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A Role for the AKT1 Potassium Channel in Plant Nutrition

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Science  08 May 1998:
Vol. 280, Issue 5365, pp. 918-921
DOI: 10.1126/science.280.5365.918

Abstract

In plants, potassium serves an essential role as an osmoticum and charge carrier. Its uptake by roots occurs by poorly defined mechanisms. To determine the role of potassium channels in planta, we performed a reverse genetic screen and identified an Arabidopsis thaliana mutant in which the AKT1 channel gene was disrupted. Roots of this mutant lacked inward-rectifying potassium channels and displayed reduced potassium (rubidium-86) uptake. Compared with wild type, mutant plants grew poorly on media with a potassium concentration of 100 micromolar or less. These results and membrane potential measurements suggest that the AKT1 channel mediates potassium uptake from solutions that contain as little as 10 micromolar potassium.

Potassium absorption by roots is essential for plant growth. Current models, which are elaborations of classic studies (1), state that K+ absorption is mediated by cotransporters at micromolar K+concentrations and channels at higher concentrations (2-6). This notion is supported by the finding that plant genes encoding channels or cotransporters could complement yeast K+-uptake mutants (6-8), but such experiments do not address which mechanisms are operating in the plant. Here we report an in planta genetic dissection of the role of the AKT1 channel in the uptake of K+ by a root.

A transferred DNA (T-DNA) mutagenized population ofArabidopsis was screened for plants containing an insertional mutation in the root-specific K+-channel geneAKT1 by using the polymerase chain reaction (PCR)–based, reverse genetic method of Krysan et al. (9, 10). From a population of 14,200 different T-DNA lines, containing about 20,000 independent insertional events, we identified and isolated a single mutant plant (akt1-1) with a T-DNA insertion inAKT1. Southern blot analysis of the akt1-1 locus (11) revealed a T-DNA insertion within the last exon of the coding region (Fig. 1). Sequence analysis reveals the T-DNA insertion site to be 4071 bases downstream of the start codon, and Northern blot analysis confirms that the mutation truncates the transcript by about 400 bases (12).

Figure 1

Analysis of the AKT1 gene. (A) Southern blot analysis of genomic DNA digested with Bam HI (lanes B), Hind III (lanes H), or Eco RI (lanes E) and hybridized with radiolabeled DNA corresponding to the AKT1coding region. (B) Restriction map of AKT1genomic DNA based on Southern analysis (A) and sequence analysis (12). Large and small boxes represent exons and introns, respectively. Structure of the T-DNA insertion 3′ to the Bam HI site is undefined. T-DNA is not drawn to scale.

We examined the K+ conductance of the plasma membrane in root cells, where high expression of AKT1 was previously found (13). Microelectrodes inserted into cells approximately 150 μm from the apex of roots, which were bathed in 10 μM K+ (14), revealed very negative resting membrane potentials (V m) in both wild-type andakt1-1 seedlings (Fig. 2B). A 10-fold increase in the extracellular K+ concentration significantly depolarized the membrane in wild-type roots but had no effect on V m in akt1-1 seedlings (Fig. 2). These results indicate that AKT1 is responsible for the K+ permeability of these apical root cells. Also, using 80 mM as the cytoplasmic K+concentration (4, 15) in the Nernst equation, it follows that a V m of at least −230 mV is required for K+ uptake to occur passively through a channel. Twenty percent of wild-type cells met this condition without making corrections for the fact that intracellular microelectrodes underestimate V m (16). Our results indicate that in wild-type seedlings passive uptake of K+by AKT1 could occur from extracellular solutions as dilute as 10 μM.

Figure 2

Membrane potential (V m) in apical root cells. (A) Representative recordings of shifts in V m in response to the indicated changes in extracellular [K+] indicated a significant K+ permeability of the wild-type plasma membrane. The much smaller shifts in akt1-1 roots indicate that the K+ permeability was greatly reduced by the mutation. (B) The average steady-stateV m in millivolts (ordinate) obtained at each extracellular [K+] (n = 10 for both wild type and akt1-1). Qualitatively similar results were obtained in 25 additional experiments that are not included here because of slight differences in the ionic conditions used.

Whole-cell recording from wild-type root protoplasts (Fig.3A) showed that voltage steps from −10 mV to positive membrane potentials elicited outward, time-dependent currents (17). Steps to negative voltages always elicited inward currents that in some cells were largely time dependent and in others were only partly so. Evidence that the inward currents in Fig.3A were carried by K+ was obtained by the use of largely impermeant anions in the patch pipette, by their disappearance when extracellular K+ was replaced by Cs+(18), and by the analysis of tail currents shown in Fig. 3B (19). The current-voltage (I-V) relationship for the tail currents reversed within 5 mV of the theoretical value for a K+-selective channel exposed to a threefold K+gradient (E K = −27 mV) (Fig. 3B). Individual 16 pS K+-selective channels in patches of membrane excised from wild-type protoplasts (Fig. 3C) displayed a voltage dependence and activation threshold consistent with their being responsible for the inward currents (Fig. 3, A and B) and the resting K+permeability determined in planta (Fig.2).

Figure 3

K+ currents in wild-type and akt1-1 root cells. (A) Whole-cell recording of K+ currents in wild-type root protoplasts. Voltage-dependent inward and outward currents were observed. (B) Tail current analysis of inward currents in a wild-type protoplast. The magnitude of current flowing at each of the test voltages was measured at the first point where relaxation could be discerned (asterisk) and was plotted versus the test voltage to construct the I-V curve shown. The close agreement between the zero-current voltage (reversal potential) of the I-Vcurve and the equilibrium potential for K+(E K) indicates that the inward-rectifying currents were carried by K+. The experiment shown is representative of three independent trials. (C) Single K+ channels in outside-out patches of plasma membrane excised from wild-type root cells. Open-channel current amplitudes were measured and plotted versus the clamped voltage to construct theI-V curve. The close agreement between the reversal potential and E K indicates that the inward-rectifying currents were carried by K+. The currents were low-pass filtered at 0.5 kHz and digitized at 1 kHz. (D) Whole-cell recording of K+ currents inakt1-1 root protoplasts. Inward currents were absent but outward currents were the same as in wild-type root protoplasts. (E) Steady-state, whole-cell I-V curves reveal the absence of inward currents in akt1-1. The raw currents were normalized relative to the value at +120 mV, averaged (n = 10 for wild type and 6 for akt1-1), and plotted versus the clamp potential. No leak subtraction or other manipulation of the currents was performed. In (A), (D), and (E), voltage steps ranged from +120 to −180 mV in 20-mV increments.

Consistent with the data in Fig. 2, patch-clamp recordings ofakt1-1 root cells revealed no inward currents, although the outward currents were normal (Fig. 3D). The normalized steady-stateI-V relationships shown in Fig. 3E illustrate that theakt1-1 mutation selectively abolished the inward component of the K+ conductance of the membrane. From the electrophysiological results presented in Figs. 2 and 3, we conclude that AKT1 encodes the inward-rectifying channel responsible for the resting K+ permeability of cells in the root apex.

Growth of akt1-1 plants on many nutrient media was indistinguishable from that of wild type. However, growth ofakt1-1 plants on media containing ≤100 μM K+(20) was significantly inhibited compared with wild type in the presence of NH4 + (Fig.4, A and B). At 10 μM, mostakt1-1 seeds failed to fully emerge from the seed coat (Fig.4A), but they resumed normal growth after transplantation to media with high K+ concentration (12). Growth ofakt1-1 seedlings on 1 mM K+ was only slightly reduced relative to wild type (Fig. 4, A and B).

Figure 4

Dependence of seedling growth on K+. (A) Wild-type and akt1-1 plants grown for 9 days on media containing 2 mM NH4H2PO4 and KCl at the concentrations indicated. (B) Fresh weight of wild-type andakt1-1 7-day old seedlings. Each value is the mean fresh weight (n = 4) of 25 seedlings ± SEM.

To determine whether the T-DNA insertion cosegregated with the mutant phenotype, we crossed homozygous akt1-1 plants to wild-typeArabidopsis of the same ecotype (WS). Self-crossed F2 seedlings were grown as described (20). The wild-type (normal growth) or mutant (poor growth) phenotype segregated as a single, recessive gene (21). For cosegregation analysis, the genotype of segregating F2 plants was analyzed by PCR using AKT1- (10) and T-DNA-specific primers (9). Twenty of 21 homozygousakt1-1 plants grew poorly, and 31 of 36 plants that were heterozygous or homozygous wild type grew normally. Several homozygous wild-type and homozygous mutant F2 plants were self-crossed; phenotypic analysis demonstrated that >90% of the progeny from the akt1-1 homozygous parents were phenotypically mutant, and 90% of the progeny from wild-type parents were phenotypically wild type. This analysis demonstrates that the mutant phenotype shows >90% penetrance and is genetically linked to the akt1-1 locus.

To determine the effect of the akt1-1 mutation on K+ absorption by roots, we performed86Rb+ tracer flux analysis in roots obtained from plants grown in a high concentration of external K+(22). Uptake rates from media containing various amounts of K+ and NH4 + are shown in Table1. The akt1-1 roots showed less 86Rb+ uptake than wild type. Loss of channel activity in akt1-1 root cells (Fig.3) is thus associated with reduced rates of 86Rb+ uptake from solutions containing only 10 μM Rb+.

Table 1

Radioactive tracer flux analysis of Rb+uptake in akt1-1 and wild-type roots. The uptake solution (22) contained Rb(86Rb)Cl at the concentrations indicated. Each value is the mean rate of uptake (n = 4) ± SEM.

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The notion that a passive transporter such as the AKT1 channel mediates what has previously been termed high-affinity uptake has been suggested (23). Our measurements of membrane potential indicate that it is energetically feasible in the root cells studied here. Dependence of the akt1-1 growth phenotype on NH4 + suggests that this cation inhibits parallel, non-AKT1 K+ uptake pathways, making growth dependent on AKT1. This is consistent with our observation as well as that of others that NH4 + inhibited86Rb+ uptake in wild-type roots (12,24). As reverse genetic strategies identify plants with disruptions in other K+ transporters, analyses of double and triple mutant combinations will directly test this hypothesis.

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