Rapid, futile K+ cycling and pool-size dynamics define low-affinity potassium transport in barley

Abstract

Using the short-lived radiotracer 42K+, we present a comprehensive subcellular flux analysis of low-affinity K+ transport in plants. We overturn the paradigm of cytosolic K+ pool-size homeostasis and demonstrate that low-affinity K+ transport is characterized by futile cycling of K+ at the plasma membrane. Using two methods of compartmental analysis in intact seedlings of barley (Hordeum vulgare L. cv Klondike), we present data for steady-state unidirectional influx, efflux, net flux, cytosolic pool size, and exchange kinetics, and show that, with increasing external [K+] ([K+]ext), both influx and efflux increase dramatically, and that the ratio of efflux to influx exceeds 70% at [K+]ext > or = 20 mm. Increasing [K+]ext, furthermore, leads to a shortening of the half-time for cytosolic K+ exchange, to values 2 to 3 times lower than are characteristic of high-affinity transport. Cytosolic K+ concentrations are shown to vary between 40 and 200 mm, depending on [K+]ext, on nitrogen treatment (NO3- or NH4+), and on the dominant mode of transport (high- or low-affinity transport), illustrating the dynamic nature of the cytosolic K+ pool, rather than its homeostatic maintenance. Based on measurements of trans-plasma membrane electrical potential, estimates of cytosolic K+ pool size, and the magnitude of unidirectional K+ fluxes, we describe efflux as the most energetically demanding of the cellular K+ fluxes that constitute low-affinity transport.

Figure 1.

Comparison of ⁴²K⁺ efflux patterns, as determined by tracer elution, in roots of barley seedlings grown under five K⁺ concentrations and two N treatments, NH₄⁺ (A) and NO₃⁻ (B). Plots have been corrected for differences in root mass and tracer activity (to the arbitrary value of 2 × 10⁵ cpm μmol⁻¹). Each point is the mean ± SEM of six to 17 replicates (SEM was, on average, <11% of the mean). Cytosolic exchange half-times are listed in parentheses (SEM < 15% of the mean).

Figure 2.

Comparison of ⁴²K⁺ accrual in growth solution surrounding roots of barley seedlings grown under five K⁺ concentrations and two N treatments, NH₄⁺ (A) and NO₃⁻ (B). Plots have been corrected for differences in root mass and tracer activity (to the arbitrary value of 2 × 10⁵ cpm μmol⁻¹). Each point is the mean ± SEM of three to 12 replicates (SEM was, on average, <10% of the mean). Cytosolic exchange half-times are listed in parentheses (SEM was <15% of the mean).

Figure 3.

Comparison of K⁺ component fluxes, as determined by elution (A) and subsampling (B), at five external K⁺ concentrations (as indicated, in mm) with two N sources (10 mm NH₄⁺ or NO₃⁻). Bars are divided into net flux (black segments) and efflux (white segments), which together comprise the influx term. Error bars refer to ±SEM of three to 17 replicates.

Figure 4.

Ratio of ⁴²K⁺ captured in the initial eluate during a subsampling protocol to tracer remaining in roots after elution. Values are corrected for root mass and tracer activity. Black squares refer to seedlings grown with NH₄⁺; white squares refer to NO₃⁻. Each point is the mean ± SEM of three to 16 replicates.

Figure 5.

K⁺ tissue content of seedlings grown at five external K⁺ concentrations (as indicated, in mm) and two N sources (10 mm NH₄⁺ or NO₃⁻). Root content is represented by white bars, shoot content by gray bars, and whole-plant content by black bars. Error bars refer to ±SEM of six to 12 replicates. Root contents are expressed per gram root, shoot contents are expressed per gram shoot, and whole-plant contents are expressed per gram total tissue.

Figure 6.

K⁺ concentration of root cytosolic compartment ([K⁺]_cyt) for barley seedlings grown at five external K⁺ concentrations (as indicated, in mm) and two N sources (10 mm NH₄⁺ or NO₃⁻), determined by either elution (A) or subsampling (B) protocols. Error bars refer to ±SEM of six to 17 replicates (A) and ±SEM of three to six replicates (B). Different letters refer to significantly different means (P < 0.05, tested by ANOVA).

Figure 7.

Energy necessary to drive K⁺ efflux from barley root cells, as determined by either elution (A) or subsampling (B) protocols. Negative values correspond to passive efflux, while positive values correspond to an active efflux. Black squares refer to NH₄⁺-grown plants, and white squares refer to NO₃⁻-grown plants.