The potassium transporter AtHAK5 functions in K(+) deprivation-induced high-affinity K(+) uptake and AKT1 K(+) channel contribution to K(+) uptake kinetics in Arabidopsis roots

Abstract

Potassium is an important macronutrient and the most abundant cation in plants. Because soil mineral conditions can vary, plants must be able to adjust to different nutrient availabilities. Here, we used Affymetrix Genechip microarrays to identify genes responsive to potassium (K(+)) deprivation in roots of mature Arabidopsis (Arabidopsis thaliana) plants. Unexpectedly, only a few genes were changed in their expression level after 6, 48, and 96 h of K(+) starvation even though root K(+) content was reduced by approximately 60%. AtHAK5, a potassium transporter gene from the KUP/HAK/KT family, was most consistently and strongly up-regulated in its expression level across 48-h, 96-h, and 7-d K(+) deprivation experiments. AtHAK5 promoter-beta-glucuronidase and -green fluorescent protein fusions showed AtHAK5 promoter activity in the epidermis and vasculature of K(+) deprived roots. Rb(+) uptake kinetics in roots of athak5 T-DNA insertion mutants and wild-type plants demonstrated the absence of a major part of an inducible high-affinity Rb(+)/K(+) (K(m) approximately 15-24 microm) transport system in athak5 plants. In comparative analyses, uptake kinetics of the K(+) channel mutant akt1-1 showed that akt1-1 roots are mainly impaired in a major transport mechanism, with an apparent affinity of approximately 0.9 mm K(+)(Rb(+)). Data show adaptation of apparent K(+) affinities of Arabidopsis roots when individual K(+) transporter genes are disrupted. In addition, the limited transcriptome-wide response to K(+) starvation indicates that posttranscriptional mechanisms may play important roles in root adaptation to K(+) availability in Arabidopsis. The results demonstrate an in vivo function for AtHAK5 in the inducible high-affinity K(+) uptake system in Arabidopsis roots.

Figure 1.

Potassium content in roots and shoots of hydroponically grown Arabidopsis after up to 6 d of K⁺ starvation. Arrows indicate time points when root RNA samples were harvested for microarray analyses (see Fig. 2). n = 5 to 6 independent root or shoot samples per data point ± se.

Figure 2.

Expression overview for Affymetrix Genechip experiments (A and B, AG1-Genechip; C and D, ATH1-Genechips) of control plants (x axis) and plants subjected to K⁺ starvation for the indicated periods of time (y axis). Diagonals indicate a 2-fold change in gene expression for visual reference. Data points in A and B represent the mean signal intensity from two independent experiments. Genes that were assigned a present call (P) by the MAS 5.0 algorithm in both control or both starvation replicates are represented by black symbols, and genes that did not meet these criteria are represented by gray symbols. ru, Relative units.

Figure 3.

RT-PCR experiments confirm induction of AtHAK5 mRNA by K⁺ starvation. PCR was performed using cDNA isolated from roots of wild-type (Col-0) plants transferred to K⁺-free nutrient solution for the times indicated. Elongation factor 1α (EF1α) was used as a control.

Figure 4.

Roots of transgenic plants expressing the GUS and GFP reporter gene driven by the AtHAK5 promoter. A and B, Roots of plants expressing the GUS reporter gene under the control of the AtHAK5 promoter. GUS activity is strongest in the roots of K⁺-starved plants (A) and decreases after resupply of potassium to the nutrient solution in roots of the same plants (B). C and D, Confocal microscopy reveals that the AtHAK5 promoter activity (GFP localization) in starved roots is highest in the epidermis of main (C) and lateral roots (D) and in the stele of main roots (C).

Figure 5.

Isolation of two homozygous T-DNA insertion mutants in the AtHAK5 gene. A, The cartoon shows the positions of the two T-DNA insertion lines, which were verified by sequencing of the left border PCR products. B, RT-PCR using cDNA from roots of wild-type (Col-0) and athak5-1 plants starved for 4 d. Primer pairs anneal to cDNA as indicated in A. AtHAK5 mRNA is absent downstream of the insertion site in athak5-1 plants.

Figure 6.

K⁺ and Rb⁺ content in Col-0 and athak5-1 plants 4 d after transfer of mature plants to nutrient solutions supplemented with either (A) 1.75 mm K⁺ (control solution), (A) 40 μm K⁺, or (B) 40 μm Rb⁺. Different letters indicate significantly different values (P < 0.01). n = 9 independent root samples/bar, mean ± se.

Figure 7.

A, Time-dependent ⁸⁶Rb⁺ uptake in roots of wild-type (Col-0, black circles), athak5-1 (black triangles), and athak5-2 (white triangles) plants after 4 d of K⁺ starvation; Rb⁺ concentration in the uptake solution was 100 μm. n = 4 root samples/data point. Mean ± se. B and C, Concentration-dependent ⁸⁶Rb⁺ uptake in roots of wild-type (Col-0) and intact athak5-1 and athak5-2 plants after 4 d of K⁺ starvation. B shows the concentration range from 0 to 200 μm (high-affinity) and C the range from 0 to 5 mm. Curves in C are plotted using the results from Table I (two-term Michaelis-Menten fit for Col-0, one-term for athak5 null mutants). n = 8 to 12 independent root samples (Col-0, athak5-1) and n = 6 from 3 experiments (athak5-2). Mean ± se. D, Michaelis-Menten kinetics for AtHAK5 calculated by fitting a curve to the difference in uptake between wild-type (Col-0) and athak5-1 roots. Inset shows Lineweaver-Burk plot of AtHAK5 uptake kinetic. Fits to the data including and excluding the relatively large Rb⁺ uptake rate at 50 μm are shown as labeled and result in nearly identical values for K_m of 14.1 and 14.5 μm and V_max of 2.4 and 2.2 μmol gFW⁻¹ h⁻¹, respectively. E and F, Concentration-dependent ⁸⁶Rb⁺ uptake in roots of intact wild-type (WS, black inverted triangles) and akt1-1 (white diamonds) plants after 4 d of K⁺ starvation. E shows the complete range from 0 to 5 mm and F the concentration range from 0 to 200 μm. Curves in E are plotted using the results from Table I (two-term Michaelis-Menten fit). n = 6 independent root samples. Mean ± se.

Figure 8.

Rb⁺ uptake rates for AKT1. The difference in Rb⁺ uptake rates between wild-type (WS) and akt1-1 roots is shown. Fits of Michaelis-Menten kinetics to the data including and excluding the Rb⁺ uptake rates from 5 to 100 μm are shown as labeled and result in similar values for the K_m of 0.88 (R² = 0.81) and 1.1 mm (R² = 0.91) and V_max of 4.8 and 5.0 μmol gFW⁻¹ h⁻¹, respectively.