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Mol Genet Genomics (2001) 265: 2±13
DOI 10.1007/s004380000400
O R I GI N A L P A P E R
J. Lohrmann á U. Sweere á E. Zabaleta á I. BaÈurle
C. Keitel á L. Kozma-Bognar á A. Brennicke
E. SchaÈfer á J. Kudla á K. Harter
The response regulator ARR2: a pollen-speci®c transcription factor
involved in the expression of nuclear genes for components
of mitochondrial Complex I in Arabidopsis
Received: 8 August 2000 / Accepted: 24 October 2000 / Published online: 3 January 2001
Ó Springer-Verlag 2001
Abstract Two-component signal systems regulate a variety of cellular activities. They involve at least two
common signalling molecules: a signal-sensing kinase
and a response regulator that mediates the output response. Multistep systems also require proteins containing phosphotransfer domains. Here we report that
the response regulator ARR2 from Arabidopsis is predominantly expressed in pollen and is localized in the
nuclear compartment of the plant cell. Furthermore,
ARR2 is transcriptionally active in yeast and binds to
the promoters of nuclear genes for several components
of mitochondrial respiratory chain complex I (nCI) from
Arabidopsis. The nuclear nCI genes are up-regulated in
pollen during spermatogenesis. The transcription factor
functions of ARR2 are mediated by its C-terminal
output domain. Our data identify ARR2 as the ®rst
eukaryotic response regulator which functions as a
transcription factor at a known promoter sequence.
Yeast two-hybrid analysis and in vitro interaction
studies suggest that ARR2 very probably forms part of a
multistep two-component signalling mechanism that
includes HPt proteins like AHP1 or AHP2. These ®ndings point to an as yet unidenti®ed signal transduction
Communicated by R. Hagemann
J. Lohrmann á U. Sweere á I. BaÈurle á C. Keitel
E. SchaÈfer á K. Harter (&)
Institut fuÈr Biologie II/Botanik, UniversitaÈt Freiburg,
SchaÈnzlestrasse 1, 79104 Freiburg, Germany
E-mail: [email protected]
Fax: +49-761-2032612
E. Zabaleta
Instituto de Investigaciones BiotecnoloÂgicas,
IIB/INTECH (CONICET/UNSAM) C.C. 164,
7130 ChascomuÂs, Argentina
L. Kozma-Bognar
Institute of Plant Biology, Biological Research Center,
P.O. Box 521, 6701 Szeged, Hungary
A. Brennicke á J. Kudla
Institut fuÈr Allgemeine Botanik, UniversitaÈt Ulm,
Albert-Einstein-Allee 11, 89069 Ulm, Germany
system that may regulate aspects of ¯oral and mitochondrial gene expression.
Key words Two-component system á Response
regulator á HPt protein á Pollen-speci®c transcription
factor á Mitochondrial Complex I
Introduction
Prokaryotic and eukaryotic organisms have evolved
sophisticated sensing and signalling systems which elicit
a variety of responses to alterations in environmental
conditions (for review see Parkinson 1993; Loomis et al.
1997). Among them the so-called two-component systems typically involve two signal transduction proteins,
a sensor kinase and a response regulator (Stock et al.
1989, 1990; Parkinson and Kofoid 1992). The sensor
kinases monitor environmental parameters, such as nutrient status, attractants, repellents, osmotic pressure
and light, and modulate the activity of their cognate
response regulators accordingly, via a His-to-Asp
phosphorelay (for review see Stock et al. 1989; Parkinson and Kofoid 1992; Parkinson 1993; Wurgler-Murphy
and Saito 1997; Chang and Stewart 1998). The phosphorylation of the receiver module within the response
regulator molecule alters the activity of its output domain, which eventually leads to alterations in cellular
functions including gene expression.
The best characterized eukaryotic two-component
system is the osmo-responsive pathway in yeast (for
review see Wurgler-Murphy and Saito 1997; Chang and
Stewart 1998). Additional examples have been discovered in Neurospora crassa (Alex et al. 1996) and in
Dictyostelium (Schuster et al. 1996; Wang et al. 1996).
Frequently the two-component systems of these eukaryotic systems are of the multistep type, and require
additional HPt proteins which mediate phosphorelay
signal transfer from hybrid kinases to their cognate response regulators (Chang and Stewart 1998; D'Agostino
and Kieber 1999). In higher plants, the identi®cation of
3
several sensor kinases has been reported; these are involved in sensing of ethylene, cytokinins and osmotic
pressure (Kieber 1997; Chang and Stewart 1998; Urao
et al. 1999). Very recently, genes coding for response
regulators (ARRs) as well as for HPt proteins (AHPs)
have been described in Arabidopsis thaliana (for review
see D'Agostino and Kieber 1999; Imamura et al. 1999).
The multiple isoforms of HPt proteins and response
regulators thus increase the complexity of two-component signalling systems and the degree of integration
required between them.
Based on the amino acid sequences of the receiver
modules, the response regulator family from A. thaliana
can be subdivided into two distinct subclasses, termed
type-A and type-B (D'Agostino and Kieber 1999;
Imamura et al. 1999). Type-A proteins lack a C-terminal
extension and their genes are rapidly induced by cytokinin (Brandstatter and Kieber 1998; Taniguchi et al.
1998; Kiba et al. 1999). Accordingly, they are regarded
as primary response genes in cytokinin signalling. In
contrast, the expression of type-B ARR genes is not
a€ected by exogenous application of plant hormones
(Kiba et al. 1999; Lohrmann et al. 1999). Response
regulators of type-B are characterized by the presence of
a large C-terminal extension. Several lines of evidence
suggest that these extensions could mediate transcription
factor functions, since an 80-amino acid stretch of this
region (the B-motif) is similar to a Myb-related motif
that is potentially capable of binding to DNA (Sakai
et al. 1998; Imamura et al. 1999). Similarity to basic
helix-loop-helix (bHLH) transcription factors has also
been noted (Lohrmann et al. 1999) and some of the Cterminal extensions in type-B proteins are rich in proline
and glutamine residues, a feature often observed in eukaryotic activation domains (Tjian and Maniatis 1994).
The C-terminal domain of the type-B response regulator
ARR11 indeed activates transcription in yeast when
fused to the GAL4-DNA-binding domain (Lohrmann
et al. 1999). The C-terminal extensions also contain
putative nuclear localization sequences (NLSs; Sakai
et al. 1998), and fusion of green ¯uorescence protein
(GFP) to full-length ARR11 or to its C-terminal domain
results in localization of each derivative to the nucleus in
transiently transformed plant protoplasts (Lohrmann
et al. 1999). Although these data strongly suggest that
B-type ARRs may function as transcription factors in
plants, none of their target genes/promoters have yet
been identi®ed and their precise biological function has
remained elusive.
Here we report that the nuclear type-B response
regulator ARR2 is predominantly expressed in pollen of
Arabidopsis ¯owers, and binds to the promoters of several nuclear genes for components of mitochondrial respiratory chain complex I (nCI) from Arabidopsis, when
tested in a yeast one hybrid system as well as in vitro.
Furthermore, ARR2 activates transcription in yeast.
Both DNA binding and transactivation are mediated by
the C-terminal output domain of ARR2. Based on these
data we conclude that ARR2 acts as a transcription
factor. As suggested by two-hybrid assays in yeast and
by in vitro interaction studies, ARR2 is very probably
part of a plant two-component mechanism that includes
HPt proteins like AHP1 or AHP2. These results indicate
that ARR2 is involved in a new type of two-component
system that regulates the expression of nuclear genes for
mitochondrial proteins in pollen grains.
Materials and methods
Plant materials, electroporation of parsley protoplasts,
and microscopic techniques
A. thaliana (L.) ecotype Columbia plants were grown on soil in10cm pots on a 16 h light/8 h dark cycle. Harvested tissues and organs
were immediately frozen in liquid nitrogen. Protoplasts were prepared from parsley (Petroselinum crispum L.) cell cultures 6 days
after subculturing as described previously (Frohnmeyer et al. 1994).
Transient transformation of protoplasts by electroporation, and
the microscopic techniques used, were carried out according to
Kircher et al. (1999). Preparation of samples for sectioning was
performed as described by Fischer and Neuhaus (1996). For
graphics and image processing the Micrografx Graphics Suite
(Micrografx) and the Microsoft Oce (Microsoft) software packages were used.
Isolation of cDNAs and plasmid construction
Polymerase chain reactions (PCRs) were performed using 2.5 ll of
phage suspension from an ampli®ed cDNA library (Kieber et al.
1993) with speci®c primers in order to isolate the ARR2 (GenBank
Accession No. AJ005196), AHP1 (U. Sweere and K. Harter,
manuscript in preparation; Suzuki et al. 1998) and AHP2 (Suzuki
et al. 1998) sequences. The PCR products were digested with the
appropriate restriction enzymes and subcloned into pBS-KS
(Stratagene). Using these constructs as templates, further PCRs
were carried out to generate cDNAs encoding the receiver module
(aa 1 to aa 164) and the C-terminal domain of ARR2 (aa 165 to
aa 664). To isolate the ARR2 promoter (nucleotide positions )3032
to )1), PCR was done with speci®c primers and genomic DNA
from A. thaliana (L.) ecotype Columbia as template. The ARR4
and ARR11 constructs have been described elsewhere (Lohrmann
et al. 1999). All cDNAs and genomic sequences generated by PCR
were sequenced. Primer sequences can be obtained on request.
For construction of the di€erent expression plasmids, the appropriate cDNAs were cloned into the Escherichia coli expression
vector pASK-IBA2 (IBA) to produce ARR4-Strep, ARR2-Strep,
and ARR2receiver-Strep, into the E. coli expression vector
pET24b(+) (Calbiochem-Novabiochem) to generate AHP1-(His)6,
AHP2-(His)6, and ARR2output-(His)6, into the plant expression
vector pMAV4 (Kircher et al. 1999) to produce ARR2-green ¯uorescence protein (GFP), into the yeast two-hybrid vector pGBT9
(Clontech) to generate the GAL4 binding domain (BD) fusions of
ARR2, ARR2receiver, ARR2output, AHP1 and AHP2, into the yeast
two-hybrid vector pGAD424 (Clontech) to produce GAL4
activation domain (AD) fusions of ARR2, ARR2receiver and
ARR2output, and into the yeast expression vector pJR3611
(Yalovsky et al. 1997) to generate pJR3611-ARR2.
Generation of transgenic Arabidopsis lines and determination
of uidA reporter gene activity
The ARR2 promoter was cloned into the binary vector pGPTVBAR (Becker et al. 1992) upstream of the uidA gene to form a
ARR2 promoter-glucuronidase (GUS) reporter gene. This construct, as well as a promoter-less uidA construct, was transformed
into the Agrobacterium tumefaciens strain GV3101. Arabidopsis
4
plants were transformed by in planta in®ltration (Bechthold et al.
1993). Seeds of in®ltrated plants were sown in soil and grown under
continuous white light for 20 days. Plants were sprayed twice
within 72 h with a solution of 0.1% glufosinate-ammonium
(Agrevo) in 0.1% Tween 20. Tissues of glufosinate-resistant plants
were employed for the determination of uidA reporter gene activity
using histochemical assay for GUS activity with 5-bromo-4-chloro3-indolyl-b-D-galactopyranoside as substrate (Kretsch et al. 1995).
RNA extraction and Northern analysis
Extraction of total RNA from Arabidopsis tissues and Northern
blotting were performed as described in Lohrmann et al. (1999).
After UV-crosslinking and baking for 1 h at 80 °C, the membrane
was prehybridized for 5 h at 42 °C in hybridization bu€er (50 mM
sodium phosphate pH 6.5, 50% formamide, 5´ Denhardt's solution, 0.1 mg/ml denatured salmon sperm DNA) and hybridized
with an ARR2-speci®c, 32P-labelled cDNA probe (see below) in
hybridization bu€er for 16 h at 42 °C. The membrane was washed
twice in 2´ SSC, 0.2% SDS for 10 min each at 42 °C and 60 °C,
respectively, and then exposed to BioMax MS X-ray ®lms (Kodak).
The ARR2-speci®c hybridization probe was derived from the 3¢-end
of the ARR2 cDNA by restriction digestion with XhoI and was
labelled with [a-32P]dCTP (3000 Ci/mmol; Amersham) using the
Megaprime DNA Labelling System according to the manufacturer's protocol (Amersham). As a loading control we used a 32Plabelled ubiquitin probe hybridized to the same membrane.
One-hybrid assay
To generate the target construct for the one-hybrid assay, the )235/
)126 bp fragment derived from the PSST promoter of A. thaliana
(Zabaleta et al. 1998) was ampli®ed by PCR using speci®c primers.
The PCR product was cloned into the pHISi vector (Clontech)
upstream of the minimal promoter of the HIS3 locus (PminHIS3) and
the HIS3 reporter gene, and sequenced. Constructs that contained
two copies of the PSST promoter fragment in tandem array were
designated p62T and used for further studies. The control plasmid
p53 (Clontech) contained three tandem copies of the consensus p53
binding site (BSp53) inserted upstream of PminHIS3. Two yeast
reporter strains derived from strain YM4271 (Clontech) were
generated by integrating the plasmids p62T and p53 into the
chromosomal HIS3 locus. Transformants containing the chromosomally integrated plasmids were selected on complete synthetic
medium without uracil (CSM-Ura; BIO101). The p62T and p53
strains were transformed with the AD fusion constructs indicated
below. Transformants were plated on CSM-Leu medium and
incubated for 5 days at 30 °C. Colonies were restreaked on CSMLeu medium and, in parallel, transferred to CSM-Leu, His medium
supplemented with 50 mM 3-aminotriazole (3-AT), and incubated
at 30 °C for 5 and 7 days, respectively.
Protein expression and extraction, protein assay and SDS-PAGE
Constructs expressing the various fusion proteins were maintained
in the E. coli strain BL21(DE3). Protein expression was induced
with 1 mM isopropyl-b-D-thiogalactopyranoside (pET24b constructs) or with 200 lg/l of anhydrotetracycline (pASK-IBA2
constructs) for 3 h. Extracts containing the Strep fusions of ARR4,
ARR2 and ARR2receiver expressed from the pASK-IBA2 plasmids
were prepared at 4 °C in bu€er S (100 mM TRIS-HCl pH 8.0,
1 mM EDTA) supplemented with a protease inhibitor mix (Complete; Boehringer), by disrupting the cells in a French Press. The
Strep-tagged proteins were puri®ed on StrepTactin resin as
described by the manufacturer (IBA). The (His)6 fusion deriviatives
of ARR2output, AHP1 and AHP2 were extracted and puri®ed under
denaturing conditions on nickel-nitrilotriacetic acid (Ni-NTA)
resin according to the manufacturer's protocol (Qiagen). Proteins
were renatured by overnight dialysis against dialysis bu€er (10 mM
TRIS-HCl pH 7.4, 150 mM NaCl, 0.1 mM DTT) at 4 °C. Puri®ed
proteins were visualized on SDS-PAGE gels by staining with
Coomassie blue.
In vitro DNase protection and electrophoretic
mobility shift assays
For DNase I protection analysis, a PSST promoter fragment
()268 bp to )129 bp) from A. thaliana was cloned into the HindIII
and XhoI sites of the vector pBS-KS. To prepare the labelled probe,
the construct (10 lg of plasmid DNA) was linearized with XhoI,
end-labelled with Klenow polymerase and [a-32P]dCTP, and the
insert was excised by digestion with XbaI. The probe was puri®ed
by electrophoresis on a 5% non-denaturing polyacrylamide gel and
isolated from the gel. Aliquots (3 ng; 30,000 cpm) of the labelled
probe were incubated on ice for 10 min in 1´ binding bu€er (12 mM
HEPES-25 mM TRIS-HCl pH 7.9, 60 mM KCl, 1 mM EDTA,
1 mM DTT, 12% glycerol) with 2.0 lg (15 ll) of ARR2-Strep or
without protein in a ®nal volume of 20 ll. The sample was partially
digested with DNase I (Calbiochem-Novabiochem) for 1 min by
adding 2 ll of DNase I solution (50±100 lg/ml DNase I in 25 mM
MgCl2). Digestion was stopped by adding 10 ll of 0.2 M EDTA
pH 8.0. Then 70 ll of extraction bu€er (6 M urea, 0.36 M NaCl,
1% SDS, 10 mM TRIS-HCl pH 8.0), 2 ll of 10 mg/ml tRNA and
12 ll of 7.5 M ammonium acetate was added to each sample. After
mixing, the samples were extracted with 120 ll of phenol:chloroform (1:1) and precipitated with ethanol. The precipitated DNA
was resuspended in 4 ll of formamide dye mix (80% deionized
formamide, 1´ TBE, 0.1% bromophenol blue, 0.1% xylene-cyanol). The sample was boiled for 3 min., cooled on ice and applied to
a 7% sequencing gel. After electrophoresis, gels were dried under
vacuum and radioactive signals were detected and processed using
the PhosphorImager 445 SI (Molecular Dynamics).
For electrophoretic mobility shift assay (EMSA), DNA probes
including the pollen-box motifs of the PSST (nucleotide )215 to
)171), TYKY (nucleotide ±144 to ±100) and 55 kDa protein (nucleotide ±176 to ±132) promoters from Arabidopsis (Zabaleta et al.
1998) were obtained by annealing two oligonucleotides covering
these sequence stretches. Preparation of the radioactively labelled
probes, as well as experimental conditions for EMSA, were
described previously (Harter et al. 1994).
Yeast transformation, GAL4-based one-hybrid assay
and two-hybrid interaction assay
For the GAL4-based one-hybrid assay and the two-hybrid interaction assay the yeast strain PJ69-4A (genotype: MATa; trp1-901;
leu2-3,112; ura3-52; his3-200; gal4D; gal80D; GAL2-2ADE;
LYS2::GAL1-HIS3; met2::GAL7-lacZ) was used (James et al.
1996). Yeast transformation was performed using polyethylene
glycol/lithium acetate as described previously (Lohrmann et al.
1999). For one-hybrid assays the strain was transformed with the
various BD constructs (see below). Transformants were plated on
CSM-Trp and incubated for 3 days at 30 °C. Colonies were tested
for the presence of b-galactosidase by quantitative o-nitrophenylgalactoside (oNPG) assay (Lohrmann et al. 1999). For twohybrid interaction assays, selected plasmid pairs were introduced
into yeast PJ69-4A (see below). Expression of the HIS3 and ADE2
reporter genes was determined by assaying for growth of transformants on CSM-Leu, Trp, Ade and CSM-Leu, Trp, His. Assays
of lacZ reporter gene expression were performed with yeast
colonies grown in CSM-Leu, Trp. b-Galactosidase activity
was calculated using the following formula: OD420nm of the
supernatants ´ 1000/reaction time (min) ´ culture volume used for
the assay (ml) ´ OD600nm of the culture (Lohrmann et al. 1999).
In vitro protein-protein interaction assay
For ARR2/AHP interaction studies a polyclonal antiserum raised
against the C-terminal domain of ARR2 was produced in rabbits
(Eurogentec). An aliquot of ARR2 antiserum was bound to 20 ll
5
of Protein A resin (Amersham) and incubated with 1 lg of Streptagged ARR2. Samples were incubated for 1 h on ice, pelleted, and
washed once with bu€er S. Then 1 lg of either AHP1(His)6 or
AHP2(His)6 was added, and the mixtures were incubated for 2 h on
ice. The samples were washed three times with bu€er S. Bound
protein complexes were eluted with 30 ll of 10 mM glycine, pH 3.0
into tubes containing 3 ll of 1 M TRIS-HCl pH 8.0. All samples
were mixed with 10 ll of protein sample bu€er, boiled, fractionated
on SDS-PA gels and transferred to PVDF membrane (Millipore).
Strep-tagged ARR2 and the (His)6-tagged AHPs were detected
with streptavidin-alkaline phosphatase conjugate (SA-AP;
Amersham) or with Ni-NTA-AP (Qiagen), respectively, according
to the manufacturer's protocols.
Results
Isolation, characterization and expression analysis
of ARR2
To isolate cDNAs encoding potential plant response
regulators, we searched the Arabidopsis genome and
EST databases with the nucleotide sequence of the gene
for the E. coli response regulator CheY (Matsumura
et al. 1984). The derived protein sequences showing high
similarity to CheY were further analysed for the presence of speci®c invariant amino acids. Using this approach we identi®ed and isolated three sequences that
encoded proteins which showed similarity to bacterial
response regulators; these were named ARR2, ARR10
and ARR11 (this work and Lohrmann et al. 1999).
Analysis of the encoded protein sequences revealed that
ARR2, ARR10 and ARR11 belong to the B-type of
response regulators in A. thaliana (D'Agostino and
Kieber 1999; Imamura et al. 1999; Lohrmann et al.
1999). Like the previously characterized ARR10 and
ARR11 (Lohrmann et al. 1999), ARR2 is relatively large
(predicted molecular weight of 72.6 kDa) and is composed of the two di€erent domains typical of response
regulators. Adjacent to the N-terminal receiver module,
the ARR2 protein contains a C-terminal output domain,
with three putative nuclear localization sequences
(NLSs; Fig. 1). The output domain also contains the
conserved B-motif and several potentially transactivating, P/Q-rich, amino acid sequences (Fig. 1).
Northern analysis of total RNA extracted from adult
Arabidopsis plants revealed that ARR2 is predominantly
Fig. 1 Schematic representation of the Arabidopsis response
regulator ARR2. The black bar identi®es the receiver module.
The adjacent C-terminal output domain contains three SV40-like
nuclear localization sequences (NLS, meshed bars). The B-motif ± a
potential DNA binding domain ± as well as the P/Q-rich putative
transactivation domain are depicted by the di€erently hatched bars.
Numbers indicate representative amino acid positions
expressed in ¯owers (Fig. 2A). Low amounts of the
ARR2 transcript were also found in leaves and stems but
were not detected in roots (Fig. 2A). To analyse ARR2
expression in more detail, the ARR2 promoter (±3032 to
±1) was ampli®ed from genomic DNA and cloned upstream of the uidA reporter gene in a binary vector.
After transformation into A. thaliana, expression of the
reporter construct was measured by histochemical GUS
staining. Strong GUS activity was detectable in anthers
but not in other ¯oral organs of transgenic plants harbouring the ARR2 promoter/uidA construct (Fig. 2B,
panels I and III). No GUS activity was observed in
leaves, stems or roots of plants transformed with the
ARR2 promoter/uidA construct (data not shown) or in
stamens of plants transformed with a promoter-less uidA
cassette (Fig. 2B, panel II). A more detailed microscopic
study revealed that ARR2-GUS expression is predominantly found in pollen grains (Fig. 2C). We conclude
from this expression analysis that the response regulator
ARR2 may be preferentially active in pollen.
ARR2 interacts with a PSST promoter
fragment in vivo
The tissue-speci®c expression of ARR2 prompted us to
investigate, in a yeast one-hybrid system, the possibility
that ARR2 binds to promoters of genes that are preferentially expressed in pollen. As an example we chose
the promoter of the nuclear PSST gene from A. thaliana,
which is up-regulated in anthers during sporogenesis and
codes for an iron-sulphur protein of the mitochondrial
Complex I (nCI; Zabaleta et al. 1998). For this purpose,
the full-length cDNAs encoding ARR2 and, as controls,
those specifying the B-type response regulator ARR11
(Lohrmann et al. 1999) and the A-type response regulator ARR4 (Brandstatter and Kieber 1998; Imamura
et al. 1998) were fused to the sequence coding for the
GAL4 activation domain (AD). These constructs were
then transformed into the yeast strain YM7241 carrying
either the p62T target promoter or the p53 control
promoter (see Fig. 3A for constructs). The p62T construct consists of a tandem repeat of the )235/)126 bp
PSST promoter fragment inserted upstream of the
minimal promoter of the HIS3 locus (PminHIS3). The p53
control construct contains three tandem copies of the
consensus p53 binding site (BSp53) in front of PminHIS3.
Transformants were plated on medium selective for
DNA/protein interaction (CSM-Leu, His supplemented
with 50 mM 3-AT) or on non-selective medium (CSMLeu). As shown in Fig. 3B, all yeast transformants grew
on non-selective medium regardless of the chromosomally integrated reporter gene tested. In contrast, on
medium selective for DNA/protein interaction only
clones harbouring p62T grew, which expressed
AD-ARR2 (Fig. 3B). This suggests that the activation of
the HIS3 reporter gene by ARR2 depends on interaction
with the PSST promoter fragment. The DNA/protein
interaction observed in vivo is speci®c for ARR2, since
6
neither ARR11 nor ARR4 was able to induce reporter
gene expression (Fig. 3B).
ARR2 binds to nCI gene promoters in vitro
To further characterize the DNA binding activity of
ARR2 and to determine its binding sites within the anther/pollen-speci®c PSST promoter fragment, we performed an in vitro DNase I protection analysis.
Recombinant Strep-tagged ARR2 was puri®ed from
E. coli (Fig. 4, lane 2) and incubated with the monomeric
PSST promoter fragment extending from nucleotide
±268 to ±129. As shown in Fig. 5A, the DNase I protection analysis de®nes two regions within the PSST
promoter fragment which are protected from digestion
by DNase I. Site 1 extends from nucleotide ±224 to ±164
and Site 2 from nucleotide ±259 to ±240 (Fig. 5A). Site 1
includes the entire pollen-box (Zabaleta et al. 1998). The
DNase I protection pattern suggests that ARR2 binds to
the PSST promoter fragment in vitro in a complex
pattern (see Discussion for further details).
In addition, we performed EMSA with puri®ed
Strep-tagged ARR2 and Strep-tagged ARR4 (Fig. 4,
lane 1 for recombinant proteins), using as a probe a
PSST promoter fragment that contains the entire pollen
box and extends from nucleotide ±215 to ±171 (see
Fig. 5A, probe I). ARR2 clearly bound to this promoter
fragment (Fig. 5B, lane 1), whereas ARR4 did not
(Fig. 5B, lane 2). The ARR2-induced DNA/protein
complex could be eciently competed out by a 50-fold
molar excess of the same unlabelled probe (Fig. 5C, lane
3). In contrast, an unlabelled probe that covers the
PSST promoter fragment from nucleotide ±150 and ±
129 (see Fig. 5A, probe II), and contains no DNase Iprotected sites, competed only very weakly (Fig. 5C,
lane 4). However, all probes derived from the PSST
promoter fragment that contained at least one of the
binding sites were able to compete for binding of ARR2
in EMSAs (data not shown). To test whether ARR2
binds to promoter fragments of other Arabidopsis nCI
genes, we included DNA probes derived from the promoters of the genes TYKY and 55 kDa protein (Zabaleta
et al. 1998), which show homology to the PSST probe,
in our EMSA analysis. As shown in Fig. 5C, lanes 5±7,
b
Fig. 2A±C Expression pattern of ARR2 in Arabidopsis. A
Northern analysis of ARR2 transcripts in various tissues of
A. thaliana plants. Each lane was loaded with 15 lg of total
RNA prepared from the indicated tissue. The blot was probed with
an ARR2-speci®c, 32P-labelled cDNA fragment (ARR2). As a
control, the same membrane was hybridized with a 32P-labelled
ubiquitin cDNA clone (Ubi). B GUS staining of a ¯ower (I) and of
stamens (III) derived from a representative transgenic plant
transformed with an ARR2 promoter/uidA gene fusion and from
a transgenic plant carrying a promoter-less uidA construct (II).
C GUS-stained section of two di€erent anthers (I, 8 lm and II,
16 lm section) derived from a transgenic plant transformed with an
ARR2 promoter/uidA gene fusion. The arrows indicate representative stained pollen grains
7
Fig. 4 Puri®cation of various recombinant polypeptides used in
this study. Recombinant polypeptides were puri®ed via their
respective tags as described in Materials and methods. Between
0.5 and 1.0 lg of tagged protein were electrophoresed on a
SDS-PAGE gel and stained with Coomassie blue. Lane 1, ARR4Strep; lane 2, ARR2-Strep; lane 3, ARR2output-(His)6; lane 4,
ARR2receiver-Strep; lane 5, AHP1-(His)6; lane 6, AHP2-(His)6.
Molecular size markers are indicated in kDa on the left
fusion peptides (see Fig. 4, lanes 3 and 4) were then
tested for PSST promoter-binding activity in EMSAs.
Whereas the receiver module showed no interaction with
the DNA-probe (Fig. 5D, lane 1), the C-terminal peptide
bound to the PSST promoter fragment even more
strongly than full-length ARR2 (Fig. 5D, compare lane
2 with lane 3). These results demonstrate that the
C-terminal region mediates the DNA-binding activity
of ARR2 and, therefore, serves as bona ®de output
domain.
ARR2 activates transcription in vivo
Fig. 3A, B ARR2 binds to a PSST promoter fragment in vivo in a
yeast one-hybrid system. A Schematic representation of one-hybrid
reporter constructs integrated into the chromosome of the yeast
strain YM4271. p62T contains a tandem repeat of the ±235/±126 bp
PSST promoter fragment inserted upstream of PminHIS3 and the
HIS3 reporter gene. p53 is similar to p62T but contains three
tandem copies of BSp53. The numbers indicate nucleotide positions.
B Yeast YM4271 was transformed with these plasmids as indicated
in the scheme. Transformants were grown on CSM-Leu medium,
which does not select for DNA/protein interaction (CSM-Leu), or
on selective CSM-Leu, His medium supplemented with 50 mM
3-AT [CSM-Leu, His (50 mM 3-AT)]
ARR2 eciently associated with the pollen box-containing promoter fragments of all tested nCI genes.
Taken together, the results of in vitro protection analysis
as well as the EMSA data indicate that the B-type response regulator ARR2 functions as a sequence-speci®c
DNA-binding protein at nCI gene promoters.
As shown in Fig. 1, ARR2 has a long C-terminal
extension which may mediate DNA binding and may
function as an output domain. We therefore expressed
the N-terminal receiver module of ARR2 as a Streptagged and the C-terminal region as a (His)6-tagged
version in E. coli. The anity-puri®ed recombinant
To investigate the transactivating potential of ARR2,
we used two di€erent yeast one-hybrid systems (Lohrmann et al. 1999). The ARR2 cDNA was cloned into
the yeast vector pJR3611 to permit expression of the
ARR2 protein without any tag. This construct, as well
as the empty vector, were transformed into the yeast
strain YM7241 carrying the p62T target promoter (see
Fig. 3A for the p62T promoter construct). In contrast
to the empty vector, pJR-ARR2 activated transcription
of the HIS3 reporter gene, indicating that ARR2
contains a functional activation domain (Fig. 6A). To
determine the sequence requirements for the transcriptional activation competence of ARR2, cDNAs encoding full-length ARR2, the receiver module and the
output domain were fused separately to the DNAbinding domain (BD) of the yeast transcription factor
GAL4. As controls, BD fusions of the ARR11 and
ARR4 genes were generated. These plasmids were
transformed into the yeast strain PJ69-4A, which harbours a chromosomally integrated GAL promoter/lacZ
reporter gene (James et al. 1996). Reporter gene activity
was then monitored by quantitative oNPG assay. As
shown in Fig. 6B, the empty BD vector and BD-ARR4
induced only negligible levels of b-galactosidase activity. By contrast, the yeast clones expressing BD-ARR2
displayed about 20-fold higher lacZ expression levels,
8
9
c
Fig. 5A±D Characterization of the ARR2 DNA binding activity
in vitro. A In vitro DNase I protection analysis. The 32P-labelled ±
268/±129 bp PSST promoter fragment was incubated with 15 ll
(2.0 lg) of ARR2-Strep (ARR2) or 15 ll bu€er (no protein). The
reactions were analyzed on a sequencing gel and processed in a
phosphoimager. Protected nucleotides are indicated by the arrowheads. Regions characterized by an accumulation of protected
nucleotides are indicatted by the black bars (Site 1 and 2). The
corresponding PSST promoter sequence is given on the left, and
the pollen-box motif within Site 1 is indicated in bold italics. I and
II indicate PSST promoter stretches used as 32P-labelled probes
and for competition experiments in EMSA. The numbers indicate
nucleotide positions. B Analysis of in vitro DNA binding activity
of recombinant ARRs by EMSA. Aliquots (250 ng) of the
indicated recombinant ARR-Strep proteins were mixed with 1 ng
of a 32P-labelled PSST promoter probe (see A, probe I), incubated
for 1 h on ice and electrophoresed on an EMSA gel. The arrow
indicates the position of the shifted DNA/ARR2 complex. In lane 3
no protein was added. C The in vitro DNA binding activity of
recombinant ARR2 is sequence-speci®c and is not restricted to the
PSST promoter fragment. Aliquots (250 ng) of ARR2-Strep were
mixed with 1 ng of the 32P-labelled PSST promoter probe I (±215/±
171 bp). Subsequently, a 50-fold molar excess of unlabelled probe I
(lane 3) or unlabelled probe II (lane 4; see A) or no competitor
DNA (lane 2) was added. Lanes 5±7 show the binding of ARR2 to
the promoter fragments of the nCI genes TYKY and 55 kDa
protein. The regions homologous to the promoter probe I of the
PSST gene were used as probes. The reactions were processed as
described for B. The arrows indicate the position of shifted DNA/
ARR2 complexes. No protein was added in lane 1. D DNA binding
by ARR2 is mediated by its C-terminal output domain. Aliquots
(250 ng) of the indicated recombinant ARR2 polypeptides were
mixed with 1 ng of the 32P-labelled PSST promoter probe I and the
EMSA was performed as in B. The arrows indicate the positions of
shifted DNA/polypeptide complexes. Protein was omitted from
lane 4. rec, receiver module; out, output domain
comparable to those seen with BD-ARR11 (Fig. 6B;
Lohrmann et al. 1999), indicating that the transactivation domain of ARR2 is functional even when fused
to a heterologous DNA-binding motif. In addition,
cells expressing the output domain of ARR2 expressed
higher levels of b-galactosidase activity than those observed with the full-length protein, whereas the receiver
module showed no activation of the lacZ reporter gene
(Fig. 6C). Taken together, these results indicate that the
C-terminal output domain of ARR2 mediates not only
DNA binding but also transactivation of the target
genes.
ARR2 is a nuclear protein
Localization of ARR2 inside the nucleus is a prerequisite for its function as a transcription factor. As shown
in Fig. 1, the output domain of ARR2 contains at least
three putative SV40-like NLSs, suggesting a nuclear localization for the response regulator. To examine the
intracellular distribution of ARR2 we fused the entire
coding region to the GFP gene. Parsley protoplasts were
transiently transformed with the ARR2-GFP construct,
and the localization of the GFP fusion protein was analyzed by epi¯uorescence and confocal microscopy. As
shown in Fig. 7, GFP ¯uorescence is detected exclusively
Fig. 6A±C Transcriptional activation properties of ARR2. A
Determination of the transcriptional activation capacity of ARR2
in the yeast strain YM4271 carrying the p62T reporter construct
(see Fig. 3A). The cells were transformed with either pJR3611ARR2 (pJR-ARR2) or the empty pJR3611 vector (pJR). Transformants were grown on CSM-Leu medium that does not select for
DNA/protein interaction (CSM-Leu) or on selective CSM-Leu, His
medium supplemented with 25 mM 3-AT [CSM-Leu, His (25 mM
3-AT)]. B Determination of the transcriptional activation of ARR2
in a GAL4-based yeast one-hybrid system. The constructs encoding
the indicated BD fusion proteins were transformed into the yeast
strain PJ69-4A. Induction of b-galactosidase activity (units) was
determined by oNPG assay. The mean values and standard
deviations of at least three independent clones per construct are
shown. C Transcriptional activity of BD fusions with full-length
ARR2 (BD-ARR2), with the ARR2 output domain (BDARR2out), and the ARR2 receiver module (BD-ARR2rec), respectively, in a GAL4-based yeast one-hybrid system. Transactivation
was assayed as described for B
inside the nucleus, indicating that ARR2 is a nuclear
protein in plant cells.
ARR2 interacts with the Arabidopsis HPt proteins
AHP1 and AHP2
As response regulators usually mediate the ®nal output
response of two-component pathways (e.g. transcrip-
10
Fig. 7A±C ARR2-GFP is localized to the nucleus in transiently
transformed plant protoplasts. Epi¯uorescence (A) and bright-®eld
light microscopic (B) images as well as a confocal section (C) of
representative parsley protoplasts transiently transformed with a
ARR2-GFP construct are shown. The GFP ¯uorescence in the
confocal image is presented in red. The epi¯uorescence and light
microscopic images show the same protoplast. The confocal section
shows a di€erent cell with the nucleus in a di€erent position. cy,
cytosol; nu, nucleus; vc, vacuole
tional regulation; Stock et al. 1990; Chang and Stewart
1998), we were interested in de®ning signalling elements
upstream of ARR2 ± such as a cognate sensor kinase.
However, all plant sensor kinases described so far are
localized in extranuclear membrane systems and are ±
with few exceptions ± of the hybrid type (Chang and
Stewart 1998; Urao et al. 1999). Thus, direct physical
interaction between ARR2 in the nucleus and one of
these hybrid kinases appears unlikely. Indeed, ARR2
does not directly interact with several sensor kinases
from plants (J. Lohrmann, I. BaÈurle, K. Harter, unpublished). However, recently HPt proteins were identi®ed in Arabidopsis (Suzuki et al. 1998), which ± as in
yeast and bacteria ± may act as molecular adaptors
mediating the phosphorelay from hybrid kinases to their
cognate response regulators (Maeda et al. 1994;
Georgellis et al. 1997). Furthermore, due to their size,
HPt proteins can shuttle between the cytosolic and nuclear compartments to transfer the signal from their
cognate hybrid kinases to the nucleus.
c
Fig. 8A±D ARR2 interacts with the HPt proteins AHP1 and
AHP2. A Interaction of ARR2 with AHP1 and AHP2 in yeast
cells. The indicated BD and AD constructs were transformed into
yeast strain PJ69-4A. Transformants were grown on either nonselective medium (CSM-Leu, Trp) or on media selecting for
protein-protein interactions (CSM-Leu, Trp, His; CSM-Leu, Trp,
Ade). B oNPG assay for the determination of b-galactosidase
activity (units) of yeast clones transformed with the pairs of BD/
AD constructs indicatted in A. Mean values and standard
deviations of at least three independent clones per construct are
shown. C In vitro interaction of ARR2 with AHP1 and AHP2.
Recombinant ARR2-Strep and AHP-(His)6 proteins were coincubated for 2 h on ice. Co-puri®cation was carried out using an
ARR2-speci®c antiserum bound to Protein A resin, and proteins
were eluted with 10 mM glycine, pH 3.0. Samples (15 ll each) were
fractionated on a SDS-PAGE gel and transferred to a membrane
®lter. Detection of tagged proteins was performed with SA-AP (a
Strep) for ARR2-Strep and Ni-NTA-AP (a His) for AHP1-(His)6
and AHP2-(His)6. As a control, the assay was also done with
bovine serum albumin (BSA). D Demonstration of the interaction
of the ARR2 output domain (ARR2out) and the ARR2 receiver
module (ARR2rec) with AHP2 by oNPG assay. The assay was
performed as described in B
We therefore investigated whether ARR2 can interact
with two distinct members of the HPt protein family
from Arabidopsis in the yeast two-hybrid system. AHP1
and AHP2 were expressed as BD fusions, while fulllength ARR2 was fused to AD. The protein interaction
was monitored by assaying for growth of the transformants on selective media and by quantitative oNPG
assay. None of the controls grew on selective media and
showed only background b-galactosidase activity
(Fig. 8A, B). However, the results of the growth tests
11
and enzyme activity assays indicated that ARR2 interacts with both AHP1 and AHP2 in vivo (Fig. 8A and B).
The interactions were also seen in vitro with Strep-tagged ARR2 and (His)6-tagged AHP1 or (His)6-tagged
AHP2, respectively (Fig. 8C; see Fig. 4, lanes 5 and 6 for
puri®ed tagged proteins). To determine the sequence
requirements for the ability of ARR2 to interact with
HPt proteins, the receiver module and the output domain fused to BD were co-expressed with AHP2.
Whereas the controls, as well as the output domain of
ARR2, failed to activate the lacZ reporter gene, the
receiver module clearly interacted with AHP2 (Fig. 8D).
These results suggest that ARR2 could be an element of
a plant two-component signalling system that includes at
least one HPt protein.
The binding patterns observed in the DNase I
protection assay and the competition experiments in
EMSA point to two di€erent binding sites for ARR2
within the PSST promoter fragment. This pattern could
be explained by a coordinate binding of several ARR2
polypeptides to each PSST promoter fragment. Interestingly, Site 1 (see Fig. 5A) is also recognized by the
APFI factor that de®nes a new type of response regulator-like proteins (E. Zabaleta, M. Perales, J. Lohrmann, M. Walter, A. Brennicke, K. Harter, J. Kudla,
submitted), indicating that more than one factor can
bind to the same regions of the PSST promoter.
Whether ARR2 and APFI compete for the same binding
sites or form a heterodimeric protein complex awaits
further elucidation.
Discussion
ARR2: a signalling factor in a novel plant
two-component system?
ARR2 functions as a pollen-speci®c
transcription factor
Although structural features had suggested that ARR2
might be a plant response regulator with transcription
factor characteristics, no experimental data were previously available to support this prediction. Here we report that the B-type plant response regulator ARR2 can
indeed function as a transcription factor. Furthermore,
we identi®ed a target DNA sequence within the promoters of nCI genes, which are preferentially expressed
in anthers/pollen, to which ARR2 can bind in vivo as
well as in vitro. Binding of ARR2 to this DNA sequence
is speci®c, as indicated by competition experiments. The
observation that neither ARR11, another nuclear B-type
response regulator with transcriptional activity (Lohrmann et al. 1999), nor the type A response regulator
ARR4 interacts with the nCI promoter fragments, further argues for the speci®city of this interaction. The
nuclear localization of ARR2-GFP in transformed
parsley protoplasts, as well as the transcriptional activity
of ARR2 in two independent yeast transactivation systems, provides additional evidence for its function as a
transcription factor.
Like many prokaryotic response regulators, DNA
binding and transactivation are mediated by the Cterminal output domain of ARR2. However, whereas
the P/Q-rich stretches within the ARR2 output domain
are reminiscent of eukaryotic transactivation motifs, it
is dicult to identify any obvious DNA binding sequence as for ``classical'' eukaryotic transcription factors, and the output domain of ARR2 may therefore
contain a unique DNA-binding motif. It is interesting
to note that the DNA binding and transactivation
activities of the ARR2 output domain alone are higher
than those observed when the full-length protein is
used. This might suggest that the receiver module has
a regulatory in¯uence on the activity of the output
domain, possibly depending on its phosphorylation
state.
The results presented in this study suggest that ARR2
is most probably part of a novel two-component signalling system that also involves at least one HPt
protein. AHP1, AHP2 and/or other members of the
plant HPt protein family may act as molecular adaptors that temporarily link a hybrid sensor kinase with
ARR2. Though we have not yet identi®ed the cognate
sensor kinase of ARR2 and, therefore, are not able to
reconstruct this multistep phosphorelay in vitro, this
plant system is reminiscent of the osmosensing
SLN1:YPD1:SSK1 two-component pathway in yeast.
There, SLN1 represents a hybrid kinase, which, after
autophosphorylation, relays the phosphate residue to
the HPt protein YPD1. YPD1 then transmits the
phosphate to the receiver module of the response regulator SSK1 (Maeda et al. 1994). In yeast, this multistep phosphorelay is located at the membrane or in the
soluble phase of the cytosol (Maeda et al. 1994).
However, for the plant system studied here, we propose
that an AHP protein shuttles between an extranuclear
and membrane-bound hybrid sensor kinase and the
nuclear protein ARR2 to form a multistep phosphorelay. The cytoplasmic and nuclear distributions of
AHP-GFP fusion proteins in parsley protoplasts are
in good agreement with such a shuttling function
(J. Lohrmann, U. Sweere, I. BaÈurle, K. Harter,
unpublished). Interestingly, the HPt proteins from
Arabidopsis so far tested are able to rescue the lethal
phenotype of a yeast strain in which the endogenous
HPt gene (YPD1) has been disrupted (Suzuki et al.
1998). This observation indicates that the AHPs are
indeed functional phospho-transfer proteins in vivo.
Furthermore, for some members of the AHP and ARR
protein families, namely AHP1 and ARR4, their
function has, in principle, been demonstrated in an
in vitro phosphate-transferring system (Suzuki et al.
1998). Experiments are currently in progress to determine the role of a possible phosphorylation of ARR2
in the regulation of its activity.
12
Physiological implications for the function
of ARR2 as a pollen-speci®c transcription factor
The nCI genes, from which the promoter fragments used
as probes were derived, encode proteins that are targeted
to mitochondria. In concert with more than thirty nucleus-encoded polypeptides and nine proteins speci®ed
by the mitochondrial genome, the PSST, TYKY and
55 kDa proteins form the nCI in the inner mitochondrial
membrane. This large multisubunit complex translocates
protons to generate the proton-motive force for ATP
synthesis (Weiss et al. 1991). During sporogenesis ± the
most energy-demanding process known in plants ± the
expression of nCI genes including PSST, TYKY and 55
kDa protein is di€erentially and coordinately up-regulated, resulting in a six to ten-fold higher level of steadystate transcripts in anthers and pollen than in leaves and
roots (Grohmann et al. 1996; Heiser et al. 1996; SchmidtBleek et al. 1997). In combination with other intracellular
processes, this increase in the amounts of nCI complexes
in the inner mitochondrial membrane increases the
capacity for ATP synthesis (Heiser et al. 1997). The
preferential expression of the nCI genes in anthers and
pollen is conferred by their ±250/±100 bp promoter
regions (Zabaleta et al. 1998) ± the region analysed in this
study. Within these regions a ``pollen box'' has been
de®ned, although adjacent cis-acting elements are also
crucial for tissue-speci®c expression (Zabaleta et al.
1998). For the PSST gene these cis-acting elements were
identi®ed by linker-scanning mutagenesis of ()253/
)126 bp)-promoter/GUS fusions expressed in transgenic
Arabidopsis plants (E. Zabaleta, M. Perales, J. Lohrmann,
M. Walter, A. Brennicke, K. Harter, J. Kudla, submitted). Interestingly, the interaction sites of ARR2 within
the PSST promoter fragment match these cis-acting
elements. This correlation and the tissue-speci®c
expression pattern suggest that ARR2 may well be one
of the factors involved in the regulation of nCI gene
expression in pollen.
The identi®cation of ARR2 as a regulator of nCI
genes for proteins of the mitochondrial respiratory chain
now provides a means of investigating the presumably
complex signalling network between nucleus and mitochondria in the plant cell.
Acknowledgements We are grateful to I. Abel, S. Feigl, A. Probst,
I. Boschke and S. Kircher for excellent technical and methodical
assistance. We also thank Dr. P. James, Dr. B. Schulz and the
Arabidopsis Biological Resource Center, Ohio, for providing the
PJ69-4A yeast strain, the pGPTV-BAR vector and cDNA libraries,
respectively. The work was in part supported by grants from the
Ulmer UniversitaÈtsgesellschaft to J.K., from the Volkswagenstiftung to E.Z. and J.K., from the Deutsche Forschungsgemeinschaft
(SFB388) to E.S. and K.H. and from the Human Frontiers Science
Program to K. H. (RG0043).
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