QTL analysis of cadmium and zinc accumulation in the heavy metal hyperaccumulator Thlaspi caerulescens

Thlaspi caerulescens (Tc; 2n = 14) is a natural Zn, Cd and Ni hyperaccumulator species belonging to the Brassicaceae family. It shares 88% DNA identity in the coding regions with Arabidopsis thaliana (At) (Rigola et al. 2006). Although the physiology of heavy metal (hyper)accumulation has been intensively studied, the molecular genetics are still largely unexplored. We address this topic by constructing a genetic map based on AFLP markers and expressed sequence tags (ESTs). To establish a genetic map, an F(2) population of 129 individuals was generated from a cross between a plant from a Pb/Cd/Zn-contaminated site near La Calamine, Belgium, and a plant from a comparable site near Ganges (GA), France. These two accessions show different degrees of Zn and, particularly, Cd accumulation. We analyzed 181 AFLP markers (of which 4 co-dominant) and 13 co-dominant EST sequences-based markers and mapped them to seven linkage groups (LGs), presumably corresponding to the seven chromosomes of T. caerulescens. The total length of the genetic map is 496 cM with an average density of one marker every 2.5 cM. This map was used for Quantitative Trait Locus (QTL) mapping in the F(2). For Zn as well as Cd concentration in root we mapped two QTLs. Three QTLs and one QTL were mapped for Zn and Cd concentration in shoot, respectively. These QTLs explain 23.8-60.4% of the total variance of the traits measured. We found only one common locus (LG6) for Zn and Cd (concentration in root) and one common locus for shoot and root concentrations of Zn (LG1) and of Cd (LG3). For all QTLs, the GA allele increased the trait value except for two QTLs for Zn accumulation in shoot (LG1 and LG4) and one for Zn concentration in root (LG1).     

Assessing the potential for zinc and cadmium phytoremediation with the hyperaccumulator Thlaspi caerulescens

Thlaspi caerulescens is a Zn and Cd hyperaccumulator, and has been tested for its phytoremediation potential. In this paper we examine the relationships between the concentrations of Zn and Cd in soil and in T. caerulescens shoots, and calculate the rates of Zn and Cd extraction from soil. Using published data from field surveys, field and pot experiments, we show that the concentrations of Zn and Cd in the shoots correlate with the concentrations of Zn and Cd in soils in a log-linear fashion over three orders of magnitude. There is little systematic difference between different populations of T. caerulescens in the relationship between soil and plant Zn concentrations. In contrast, populations from southern France are far superior to those from other regions in Cd accumulation. Bioaccumulation factors (plant to soil concentration ratio) for Zn and Cd decrease log-linearly with soil metal concentration. Model calculations show that phytoremediation using T. caerulescens is feasible when soil is only moderately contaminated with Zn and Cd, and the phytoremediation potential is better for Cd than for Zn if the populations from southern France are used. Recent progress in the understanding of the mechanisms of Zn and Cd uptake by T. caerulescens is also reviewed.     

Influence of iron status on cadmium and zinc uptake by different ecotypes of the hyperaccumulator Thlaspi caerulescens

We have previously identified an ecotype of the hyperaccumulator Thlaspi caerulescens (Ganges), which is far superior to other ecotypes (including Prayon) in Cd uptake. In this study, we investigated the effect of Fe status on the uptake of Cd and Zn in the Ganges and Prayon ecotypes, and the kinetics of Cd and Zn influx using radioisotopes. Furthermore, the T. caerulescens ZIP (Zn-regulated transporter/Fe-regulated transporter-like protein) genes TcZNT1-G and TcIRT1-G were cloned from the Ganges ecotype and their expression under Fe-sufficient and -deficient conditions was analyzed. Both short- and long-term studies revealed that Cd uptake was significantly enhanced by Fe deficiency only in the Ganges ecotype. The concentration-dependent kinetics of Cd influx showed that the V(max) of Cd was 3 times greater in Fe-deficient Ganges plants compared with Fe-sufficient plants. In Prayon, Fe deficiency did not induce a significant increase in V(max) for Cd. Zn uptake was not influenced by the Fe status of the plants in either of the ecotypes. These results are in agreement with the gene expression study. The abundance of ZNT1-G mRNA was similar between the Fe treatments and between the two ecotypes. In contrast, abundance of the TcIRT1-G mRNA was greatly increased only in Ganges root tissue under Fe-deficient conditions. The present results indicate that the stimulatory effect of Fe deficiency on Cd uptake in Ganges may be related to an up-regulation in the expression of genes encoding for Fe(2+) uptake, possibly TcIRT1-G.     

Uptake and transport of zinc in the hyperaccumulator Thlaspi caerulescens and the non-hyperaccumulator Thlaspi ochroleucum

Growth and zinc uptake of the hyperaccumulator species Thlaspi caerulescens J. & C. Presl and the non-hyperaccumulator species Thlaspi ochroleucum Boiss. & Heldr. were compared in solution culture experiments. T. caerulescens was able to tolerate 500 mmol m^?3 (32.5 g m^?3) Zn in solution without growth reduction, and up to 1000 mmol m^?3 (65 g m^?3) Zn without showing visible toxic symptoms but with a 25% decrease in dry matter (DM) yield. Up to 28 g kg^?1 of Zn in shoot DM was obtained in healthy plants of T. caerulescens. In contrast, T. ochroleucum suffered severe phytotoxicity at 500 mmol m^?3 Zn. Marked differences were shown in Zn uptake, distribution and redistribution between the two species. T. caerulescens had much higher concentrations of Zn in the shoots, whereas T. ochroleucum accumulated higher concentrations of Zn in the roots. When an external supply of 500 mmol m^?3 Zn was withheld, 89% of the Zn accumulated previously in the roots of T. caerulescens was transported to the shoots over a 33 d period, whereas in T. ochroleucum only 32% was transported. T. caerulescens was shown to have a greater internal requirement for Zn than other plants. Increasing the supply of Zn from 1 to 10 mmol m^?3 gave a 19% increase in the total DM of this species. liven the shoots from the 1 mmol m^?3 Zn treatment which showed Zn deficiency contained 10 times greater Zn concentrations than the widely reported critical value for Zn deficiency to occur in many other plant species. The results obtained suggest that strongly expressed constitutive sequestration mechanisms exist in the hyperaccumulator T. caerulescens, which detoxify the large amount of Zn present in shoot tissues and decrease its physiological availability in the cytosol. Both T. caerulescens and T. ochroleucum had constitutively high concentrations of malate in shoots, which were little affected by different Zn treatments. Although malate may play a role in Zn chelation because of the high concentrations present, it cannot explain the species specificity of Zn tolerance and hyperaccumulation.     

Solubility of zinc and interactions between zinc and phosphorus in the hyperaccumulator Thlaspi caerulescens

The relationship between Zn and P in the Zn hyperaccumulator Thlaspi caerulescens J. & C. Presl was investigated using hydroponic culture. Total concentrations of Zn in the shoots increased from 0·2 to 27 g kg^–1 dry mass when solution Zn increased from 1 to 1000 mmol m^–3. Water-soluble Zn accounted for > 80% of the total Zn in the shoots containing > 5 g Zn kg^–1 dry mass. Total P was maintained at about 3 g kg^–1 dry mass in the shoots containing < 20 g Zn kg^–1 dry mass, but significantly decreased with higher Zn concentrations. Linear regression between insoluble P and insoluble Zn in the shoots produced a small slope, suggesting that co-precipitation of Zn and P was not an important detoxification mechanism in the shoots. In contrast, there was a strong correlation between insoluble P and insoluble Zn in the roots, with a linear slope of 0·3 — close to the P:Zn ratio in Zn₃(PO₄)₂. Foliar sprays of phosphate did not affect shoot dry mass significantly, but decreased root length and root dry mass significantly at Zn concentrations in solution from 10 to 3000 mmol m^–3. Foliar P was translocated to roots to enhance co-precipitation of Zn and P, although this did not enhance Zn tolerance. The results suggest that T.caerulescens possesses mechanisms which allow it to accumulate and sequester huge amounts of Zn in the shoots without causing P deficiency.     

Effect of nitrogen form on the rhizosphere dynamics and uptake of cadmium and zinc by the hyperaccumulator Thlaspi caerulescens

Information about root distribution is important for characterization and modeling of water and nutrient uptake, biomass, and yield. Due to the heterogeneous distribution of roots in row crops, the reliability and representativeness of estimates of RL and root morphology using core sampling depend on the number of samples and their location. The objectives of this study were to evaluate errors when assessing RLD and root morphology from auger core sampling schemes, to estimate 2D distributions of RLD by auger core sampling, and to assess the number of samples necessary for representative estimates of RLD and RD classes. The reference dataset utilized in this study is based on completely sampled 3D soil monoliths under maize, taken at three different dates (55, 78, and 104 days after planting) and two different row spacings (75 and 37.5 cm). A hypothetical auger core sampling scheme with one core within the row and another midway between rows mostly overpredicted total RLD. Bias was lower when using an 1:3 weighting scheme. Estimation of 2D vertical RLD distribution by calculating the ratio of RLD in the plant row to RLD midway between rows yielded reasonable estimates only when the average of eight cores was used. An assessment of the proportions of RD classes yielded high bias values, even when using the average of eight cores. An analysis of sampling errors using successively more cores revealed that for total RLD, to attain a bias <20%, more than ten samples would be necessary. This suggests that the number of core samples taken in many root studies could be too low. This bias was even higher when taking core samples for estimation of proportions of RD classes, where a reasonably low bias between estimated and “true” values could not be attained in this study even with ten core samples. Consequently, when taking samples for measurement of root morphological parameters, more detailed and site-adapted sampling schemes have to be devised. For estimating total averaged RLD, two core samples (within-row and midway between rows) could be sufficient when using a 1:3 weighting scheme for calculating the average RLD. When samples are taken in a more random manner and no weighting is applied, the necessary number of samples would be at least ten.     

Characteristics of cadmium uptake in two contrasting ecotypes of the hyperaccumulator Thlaspi caerulescens

Uptake of Cd and Zn by intact seedlings of two contrasting ecotypes of the hyperaccumulator Thlaspi caerulescens was characterized using radioactive tracers. Uptake of Cd and Zn at 2 degrees C was assumed to represent mainly apoplastic binding in the roots, whereas the difference in uptake between 22 degrees C and 2 degrees C represented metabolically dependent influx. There was no significant difference between the two ecotypes in the apoplastic binding of Cd or Zn. Metabolically dependent uptake of Cd was 4.5-fold higher in the high Cd-accumulating ecotype, Ganges, than in the low Cd-accumulating ecotype, Prayon. By contrast, there was only a 1.5-fold difference in the Zn uptake between the two ecotypes. For the Ganges ecotype, Cd uptake could be described by Michaelis-Menten kinetics with a V(max) of 143 nmol g(-1) root FW h(-1) and a K(m) of 0.45 microM. Uptake of Cd by the Ganges ecotype was not inhibited by La, Zn, Cu, Co, Mn, Ni or Fe(II), and neither by increasing the Ca concentration. By contrast, addition of La, Zn or Mn, or increasing the Ca concentration in the uptake solution decreased Cd uptake by Prayon. Uptake of Ca was larger in Prayon than in Ganges. The results suggest that Cd uptake by the low Cd-accumulating ecotype (Prayon) may be mediated partly via Ca channels or transporters for Zn and Mn. By contrast, there may exist a highly selective Cd transport system in the root cell membranes of the high Cd-accumulating ecotype (Ganges) of T. caerulescens.     

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。     

Complexation of cadmium in seeds and vegetative tissues of the cadmium hyperaccumulator Thlaspi praecox as studied by X-ray absorption spectroscopy

The cadmium hyperaccumulator Thlaspi praecox Wulfen (Brassicaceae) can accumulate unusually high amounts of Cd (>1,000 μg g^?1 dry weight) in its seeds without drastically affecting seed viability. As embryonic tissues are the most sensitive to Cd toxicity, the aim of this study was to investigate the Cd coordination and ligand environment in seeds of field collected T. praecox using extended X-ray absorption fine structure (EXAFS), and to compare the Cd ligand environment to that in the vegetative tissues of the plant. In intact seeds and isolated embryos, almost two thirds of the Cd ligands were thiol groups (Cd-S-C-). In addition, there was coordination to phosphate groups via bridging oxygens (Cd-O-P-), as for phytate, although this ligand was not observed in the vegetative organs and tissues. In roots and shoots up to 80% of the Cd ligands were oxygen ligands that are provided by the cell walls and by organic acids stored in vacuoles. In leaf epidermis only a slightly higher percentage of oxygen ligands was detected, as compared to the mesophyll, making vacuolar compartmentation and binding to the cell walls the main detoxification mechanisms in both of these leaf tissues.     

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.     

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.     

Tandem quadruplication of HMA4 in the zinc (Zn) and cadmium (Cd) hyperaccumulator Noccaea caerulescens

Zinc (Zn) and cadmium (Cd) hyperaccumulation may have evolved twice in the Brassicaceae, in Arabidopsis halleri and in the Noccaea genus. Tandem gene duplication and deregulated expression of the Zn transporter, HMA4, has previously been linked to Zn/Cd hyperaccumulation in A. halleri. Here, we tested the hypothesis that tandem duplication and deregulation of HMA4 expression also occurs in Noccaea.A Noccaea caerulescens genomic library was generated, containing 36,864 fosmid pCC1FOS™ clones with insert sizes ∼20–40 kbp, and screened with a PCR-generated HMA4 genomic probe. Gene copy number within the genome was estimated through DNA fingerprinting and pooled fosmid pyrosequencing. Gene copy numbers within individual clones was determined by PCR analyses with novel locus specific primers. Entire fosmids were then sequenced individually and reads equivalent to 20-fold coverage were assembled to generate complete whole contigs.Four tandem HMA4 repeats were identified in a contiguous sequence of 101,480 bp based on sequence overlap identities. These were flanked by regions syntenous with up and downstream regions of AtHMA4 in Arabidopsis thaliana. Promoter-reporter β-glucuronidase (GUS) fusion analysis of a NcHMA4 in A. thaliana revealed deregulated expression in roots and shoots, analogous to AhHMA4 promoters, but distinct from AtHMA4 expression which localised to the root vascular tissue.This remarkable consistency in tandem duplication and deregulated expression of metal transport genes between N. caerulescens and A. halleri, which last shared a common ancestor >40 mya, provides intriguing evidence that parallel evolutionary pathways may underlie Zn/Cd hyperaccumulation in Brassicaceae.     

Adaptative evolution of metallothionein 3 in the Cd/Zn hyperaccumulator Thlaspi caerulescens

Laboratoire de Physiologie et Génétique Moléculaire des Plantes, Université Libre de Bruxelles, Campus Plaine (CP 242) Bd du triomphe, B-1050 Brussels, Belgium.     

Variation in root-to-shoot translocation of cadmium and zinc among different accessions of the hyperaccumulators Thlaspi caerulescens and Thlaspi praecox

Efficient root-to-shoot translocation is a key trait of the zinc/cadmium hyperaccumulators Thlaspi caerulescens and Thlaspi praecox, but the extent of variation among different accessions and the underlying mechanisms remain unclear. Root-to-shoot translocation of Cd and Zn and apoplastic bypass flow were determined in 10 accessions of T. caerulescens and one of T. praecox, using radiolabels (109)Cd and (65)Zn. Two contrasting accessions (Pr and Ga) of T. caerulescens were further characterized for TcHMA4 expression and metal compartmentation in roots. Root-to-shoot translocation of (109)Cd and (65)Zn after 1 d exposure varied 4.4 to 5-fold among the 11 accessions, with a significant correlation between the two metals, but no significant correlation with uptake or the apoplastic bypass flow. The F(2) progeny from a cross between accessions from Prayon, Belgium (Pr) and Ganges, France (Ga) showed a continuous phenotype pattern and transgression. There was no significant difference in the TcHMA4 expression in roots between Pr and Ga. Compartmentation analysis showed a higher percentage of (109)Cd sequestered in the root vacuoles of Ga than Pr, the former being less efficient in translocation than the latter. Substantial natural variation exists in the root-to-shoot translocation of Cd and Zn, and root vacuolar sequestration may be an important factor related to this variation.     

Evidence for copper homeostasis function of metallothionein (MT3) in the hyperaccumulator Thlaspi caerulescens

Metallothioneins chelate metals and consequently may be a control point of metal homeostasis. Homologous to type 3 metallothioneins, TcMT3 cDNA was identified in the Cd/Zn hyperaccumulator, Thlaspi caerulescens. TcMT3 amino acid sequence showed modifications in the Cys positions when compared with its Arabidopsis orthologue. A structural model established that the MT3 carboxyterminal domain is similar to the beta domain of animal metallothioneins and predicts a smaller cavity to chelate metals for A. thaliana than for T. caerulescens. Functional testing in yeast and Northern blot analysis added further evidence for adaptative variations of MT3 for the maintenance of Cu homeostasis in a metal hyperaccumulator.