Silver ions disrupt K⁺ homeostasis and cellular integrity in intact barley (Hordeum vulgare L.) roots

Abstract

The heavy metals silver, gold, and mercury can strongly inhibit aquaporin-mediated water flow across plant cell membranes, but critical examinations of their side effects are rare. Here, the short-lived radiotracer (42)K is used to demonstrate that these metals, especially silver, profoundly change potassium homeostasis in roots of intact barley (Hordeum vulgare L.) plants, by altering unidirectional K(+) fluxes. Doses as low as 5 μM AgNO(3) rapidly reduced K(+) influx to 5% that of controls, and brought about pronounced and immediate increases in K(+) efflux, while higher doses of Au(3+) and Hg(2+) were required to produce similar responses. Reduced influx and enhanced efflux of K(+) resulted in a net loss of >40% of root tissue K(+) during a 15 min application of 500 μM AgNO(3), comprising the entire cytosolic potassium pool and about a third of the vacuolar pool. Silver also brought about major losses of UV-absorbing compounds, total electrolytes, and NH(4)(+). Co-application, with silver, of the channel blockers Cs(+), TEA(+), or Ca(2+), did not affect the enhanced efflux, ruling out the involvement of outwardly rectifying ion channels. Taken together with an examination of propidium iodide staining under confocal microscopy, the results indicate that silver ions affect K(+) homeostasis by directly inhibiting K(+) influx at lower concentrations, and indirectly inhibiting K(+) influx and enhancing K(+) efflux, via membrane destruction, at higher concentrations. Ni(2+), Cd(2+), and Pb(2+), three heavy metals not generally known to affect aquaporins, did not enhance K(+) efflux or cause propidium iodide incorporation. The study reveals strong and previously unknown effects of major aquaporin inhibitors and recommends caution in their application.

Fig. 1.

Response of NH₃/NH₄⁺ influx into roots of intact barley (Hordeum vulgare L.) seedlings to 10 min incubation in 500 μM Ag⁺. Inset: response of ¹³NH₃/¹³NH₄⁺ efflux from roots of intact barley seedlings to sudden provision (at elution time=15 min, see arrow) of 500 μM Ag⁺. Asterisk represents a significantly different mean from control (t-test, P <0.05). Each treatment represents the mean of three replicates. Error bars indicate ±SEM.

Fig. 2.

Response of K⁺ influx into roots of intact barley (Hordeum vulgare L.) seedlings to 10 min incubation in Ag⁺, Hg²⁺, and Au³⁺ (at 5, 50, and 500 μM), and Pb²⁺, Cd²⁺, and Ni²⁺ (at 500 μM). Asterisks represent significantly different means from control (one-way ANOVA with Dunnett's post-test). Each treatment represents the mean of 4–7 replicates. Error bars indicate ±SEM.

Fig. 3.

Response of ⁴²K⁺ efflux from roots of intact barley (Hordeum vulgare L.) seedlings to sudden provision (see arrow) of (a) Ag⁺, Hg²⁺, and Au³⁺ (at 5, 50, and 500 μM), and 160 mM Na⁺; (b) 10 μM acetazolamide, zonisamide, and forskolin.; (c) 500 μM Pb²⁺, Cd²⁺, and Ni²⁺. In the internal legend, the numbers in parentheses indicate the amount of K⁺ released during a specific treatment (t=16.5–29.5 min), in μmol g (root FW)⁻¹. Each plot represents the mean of 3–8 replicates. Error bars indicate ±SEM. (This figure is available in colour at JXB online.)

Fig. 4.

Response of ⁴²K⁺ efflux from roots of intact barley (Hordeum vulgare L.) seedlings to sudden provision (see arrow) of 500 μM Ag⁺ in combination with the channel inhibitors Cs⁺ (10 mM, as Cs₂SO₄), TEA⁺ (10 mM, as TEA-NO₃), and Ca²⁺ (5 mM, as CaSO₄). Each plot represents the mean of 3–8 replicates. Error bars indicate ±SEM. (This figure is available in colour at JXB online.)

Fig. 5.

Illustration of the integration technique employed to quantify total K⁺ released during treatment with 500 μM Ag⁺. As indicated in the equation, K⁺ released is determined by the summation of counts released per gram during Ag⁺ treatment (shaded area), divided by the corrected specific activity of the cytosol, where SA_o represents the specific activity of the loading solution, k the rate constant representing the slope of the semi-logarithmic regression line of the slowest exchanging (cytosolic) phase, t the loading time (60 min), and t' the time between the start of the elution series and the beginning of the treatment (15.5 min). Based on eight replicates, 4.38±0.22 μmol K⁺ g⁻¹ was released from seedlings treated with 500 μM Ag⁺ for 15 min. Note that this graph is prematurely truncated; however, a few experiments involving longer term (45 min) treatment and elution were conducted, and showed that ∼25% additional K⁺ was available for release.

Fig. 6.

Confocal micrographs showing propidium iodide staining of the cell wall and nuclei of damaged cells from lateral root tips of intact barley (Hordeum vulgare L.) seedlings treated for 15 min in (A) control, (B) 5 μM Ag⁺, (C) 500 μM Ag⁺, (D) 500 μM Hg²⁺, (E) 500 μM Au³⁺, (F) 500 μM Cd²⁺, (G) 500 μM Ni²⁺, (H) 500 μM Pb²⁺, and (I) 160 mM Na⁺. Scale bars represent 20 μm. (This figure is available in colour at JXB online.)