Does zinc move apoplastically to the xylem in roots of Thlaspi caerulescens?

Thlaspi caerulescens can accumulate very large amounts of zinc in its tissues, which is of significant interest in commercial strategies for obtaining plants able to remove Zn from contaminated land – phytoremediation (Chaney, 1993; Salt et al., 1998; Schat et al., 2000). An understanding of the mechanisms controlling Zn entry into the root and the accumulation of Zn within the shoot of such hyperaccumulators can inform both genetic modification and conventional breeding strategies to obtain plants with improved phytoremediation potential (Lasat & Kochian, 2000). However, at present these mechanisms are not clear. Zinc may reach the xylem (and hence the shoot) either through the symplast or apoplast. Current hypotheses suggest that all Zn reaches the xylem through a symplastic pathway and that hyperaccumulation in T. caerulescens is the result of enhanced unidirectional influx of Zn2+ into root cells, coupled to a greater Zn efflux to the xylem. Here, two arguments are presented that suggest that Zn movement to the xylem cannot be solely symplastic when roots are exposed to high Zn concentrations in the rhizosphere ([Zn]ext). First, the rate of delivery of Zn to the xylem may exceed Zn influx to root cells. Second, Zn influx to root cells cannot supply sufficient Zn for hyperaccumulation with the combination of relative growth rates and shoot : root ratios observed in T. caerulescens. A Zn hyperaccumulator is defined as a plant with a shoot Zn content (tissue concentration) in excess of 10 mg Zn g−1 d. wt when growing in its natural habitat (Baker & Brooks, 1989). They appear to have unusually active mechanisms for Zn uptake and translocation to the shoot, as well as the ability to detoxify excessive Zn2+ within the shoot. Eleven Zn hyperaccumulators have been recorded, of which Thlaspi caerulescens is the most studied (Baker et al., 1994; Brooks, 1998; Reeves & Baker, 2000; Broadley et al., 2001). This short-lived perennial occurs mainly on calamine soils (enriched with Zn, Pb and often Cd), but also occurs on serpentine (enriched in Co, Cr, Fe, Mg, Ni) and nonmineralised soils. It is extremely tolerant of Zn and, when grown hydroponically, can accumulate up to 25–30 mg Zn g−1 shoot d. wt without showing toxicity symptoms (Baker et al., 1994; Brown et al., 1995; Pollard & Baker, 1996; Shen et al., 1997; Zhao et al., 1998). It is thought that T. caerulescens detoxifies Zn2+ in the shoot by decreasing its physiological availability within the cytoplasm, either by chelation or by sequestration within the vacuole (Shen et al., 1997). Studies using energy-dispersive X-ray microanalysis to determine the spatial distribution of Zn within leaves of T. caerulescens concur with these conclusions (Frey et al., 2000). The characteristics of Zn tolerance and Zn hyperaccumulation in T. caerulescens are genetically determined and genetically independent (Baker et al., 1994; Pollard & Baker, 1996; Meerts & Van Isacker, 1997; Schat et al., 2000). Thus, the selection and breeding for optimal combinations of these traits could lead to improved varieties for phytoremediation. Zinc may traverse the root to the xylem either through the cytoplasmic continuum of root cells linked by plasmodesmata (symplast) or through the extracellular spaces between cells (apoplast). The symplastic pathway selects between cations, since it involves specific transporters in the plasma membrane of root cells. The apoplastic cation flux is largely determined by the cation exchange properties of the cell wall and by water flows (Sattelmacher, 2001). Despite decades of research, remarkably little is known about the relative contribution of the symplastic and apoplastic pathways to the delivery of particular cations to the xylem (White, 2001). Recently, Lasat & Kochian (2000) presented a schematic model of Zn fluxes across root cell membranes to account for the phenomenon of hyperaccumulation in T. caerulescens (Box 1). This model contrasts the magnitude of the Zn fluxes across the cell membranes of T. caerulescens with those across the cell membranes of the related nonhyperaccumulator species T. arvense. It is postulated that the entry point for Zn accumulation in the plant is across the plasma membrane of root cells and that all Zn reaches the xylem through a symplastic pathway in both species (Lasat et al., 1996; Lasat & Kochian, 2000). It is proposed that hyperaccumulation can be explained by a fourfold greater unidirectional influx (φoc) of Zn2+ into root cells, coupled to a greater Zn efflux to the xylem (φcx), in T. caerulescens than in T. arvense. In the xylem sap of T. caerulescens much Zn is complexed to organic acids (Lasat et al., 1998; Salt et al., 1999) or histidine (Schat et al., 2000); Schat et al. (2000) speculate that interspecific variation in the xylem concentrations of these solutes may also contribute to the trait of Zn hyperacumulation. In addition, it is proposed that T. arvense sequesters more Zn in the vacuoles of root cells, having a greater unidirectional influx (φcv) and reduced unidirectional efflux (φvc) from the vacuole than T. caerulescens. The unidirectional efflux (φco) of Zn from the root cytoplasm to the solution was found to be similar in the two species. Nomenclature and schematic representation of Zn fluxes across the root of Thlaspi caerulescens and Thlaspi arvense according to the model of Lasat & Kochian (2000). (a) The symplastic pathway across the root can be represented by unidirectional (φ) and net (J) fluxes across the plasma membrane between the cytoplasm (c) and the external solution (o) or xylem (x) and across the tonoplast between the cytoplasm (c) and the vacuole (v) of root cells. The apoplast of cortex and stele are hydraulically separated by the Casparian band. (b,c) The trait of hyperaccumulation is thought to result from greater Zn influx across the plasma membrane (φoc), reduced Zn influx to the vacuole (φcv) and greater Zn efflux to the xylem (φcx) in roots of T. caerulescens than in roots of T. arvense. The model presented by Lasat & Kochian (2000) considers only the symplastic movement of Zn across the root. However, Zn may also reach the xylem via an apoplastic pathway. It is noteworthy that, when supplied with Zn, the Zn content of shoots of T. caerulescens reaches that of major nutrient elements, such as Ca. Furthermore, like Ca2+, a low cytoplasmic Zn2+ concentration in root cells is maintained (Brune et al., 1994; Reid et al., 1996). The total cytoplasmic Zn concentration in plant cells appears to be homeostatically maintained at approximately 55 µM (Brune et al., 1994; Reid et al., 1996). To deliver Zn to the xylem stream, which may contain in excess of 500 µM Zn2+ (Lasat et al., 1998), requires active transport across the plasma membrane of cells within the stele. This may be effected by a CPx-ATPase or by a H+/Zn2+ antiport (Williams et al., 2000; Clemens, 2001). It has been argued that such mechanisms are kinetically challenged for Ca2+ delivery to the xylem (White, 1998, 2001), and this is likely also to be true for Zn2+. Thus, by analogy with Ca2+, it is likely that an apoplastic pathway contributes to the delivery of Zn2+ to the xylem. If this is the case, then differences in the apoplastic movement of Zn across the root may impact on the trait of hyperaccumulation. Here, a simple test of the hypothesis that all Zn is delivered to the xylem by a symplastic pathway is presented: if the symplastic pathway operates exclusively, it is impossible for the Zn flux to the xylem (φcx) to exceed the unidirectional Zn influx to root cells (φoc). This test employs published data. In addition, the physiological characteristics (relative growth rate, RGR, and shoot : root ratio) that would be required if all the Zn accumulated by the shoot of T. caerulescens were delivered to the xylem by a symplastic pathway across the root have been determined. These are compared with published RGR and shoot : root ratios for T. caerulescens. Unidirectional influx (φoc) has been estimated by allowing intact plants to accumulate Zn from solutions containing the radioisotope 65Zn for a period of 20 min and then washing briefly in solutions containing nonradioactive Zn (Lasat et al., 1996; Pence et al., 2000; Lombi et al., 2001). Following this procedure, the dependence of Zn accumulation by T. caerulescens (A) on the solution Zn2+ concentration ([Zn]ext) can be fitted to the sum of a saturatable (Michaelis–Menten) and a linear component: A = ((Vmax[Zn]ext)/(Km + [Zn]ext)) + k[Zn]ext (Km, the Michaelis constant; Vmax, the maximal rate of Zn influx through the Michaelian component; and k, the proportionality constant of the linear component.) It is argued that the linear component reflects 65Zn bound to the cell wall rather than influx to the root symplast (Lasat et al., 1996; Lombi et al., 2001). Thus, only the Michaelian component reflects Zn influx (φoc) to the root symplast. The Michaelian component saturates at low micromolar [Zn]ext (Table 1). This is a significant observation when one considers that Zn translocation to the shoot does not saturate even at millimolar [Zn]ext when plants are grown hydroponically (Brown et al., 1995; Tolràet al., 1996, 2001; Shen et al., 1997; Zhao et al., 1998; Schat et al., 2000) or with 5 mg g−1 extractable Zn in the soil (Robinson et al., 1998; Küpper et al., 1999). (Since experiments suggest a maximum value of about 1–3% for shoot Zn accumulation in T. caerulescens, it should be noted that the [Zn]ext at which the shoot approaches this maximal content will be determined by growth characteristics, such as RGR and shoot : root ratio, in addition to the kinetics of Zn translocation from the root (Fig. 2): this may explain the observations of Knight et al. (1997), who reported saturation of Zn translocation to the shoot at lower [Zn]ext.) Since the Vmax for Zn influx (φoc) to roots decreases in plants grown at higher [Zn]ext (Table 1), the apparent Km is even lower under steady state conditions (Fig. 1). The relationships between the maximum shoot Zn content ([Zn]shoot) of plants with relative growth rates of 0.03, 0.05 and 0.15 d−1 and (a) shoot f. wt : root f. wt ratio, assuming Zn influx to roots (φoc) of 80 nmol h−1 g−1 f. wt root, or (b) Zn influx to roots (φoc), assuming a shoot f. wt : root f. wt ratio of 3. The minimal Zn content of 32 µmol g−1 f. wt shoot for a Zn hyperaccumulating Thlaspi caerulescens is indicated by the horizontal line. The relationship between unidirectional Zn influx (φoc) to roots, or Zn translocation to the shoot (φcx), in intact Thlaspi caerulescens growing hydroponically and the Zn concentration of the solution ([Zn]ext). Values for Zn influx (o) were calculated from the data presented in Table 1 from studies by Lasat et al. (1996) and Pence et al. (2000). The curve for Zn influx is interpolated to 1 µM [Zn]ext based on a Km of 7 µM and a Vmax of 270 nmol g−1 f. wt h−1 and extrapolated from 50 µM [Zn]ext based on a Km of 5 µM and a Vmax of 76 nmol g−1 f. wt h−1. Values for Zn translocation to the shoot (closed circles) were calculated from the data supplied by Lasat et al. (1996) as described in the text. In addition to studying Zn influx to roots, Lasat et al. (1996) obtained data (i) on the accumulation of Zn in the shoot of 35 day-old T. caerulescens seedlings transferred to a solution containing 10 µM [Zn]ext over a 96-h period (14.7 nmol h−1 g−1 f. wt shoot), which allows an estimate of the rate of Zn translocation to the shoot at this [Zn]ext, and (ii) on the Zn content of the shoot of 32-d-old T. caerulescens seedlings that had been transferred to solutions containing various [Zn]ext for a 10-d period. The latter data allow the calculation of the rate of Zn translocation to the shoot at other [Zn]ext provided that the shoot mass, shoot f. wt : root f. wt ratio and tissue f. wt : d. wt ratios did not change with [Zn]ext, which seems reasonable under the conditions of these experiments (Brown et al., 1995; Shen et al., 1997; Zhao et al., 1998). Assuming a shoot f. wt : root f. wt ratio of 3.1 for T. caerulescens growing hydroponically (S. N. Whiting, unpublished), the rate of Zn translocation to the shoot at 10 µM [Zn]ext approximates 45.6 nmol h−1 g−1 FW root (Fig. 1), and increases substantially at higher [Zn]ext. This is consistent with direct experimental observations (Brown et al., 1995; Tolràet al., 1996, 2001; Shen et al., 1997; Zhao et al., 1998; Schat et al., 2000). At [Zn]ext greater than about 27 µM the predicted rate of Zn translocation to the shoot exceeded that of Zn influx to the roots (Fig. 1). Thus, at [Zn]ext > 27 µM an apoplastic pathway must contribute to the delivery of Zn to the xylem. For comparison, soils from seven European sites contaminated by industrial activity or the disposal of sewage sludge had up to 20 µM Zn in solution when determined at 80% field water holding capacity (Knight et al., 1997). If additional, low-affinity Zn transporters contributed to Zn influx across the plasma membrane of root cells, then these calculations would underestimate the potential symplastic Zn flux to the xylem, especially at higher [Zn]ext. It is possible that part of the (substantial) linear component to the dependence of Zn accumulation by roots of intact plants and [Zn]ext (Lasat et al., 1996; Lombi et al., 2001) is due to low-affinity Zn influx into root cells. However, this linear component principally suggests the presence of a large apoplastic reservoir for Zn, which does not differ between T. caerulescens and T. arvense (Lasat et al., 1996). It would seem worthwhile therefore to probe the relationship between this component and any apoplastic pathway for Zn movement to the xylem. Since the symplastic pathway selects between divalent cations, but the apoplastic pathway does not, an estimate of the relative importance of symplastic and apoplastic pathways can be obtained by comparing the accumulation factors (shoot cation content/solution cation concentration) for different divalent cations. At low [Zn]ext (< 20 µM) the accumulation factor for Zn was far greater than for Ca (Knight et al., 1997). However, the Zn concentration factor decreased as [Zn]ext increased (Knight et al., 1997), and the accumulation ratio for Zn approached the accumulation ratio for Ca in plants grown hydroponically at [Zn]ext between 100 and 500 µM (Tolràet al., 1996). These data suggest an increasing contribution of the apoplastic pathway to the delivery of Zn to the xylem as [Zn]ext increases. An assessment of the contribution of the apoplastic pathway to the delivery of Zn to the xylem in roots of T. caerulescens can also be approached from a separate perspective. The physiological characteristics of T. caerulescens that would be required for a given shoot Zn content ([Zn]shoot) if all the Zn were delivered to the xylem by a symplastic pathway can be determined using simple allometric relationships. If it is assumed that the root Zn content of T. caerulescens growing exponentially at a constant [Zn]ext does not change, and that all the Zn destined for the xylem traverses the root through the symplastic pathway, then the maximal rate at which Zn could be translocated to the shoot (φcx), in the absence of the recirculation of Zn within the plant, equals the rate of Zn influx (φoc) to the root symplast (Box 1). Both φoc and φcx are conventionally expressed on the basis of root FW (R). At a given [Zn]ext, the rate at which Zn is translocated to the shoot (T) at time t is given by the equation: Tt = φcxRt = φocRt If it is also assumed that φcx is constant during exponential growth, then the accumulation of Zn in the shoot follows the exponential equation: Znt = Zn0ebt (Zn0, initial amount of Zn in the shoot; Znt, amount of Zn in the shoot after an interval t; and b, the relative accumulation rate of Zn in the shoot.) The rate at which Zn is translocated to the shoot in the absence of Zn recirculation within the plant equals the rate at which Zn is accumulated in the shoot and is given by: Tt= bZnt= bZn0ebt If it is assumed that both root and shoot have identical relative growth rates (RGR) and that these are identical to the relative accumulation rate for Zn in the shoot (i.e. that the root f. wt : shoot f. wt ratio, R/S, and the shoot Zn content are constant) then: Tt = φcxRt = φcxR0ebt = φcxR/SS0ebt Finally, assuming that the shoot Zn content remains constant ([Zn]shoot= Zn0/S0), then: [Zn]shoot= (R/S)(φoc/b) Using this equation, the effect of shoot f. wt : root f. wt ratio, RGR and φoc on the maximal [Zn]shoot supported by exclusively symplastic Zn transport across the root can be assessed in T. caerulescens. Under most soil conditions it is unlikely that φoc exceeds 80 nmol g−1 f. wt h−1 (Fig. 1; Pence et al., 2000) and this value has been used for the calculations shown in Fig. 2(a). The shoot FW : root FW ratio of T. caerulescens growing hydroponically at low [Zn]ext approximates 3.1 (S. N. Whiting, unpublished). This value is consistent with the shoot d. wt : root d. wt ratios observed in T. caerulescens growing hydroponically at [Zn]ext ≤ 500 µM (2.7–4.8; Shen et al., 1997; Zhao et al., 1998, 2001), although the shoot d. wt : root d. wt ratio increases markedly at higher [Zn]ext and the shoot d. wt : root d. wt ratio of plants growing in contaminated soil may be more than triple this value (McGrath et al., 1997; Whiting et al., 2000). (The f. wt : d. wt ratio of shoots approximates 4.8 and the f. wt : d. wt ratio of roots approximates 5.7 in T. caerulescens growing hydroponically at low [Zn]ext (S. N. Whiting, unpublished)). The RGR of T. caerulescens growing hydroponically can be calculated from the data of Shen et al. (1997) as lying between 0.049 and 0.055 d−1, which is consistent with the RGR of slow-growing, stress-tolerant plant species (Grime & Hunt, 1975). The RGR of plants growing under suboptimal field conditions may, of course, be lower. Based on the definition that a Zn hyperaccumulator has a shoot content in excess of 10 mg Zn g−1 d. wt (Baker & Brooks, 1989) and assuming a f. wt : d. wt ratio of 4.8 for shoots of T. caerulescens grown hydroponically, a possible shoot Zn content of 32 µmol g−1 FW for T. caerulescens growing in Zn rich soils can be calculated. This shoot Zn content can be obtained only in plants combining a low shoot f. wt : root f. wt ratio with a low RGR (Fig. 2a), which may be the reason why metal hyperaccumulators generally grow slowly. However, published data suggest that T. caerulescens does not possess an appropriate combination of shoot f. wt : root f. wt ratio and RGR to allow exclusively symplastic delivery of Zn to the xylem at high [Zn]ext. Indeed, a maximal φoc of 80 nmol g−1 FW h−1 (Fig. 1; Pence et al., 2000) could not sustain the symplastic Zn flux required for Zn hyperaccumulation in plants with a shoot FW : root FW ratio of 3.0 even at RGR as low as 0.03 d−1 (Fig. 2b). An RGR of < 0.02 d−1 would be required if plants with a shoot f. wt : root f. wt ratio of 3.0 and a φoc of 80 nmol g−1 f. wt h−1 were to hyperaccumulate Zn through a symplastic pathway. Thus, the perfectly controlled and selective hyperaccumulation of Zn in the shoot of T. caerulescens seems improbable. Some apoplastic transport of Zn to the xylem is likely to occur at high [Zn]ext and future studies should attempt to ascertain the relative contributions of the symplastic and apoplastic pathways to the delivery of Zn to the xylem in roots of T. caerulescens. Previously, it was assumed that all Zn entering the xylem had traversed the root through a symplastic pathway. The arguments presented here challenge this assumption and suggest that some Zn may reach the xylem through an apoplastic pathway, especially at higher [Zn]ext. This has important implications for identifying the targets for improving phytoremediation potential: targets of the symplastic pathway are the Zn transporters in cell membranes, but targets of the apoplastic pathway may be more diverse. Molecular targets for the manipulation of symplastic Zn fluxes across the root to the xylem are beginning to be identified. Genes for several Zn transporters likely to mediate Zn influx to root cells of T. caerulescens have been cloned. These include the high-affinity Zn transporters ZNT1 and ZNT2 (Pence et al., 2000; Assunção et al., 2001). The of both ZNT1 and ZNT2 is by Zn which is consistent with an in the Vmax for Zn influx to roots of plants with a reduced Zn (Table 1), and far greater in T. caerulescens than in T. which is consistent with the greater Zn influx capacity of T. caerulescens (Table 1). The efflux of Zn to the xylem across the plasma membrane of cells within the stele of the root is likely to be by a CPx-ATPase or by a H+/Zn2+ antiport (Williams et al., 2000; Clemens, 2001), but little is known about these transport Molecular and physiological targets for the manipulation of apoplastic Zn fluxes to the xylem have to be identified. However, experimental strategies for the of such targets could be based on the apparent of cation and between cations a apoplastic to the xylem (White, 2001). The trait of cation could be with appropriate such as the of the within a Broadley et al., 2001). trait on Zn hyperaccumulation could be in for this between in Zn hyperaccumulation & Baker, 1996; Meerts & Van Isacker, 1997). These approaches would independent of the cations an apoplastic pathway, into the of apoplastic cation fluxes and on the trait of Zn hyperaccumulation. This was supported by the and and for