The role of free histidine in xylem loading of nickel in Alyssum lesbiacum and Brassica juncea

Abstract

Exposure of the hyperaccumulator Alyssum lesbiacum to nickel (Ni) is known to result in a dose-dependent increase in xylem sap concentrations of Ni and the chelator free histidine (His). Addition of equimolar concentrations of exogenous L-His to an Ni-amended hydroponic rooting medium enhances Ni flux into the xylem in the nonaccumulator Alyssum montanum, and, as reported here, in Brassica juncea L. cv Vitasso. In B. juncea, reducing the entry of L-His into the root by supplying D-His instead of L-His, or L-His in the presence of a 10-fold excess of L-alanine, did not affect root Ni uptake, but reduced Ni release into the xylem. Compared with B. juncea, root His concentrations were constitutively about 4.4-fold higher in A. lesbiacum, and did not increase within 9 h of exposure to Ni. Cycloheximide did not affect root His or Ni concentrations, but strongly decreased the release of His and Ni from the root into the xylem of A. lesbiacum, whereas xylem sap concentrations of Ca and Mg remained unaffected. Near-quantitative chelation of Ni with nitrilotriacetate in the rooting medium did not enhance Ni flux into the xylem of A. lesbiacum and B. juncea, suggesting the absence of a significant apoplastic pathway for Ni entry into the xylem. The data suggest that in B. juncea roots, Ni(2+) uptake is independent of simultaneous uptake of His. In both species, enhanced release of Ni into the xylem is associated with concurrent release of His from an increased root free His pool.

Figure 1

Time course of the effect of Ni exposure on Ni and His concentrations in the xylem sap of A. lesbiacum. Values are arithmetic means ± sd of n = 3 replicates. The hydroponic root medium of 8-week-old plants was supplemented with 300 μm Ni for between 0 and 8 h. Subsequently, the shoots were cut off to collect xylem sap as root pressure exudates for 5 h, while Ni exposure continued. Each replicate value corresponds to pooled xylem sap from three plants in one culture vessel. Data are from one experiment representative of a total of two independent experiments, with three replicate culture vessels per treatment in each experiment.

Figure 2

Effect of CHX on Ni and His concentrations in xylem sap (a) and in root fresh biomass (b; fresh weight) of A. lesbiacum. Where indicated, the hydroponic root medium of 8-week-old plants was supplemented with 5 μm CHX for 1 h before addition of 300 μm Ni (controls correspond to no additions). Values are arithmetic means ± sd of n = 6 replicates. Each replicate value corresponds to pooled samples from three plants in one culture vessel. For each compound analyzed, different characters indicate that mean values are significantly different at P < 0.05 (uppercase for His and lowercase for Ni). Data are from two independent experiments with three replicate culture vessels per treatment in each experiment. n.d., Not detectable.

Figure 3

Ni and His concentrations in xylem sap (a) and in root fresh biomass (b; fresh weight) of B. juncea. The hydroponic root medium of 6-week-old plants was supplemented with various solutes, i.e. none (control), 300 μm Ni, 300 μm Ni and 300 μm l-His, 300 μm Ni preceded by a pre-exposure to 1 mm l-His for 4 h (Pre-l-His + Ni), 300 μm Ni and 300 μm d-His, or 300 μm Ni and 300 μm l-His with an excess of 3 mm Ala. Values are arithmetic means ± sd of n = 6 replicates, and means ± sd of n = 3 replicates for the Ni + l-His + Ala and the Ni + d-His treatments. Each replicate value corresponds to pooled samples from three plants in one culture vessel. For each compound analyzed, different characters indicate that mean values are significantly different at P < 0.05 (uppercase for His and lowercase for Ni). Two independent experiments were performed with three replicate culture vessels per treatment in each experiment, except for the Ni + l-His + Ala treatment, which was not repeated in an independent experiment. n.a., Not analyzed; n.d., not detectable.

Figure 4

Effect of addition of NTA to the hydroponic root medium on Ni concentration in xylem sap (a) and in root fresh biomass (b; fresh weight) of B. juncea (left) and A. lesbiacum (right). The hydroponic medium was supplemented with 300 μm Ni, 300 μm Ni + 300 μm l-His, 300 μm Ni + 300 μm NTA, 600 μm Ni + 300 μm l-His + 300 μm NTA (Ni + L-His + Ni + NTA), or 300 μm Ni + 300 μm NTA + 300 μm l-His (Ni + NTA + L-His). Values are arithmetic means ± sd of n = 3 replicates for B. juncea and n = 6 replicates for A. lesbiacum. Each replicate value corresponds to pooled samples from three plants per culture vessel. Different characters indicate means that are significantly different at P < 0.05. Data are from two independent experiments for A. lesbiacum and from one for B. juncea, with three replicate culture vessels per treatment in each experiment. n.a., Not analyzed; n.d., not detectable.