Citrate-permeable channels in the plasma membrane of cluster roots from white lupin

Abstract

White lupin (Lupinus albus) is well adapted to phosphorus deficiency by developing cluster roots that release large amounts of citrate into the rhizosphere to mobilize the sparingly soluble phosphorus. To determine the mechanism underlying citrate release from cluster roots, we isolated protoplasts from different types of roots of white lupin plants grown in phosphorus-replete (+P) and phosphorus-deficient (-P) conditions and used the patch-clamp technique to measure the whole-cell currents flowing across plasma membrane of these protoplasts. Two main types of anion conductance were observed in protoplasts prepared from cluster root tissue: (1) an inwardly rectifying anion conductance (IRAC) activated by membrane hyperpolarization, and (2) an outwardly rectifying anion conductance (ORAC) that became more activated with membrane depolarization. Although ORAC was an outward rectifier, it did allow substantial inward current (anion efflux) to occur. Both conductances showed citrate permeability, with IRAC being more selective for citrate3- than Cl- (PCit/PCl = 26.3), while ORAC was selective for Cl- over citrate (PCl/PCit = 3.7). Both IRAC and ORAC were sensitive to the anion channel blocker anthracene-9-carboxylic acid. These currents were also detected in protoplasts derived from noncluster roots of -P plants, as well as from normal (noncluster) roots of plants grown with 25 microm phosphorus (+P). No differences were observed in the magnitude or frequency of IRAC and ORAC currents between the cluster roots and noncluster roots of -P plants. However, the IRAC current from +P plants occurred less frequently than in the -P plants. IRAC was unaffected by external phosphate, but ORAC had reduced inward current (anion efflux) when phosphate was present in the external medium. Our data suggest that IRAC is the main pathway for citrate efflux from white lupin roots, but ORAC may also contribute to citrate efflux.

Figure 1.

Root system of 4-week-old white lupin, grown in the absence of phosphorus, showing the mature cluster root with numerous rootlets and the noncluster root tissue used for the isolation of protoplasts.

Figure 2.

Three types of current detected in lupin roots. Typical examples of the inwardly rectifying current (A), outwardly rectifying current (C), and the pump current (E) in protoplasts from cluster roots of −P plants. Data show superimposed current traces in response to voltage pulses ranging from −151 to 69 mV (A), from −171 to 69 mV (C), and from −191 to 49 mV (E) in 20-mV intervals. Insets in A and C show the activation of the two types of current elicited by hyperpolarizing from 9 to −131 mV (inset A) and from −31 to −171 mV (inset C). D–F, Steady current-voltage curves for currents presented in A, C, and E. Pipette solution was type I; bathing solution was 10 mm CaCl₂, 5 mm MES, pH 6.0.

Figure 3.

Types of current observed in a single protoplast can change. Data show the changes in conductance from the IRAC (A) to ORAC (C) in a single protoplast derived from −P cluster roots when the identical pulse protocols were applied. Recordings were made 5 min (A) and 16 min (C) after the formation of a whole-cell configuration. B, D shows the current-voltage curves constructed using the steady-state currents in A and C, respectively. Pipette and bath solutions were identical to those shown in Figure 2.

Figure 4.

Effects of Gd³⁺ (A) and external CaCl₂ (B) on IRAC. Data show the mean ± se of four protoplasts (A) and three protoplasts (B). Pipette solution was type II; bath solution was the same as in Figure 2.

Figure 5.

Hyperpolarization-activated inward current for a −P cluster root protoplast with the pipette solution containing no Cl⁻. Current traces elicited by voltage pulses ranging from −178 to 58 mV in 20-mV intervals from a holding potential of −2 mV (A). B, Steady-state current-voltage curve for three protoplasts showing the currents in A. Pipette solution was type III; bath solution was the same as in Figure 2.

Figure 6.

Effect of A-9-C on the hyperpolarization-activated inward current in −P cluster root protoplasts. Data show the superimposed current traces in response to voltage pulses ranging from −151 to 49 mV in 20-mV intervals from a holding potential of −1 mV before (A) and 5 min after (B) addition of 100 μm A-9-C. Current-voltage curves from the steady currents recorded before and after the treatment are shown as well as the subtracted current shown as a dashed line (C). Data show the mean ± SEM from four protoplasts. Pipette and bath solutions were identical to those in Figure 4.

Figure 7.

Outwardly rectifying currents in protoplasts from the cluster roots of a −P plant. Data show the superimposed current traces in response to voltage pulses ranging from −171 to 89 mV in 20-mV intervals from a holding potential of −171 mV (A). Tail currents were generated when the membrane was returned to the holding voltage from positive pulses. The tail currents were fitted with a double exponential time course and the time-zero current amplitudes, indicated by arrows, were extrapolated as shown in inset. The downward deflections of the tail current were removed for curve fitting. Relative conductance of the tail currents was plotted against voltage and fitted with the Boltzmann function described by Equation 1 (B). Data show the mean ± SEM from four protoplasts. Pipette and bath solutions were identical to those used in Figure 2.

Figure 8.

Effects of external Cl⁻ and concentrations on the outwardly rectifying current. Data in A and B show the superimposed current traces from a single cluster root protoplast elicited by voltage pulses between −171 to 59 mV in 20-mV intervals from a holding potential of −1 mV. External solutions contained 10 mm CaCl₂ (A) and 1 mm CaCl₂ (B). Steady current-voltage curves from A and B are presented in C. Data in D and E show the superimposed current traces from a single −P cluster root protoplast in response to voltage pulses ranging from −173 to 57 mV in 20-mV intervals from a holding potential of −3 mV. External solutions contained 1 mm Ca-(gluconate)₂ together with either 10 mm KCl (D) or 10 mm KH₂PO₄ (E). Steady current-voltage curves from D and E are presented in F. Pipette solution was type I.

Figure 9.

Effects of A-9-C on the ORAC in protoplasts from cluster roots of −P plants. Data show the superimposed current traces in response to voltage pulses ranging from −151 to 49 mV in 20-mV intervals from a holding potential of −1 mV before (A) and 5 min after (B) addition of 100 μm A-9-C. Current-voltage curves from the steady currents recorded before and after the treatment are shown in (C). Data show the mean ± SEM from four protoplasts.

Figure 10.

K⁺-dependent currents in lupin roots. Superimposed current traces collected from a single cluster root protoplast elicited by voltage pulses ranging from −183 to 37 mV in 20-mV intervals from a holding potential of −43 mV (A). The external solution (mm) was 5 K₂-malate, 1 Ca-(gluconate)₂, 5 MES, pH 6.0. B, Steady-state current-voltage curves measured with 5 and 50 mm K₂-malate in the bath solutions. Data show the mean ± SEM for four to five protoplasts. Pipette solution (mm), 10 KCl, 90 K-Glu, 2 CaCl₂, 2 Na₂ATP, 2 MgCl₂, 10 EGTA, 10 HEPES, adjusted to pH 7.2 with Tris, and osmolality adjusted to 720 mosmol kg⁻¹ with Suc.