A Transmembrane Hybrid-Type Histidine Kinase in Arabidopsis Functions as an Osmosensor

Correspondence to: Kazuo Shinozaki, sinozaki{at}rtc.riken.go.jp (E-mail), 81-298-36-9060 (fax)

	ABSTRACT

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Water deficit and the resulting osmotic stress affect plant growth. To understand how plant cells monitor and respond to osmotic change from water stress, we isolated a cDNA from dehydrated Arabidopsis plants. This cDNA encodes a novel hybrid-type histidine kinase, ATHK1. Restriction fragment length polymorphism mapping showed that the ATHK1 gene is on chromosome 2. The predicted ATHK1 protein has two putative transmembrane regions in the N-terminal half and has structural similarity to the yeast osmosensor synthetic lethal of N-end rule 1 (SLN1). The ATHK1 transcript was more abundant in roots than other tissues under normal growth conditions and accumulated under conditions of high or low osmolarity. Histochemical analysis of ß-glucuronidase activities driven by the ATHK1 promoter further indicates that the ATHK1 gene is transcriptionally upregulated in response to changes in external osmolarity. Overexpression of the ATHK1 cDNA suppressed the lethality of the temperature-sensitive osmosensing-defective yeast mutant sln1-ts. By contrast, ATHK1 cDNAs in which conserved His or Asp residues had been substituted failed to complement the sln1-ts mutant, indicating that ATHK1 functions as a histidine kinase. Introduction of the ATHK1 cDNA into the yeast double mutant sln1 sho1, which lacks two osmosensors, suppressed lethality in high-salinity media and activated the high-osmolarity glycerol response 1 (HOG1) mitogen-activated protein kinase (MAPK). These results imply that ATHK1 functions as an osmosensor and transmits the stress signal to a downstream MAPK cascade.

	INTRODUCTION

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Plants have multiple physiological and biochemical systems that enable them to tolerate environmental stresses. Water deficit is the most serious factor limiting plant growth and productivity, and it occurs not only during drought but also with high salinity and low temperature. A change in osmotic potential in cells caused by water loss triggers various molecular responses in plants (Bray 1997 ). To date, many genes induced by drought, salinity, or cold stress have been identified and studied (Ingram and Bartels 1996 ; Shinozaki and Yamaguchi-Shinozaki 1996 , Shinozaki and Yamaguchi-Shinozaki 1997 ). However, little is known about how plant cells detect water deficits.

In bacteria, histidine kinases function as sensor molecules that transduce extracellular signals (including chemotactic factors, changes in osmolarity, and nutrient deficiency) to the cytoplasm. This transduction is mediated by phosphotransfer to the cognate response regulator (Parkinson and Kofoid 1992 ; Parkinson 1993 ; Alex and Simon 1994 ; Swanson et al. 1994 ; Chang and Meyerowitz 1995 ; Mizuno et al. 1996 ; Mizuno 1997 , Mizuno 1998 ; Wurgler-Murphy et al., 1997; Chang and Stewart 1998 ). This simple signaling unit is called a two-component system. Typically, the two-component system is composed of two types of proteins, a sensory histidine kinase and a response regulator. A typical histidine kinase contains an N-terminal input domain and a C-terminal transmitter domain with an invariant histidine residue. A typical response regulator contains an N-terminal receiver domain with an invariant aspartate residue and a C-terminal output domain. The input domain of the sensory histidine kinase detects environmental variables and selectively promotes autophosphorylation of a histidine residue within its transmitter domain. Following autophosphorylation, the phosphoryl group is transferred to the invariant aspartate residue in the receiver domain of the cognate response regulator. A change in the phosphorylation state of the response regulator alters the effector activity of the output domain to control the transcription of signal-responsive genes.

Recently, several histidine kinase–encoding genes have been cloned in eukaryotes. Some of these have been shown to be involved in osmosensing (Ota and Varshavsky 1993 ; Maeda et al. 1994 , Maeda et al. 1995 ; Posas et al. 1996 ; Wang et al. 1996 ), development (Alex et al. 1996 ; Schuster et al. 1996 ), and plant hormone responses (Chang et al. 1993 ; Hua et al. 1995 , Hua et al. 1998 ; Wilkinson et al. 1995 ; Kakimoto 1996 ; Payton et al. 1996 ; Vriezen et al. 1997 ; Lashbrook et al. 1998 ; Sakai et al. 1998 ). It is thus apparent that the two-component system is not confined to prokaryotes. A two-component histidine kinase functions as an osmosensor in yeast (Maeda et al. 1994 , Maeda et al. 1995 ); however, all of the plant histidine kinases described to date are involved in perception of the plant hormones ethylene and cytokinin (Chang et al. 1993 ; Hua et al. 1995 , Hua et al. 1998 ; Wilkinson et al. 1995 ; Kakimoto 1996 ; Payton et al. 1996 ; Vriezen et al. 1997 ; Lashbrook et al. 1998 ; Sakai et al. 1998 ). These observations prompted us to isolate histidine kinases and to investigate their roles in osmosensing by plants. In this report, we provide evidence, using yeast mutants, that a novel Arabidopsis histidine kinase has a potential function as an osmosensor. We also present an analysis of the upregulation of the histidine kinase gene by changes in external osmolarity. Possible functions of the histidine kinase in osmosensing in plants are discussed.

	RESULTS

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ATHK1 Is an Aradidopsis Hybrid-Type Histidine Kinase
To isolate histidine kinases, we used a polymerase chain reaction (PCR) strategy with degenerate oligonucleotide primers and cDNAs prepared from dehydrated Arabidopsis rosette plants. One PCR fragment that encodes a novel histidine kinase was identified and then used to screen a cDNA library. The cDNA clone with the longest insert (~4 kb), designated ATHK1 (for Arabidopsis thaliana histidine kinase 1), encoded a polypeptide of 1207 amino acid residues with a predicted molecular weight of 135 kD (Figure 1A). We also screened a genomic library using the cDNA clone as a probe and isolated a genomic clone that contains the 1.6-kb upstream region of the ATHK1 gene. The predicted protein, ATHK1, contains both a transmitter and a receiver domain with a conserved histidine residue in the transmitter domain and a conserved aspartate residue in the receiver domain (Figure 1A). Hydrophobicity analysis revealed that ATHK1 has two hydrophobic regions in the N-terminal half (Figure 1B). This suggests that ATHK1 is a transmembrane protein.

View larger version (46K): [in this window] [in a new window]   Figure 1. ATHK1 Is a Hybrid-Type Histidine Kinase. (A) Amino acid sequence of ATHK1 deduced from the nucleotide sequence. Two putative transmembrane regions are indicated by boldface underlines. The transmitter and receiver domains are indicated by light underlines. The potential phosphorylation sites, His-508 and Asp-1074, are shown by asterisks. The nucleotide sequence appears in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases as accession number AB010914. (B) Structure of ATHK1. The hydrophobicity plot was calculated by the hydropathy algorithm (Kyte and Doolittle 1982 ). An open box and oval indicate transmitter and receiver domains, respectively. Two filled boxes represent putative transmembrane regions. a.a., amino acids; D, aspartate residue; H, histidine residue.

Comparisons of amino acid sequences of the transmitter and receiver domains of ATHK1 with those of known histidine kinases are shown in Figure 2A and Figure 2B. The transmitter domain of ATHK1 shows the highest degree of similarity to that of the Arabidopsis cytokinin independent 1 (CKI1) protein (30%), and the receiver domain shows the highest degree of similarity with that of synthetic lethal of N-end rule 1 (SLN1) protein from Saccharomyces cerevisiae (29%). Thus, although three putative ethylene receptors, ethylene resistant 1 (ETR1), ethylene response sensor (ERS), and never-ripe (NR), exhibit a high degree of similarity (70 to 80%) with each other (Hua et al. 1995 ; Wilkinson et al. 1995 ), ATHK1 exhibits no marked similarity to particular histidine kinases found in plants. However, the significant structural similarity to the yeast osmosensor SLN1 suggests that ATHK1 may be an osmosensor in plants.

View larger version (66K): [in this window] [in a new window]   Figure 2. Comparison of Amino Acid Sequences with Known Two-Component System Molecules. (A) Alignment of amino acid sequences of the transmitter domain. (B) Alignment of amino acid sequences of the receiver domain. Predicted amino acid sequence of ATHK1 aligned with those of S. cerevisiae SLN1 (Ota and Varshavsky 1993 ), Arabidopsis ETR1 (Chang et al. 1993 ), Arabidopsis ERS (Hua et al. 1995 ), tomato NR (Wilkinson et al. 1995 ), Arabidopsis CKI1 (Kakimoto 1996 ), S. cerevisiae SSK1 (Maeda et al. 1994 ), E. coli BarA (Nagasawa et al. 1992 ), E. coli RcsC (Stout and Gottesman 1990 ), Pseudomonas syringae LemA (Hrabak and Willis 1992 ), Neurospora crassa NikA (Alex et al. 1996 ), Dictyostelium discoideum DokA (Schuster et al. 1996 ), and D. discoideum DhkA (Wang et al. 1996 ). Conserved amino acid residues are shown with a black background; dashes indicate gaps introduced to maximize alignment. Asterisks indicate potential phosphorylation sites.

Genomic DNA gel blot analysis revealed no extra bands that hybridized with the ATHK1 cDNA probe under high-stringency conditions, suggesting that the ATHK1 gene is present as a single copy (Figure 3). However, under low-stringency conditions, a few extra bands, shown by arrowheads, were detected, suggesting that there may be a few related genes in the Arabidopsis genome (Figure 3).

View larger version (47K): [in this window] [in a new window]   Figure 3. Genomic DNA Gel Blot Analysis of the ATHK1 Gene. Genomic DNA (5 µg) was digested with BamHI (B), EcoRI (E), and HindIII (H), fractionated in a 0.7% agarose gel, and transferred to a nitrocellulose membrane. The membrane was hybridized with 32P-labeled PCR fragment and washed with 0.1 x SSC, 0.1% SDS, at 50°C (high-stringency conditions) or 2 x SSC, 0.5% SDS, at 37°C (low-stringency conditions). DNA markers are indicated at right in kilobases.

We determined the chromosomal position of the ATHK1 gene by restriction fragment length polymorphism (RFLP) mapping using P1 clones. When restriction fragments of genomic DNA were probed with a P1 plasmid DNA (MZL17) carrying the ATHK1 gene, HindIII-digested bands revealed a polymorphism between the ecotypes Landsberg erecta and Columbia. Therefore, we analyzed the RFLP segregation patterns of genomic DNA digested with HindIII from 99 recombinant inbred lines. Figure 4 shows the approximate chromosomal position of the ATHK1 locus on chromosome 2, flanked by the markers mi139 and m216. This map position is consistent with the report that the bacterial artificial chromosome clone T17A5, which also carries the ATHK1 gene, maps to yeast artificial chromosome clone CIC8B10 on chromosome 2 (http://nucleus. cshl.org/protarab/).

View larger version (10K): [in this window] [in a new window]   Figure 4. Location of the ATHK1 Gene on Chromosome 2. Mapping of the ATHK1 gene was achieved by RFLP linkage analysis with the F8 progeny of 99 recombinant inbred lines generated between Landsberg erecta and Columbia ecotypes.

ATHK1 Acts as a Histidine Kinase in Yeast
Because ATHK1 is structurally related to the yeast osmosensor SLN1, we analyzed its biological function by using yeast sln1 mutants. First, we introduced the ATHK1 cDNA into the temperature-sensitive sln1 mutant sln1-ts under the control of the galactose-inducible GAL1 promoter to examine the catalytic activity of ATHK1. The sln1-ts mutation is lethal because of the constitutive activation of the high-osmolarity glycerol response 1 (HOG1) mitogen-activated kinase (MAPK) cascade at a restrictive temperature, 37°C (Maeda et al. 1994 ). Overexpression of the ATHK1 cDNA (ATHK1FL) suppressed the phenotype of sln1-ts mutant in a galactose-dependent manner (Figure 5). By contrast, cDNAs in which either putative phosphorylation site had been substituted—His-508 (ATHK1H508V; His-508 to Val) or Asp-1074 (ATHK1D1074E; Asp-1074 to Glu)—failed to complement the sln1-ts mutation (Figure 5). These results suggest that ATHK1 acts as a histidine kinase in yeast.

View larger version (35K): [in this window] [in a new window]   Figure 5. Complementation of the sln1-ts Mutation by Expression of the ATHK1 cDNA. (A) Constructs used in this experiment. D, aspartate residue; E, glutamate residue; H, histidine residue; V, valine residue. (B) Strains used in this experiment. (C) and (D) The sln1-ts mutants carrying either vector alone, the indicated expression plasmids of ATHK1 and its mutants, ETR1, or SLN1, were cultured on plates supplemented with 2% glucose (C) or 2% galactose (D) for 3 days at 37°C.

Similarly, deletions of both the N-terminal half (ATHK1SOL) and the predicted extracellular domain between the two hydrophobic regions (ATHK1dExD) resulted in failure to complement the sln1-ts mutation. This indicates that ATHK1 contains an extracellular domain that plays an important role in the activation of ATHK1 (Figure 5). The deletion of the extracellular domain may affect the activity of ATHK1 due to a structural change. Interestingly, the lethality of the sln1-ts mutants was not complemented by the expression of ETR1, an ethylene receptor in Arabidopsis (Chang et al. 1993 ). To determine whether the suppression of the sln1-ts mutation by ATHK1 requires SLN1 to be active in the sln1-ts mutants, we introduced the ATHK1 cDNA into an sln1 deletion mutant (sln1). ATHK1 was able to complement sln1, indicating that ATHK1 can functionally substitute for SLN1 (data not shown). Thus, ATHK1 seems to be active, at least in yeast, without any protein modifications or a requirement for external signals. Apparently, ATHK1 is capable of inactivating the HOG1 MAPK cascade by performing phosphotransfer to the downstream signal transduction components that ordinarily are modified by SLN1.

ATHK1 Confers High-Osmolarity Tolerance to the sln1 sho1 Yeast Double Mutant
S. cerevisiae contains two osmosensors: synthetic high-osmolarity-sensitive 1 (SHO1), a protein that contains transmembrane domains, and SLN1. Mutants lacking both SLN1 and SHO1 are lethal under high-osmolarity conditions. If ATHK1 expressed in yeast becomes inactive in response to increases in osmolarity, mutants lacking both SLN1 and SHO1 should be able to grow upon activation of the HOG1 MAPK cascade. To analyze the function of ATHK1 as an osmosensor in yeast, we introduced the ATHK1 cDNA into the sln1 and sho1 deletion mutants (sln1 sho1) and then examined their ability to grow on high-osmolarity media. As shown in Figure 6, the sln1 sho1 double mutant expressing either ATHK1 (sln1 sho1 ATHK1) or SLN1 (sln1 sho1 SLN1) grew as well as wild-type cells on YPD medium containing 0.9 M NaCl, but the hog1 deletion mutants (hog1) showed reduced growth under these conditions. However, introduction of ATHK1D1074E did not confer high-osmolarity tolerance to the sln1 sho1 double mutant (sln1 sho1 ATHK1D1074E). This suggests that the catalytic activity of ATHK1 is required for growth under high-salinity conditions. The growth of the sln1 mutants expressing either ATHK1 (sln1 ATHK1) or SLN1 (sln1 SLN1) was not affected by increases in osmolarity in the medium (Figure 6). These results imply that the activity of ATHK1 was stopped in response to increases in external osmolarity, and consequently the HOG1 MAPK cascade was activated.

View larger version (35K): [in this window] [in a new window]   Figure 6. Sensitivity to High Osmolarity of the sln1 sho1 Double Mutant Transformed with the ATHK1 cDNA. (A) Strains used in this experiment. WT, wild-type; hog1, hog1 deletion mutant; sln1 ATHK1, sln1 deletion mutant expressing ATHK1; sln1 SLN1, sln1 deletion mutant expressing SLN1; sln1 sho1 ATHK1, sln1 sho1 double deletion mutant expressing ATHK1; sln1 sho1 ATHK1D1074E, sln1 sho1 double mutant expressing ATHK1D1074E; and sln1 sho1 SLN1, sln1 sho1 double mutant expressing SLN1. (B) Yeast strains cultured on YPD media containing 0.9 M NaCl for 3 days at 30°C.

ATHK1 Senses and Transduces Changes in External Osmolarity to the HOG1 MAPK Cascade in Yeast
To confirm that the tolerance of the sln1 sho1 ATHK1 cells to high osmolarity was due to activation of the HOG1 MAPK cascade, we analyzed tyrosine phosphorylation of the HOG1 MAPK by immunoblot analysis with an antiphosphotyrosine antibody. Treatment of the sln1 sho1 ATHK1 cells, the wild-type cells, and the sln1 sho1 SLN1 cells with 0.4 M NaCl induced tyrosine phosphorylation of HOG1 (Figure 7A). No phosphorylated bands were detected at the same position in the hog1 cells, indicating that the corresponding band was tyrosine-phosphorylated HOG1 (Figure 7A). Tyrosine phosphorylation of HOG1 also was not detected in a double mutant lacking both the suppressor of sensor kinase 1 (SSK1) and SHO1 (ssk1 sho1). This indicates that the NaCl-dependent tyrosine phosphorylation of HOG1 in the sln1 sho1 ATHK1 cells was mediated by ATHK1. Furthermore, the amount of phosphorylation of HOG1 in the sln1 sho1 ATHK1 cells depended on the concentration of NaCl; phosphorylation rapidly increased within 5 min after exposure to high osmolarity and returned to a basal level after 60 min (Figure 6B). This response was similar to that observed in the sln1 sho1 SLN1 cells (Figure 6B). Thus, ATHK1 seems to sense changes in external osmolarity, as does SLN1, and then transduces the information to the downstream cytoplasmic targets in yeast.

View larger version (55K): [in this window] [in a new window]   Figure 7. ATHK1 Senses and Transduces Changes in External Osmolarity in Yeast. (A) NaCl-dependent tyrosine phosphorylation of the HOG1 MAPK by ATHK1. Wild-type (WT) cells, the ssk1 sho1 deletion mutant (ssk1 sho1), the sln1 sho1 double mutant expressing either ATHK1 (sln1 sho1 ATHK1) or SLN1 (sln1 sho1 SLN1), and hog1 deletion mutants (hog1) were cultured in YPD medium. The cells were treated without (-) or with (+) 0.4 M NaCl for 10 min and subjected to SDS-PAGE. The tyrosine phosphorylation of HOG1 was analyzed by immunoblotting with the anti-phosphotyrosine antibody 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY). Arrowheads indicate the tyrosine-phosphorylated HOG1. The sizes of protein markers are indicated at left in kilodaltons. (B) Transient tyrosine phosphorylation of HOG1 MAP kinase in the sln1 sho1 double mutant expressing ATHK1 in response to high osmolarity. sln1 sho1 mutants expressing either ATHK1 (sln1sho1ATHK1) or SLN1 (sln1sho1SLN1) were cultured in YPD medium. The cells were treated with 0.4 M NaCl for the indicated periods (top) or for 10 min with the indicated concentrations of NaCl (bottom). The tyrosine phosphorylation of HOG1 was analyzed by immunoblotting with an anti-phosphotyrosine antibody.

The ATHK1 mRNA Is More Abundant in Roots and Accumulates under High- and Low-Osmolarity Conditions
We examined the expression of the ATHK1 gene in normally grown Arabidopsis organs using RNA gel blot analysis. The ATHK1 mRNA was more abundant in roots than in other organs, suggesting a functional importance of ATHK1 in root tissue (Figure 8A). We then examined the induction of the ATHK1 gene under a variety of stress conditions. ATHK1 mRNA significantly accumulated under conditions of high salinity and low temperature (Figure 8B). Moreover, treatment with distilled water and dehydration also slightly stimulated the induction of the ATHK1 gene (Figure 8B). These results suggest that a change in osmolarity may trigger the induction of the ATHK1 gene. Extra transcripts of ~2 kb in length were also detected in each lane. These were probably degraded or alternatively spliced products. We also examined the protein level of ATHK1 after stress treatments by protein gel blotting with an antiserum raised against recombinant ATHK1 proteins. However, we could not detect a specific band due to very low abundance of the endogenous ATHK1 in wild-type Arabidopsis (data not shown).

View larger version (82K): [in this window] [in a new window]   Figure 8. Expression of the ATHK1 Gene in Organs and under Stress Conditions. (A) Each lane was loaded with 30 µg of total RNA prepared from flowers (F), stems (St), roots (R), developing seeds (Sd), and leaves (L) from whole rosette plants. (B) Plants were treated with 250 mM NaCl (NaCl), distilled water (Dw), dehydration, low temperature at 4°C (Cold), or 100 µM abscisic acid (ABA). The blots were probed with the 32P-labeled ATHK1 cDNA.

We then constructed a chimeric gene consisting of the ATHK1 promoter fused to a ß-glucuronidase (GUS) reporter gene (ATHK1p–GUS) to analyze the distribution of expression of the GUS activity driven by the ATHK1 promoter in transgenic Arabidopsis plants (Figure 9A). Strong GUS activity was detected in leaf bases and roots of rosette plants when the transgenic plants were exposed to either high- or low-osmolarity solutions (Figure 9C and Figure 9D). By contrast, weak GUS activity was observed in transgenic plants that had been exposed to isotonic solution (GM solution) and in untreated plants (Figure 9B and Figure 9G). These results further suggest that the expression of the ATHK1 gene is transcriptionally regulated in response to changes in external osmolarity.

View larger version (82K): [in this window] [in a new window]   Figure 9. Histochemical Localization of GUS Activity in Transgenic Arabidopsis Plants. (A) Shown is the construct used in this experiment. A PCR fragment containing 1.6 kb of the upstream region from the initiation site of translation (ATG) of the ATHK1 gene was fused to the coding region of a GUS reporter gene, as described in Methods. (B) to (G) Histochemical localization of GUS activity in transgenic Arabidopsis plants. (B) Plant grown under normal conditions. (C) Plant exposed to 250 mM NaCl. (D) Plant exposed to distilled water. (E) Dehydrated plant. (F) Plant exposed to 4°C. (G) Plant exposed to isotonic solution.

	DISCUSSION

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In this study, we isolated a cDNA for a hybrid-type histidine kinase that has structural similarity to the yeast osmosensor SLN1. The ATHK1 protein contains both a transmitter and a receiver domain with conserved histidine and aspartate residues, respectively, and a transmembrane domain with two hydrophobic regions toward the N terminus, as does SLN1 (Figure 1B). ATHK1 complemented both sln1-ts and sln1 mutants, whereas ATHK1 that was mutated at the invariant His or Asp positions did not (Figure 5). These results indicate that ATHK1 has a histidine kinase activity. ATHK1 also complemented sln1 sho1 double deletion mutants that lacked two osmosensors, SLN1 and SHO1, whereas ATHK1 mutated at an Asp did not (Figure 6). Moreover, high-salinity-dependent tyrosine phosphorylation of the HOG1 MAP kinase was detected in sln1 sho1 double deletion mutants (Figure 7). These results indicate that ATHK1 became inactive in response to high salinity and activated the HOG1 MAPK cascade, as does SLN1 in yeast. Thus, we showed, by analyzing both sensing (input) and catalytic (output) activities with yeast osmosensing-defective mutants, that Arabidopsis ATHK1 can act as an osmosensor in yeast in vivo.

To date, histidine kinases that play roles in the perception of plant hormone signals have been found in plants (Chang et al. 1993 ; Hua et al. 1995 , Hua et al. 1998 ; Wilkinson et al. 1995 ; Kakimoto 1996 ; Payton et al. 1996 ; Vriezen et al. 1997 ; Lashbrook et al. 1998 ; Sakai et al. 1998 ). However, two-component systems are thought to be involved in sensing osmotic stress, which is a common environmental signal not only for unicellular organisms, such as bacteria and yeast, but also for higher plants. In S. cerevisiae, SLN1 acts as an osmosensory protein, sequentially phosphorylating a phosphorelay intermediater, tyrosine phosphatase dependent 1 (YPD1), and the response regulator SSK1, under conditions of normal osmolarity (Posas et al. 1996 ). These three protein factors perform a four-step phosphorelay (His-Asp-His-Asp). Recently, several cDNAs encoding response regulators (Brandstatter and Kieber 1998 ; Imamura et al. 1998 ; Sakakibara et al. 1998 ; Urao et al. 1998 ) and three cDNAs encoding phosphorelay intermediaters that possess a histidine-containing phosphotransfer domain (Miyata et al. 1998 ; Suzuki et al. 1998 ) have been found in Arabidopsis and maize. These observations further suggest that plants have an osmosensing and signaling system similar to that of yeast. In addition, a MAPK cascade that consists of SSK2/SSK22 (MAPK kinase kinase [MAPKKK]), PBS2 (MAPKK), and HOG1 (MAPK) operates downstream of the SLN1–YPD1–SSK1 two-component osmosensing system in S. cerevisiae. At high osmolarity, unphosphorylated SSK1 activates SSK2/SSK22, resulting in the activation of PBS2 by Ser-Thr phosphorylation. Then, phosphorylated PBS2 activates HOG1 by Thr-Tyr phosphorylation. A MAPK cascade is also thought to be involved in osmotic stress response in higher plants (Hirt 1997 ; Mizoguchi et al. 1997 ). Expression of genes for MAPKs of various plants, such as ATMPK3 in Arabidopsis and MMK4 in alfalfa, is transcriptionally induced by drought, low temperature, and mechanical stress. Moreover, two genes for protein kinases, MAPKKK (ATMEKK1) and ribosomal S6 kinase (ATPK19), are induced by similar stresses (Mizoguchi et al. 1996 ). Recently, activation of MMK4 at the post-translational level by these same stresses was shown (Jonak et al. 1996 ). These observations indicate that a MAPK cascade analogous to the yeast HOG1 pathway might function in plants in a signal transduction cascade in response to osmotic stress.

RNA gel blot analysis revealed that the ATHK1 transcript was more abundant in roots than in other tissues under normal growth conditions and that it accumulated under conditions of high or low osmolarity (Figure 8). Roots represent the interface between a plant and its soil environment and are involved in nutrient acquisition and responses to environmental conditions, such as gravity, light, water, and temperature (Jackson 1997 ). Several ion transporter genes, which are involved in nutrient uptake, are expressed predominantly in roots. Interestingly, the levels of the transcripts are regulated by each of their substrates (Lauter et al. 1996 ; Muchhal et al. 1996 ). The expression of the GUS activity driven by the ATHK1 promoter was strongly induced in roots in response to changes in external osmolarity (Figure 9). These results further indicate that the expression of the ATHK1 gene is transcriptionally upregulated by changes in external osmolarity.

A possible advantage of the upregulation of sensor molecules is speculated as follows. Increased osmotic pressure may cause a conformational change in ATHK1; consequently, its kinase activity may be inactivated. The newly produced ATHK1 under high-osmolarity conditions would also be inactive in the membrane. The accumulation of the inactive ATHK1 may be required to enhance the response or to reduce the response quickly through the reactivation of the kinase activity of ATHK1 when osmotic stress is relieved. Recently, the Arabidopsis ethylene receptors ERS, ERS2, and ETR2 have been shown to be upregulated by ethylene (Hua et al. 1998 ). The upregulation of the ethylene receptors is suggested to be a mechanism for adaptation in ethylene responses by production of additional proteins that can repress the ethylene response (Hua and Meyerowitz 1998 ).

The mRNAs of several genes involved in signal transduction pathways, such as protein kinases, protein phosphatases, a phospholipase C, calmodulins, monomeric small GTP binding proteins, and transcription factors, also accumulate in response to environmental stimuli or stresses (Shinozaki and Yamaguchi-Shinozaki 1996 , Shinozaki and Yamaguchi-Shinozaki 1997 ). Under stress conditions, it appears that the transcriptional regulation of genes for such signal transducers confers higher sensitivity and signaling efficiency to the cellular transducing processes. Thus, a high level of expression of ATHK1 in roots and stressed plants may be necessary for efficient sensing of environmental changes. Although analysis of transgenic plants is now in progress to elucidate the physiological function of ATHK1 in plant cells, we suggest that ATHK1 senses changes in osmolarity in cells caused by dehydration, salinity, and cold stresses, and subsequently transduces the stress signals to the nucleus through a protein phosphorylation cascade, probably a MAPK cascade.

	METHODS

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Plant Materials
Plants (Arabidopsis thaliana ecotype Columbia) were grown on GM agar plates (Valvekens et al. 1988 ) under continuous illumination of ~2000 lux at 22°C for 3 to 4 weeks and were used for the construction of a cDNA library and in stress treatments. Plants used for the analysis of tissue-specific expression were grown in 15-cm pots filled with a 1:1 mixture of perlite and vermiculite and were watered with 0.1% Hyponex (Hyponex, Inc., Tokyo, Japan).

For the growth regulator and salt treatments, plants were transferred to and grown hydroponically in water of either 100 µM abscisic acid or 250 mM NaCl under dim light. For temperature treatments, plants were grown under continuous light at temperatures of 40 or 4°C. For dehydration treatments, plants were removed from the agar and desiccated in plastic dishes at 22°C and 60% humidity under dim light.

Molecular Cloning
Two oligonucleotide primers, 5'-CAC/TGAG/AATGA/CGIACICC-3' and 5'-ATIGCIAG/AICCIAG/AICCIG/CT/AICCICC-3', corresponding to the amino acid sequences HEMRTP (H box) and GGSGLGLAI (G2 box), respectively, were synthesized and phosphorylated with T4 polynucleotide kinase, as described by Sambrook et al. 1989 . Polymerase chain reaction (PCR) was performed in a 100-µL reaction mixture containing 10 mM Tris-HCl, pH 8.9, 80 mM KCl, 1.5 mM MgCl₂, 0.5 mg mL^-1 BSA, 0.1% deoxycholate, 0.1% Triton X-100, 0.2 mM deoxynucleotide triphosphates, 1 nmol of primer, 10 ng of cDNA, and 2.0 units of Tth DNA polymerase (Toyobo, Ltd., Tokyo, Japan). The reaction mixture was overlaid with 100 µL of liquid paraffin and subjected to 30 cycles of amplification at 94°C for 1 min (denaturation), 40°C for 2 min (annealing), and 72°C for 2 min (polymerization) followed by final incubation at 72°C for 7 min. cDNA templates for amplification by PCR were prepared from poly(A)⁺ RNA by using the cDNA synthesis tool System Plus (Amersham International, Little Chalfont, UK).

cDNA Library Screening
The PCR fragments were purified on a 5% polyacrylamide gel and cloned into the SmaI site of pBluescript II SK- (Stratagene, La Jolla, CA). DNA sequences were determined by the dye–primer cycle sequencing method using a DNA sequencer (model 373A; Applied Biosystems, San Jose, CA). Gene Works software (InteriGenetics, Inc., Mountain View, CA) was used for the analysis of DNA and amino acid sequences. To isolate full-length cDNA, we screened by plaque hybridization an Arabidopsis cDNA library (constructed with ZAPII), prepared from rosette plants that had been dehydrated for 1 hr (Kiyosue et al. 1994 ), as described by Sambrook et al. 1989 . Positive plaques were purified, and the cDNA inserts were subcloned into a pBluescript phagemid by an in vivo excision process, according to the manufacturer's instructions (Stratagene).

Restriction Fragment Length Polymorphism Mapping
The Arabidopsis P1 library (Liu et al. 1995 ) was screened using a DNA fragment of ATHK1 as a probe. A P1 plasmid DNA carrying the ATHK1 gene was sheared by vigorous vortexing, labeled by random priming using a ³²P-dCTP labeling kit (BCA Best; Takara, Kyoto, Japan) according to the manufacturer's protocol, and used for restriction fragment length polymorphism (RFLP) mapping with the recombinant inbred lines that had been generated between two ecotypes, Landsberg erecta and Columbia (Lister and Dean 1993 ), as described by Liu et al. 1995 , Liu et al. 1996 . Segregation data for RFLP markers on 99 recombinant inbred lines (Lister and Dean 1993 ) were analyzed using the Mapmaker mapping program (Lander et al. 1987 ), as described by Liu et al. 1996 . The scoring data for 67 markers (provided by C. Dean, John Innes Research Institute, Colney, UK) and those of 129 mi markers (Liu et al. 1996 ) were used for the calculation.

Complementation Analysis of the Temperature-Sensitive SLN1 Mutants
Yeast (Saccharomyces cerevisiae) strain HS13 with a temperature-sensitive SLN1 allele (Maeda et al. 1994 ) was used for complementation experiments. Expression plasmids used in this experiment were constructed with the multicopy expression vector pNV7, in which expression is under the control of the galactose-inducible GAL1 gene promoter (Jonston 1987 ). The ATHK1 expression plasmid pNV-ATHK1FL was constructed by subcloning a BamHI fragment containing the complete open reading frame from the ATHK1 cDNA into the BamHI site of pNV7.

To construct the catalytically inactive mutants pNV-ATHK1H508V and pNV-ATHK1D1074E, we subcloned BamHI-XbaI and SpeI fragments from the ATHK1 cDNA containing His-508 and Asp-1074 residues, respectively, into pBluescript II SK-. Nucleotide substitutions were generated by a site-directed mutagenesis kit (Chameleon; Stratagene) with oligonucleotides 5'-pTTGGCAAATATGAGTGTTGAGTTGAGGACACCA-3' (H508V; His [CAT] replaced by Val [GTT]) and 5'-pTTTTTGGCATCTGGCACTCCATGAGGATCAAG-3' (D1074E; Asp [GAC] replaced by Glu [CTC]). Each mutation was confirmed by DNA sequencing. Subsequently, the mutated fragments were cloned into their parental plasmids; then each BamHI fragment containing the complete open reading frame was subcloned into the BamHI site of pNV7.

To construct the deletion mutant of the extracellular domain, we subcloned pNV-ATHK1dExD, a BamHI-XbaI fragment from the ATHK1 cDNA, into pBluescript II SK- and then mutated it with the oligonucleotide 5'-pGCCTTAGATACGAAGATCTTCAGCGTCCAGTC-3' (GCT replaced by AGA, resulting in a BglII site [AGATCT]) to introduce an additional BglII site at amino acid 122. The plasmid was digested with BglII to remove an internal BglII fragment (amino acids 122 to 231) and religated, resulting in an in-frame deletion. The fragment encoding the protein in which the extracellular domain had been deleted was excised with XhoI and XbaI and used to replace the XhoI- XbaI fragment of pNV-ATHK1FL. To construct the N-terminal-half deletion mutant pNV-ATHK1SOL, a cDNA fragment (amino acids 477 to 1207) was amplified by PCR by using oligonucleotides incorporating BamHI or KpnI sites at the 5' ends (5'-CAGTGGATCCAT-GAAGCTAAGAGCAGAACTGATAAGG-3' [for amino acids 477 to 485; the BamHI site is underlined] and 5'-ATAGGGTACCGTTAGATTTTTACCCGATTCTTCA-3' [for amino acids 1200 to 1207; the KpnI site is underlined]). The PCR fragments were digested with BamHI and KpnI and then cloned into the BamHI-KpnI site of pNV7.

The ETR1 expression plasmid pNV-ETR1 was constructed by insertion of a BamHI fragment from the ETR1 cDNA into the BamHI site of pNV7. The SLN1 expression plasmid cloned in pRS414, pPD2146 (Maeda et al. 1994 ), was used as a positive control. These plasmids were introduced into yeast cells by using a lithium-acetate method (Ito et al. 1983 ).

Complementation Analysis of the sln1 and sho1 Deletion Mutant (sln1 sho1)
To construct the sln1 deletion mutant (sln1), strain TM141 (MAT , ura3, leu2, trp1, his3) (Maeda et al. 1994 ) was transformed with the PTP2 expression vector under the control of the GAL1 promoter (pGP22; GAL1p::PTP2 in pRS413) (Maeda et al. 1994 ). The resulting transformants were mated with the strain TM205 (MAT , ura3, leu2, his3, sln1::LEU2, ssk1::URA3) (Maeda et al. 1994 ). The heterozygous diploid cells were induced to sporulate, and sln1 deletion mutants (sln1::LEU2, GAL1p::PTP2) were obtained by random spore analysis, as described by Ausubel et al. 1987 . The sln1 deletion mutants were transformed with either YEp-ATHK1FL or pRS-SLN1, and their ability to grow on YPD medium (1% yeast extract, 2% polypeptone, 2% glucose [rather than galactose]) was examined.

Expression plasmids used in this experiment were constructed with a multicopy expression vector, YEpGAP, in which expression is under the control of the constitutive GAP promoter (Rosenberg et al. 1990 ). The plasmids YEp-ATHK1FL and YEp-ATHK1D1074E were constructed by insertion of BamHI fragments containing the complete open reading frame from pNV-ATHK1FL and pNV-ATHK1D1074E, respectively, into the BamHI site of YEpGAP. The hog1 deletion mutant (hog1) was constructed by introduction of the hog1::LEU2 disruption vector pHG11 (Maeda et al. 1994 ) into TM141. The disruption of hog1::LEU2 was confirmed by the failure of growth on YPD medium containing 0.9 M NaCl. The sln1 sho1 deletion double mutant (sln1 sho1) was constructed by transformation of the sln1 deletion mutant with the sho1::URA3 disruption vector pDOS82 (Maeda et al. 1995 ). The disruption of sho1 by URA3 was confirmed by PCR amplification using the oligonucleotides 5'-TCTGGATTCTGAACTACGGCG-3' and 5'-ATCATCGTCATCAGCGTCG-3' corresponding to both flanking sequences at the URA3 insertion point and the genomic DNA isolated from Ura⁺ transformants. The sln1 and sho1 deletion mutants (sln1 sho1) were then transformed with YEp-ATHK1FL, YEp-ATHK1D1074E, or pRS-SLN1. The ssk1 and sho1 deletion strain TM301 (MAT, ura3 leu2 his3 trp1 ssk1::HIS3 sho1::TRP1) was kindly provided by T. Maeda (Cancer Institute, Tokyo, Japan) and used as a negative control.

Analysis of the Tyrosine Phosphorylation of HOG1 MAP Kinase
The yeast strains were cultured on YPD medium. When the cell density of the culture reached 10⁷ cells per mL, NaCl was added and incubation was continued for various periods. The cells (in 1.5 mL of culture medium) were harvested by centrifugation, resuspended in 40 µL of SDS sample buffer, immediately boiled, and then resolved by 10% SDS-PAGE. Proteins were transferred to a polyvinylidine difluoride membrane (Immobilon-P; Millipore Ltd., Tokyo, Japan), and the blot was blocked in TBST buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Triton X-100) containing 3% BSA and then incubated with antiphosphotyrosine monoclonal antibody 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY) in TBST buffer containing 1% BSA. The blot was washed three times with TBST buffer and then incubated with anti–mouse IgG antibody conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in TBST buffer containing 1% BSA. After three washes with TBST buffer, the immune complexes were visualized by using the enhanced chemiluminescence detection system (Amersham International).

RNA Gel Blot Analysis
Total RNA was isolated by the phenol-SDS method described by Ausubel et al. 1987 . Thirty micrograms of total RNA was fractionated on a 1% agarose gel containing formaldehyde and blotted onto a nylon membrane (Hybond-N; Amersham International). The membrane was hybridized with the ³²P-labeled PCR fragment in 50% formamide, 5 x SSC (1 x SSC is 0.15 M NaCl and 0.015 M sodium citrate), 25 mM sodium phosphate buffer, pH 6.5, 10 x Denhardt's solution (1 x Denhardt's solution is 0.02% Ficoll, 0.02% PVP, and 0.02% BSA), and 250 µg mL^-1 of denatured salmon sperm DNA at 42°C. The membrane was washed twice with 0.1 x SSC and 0.1% SDS at 60°C for 15 min and subjected to autoradiography.

Histochemical Analysis of GUS Activity
A genomic DNA library (Clontech, Palo Alto, CA) was screened using the full-length cDNA clone as a probe. Based on DNA gel blot analysis of the purified DNA digested with several enzymes, a 3.2-kb XbaI-SacI DNA fragment was subcloned, and part of it was sequenced. A DNA fragment containing 1.6 kb of the upstream region of the ATHK1 gene was amplified by PCR by using oligonucleotides incorporating SalI or BamHI sites at the 5' ends (5'-GAGGTCGACGGTATCGATAAGCT-3' [the SalI site is underlined] and 5'-TCGGATCCGTTTCAGATGATCCCAAATCAT-3' [the BamHI site is under-lined]). The PCR fragments were digested with SalI and BamHI and then cloned into the SalI-BamHI site of a promoterless ß-glucuronidase (GUS) expression vector, pBI101.1 (Clontech). The resulting construct, ATHK1p–GUS, was transferred from Escherichia coli DH5 into Agrobacterium tumefaciens C58C1Rif by triparental mating with E. coli that contained plasmid pRK2013. Arabidopsis was transformed by vacuum infiltration with Agrobacterium that contained ATHK1p–GUS, as described by Bechtold et al. 1993 . Histochemical localization of GUS activities in the transgenic plants was performed by incubating the transgenic unbolted plants in 5-bromo-4-chloro-3-indolyl glucuronide (X-gluc) buffer (50 mM sodium phosphate buffer, pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 2% DMSO, 0.5 mM potassium ferrocyanide, 2 mg mL^-1 X-gluc) at 37°C for 6 to 12 hr. The stained plants were fixed in 5% formaldehyde, 5% acetic acid, and 20% ethanol and then washed for 30 min with 50 to 100% ethanol to remove the chlorophyll.

	ACKNOWLEDGMENTS

We thank Drs. Tatsuya Maeda (Cancer Institute, Tokyo, Japan), and Haruo Saito (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA) for providing us with yeast strains and plasmids and for helpful discussions. We also thank Drs. Tatsuo Kakimoto (Osaka University, Osaka, Japan) and Takeshi Mizuno (Nagoya University, Nagoya, Japan) for helpful discussions. This work was supported in part by a grant from the Program for Promotion of Basic Research Activities for Innovative Bioscience to K.Y.-S., by the Special Coordination Fund of the Science and Technology Agency of the Japanese Government, and by a Grant-in-Aid from the Ministry of Education, Science, and Culture of Japan and the Human Frontier Science Program to K.S. T.U. is supported by a fellowship from the Science and Technology Agency of Japan.

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