Altered Zn compartmentation in the root symplasm and stimulated Zn absorption into the leaf as mechanisms involved in Zn hyperaccumulation in thlaspi caerulescens

Abstract

We investigated Zn compartmentation in the root, Zn transport into the xylem, and Zn absorption into leaf cells in Thlaspi caerulescens, a Zn-hyperaccumulator species, and compared them with those of a related nonaccumulator species, Thlaspi arvense. 65Zn-compartmental analysis conducted with roots of the two species indicated that a significant fraction of symplasmic Zn was stored in the root vacuole of T. arvense, and presumably became unavailable for loading into the xylem and subsequent translocation to the shoot. In T. caerulescens, however, a smaller fraction of the absorbed Zn was stored in the root vacuole and was readily transported back into the cytoplasm. We conclude that in T. caerulescens, Zn absorbed by roots is readily available for loading into the xylem. This is supported by analysis of xylem exudate collected from detopped Thlaspi species seedlings. When seedlings of the two species were grown on either low (1 &mgr;M) or high (50 &mgr;M) Zn, xylem sap of T. caerulescens contained approximately 5-fold more Zn than that of T. arvense. This increase was not correlated with a stimulated production of any particular organic or amino acid. The capacity of Thlaspi species cells to absorb 65Zn was studied in leaf sections and leaf protoplasts. At low external Zn levels (10 and 100 &mgr;M), there was no difference in leaf Zn uptake between the two Thlaspi species. However, at 1 mM Zn2+, 2.2-fold more Zn accumulated in leaf sections of T. caerulescens. These findings indicate that altered tonoplast Zn transport in root cells and stimulated Zn uptake in leaf cells play a role in the dramatic Zn hyperaccumulation expressed in T. caerulescens.

Figure 1

Short-term efflux of ⁶⁵Zn from roots of T. arvense and T. caerulescens seedlings. After a 24-h incubation in an uptake solution containing 20 μm ⁶⁵Zn²⁺, roots were rinsed in deionized water, and, to initiate ⁶⁵Zn efflux, the radioactive uptake solution was replaced with an identical, nonlabeled solution containing 20 μm Zn²⁺. Efflux of ⁶⁵Zn from roots into the external solution was subsequently monitored for a 6-h period. Lines represent regressions of the linear portion of each curve extrapolated to the y axis. The curve shown in B was derived by subtracting the linear component in A from the data points in A. The curve in C was similarly derived from the curve in B. Data points in A represent means ± se of four replicates.

Figure 2

Long-term efflux of ⁶⁵Zn from roots (A) and ⁶⁵Zn translocation to shoots (B) of T. arvense and T. caerulescens seedlings. Bundles of four T. arvense and T. caerulescens seedlings were immersed with roots in a 20 μm ⁶⁵Zn²⁺ uptake solution. After a 24-h loading period, roots were rinsed in deionized water, and the radioactive uptake solution was replaced with an identical, nonlabeled solution containing 20 μm Zn²⁺. At different time intervals up to 46 h, one bundle of each Thlaspi species was harvested, roots were excised, blotted, and weighed, and gamma activity was measured. Data points represent means ± se of four replicates. wt, Weight.

Figure 3

Time course of ⁶⁵Zn accumulation in leaf sections of the two Thlaspi species. Leaves of T. arvense and T. caerulescens seedlings were cut into 10- to 20-mm² sections and immersed in an uptake solution containing 2 mm Mes-Tris buffer, pH 6.0, 0.5 mm CaCl₂ and 10 μm (A), 100 μm (B), or 1000 μm (C) ⁶⁵ZnCl₂. After exposures for up to 48 h, the radioactive uptake solution was replaced with a solution consisting of 5 mm Mes-Tris, pH 6.0, 5 mm CaCl₂, and 100 μm ZnCl₂ and allowed to desorb for 15 min. Leaf sections were then harvested, blotted, and weighed, and the gamma activity was measured. Data points represent means ± se of four replicates.

Figure 4

Time course of ⁶⁵Zn accumulation in protoplasts isolated from the two Thlaspi species. Protoplasts isolated from T. arvense and T. caerulescens leaves were suspended in an uptake buffer containing 10 μm (A), 100 μm (B), or 1000 μm (C) ⁶⁵ZnCl₂. At different time intervals up to 12 min, an aliquot of the uptake suspension was collected and placed on top of a discontinuous gradient consisting of 50 μL of 10% (v/v) HClO₄ on top of 400 μL of silicon oil, and the protoplasts were pelleted by centrifugation. The tube was then frozen in liquid N₂ and the tip containing the pellet was cut and placed in a vial, and the gamma activity was measured. For the experiments with CCCP, protoplasts were exposed to 10 μm CCCP for 30 min before the uptake experiment.

Figure 5

Time course of ⁶⁵Zn uptake in intact and ruptured T. arvense protoplasts. Protoplasts were ruptured by freezing in liquid N₂. The uptake solution contained 10 μm ⁶⁵Zn²⁺. At different time intervals up to 12 min, an aliquot of the uptake suspension was removed and placed on top of a discontinuous gradient consisting of 50 μL of 10% (v/v) HClO₄ on top of 400 μL of silicon oil, and the protoplasts were pelleted by centrifugation. The tube was then frozen in liquid N₂ and the tip containing the pellet was cut and placed in a vial, and the gamma activity was measured.