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Novel Family of Sensor Histidine Kinase Genes in Arabidopsis thaliana

Article  in  Plant and Cell Physiology · February 2001

DOI: 10.1093/pcp/pce015

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Plant Cell Physiol. 42(2): 231–235 (2001)
JSPP © 2001

Novel Family of Sensor Histidine Kinase Genes in Arabidopsis thaliana

Chiharu Ueguchi 1, 2, 3, Hiromi Koizumi 2, Tomomi Suzuki 2 and Takeshi Mizuno 2
1
Bioscience Center, Nagoya University, Chikusa-ku, Nagoya, 464-8601 Japan
2
School of Agriculture, Nagoya University, Chikusa-ku, Nagoya, 464-8601 Japan

;
We identified three novel, highly homologous, sensor search with several EST databases was carried out at the DDBJ
histidine kinases that possibly function in the plasma mem- web site (National Institute of Genetics) using the TBLASTN
brane of Arabidopsis thaliana, i.e. AHK2, 3 and 4. While program (Altschul et al. 1990). The amino acid sequences used
AHK2 and 3 are expressed in several organs, AHK4 is for the search were AHK2 (amino acids 259–536), AHK3
mainly expressed in roots. AHK3 suppresses a sensor histi- (amino acids 120–399), and AHK4 (amino acids 155–421).
dine kinase mutant of yeast. Construction of a phylogenetic tree was carried out by the

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neighbor-joining method (Saitou and Nei 1987). The amino
Key words: Phosphorelay — Sensor histidine kinase — Sig- acid sequences of the transmitter domains of Arabidopsis sen-
nal transduction. sor kinases were aligned using the CLUSTAL W program
(Thompson et al. 1994) at the DDBJ web site and then the tree
was drawn by TreeView program (Page 1996). Note that all the
amino acid sites where gaps exist in the alignment were
The perception of extracellular stimuli and subsequent excluded from the calculation.
signal transduction are essential for the acclimation (and Isolation of cDNA clones was performed as follows. First,
survival) of organisms to harsh environmental conditions. Sen- we obtained probe DNAs, each corresponding to a putative
sor histidine kinases play an important part in signal percep- transmitter domain, by means of genomic PCR. Using genomic
tion and are widely used as components of the His-to-Asp DNA prepared from the Columbia ecotype of A. thaliana (L.)
phosphorelay systems in prokaryotic signal transduction sys- Heynh as a template, PCR (for 40 cycles of 0.5 min at 94
C,
tems (for a review, see Parkinson and Kofoid 1992). Recent 0.5 min at 55
C, and 3 min at 72
C in 50 l reaction mixture)
genetic analyses have revealed that sensor histidine kinases was performed using the following gene-specific primers; 5-
play important roles in several eukaryotic species including ATGAGGGAACTCAAAGCTC-3 and 5-AACTCCAGTAAA-
yeasts, fungi, slime molds and higher plants (Chang et al. 1993, TGAAAAAG-3 for AHK2, 5-ATGAAGCAGCTCAAGAAA-
Wurgler-Murphy and Saito 1997, Chang and Stewart 1998, and AAGG-3 and 5-TACAGCAGTAAATGTGAATG-3 for AHK3,
references therein). and 5-ATGCAAGAGCTTAAAGTTCG-3 and 5-ATCGCAT-
Several sensor kinases in Arabidopsis thaliana have been TTCTCTAAAACAGC-3 for AHK4. Using the resultant DNA
identified. Five of them, ETR1, ETR2, EIN4, ERS1 and ERS2, fragments as probes, we screened a gt11-bank of Arabidopsis
are involved in the control of the ethylene signal transduction cDNA as described previously (Suzuki et al. 1998). Cloning of
pathway (Hua and Meyerowitz 1998, and references therein). the 5 portion of each cDNA was carried out by reverse tran-
CKI1 sensor kinase is presumably involved in the cytokinin- scriptase-PCR (RT-PCR). Note that the nucleotide sequences of
signaling pathway (Kakimoto 1996). AtHK1 sensor has been the primers used for RT-PCR were; 5-ATGTCTATAACTTGT-
implicated in the osmotic response (Urao et al. 1999). These GAGCTCTTGA-3 and 5-ACAAGGTTCAAAGAATCTTGC-
observations strongly suggested that sensor histidine kinase TACC-3 for AHK2, 5-GGTTGATCGTGTATTCAAGTG-
families are important components for the adaptive responses GTGG-3 and 5-ATCAGGGGGAGATGGTTCAAAGC-3 for
of higher plants. The plant genome is likely to include other AHK3, and 5-ATGAGAAGAGATTTTGTGTA-3 and 5-
sensor histidine kinase families. ATCGCATTTCTCTAAAACAGC-3 for AHK4. RT-PCR with
A similarity search with Arabidopsis genomic sequences 1 g of total RNA prepared from Arabidopsis shoots or roots
deposited in GenBank was carried out at the KAOS web site was carried out with an RNA PCR kit (Takara Shuzo Co.,
(Kazusa DNA Research Institute) using the TBLASTN pro- Japan). After reverse transcription at 50
C for 1 h in 20 l reac-
gram (Altschul et al. 1990). The query sequences were the tion mixture, PCR was performed for 40 cycles of 0.5 min at
transmitter domains of ETR1 (amino acids 332–579; Chang et 94
C, 0.5 min at 55
C, and 3 min at 72
C in 100 l reaction
al. 1993), CKI1 (amino acids 384–666; Kakimoto 1996) and mixture. The resultant DNA fragments were cloned into the
AtHK1 (amino acids 501–744; Urao et al. 1999). A similarity pT7 Blue T-vector (Novagen) and sequenced with an auto-
3
Corresponding author: E-mail, [email protected]; Fax, +81-52-789-5214; Phone, +81-52-789-5512.

231
232 Novel family of sensor histidine kinase genes

mated DNA sequencer (Applied Biosystems, model 373A),

with the recommended sequencing kit, according to the manu-
facturer’s instructions. RT-PCR, to examine the expression of
AHKs in plants, was carried out using total RNA prepared from
roots, leaves, stems and flowers, as described previously
(Suzuki et al. 1998). One g of each total RNA was used for
RT-PCR analysis as described above. The primer sets corre-
sponding to the respective transmitter domains were the pairs
used for the genomic PCR described above. Two yeast strains,
TM182 (Maeda et al. 1994) and CUY1 (Suzuki et al. 1998),
were used for suppression analysis. The plasmids used in this
study were constructed as follows. The full-length AHK3
cDNA was cloned into pCUY315 (C. Ueguchi, unpublished), a
yeast expression vector carrying CEN6, the ADH1 promoter,
and the LEU2 marker, to yield pCUY41. pCUY65 is a deriva-
tive of pCUY41, carrying the H460L mutation of AHK3. The

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mutation was introduced by site-directed mutagenesis with a
Mutan-K kit (Takara Shuzo Co., Japan). The sequence of the
oligonucleotide used was 5-CTTGCCACTGTTTCACTT-
GAAATCAGAACTCCA-3, with one base change (under-
lined). pCU64 is also a derivative of pCU41, carrying the
URA3 marker instead of LEU2. pCUY1 (Suzuki et al. 1998) Fig. 1 Primary structures of AHK proteins. (A) The primary struc-
tures of AHK proteins are represented schematically. Closed and
carrying the YPD1 gene was also used as an appropriate control
hatched rectangles indicate transmembrane segments (TM) and extra-
plasmid. Yeast transformation was carried out by the lithium cytoplasmic domains (Ex), respectively. Rounded rectangles and ovals
acetate method (Rose et al. 1990). Overnight cultures of the with thin and thick lines indicate transmitter (T), receiver-like (RL),
transformants in synthetic complete medium (SC; Rose et al. and receiver (R) domains, respectively. The conserved motifs in the
1990) supplemented with 2% galactose were diluted 10–1-fold latter domains are indicated within the symbols. Underlined letters
denote presumed phosphorylation sites. (B) Phylogenetic relation-
with TE buffer (10 mM Tris-HCl [pH 7.5], 1 mM EDTA) and
ships of the amino acid sequences of the transmitter domain of sensor
then a 10 l portion of each culture was spotted onto an SC histidine kinases in Arabidopsis. Numbers at the branch points repre-
plate supplemented with either 2% galactose or glucose. The sent the bootstrap values (%) of 1,000 replicate trees.
plates were incubated at 30
C for 3 d.
To identify novel sensor histidine kinase genes in Arabi-
dopsis, we carried out a similarity search using the amino acid
sequences of the transmitter domains of several sensor histidine mitter domain and a receiver domain (“T” and “R” in Fig. 1A),
kinases (e.g. ETR1, CKI1 and AtHK1) as probes. The screen- indicating that they each represent a hybrid-type of sensor his-
ing in silico revealed three putative genes, described below, tidine kinase. As shown in Fig. 2A and B, they possess invari-
each potentially encoding a novel sensor histidine kinase (Gen- ant histidine residues in the transmitter domain and aspartate
Bank accession numbers AB011485, AC004557 and residues in the receiver domain. Furthermore, they contain
AC007069). To determine their putative amino acid sequences, characteristic consensus motifs (Parkinson and Kofoid 1992)
we isolated the relevant cDNAs. Because the cDNA clones H, N, G1, F and G2 in the transmitter domain, and the DD, D
thus obtained appeared to lack the 5 portion, RT-PCR were and K in the receiver domain. The primary structures of the
therefore performed to obtain full-length cDNA clones. signaling domains of the newly identified proteins are quite
Sequencing analyses of the entire cDNA clones thus obtained similar to each other. The sequence identity between two given
revealed that they encode novel sensor histidine kinases corresponding sequences in each domain is over 60%. To con-
expressed in the plant. We designated these genes of firm this further, we constructed a phylogenetic tree using the
AB011485, AC004557 and AC007069 as AHK (Arabidopsis amino acid sequences of the transmitter domains of Arabidop-
histidine kinase) 2, 3 and 4, respectively (the cDNA nucleotide sis sensor kinases by means of the neighbor-joining method
sequences of AHK2, 3 and 4 have been deposited in the DDBJ/ (Saitou and Nei 1987). The results also showed that AHK2, 3
EMBL/GenBank databases, under accession numbers and 4 belong to a novel family of sensor histidine kinase genes
AB046869, AB046870 and AB046871, respectively). in Arabidopsis (Fig. 1B). Hydrophobicity analyses revealed
The nucleotide sequences revealed that AHK2, 3 and 4 putative transmembrane segments in each of the N-terminal
encode open reading frames of 1,176, 1,036 and 1,080 amino parts (closed rectangles in Fig. 1A), three in AHK2 and 3, and
acid residues with predicted molecular masses of 132, 116 and two in the case of AHK4. Use of the PSORT program (Nakai
121 kDa, respectively. Each of them contains a typical trans- and Horton 1999) at the GenomeNet web site (Institute for
 Novel family of sensor histidine kinase genes 233

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Fig. 2 Alignment of the deduced amino acid sequences of AHKs with other related sequences. (A) Alignment of the amino acid sequences of
transmitter domains. The predicted amino acid sequences of the transmitter domains of AHKs are aligned with those of ETR1 (Chang et al. 1993)
and CKI1 (Kakimoto 1996). The five conserved motifs (Parkinson and Kofoid 1992) are indicated above the alignment. The numbers in parenthe-
ses are numbers of amino acid residues in the gaps between the motifs. The amino acid residues conserved among the AHKs are highlighted. The
alighment was constructed by CLUSTAL W program (Thompson et al. 1994). (B) Alignment of the amino acid sequences of receiver (R) as well
as receiver-like (RL) domains, as described in panel (A). The three conserved motifs (Parkinson and Kofoid 1992) are indicated above the align-
ment. The numbers in parentheses are the numbers of amino acid residues in gaps. (C) Alignment of the amino acid sequences of extracytoplas-
mic domains. The predicted amino acid sequences of the extracytoplasmic domains of AHKs with several EST sequences are aligned, as
described in panel (A). The GenBank accession numbers used here are: A. AI974236 (Medicago truncatula), B. BE417613 (Triticum aestivum),
C. AW585576 (Medicago truncatula), D. AA660817 (Medicago truncatula), and E. AU062638 (Oryza sativa).
234 Novel family of sensor histidine kinase genes

Fig. 4 Suppression of the sln1
mutation by AHK3. Yeast cells,

TM182 and CUY1 each carrying sln1
and ypd1
mutations, respec-
tively, were transformed with plasmids carrying the indicated genes
Fig. 3 Expression of AHKs in several plant organs. RT-PCR analysis under the ADH1 promoter control. Overnight cultures of the trans-

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involving total RNA prepared from roots (R), leaves (L), stems (S), formants in synthetic complete (SC) medium supplemented with 2%
and flowers (F) was carried out. The PCR products with cDNA (c) and galactose were appropriately diluted and then aliquots were spotted
genomic DNA (g) as templates are also shown. Arrows and arrow- onto SC plate supplemented with either 2% galactose (Gal.) or glu-
heads indicate the positions of PCR products generated from mRNA cose (Glc.). The plates were incubated at 30C for 3 d.
and contaminating genomic DNA, respectively.

Chemical Research, Kyoto University) indicated that they are Finally, we examined whether or not the putative AHKs
located in the plasma membrane (note that the certainty of the are able to function as histidine kinases by means of a yeast
prediction for localization in the plasma membrane was 0.600 genetic system. Budding yeast gene SLN1 encodes a sensor his-
in all cases). tidine kinase, the deletion of which is lethal due to constitutive
In addition to the signaling modules, another characteristic activation of the HOG1 MAP kinase cascade (Maeda et al.
sequence segment consisting of about 280 amino acids is con- 1994). Yeast strain TM182 (Maeda et al. 1994) carries the
served in all the newly identified AHK family members. This sln1, mutation and a plasmid harboring PTP2. The latter
is located between the two transmembrane segments (second encodes a phospho-tyrosine phosphatase under GAL promoter
and third ones of AHK2 and 3, and first and second ones of control. TM182 cells can grow on galactose- but not on
AHK4), and is presumably located in the extracytoplasmic glucose-containing medium due to the galactose-dependent
space (“Ex” in Fig. 1A, 2C). Finally, in all three AHKs, the expression of PTP2 (inactivation of the HOG1 MAP kinase
amino acid residues between the transmitter and receiver cascade). The AHK3 cDNA was cloned into pCUY315 (C.
domains show some similarity those of typical receiver, and Ueguchi, unpublished), a yeast expression vector carrying
thus we named them receiver-like domains (“RL” in Fig. 2B, CEN6, the ADH1 promoter, and the LEU2 marker. The result-
upper panel). The potential phospho-accepting aspartate resi- ant construct was then introduced into TM182 cells and AHK3
due in the RL domain of AHK2 is conserved, but the corre- expression was induced constitutively with the ADH1 pro-
sponding residues in AHK3 and 4 are not; they are substituted moter. As shown in Fig. 4, TM182 harboring AHK3 cDNA
with glutamate (Fig. 2B), suggesting that the RL domains of grew well on either galactose or glucose medium. In contrast,
AHK3 and 4 may not be functional in terms of the phosphore- the introduction of pCUY65 carrying a mutated AHK3 gene, in
lay reaction. which the His-460 residue in the transmitter domain was sub-
To examine the expression of AHKs in plants, a series of stituted with Leu (H460L), was unable to suppress the growth
RT-PCRs was carried out using total RNA prepared from repre- defect on glucose (Fig. 4). To determine whether or not the
sentative plant organs, including roots, leaves, stems and flow- suppression ability of AHK3 depends on the Ypd1 function (the
ers. As shown in Fig. 3, the transcript levels of AHK3 and 2 yeast HPt factor functioning just downstream of Sln1; Posas et
were determined in all the organs examined. While AHK3 al. 1996), AHK3 cDNA was introduced into CUY1 cells carry-
expression was observed significantly in all organs tested, the ing the ypd1, mutation (Suzuki et al. 1998). As shown in Fig.
AHK2 signal was relatively weak (Fig. 3). Interestingly, the 4, AHK3 failed to suppress this particular mutation. These data
expression of AHK4 appears to be organ-specific. A signifi- clearly indicated that AHK3 could function as a histidine
cant amount of the AHK4 transcript was observed in roots, kinase, at least in yeast. Notably, unlike in the case of AHK3,
whereas it was hardly detected in stems or flowers, and not at the introduction of AHK2 cDNA did not suppress the mutated
all in leaves (Fig. 3). sln1, strain (data not shown) for an as yet unknown reason.
 Novel family of sensor histidine kinase genes 235

The results described here revealed that Arabidopsis con- Hua, J. and Meyerowitz, E.M. (1998) Ethylene responses are negatively regu-
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similarity of their primary structures suggests that they may signal transuction. Science 274: 982–985.
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somewhat supported by the presence of a conserved extracyto- tem that regulates an osmosensing MAP kinase cascade in yeast. Nature 369:
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Harbor, NY.
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(Received Septeber 13, 2000; Accepted November 12, 2000)

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