Journal of Experimental Botany, Vol. 51, No. 342, pp. 71-79,
January 2000
© 2000 Oxford University Press
Molecular physiology of zinc transport in the Zn hyperaccumulator Thlaspi caerulescens
US Plant, Soil and Nutrition Laboratory, USDA-ARS, Cornell University, Ithaca, NY 14853, USA
Received 19 January 1999; Accepted 4 May 1999
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Abstract |
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 Top Abstract IntroductionPhysiological investigations of...Molecular basis of zinc...SummaryReferences |
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In this manuscript, recent research from this laboratory into physiological and molecular aspects of heavy metal (Zn) transport in the hyperaccumulating plant species, Thlaspi caerulescens is reviewed. This research is aimed at elucidating the processes that underlie the accumulation of extraordinarily high levels of Zn in the T. caerulescens shoot (up to 3% Zn dry wt.) without any associated toxicity symptom. Physiological studies focused on the use of radiotracer flux techniques (65Zn2+) to characterize zinc transport and compartmentation in the root, and translocation and accumulation in the shoot of T. caerulescens in comparison with a related non-accumulator, T. arvense. These studies indicated that Zn transport was stimulated at a number of sites in T. caerulescens, contributing to the hyperaccumulation trait. The transport processes that were stimulated included Zn influx into both root and leaf cells, and Zn loading into the xylem. The 4- to 5-fold stimulation of Zn influx into the root was hypothesized to be due to an increased abundance of Zn transporters in T. caerulescens root cells. Additionally, compartmental analysis (radiotracer wash out or efflux techniques) was used to show that Zn was sequestered in the vacuoles of T. arvense root cells which retarded Zn translocation to the shoot in this non-accumulator species. Molecular studies have focused on the cloning and characterization of Zn transport genes in T. caerulescens. Complementation of a yeast Zn transport-defective mutant with a T. caerulescens cDNA library resulted in the recovery of a cDNA, ZNT1, that encodes a Zn transporter. Sequence analysis of ZNT1 indicated it is a member of a recently discovered micronutrient transport gene family which includes the Arabidopsis Fe transporter, IRT1, and the ZIP Zn transporters. Expression of ZNT1 in yeast allowed for a physiological characterization of this transporter. It was shown to encode a high affinity Zn transporter which can also mediate low affinity Cd transport. Northern analysis of ZNT1 and its homologue in the two Thlaspi species indicated that enhanced Zn transport in T. caerulescens results from a constitutively high expression of the ZNT1 gene in roots and shoots. In T. arvense, ZNT1 is expressed at far lower levels and this expression is stimulated by imposition of Zn deficiency.
Key words: Thlaspi caerulescens, Zn hyperaccumulation, Zn transport, ZNT1, Zn transport genes.
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Introduction |
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TopAbstractIntroductionPhysiological investigations of...Molecular basis of zinc...SummaryReferences |
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Recently, there has been an increase in research focusing on the use of higher plants to clean up soils contaminated with heavy metals (Chaney, 1983
, 1993; Benemann et al., 1994; Cunningham et al., 1995; Salt et al., 1995; Nanda Kumar et al., 1995; Cunningham and Ow, 1996). This approach exploits the ability of terrestrial plants to absorb contaminants from the rhizosphere and translocate them to the shoot. Contaminants are subsequently removed by harvesting the above-ground shoot biomass for volume reduction and storage. The basic concepts for this technology, termed phytoremediation, are not new and cannot be traced to a specific reference; however the use of phytoremediation as an environmental clean-up technology has only recently been seriously considered (Chaney, 1983). The identification of over 200 terrestrial species that can both tolerate and accumulate high levels of heavy metals in the shoot (Zn, Ni, Co, Cu) (Baker and Brooks, 1989) indicates that the genetic potential for phytoremediation exists. Unfortunately, most of these hyperaccumulator species are small and slow growing, severely limiting their potential for large-scale decontamination of polluted soils (Ebbs et al., 1997). Transferring the genes conferring the hyperaccumulating phenotype to plants that produce more shoot biomass has been suggested as a potential avenue for making phytoremediation a viable commercial technology (Brown et al., 1995a). However, progress towards this goal has been hindered by a lack of understanding of the basic biochemical, physiological and molecular mechanisms involved in heavy metal hyperaccumulation.
Possibly one of the best known heavy metal hyperaccumulator plant species is Thlaspi caerulescens J&C Presl, which can accumulate up to 3% Zn in dry shoots without showing any sign of toxicity (Brown et al., 1995b). This feature makes T. caerulescens a very interesting experimental system for studying mechanisms of heavy metal transport and accumulation in plants as they relate to phytoremediation.
The topic of plant Zn transport has not been extensively studied, though several reports have dealt with general aspects of Zn uptake in roots and translocation to shoots (for reviews see Kochian, 1991, 1993). Studies on the concentration-dependent kinetics of root Zn absorption over a 0–10 µM concentration range showed that Zn uptake followed Michaelis–Menten kinetics with Km values of 3 and 1.5 µM for barley (Hordeum vulgare L.) and maize (Zea mays L.), respectively (Veltrup, 1978; Mullins and Sommers, 1986). The existence of this saturable transport component suggests that Zn transport into the root cytosol is via a protein-mediated transport system with a fairly high affinity for Zn. Recent advances in our understanding of molecular aspects of plant micronutrient transport have opened up new avenues for examining the mechanisms plants use to acquire and accumulate heavy metals, many of which are also essential micronutrients. For example, complementation of a yeast Zn transport-defective mutant has been used to clone four cDNAs encoding Zn transporters in Arabidopsis (Grotz et al., 1998). When expressed in yeast, these transporters, named ZIP1 through ZIP4 (for zinc inducibleprotein) exhibited Zn transport properties similar to those seen in roots of intact plants. Additionally, it was shown that expression of the ZIP genes was influenced by plant Zn status.
Currently, there is little fundamental insight on both physiological and molecular mechanisms of Zn transport in hyperaccumulator plant species. High Zn accumulation in T. caerulescens has been previously reported (Rascio, 1977; Reeves and Brooks, 1983; Reeves and Baker, 1984). More recently, it has been shown that, from culture solution, T. caerulescens accumulated more than 25 000 ppm Zn in the shoots before any yield reduction occurred (Brown et al., 1995b). This study, however, provided little information on the mechanisms of uptake and translocation resulting in Zn hyperaccumulation in T. caerulescens.
In this article, recent research from this laboratory into fundamental aspects of the physiology and molecular biology of Zn hyperaccumulation in T. caerulescens in comparison with a related non-accumulator species, T. arvense has been reviewed.
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Physiological investigations of Zn hyperaccumulation |
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TopAbstractIntroductionPhysiological investigations of...Molecular basis of zinc...SummaryReferences |
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Zn accumulation and tolerance in hyperaccumulator and non-accumulator species of Thlaspi
After 10 d of growth in a nutrient solution supplemented with different Zn2+ concentrations (1–100 µM), the non-accumulator T. arvense accumulated more Zn2+ in roots, while in the hyperaccumulator T. caerulescens, most of the absorbed zinc was translocated to the shoot. From a growth solution containing 100 µM Zn2+, 4.6-fold more Zn was accumulated in the shoots of T. caerulescens than in T. arvense, while 1.9-fold more Zn accumulated in roots of T. arvense (Table 1
). The greater zinc accumulation did not affect the chlorophyll content of T. caerulescens shoots. In contrast, accumulation of lower Zn levels in T. arvense shoots resulted in dramatic leaf chlorosis as indicated by a significant reduction in relative chlorophyll content (Table 1). To investigate in more detail differences in Zn transport between the two Thlaspi species, radiotracer (65Zn) flux techniques were used. Short-term (3 h) 65Zn transport studies showed that Zn accumulation was greater in roots of T. caerulescens (Fig. 1A, inset) indicating a greater Zn influx into the root cells of T. caerulescens. However, following longer 65Zn uptake periods (>48 h), more 65Zn was accumulated in T. arvense roots (Fig. 1A), while up to 10-fold more 65Zn was translocated to T. caerulescens shoots (Fig. 1B).
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In some hyperaccumulators such as the Ni accumulator, T. goesingense, metal hyperaccumulation appears to be due primarily to the high level of metal tolerance without any changes in metal transport (Krämer et al., 1997
). The results presented above (Table 1; Fig. 1) suggest that, in addition to the extraordinary tolerance to Zn in the shoots, a complex alteration of zinc transport processes is also likely to play a significant role in Zn hyperaccumulation in T. caerulescens. To investigate this, experiments were designed to look specifically into the characteristics of 65Zn transport and accumulation in roots and shoots of the two Thlaspi species.
65Zn transport in roots of T. arvense and T. caerulescens
To investigate the capacity for zinc transport across the root cell plasma membrane and the affinity of the transport system for Zn, the concentration-dependent kinetics of root 65Zn influx was investigated. In such studies, it is important to select a relatively short uptake period that will minimize the possibility that absorbed 65Zn is effluxed back from the cytosol into the external solution during the measurement of unidirectional Zn influx. As the rate of root Zn accumulation was linear for up to 180 min (Fig. 1A, inset), a 20 min radiotracer uptake period followed by 15 min desorption (to remove cell wall-bound 65Zn) was used to quantify 65Zn2+ uptake that primarily reflected unidirectional zinc influx across the root cell plasma membrane.
In both species, concentration-dependent Zn uptake kinetics were characterized by smooth non-saturating curves (Fig. 2A), which could be graphically resolved into saturable and linear components (Fig. 2B). A number of different experimental approaches were used to demonstrate that the linear component represented cell wall-bound 65Zn2+ remaining in roots after desorption (Lasat et al., 1996), while the saturable component represented true Zn transport across the plasma membrane. The apparent Km values (derived from Lineweaver-Burk data transformations) for the saturable components were 8 and 6 µM for T. caerulescens and T. arvense, respectively, while the Vmax values were 270 and 60 nmol Zn2+ g-1 fresh weight h-1 for T. caerulescens and T. arvense. These results suggest that in both Thlaspi species, zinc transport across the plasma membrane is mediated by proteins with similar Zn2+ affinities (possibly similar transporters), but the capacity for influx is much greater in T. caerulescens roots. A significantly larger Vmax value (4-fold higher) may suggest that there are more transporters expressed at the plasma membrane of T. caerulescens root cells. This hypothesis is addressed in the molecular section of this review.
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Despite greater Zn influx into T. caerulescens roots (Figs 1, 2), more Zn was accumulated in roots of T. arvense indicating that a significant fraction of the absorbed Zn was translocated to the shoots in T. caerulescens. Another interpretation of these results is that in T. arvense (and other ‘normal’ non-accumulator plants) much more of the absorbed Zn was sequestered in the root, possibly via storage in the vacuole, and rendered unavailable for translocation to the shoot. To investigate this, both Zn accumulation in the xylem sap and Zn compartmentation in roots of the two Thlaspi species were studied. Approximately 5-fold more Zn was accumulated in the xylem sap of T. caerulescens than in T. arvense (Lasat et al., 1998
), indicating a higher Zn mobility across the root to the xylem in T. caerulescens. To investigate the Zn compartmentation in roots, 65Zn flux (compartmentation) experiments were conducted with roots of intact Thlaspi plants labelled to steady-state with 65Zn. Despite the limitations associated with the application of this technique to multicellular organs such as roots (for a review see Lasat et al., 1998), no other experimental method is currently available to investigate ion compartmentation and fluxes between subcellular compartments (i.e. the cell wall, cytoplasm, and vacuole) in a semi-quantitative manner. Moreover, this approach has been previously used to provide valuable information on the efflux and compartmentation of a number of mineral ions such as Na+ (Pierce and Higinbotham, 1970), K+ (Kochian and Lucas, 1982), Cd2+ (Rauser, 1987), Co2+ (Macklon and Sim, 1987), Zn2+ (Santa Maria and Cogliatti, 1988), NH+4 (Macklon et al., 1990), and Cu2+ (Thornton, 1991).
A representative efflux experiment conducted with roots of the two Thlaspi species is shown in Fig. 3. Plots which represent a first-order kinetic transformation of Zn efflux (log 65Zn remaining in root as a function of time) could be dissected into three linear phases representing Zn efflux from three compartments in series: vacuole, cytoplasm and cell wall. The straight line estimating the slowest exchanging phase (180–360 min) was interpreted to represent 65Zn efflux from the vacuole (Fig. 3A). Subtraction of this linear component from the total efflux data yielded a second curve that was analysed similarly and was interpreted to represent 65Zn efflux from the cytoplasm and cell wall (Fig. 3B). Efflux from the cell wall (Fig. 3C) was obtained after subtracting the linear phase for cytoplasmic efflux (30–60 min) from the data points plotted in Fig. 3B.
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The slope of the linear components was used to calculate the half-time (t1/2) for 65Zn efflux from the subcellular compartments (Table 2
). Furthermore, the y-axis intercept of the linear components provided an estimate of the amount of 65Zn accumulated in the corresponding subcellular compartment at the end of the radioisotope loading period and was used to calculate the distribution of 65Zn in root cells at the termination of the loading period (Table 2).
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At the end of a 24 h radioisotope-loading period, comparable amounts of 65Zn were accumulated in the roots of the two Thlaspi species (first pair of data points in Fig. 3A). The Zn compartmentation data summarized in Table 2 indicate that similar amounts of Zn were accumulated in the root cell wall of the two Thlaspi species. This argues against Zn sequestration in the root cell wall of T. arvense as a mechanism of Zn tolerance as suggested by Peterson (1969)
in Agrostis tenuis. Comparable Zn levels were also accumulated in T. arvense and T. caerulescens root cell cytoplasm. However, there were significant differences in the level of vacuolar Zn and the rate of Zn efflux back out of the vacuole. As shown in Table 2, almost 2.5 times more zinc was stored in the vacuoles of T. arvense roots, and the rate of Zn efflux out of the vacuole was about 2-fold slower than in T. caerulescens. Because of significantly different vacuolar efflux rates, the difference in root 65Zn content between T. arvense and T. caerulescens should increase after longer efflux periods. In support of this, after a 46 h efflux period, approximately 6-fold more 65Zn remained in T. arvense roots compared with T. caerulescens (Fig. 4). These results confirmed that Zn is sequestered in the vacuoles of T. arvense root.
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65Zn transport in shoots of T. arvense and T. caerulescens
Because T. caerulescens has the ability to accumulate high Zn levels in shoots (Table 1; Fig. 1B), the possibility was investigated that Zn influx across the leaf cell plasma membrane was also stimulated in the hyperaccumulator species.
The time-course of Zn accumulation into leaf sections was approximately linear for up to 48 h (Fig. 5). At the lower Zn2+ concentration in the leaf uptake solution (10 µM), there were no significant differences in leaf Zn accumulation between the two Thlaspi species (Fig. 5A). Similar results were obtained at external Zn2+ concentration of 100 µM (data not shown). However, at the highest Zn2+ concentration (1 mM), which is more representative of the Zn concentration in the xylem sap of T. caerulescens, approximately 2.5-fold more zinc was accumulated in T. caerulescens leaf sections (Fig. 5B). These findings are interesting, because they suggest that Zn transport is stimulated at both the root cell and leaf cell plasma membrane in the hyperaccumulator species. However, if this is the case, the Zn transporters have different kinetic properties in roots versus shoots, as stimulated root Zn2+ influx was observed in T. caerulescens at external Zn2+ concentrations as low as 1 µM (Lasat et al., 1996), while in leaves, transport stimulation was observed only at much higher Zn2+ concentrations (>100 µM). It should also be noted that enhanced Zn transport into leaf cells could reflect a combination of stimulated transport systems operating at both the leaf cell plasma membrane and tonoplast (to effectively store Zn in the vacuole). In support of this, Zn storage in the leaf vacuole has been previously documented (Vázquez et al., 1994).
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To avoid the confounding effect of the cell wall (which physically binds Zn2+ to negatively charged sites), and leaf cuticle (which prevents 65Zn penetration into leaf tissue), accumulation of 65Zn2+ was also investigated in protoplasts isolated from leaves of the two Thlaspi species. The time dependence of 65Zn uptake into protoplasts was studied using uptake solutions containing 10 µM (Fig. 6A
) and 1 mM 65Zn2+ (Fig. 6B). As observed with leaf sections, when the external 65Zn2+ concentration was low (10 µM), Zn uptake was identical in protoplasts isolated from T. arvense and T. caerulescens leaves. From an uptake solution containing 1 mM 65Zn2+, however, there was a tendency for higher zinc accumulation in protoplasts isolated from T. caerulescens leaves, although this difference in Zn uptake between the two Thlaspi species was much smaller than in leaf sections (Fig. 6B). With protoplasts, the time-dependence for Zn uptake was smooth and non-saturating. Initially the uptake was rapid and was followed by a second component with a slower rate of uptake. The initial rapid phase over the first 30–45 s was interpreted to represent binding to negatively charged sites associated with the external face of the plasma membrane. The subsequent slower component was interpreted to represent influx of radiozinc across the plasma membrane into the cytosol. To confirm this interpretation, an uptake study was conducted with protoplasts pretreated with CCCP, an anionic protonophore and uncoupler of mitochondrial oxidative phosphorylation. Thus, CCCP can inhibit transmembrane ion transport in a number of ways including a general inhibition of metabolism (reduction in cellular ATP and NADH/NADPH), and the abolition of H+ gradients across the plasma membrane and tonoplast. Exposure to CCCP has also been previously shown dramatically to depolarize the transmembrane electrical potential across the plasma membrane of plant cells (DiTomaso et al., 1992), which should reduce the electrical driving force for divalent cation uptake. Exposure of protoplasts to CCCP (10 µM) elicited a large (>50%) inhibition of 65Zn2+ uptake into Thlaspi protoplasts (Fig. 6A). This observation indicates that the slower phase of Zn accumulation does represent true transmembrane Zn2+ transport into the Thlaspi protoplasts.
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Molecular basis of zinc hyperaccumulation in Thlaspi caerulescens |
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TopAbstractIntroductionPhysiological investigations of...Molecular basis of zinc...SummaryReferences |
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An important component of our research programme is the molecular characterization of Zn transport via cloning of the relevant transporters from the two contrasting Thlaspi species. At least one Zn transport cDNA from T. caerulescens has recently been successfully cloned, which opens up many opportunities for understanding the molecular basis of increased Zn uptake in T. caerulescens.
Cloning of Zn transport cDNAs from T. caerulescens
In order to isolate Zn transporters, a strategy focusing on functional complementation of a yeast mutant defective in Zn uptake (zhy3; provided by Dr David Eide, University of Missouri) was employed. In short, this strategy relied on the construction of a T. caerulescens cDNA library in a yeast expression vector. After transformation of zhy3 with this library, cDNAs of interest were identified as those that permitted growth of the yeast strain on a Zn-limited medium. mRNA for library construction was isolated from both T. caerulescens roots and shoots and from plants grown under both Zn- deficient and Zn-sufficient conditions. This was done to identify Zn transporter cDNAs occurring throughout the plant. Additionally, since it was not known if expression of these genes was going to be regulated by Zn status, it was felt that a library representing mRNA transcripts from plants grown under several different Zn regimes would enhance the probability of recovering Zn transporter cDNAs.
To construct the library, a commonly used yeast expression vector, pFL61, was employed. This vector has been successfully used to isolate other plant genes by functional complementation of yeast metabolic mutants that can be used to complement biochemical pathways in yeast. These include genes important for amino acid biosynthesis and genes encoding nutrient transporters, such as the Arabidopsis K+ transporter cDNA, AKT1. cDNA from the aforementioned T. caerulescens mRNA was synthesized and subsequently size-selected for products that were greater than 1 kb in length. This cDNA was ligated into pFL61 and a primary library consisting of 3x105 independent clones with an average insert size of 1.3 kb was generated.
In order to identify genes that permitted zhy3 to grow on normally restrictive levels of Zn, a screening protocol was devised. It was determined that zhy3 was incapable of growth on synthetic dextrose (SD) standard media (-uracil, +all amino acids) that was supplemented with 1000 µM EDTA and 750 µM ZnCl2. The chelate was added to scavenge free Zn contributed by the SD medium, maintain the Zn-limiting conditions imposed, and ensure that only transformants of interest were capable of growth. Screening of 3x105 yeast transformants resulted in the identification of 20 colonies that grew on Zn-limiting media. Subsequent screening confirmed that, of these colonies, seven were capable of restoration of some degree of growth on low Zn. DNA sequence analysis revealed that five of the seven clones represent the same gene which was designated ZNT1.
Genetic analysis has confirmed that ZNT1 mediates Zn uptake and therefore represents a Zn transporter. It was found that ability to grow on low Zn was dependent upon the ZNT1 plasmid construct (pZNT1) and not a second site alteration in the yeast genome. Insertion of pZNT1 into zhy3 resulted in the restoration of growth on low Zn further demonstrating the dependence of this phenotype on the presence of pZNT1 (Fig. 7). Sequence analysis of ZNT1 cDNA revealed that it is a member of a recently identified micronutrient transporter gene family and shares significant sequence homology with IRT1, a putative Fe transporter isolated from Arabidopsis (Eide et al., 1996), ZRT1, a high affinity Zn transporter isolated from yeast (Zhao and Eide, 1996), and ZIP4, one of four zinc transporters recently isolated from Arabidopsis (Grotz et al., 1998). The predicted amino acid sequence of the ZNT1 protein is 36% identical to IRT1 and 88% identical to ZIP4.
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A molecular physiological characterization of ZNT1
Expression of this T. caerulescens Zn transporter in unicellular yeast lacking endogenous Zn transporters provides an ideal research system to study the transport properties of the ZNT1 protein. Radiotracer (65Zn) flux techniques were employed to quantify unidirectional Zn2+ influx both in zhy3 and in zhy3 expressing ZNT1. As shown in Fig. 8A
, expression of ZNT1 in yeast conferred a greatly increased ability to transport Zn into yeast cells over a wide concentration range. Subtraction of the concentration-dependent Zn2+ influx into zhy3 from Zn2+ influx into cells expressing ZNT1 indicated that the ZNT1 gene encodes a saturable Zn2+ transporter that has kinetic properties very similar to the Zn transporter characterized in T. caerulescens roots (compare Fig. 2B and Fig. 8A). The Km for Zn2+ transport via ZNT1 was 7.5 µM, which is very similar to that determined for Zn2+ influx into T. caerulescens roots (8 µM; compare Figs 2B and 8A). Thus, it is likely that ZNT1 is involved in the stimulated Zn influx into roots of T. caerulescens. It has often been suggested that the toxic heavy metal Cd is transported into and within plants via endogenous Zn transporters (see, for example, Hart et al., 1998). As shown in Fig. 8B, ZNT1 mediates Cd2+ influx as well as Zn2+ uptake, although with a much lower affinity. This is the first direct evidence that Cd2+ is transported in plants via the native Zn transporters.
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), zhy3 containing the yeast expression vector pFL61; (•), zhy3 containing the ZNT1 cDNA in pFL61. For Zn, ZNT1-dependent uptake displayed saturable Michaelis–Menten kinetics (Vmax=2.2 pmol Zn min-1 10-6 cells; Km=7.5 µM). Cd influx did not conform to Michaelis–Menten kinetics. Data points and error bars represent means and SEs of four replicates.Based on the 4- to 5-fold increase in the Vmax for Zn transport observed in T caerulescens roots compared with T. arvense (Fig. 2), the authors have speculated that the enhanced Zn uptake in roots of T. caerulescens is due to an increased abundance of Zn transporters in root cells. Molecular evidence in support of this was obtained from a Northern analysis (Fig. 9
), where it is apparent that ZNT1 mRNA abundance is much higher in both roots and shoots of T. caerulescens compared with T. arvense. This pattern was obtained regardless whether Northern analysis was conducted using T. caerulescens ZNT1 cDNA (Fig. 9), or a probe representing a ZNT1 homologue from T. arvense (data not shown). Several important pieces of information can be gleaned from the data in Fig. 9. First, Zn transporters are expressed to much higher levels in both roots and shoots of T. caerulescens, and this may be a major reason for the stimulated Zn uptake in roots and shoots of this Thlaspi species. Second, in the ‘normal’ (non-accumulator) plants (represented by T. arvense), Zn transporter genes are expressed at very low levels in Zn sufficient plants. Imposition of Zn deficiency induces an increased expression of these genes thereby facilitating enhanced Zn absorption. In the Zn hyperaccumulator, T. caerulescens, these Zn transporter genes are expressed at very high levels irrespective of the plant Zn status. Clearly there is an alteration of the signal transduction system linking Zn status to expression of Zn transporter genes in T caerulescens, resulting in increased transporter abundance and, presumably, stimulated Zn uptake. The mechanistic basis of the Zn regulation of transporter gene expression and the nature of the alterations in this system in T. caerulescens are currently being investigating.
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Summary |
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TopAbstractIntroductionPhysiological investigations of...Molecular basis of zinc...SummaryReferences |
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The physiological investigations of Zn hyperaccumulation in the two Thlaspi species have shown that a number of Zn transport sites are stimulated or altered in T. caerulescens, contributing to Zn hyperaccumulation. The stimulated transport processes include Zn influx into both root and leaf cells, and Zn loading into the xylem. Additionally, compartmental analysis showed that Zn was sequestered in the root vacuole of T. arvense, which retarded Zn translocation to the shoot in this nonaccumulator species.
Molecular studies focused on the cloning and characterization of Zn transport genes in T. caerulescens. Complementation of a yeast Zn transport-defective mutant with a T. caerulescens cDNA library constructed in a yeast expression vector resulted in the cloning of a Zn transport cDNA, ZNT1. Sequence analysis of ZNT1 indicated it is a member of a recently discovered micronutrient transport gene family which includes the Arabidopsis Fe transporter, IRT1, and the ZIP Zn transporters. Expression of ZNT1 in yeast permitted a physiological characterization of this transporter. ZNT1 was shown to encode a high affinity Zn transporter which can also mediate low affinity Cd transport. Northern analysis indicated that enhanced Zn transport in T. caerulescens results from a constitutively high expression of ZNT1 in roots and shoots. By contrast, in T. arvense the ZNT1 homologue is expressed to much lower levels and this expression is stimulated by the imposition of Zn deficiency. These studies indicate that alterations in the normal regulation of Zn transporter genes by plant Zn status plays an important role in Zn hyperaccumulation in T. caerulescens. It is likely that this alteration is linked to the tolerance mechanism(s) employed in this species. The mechanisms of Zn tolerance in T. caerulescens are a focus of ongoing research in the laboratory.
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1 To whom correspondence should be addressed. Fax: +1 607 255 1132. E-mail: lvk1@cornell.edu
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