AtKC1, a silent Arabidopsis potassium channel alpha -subunit modulates root hair K+ influx

Abstract

Ion channels in roots allow the plant to gain access to nutrients. The composition of the individual ion channels and the functional contribution of different alpha-subunits is largely unknown. Focusing on K(+)-selective ion channels, we have characterized AtKC1, a new alpha-subunit from the Arabidopsis shaker-like ion channel family. Promoter-beta-glucuronidase (GUS) studies identified AtKC1 expression predominantly in root hairs and root endodermis. Specific antibodies recognized AtKC1 at the plasma membrane. To analyze further the abundance and the functional contribution of the different K(+) channels alpha-subunits in root cells, we performed real-time reverse transcription-PCR and patch-clamp experiments on isolated root hair protoplasts. Studying all shaker-like ion channel alpha-subunits, we only found the K(+) inward rectifier AtKC1 and AKT1 and the K(+) outward rectifier GORK to be expressed in this cell type. Akt1 knockout plants essentially lacked inward rectifying K(+) currents. In contrast, inward rectifying K(+) currents were present in AtKC1 knockout plants, but fundamentally altered with respect to gating and cation sensitivity. This indicates that the AtKC1 alpha-subunit represents an integral component of functional root hair K(+) uptake channels.

Figure 1

(A) Unrooted phylogenetic tree from all members of the shaker-like channel family of Arabidopsis thaliana. Multiple alignment and distance calculations were performed by the WISCONSIN PACKAGE V.10.0 (Genetics Computer Group, Madison, WI) with default parameters. Graphical presentation was done with TREEVIEW 1.6.5 (http://taxonomy.zoology.gla.ac.uk/rod/rod.html). AKT1 [Munich Information Center for Protein Sequences Arabidopsis thaliana database (MAtDB) protein entry code, At2g26650; AKT2/3 (At4g22200), AKT5 (At2g25600), AKT6 (At4g32500), AtKC1 (At4g32650, also named KAT3 or AKT4), KAT1 (At5g46240), KAT2 (At4g18290), SKOR (At3g02850), GORK (At5g37500, also assigned as SKOR2)]. (B) Position of the footprint mutation in plant line Atkc1-f. The diagram depicts the genomic organization of the AtKC1 gene (accession no. U73325) with exons (black boxes) and introns (lines). The sequence is given in detail below. The four are as follows: the derived amino acid and the nucleotide sequence (DNA) of the AtKC1 wild-type allele (wt) and the nucleotide and the derived amino acid sequence of the Atkc1-f mutant allele (footprint, fp). Gaps in the nucleotide sequence, which were introduced for optimal alignment, are indicated by a period (.). The splice site is indicated by an arrow. The primers for footprint sequencing (At-1, At-2) are indicated.

Figure 2

AtKC1 expression. (A) Northern Blot analysis of AtKC1 transcripts in different Arabidopsis tissues: Si, silique; F, flower; yL, young leaf; oL, old leaf; St, Stem; R, root. Actin controls (data not shown) confirmed that the gels were identically loaded. (B and C) Analysis of AtKC1 promoter activity in transgenic A. thaliana plants. Upon staining with X-gluc, blue-colored tissue represented areas of AtKC1 promoter activity. Staining can be observed in seedlings throughout the whole root (B, Upper Left) and with high intensity in root hairs (B, Upper Right). On semithin cross sections through the root, staining intensity increased from the epidermis (ep) through cortex (co) to the endodermis (en) (B Lower). (C) AtKC1 and AKT1 represent the major K⁺-uptake channel transcripts in roots and root hairs. Quantitative RT-PCR was used on total RNA isolated from either root tissue (black bars) or root-hair protoplasts (gray bars) with specific primers for all inward rectifying members of the plant shaker-like potassium channels. The numbers above the bars represent the calculated numbers of cDNA molecules in the individual probes according to (29).

Figure 3

(A) The diagram depicts the absorbance at a wavelength λ = 280 nm of fractions obtained by free-flow electrophoresis. According to marker enzyme analysis (not shown), single fractions were pooled into A, plasma membrane; C, endoplasmic reticulum and mitochondria; and E, tonoplast as well as three additional intermediate pools (B, D, and F). (B) Western blot probed with an anti-AtKC1 antibody (1:5,000 dilution) with the main signal in the plasma membrane fraction and a weaker signal in the intermediate fraction. (C) Western blot analysis of root microsomes isolated from wildtype (wt) and Atkc1-f Arabidopsis plants. The blot was probed as in B.

Figure 4

Electrophysiological analyses of whole-cell K⁺ currents in Arabidopsis root hair protoplasts. In the whole-cell configuration, voltage pulses (A and B) were applied from a holding potential of −48 mV in 20-mV decrements in the range from +52 to −188 mV, with subsequent voltage steps to −88 mV. (A) Voltage- and time-dependent of inward and outward K⁺ currents in root hair protoplasts from Arabidopsis wild type. (B) Representative outward K⁺ currents in root hair protoplasts from the Arabidopsis akt1–1 knockout mutant. Voltage- and time-dependent inward K⁺ currents in root hair protoplasts from Arabidopsis wildtype (C, E, G, I, and K) and Atkc1-f knockout mutant (D, F, H, J, and L). The voltage-current characteristics of inward K⁺ currents in wild-type (C and E) and Atkc1-f knockout protoplasts (D and F) in the presence of 20 mM (●) or 1 mM (○) CaCl₂. Current amplitudes were sampled at the end of 1-s pulses to different voltages in the range from +12 to −188 mV, applied from a holding potential of −48 mV. The data points represent mean values ± SD for n = 10 protoplasts. Representative whole-cell K⁺ currents in wild-type (C) and Atkc1 knockout (D) protoplasts were measured in an external solution containing 30 mM K-gluconate, 10 mM Mes/Tris (pH 5.6), and 20 mM CaCl₂. Voltage pulses were applied from a holding potential of −48 mV in 20-mV decrements in the range from +12 to −188 mV. Steady-state currents were normalized to currents at −168 mV. (G and H) Effect of pH on inward K⁺ currents in root hair protoplasts from Arabidopsis wildtype and the Atkc1-f mutant. Whole-cell K⁺ currents in wild-type (G) and knockout protoplasts (H) were elicited by voltage pulse to −188 mV from a holding potential −48 mV. External solutions contained 1 mM CaCl₂, 30 mM K-gluconate, and 10 mM Mes/Tris (pH 5.6) or Hepes/Tris (pH 7.0). Current traces represent typical recordings obtained in nine independent experiments. Under these conditions, changes in steady-state current amplitudes in response to alkalization of the bathing medium where −26 ± 5.25% for wildtype and +15 ± 4.24% for Atkc1-f, respectively (values are given in means ± SE). Note the different scales of the current axis. (I–L) Block of inward K⁺ currents by Rb⁺ in root hair protoplasts from Arabidopsis wild-type and the Atkc1-f mutant. Whole-cell K⁺ currents in wild-type (I and K) and Atkc1-f protoplasts (J and L) were elicited by voltage pulses from a holding potential of −48 mV in 20-mV decrements in the range from +12 to −188 mV. (I and J) External solution contained 1 mM CaCl₂, 30 mM K-gluconate, and 10 mM Mes/Tris (pH 5.6). (K and L) RbCl (1 mM) was added to external solution.