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First published online February 25, 2005; 10.1104/pp.104.057216 Plant Physiology 137:1105-1114 (2005) © 2005 American Society of Plant Biologists The Potassium Transporter AtHAK5 Functions in K+ Deprivation-Induced High-Affinity K+ Uptake and AKT1 K+ Channel Contribution to K+ Uptake Kinetics in Arabidopsis Roots1,[w]Division of Biological Sciences, Cell and Developmental Biology Section and Center for Molecular Genetics, University of California San Diego, La Jolla, California 92093–0116
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-  -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+ (Km approximately 15–24 µM) 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.
Potassium is the most abundant cation in plants and has important functions as a major osmolyte in vacuoles, in turgor-driven movements, as a cofactor for enzymes, and for maintaining the plasma membrane potential. Root potassium uptake has been described in classical experiments as a biphasic process (Epstein et al., 1963  ) with mechanism I, also referred to as the high-affinity system, showing apparent affinities of approximately 20 µM Rb+. Mechanism II, the low-affinity system (Km in the millimolar range), provides an increasing contribution from >200 µM to millimolar external K+ concentrations. Early analyses of the K+ uptake systems in plants have served as models for many other nutrients that also show more than one kinetic uptake phase (Epstein and Rains, 1965; Epstein, 1966; Welch and Epstein, 1968; Kochian and Lucas, 1982; Siddiqi and Glass, 1983; Kochian et al., 1985; Siddiqi et al., 1990).
In Arabidopsis (Arabidopsis thaliana), several molecular components have been shown to contribute to potassium uptake into plant roots. AKT1, a shaker-family potassium channel, is expressed in root tissues and mediates potassium uptake over a wide range of external potassium concentrations (Sentenac et al., 1992
Members of the KUP/HAK/KT family of potassium transporters in Arabidopsis were suggested to be involved in high-affinity K+ uptake because several gene family members transport K+ in the low micromolar range as demonstrated by heterologous expression in yeast (Santa-Maria et al., 1997 Since potassium is one of the most important macronutrients, plants must be able to adjust their uptake systems rapidly to a varying supply to maintain growth and development. In this respect, high-affinity potassium uptake becomes particularly important for plants growing in soils with limited potassium supply. Here, we describe the response of Arabidopsis roots to potassium starvation based on transcriptome analyses, K+ and Rb+ uptake measurements, and genetic disruption of the K+ starvation-induced K+ transporter AtHAK5.
To investigate the response of Arabidopsis roots to potassium starvation, a hydroponic system was used that allowed analyses of individual plants. Plants were grown for 21 d in a K+ replete (1.75 mM) medium and subsequently the roots were washed and the plants transferred to nominally potassium-free medium. The potassium content in root and shoot tissue had already started to decrease after 6 h of growth on potassium free solution and continued to decline with progressing starvation time (Fig. 1), with root K+ content being reduced by 60% after 96 h of K+ starvation. K+ concentrations were initially higher in roots than in shoots, whereas after  96 h of starvation they were similar (Fig. 1).
Removal of K+ from the growth medium causes rapid changes in the membrane potential of root cells (Rubio et al., 1996 ; Hirsch et al., 1998; Wang et al., 1998). To investigate the plant's response to K+ starvation at the molecular level, we performed microarray analyses of total root mRNA. The transcriptome-wide changes in expression of individual genes in response to different periods of potassium starvation are shown in Figure 2. Expression levels were detected by hybridizing labeled cRNA from roots to Affymetrix Microarray Chips spotted with oligonucleotides representing approximately 8,300 genes (AG1-Genechip). In addition, experiments after 48 and 96 h of starvation were performed using the ATH1 Genechip (approximately 23,000 genes). Remarkably, no dramatic changes were observed. Genes that were reliably expressed were analyzed using the statistical comparison algorithm of the MAS 5.0 software. Interestingly, less than 1% of the genes showed a significant change in expression levels compared to nonstarved roots after 6 h (21 genes) and 48 h (83 genes) of potassium starvation in both of the replicate experiments (Supplemental Tables III and V). None of those genes was changed over 2-fold in its transcript abundance after 6 h of starvation and only 1 gene (AtHAK5) was changed over 2-fold after 48 h of starvation in both experiments (Supplemental Tables III and V). For the 48 h starvation time point, the comparison of results obtained with AG1 and ATH1 chips showed the same general observation of only a small number of mRNAs that showed significant and over 2-fold changed expression levels (Fig. 2, B and C; Supplemental Tables V and VII).
The gene most strongly and most consistently affected in all experiments for 48- and 96-h K+ starvation was the potassium transporter AtHAK5 (arrows in Fig. 2), a member of the KUP/HAK/KT family. Interestingly, analysis of all of the K+ transporter and K+ channel genes represented on the chips showed that AtHAK5 was the only K+ translocator responsive to low external K+ concentrations under the imposed conditions (Supplemental Table I). Under starvation conditions, AtHAK5 was among the most highly expressed K+ transporter genes in roots, which include AtHAK5, AtHAK6, AtHAK8, AKT1, and KAT3 (Supplemental Table I). Note that other growth conditions and developmental stages can cause induction of other transporters. For example, AtKUP3 mRNA levels are elevated in seedling roots after 2 to 3 weeks of growth on 40 µM K+ (Kim et al., 1998 The increased transcript level of the AtHAK5 gene in response to potassium starvation was confirmed by semiquantitative RT-PCR for the time points tested on Affymetrix chips and also for a longer starvation period of 7 d, showing a continuing induction of AtHAK5 transcripts (Fig. 3).
The AtHAK5 expression pattern was analyzed by generating transgenic plants expressing both the -glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes under the control of the promoter region of the AtHAK5 gene (2 kb upstream of ATG). Analysis of GUS staining in roots of K+-starved plants revealed AtHAK5 promoter activity in the cortex and stele of mature roots and even stronger staining in lateral roots (Fig. 4A). Very low or no activity was detectable in root tips (Fig. 4, A and B). Seven days after resupply of K+ to roots of the same plants, GUS staining in mature and lateral roots was barely detectable and only visible in the root vasculature (Fig. 4B). The pCAMBIA1303 cassette allowed GUS activity and GFP synthesis to be controlled by the AtHAK5 promoter at the same time. Analysis of GFP localization in transgenic K+-starved roots by confocal microscopy gave a more detailed view of AtHAK5 promoter activity (Fig. 4, C and D). In optical cross sections through K+-starved main roots, GFP was detectable in the root epidermis and in the stele (Fig. 4C), whereas in lateral roots GFP was only visible in the epidermis (Fig. 4D).
To characterize the contribution of AtHAK5 to plant potassium homeostasis, AtHAK5 T-DNA insertion mutants were identified from the sequence-indexed SIGNAL database (http://signal.salk.edu/cgi-bin/tdnaexpress), and two homozygous lines, athak5-1 (Salk_014177) and athak5-2 (Salk_05604), were isolated (Fig. 5A). In both homozygous lines, the T-DNA insertion is located in the fifth intron, at positions 3,143 (athak5-1) and 3,159 (athak5-2; Fig. 5A). The insertion sites were verified and two independent lines were confirmed by sequencing the left border PCR products with primers used to identify the T-DNA mutants from the seed population supplied by the Arabidopsis Biological Resource Center (ABRC). RT-PCR using mRNA from K+-starved roots did not detect any full-length AtHAK5 transcript but only a truncated mRNA (Fig. 5B). The resulting protein would be disrupted after the third of 13 predicted transmembrane regions, making it unlikely to produce a functional transporter even if translated.
When mature, 3-week-old athak5-1 plants were subjected to 4 d of potassium deprivation at 40 µM K+, plants showed a slightly though significantly lower potassium content compared to wild-type Columbia (Col-0) plants (Fig. 6A; P < 0.01). When K+ was replaced with 40 µM Rb+ (used as a pulse-chase substitute for K+) in the starvation media, the difference in tissue Rb+ content between wild-type (Col-0) and athak5-1 plants was clearly resolved (Fig. 6B; P < 0.01), demonstrating reduced K+(Rb+) uptake in athak5-1 plants. As expected, Rb+(K+) uptake did not drop to 0 in athak5-1 plants given the many K+ transporter and K+ channel genes expressed in roots (Supplemental Table I).
Rubidium uptake kinetics of athak5 and wild-type (Col-0) plants were directly analyzed by measuring time- and concentration-dependent 86Rb+ uptake into roots of K+-starved plants. The initial uptake of 86Rb+ was linear in wild-type and mutant roots for at least 25 min, with uptake being dramatically higher for Col-0 than for both athak5 mutant alleles (Fig. 7A). Concentration-dependent 86Rb+ uptake was subsequently measured for 20 min at a range of external Rb+ concentrations. Uptake rates for 86Rb+ revealed a significant difference between wild-type (Col-0) and both athak5 alleles at low Rb+ concentrations (Fig. 7B). At high Rb+ concentrations greater than 500 µM, a difference in uptake kinetics could not be resolved between wild-type and athak5 alleles (Fig. 7C). Uptake by wild-type (Col-0) roots at external concentrations below 200 µM revealed that the high-affinity component was greatly reduced in athak5 plants (Fig. 7B).
As it has been demonstrated that the potassium channel gene AKT1 contributes to root K+ uptake, 86Rb+ uptake kinetics were also analyzed for the mutant akt1-1 and the corresponding wild-type (ecotype Wassilewskija [WS]) plants (Fig. 7, E and F). The effect of the akt1-1 mutation on 86Rb+ uptake is strongest at concentrations 500 µM, largely reducing Rb+ uptake in the low-affinity range (Fig. 7E). At concentrations  200 µM, the Rb+ uptake rates in akt1-1 plants were smaller than in wild-type (WS) plants at some, but not consistently at all, concentrations (Fig. 7F). The 86Rb+ uptake rate differences between wild-type (WS) and akt1-1 plants could be described by Michaelis-Menten kinetics, indicating an apparent affinity for the component missing in akt1 roots of approximately 0.9 to 1 mM (Fig. 8).
In wild-type (Col-0) roots, Rb+ uptake rates were best described by Michaelis-Menten uptake kinetics, indicating two major phases of Rb+ transport with Km values of 24 µM for mechanism I and 4 mM for mechanism II, similar to the Rb+ affinities found in barley (Hordeum vulgare) roots (Epstein et al., 1963 ; Table I). The high-affinity (Km approximately 24 µM Rb+) component disappeared in the two athak5 disruption lines (Table I). Michaelis-Menten kinetics using 2 major phases also gave better curve fitting results for wild-type (WS) and akt1-1 roots (R2 closer to 1; Table I). Curve-fitting results for data extracted from Figure 1 in Epstein et al. (1963) are also included in Table I, showing that both for K+-starved barley roots (Epstein et al., 1963) and for K+-starved wild-type Arabidopsis roots, two uptake phases describe the data better than a single phase. In contrast, Rb+ uptake kinetics in athak5 plants can be described as dominated by only 1 component with an intermediate affinity (approximately 350 µM; Table I). Based on subtraction of athak5 Rb+ uptake from wild-type Col-0 measurements, the component missing in roots of athak5 null mutants has an estimated Km of approximately 14 µM and a Vmax of approximately 2.4 µmol gFW–1 h–1 (Fig. 7D). These data suggest that the AtHAK5 gene encodes an important component that functions in K+ starvation-induced high-affinity K+ uptake in roots.
Limited Genome-Wide K+ Starvation Effect on mRNA Levels
Being rooted in one place, plants need to concentrate nutrients from the soil and to adapt to environmental changes. Here, we analyzed the response of Arabidopsis to potassium deprivation by starving mature plants in nominally K+-free hydroponic medium. The lack of available K+ to Arabidopsis roots was detectable through a decrease in root K+ content as early as 6 h after the start of the starvation period (Fig. 1). A decrease in tissue [K+] has recently been observed in mature roots after 1 d of starvation (Ahn et al., 2004
Interestingly, the rapidly decreasing root K+ tissue content resulted in a significant and 2-fold change in transcript abundance of only few of the genes represented on the Genechips analyzed here after 6, 48, and 96 h of K+ starvation (Fig. 2; Supplemental Tables III, V, VII, and IX). This is surprising with regard to the many essential functions of K+ in plant metabolism and the high demand of plants for this ion. Rapid and massive changes in gene expression levels on a genomic scale have been reported when other major plant nutrients including nitrogen or phosphorus were supplied in a different ionic form (Wang et al., 2003
K+ channels and K+ transporters allow plant cells to respond instantaneously to K+ concentration changes by means of membrane potential changes (Grabov, 1990
The K+-starvation induction of the AtHAK5 mRNA levels observed in this study is in agreement with recently reported results (Ahn et al., 2004
However, a repression of the AtHAK5 transcript was observed in plate-grown seedling roots when K+ was withheld from the growth media (Rubio et al., 2000
The AtHAK5 gene is a member of the KUP/HAK/KT family of K+ transporters, comprised of 13 members in Arabidopsis (Mäser et al., 2001
The function of AtHAK5 in roots has not been analyzed to date. Furthermore, detailed kinetic Rb+ uptake isotherms in intact roots of K+ transporter and K+ channel mutants have not yet been reported with exception of AKT1 at 3 Rb+ concentrations (Hirsch et al., 1998 Experiments characterizing 86Rb+ transport in roots of intact plants revealed that a high-affinity component was strongly reduced in athak5 mutants but not completely absent (Fig. 7, B and D). In addition, Michaelis-Menten analyses indicated a change in the apparent Km of a major Rb+ transport component from 24 µM in wild-type (Col-0) roots to 300 to 360 µM in athak5 roots (Table I). These results demonstrate an important physiological contribution of AtHAK5 to high-affinity Rb+/K+ uptake at limiting external K+ availability.
Constitutively expressed root K+ transporters include the K+ channel AKT1, which is expressed in the root epidermis (Lagarde et al., 1996 Subtraction of 86Rb+ uptake kinetics in roots of K+ transporter mutants from the respective wild-type reflect the change in transport properties on the whole root level including tuning of kinetic characteristics of other transporters present in mutant roots. For example, K+ channels and transporters affect the membrane potential, which in turn affects uptake via other transporters. Taking this into account, the athak5 mutation has a stronger effect on the high-affinity mechanism, whereas the akt1 mutation affects the low-affinity component most strongly (Figs. 7D and 8).
For AtHAK5, the permeability of Rb+ and K+ has been found to be very similar (Rubio et al., 2000
For HvHAK1, the barley homolog of AtHAK5, ammonium sensitivity has been demonstrated by yeast mutant complementation studies (Santa-Maria et al., 2000 In conclusion, we show here that K+ deprivation causes only a limited transcriptional response in Arabidopsis roots and that AtHAK5 functions as a major K+ starvation-induced high-affinity (mechanism I) K+ uptake transporter in Arabidopsis roots. Furthermore, detailed kinetic 86Rb+ flux analyses provide molecular genetic, in planta, support for the classical model in which major contributions of high-affinity and low-affinity uptake components can be mechanistically and genetically separated. Furthermore, apparent root K+(Rb+) uptake affinities adapt to transporter impairment.
Plant Culture Seeds of Arabidopsis (Arabidopsis thaliana) ecotype Col-0 were surface sterilized, washed, and stored in water for 4 d at 4°C. Seeds were then germinated on foam plugs inserted into Magenta box rafts (Sigma, St. Louis) and grown in hydroponic medium (100 mL) under a 16/8 h day/night cycle for 4 weeks. The hydroponic solution contained: 1.0 mM Ca(NO3)2; 0.5 mM KH2PO4; 1.25 mM KNO3; 0.5 mM MgSO4; 20 µM FeNaEDTA; 0.5 µM CuSO4; 0.5 µM MnSO4; 10 µM H3BO3; 0.05 µM Na2MoO4; 0.25 µM NaCl; and 0.5 µM ZnSO4. In the potassium-free solution, KNO3 and KH2PO4 were substituted by HNO3 and H3PO4 and the pH was adjusted with N-methyl-glucamine to 5.5. The solution was renewed after 1, 2, and 3 weeks, and on the second day preceding the start of the starvation period. At the beginning of the starvation period, plants were washed for 5 min in deionized water and then transferred to either potassium-free or control medium. During the experiment, the solutions were exchanged every 24 h. Plants were always harvested 8 h after daybreak. For determination of Rb+ tissue content and 86Rb+ uptake kinetics, plants were cultivated in the same media on a slightly modified hydroponic system consisting of plastic containers holding 4 L of aerated nutrient solution. Containers were covered with plastic lids supporting nine plants with roots growing through foam plugs into the hydroponic solution.
For all experiments, Affymetrix Genechips were used (Affymetrix, Santa Clara, CA); AG1 chips represented approximately 8,300 genes for 6 and 48 h of starvation and ATH1 chips for 48 and 96 h of starvation. For AG1 Genechip analyses, annotation was used as described in Ghassemian et al. (2001) Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA). Two independently isolated mRNA samples were pooled for each Genechip hybridization experiment. AG1 experiments at 6 and 48 h of K+ starvation were carried out in duplicate (total of 8 hybridizations). For ATH1-chips, a total of 4 hybridizations were performed. cRNA labeling and hybridizations were performed by the DNA Microarray Facilities at University of California, Irvine, and University of California, San Diego, according to the Affymetrix manual. Expression data sets will be deposited at The Arabidopsis Information Resource (TAIR; http://www.arabidosis.org).
Expression data were analyzed with Microarray Suite 5.0 (Affymetrix) using default settings to calculate raw data values. The detection signal value was calculated as a measure of the relative transcript abundance, and a detection P-value was computed for each probe set from independent control and starvation experiments and was used to evaluate whether or not a transcript was reliably expressed. Only genes that received a present call (P) and a detection P-value of
Roots of plants were washed for 5 min in 0.2 mM CaSO4, separated into roots, leaves, and stems, and surface dried by blotting with ash-free filter paper. The fresh weight was determined prior to drying the samples for 24 h at 75°C. An aliquot of dry matter was digested in HNO3 (concentrated) at 95°C for 1 h. After complete digestion, the acid concentration was adjusted to 5% by dilution with double deionized water. Mineral element concentrations in the solutions were determined by ICP-OES (Perkin Elmer Optima 3000 DV, Boston) at the Scripps Institution of Oceanography (University of California, San Diego) analysis facility.
cDNA was synthesized from 2.5 µg of root total RNA using the Amersham-Biosciences First Strand cDNA kit (Uppsala). The three-step PCR reaction was run for a total of 30 cycles and aliquots were removed after 27 cycles. Primers used to validate the expression pattern of AtHAK5 were S15 (5'-gctcagaagagcccatatgatca-3') and AS20 (5'-agctcgaacactgaagtgacctaga-3').
Primers used to check for the presence of AtHAK5 transcript in Col-0 and athak5 null mutants are given below. The PCR product obtained for elongation factor 1
Putative T-DNA insertion lines were identified by searching the SIGNAL database (http://signal.salk.edu/cgi-bin/tdnaexpress). Plants of the identified seed populations (obtained from the ABRC) were screened for homozygous individuals by PCR using primers annealing up and downstream of the putative insertion sites (S15, AS20). Positively identified plants were subsequently tested with PCR using the T-DNA left border primer (LBa1: 5'-tggttcacgtagtgggccatcg-3') and a gene-specific primer. PCR products were cloned into pGEM T-easy (Promega, Madison, WI) and sequenced to verify the insertion site.
86Rb+ was used as a tracer to analyze kinetic properties of Rb+(K+) uptake, as previous studies have shown substantial Rb+ permeabilities of KUP/HAK/KT transporters and inward K+ channels (Schachtman et al., 1992
The 2-kb DNA fragment upstream of the AtHAK5 start codon was amplified by PCR from Col-0 genomic DNA using a proofreading DNA polymerase (Pwo, Roche, Indianapolis) and cloned into the PstI/NcoI sites of the binary vector pCAMBIA 1303 replacing the cauliflower mosaic virus 35S promoter. The 3' NcoI site was introduced by a mismatch in the antisense primer (5'-cctcaccatccatGGtttgctgtgtt-3'), while the 5' PstI site was present in the native DNA sequence (5'-cttacactgctgcagcctcggctt-3'). Sequence integrity was verified by sequencing the cloned product. Arabidopsis ecotype Col-0 plants were transformed by the floral dip method (Clough and Bent, 1998
Seeds of the T1 generation were surface sterilized and the population screened for hygromycin resistant individuals on one-half strength Murashige and Skoog agar plates (Murashige and Skoog, 1962 GFP-fluorescence was monitored by spinning-disc confocal microscopy (QLC100 confocal scanning unit from Solamere Technology Group, Salt Lake City attached to a NIKON Eclipse TE 2000-U bright field microscope; Tokyo) in intact roots using an argon laser (500 M Select, Laserphysics, West Jordan, UT, excitation wavelength filter at 488 nm and emission filter 500–550 nm). Images were captured by a CCD-camera (CoolSnap-HQ, Photometrics, Tucson, AZ) using Metamorph software (Universal Imaging, Downington, PA). Upon request, all novel material described in this publication will be made available in a timely manner for non-commercial research purposes, subject to the requisite permission from any third-party owners of all or part of the material. Obtaining permission will be the responsibility of the requestor.
Sequence-indexed Arabidopsis T-DNA insertion mutants athak5-1 (Salk_014177) and athak5-2 (Salk_05604) were generated by the Salk Institute Genomic Analysis Laboratory and seeds were obtained from ABRC (Alonso et al., 2003 ). Seeds of the akt1-1 mutant were kindly provided by Mike Sussman (University of Wisconsin). Radiotracer uptake assays were in part performed at the University of Cologne in the laboratory of U.I. Fluegge. K+ deprivation-dependent microarray expression data are available at TAIR (www.arabidopsis.org). Received November 24, 2004; returned for revision December 29, 2004; accepted December 29, 2004.
1 This work was supported by the Department of Energy (grant no. DE–FG02–03ER15449), by the National Science Foundation (grant no. DBI–0077378 to J.I.S.), by the Alexander von Humboldt Foundation (a Feodor-Lynen Fellowship to M.G.), and by the Human Frontier Science Program (fellowship to P.M.). 
2 Present address: University of Cologne, Institute of Botany II, Gyrhofstr. 15, 50931 Köln, Germany.
3 Present address: University of Bern, Institute of Cell Biology, Baltzerstr. 4, CH–3012 Bern, Switzerland.
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.057216. * Corresponding author; julian{at}biomail.ucsd.edu; fax 1–858–534–7108.
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ABSTRACT
RESULTS
-subunits of AtKC1, another shaker-like potassium channel expressed in root hair cells and in the root endodermis, do not form a functional channel but rather seem to influence the transport properties of other root potassium channels like AKT1 (Reintanz et al., 2002
