in soils by the hyperaccumulator T. caerulescens.     

Heavy metal uptake and chemical changes in the rhizosphere of Thlaspi caerulescens and Thlaspi ochroleucum grown in contaminated soils

Thlaspi caerulescens (J. and C. Presl) and Thlaspi ochroleucum (Boiss. ex Heldr) were grown in three different soils containing moderate to high amounts of heavy metals in a pot experiment, using a rhizobag technique. T. caerulescens accumulated significantly more Zn in the shoots than T. ochroleucum. The concentrations of Zn in the shoots of T. caerulescens ranged from 3100 to 8100 mg kg^-1 dry matter (DM), but only from 800 to 1600 mg kg^-1 DM in T. ochroleucum. Total uptake of Zn in the shoots of T. caerulescens was about 5 times that of T. ochroleucum. In contrast, the differences between the two species in the uptake of Cd, Cr, Cu, Ni and Pb were generally small. Concentrations of mobile Zn (extractable with 1M NH₄NO₃) in the rhizosphere and non-rhizosphere soils decreased considerably after growth of both plants, but the decreases were greater with T. caerulescens than with T. ochroleucum. The decreases in the mobile fraction accounted for less than 10% of the total Zn uptake by T. caerulescens indicating that this species was effective in mobilising Zn from less soluble fractions in the soils. The rhizosphere soils tended to have higher concentrations of mobile Zn than the non-rhizosphere soils, probably because of the lower pH in the rhizosphere. The pH in the rhizosphere soils was 0.2-0.4 units lower than that in the non-rhizosphere soils at the end of the experiment. However, there were no significant differences between the two species in the degree of rhizosphere acidification. The results suggest that T. caerulescens has potential for removing Zn from moderately to highly contaminated soils, but that this ability was not related to the pH changes in the rhizosphere.     

Soil microbial diversity as affected by the rhizosphere of the hyperaccumulator Thlaspi caerulescens under natural conditions

It is hypothesized that metal hyperaccumulator plants have specific rhizosphere conditions, potentially modifying the bioavailability of soil metals. This article aims to further the knowledge about the rhizosphere of the hyperaccumulator Thlaspi caerulescens, focusing on its microflora isolated from metalliferous soils collected in situ where the plants grow naturally. We characterized the cultivable microbial communities isolated from the rhizosphere of one population of this Ni hyperaccumulator species grown on a serpentine soil. The rhizosphere soil harbored a wide variety of microorganisms, predominantly bacteria, confirming the stimulatory effect of the T. caerulescens rhizosphere on microbial growth and proliferation. We tested the hypothesis that the rhizosphere of T. caerulescens influences (1) the metabolic diversity of the bacterial community and (2) the bacterial resistance to metals. The principal component analysis of the Biolog plate's data confirmed a structural effect of the rhizosphere of T. caerulescens on bacterial communities. The percentage of Ni-resistant bacteria was higher in the rhizosphere than in the bulk soil, suggesting a direct effect of the rhizosphere on Ni tolerance, reflecting a greater bacterial tolerance to Ni in the rhizosphere.     

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.     

Cadmium accumulation in populations of Thlaspi caerulescens and Thlaspi goesingense

The capacity to accumulate cadmium (Cd) and zinc (Zn) was compared in Thlaspi goesingense and four populations of Thlaspi caerulescens . Two populations of T. caerulescens were grown in hydroponics at five concentrations of Cd. In addition, plants were grown in pots containing compost in which three different concentrations of Cd and two concentrations of Zn were added. A field trial was conducted to compare Zn and Cd uptake by three populations of T. caerulescens on nine selected plots of the Woburn Market Garden Experiment (UK) which had been contaminated to different degrees with heavy metals owing to past applications of sewage sludge. Results show that the four populations of T. caerulescens had the same ability to hyperaccumulate Zn but were significantly different in terms of Cd accumulation. Two populations of T. caerulescens from Southern France accumulated much more Cd than the populations from Prayon (Belgium) and Whitesike (UK). Generally, uptake of Cd was not decreased by increased concentrations of Zn in the substrate. These results indicate that the mechanisms of Cd and Zn hyperaccumulation are not identical in this species. This is the first report of hyperaccumulation of Cd by T. goesingense , but the growth of this species was markedly reduced by the large concentrations of Zn in the substrate. Future work should focus on the differences between Cd and Zn uptake in hyperaccumulator plants at the species and population level.     

Cadmium-induced inhibition of photosynthesis and long-term acclimation to cadmium stress in the hyperaccumulator Thlaspi caerulescens

Acclimation of hyperaccumulators to heavy metal-induced stress is crucial for phytoremediation and was investigated using the hyperaccumulator Thlaspi caerulescens and the nonaccumulators T. fendleri and T. ochroleucum. Spatially and spectrally resolved kinetics of in vivo absorbance and fluorescence were measured with a novel fluorescence kinetic microscope. At the beginning of growth on cadmium (Cd), all species suffered from toxicity, but T. caerulescens subsequently recovered completely. During stress, a few mesophyll cells in T. caerulescens became more inhibited and accumulated more Cd than the majority; this heterogeneity disappeared during acclimation. Chlorophyll fluorescence parameters related to photochemistry were more strongly affected by Cd stress than nonphotochemical parameters, and only photochemistry showed acclimation. Cd acclimation in T. caerulescens shows that part of its Cd tolerance is inducible and involves transient physiological heterogeneity as an emergency defence mechanism. Differential effects of Cd stress on photochemical vs nonphotochemical parameters indicate that Cd inhibits the photosynthetic light reactions more than the Calvin-Benson cycle. Differential spectral distribution of Cd effects on photochemical vs nonphotochemical quenching shows that Cd inhibits at least two different targets in/around photosystem II (PSII). Spectrally homogeneous maximal PSII efficiency (F(v)/F(m)) suggests that in healthy T. caerulescens all chlorophylls fluorescing at room temperature are PSII-associated.     

超富集植物遏蓝菜对重金属吸收、运输和累积的机制

遏蓝菜Thlaspi caerulescens可以在其地上部累积大量重金属如锌、镉等,是公认的超富集植物。由于该植物生物量小,不宜直接用于重金属污染的土壤植物修复,而被广泛作为一种模式植物来进行重金属富集机制研究。遏蓝菜对重金属离子的累积大致经过螯合剂解毒、地上部长距离运输以及在液泡中的储存等生理过程。已经发现的植物体内的金属螯合剂——有机酸、氨基酸、植物络合素(PCs)、金属硫蛋白(MT)和尼克烟酰胺NA等,区室化以及长距离运输相关的转运蛋白——ZIP(ZRT/IRTlike protein)、CDF(Cation diffusion facilitator)、Nramp(Natural resistance and macrophage protein)和HMA(Heavy metal ATPase)等家族,以上各种基因、多肽与蛋白等共同参与了植物对金属累积与耐受过程并发挥各自重要的作用。以下主要介绍了遏蓝菜重金属超富集相关的基因、多肽和蛋白,以及它们在重金属螯合作用和运输过程中的功能。     

Influence of the zinc hyperaccumulator Thlaspi caerulescens J. & C. Presl. and the nonmetal accumulator Trifolium pratense L. on soil microbial populations

Metal hyperaccumulator plants like Thlaspi caerulescens J. & C. Presl. are used for phytoremediation of contaminated soils. Since little is known about the rhizosphere of hyperaccumulators, the influence of T. caerulescens was compared with the effects of Trifolium pratense L. on soil microbes. High- and low-metal soils were collected near a zinc smelter in Palmerton, Penn. Soil pH was adjusted to 5.8 and 6.8 by the addition of Ca(OH)2. Liming increased bacterial populations and decreased metal toxicity to levels allowing growth of both plants. The effects of the plants on total (culturable) bacteria, total fungi, as well as cadmium- and zinc-resistant populations were assessed in nonrhizosphere and rhizosphere soil. Both plants increased microbial populations in rhizosphere soil compared with nonrhizosphere soil. Microbial populations were higher in soils planted with T. pratense, but higher ratios of metal-resistant bacteria were found in the presence of T. caerulescens. We hypothesize that T. caerutescens acidifies its rhizosphere. Soil acidification in the rhizosphere of T. caerulescens would affect metal uptake by increasing available metals around the roots and consequently, increase the selection for metal-resistant bacteria. Soil acidification may be part of the hyperaccumulation process enhancing metal uptake from soil.     

Physiological Characterization of Root Zn2+ Absorption and Translocation to Shoots in Zn Hyperaccumulator and Nonaccumulator Species of Thlaspi

Radiotracer techniques were employed to characterize 65Zn2+ influx into the root symplasm and translocation to the shoot in Thlaspi caerulescens, a Zn hyperaccumulator, and Thlaspi arvense, a nonaccumulator. A protocol was developed that allowed us to quantify unidirectional 65Zn2+ influx across the root-cell plasma membrane (20 min of radioactive uptake followed by 15 min of desorption in a 100 [mu]M ZnCl2 + 5 mM CaCl2 solution). Concentration-dependent Zn2+ influx in both Thlaspi species yielded nonsaturating kinetic curves that could be resolved into linear and saturable components. The linear kinetic component was shown to be cell-wall-bound Zn2+ remaining in the root after desorption, and the saturable component was due to Zn2+ influx across the root-cell plasma membrane. This saturable component followed Michaelis-Menten kinetics, with similar apparent Michaelis constant values for T. caerulescens and T. arvense (8 and 6 [mu]M, respectively). However, the maximum initial velocity for Zn2+ influx in T. caerulescens root cells was 4.5-fold higher than for T. arvense, indicating that enhanced absorption into the root is one of the mechanisms involved in Zn hyperaccumulation. After 96 h 10-fold more 65Zn was translocated to the shoot of T. caerulescens compared with T. arvense. This indicates that transport sites other than entry into the root symplasm are also stimulated in T. caerulescens. We suggest that although increased root Zn2+ influx is a significant component, transport across the plasma membrane and tonoplast of leaf cells must also be critical sites for Zn hyperaccumulation in T. caerulescens.     

Heavy metal tolerance and accumulation in metallicolous and non-metallicolous populations of Thlaspi caerulescens from continental Europe

In continental Europe, the heavy metal hyperaccumulator Thlaspi caerulescens occurs both on heavy-metal polluted soils (subsp. calaminare) and on soils with normal heavy metal content (subsp. caerulescens). In order to assess the extent and partitioning of variation in heavy metal tolerance and foliar mineral composition, twelve families from two populations of each subspecies were grown in pots in four soil treatments differing in heavy metal (Zn, Pb) and macronutrient concentrations. The two subspecies differed systematically in many respects. Subsp. calaminare had a higher survival at high levels of heavy metals and a higher tolerance index in all treatments. It also had three times lower foliar zinc and lead concentrations when grown at moderate levels of heavy metals. This, together with a negative correlation of foliar Pb concentration with growth in subsp. caerulescens, suggests that heavy metal accumulation per se is not a mechanism of tolerance in this species. Variation among families within populations accounted for a larger proportion of total variance in growth and mineral composition than variation between populations. Additionally, within population variation in heavy metal tolerance and accumulation was significantly lower in subsp. calaminare. This suggests that, adding to a background constitutive tolerance at the species level, natural selection has increased heavy metal tolerance in metallicolous populations of Thlaspi caerulescens.     

Life history traits of the pseudometallophyte Thlaspi caerulescens in natural populations from Northern Europe

We examined recruitment, survival, life cycle and fecundity of two metallicolous (M, on metalliferous calamine soils) and two non-metallicolous (NM, on normal soils) populations of Thlaspi caerulescens in Belgium and Luxemburg. In each population, permanent plots were monitored over two reproductive seasons. In M populations, plots were located in two contrasting environments (grass versus grove) in order to test the influence of vegetation cover on life strategy. Our results show that the monocarpic life cycle is dominant in all populations of T. caerulescens. However the length of the pre-reproductive period varies from several months (winter annuals) to 1 year or more (perennials), and is partly related to plant origin (M versus NM). Most plants growing in metalliferous environments were annuals, whereas NM plants were mostly perennials. These differences in life cycle were related to differences in survival during summer, which was better in NM than in M populations. Within each M population, different survival conditions and life cycles were observed according to vegetation cover. Plants growing in grass areas were mostly annuals and had a low survival rate in summer whereas grove plants were mostly perennials and survived better in summer. Our results suggest the selection of stress avoiders (shortening of life cycle) in M populations of T. caerulescens but only for individuals growing in grass areas. Summer survival seems to play a key role in selection of life strategy in T. caerulescens.     

Phytochelatin synthesis is not responsible for Cd tolerance in the Zn/Cd hyperaccumulator Thlaspi caerulescens (J. & C. Presl)

Thlaspi caerulescens (J. & C. Presl, "Prayon") is a heavy-metal hyperaccumulator that accumulates Zn and Cd to high concentrations (40,000 and 4,000 mg kg DW-1 respectively) without phytotoxicity. The mechanism of Cd tolerance has not been characterized but reportedly involves vacuolar sequestration. The role of phytochelatins (PCs) in metal tolerance in T. caerulescens and the related non-accumulator T. arvense was examined. Although PCs were produced by both species in response to Cd, these peptides do not appear to be involved in metal tolerance in the hyperaccumulator. Leaf and root PC levels for both species showed a similar positive correlation with tissue Cd, but total PC levels in the hyperaccumulator were generally lower, despite correspondingly higher metal concentrations. The lack of a role for PCs in the hyperaccumulator's response to metal stress suggests that other mechanisms are responsible Cd tolerance. The lower level of leaf PCs in T. caerulescens also implies that Cd in the shoot is sequestered in a compartment or form that does not elicit a PC response.