Identification of Thlaspi caerulescens genes that may be involved in heavy metal hyperaccumulation and tolerance. Characterization of a novel heavy metal transporting ATPase

Thlaspi caerulescens is a heavy metal hyperaccumulator plant species that is able to accumulate extremely high levels of zinc (Zn) and cadmium (Cd) in its shoots (30,000 microg g(-1) Zn and 10,000 microg g(-1) Cd), and has been the subject of intense research as a model plant to gain a better understanding of the mechanisms of heavy metal hyperaccumulation and tolerance and as a source of genes for developing plant species better suited for the phytoremediation of metal-contaminated soils. In this study, we report on the results of a yeast (Saccharomyces cerevisae) complementation screen aimed at identifying candidate heavy metal tolerance genes in T. caerulescens. A number of Thlaspi genes that conferred Cd tolerance to yeast were identified, including possible metal-binding ligands from the metallothionein gene family, and a P-type ATPase that is a member of the P1B subfamily of purported heavy metal-translocating ATPases. A detailed characterization of the Thlaspi heavy metal ATPase, TcHMA4, demonstrated that it mediates yeast metal tolerance via active efflux of a number of different heavy metals (Cd, Zn, lead [Pb], and copper [Cu]) out of the cell. However, in T. caerulescens, based on differences in tissue-specific and metal-responsive expression of this transporter compared with its homolog in Arabidopsis (Arabidopsis thaliana), we suggest that it may not be involved in metal tolerance. Instead, we hypothesize that it may play a role in xylem loading of metals and thus could be a key player in the hyperaccumulation phenotype expressed in T. caerulescens. Additionally, evidence is presented showing that the C terminus of the TcHMA4 protein, which contains numerous possible heavy metal-binding His and Cys repeats residues, participates in heavy metal binding. When partial peptides from this C-terminal domain were expressed in yeast, they conferred an extremely high level of Cd tolerance and Cd hyperaccumulation. The possibilities for enhancing the metal tolerance and phytoremediation potential of higher plants via expression of these metal-binding peptides are also discussed.     

Growth and mineral element composition in two ecotypes of Thlaspi caerulescens on Cd contaminated soil

The heavy metal hyperaccumulator Thlaspi caerulescens occurs both on heavy metal polluted soils (metallicolous ecotype MET) and on soils with normal heavy metal content (non-metallicolous ecotype: NMET). In order to assess the extent and structure of variation in growth, shoot accumulation of Cd, Zn and mineral element (Ca, Mg, K, Fe), a MET ecotype from Belgium and a NMET ecotype from Luxembourg were studied. Seven maternal families from two populations of each ecotype were grown on both Cd and Zn contaminated soil. Although both ecotypes presented a similar heavy metal tolerance in the experimental conditions tested, they differed in several points. The MET populations had markedly higher biomass and higher root:shoot ratio compared to NMET populations. The Zn, and at lesser extent, the Cd hyperaccumulation capacity tended to be higher in the NMET populations. The same trend was observed for the foliar concentrations of Mg, Ca and Fe with NMET populations having higher concentrations compared to MET ones. Cd and Zn concentrations were negatively correlated with the biomass of both ecotype. However, the negative correlation between the Zn and biomass was much lower in MET ecotype suggesting a tighter control of internal Zn concentration in this ecotype. Finally, although the Cd phytoextraction capacity was similar in both ecotype, a higher Zn phytoextraction capacity was detected in NMET ecotype when these plants grow on moderate Cd and Zn concentrations.     

Evolution of metal hyperaccumulation and phytoremediation hype

Cadmium accumulation and tolerance are discussed in a New Phytologist article by Krämer (2000), which comments on a paper by Lombi et al . (2000). In this context, a number of additional points should be made, putting the role of humans in the evolution of metal resistance into context and emphasizing what is the 'hype' of phytoremediation.
It is important that sites created by humans should not be overemphasized in considering the evolution of metal resistance. Plants resistant to heavy metals have their primary sites not on these, but on soils where ores are outcropping, the so-called metalliferous or orogenic soils (Ernst, 1974). Over thousands of years, natural exposure to a surplus of various metals, depending on the mineralization process, has driven the evolution of metal resistance in many plant species under the local environmental conditions. Many publications have shown that Thlaspi caerulescens can hyperaccumulate Zn (e.g. Vázquez et al ., 1992), and accumulate other heavy metals such as Cu and Pb depending on soil chemistry (e.g. Baker et al ., 1994). It has been known for more than 30 years that T. caerulescens gives a good response to experimentally supplied high Zn levels (Ernst, 1968). One of the ecological effects of hyperaccumulation of heavy metals is a defence against herbivorous insects (Boyd & Martens, 1994). This effect is enhanced by a preferential accumulation of heavy metals in the epidermal leaf layer (Heath et al ., 1997).     

Colonization of pennycresses (Thlaspi spp.) of the Brassicaceae by arbuscular mycorrhizal fungi

Members of the Brassicaceae are generally believed to be non-mycorrhizal. Pennycress (Thlaspi) species of this family from diverse locations in Slovenia, Austria, Italy and Germany were examined for their colonisation by arbuscular mycorrhizal fungi (AMF). Meadow species (T. praecox, T. caerulescens and T. montanum) were sparsely but distinctly colonised, as indicated by the occurrence of intraradical hyphae, vesicles, coils, and occasionally arbuscules. Species from other locations were poorly colonised, but arbuscules were not discernible. The genus Thlaspi comprises several heavy metal hyperaccumulating species (T. caerulescens, T. goesingense, T. calaminare, T. cepaeifolium). All samples collected from heavy metal soils were at best poorly colonized. Thus the chance is small to find a "hypersystem" in phytoremediation consisting of an AM fungus which prevents the uptake of the major part of the heavy metals and of a Thlaspi species which effectively deposits the residual heavy metals inevitably taken up into its vacuoles. In two different PCR approaches, fungal DNA was amplified from most of the Thlaspi roots examined, even from those with a very low incidence of AMF colonization. Sequencing of the 28S- and 18S-rDNA PCR-products revealed that different Thlaspi field samples were colonized by Glomus intraradices and thus by a common AM fungus. However, none of the sequences obtained was identical to any other found in the present study or deposited in the databanks, which might indicate that a species continuum exists in the G. intraradices clade. An effective colonization of Thlaspi by AMF could not be established in greenhouse experiments. Although the data show that Thlaspi can be colonized by AMF, it is doubtful whether an effective symbiosis with the mutual exchange of metabolites is formed by both partners.     

Thlaspi caerulescens on nonmetalliferous soil in Luxembourg: ecological niche and genetic variation in mineral element composition

Forty-seven populations of Thlaspi caerulescens in Luxembourg were characterised for population size, soil mineral element composition and other habitat characteristics. Foliar concentrations of eight elements were assessed in 15 populations in the field and in eight populations cultivated in zinc (Zn)-cadmium (Cd)-nickel (Ni)-enriched soil. T. caerulescens favoured stony soil developed on steep, south-facing Emsian shale outcrops. All soil samples were nonmetalliferous. Soil pH ranged from 4.2 to 6.9. Field-growing plants had very high concentrations of heavy metals in the leaves (Zn, 3000-13 000 mg kg(-1); Cd, 11-44 mg kg(-1); Ni, 38-473 mg kg(-1)). Positive soil-plant correlations existed for Zn and Mn. In cultivation, significant genetic variation was found for biomass and six of eight mineral elements. For Cd and Zn, variation range among 48 half-sib families was two-fold (Cd, 183-334 mg kg(-1); Zn, 8030-16 295 mg kg(-1)). Most of the variation occurred among populations, consistent with the selfing mating system of those populations. There was a tight Zn-Cd genetic correlation (r = +0.83, P < 0.0001). The significance of the results to the conservation of T. caerulescens in Luxembourg is briefly discussed.     

Cadmium hyperaccumulation and reproductive traits in natural Thlaspi caerulescens populations

During the last decade, the metal hyperaccumulating plants have attracted considerable attention because of their potential use in decontamination of heavy metal contaminated soils. However, in most species, little is known regarding the function, the ecological and the evolutionary significances of hyperaccumulation. In our study, we investigated the parameters influencing the Cd concentration in plants as well as the biological implications of Cd hyperaccumulation in nine natural populations of Thlaspi caerulescens. First, we showed that Cd concentration in the plant was positively correlated with plant Zn, Fe, and Cu concentrations. This suggested that the physiological and/or molecular mechanisms for uptake, transport and/or accumulation of these four heavy metals interact with each other. Second, we specified a measure of Cd hyperaccumulation capacity by populations and showed that T. caerulescens plants originating from populations with high Cd hyperaccumulation capacity had better growth, by developing more and bigger leaves, taller stems, and produced more fruits and heavier seeds. These results suggest a tolerance/disposal role of Cd hyperaccumulation in this species.     

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。     

Systematics and evolutionary history of heavy metal tolerant Thlaspi caerulescens in Western Europe: evidence from genetic studies based on isozyme analysis

Thlaspi caerulescens is distributed in Europe on metalliferous and not metalliferous soils. Individuals from populations growing on heavy metal contaminated soils are well known as hyperaccumulators of zinc and cadmium. The taxonomical treatment of subspecies of Thlaspi caerulescens is unsettled. We investigated the degree of genetic variation among 28 populations of Thlaspi caerulescens from Europe with isozyme analysis to compare inter- and intrapopulational diversity. British material from heavy metal contaminated environments recognized as Thlaspi sylvestre and T. occitanicum are quite similar to each other on the level of isozyme polymophisms, but they are more closely related to populations from non-contaminated stands from Scandinavia and Middle Europe than to metallophytes distributed in Continental Europe. Our findings indicate that a taxonomical subdivision of T. caerulescens is not possible and, furthermore, heavy metal tolerance might have evolved twice in populations of Thlaspi caerulescens from different areas. The trait of zinc tolerance and hyperaccumulation is frequently found in numerous relatives of Thlaspi caerulescens, and it is suggested that this trait has been established and manifested in populations from metalliferous sites during postglacial colonization. From Scandinavia only non-metallophytes are known. These populations are very similar to each other on the isozyme level. This fits to the hypothesis that Thlaspi caerulescens was introduced to Scandinavia in recent times by human activity. Despite full self-compatibility we estimated varying outcrossing rates up to 0.88 in the metallophytes and 0.658 in the non-metallophytes depending on population size and structure.     

Hyperaccumulation of metals by Thlaspi caerulescens as affected by root development and Cd-Zn/Ca-Mg interactions

The aim of this work was to study, in a rhizobox experiment, the phytoextraction of metals by the hyperaccumulator plant Thlaspi caerulescens in relation to the heterogeneity of metal pollution. Six treatments were designed with soils containing various levels of metals. Homogeneous soils and inclusions of soils in other soil matrices were prepared in order to vary metal concentration and localization. Growth parameters of the plant (rosette diameter and shoot biomass) and localization of roots and shoot uptake of Zn, Cd, Ca, and Mg were determined after 10 weeks of growth. The plants grown on the polluted industrial soils provided a larger biomass and had lower mortality rates than those grown on the agricultural soil. Moreover, these plants accumulated more Zn and Cd (up to 17,516 and 375 mg kg(-1) DM, respectively) than plants grown on the agricultural soil (up to 7300 mg Zn kg(-1) and 83 mg Cd kg(-1) DM). The roots preferentially explored metal-contaminated areas. The exploration of polluted soil inclusions by the roots was associated with a higher extraction of metals. Zinc and Cd in the shoots of Thlaspi caerulescens were negatively correlated with Ca and Mg concentrations; however, the soil supply for these two elements was identical. This suggests that there is competition for the uptake of these elements and that Zn is preferentially accumulated.     

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 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.     

Plant Cd2+ and Zn2+ status effects on root and shoot heavy metal accumulation in Thlaspi caerulescens

* In this study we address the impact of changes in plant heavy metal, (i.e. zinc (Zn) and cadmium (Cd)) status on metal accumulation in the Zn/Cd hyperaccumulator, Thlaspi caerulescens. * Thlaspi caerulescens plants were grown hydroponically on both high and low Zn and Cd regimes and whole-shoot and -root metal accumulation, and root (109)Cd(2+) influx were determined. * High-Zn-grown (500 microm Zn) plants were found to be more Cd-tolerant than plants grown in standard Zn conditions (1 microm Zn). Furthermore, shoot Cd accumulation was significantly greater in the high-Zn-grown plants. A positive correlation was also found between shoot Zn accumulation and increased plant Cd status. Radiotracer (109)Cd root flux experiments demonstrated that high-Zn-grown plants maintained significantly higher root Cd(2+) influx than plants grown on 1 microm Zn. It was also found that both nickel (Ni) and copper (Cu) shoot accumulation were stimulated by high plant Zn status, while manganese (Mn) accumulation was not affected. * A speculative model is presented to explain these findings, suggesting that xylem loading may be one of the key sites responsible for the hyperaccumulation of Zn and Cd accumulation in Thlaspi caerulescens.     

Native plant communities in an abandoned Pb-Zn mining area of northern Spain: implications for phytoremediation and germplasm preservation

Plants growing on metalliferous soils from abandoned mines are unique because of their ability to cope with high metal levels in soil. In this study, we characterized plants and soils from an abandoned Pb-Zn mine in the Basque Country (northern Spain). Soil in this area proved to be deficient in major macronutrients and to contain toxic levels of Cd, Pb, and Zn. Spontaneously growing native plants (belonging to 31 species, 28 genera, and 15 families) were botanically identified. Plant shoots and rhizosphere soil were sampled at several sites in the mine, and analyzed for Pb, Zn and Cd concentration. Zinc showed the highest concentrations in shoots, followed by Pb and Cd. Highest Zn concentrations in shoots were found in the Zn-Cd hyperaccumulator Thlaspi caerulescens (mean = 18,254 mg Zn kg(-1) DW). Different metal tolerance and accumulation patterns were observed among the studied plant species, thus offering a wide germplasm assortment for the suitable selection of phytoremediation technologies. This study highlights the importance of preserving metalliferous environments as they shelter a unique and highly valuable metallicolous biodiversity.     

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.     

Zinc tolerance and hyperaccumulation in F1 and F2 offspring from intra and interecotype crosses of Thlaspi caerulescens

The relationship between zinc (Zn) tolerance and hyperaccumulation in Thlaspi caerulescens was investigated from F1 and F2 crosses within and among metallicolous and nonmetallicolous Mediterranean populations. F1 offspring were grown on increasingly Zn-enriched soils to test Zn enrichment effects, and many families of F2 offspring were grown on a Zn-rich soil. Tolerance of F1 offspring depended on stress intensity. Tolerance of interecotype crosses was intermediate between that of the intraecotype crosses. No difference in Zn accumulation appeared among the F1 offspring from the three crosses involving metallicolous parents. Otherwise, none of these offspring exceeded the Zn hyperaccumulation threshold (10,000 mg kg(-1)), unlike the nonmetallicolous ones. The latter also showed the highest mortality. In some F2 families from interecotype crosses, hyperaccumulation values exceeded 15,000 mg kg(-1) in nontolerant offspring, whereas tolerant offspring displayed lower values (c. 10,000 mg kg(-1)). There was no difference between tolerant and nontolerant offspring when they showed low hyperaccumulation. Therefore, the relationship between tolerance and hyperaccumulation in F1 and F2 crosses depended on the hyperaccumulation level of plants.     

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 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.     

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.