AKT1 and TRH1 are required during root hair elongation in Arabidopsis

Journal of Experimental Botany, Vol. 54, No. 383, pp. 781±788, February 2003 DOI: 10.1093/jxb/erg066 RESEARCH PAPER AKT1 and TRH1 are required during root hair elongation in Arabidopsis Guilhem Desbrosses 1, Caroline Josefsson 1, Stamatis Rigas 2, Polydefkis Hatzopoulos 2 and Liam Dolan 1,3 1 Department of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, UK 2 Laboratory of Molecular Biology, Agricultural University of Athens, 118 55 Athens, Greece Received 13 September 2002; Accepted 8 October 2002 Abstract TRH1 is a member of the AtKT/AtKUP/AtHAK family of potassium carriers that is required for root hair elongation and AKT1 is an inward rectifying potassium channel expressed in the root epidermis, endodermis and cortex of Arabidopsis thaliana. Plants homozygous for the trh1-1 mutation form short root hairs. The Trh1 ± phenotype cannot be suppressed by growing plants homozygous for the trh1-1 mutation in the presence of high external KCl concentration. This indicates an absolute requirement for TRH1 in root hair tip growth. Plants homozygous for the akt1-1 mutation develop longer root hairs than the wild type when grown in 0 mm external potassium, but develop shorter hairs than the wild type when grown in higher concentrations [>10 mm] of potassium. These data indicate that both TRH1 and AKT1 are active in the root hair over a wide range of external potassium concentrations, but suggest they have different functions in the growing hair cell. Key words: AKT1, Arabidopsis, potassium, root hairs, TRH1. Introduction Root hairs in Arabidopsis thaliana are tubular projections that emerge from the base of specialized epidermal cells, the trichoblasts (Dolan et al., 1993). During the elongation of root hairs, expansion of the vacuole is coupled with the deposition of new membrane and wall at a restricted area at the surface of the cell, the tip (Galway et al., 1997). The growth of the root hair can be divided into three distinct phases. The rst is initiation and involves the formation of a bulge and there is a localized increase in cytoplasmic ph (Bibikova et al., 1997, 1998; Leavitt, 1904). In the second phase, a localized area of the bulge undergoes a slow elongation (<5 mm h ±1 ) (Dolan et al., 1994), a local Ca 2+ gradient forms as a result of the activity of inward rectifying calcium channels (Very and Davies, 2000; Wymer et al., 1997; Schiefelbein et al., 1992). During the third phase, elongation accelerates to a rate of 1±3 mm min ±1 (Dolan et al., 1994; Schiefelbein et al., 1992). Hairs at this stage of growth have a characteristic `tip growth' cytoplasmic organization, with Golgi-derived vesicles at the growing tip, behind which is a zone rich in endoplasmic reticulum, Golgi apparatus and mitochondria (Schempf, 1986; Sievers and Schnepf, 1981). Plants homozygous for the complete loss of function tiny root hair1-1 (trh1-1) mutation have short root hairs and frequently initiate more than one root hair per trichoblast (Rigas et al., 2001). TRH1 encodes a protein of the AtKT (for Arabidopsis thaliana K + Transport) family, and was previously designated AtKUP4 (Kim et al., 1998). There are 13 members of the KT/KUP/HAK ion transporters in Arabidopsis and they are similar to the KUP potassium carrier of E. coli (Rubio et al., 2000; Schleyer and Bakker, 1993). It is likely that they function as H + and K + symporters with a capacity to transport potassium with both low and high af nity (Rodriguez-Navarro, 2000; De Boer, 1999; Fu and Luan, 1998). Mutations in other members of the Arabidopsis KUP/KT/HAK family have been identi ed. For example shy3-1 mutants have defective hypocotyl elongation and result from mutations in the AtKT2/KUP2 gene (Elumalai et al., 2002; Reed et al., 1998). The Shy3 ± and Trh1 ± phenotypes suggest that proteins in the KUP/KT/HAK family are active in ion transport events involved in cell expansion (Elumalai et al., 2002). A number of other potassium transport systems have been identi ed in the root. These include the AKT1 and 3 To whom correspondence should be addressed. Fax: +44 (0)1603 450 022. e-mail: liam.dolan@bbsrc.ac.uk Journal of Experimental Botany, Vol. 54, No. 383, ã Society for Experimental Biology 2003; all rights reserved

782 Desbrosses et al. SKOR1 (Gaymard et al., 1998; Sentenac et al., 1992). AKT1 and SKOR1 are members of the shaker family of potassium channels (Gaymard et al., 1998; Sentenac et al., 1992). SKOR1 encodes an outward rectifying potassium channel expressed in the stele of the root and is likely to be required for the loading of potassium into the xylem sap (Gaymard et al., 1998). AKT1 encodes an inward rectifying potassium channel (Gaymard et al., 1998; Sentenac et al., 1992) located in the plasma membrane and is expressed in the elongation and differentiation zones of the epidermis, cortex and endodermis (Lagarde et al., 1996). Plants homozygous for akt1-1 loss of function mutations are very small when grown in media containing no potassium and 2 mm ammonium phosphate, demonstrating that AKT1 transports potassium at external concentrations between 10 mm and 100 mm (Hirsch et al., 1998; Gaymard et al., 1996). In addition, rubidium uptake, growth and electrical measurements on the akt1-1 mutant grown in the presence of various concentrations of ammonium, sodium and potassium, suggest that the root uptake of potassium is achieved by AKT1 and at least one additional unknown potassium transport system (Spalding et al., 1999). In conclusion, potassium transport in the root is complex and involves various components, many of which are unknown. As AKT1 is expressed in the root epidermis, it is hypothesized that a plant homozygous for the akt1-1 mutation might have a short root hair phenotype. Here, the function of both TRH1 and AKT1 during root hair elongation was investigated by examining the phenotypes of trh1-1, akt1-1 and of trh1-1 akt1-1 mutant plants grown in different concentrations of external potassium. These data demonstrate that both TRH1and AKT1 are involved in root hair elongation, but it is likely that these proteins are required for different aspects of ion transport during cell growth. Materials and methods Media preparation and growth condition Arabidopsis thaliana WS ecotypes were grown in Murashige and Skoog media (Murashige and Skoog, 1962) supplemented with 1% sucrose and 1% phytagel (Sigma, St Louis, USA). To study the effect of KCl on the length of the wild-type and trh1-1 root hairs, plants were grown in an ammonium-free media composed of 2.5 mm NaNO 3, 2.5 mm Ca(NO 3 ) 2, 2 mm MgSO 4, 1,08 mm Ca(H 2 PO 4 ), 0.1 mm NaFeEDTA, 25 mm CaCl 2,25mMH 3 BO 3,22mM ZnSO 4, 2 mm MnSO 4, 0.5 mm CuSO 4, 0.5 mm Na 2 MoO 4, 0.01 mm CoCl 2, 0.5% sucrose, 2.5 mm Mes, ph 5.7, 0.8% puri ed agarose (Sigma, St Louis, USA) and autoclaved for 10 min (Spalding et al., 1999). Seeds were incubated for 3 d in the dark at 4 C and then transferred to 25 C under continuous light and laid horizontally. Measurement of root hair growth Three days after germination, root hairs growing in media were measured using an ocular micrometer on a wild M 7 Z stereomicroscope (Leica Microsystems, Heidelberg, Germany). For all experiments, the length of 10 fully expanded root hairs per root was measured in the differentiation zone. Ten roots were used for the wild type, trh1-1, akt1-1 single mutants, and the trh1-1-akt1-1 double mutant. Measurements were repeated on day 4. The experiment was repeated on two independent preparations of media with the exception of the akt1-1:trh1-1 double mutant, where the experiment was performed on one media preparation. T-tests for statistical analysis were performed online on www. graphpad.com. Root hairs were imaged on an Olympus, AX70 (Japan) light microscope. Photomicrograps were made with a CCD3 camera (Sony, Japan) and digitized with image analysis software (sis GmbH, MuÈnster, Germany). RNA extraction, reverse transcriptase PCR and competitive PCR To study TRH1 expression, 3-d-old seedlings were transferred to ammonium-free liquid media containing 20 mm KCl. KCl was omitted from the control. Plant were harvested at different times after the beginning of the treatment, dried quickly between two sheets of towel, frozen in liquid nitrogen and stored at ±70 C. For RNA extraction, approximatively 0.5 g fresh weight material was ground. Total RNA was puri ed using the RNeasy kit (Qiagen, Crawley, England) following the manufacturer's recommendations. For competitive PCR, genomic contamination was removed by incubating the puri ed total RNA with DNaseI (Roche Diagnostics GmbH, Mannheim, Germany) following the manufacturer's recommendations. Total RNA was then precipitated using LiCl and resuspended in the same volume of water used initially. The amount of RNA was measured spectrophotometrically twice. The reverse transciptase experiment was performed by incubating 1 mg of total RNA with RT superscript II for 50 min following the manufacturer's recommendations. For the TRH1 competitive PCR, a DNA competitor was made using primers that ank a small intron of 72 bp within the TRH1 sequence (primers TRH1-3; 5 -TGCTATGCAT- TGGGATGCTTC-3 and TRH1-2; 5 -CGTCAATCGAACGGTCA- GTGT-3 ). Using genomic DNA as a template, a product of 882 bp was ampli ed, while using TRH1 cdna as a template, a product of 810 bp was ampli ed. For the AKT1 competitive PCR, a DNA competitor was prepared using primers that ank the second intron of AKT1 with a size of 70 bp (primers AKT1-4; 5 -GATATG- GGAGGCTTTCTTAG-3 and AKT1-5; 5 -AACGAGTTTTGCGC- ATCGGA-3 ). A 480 bp product was ampli ed when using genomic DNA as a template, while a 410 bp product was ampli ed when using the AKT1 cdna as a template. A large amount of competitor was prepared, puri ed and quanti ed by spectrophotometry. The competitor was diluted to a nal concentration of 1 ng ml ±1. This stock solution was then further diluted as described in Fig. 4 and used as a competitor. To check the cdna preparation for genomic DNA competition and for the competitive PCR itself, 1 ml of the RT- PCR was used with 1 ml of competitor in a nal volume of 50 ml with the following programme: denaturation 94 C 15 s, annealing 55 C 30 s, elongation 68 C 1 min; for 40 cycles. The PCR product where subsequently separated by electrophoresis through a 3% agarose gel. PCR identi cation of a trh1-1 akt1-1 double mutant Both trh1-1 and akt1-1 mutations are in the WS background. Crosses were made between two homozygous mutants. F 1 seeds were grown and allowed to self to obtain an F 2 population. Because the TRH1 and AKT1 genes are unlinked and the Trh1 ± phenotype is easy to select, the F 2 offspring were screened for the Trh1 ± short root hair phenotype. One hundred seeds from the F 2 population were plated on media containing 50 mm KCl. In this media the difference in length of the root hair between the wild type and trh1-1 is maximized. Twenty-three plants showed a clear Trh1 ± phenotype and 10 of these plants were randomly selected. Since the akt1-1 mutation is caused by the insertion of a T-DNA into the open reading

frame of the AKT1 gene, the akt1 mutation was screened for by PCR. Primers within the AKT1 sequence (AKT1-2; 5 -ACCCAAT- TCTAGCAACTCCTTGAAACTCC-3 ; AKT1-3; 5 -AACACCTC- TGGTCCGTCAAG-3 ) and the T-DNA sequence (T-DNA-l; 5 - GATGCACTCGAAATCAGCCAATTTTAGAC-3 ) were used. plant material was prepared for PCR according to Klimyuk et al. (1993) and the following PCR programme was used: denaturation 2 min 94 C, annealing 30 s at 55 C, elongation 2 min at 72 C with 30 cycles. Six of the 10 plants gave three PCR products corresponding to a non-speci c product, a wild-type copy (900 bp) and a mutant copy of the AKT1 gene (700 bp). These plants are heterozygous for the akt1-1 mutation. Two plants gave a single PCR product (900 bp) corresponding to the wild-type AKT1 gene. Finally, three plants gave only one PCR product of 700 bp corresponding to the akt1-1 mutation. These plants were homozygous for the akt1-1 mutation. They were allowed to self pollinate and seeds collected. The presence of the trh1-1 mutation was con rmed by visual screening and the akt1-1 mutation was con rmed by PCR on the F 3 plants. Role of AKT1 and TRH1 in root hair growth 783 Results The Trh1 ± phenotype is not suppressed by elevated concentrations of external potassium If TRH1 were a major inward potassium transporter located on the plasma membrane, it is likely that the phenotype of plants homozygous for the trh1-1 mutation would be suppressed by the addition of external potassium. To test this hypothesis, the elongation of trh1-1 root hairs was examined when seedlings were grown in media containing different concentrations of potassium. Plants were grown on media containing 0, 10, 50 or 100 mm KCl. The contaminating potassium concentration was 100 mm (A Grabov, personal communication). While 50 mm and 100 mm external KCl represent non-physiological growing conditions, phenotypic comparisons of mutant and wild type grown at these concentrations will be instructive in ascribing function to TRH1. Wild-type root hairs grow in the presence of 0, 10, 50, and 100 mm KCl (Fig. 1). However, root hair length decreases with increased external KCl concentration (Fig. 1). The trh1-1 root hairs are shorter than the wild type at all KCl concentrations (Fig. 1). In the absence of external potassium, trh1-1 root hairs are 65% shorter than the wild type (P <0.0001, n=200) (Figs 1, 2A, B). At 10 mm KCl, the trh1-1 root hairs are 50% shorter than the wild type (P <0.0023, n=200). This is not a partial suppression of the Trh1 ± phenotype since trh1-1 root hair length remains constant while wild-type root hairs are 30% shorter, however, this decrease is not statistically signi cant. At 50 mm KCl, trh1-1 root hairs are 70% shorter than the wild type (P <0.0001, n=200) (Figs 1, 2C, D). Hairs do not grow on trh1-1 mutants cultured on 100 mm KCl, while wild-type hairs grow at this concentration. In conclusion, the Trh1 ± phenotype is not even partially suppressed by the addition of potassium. TRH1 activity is absolutely required for root hair tip growth over a wide range of external potassium concentration. In addition, Fig. 1. The root hair lengths of wild type trh1-1, akt1-1, and akt1-1 trh1-1 double mutant vary as a function of external potassium concentration. Wild type, trh1-1, akt1-1, and akt1-1 trh1-1 were grown on ammonium-free media supplemented with various concentration of potassium. 3 d after germination fully elongated root hairs of the wild type ( lled circles), trh1-1 (open circles), akt1-1 ( lled squares) and of akt1-1 trh1-1 double mutant (open squares) were measured as described in the Materials and methods. thr1-1 root hair length decreased constantly between 10 mm and 50 mm external KCl unlike the wild type (Fig. 1). Therefore, the trh1-1 root hairs are hypersensitive to increased external KCl. The Trh1 ± phenotype is enhanced by increased external osmolarity The effect of high concentrations of external KCl on trh1-1 root hair tip growth was investigated. It may result directly from the high concentration of potassium or chloride ions in the media or indirectly from the increased osmolarity of the media. To test these hypothesis, wild-type and trh1-1 mutants were grown in the presence of 50 mm NaCl or 100 mm mannitol instead of 50 mm KCl. Root hair length is the same in trh1 ± seedlings when grown on either 50 mm NaCl or 50 mm KCl (Fig. 2D, E), while wild-type root hairs are the same length as those grown in the absence of potassium (data not shown). Therefore, the hypersensitivity of trh1-1 root hairs is not speci c to potassium, but may be due to chloride. However, in the presence of 100 mm mannitol, which mimics the osmotic conditions of 50 mm KCl or NaCl, root hairs of trh1-1 mutants are the same length as those of mutants grown in the presence of 50 mm KCl or NaCl (Fig. 2D, E, F). In conclusion, these

784 Desbrosses et al. Fig. 2. NaCl and mannitol affect root hair elongation in trh1-1 mutants. Wild-type WS ecotype (A, C) and trh1-1 (B, D, E, F) seeds were grown on ammonium-free media supplemented with 10 mm KCl (A, B) or 50 mm KCl (C, D) or 50 mm NaCl (E) or 100 mm mannitol (F). Seedlings were grown in media for 4 d, before the photographs were taken (scale bar: 0.4 mm). observations indicate that the Trh1 ± phenotype is enhanced by an increased osmolarity. This is consistent with a defect in potassium import into the cell. AKT1 is required for root hair elongation at high external potassium concentrations AKT1 is active in root epidermal cells (Hirsch et al., 1998; Lagarde et al., 1996). Therefore, it might be predicted that the akt1-1 mutant would have a defect in root hair elongation. This hypothesis was tested by growing akt1-1 plants in the presence of different concentrations of potassium. The akt1-1 root hairs are longer than the wild type when grown in the absence of potassium (Fig. 1, P <0.005, n=150). In these conditions AKT1 negatively regulates root hair elongation. Hair length of akt1-1 and the wild type are identical when grown in the presence of 10 mm KCl, suggesting that AKT1 activity is not required for root hair elongation in these conditions. Root hairs of akt1-1 mutants are shorter than the wild type when grown in 50 mm KCl (Fig. 1, P <0.0413, n=150) and 100 mm KCl (Fig. 1, P <0.0081, n=150). AKT1 is, therefore, required for root hair tip growth in the range of 50±100 mm KCl and may repress hair tip growth at low external KCl concentrations. Taken altogether, these data suggest that AKT1 is required for root hair elongation. Root hair phenotype of the trh1-1 akt1-1 double mutant To characterize further TRH1 and AKT1 function during root hair elongation, a trh1-1 akt1-1 double mutant was made. Root hair length of this double mutant was measured in the presence of various external concentrations of KCl. In the absence of potassium, hairs of the trh1-1 akt1-1 double mutant are longer than trh1-1 single mutant, but

shorter than the akt1-1 single mutant (Fig. 1, P <0.05, n=120; P <0.0005, n=120). At 10 mm KCl, root hairs of the double mutant are similar in length to trh1-1 and shorter than hairs of the double mutants grown in the absence of potassium (P <0.0069, n=120), i.e. the akt1-1 mutation does not suppress the Trh1 ± phenotype at 10 mm external potassium. At 50 mm KCl, root hairs of the akt1-1 trh1-1 double mutant are similar in length to hairs of the trh1-1 mutant. Finally, at 100 mm KCl, hair length of the double mutant is identical to trh1-1 and akt1-1 single mutants. In conclusion, at low potassium concentration, the akt1-1 mutation partially suppresses the Trh1 ± phenotype, while at higher potassium concentrations the double mutant is essentially showing the Trh1 ± phenotype. Therefore, the double mutant displays characteristics of each single mutant. trh1-1, akt1-1 and the trh1-1 akt1-1 double mutant have a shoot phenotype when grown in the presence of high external KCl TRH1 is expressed in the root and shoot, however the trh1-1 mutant does not exhibit a shoot phenotype (Rigas et al., 2001). AKT1 is predominantly expressed in roots. A defect of AKT1 activity in the homozygous akt1-1 mutant results in a smaller shoot size (Hirsch et al., 1998). It is possible that in an akt1-1 mutant background the trh1-1 mutation may result in a shoot phenotype. The plant weight of trh1-1, akt1-1 and trh1-1 akt1-1 plants was determined when grown in the presence of various concentrations of potassium. When grown in 0 and 10 mm KCl, there is no signi cant difference in weight between any of the mutant lines and the wild type (Fig. 3B). However, when grown in 50 mm KCl, akt1-1 mutants are smaller than the wild type (P <0.05 n=8). This is even more dramatic in the presence of 100 mm KCl. This suggests that AKT1 positively regulates rosette weight in the presence of high concentrations of external potassium. At 50 mm the size of a trh1-1 plant is not signi cantly different from the wild type. However, at 100 mm, the rosettes of trh1-1 mutants are larger than wild type (Fig. 3A, B) (P <0.05; n=8). This suggests that TRH1 is a negative genetic regulator of rosette size in the plants grown in 100 mm external KCl. Taken together, these observations demonstrate that TRH1 and AKT1 are both active in controlling the size of the rosette at high external potassium concentrations, but in a antagonistic fashion. At 0 and 10 mm KCl, rosette size of the double mutant is similar to the wild type, akt1-1 and trh1-1 single mutants (Fig. 3B). At 50 mm the rosette size of the double mutant, is similar to the wild type and to trh1-1, but larger than akt1-1. At 100 mm KCl, The trh1-1 akt1-1 double mutant rosette size is smaller than trh1-1 single mutant, although not signi cantly different (Fig. 3A, B) and is greater than the wild type (P <0.05; n=8) and akt1-1 (P <0.05; n=8). Therefore, the trh1-1 mutation partially suppresses the Role of AKT1 and TRH1 in root hair growth 785 Fig. 3. trh1-1, akt1-1, and the trh1-1akt1-1 double mutant have a shoot phenotype. Wild-type WS ecotype (black bars), trh1-1 (white bars), akt1-1 (striped bars) and akt1-1 trh1-1 (blue bars) were grown on ammonium-free media supplemented with 100 mm KCl. Two weeks after germination an image of a typical experiment is shown in (A) and the rosettes were collected and weighed individually. The relative weight of the mutants compared to the wild type in the absence of external KCl is presented in (B). Akt1 ± phenotype at high external potassium concentration. Additional shoot developmental phenotypes (e.g. trichome) for the double mutant have not been noticed. The steady-state level of TRH1 mrna is not dependent on external KCl The phenotypic characterization of akt1-1 and trh1-1 mutants indicates that the respective wild-type proteins are required over a wide range of potassium concentrations. It was therefore predicted that both genes would be transcribed in these growth conditions at low (0 mm) and high (20 mm) external potassium concentrations. TRH1 mrna was quanti ed from seedlings grown in the presence or absence of KCl using competitive PCR. As shown (Fig. 4A), 1 pg of competitor is required to amplify both the competitor and TRH1 cdna when using cdna samples prepared from plants at the beginning of the treatment, i.e. grown in 20 mm external KCl. One pg is also suf cient for co-ampli cation of TRH1 and as a

786 Desbrosses et al. external potassium and is, therefore, likely to be constitutive. In conclusion, steady-states levels of TRH1 and AKT1 mrna are constant over a range of external potassium concentration as predicted by the phenotypic analysis. Discussion It is reported here that both the putative potassium carrier TRH1 and the potassium channel AKT1 are required for root hair tip growth. Phenotypic characterization of the single trh1-1 and akt1-1 mutants indicates that these transporters are active over a wide range of external potassium concentrations. Consistent with this observation it was con rmed that the steady-state level of TRH1 and AKT1 is not affected by external potassium concentrations and both transporters are constitutively expressed. Plants lacking both TRH1 and AKT1 activity display an intermediate root hair length phenotype compared to either single mutant. The shoot phenotypes suggest that both AKT1 and TRH1 antagonistically regulate rosette size when plants are grown in the presence of high concentrations of external KCl. Taken together these observations suggest that AKT1 and TRH1 have different functions in planta. Fig. 4. Steady-state levels of TRH1 and AKT1 mrna are independent of external KCl concentrations. 3-d-old seedlings were grown in liquid media with or without 20 mm KCl. At various times, total mrna was extracted and cdna was prepared as described in the Materials and methods. Competitive PCR were performed to quantify the TRH1 cdna (A, B) and the AKT1 cdna (C). (A) Decreasing concentrations of competitor (ng ml ±1 ; 882 bp PCR product) were used to quantify TRH1 cdna (810 bp PCR product) from plants subcultured with 20 mm KCl. One pg of competitor is required to have coampli cation with the TRH1 cdna. (B) Abundance of TRH1 cdna after plants were subcultured between 0 h and 16 h without KCl. (C) Abundance of AKT1 cdna after plants were subcultured for 0 h and 16 h without KCl. The amount of AKT1 competitor required (480 bp PCR product) to have a co-ampli cation with the AKT1 cdna (410 bp PCR product) is 3 fg. competitor with cdna samples prepared from plants subcultured onto 0 mm KCl for 2±16 h (Fig. 4B). Therefore, TRH1 mrna levels are not affected by changes in external potassium concentration and the gene is likely to be constitutively expressed. The steady-state level of AKT1 gene expression was examined (Fig. 4C) in the same cdna preparations that were used to examine TRH1 expression. The AKT1 cdna is less abundant than the TRH1 cdna (Fig. 4). The AKT1 mrna levels are identical in seedlings grown in 0 and 20 mm KCl for 16 h. In addition, AKT1 expression does not vary throughout the 16 h of the incubation (data not shown). Therefore, the steady-state level of AKT1 gene expression is not dependent on the concentration of TRH1 is absolutely required during root hair tip growth trh1-1 mutants have short hairs and TRH1 is a putative potassium transporter (Rigas et al., 2001). It was therefore predicted that a suf cient external supply of potassium would suppress the Trh1 ± phenotype. This potassium could be transported into root epidermis cells by any number of other potassium uptake systems that are known to operate at the plasma membrane of the root hair (such as AKT1). Seedlings were grown in media containing a range of increasing external potassium concentrations. At all potassium concentrations tested, it was not possible to restore root hair tip growth in trh1-1. It was also shown that the trh1-1 mutant is hypersensitive to high osmolarity in the external mediumðhairs are very short in these conditions. This hypersensitivity was interpreted as being the result of a potassium transport defect in the mutant. To maintain normal elongation rates in a high osmolarity medium, plant cells import more osmotically active ions such as potassium, than cells grown in low osmolarity conditions. The increased sensitivity of trh1-1 mutants to osmolarity, suggests that these mutants are unable to compensate for the high osmolarity of the external medium, because the trh1 mutants take up only 40% the wild-type amount of rubidium, a tracer used to transport potassium (Rigas et al., 2001). How can the thr1-1 phenotype be explained, given that AKT1 is active and transporting large amounts of potassium into the cell? There are at least two possible explanations. One is that the potassium is transported

across the plasma membrane by proteins such as AKT1, but does not cross the tonoplast in the trh1-1 mutant. As a consequence trh1-1 has no storage capacity and accumulates less potassium, thereby reducing the growth of the vacuole. This hypothesis predicts that TRH1 is located on the tonoplast where it would control the ux of potassium between the cytoplasm and the vacuole. An alternative hypothesis is that TRH1 is located in the plasma membrane where it transports another molecule into the cell in addition to potassium, and this other molecule is not transported into the cell by AKT1. Characterization of the transport speci city of TRH1 will be instructive in distinguishing between these two hypotheses. AKT1 is required for root hair growth and is the low af nity component of potassium transport Root hairs of akt1-1 mutants do not elongate when grown in the presence of 100 mm external KCl. Since, the akt1-1 mutants lacks all AKT1 potassium transport activity it is likely that the growth defect observed at 100 mm external KCl is due to the inability of the mutants to accumulate suf cient potassium for growth. In conclusion, AKT1 activity is required for root hair elongation. It was shown that, in the presence of high external potassium, the akt1-1 shoot is signi cantly smaller than the wild type. Taken together with the conclusions from the analysis of the trh1-1, it con rms that cellular potassium is critical for cell elongation (Leigh and Wyn-Jones, 1984). It is also consistent with previous assumptions that AKT1 is a component of the low af nity potassium uptake system of the plant (Maathuis and Sanders, 1994). Finally, it suggests that AKT1 is the major potassium uptake system when the external potassium concentration is high, which is in agreement with the classical model describing the uptake of potassium in plants (Epstein et al., 1963). Root hairs of akt1-1 mutants grown on 10 and 50 mm potassium are indistinguishable from wild-type hairs. Therefore, AKT1 might not be active in this range of potassium concentrations or another transporter can transport ions in the absence of AKT. It is possible that TRH1 may be such a carrier. If this hypothesis were correct then the Trh1 ± phenotype could be expected to be observed when akt1-1 trh1-1 double mutants roots are grown on 10 and 50 mm potassium. Indeed, the phenotypes of trh1-1 and the akt1-1 trh1-1 double mutant are indistinguishable when grown on 10 and 50 mm external potassium. This may indicate that TRH1 function can compensate for the loss of AKT1 function at certain external potassium concentrations. AKT1 and TRH1 have different functions in planta In the absence of external potassium, root hairs of the trh1-1 mutant are short. TRH1, therefore, positively regulates root hair tip growth. Root hairs of akt1-1 mutants grown in the absence of potassium are longer than the wild type. Role of AKT1 and TRH1 in root hair growth 787 This suggests that AKT1 negatively regulates root hair elongation when grown in the presence of low external potassium. Therefore, TRH1 and AKT1 have opposite functions at low concentrations of potassium. A possible interpretation for the unexpected long root hair phenotype of akt1-1, is that AKT1 could be responsible for the movement of potassium out of the root hair cell when the plants are grown in very low external potassium conditions. This movement of potassium out of the cell in wildtype AKT1 plants would result in a decrease in hair cell elongation and the formation of very short hairs. Such outward movement of potassium would not occur in plants homozygous for the complete loss of function in akt1-1 mutation, allowing them to grow longer than the wild type. This hypothesis could be tested by comparing 86 Rb extrusion in wild-type and akt1-1 mutant plants. The akt1-1 mutants would be predicted to lose more 86 Rb tracer to the surrounding medium than wild-type plants. Consistent with this hypothesis is the nding that the AKT2/AKT3 potassium channel, which is similar to AKT1, has been shown to be a weak outward recti er in oocytes (Lacombe et al., 2000). The shoot of the trh1-1 mutant is larger than the wild type when grown in the presence of high concentrations of external potassium, while the akt1-1 mutant shoots are smaller than the wild type. This again suggests that in planta, TRH1 and AKT1 have distinct functions. That the root hair phenotype of the trh1-1 akt1-1 double mutant exhibits characteristics of both single mutants, is consistent with this interpretation. This absence of an epistatic interaction suggests that the transporters act independently of each other. The distinct roles of AKT1 and TRH1 in cell expansion revealed by the phenotypes of the single and double mutants may simply result from the different functional properties for AKT1 and TRH1, i.e. AKT1 is a channel, while TRH1 is a carrier, and both proteins are located on the plasma membrane. An alternative explanation is that the distinct roles of these proteins is the result of their being located in different parts of the cell. Since it is known that AKT1 is located on the plasma membrane, TRH1 may be located on the tonoplast where it participates in ion transport into or out of the vacuole. In agreement with such interpretation, genetic studies and biochemical analyses have suggested that other members of the KUP/ HAK/KT family may be located on the tonoplast (Rubio et al., 2000; Senn et al., 2001; Elumalai et al., 2002). This hypothesis is currently being tested. Acknowledgements The authors would like to thank Professsor Claude Grignon (INRA- Montpellier France), Dr Anne-Garlonn Desbrosses-Fonrouge (JIC, UK), Dr Alisdair Fernie (MPI-MP-Golm, Germany), members of the Developmental Genetics group in the Department of Cell and

788 Desbrosses et al. Developmental Biology (JIC, UK) for stimulating discussion and the Arabidopsis Biological Resource Center (Columbus, OH, USA) for akt1-1 mutant seeds. This research was funded by INRA, BBSRC, the Greek IKY fellowship, and the European Union. References Bibikova TN, Jacob T, Dahse I, Gilroy S. 1998. Localized changes in apoplastic and cytoplasmic ph are associated with root hair development in Arabidopsis thaliana. Development 125, 2925±2934. Bibikova TN, Zhigilei A, Gilroy S. 1997. Root hair growth in Arabidopsis thaliana is directed by calcium and an endogenous polarity. Planta 203, 495±505. De Boer A. 1999. Potassium translocation into the root xylem. Plant Biology 1, 36±45. Dolan L, Duckett CM, Grierson C, Linstead P, Schneider K, Lawson E, Dean C, Poethig S, Roberts K. 1994. Clonal relationships and cell patterning in the root epidermis of Arabidopsis. Development 120, 2465±2474. Dolan L, Janmaat K, Willemsen V, Linstead P, Poethig S, Roberts K, Scheres B. 1993. Cellular organisation of the Arabidopsis thaliana root. Development 119, 71±84. Elumalai RP, Nagpal P, Reed JW. 2002. A mutation in the Arabidopsis KT2/KUP2 potassium transporter gene affects shoot cell expansion. The Plant Cell 14, 119±131. Epstein E, Rains DW, Elzam OE. 1963. Resolution of dual mechanisms of potassium absorption by barley roots. Proceedings of the National Academy of Sciences, USA 49, 684±692. Fu HH, Luan S. 1998. AtKuP1: a dual-af nity K + transporter from Arabidopsis. The Plant Cell 10, 63±73. Galway ME, Heckman Jr JW, Schiefelbein JW. 1997. Growth and ultrastructure of Arabidopsis root hairs: the rhd3 mutation alters vacuole enlargement and tip growth. Planta 201, 209±218. Gaymard F, Cerutti M, Horeau C, Lemaillet G, Urbach S, Ravallec M, Devauchelle G, Sentenac H, Thibaud JB. 1996. The baculovirus/insect cell system as an alternative to Xenopus oocytes. First characterization of the AKT1 K + channel from Arabidopsis thaliana. Journal of Biological Chemistry 271, 22863±22870. Gaymard F, Pilot G, Lacombe B, Bouchez D, Bruneau D, Boucherez J, Michaux-Ferriere N, Thibaud JB, Sentenac H. 1998. Identi cation and disruption of a plant shaker-like outward channel involved in K + release into the xylem sap. Cell 94, 647±655. Hirsch RE, Lewis BD, Spalding EP, Sussman MR. 1998. A role for the AKT1 potassium channel in plant nutrition. Science 280, 918±921. Kim EJ, Kwak JM, Uozumi N, Schroeder JI. 1998. AtKUP1: an Arabidopsis gene encoding high-af nity potassium transport activity. The Plant Cell 10, 51±62. Klimyuk VI, Carroll BJ, Thomas CM, Jones JD. 1993. Alkali treatment for rapid preparation of plant material for reliable PCR analysis. The Plant Journal 3, 493±494. Lacombe B, Pilot G, Michard E, Gaymard F, Sentenac H, Thibaud JB. 2000. A shaker-like K (+) channel with weak recti cation is expressed in both source and sink phloem tissues of Arabidopsis. The Plant Cell 12, 837±851. Lagarde D, Basset M, Lepetit M, Conejero G, Gaymard F, Astruc S, Grignon C. 1996. Tissue-speci c expression of Arabidopsis AKT1 gene is consistent with a role in K + nutrition. The Plant Journal 9, 195±203. Leavitt RG. 1904. Trichomes of the root in vascular cryptogams and angiosperms. Proceeding of the Boston Society of Natural History 31, 273±313. Leigh RA, Wyn Jones RG. 1984. A hypothesis relating critical potassium concentrations for growth to the distribution and functions of this ion in the plant cell. New Phytologist 97, 1±13. Maathuis FJ, Sanders D. 1994. Mechanism of high-af nity potassium uptake in roots of Arabidopsis thaliana. Proceedings of the National Academy of Sciences, USA 91, 9272±9276. Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15, 473±497. Reed JW, Elumalai RP, Chory J. 1998. Suppressors of an Arabidopsis thaliana phyb mutation identify genes that control light signalling and hypocotyl elongation. Genetics 148, 1295±1310. Rigas S, Debrosses G, Haralampidis K, Vicente-Agullo F, Feldmann K, Grabov A, Dolan L, Hatzopoulos P. 2001. Trh1 encodes a potassium transporter required for tip growth in Arabidopsis root hairs. The Plant Cell 13, 139±151. Rodriguez-Navarro A. 2000. Potassium transport in fungi and plants. Biochimica et Biophysica Acta 1469, 1±30. Rubio F, Santa-Maria G, Rodriguez-Navarro A. 2000. Cloning of Arabidopsis and barley cdnas encoding HAK potassium transporters in root and shoot cells. Physiologia Plantarum 109, 34±43. Schempf E. 1986. Cellular polarity. Annual Review of Plant Physiology 37, 23±47. Schiefelbein JW, Shipley A, Rowse P. 1992. Calcium in ux at the tip of growing root-hair cells of Arabidopsis thaliana. Planta 187, 455±459. Schleyer M, Bakker EP. 1993. Nucleotide sequence and 3 -end deletion studies indicate that the K (+) -uptake protein kup from Escherichia coli is composed of a hydrophobic core linked to a large and partially essential hydrophilic C terminus. Journal of Bacteriology 175, 6925±6931. Senn ME, Rubio F, Banuelos MA, Rodriguez-Navarro A. 2001. Comparative functional features of plant potassium HvHAK1 and HvHAK2 transporter. Journal of Biological Chemistry 276, 44563±44569. Sentenac H, Bonneaud N, Minet M, Lacroute F, Salmon JM, Gaymard F, Grignon C. 1992. Cloning and expression in yeast of a plant potassium ion transport system. Science 256, 663±665. Sievers A, Schnepf E. 1981. Morphogenesis and polarity of tubular cells with tip-growth. In: Kiermayer O, ed. Cytomorphogenesis in plants. Wien: Springer, 265±299. Spalding EP, Hirsch RE, Lewis DR, Qi Z, Sussman MR, Lewis BD. 1999. Potassium uptake supporting plant growth in the absence of AKT1 channel activity: inhibition by ammonium and stimulation by sodium. Journal of General Physiology 113, 909±918. Very AA, Davies JM. 2000. Hyperpolarization-activated calcium channels at the tip of arabidopsis root hairs. Proceedings of the National Academy of Sciences, USA 97, 9801±9806. Wymer CL, Bibikova TN, Gilroy S. 1997. Cytoplasmic free calcium distributions during the development of root hairs of Arabidopsis thaliana. The Plant Journal 12, 427±439.