RESEARCH ARTICLE
627
Development 136, 627-636 (2009) doi:10.1242/dev.028365
The polarly localized D6 PROTEIN KINASE is required for
efficient auxin transport in Arabidopsis thaliana
Melina Zourelidou1,*, Isabel Müller1,*, Björn C. Willige1,*, Carola Nill1, Yusuke Jikumaru2, Hanbing Li1,† and
Claus Schwechheimer1,*,‡
The phytohormone auxin is a major determinant of plant growth and differentiation. Directional auxin transport and auxin
responses are required for proper embryogenesis, organ formation, vascular development, and tropisms. Members of several
protein families, including the PIN auxin efflux facilitators, have been implicated in auxin transport; however, the regulation of
auxin transport by signaling proteins remains largely unexplored. We have studied a family of four highly homologous AGC protein
kinases, which we designated the D6 protein kinases (D6PKs). We found that d6pk mutants have defects in lateral root initiation,
root gravitropism, and shoot differentiation in axillary shoots, and that these phenotypes correlate with a reduction in auxin
transport. Interestingly, D6PK localizes to the basal (lower) membrane of Arabidopsis root cells, where it colocalizes with PIN1, PIN2
and PIN4. D6PK and PIN1 interact genetically, and D6PK phosphorylates PIN proteins in vitro and in vivo. Taken together, our data
show that D6PK is required for efficient auxin transport and suggest that PIN proteins are D6PK phosphorylation targets.
INTRODUCTION
Plant development is to a large extent dependent on the proper
distribution of the hormone auxin (indole-3-acetic acid, IAA). IAA
can enter plant cells via membrane diffusion in its protonated form
(IAAH) or via active transport in its anionic form (IAA–); IAA–, the
predominant form of IAA in cells, is exported via auxin efflux
carriers (Kramer and Bennett, 2006). The permease-like AUXINRESISTANT 1 and LIKE-AUX1 (AUX1 and LAX) proteins are
auxin influx (Bainbridge et al., 2008; Marchant et al., 1999; Yang et
al., 2006) and the PIN-FORMED (PIN) proteins are auxin efflux
facilitators (Kramer and Bennett, 2006; Petrasek et al., 2006;
Teale et al., 2006; Vieten et al., 2007). AUX1 and PIN proteins have
been shown to promote auxin influx and efflux, respectively
(Chen et al., 1998; Luschnig et al., 1998; Petrasek et al., 2006; Yang
et al., 2006). In addition, it has been proposed that PIN proteins
in planta act together with MULTIDRUG RESISTANCE/
PHOSPHOGLYCOPROTEIN (MDR/PGP) ATP-binding cassette
transporters (Bandyopadhyay et al., 2007; Blakeslee et al., 2007;
Geisler and Murphy, 2006).
The directionality of auxin transport within the plant is achieved
by the differential and often polar localization of the AUX1/LAX
and PIN transport facilitators (Benkova et al., 2003; Friml et al.,
2002a; Friml et al., 2003; Friml et al., 2002b; Galweiler et al., 1998;
Kleine-Vehn et al., 2006; Muller et al., 1998; Sauer et al., 2006;
Swarup et al., 2001; Wisniewska et al., 2006). Mutations in
AUX1/LAX genes have been reported to affect gravitropism, lateral
root formation, and phyllotaxy (Bainbridge et al., 2008; Bennett et
1
Tübingen University, Center for Plant Molecular Biology, Department of
Developmental Genetics, Auf der Morgenstelle 5, 72076 Tübingen, Germany.
RIKEN, Plant Science Center, Suehiro-cho 1-7-22, Tsurumi-ku, Yokohama,
Kanagawa 230-0045, Japan.
2
*Present address: Technische Universität München, Department of Plant Systems
Biology, Am Hochanger 4, 85354 Freising, Germany
†
Present address: 1 University of Missouri-Columbia, Department of Biochemistry,
117 Schweitzer Hall, Columbia, MO 65211, USA
‡
Author for correspondence (e-mail: [email protected])
Accepted 16 December 2008
al., 1996; Marchant et al., 2002; Marchant et al., 1999; Swarup et al.,
2008). Mutations in single PIN genes affect shoot differentiation,
vascular development, lateral root development, and tropisms
(Benkova et al., 2003; Chen et al., 1998; Friml et al., 2002b;
Galweiler et al., 1998; Luschnig et al., 1998; Muller et al., 1998;
Okada et al., 1991; Scarpella et al., 2006), whereas mutants defective
in multiple PIN genes have more pronounced phenotypes and
affected embryonic development, root patterning, and lateral root
initiation (Benkova et al., 2003; Blilou et al., 2005; Friml et al.,
2003). PIN proteins have redundant functions, and the loss of one
PIN protein is compensated for by the ectopic activities of the other
PIN family members (Blilou et al., 2005; Paponov et al., 2005;
Vieten et al., 2005). Interestingly, pin mutant phenotypes can in
many cases be mimicked by the application of the auxin efflux
inhibitor naphtylphtalamic acid (NPA) (Katekar and Geissler, 1977),
indicating that this inhibitor functions in the proximity of PINs.
PIN polarity is controlled by differential PIN phosphorylation,
which appears to be the result of the antagonistic activities of the
serine-threonine kinase PINOID (PID) and a phosphatase containing
the subunit PP2A (Friml et al., 2003; Michniewicz et al., 2007). PID
overexpression leads to a shift in the polarity of at least PIN1, PIN2
and PIN4 from the basal (lower) to the apical (upper) membrane in
root cortex and lateral root cap cells (Friml et al., 2003). These PIDdependent polarity changes can be reverted by increasing the
expression of PP2A, which suggests that PIN polarity and auxin
efflux are at least in part controlled by PID-dependent
phosphorylation (Michniewicz et al., 2007).
The plant-specific AGC kinases were named on the basis of their
homology to the mammalian cAMP-dependent protein kinase A,
cGMP-dependent protein kinase G and phospholipid-dependent
protein kinase C (Bogre et al., 2003). PID together with 22 other
protein kinases forms the AGCVIII subgroup of the Arabidopsis
AGC kinase family (Bogre et al., 2003; Galvan-Ampudia and
Offringa, 2007). AGCVIII kinases are characterized by a DFG to
DFD substitution in the conserved catalytic subdomain VII, as well
as by the presence of a conserved insertion between subdomains VII
and VIII of the kinase. In addition to PID, other protein kinases of
the AGCVIII subgroup have been examined, including the blue light
DEVELOPMENT
KEY WORDS: Arabidopsis, Protein kinase, Auxin transport, PIN proteins, Lateral root
RESEARCH ARTICLE
receptors PHOTOTROPIN 1 (PHOT1) and PHOT2, and the root
growth regulators WAVY ROOT GROWTH 1 (WAG1) and WAG2
(Sakai et al., 2001; Santner and Watson, 2006). Although the precise
molecular mechanisms that control PHOT-mediated phototropism
and WAG-mediated root waving remain to be elucidated, changes
in auxin transport or auxin response may well be responsible for
these growth responses (Esmon et al., 2006; Harper et al., 2000;
Santner and Watson, 2006).
MATERIALS AND METHODS
Biological material
d6pk mutant lines (Arabidopsis thaliana Columbia) were identified in the
SIGNAL database (http://signal.salk.edu/cgi-bin/tdnaexpress) and obtained
from the Nottingham Arabidopsis Stock Centre: d6pk-1 (SALK_061847),
d6pk-2 (SAIL_242_C05), d6pkl1-1 (SALK_056618), d6pkl2-1
(SALK_005798), d6pkl2-2 (SALK_086127), d6pkl3-1 (SALK_011507),
d6pkl3-2 (SALK_047347) and d6pkl3-3 (SALK_081961). The pin1
(SALK_097144) allele was identified and obtained in the same manner. The
alleles d6pk-1, d6pkl1-1, d6pkl2-2 and d6pkl3-2 were chosen for further
biological and genetic analyses. The sequences of the primers used for
genotyping can be provided on request. DR5:GUS transgenic lines were a
gift from Tom Guilfoyle (University of Columbia, MO, USA) (Ulmasov et
al., 1997); the PIN1:GFP construct was obtained from Jiri Friml (Gent,
Belgium) (Benkova et al., 2003). To introduce the pin1 mutation and the
DR5:GUS reporter into the d6pk and YFP:D6PK backgrounds, pin1/PIN1
plants or DR5:GUS transgenic lines were crossed with d6pk/d6pk
d6pkl1/d6pkl1 d6pkl2/D6PKL2 and YFP:D6PK lines (DR5:GUS only).
Relevant homozygous backgrounds were selected from the progeny of these
crosses by phenotyping and genotyping.
Cloning procedures
To generate YFP:D6PK, the D6PK open reading frame was amplified by
PCR with the primers D6PK-FW-GW3 and D6PK-RV-GW3 and inserted
into the Gateway-compatible vector pEXTAG-YFP-GW (a gift from Jane
Parker, Cologne, Germany). 35S:D6PK and D6PK:GFP were generated
using Gateway-technology by insertion of D6PK fragments obtained with
the primers D6PK-FW-GW1 and D6PK-RV-GW1 or D6PK-RV-GW2 into
p35SGW-MYC and pMDC83 (Curtis and Grossniklaus, 2003). YFP:D6PK,
D6PK:GFP and 35S:D6PK were transformed into Arabidopsis thaliana
(Columbia) to obtain transgenic plants expressing the respective D6PK
variants. YFP:D6PK and D6PK:GFP transgenic plants have identical
morphology and the subcellular localization of D6PK:GFP is identical to the
one reported here for YFP:D6PK. YFP:D6PK was introgressed into the
d6pk d6pkl1 d6pkl2 mutant and YFP:D6PK overexpression was found to
overcome the d6pk d6pkl1 d6pkl2 phenotype, suggesting that YFP:D6PK
encodes a functional D6PK. YFP:D6PKin carries a K to E amino acid
substitution in the ATP-binding pocket of the kinase and was obtained by
PCR-based mutagenesis (Sawano and Miyawaki, 2000) of YFP:D6PK with
the primer D6PKin. D6PK promoter fragments corresponding to a 2-kb
region upstream of the ATG start codon were amplified from Arabidopsis
thaliana (Columbia) genomic DNA (primer details can be provided on
request). The fragments were cloned into the Gateway-compatible vector
pBGWFS7 (Karimi et al., 2002) and transformed into Arabidopsis thaliana
(Columbia).
The cytoplasmic loops of PIN1, PIN2, PIN3, PIN4 and PIN7 were
identified based on homology to a previously published prediction of PIN
topology (Muller et al., 1998). The corresponding gene fragments were
amplified by RT-PCR with specific PINLOOP-FW and PINLOOP-RV
primers from Arabidopsis thaliana (Columbia) mRNA. The Gatewaysystem compatible fragments were then cloned into the expression vectors
pDEST17 (Invitrogen, Carlsbad, CA, USA), to generate constructs for the
bacterial expression of His-tagged PINLOOP fusion proteins, namely
HIS:PIN1 through HIS:PIN7, and pDEST15 (Invitrogen, Carlsbad, CA,
USA), to generate GST:PIN1. GST:D6PK was obtained by insertion of the
D6PK open reading frame amplified with D6PK-FW-GW3 and D6PK-RVGW3 into pDEST15. The GST:D6PKin kinase-dead variant was derived
from GST:D6PK with the primer D6Pkin (Sawano and Miyawaki, 2000).
Development 136 (4)
Physiological experiments
Unless otherwise stated, seedlings were grown in continuous light on
standard growth medium [4.2 g/l Murashige and Skoog salts, 1% sucrose,
0.5 g/l 2-(N-morpholino)ethanesulfonic acid, 5.5 g/l agar, pH 5.8]. Older
plants were grown in the greenhouse with 16-hour light/8-hour dark cycles.
Sensitivity to 2,4D was measured in 11-day-old seedlings that had been
transferred after 4 days to medium containing 0.1 μM 2,4D. Auxin-induced
lateral root formation was examined in seedlings that had been transferred
after growth for 7 days on standard growth medium to medium
supplemented with 0.05 μM NAA or 0.05 μM 2,4D. For microscopic
analyses, plants were mounted on microscope slides with chloral
hydrate:ddH2O:glycerol (20:9:3) and examined using an Axiophot
microscope using Nomarski optics (Zeiss, Oberkochen, Germany).
Auxin transport
Following a previously published protocol, 25-mm inflorescence pieces
were cut above the rosette of 3- to 4-week-old wild-type and d6pk d6pkl1
d6pkl2 mutants and placed in inverted orientation for one hour in 30 μl
auxin transport buffer [500 pM IAA, 1% sucrose, 5 mM 2-(Nmorpholino)ethanesulfonic acid, pH 5.5] with or without 100 μM NPA. The
inflorescence pieces were subsequently transferred for 5 minutes to auxin
transport buffer containing 11 kBq (417 nM) radiolabeled [3H]IAA (GE
Healthcare, UK) and then placed into a tube containing only auxin transport
buffer. After 2 hours, 5-mm segments were dissected from the inflorescence
stem, the lowermost segment was discarded, and the remaining segments
were macerated overnight in 300 μl Hydroxide of Hyamine 10-X (Packard
Instrument Company, Meriden, CT, USA). The solution was neutralized by
adding 300 μl acetic acid and the uptake of [3H]IAA was quantified using a
Wallac WinSpectral 1414 Liquid Scintillation Counter (Perkin Elmer Life
Sciences Waltham, MA, USA). Three replicate measurements were made
for each genotype. The experiment was repeated three times with
reproducible results and the result of one experiment is shown.
Gene expression
RT-PCR-based gene expression analysis was performed on RNA extracted
from 7-day-old light-grown Arabidopsis seedlings with the RNeasy Kit
(Qiagen). Total RNA (3 μg) was reverse transcribed with an oligo-dT primer
using M-MuLV Reverse Transcriptase (Fermentas, St Leon, Germany). The
consequences of the T-DNA insertions on the expression of the D6PK fulllength transcripts were tested after 28 PCR amplification cycles. Auxininduced gene expression of IAA genes was examined by RT-PCR using the
same protocol. Primer sequences can be provided on request. D6PKp:GUS
and DR5:GUS activity was detected by histological GUS staining of 7-dayold seedlings. For GUS staining, the seedlings were fixed for 15 minutes in
heptane and stained for 30 minutes (DR5:GUS) or for 4 hours
(D6PKp:GUS) with GUS-staining solution [100 mM Na-phosphate buffer
pH 7.0, 0.5 mM K4Fe (CN)6, 0.5 mM K3Fe (CN)6, 0.1% (v/v) Triton X-100,
0.5 mg/ml 5-bromo-4-chloro-3-indolyl β-D-glucuronic acid], and
subsequently destained in 70% (v/v) ethanol. For microscopic analyses,
stained plants were mounted with chloral hydrate:ddH2O:glycerol (20:9:3)
and examined using an Axiophot (Zeiss, Oberkochen, Germany) or a Leica
MZ16 microscope (Leica Microsystems, Heerbrugg, Switzerland).
IAA measurements
Extraction and purification of IAA were carried out as previously reported
(Edlund et al., 1995) with slight modifications as follows. Liquid nitrogenfrozen 7-day-old light-grown Arabidopsis seedlings (15 mg fresh weight) were
ground in 1 ml pre-chilled (–20°C) 80% methanol containing 1% acetic acid
(v/v). The sample was extracted for 2 hours at 4°C under continuous shaking.
As an internal standard, 150 pg of d2-IAA (Sigma-Aldrich, Oakville, ON,
Canada) was added. After centrifugation (10,000 g, 5 minutes), the supernatant
was collected and concentrated in vacuo. The sample was resuspended in 1 ml
0.01N HCl and slurried for 10 minutes at 4°C under continuous shaking with
15 mg of Amberlite XAD-7HP (Organo, Tokyo, Japan). After removal of the
supernatant, the XAD-7HP was washed twice with 1% acetic acid. Acidic
compounds were then extracted with CH2Cl2 (700 μl and two times 500 μl),
and the combined CH2Cl2 fraction was passed through a 0.2 μM filter. After
concentration in vacuo, the sample was resuspended in 20 μl H2O and 10 μl
were injected into a liquid chromatography-electrospray ionisation-mass
DEVELOPMENT
628
spectrometer/mass spectrometer (LC-ESI-MS/MS). IAA was quantified on an
LC (Acquity Ultra Performance LC, Waters, Mildford, MA)-MS/MS (Q-TOF
Premier, Micromass Technologies, Manchester, UK) system using an Acquity
UPLC BEH C18 column (Waters, Mildford, MA, USA). A binary solvent
system was used consisting of H2O as solvent A and acetonitrile containing
0.05% acetic acid as solvent B. Separations were performed using an isocratic
elution with 15% solvent B at a flow rate of 0.2 ml/minutes. The retention time
of IAA and d2-IAA was 6.67 minutes. The MS/MS conditions were as follows:
capillary, 2.6 kV; source temperature, 80°C; desolvation temperature, 400°C;
cone gas flow 0 l/hour; desolvation gas flow, 500 l/hour; collision energy, 8.0;
MS/MS transition, (m/z) 176/130 (unlabeled IAA), 178/132 (d2-IAA). The
levels of IAA were determined against a calibration curve, which was obtained
by injecting a series of standard solutions that contained a fixed concentration
of d2-IAA (100 pg/ml) and varying concentrations of unlabeled IAA.
MassLynx software 4.1 (Waters, Manchester, UK) was used to calculate IAA
concentrations from the LC-MS-MS data.
Immunostaining
Immunostaining was performed on roots of 5-day-old seedlings as previously
described (Lauber et al., 1997), using mouse anti-GFP (dilution 1:300; Roche
Applied Science, Indianapolis, IN, USA) for the detection of YFP:D6PK,
rabbit anti-PIN1 [1:1000 (Paciorek et al., 2005)], rabbit anti-PIN2 [1:1000
(Abas et al., 2006)], and rabbit anti-PIN4 [1:200 (Friml et al., 2002a)] and
secondary antibodies (anti-mouse FITC conjugate, dilution 1:600; and antirabbit Cy3-conjugate, dilution 1:600; both Dianova, Hamburg, Germany).
DAPI (1 μg/ml) was used to stain nuclear DNA. For live imaging, propidium
iodide (100 μg/ml) was used to outline cell walls. The effect of Brefeldin A
(BFA; 50 μM) was analyzed following a 90-minute treatment as described
previously (Geldner et al., 2003). All images were taken with a Leica TCS SP2
confocal microscope (Leica Microsystems, Heerbrugg, Switzerland).
Spearman’s correlation coefficients were calculated using the ImageJ plugin
from http://www.cpib.ac.uk/~afrench/coloc.html, according to French et al.
(French et al., 2008).
Phosphorylation experiments
In vitro phosphorylation experiments were carried out using purified
recombinant GST:D6PK and GST:D6PKin in combination with purified
recombinant HIS:PIN proteins. The recombinant proteins were incubated
for 30 minutes in phosphorylation buffer (20 mM Tris-HCl pH 7.5, 50 mM
NaCl, 10 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, 50 μM ATP)
supplemented with 10 μCi [g-32P]ATP. The reaction was stopped by adding
5⫻Laemmli buffer, and protein extracts were separated on a 10% SDSpolyacrylamide gel (SDS-PAGE). GST:D6PK autophosphorylation and
HIS:PIN transphosphorylation were detected by autoradiography, protein
loading was controlled by immunoblots with anti-GST (GE Healthcare, UK)
and anti-HIS (Sigma-Aldrich, Taufkirchen, Germany) antibodies.
YFP:D6PK and YFP:D6PKin were immunoprecipitated as previously
described from 400 μg total protein extract from 7-day-old transgenic
seedlings (Willige et al., 2007). One third of the immunoprecipitated
YFP:D6PK and YFP:D6PKin fusion protein was used in a phosphorylation
reaction with recombinant purified GST:PIN1, as described above.
Phosphorylation experiments in protoplasts were performed by
transformation of Arabidopsis protoplasts with the DNA constructs specified
in the text. Total protein was extracted in 2⫻Laemmli buffer and resolved
on a 10% SDS-PAGE gel. For dephosphorylation, the protein extract was
prepared in 50 μl 1⫻calf intestinal alkaline phosphatase (CIAP) buffer,
incubated in the presence of 1% Nonidet-40 with 80U CIAP (Fermentas, St
Leon, Germany) for 40 minutes, and subsequently resolved on a 10% SDSPAGE gel. The D6PK antibody is a rabbit anti-peptide antibody generated
against the peptide NSKINEQGESGKSSTC (Eurogentec, Liège, Belgium).
RESULTS
d6pk mutant analysis suggests a role for D6PKs in
auxin response or transport
We have examined the biological function of an AGCVIII subfamily,
which comprises a protein that we designated D6 PROTEIN KINASE
(D6PK; At5g55910, alternative nomenclature AGC1-1/PK64) and its
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629
close homologs D6PK-LIKE1 (D6PKL1; At4g26610, AGC1-2),
D6PK-LIKE2 (D6PKL2; At5g47750, PK5) and D6PK-LIKE3
(D6PKL3; At3g27580, PK7; see Fig. S1 in the supplementary
material) (Bogre et al., 2003; Galvan-Ampudia and Offringa, 2007).
The high degree of sequence conservation between these proteins
strongly suggests that they have redundant function, and throughout
this report we will refer to these four kinases collectively as D6PKs
(see Fig. S1 in the supplementary material). By coincidence, D6PK is
the closest Arabidopsis homolog of Phaseolus vulgaris (bean)
PROTEIN KINASE-1 (PvPK-1), the first protein kinase to be isolated
from plants almost 20 years ago (Lawton et al., 1989).
To gain an understanding of the biological function of the D6PKs,
we isolated and characterized homozygous T-DNA insertion
mutants for each of the four D6PK genes (Fig. 1A). Because the
alleles d6pk-1, d6pkl2-2 and d6pkl3-2 carry in-gene in-exon
insertions and do not express detectable levels of the respective fulllength transcript, we assume that these alleles are loss-of-function
mutants (Fig. 1B). Based on the reduction of D6PKL1 expression in
d6pkl1-1, the only available D6PKL1 allele, and based on the
genetic interaction of d6pkl1-1 with other d6pk alleles (see below),
we further concluded that d6pkl1-1 is a mutant with reduced
D6PKL1 function (Fig. 1B). None of the d6pk single mutants
displayed an obvious phenotype (Fig. 1C-G), but because the high
degree of sequence conservation between the D6PKs suggested that
these proteins have redundant biochemical function (see Fig. S1 in
the supplementary material), and because their, at least in part,
overlapping expression patterns indicated that they act in the same
tissues (see Fig. S2 in the supplementary material), we decided to
generate d6pk mutant combinations (Fig. 1H-M). When we
analyzed these mutants, we observed a range of developmental
defects, particularly in d6pk d6pkl1 d6pkl2, as well as in d6pk d6pkl1
d6pkl2 d6pkl3 mutants, which were absent in the single mutants and
less pronounced in the d6pk double or the d6pk d6pkl1 d6pkl3 triple
mutants (Fig. 1H-M). Adult d6pk d6pkl1 d6pkl2 and d6pk d6pkl1
d6pkl2 d6pkl3 mutants had narrow and twisted leaves, formed fewer
axillary shoots and were almost infertile (Fig. 1L,M); at the seedling
stage, d6pk d6pkl1 d6pkl2 and d6pk d6pkl1 d6pkl2 d6pkl3 mutants
sometimes had fused or single cotyledons (10% penetrance), were
deficient in lateral root formation, and were mildly agravitropic (Fig.
2). Our observation that mutants with reduced D6PK gene dosage
display novel and increasingly stronger phenotypes supports our
hypothesis that the four D6PK genes have redundant functions. To
reduce the complexity of the subsequent experiments, further mutant
analyses were largely restricted to the d6pk d6pkl1 d6pkl2 mutant.
Fused or single cotyledons, agravitropic root growth, and reduced
lateral root formation are phenotypes that are frequently observed in
auxin response or transport mutants (Chen et al., 1998; Dharmasiri
et al., 2005; Friml et al., 2002b; Fukaki et al., 2002; Luschnig et al.,
1998; Muller et al., 1998; Scarpella et al., 2006). Auxin distribution
can be estimated with the DR5:GUS reporter, which marks the sites
of auxin (response) maxima, e.g. in lateral root founder cells and the
root tip (Fig. 3A,E) (Benkova et al., 2003; Dubrovsky et al., 2008;
Sabatini et al., 1999). In d6pk d6pkl1 d6pkl2 mutants, we found that
DR5:GUS maxima are absent at the predicted sites of lateral root
formation, and at the same time that the maximum in the root tip is
broadened and shifted above the quiescent center (Fig. 3B,F).
Because these phenotypes can, at least to some extent, be
phenocopied by NPA-treatment of wild-type seedlings (Casimiro et
al., 2001; Sabatini et al., 1999) (Fig. 3D,H), and because changes in
the DR5:GUS maximum have also been reported for pin mutants
(Friml et al., 2002a; Sabatini et al., 1999), we hypothesized that d6pk
mutants might be deficient in auxin response or auxin transport.
DEVELOPMENT
D6 protein kinases and auxin transport
RESEARCH ARTICLE
Reduced auxin transport in d6pk mutants
To distinguish between defects in auxin response and auxin transport
in d6pk mutants, we conducted a number of physiological
experiments. Each of our auxin response experiments led us to
conclude that auxin responses are not compromised in d6pk d6pkl1
Development 136 (4)
d6pkl2 mutants: auxin-induced gene expression was not affected in
the mutants (see Fig. S3A in the supplementary material); mutant
seedlings were indistinguishable from the wild type with regard to
the auxin-induced inhibition of primary root growth (see Fig. S3B in
the supplementary material); and, finally, lateral root formation along
Fig. 1. Isolation of d6pk mutants and YFP:D6PK overexpression lines. (A) Schematic representation of the four Arabidopsis D6PK genes,
names of the mutant alleles, and positions of the respective T-DNA insertions. Black triangles mark the positions of the T-DNA insertions in lines that
were used for mutant analyses. Introns are represented by lines, protein-coding exon sequences are presented as black boxes, non-coding exon
sequences as gray boxes. (B) The RT-PCR-based gene expression analysis (28 PCR amplification cycles) of D6PK gene expression in the mutant alleles
used for this study reveals the absence of full-length transcripts in d6pk-1, d6pkl2-2 and d6pkl3-2, and reduced transcript levels in d6pkl1-1. (CM) Phenotype of 6-week-old adult plants (top) and vegetative rosettes of 3-week-old plants (bottom) of d6pk single, double, triple and quadruple
mutants, as indicated. Scale bars: 6 cm. (N-P) The differentiation defects of apical meristems from lateral shoots of d6pk quadruple mutants (O) are
reminiscent of the differentiation defects observed in pin1 mutant alleles (P). Scale bar: 0.5 cm. (Q) The overexpression of YFP:D6PK results in plants
with reduced plant height and broader leaves, when compared with wild type. Scale bar: 6 cm. (R) In contrast to wild-type leaves, the leaves of
YFP:D6PK plants are shorter and broader, and in some places are uneven. Scale bar: 1 cm.
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D6 protein kinases and auxin transport
RESEARCH ARTICLE
631
Fig. 2. Arabidopsis d6pk mutants and overexpression lines have
defects in lateral root formation and gravitropism. (A-C) The
relative number of emerged lateral roots is reduced in 10-day-old lightgrown d6pk d6pkl1 d6pkl2 mutants (d6pk012) and YFP:D6PK
seedlings when compared with wild type. Scale bar: 0.5 cm. (D-F) Tenday-old light-grown d6pk d6pkl1 d6pkl2 mutants (d6pk012) grow
agravitropically when compared with wild-type and YFP:D6PK
seedlings. Scale bar: 0.5 cm. (G) Approximately 10% of the d6pk
d6pkl1 d6pkl2 (d6pk012) mutant seedlings (5 days old) have only a
single cotyledon or fused cotyledons. Scale bar: 0.5 cm. (H) Light-grown
YFP:D6PK seedlings have epinastic cotyledons. Scale bar: 0.5 cm.
(I) Dark-grown YFP:D6PK seedlings (7 days old) have a dramatically
shortened hypocotyl. The overexpression of YFP:D6PK was confirmed
by immunoblots and by fluorescence microscopy (see also Fig. 4). Scale
bar: 3 mm. (J) Relative number of lateral roots in 9-day-old d6pk
mutant and YFP:D6PK seedlings. Nomenclature: d6pk0 (d6pk-1),
d6pk01 (d6pk-1 d6pkl1-1), d6pk012 (d6pk-1 d6pkl1-1 d6pkl2-2), etc.
The number of lateral roots decreases with reduced D6PK gene dosage.
The graph shows the average root length (and standard deviation) of at
least eight seedlings for each genotype. The root length is reduced in
YFP:D6PK seedlings but is unaltered in the single d6pk mutants when
compared with wild type (compare for example A-F).
the primary root was efficiently induced in the mutants by the auxins
1-naphthalene acetic acid (1-NAA) and 2,4-dichlorophenoxyacetic
acid (2,4D; see Fig. S3C in the supplementary material).
Interestingly, we observed a difference between d6pk d6pkl1
d6pkl2 mutants and the wild type with regard to lateral root initiation
following NAA treatment. Although lateral root primordia were
formed at regular intervals in untreated and in NAA-treated wildtype seedlings, lateral root primordia were often initiated close to a
pre-existing primordium in d6pk d6pkl1 d6pkl2 mutants in response
to NAA (see Fig. S3C in the supplementary material). NAA is
known to enter cells by passive diffusion and therefore does not
require auxin influx carrier activity (Delbarre et al., 1996). When we
tested the sensitivity of d6pk mutants to the auxin transport inhibitor
D6PK overexpression phenotypes
To examine the effects of ectopic D6PK expression, we generated
constructs for the overexpression of untagged D6PK (35S:D6PK)
and of fluorescent protein-tagged D6PK (YFP:D6PK and
D6PK:GFP). Interestingly, we failed to recover any 35S:D6PK
transgenic plants, and we obtained only a small number of
YFP:D6PK and D6PK:GFP overexpression lines after repeated
rounds of transformation. Although our further experiments did not
reveal any obvious differences in the biochemical activity of these
D6PK variants (see below), we speculate that the overexpression of
untagged D6PK causes lethality. YFP:D6PK and D6PK:GFP
transgenic plants display identical phenotypes in that adult plants are
shorter than the wild type, and in that they have shorter as well as
broader, in places uneven, leaves (Fig. 1Q,R; Fig. 2I,J; data not
shown). Light-grown YFP:D6PK and D6PK:GFP seedlings have
shorter roots than wild-type seedlings, fewer lateral roots, and
epinastic cotyledons; dark-grown seedlings have a severely
shortened and thickened hypocotyl (Fig. 2C,F,H,I; data not shown).
When we introduced the YFP:D6PK transgene into d6pk d6pkl1
d6pkl2 mutants, we found that the effect of YFP:D6PK
overexpression is epistatic to the d6pk mutations and we therefore
reasoned that the YFP:D6PK fusion protein is functional. Based on
this observation, we restricted our further analysis to YFP:D6PK
transgenic plants (data not shown).
Similarly to d6pk mutants, we found YFP:D6PK seedlings to
have fewer emerged lateral roots (Fig. 2C,J). However, unlike the
d6pk mutants, which are defective in lateral root initiation,
YFP:D6PK seedlings initiate lateral roots but are defective in their
outgrowth (Fig. 3C). Furthermore, we found YFP:D6PK
overexpression to be sufficient to induce adventitious root formation
in the hypocotyls of dark-grown seedlings (Fig. 3K,L), and to cause
stronger DR5:GUS expression in leaves of YFP:D6PK plants,
notably in the uneven leaf areas (Fig. 3I,J). At the same time, the
DR5:GUS maximum was unaltered in the root tips of YFP:D6PK
seedlings (Fig. 3C,G). In summary, these findings suggest that auxin
response or auxin distribution are altered in YFP:D6PK plants.
Because our auxin response experiments largely supported the
notion that auxin responses are unaffected in YFP:D6PK plants (see
Fig. S3A-C in the supplementary material) and because we found
that YFP:D6PK seedlings are less sensitive to NPA (Fig. 3M), we
reasoned that the YFP:D6PK phenotype is most likely to result
primarily from altered auxin transport.
D6PK and PIN proteins colocalize at the basal
membrane of root cells
Despite the fact that D6PK and its homologs are devoid of sequence
motifs that would indicate that the proteins might reside in or at the
plasma membrane, we found YFP:D6PK to localize to the basal
(lower) membrane of various root cell types, specifically stele,
DEVELOPMENT
NPA, we found that d6pk d6pkl1 d6pkl2 mutant seedlings are
hypersensitive to NPA, a result that might indicate that the mutants
are auxin transport deficient (Fig. 3M). When we measured auxin
transport in wild-type and in d6pk d6pkl1 d6pkl2 mutant stem
segments using radiolabeled IAA, we revealed a strong decrease in
IAA transport in the d6pk d6pkl1 d6pkl2 mutants (Fig. 3N). Because
at the same time we did not detect any significant difference in the
auxin content between the wild-type (15.2±1.3 ng IAA/g fresh
weight; n=7) and d6pk d6pkl1 d6pkl2 mutant (14.3±3.1 ng IAA/g
fresh weight; n=6) seedlings, we concluded that the d6pk d6pkl1
d6pkl2 mutant phenotype might be caused by an auxin transport
defect.
RESEARCH ARTICLE
Development 136 (4)
Fig. 3. Altered auxin transport in d6pk mutants and YFP:D6PK
plants. (A-D) DR5:GUS auxin (response) maxima at the sites of lateral
root initiation in the primary roots of 7-day-old seedlings grown on
standard growth medium (A-C) and on medium supplemented with
1 μM NPA (D). Scale bar: 1 mm. (E-H) The DR5:GUS maxima in primary
root tips of 7-day-old seedlings is shifted above the quiescent center
(asterisks) in d6pk d6pkl1 d6pkl2 (d6pk012) mutant roots. Scale bar:
0.5 mm. (I,J) Auxin (response) maxima in leaves of wild-type (I) and
YFP:D6PK transgenic (J) plants. Maxima in the leaf surface coincide with
uneven areas of the leaf. Scale bars: 1 cm. (K,L) Staining for the activity
of DR5:GUS reveals the formation of adventitious root primordia in
hypocotyls of dark-grown YFP:D6PK seedlings (blue, L), a differentiation
event that is never observed in the wild type (K). Scale bar: 3 mm.
(M) Relative root elongation of wild type, d6pk d6pkl1 d6pkl2
(d6pk012) mutants, d6pk d6pkl1 d6pkl3 (d6pk013) mutants, and
YFP:D6PK transgenic seedlings grown on standard growth medium
supplemented with the auxin transport inhibitor NPA. The values
displayed in the graph correspond to the average and standard
deviation of at least eight replicate measurements. t-test significance:
***P<0.001, **P=0.001-0.01, *P=0.01-0.05. (N) Auxin transport in
stem segments of wild type and d6pk d6pkl1 d6pkl2 (d6pk012)
mutants, as revealed by measuring the presence of radiolabeled IAA.
The measured IAA transport is the result of active auxin transport, as it
is blocked in the presence of NPA. In each experiment, four replicate
measurements were analyzed, the experiment was repeated four times,
the average and the standard deviation of one experiment are shown.
cortex, epidermis and lateral root cap cells (Fig. 4). In turn, the
YFP:D6PK protein – although expressed from the constitutive
35SCaMV promoter – was not detectable in the root meristem or in
columella root cap cells, suggesting that YFP:D6PK might be
regulated at the posttranscriptional level (Fig. 4S).
The basal localization of YFP:D6PK in the stele is reminiscent of
the basal localization of PIN auxin efflux facilitators. Using
immunostaining, we could indeed show that YFP:D6PK colocalizes
with basally localized PINs, specifically with PIN1 in stele cells
(Fig. 4C), with PIN2 in cortex cells (Fig. 4K), and with PIN4 in
lateral root cap cells (Fig. 4O), suggesting a possible functional
relationship between these proteins in controlling auxin transport.
Unlike PID, which belongs to the same AGC kinase family as
D6PK, and whose absence and presence had been shown to
determine PIN polarity (Friml et al., 2004; Michniewicz et al.,
2007), we found PIN polarity (as well as PIN abundance) to be
unaltered in the roots of any of the D6PK genotypes examined (Fig.
4A-D,I-P). This was most obvious in the case of PIN2, which
localizes to the basal membrane in cortex cells (C) and to the apical
(upper) membrane in epidermal cells (E). In neither cell type did the
presence or absence of D6PK affect the differential polarity of PIN2
(Fig. 4I-L). We therefore reasoned that neither changes in PIN
polarity nor changes in PIN abundance are the likely cause of the
reduced auxin transport in d6pk mutants.
Next, we examined the recycling of PIN1 and YFP:D6PK in
response to auxin and the fungal toxin Brefeldin A (BFA). Auxin
was shown to inhibit PIN1 endocytosis (Paciorek et al., 2005), and
BFA treatment is known to block the recycling of PINs to the plasma
membrane and leads to the accumulation of PIN proteins in
intracellular compartments (Geldner et al., 2003). We reasoned that
D6PK might be important for PIN endocytosis or PIN recycling, and
tested the effects of auxin and BFA in the d6pk mutants and in
YFP:D6PK seedlings using previously described experimental
conditions (Geldner et al., 2003; Paciorek et al., 2005). However,
DEVELOPMENT
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D6 protein kinases and auxin transport
RESEARCH ARTICLE
633
Fig. 4. YFP:D6PK colocalizes with PIN proteins at the basal
membrane of root cells. (A-D) Immunostaining with anti-PIN1 (red
signal) reveals the lower (basal) localization of PIN1 in stele cells of roots
of wild-type, d6pk mutant and YFP:D6PK transgenic seedlings (red
arrowheads). YFP:D6PK (green signal) and PIN1 (red signal) colocalize at
the basal membrane of stele cells (green and red arrowheads).
Spearman’s correlation coefficient for the PIN1 and YFP:D6PK signal
shown in D is 0.586. (E-H) Following treatment with BFA, PIN1 (red)
accumulates in endosomal compartments in all three genotypes (red
arrowheads). YFP:D6PK (green) looses its association with the
membrane and becomes cytoplasmic following BFA treatment (G,H).
Spearman’s correlation coefficient for the PIN1 and YFP:D6PK signal
shown in H is 0.0135. (I,J) Immunostaining with anti-PIN2 (red) reveals
the apical localization of PIN2 in root epidermal cells and the basal
localization of PIN2 in cortical cells (red arrowheads). YFP:D6PK (green)
colocalizes with basal PIN2 in the cortex but not with apical PIN2 in the
epidermis (green and red arrowheads; K,L). E, epidermis; C, cortex.
Spearman’s correlation coefficient for the PIN2 and YFP:D6PK signal in
K, in cortical cells is 0.255, in epidermal cells is –0.609.
(M-P) Immunostaining with anti-PIN4 (red) reveals the basal localization
of PIN4 in cells of the root tips of wild-type, d6pk mutant and
YFP:D6PK transgenic seedlings. YFP:D6PK (green) and PIN4 (red)
colocalize at the basal membrane of lateral root cap cells (green and
red arrowheads; O,P). Spearman’s correlation coefficient for the PIN4
and YFP:D6PK signal shown in P is 0.558. (Q-S) Propidium iodide
staining of wild-type, d6pk d6pkl1 d6pkl2 mutant and YFP:D6PK roots
reveals that the different genotypes have normal root morphology.
(S) Although overexpressed, YFP:D6PK (green, live imaging) is not
detectable in the central root meristem, but it is expressed in lateral
root cap cells, cortical cells, the epidermis and stele cells (see also C,D).
PIN proteins are in vitro and in vivo
phosphorylation substrates of D6PK
We next examined whether PIN proteins are phosphorylation
substrates of D6PK. To this end, we expressed and purified the
cytoplasmic domain (cytoplasmic loop) of PIN1, PIN2, PIN3, PIN4
and PIN7 as a poly-histidine-tagged peptide from bacteria, and used
the purified proteins as in vitro phosphorylation substrates for
recombinant purified GST-tagged D6PK (Fig. 5A). Interestingly,
each of the PIN protein fragments was efficiently phosphorylated by
GST:D6PK, but not by a kinase-dead GST:D6PKin variant.
Recombinant purified GST:PIN1 was also phosphorylated by
YFP:D6PK, but not by YFP:D6PKin, after immunoprecipitation of
the D6PK fusion proteins from transgenic plants (Fig. 5B). Finally,
the overexpression of YFP:D6PK or of untagged D6PK (but not of
YFP:D6PKin) led to the phosphorylation of a functional PIN1:GFP
fusion protein in transiently transformed Arabidopsis protoplasts
(Fig. 5C). Taken together, these data suggest that PIN proteins are
phosphorylation targets of D6PKs in planta, and this finding invites
the conclusion that D6PK and PIN proteins functionally interact.
The phosphorylation reactions outlined above were not affected
when the material was treated with IAA or NPA, or when the
compounds were included in the phosphorylation reactions, which
suggests that IAA and NPA do not directly modulate D6PK activity.
Next, we tested the genetic interaction between D6PK genes and
PIN1 by introducing d6pk mutations into a pin1 mutant background.
In support of the postulated interaction between D6PK and PIN1,
we found that the pin1 mutant phenotype is enhanced in the presence
of the d6pk d6pkl1 d6pkl2 mutations and the d6pk d6pkl2 mutations,
the latter of which does not have a phenotype on its own: the pin1
d6pk d6pkl1 d6pkl2 and the pin1 d6pk d6pkl2 mutants were
significantly smaller than the respective pin1 or d6pk mutants, and
the leaves of pin1 d6pk d6pkl2 were significantly shorter than those
of pin1 and d6pk d6pkl2 mutants, respectively (Fig. 5C; see Table
S1 in the supplementary material). Because these findings indicated
an important role for D6PKs as regulators of PIN1, and because
undifferentiated pin-formed shoot apices are a hallmark phenotype
of pin1 mutants, we examined the d6pk mutants for similar defects.
DEVELOPMENT
our experiments did not reveal any alterations in the behavior of
PIN1 in the d6pk mutants or in YFP:D6PK plants (Fig. 4E-H; data
not shown). We therefore consider it unlikely that D6PKs exert their
role in auxin transport by controlling PIN1 endocytosis or recycling.
Following BFA treatment, we made the interesting observation
that YFP:D6PK looses its affinity for the plasma membrane.
However, YFP:D6PK did not accumulate with PIN1 in BFAinduced compartments but was dispersed throughout the cytoplasm
(Fig. 4E-H). This observation suggests that YFP:D6PK is retained
at the plasma membrane by a BFA-sensitive component. Because
PIN1 represents a good candidate for such a BFA-sensitive
interaction partner, we introduced YFP:D6PK in a pin1 mutant
background. We found, however, that YFP:D6PK is still plasmamembrane associated in a pin1 mutant (data not shown), and we
therefore presently rule out that PIN1 is the protein required for the
membrane association of YFP:D6PK. In addition, BFA treatments
also revealed a differential growth response between the wild type
and the D6PK genotypes. We found that BFA-induced agravitropic
growth occurs in d6pk mutants at lower concentrations than in the
wild type, and at the same time we found YFP:D6PK seedlings to
be less sensitive to BFA (see Fig. S4 in the supplementary material).
This demonstrates that altering D6PK dosage affects the BFA
sensitivity of Arabidopsis roots with regard to root gravitropism,
which is an auxin transport-dependent mechanism.
634
RESEARCH ARTICLE
Development 136 (4)
Fig. 5. Biochemical and genetic interaction between D6PK and
PIN auxin efflux facilitators. (A) Recombinant GST:D6PK but not the
kinase-dead variant GST:D6PKin phosphorylates the histidine-tagged
cytoplasmic loops of PIN1, PIN2, PIN3, PIN4 and PIN7 in vitro.
(B) Following immunoprecipitation from transgenic plants, YFP:D6PK
but not the kinase-dead variant YFP:D6PKin phosphorylates a
recombinant GST-tagged PIN1 cytoplasmic loop in vitro. (C) Untagged
D6PK or YFP:D6PK (but not the kinase-dead variant expressed from
YFP:D6PKin) phosphorylate PIN1:GFP when coexpressed in Arabidopsis
protoplasts. The phosphorylation is revealed by the appearance of
additional low mobility PIN1:GFP-specific bands, whose abundance is
diminished following treatment with calf intestinal alkaline phosphatase
(CIAP; right panel). (D) Genetic interaction between a pin1 mutant
allele and d6pk mutants. The height of pin1 d6pk d6pkl1 d6pkl2 (pin1
d6pk012) and of pin1 d6pk d6pkl2 (pin1 d6pk02) mutants is reduced
more strongly than that of the respective pin1 or d6pk mutants (see
Table S1 in the supplementary material). Numbers indicate the average
plant height and its standard deviation (cm) from at least six plants. The
shoot differentiation defects of the pin1 mutants are maintained in the
pin1 d6pk mutant combinations (not visible in the case of pin1 d6012),
but the leaf shape of pin1 d6pk d6pkl2 mutants is altered in
comparison to that of the pin1 and d6pk d6pkl2 mutants, such that the
leaves are rounder and have an uneven leaf surface.
Following a more careful analysis, we found that the d6pk d6pkl1
d6pkl2 d6pkl3 quadruple mutant frequently develops axillary shoots
with differentiation defects that were strongly reminiscent of the
DISCUSSION
In the present study, we have characterized four highly homologous
members of the Arabidopsis AGCVIIIa kinase family, which we
designated the D6PKs (see Fig. S1 in the supplementary material).
Based on the phenotypes of the d6pk mutants and the YFP:D6PK
overexpressing lines, we judge that these kinases have redundant
functions in regulating lateral root initiation, gravitropism, and shoot
differentiation in axillary shoots. In addition, we could show that
auxin transport is impaired in d6pk mutants, that DR5:GUS
expression is altered in d6pk mutant root tips, and that DR5:GUS
expression is absent at the presumed sites of lateral root initiation in
d6pk mutants. It is known that many of these defects can be
mimicked in wild-type plants by treatment with the auxin efflux
inhibitor NPA. Based on our findings and the phenotypic similarities
between d6pk mutants and NPA-treated plants, we suggest that
D6PKs regulate a protein that is close to the site of action of NPA.
Because D6PK and the PIN auxin efflux facilitators colocalize in
planta, and because D6PK phosphorylates PINs, our data also invite
the hypothesis that D6PKs interact with PIN proteins and possibly
regulate their function by phosphorylation. Although we present
several lines of evidence that include a functional link between
D6PK and PIN, including the phosphorylation of PINs by D6PK,
we are at present unable to shed light on the mechanistic interplay
between the two protein families. Based on our cell biological
analyses, we can rule out, however, that D6PKs control PINs by
changing their abundance, tissue distribution, polarity, endocytosis
or recycling.
We have been unable to detect any effects of auxin or NPA on the
expression, abundance, localization or biochemical activity of
D6PK. In this respect, it is noteworthy that we found that several
D6PK genes are expressed strongly at the sites of lateral root
initiation but that the expression of none of the D6PK genes is
induced by auxin alone. Furthermore, D6PK expression appears to
be regulated at the posttranscriptional level because YFP:D6PK –
although overexpressed – is not detected in the root meristem or the
columella root cap. Regarding YFP:D6PKs polarity, we also would
like to stress the fact that, although D6PK and PINs colocalize at the
basal membrane of several other root cell types, D6PK and PIN2 do
not colocalize in root epidermal cells, where PIN2 is the most
abundant PIN. Thus, at least in epidermal cells, the polar distribution
of D6PK is regulated independently from that of PIN2. Taking these
findings together, we propose that D6PK regulates auxin efflux
efficiency and that the biological activity D6PK is, at least in part,
determined by its polarity and by its expression domain. Based on
the observed interaction between D6PK and PINs, it could be
speculated that D6PKs enhance the PIN auxin efflux facilitator
function by phosphorylation or by another mechanism, e.g. by
influencing their ability to interact with other proteins required for
efficient auxin transport, such as the MDR/PGPs. The regulatory
mechanism responsible for altered auxin transport in d6pk mutants
is one important question that needs to be addressed in future
experiments.
For several reasons, we have focused in the present study on the
PIN proteins as putative phosphorylation targets of D6PK. First, we
observed a number of morphological phenotypes in d6pk mutants that
have also been observed in pin mutants (Benkova et al., 2003; Chen
et al., 1998; Friml et al., 2002b; Galweiler et al., 1998; Luschnig et al.,
DEVELOPMENT
differentiation defects of primary and axillary pin1 mutant shoots
(Fig. 1N-P). We thus propose that D6PKs are regulators of auxin
transport, which might, at least in part, act together with PIN auxin
efflux facilitators.
1998; Muller et al., 1998; Scarpella et al., 2006), and these defects
appear generally to be stronger – as far as can be judged from
published work – than those observed in mutants deficient in all four
AUX1/LAX genes (Bainbridge et al., 2008; Casimiro et al., 2001;
Marchant et al., 2002; Marchant et al., 1999) (Figs 1, 2). Second, we
observed a shift of the DR5:GUS expression maximum in the root tips
of d6pk mutants, and similar changes in DR5:GUS activity were also
reported in pin2 and pin4 mutants (Friml et al., 2002a; Sabatini et al.,
1999). Third, we observed a synergistic genetic interaction between
PIN1 and the D6PK genes, thereby supporting the idea that the two
proteins act in close proximity. Finally, D6PKs belong to the same
protein kinase family as PID, which phosphorylates PIN1 and whose
mutant phenocopies the pin1 mutant phenotype (Michniewicz et al.,
2007). Therefore, PINs are good candidates for D6PK
phosphorylation substrates. However, it may be that the observed
D6PK-PIN interactions are not functionally relevant in vivo or that
other auxin transport proteins are D6PK substrates. In view of the fact
that YFP:D6PK localizes to the basal membrane of root epidermal
cells, where it does not colocalize with PIN2, D6PK may well have
additional phosphorylation targets.
Because PID and D6PKs all belong to the AGCVIIIa kinase family
and because all are functionally implicated in regulating PIN activity,
it may be reasoned that these kinases regulate auxin transport in the
same manner (Galvan-Ampudia and Offringa, 2007; Michniewicz et
al., 2007). In contrast to what was observed for PID loss-of-function
and overexpression lines, our data for D6PK show that PIN polarity
is unaltered in d6pk mutants, as well as in YFP:D6PK overexpression
lines. We therefore suggest that PID and the D6PK proteins do not
have a redundant function. A differential role of D6PK and PID is also
supported by the fact that the overexpression of either kinase has a
different effect on plant growth. First, YFP:D6PK seedlings are
defective in lateral root formation and the development of dark-grown
YFP:D6PK seedlings is strongly impaired. In turn, lateral root
development is unaffected in PID overexpression lines and darkgrown seedlings have only subtle defects (Benjamins et al., 2001;
Friml et al., 2004). Second, whereas the roots of PID overexpressing
seedlings grow agravitropically and their meristems collapse,
YFP:D6PK overexpression lines do not grow agravitropically and
they have an intact meristem and normal DR5:GUS reporter activity
(Benjamins et al., 2001; Friml et al., 2004). Another difference
between D6PK and PID resides in the polar localization of the
proteins. Whereas YFP:D6PK (as well as D6PK:GFP) localize to the
basal membrane of all root cell types examined, including epidermal
cells, PID:YFP and PID:GFP have an apicobasal localization in the
epidermis (Galvan-Ampudia and Offringa, 2007; Michniewicz et al.,
2007). Taken together, these differences indicate that D6PKs and PID
have differential biochemical functions and control plant growth in a
different manner. Nevertheless, our observations reported here,
together with the observations previously reported for PID, could help
to reveal the molecular mechanisms that underlie the functions of
other AGCVIIIa kinases, such as PHOT and WAG protein kinases.
We acknowledge the members of our laboratory for critical comments on the
manuscript, and Remko Offringa and colleagues (Leiden University, The
Netherlands) for discussions and sharing unpublished data. We are grateful to
Catarina Brancato (Center for Plant Molecular Biology, Tübingen, Germany) for
the transient transformations, and the Salk Institute Genomic Analysis
Laboratory (San Diego, CA, USA) as well as the Nottingham Arabidopsis Stock
Centre (Nottingham, UK) for generating and providing the sequence-indexed
Arabidopsis T-DNA insertion mutants. The auxin measurements were carried
out in the laboratory of Shinjiro Yamaguchi (RIKEN Yokohama, Japan). This
work was supported by grants from the Deutsche Forschungsgemeinschaft
(SCHW751/5) and the Sonderforschungsbereich SFB446.
RESEARCH ARTICLE
635
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/4/627/DC1
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