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).     

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.     

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。     

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.     

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.     

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.     

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.     

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.     

Root-to-shoot long-distance circulation of nicotianamine and nicotianamine-nickel chelates in the metal hyperaccumulator Thlaspi caerulescens

Plant metal hyperaccumulator species are widely used as models to unravel the heavy metal tolerance and hyperaccumulation mechanisms. Thlaspi caerulescens is capable of tolerating and hyperaccumulating Zn, Cd, and Ni. A search for factors involved in the cellular tolerance to Ni, based on yeast screens, led to isolation of a cDNA encoding a functional nicotianamine (NA) synthase (NAS). The T. caerulescens genome appears to contain a single copy of the NAS gene named TcNAS whose expression is restricted to the leaves. The analysis of dose-response and time-course Ni treatments have revealed that the exposure to Ni triggers the accumulation of NA in the roots. Because neither TcNAS expression nor NAS activity were detected in the roots, the NA accumulation in roots is most probably the result of its translocation from the leaves. Once in the roots, NA, together with Ni, is subsequently found in the xylem, for redirection to the aerial parts. Using liquid chromatography coupled to inductively coupled plasma or electrospray ionization mass spectrometry, it has been shown that part of the Ni is translocated as a stable Ni-NA complex in the xylem sap. This circulation of NA, Ni, and NA-Ni chelates is absent in the non-tolerant non-hyperaccumulator related species T. arvense. Taken together, the results provide direct physiological and chemical evidence for NA and NA-heavy metal complex translocation in a hyperaccumulator species.     

Anthocyanin production in the hyperaccumulator plant Noccaea caerulescens in response to herbivory and zinc stress

Stomatal behavior in response to drought has been the focus of intensive research, but less attention has been paid to stomatal density. In this study, 5-week-old maize seedlings were exposed to different soil water contents. Stomatal density and size as well as leaf gas exchange were investigated after 2-, 4- and 6-week of treatment, which corresponded to the jointing, trumpeting, and filling stages of maize development. Results showed that new stomata were generated continually during leaf growth. Reduced soil water content significantly stimulated stomatal generation, resulting in a significant increase in stomatal density but a decrease in stomatal size and aperture. Independent of soil water conditions, stomatal density and length in the trumpeting and filling stages were greater than in the jointing stage. Irrespective of growth stage, severe water deficit significantly reduced stomatal conductance (G _s), decreasing the leaf transpiration rate (T _r) and net photosynthetic rate (P _n). Stomatal density was significantly negatively correlated with both P _n and T _r but more strongly with T _r, so the leaf instantaneous water use efficiency (WUE _i ) correlated positively with stomatal density. In conclusion, drought led to a significant increase in stomatal density and a reduction in stomatal size and aperture, resulting in decreased P _n and T _r. Because the negative correlation of stomatal density to T _r was stronger than that to P _n, leaf WUE _i tended to increase.     

Isolation,characterization, and identification of bacteria associated with the zinc hyperaccumulator Thlaspi caerulescens subsp. calaminaria

We investigated bacterial populations associated with the Zn hyperaccumulator Thlaspi caerulescens subsp. calaminaria grown in a soil collected from an abandoned Zn-Pb mine and smelter in Plombières, Belgium. The bacterial population of the nonrhizospheric soil consisted of typical soil bacteria, some exhibiting multiple heavy-metal resistance characteristics that often are associated with polluted substrates: 7.8% and 4% of the population survived in the presence of elevated levels of Zn (1 mM) and Cd (0.8 mM), respectively. For the bacterial population isolated from the rhizosphere, the comparable survival rates were 88 and 78%. This observation indicates a selective enrichment of the metal-resistant strains due to an increased availability of the metals in soils near the roots compared with nonrhizospheric soil. The endophytic inhabitants of the roots and shoots were isolated, identified, and characterized. Although similar endophytic species were isolated from both compartments, those from the rhizoplane and roots showed lower resistance to Zn and Cd than the endophytic bacteria isolated from the shoots. In addition, root endophytic bacteria had additional requirements. Contrary to the rootresiding inhabitants, the shoot represented a niche rich in metal-resistant bacteria and even seemed to contain species that were exclusively abundant there. These differences in the characteristics of the bacterial microflora associated with T. caerulescens might possibly reflect altered metal speciation in the different soils and plant compartments studied.     

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.     

Tissue- and age-dependent differences in the complexation of cadmium and zinc in the cadmium/zinc hyperaccumulator Thlaspi caerulescens (Ganges ecotype) revealed by x-ray absorption spectroscopy

Extended x-ray absorption fine structure measurements were performed on frozen hydrated samples of the cadmium (Cd)/zinc (Zn) hyperaccumulator Thlaspi caerulescens (Ganges ecotype) after 6 months of Zn(2+) treatment with and without addition of Cd(2+). Ligands depended on the metal and the function and age of the plant tissue. In mature and senescent leaves, oxygen ligands dominated. This result combined with earlier knowledge about metal compartmentation indicates that the plants prefer to detoxify hyperaccumulated metals by pumping them into vacuoles rather than to synthesize metal specific ligands. In young and mature tissues (leaves, petioles, and stems), a higher percentage of Cd was bound by sulfur (S) ligands (e.g. phytochelatins) than in senescent tissues. This may indicate that young tissues require strong ligands for metal detoxification in addition to the detoxification by sequestration in the epidermal vacuoles. Alternatively, it may reflect the known smaller proportion of epidermal metal sequestration in younger tissues, combined with a constant and high proportion of S ligands in the mesophyll. In stems, a higher proportion of Cd was coordinated by S ligands and of Zn by histidine, compared with leaves of the same age. This may suggest that metals are transported as stable complexes or that the vacuolar oxygen coordination of the metals is, like in leaves, mainly found in the epidermis. The epidermis constitutes a larger percentage of the total volume in leaves than in stems and petioles. Zn-S interaction was never observed, confirming earlier results that S ligands are not involved in Zn resistance of hyperaccumulator plants.