Properties of enhanced tonoplast zinc transport in naturally selected zinc-tolerant silene vulgaris

Abstract

It was demonstrated recently that isolated tonoplast vesicles derived from plants of a Zn-tolerant ecotype of Silene vulgaris accumulate more Zn than vesicles derived from a Zn-sensitive ecotype. We have now characterized the tonoplast-transport system that causes this uptake difference and demonstrated its genetic correlation to Zn tolerance using plant crosses. We conclude that the tonoplast Zn uptake system of the tolerant ecotype differs greatly in its characteristics from that of the sensitive one, with the most prominent differences being its insensitivity to protonophores and ortho-vanadate and its stimulation by Mg-GTP. These differences in characteristics are most likely due to the fact that Zn can be taken up by two or more parallel pathways, which are not present in the same proportions in both ecotypes. In both ecotypes, Zn is actively transported across the tonoplast (temperature coefficient > 1.6), most likely as a free ion, since citrate does not accumulate in vesicles. Most importantly, the uptake difference found using the ecotypes was also found between homozygous Zn-tolerant and Zn-sensitive F3 plants, proving the genetic correlation between increased tonoplast Zn transport and naturally selected Zn tolerance in S. vulgaris.

Figure 1

The effect of 0.5 mm Zn citrate on the ΔpH and Δψ of S. vulgaris tonoplast vesicles. Fluorescence (F) of acridine orange and oxonol-V were measured in arbitrary units. A, Effect of 0.5 mm Zn citrate on the ΔpH of tonoplast vesicles of a sensitive and tolerant ecotype of S. vulgaris, measured using acridine orange quenching (this graph is similar for both ecotypes). B and C, Effect of 0.5 mm Zn citrate on the Δψ of tonoplast vesicles of a Zn-sensitive (B) and a Zn-tolerant (C) ecotype was measured using oxonol-V fluorescence. In all cases, Mg-ATP was added immediately after adding the vesicles, and Zn citrate was added after 400 s. After another 100 s, NH₄Cl or gramicidin was added to disrupt the gradient.

Figure 2

The concentration of Zn and citrate in isolated tonoplast vesicles derived from sensitive (A) and tolerant (B) plants. Tonoplast vesicles were incubated with either 0.1 or 0.5 mm Zn citrate in the presence or absence of Mg-ATP. After measuring Zn with flame atomic absorption spectrophotometry, the citrate concentration of the samples was determined using GC (see Methods). Bars represent means of three to six samples. Error bars represent se values. White bars, Zinc (μmol/mg vesicle protein); shaded bars, citrate (μmol/mg vesicle protein).