|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online January 23, 2009
doi: 10.1242/10.1242/dev.028365
1 Tübingen University, Center for Plant Molecular Biology, Department of
Developmental Genetics, Auf der Morgenstelle 5, 72076 Tübingen,
Germany.
2 RIKEN, Plant Science Center, Suehiro-cho 1-7-22, Tsurumi-ku, Yokohama,
Kanagawa 230-0045, Japan.
Author for correspondence (e-mail:
claus.schwechheimer{at}wzw.tum.de)
Accepted 16 December 2008
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: Arabidopsis, Protein kinase, Auxin transport, PIN proteins, Lateral root
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
).
The permease-like AUXIN-RESISTANT 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 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 PID-dependent 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 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 |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
); 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
Gateway-system 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-RV-GW3 into pDEST15.
The GST:D6PKin kinase-dead variant was derived from GST:D6PK with the primer
D6Pkin (Sawano and Miyawaki,
2000).
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-(N-morpholino)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
full-length transcripts were tested after 28 PCR amplification cycles.
Auxin-induced 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-day-old
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 nitrogen-frozen 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 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 anti-rabbit 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
5xLaemmli buffer, and protein extracts were separated on a 10%
SDS-polyacrylamide 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 2xLaemmli buffer and resolved on a 10% SDS-PAGE gel. For dephosphorylation, the protein extract was prepared in 50 µl 1xcalf 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% SDS-PAGE gel. The D6PK antibody is a rabbit anti-peptide antibody generated against the peptide NSKINEQGESGKSSTC (Eurogentec, Liège, Belgium).
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
;
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 full-length 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.
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 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 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).
|
|
). When
we tested the sensitivity of d6pk mutants to the auxin transport
inhibitor 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.
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, 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).
|
; 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, 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.
|
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. 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 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.
|
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
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., 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 dark-grown 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.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/4/627/DC1
|
Footnotes |
|---|
* 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
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Abas, L., Benjamins, R., Malenica, N., Paciorek, T., Wisniewska,
J., Moulinier-Anzola, J. C., Sieberer, T., Friml, J. and Luschnig, C.
(2006). Intracellular trafficking and proteolysis of the
Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism.
Nat. Cell Biol. 8,249
-256.[CrossRef][Medline]
Bainbridge, K., Guyomarc'h, S., Bayer, E., Swarup, R., Bennett,
M., Mandel, T. and Kuhlemeier, C. (2008). Auxin influx
carriers stabilize phyllotactic patterning. Genes Dev.
22,810
-823.
Bandyopadhyay, A., Blakeslee, J. J., Lee, O. R., Mravec, J.,
Sauer, M., Titapiwatanakun, B., Makam, S. N., Bouchard, R., Geisler, M.,
Martinoia, E. et al. (2007). Interactions of PIN and PGP
auxin transport mechanisms. Biochem. Soc. Trans.
35,137
-141.[CrossRef][Medline]
Benjamins, R., Quint, A., Weijers, D., Hooykaas, P. and
Offringa, R. (2001). The PINOID protein kinase regulates
organ development in Arabidopsis by enhancing polar auxin transport.
Development 128,4057
-4067.
Benkova, E., Michniewicz, M., Sauer, M., Teichmann, T.,
Seifertova, D., Jurgens, G. and Friml, J. (2003). Local,
efflux-dependent auxin gradients as a common module for plant organ formation.
Cell 115,591
-602.[CrossRef][Medline]
Bennett, M. J., Marchant, A., Green, H. G., May, S. T., Ward, S.
P., Millner, P. A., Walker, A. R., Schulz, B. and Feldmann, K. A.
(1996). Arabidopsis AUX1 gene: a permease-like regulator of root
gravitropism. Science
273,948
-950.[Abstract]
Blakeslee, J. J., Bandyopadhyay, A., Lee, O. R., Mravec, J.,
Titapiwatanakun, B., Sauer, M., Makam, S. N., Cheng, Y., Bouchard, R., Adamec,
J. et al. (2007). Interactions among PIN-FORMED and
P-glycoprotein auxin transporters in Arabidopsis. Plant
Cell 19,131
-147.
Blilou, I., Xu, J., Wildwater, M., Willemsen, V., Paponov, I.,
Friml, J., Heidstra, R., Aida, M., Palme, K. and Scheres, B.
(2005). The PIN auxin efflux facilitator network controls growth
and patterning in Arabidopsis roots. Nature
433, 39-44.[CrossRef][Medline]
Bogre, L., Okresz, L., Henriques, R. and Anthony, R. G.
(2003). Growth signalling pathways in Arabidopsis and the AGC
protein kinases. Trends Plant Sci.
8, 424-431.[CrossRef][Medline]
Casimiro, I., Marchant, A., Bhalerao, R. P., Beeckman, T.,
Dhooge, S., Swarup, R., Graham, N., Inze, D., Sandberg, G., Casero, P. J. et
al. (2001). Auxin transport promotes Arabidopsis lateral root
initiation. Plant Cell
13,843
-852.
Chen, R., Hilson, P., Sedbrook, J., Rosen, E., Caspar, T. and
Masson, P. H. (1998). The Arabidopsis thaliana AGRAVITROPIC 1
gene encodes a component of the polar-auxin-transport efflux carrier.
Proc. Natl. Acad. Sci. USA
95,15112
-15117.
Curtis, M. D. and Grossniklaus, U. (2003). A
gateway cloning vector set for high-throughput functional analysis of genes in
planta. Plant Physiol.
133,462
-469.
Delbarre, A., Muller, P., Imhoff, V. and Guern, J.
(1996). Comparison of mechanisms controlling uptake and
accumulation of 2,4-dichlorophenoxy acetic acid, naphtalene-1-acetic acid, and
indole-3-acetic acid in suspension-cultured tobacco cells.
Planta 198,532
-541.
Dharmasiri, N., Dharmasiri, S., Weijers, D., Lechner, E.,
Yamada, M., Hobbie, L., Ehrismann, J. S., Jurgens, G. and Estelle, M.
(2005). Plant development is regulated by a family of auxin
receptor F box proteins. Dev. Cell
9, 109-119.[CrossRef][Medline]
Dubrovsky, J. G., Sauer, M., Napsucialy-Mendivil, S.,
Ivanchenko, M. G., Friml, J., Shishkova, S., Celenza, J. and Benkova, E.
(2008). Auxin acts as a local morphogenetic trigger to specify
lateral root founder cells. Proc. Natl. Acad. Sci. USA
105,8790
-8794.
Edlund, A., Eklof, S., Sundberg, B., Moritz, T. and Sandberg,
G. (1995). A microscale technique for gas chromatography-mass
spectrometry measurements of picogram amounts of indole-3-acetic acid in plant
tissues. Plant Physiol.
108,1043
-1047.[Abstract]
Esmon, C. A., Tinsley, A. G., Ljung, K., Sandberg, G., Hearne,
L. B. and Liscum, E. (2006). A gradient of auxin and
auxin-dependent transcription precedes tropic growth responses.
Proc. Natl. Acad. Sci. USA
103,236
-241.
French, A. P., Mills, S., Swarup, R., Bennett, M. J. and
Pridmore, T. P. (2008). Colocalization of fluorescent markers
in confocal microscope images of plant cells. Nat.
Protoc. 3,619
-628.[CrossRef][Medline]
Friml, J., Benkova, E., Blilou, I., Wisniewska, J., Hamann, T.,
Ljung, K., Woody, S., Sandberg, G., Scheres, B., Jurgens, G. et al.
(2002a). AtPIN4 mediates sink-driven auxin gradients and root
patterning in Arabidopsis. Cell
108,661
-673.[CrossRef][Medline]
Friml, J., Wisniewska, J., Benkova, E., Mendgen, K. and Palme,
K. (2002b). Lateral relocation of auxin efflux regulator PIN3
mediates tropism in Arabidopsis. Nature
415,806
-809.[Medline]
Friml, J., Vieten, A., Sauer, M., Weijers, D., Schwarz, H.,
Hamann, T., Offringa, R. and Jurgens, G. (2003).
Efflux-dependent auxin gradients establish the apical-basal axis of
Arabidopsis. Nature 426,147
-153.[CrossRef][Medline]
Friml, J., Yang, X., Michniewicz, M., Weijers, D., Quint, A.,
Tietz, O., Benjamins, R., Ouwerkerk, P. B., Ljung, K., Sandberg, G. et al.
(2004). A PINOID-dependent binary switch in apical-basal PIN
polar targeting directs auxin efflux. Science
306,862
-865.
Fukaki, H., Tameda, S., Masuda, H. and Tasaka, M.
(2002). Lateral root formation is blocked by a gain-of-function
mutation in the SOLITARY-ROOT/IAA14 gene of Arabidopsis. Plant
J. 29,153
-168.[CrossRef][Medline]
Galvan-Ampudia, C. S. and Offringa, R. (2007).
Plant evolution: AGC kinases tell the auxin tale. Trends Plant
Sci. 12,541
-547.[CrossRef][Medline]
Galweiler, L., Guan, C., Muller, A., Wisman, E., Mendgen, K.,
Yephremov, A. and Palme, K. (1998). Regulation of polar auxin
transport by AtPIN1 in Arabidopsis vascular tissue.
Science 282,2226
-2230.
Geisler, M. and Murphy, A. S. (2006). The ABC
of auxin transport: the role of p-glycoproteins in plant development.
FEBS Lett. 580,1094
-1102.[CrossRef][Medline]
Geldner, N., Anders, N., Wolters, H., Keicher, J., Kornberger,
W., Muller, P., Delbarre, A., Ueda, T., Nakano, A. and Jurgens, G.
(2003). The Arabidopsis GNOM ARF-GEF mediates endosomal
recycling, auxin transport, and auxin-dependent plant growth.
Cell 112,219
-230.[CrossRef][Medline]
Harper, R. M., Stowe-Evans, E. L., Luesse, D. R., Muto, H.,
Tatematsu, K., Watahiki, M. K., Yamamoto, K. and Liscum, E.
(2000). The NPH4 locus encodes the auxin response factor ARF7, a
conditional regulator of differential growth in aerial Arabidopsis tissue.
Plant Cell 12,757
-770.
Karimi, M., Inze, D. and Depicker, A. (2002).
GATEWAY vectors for Agrobacterium-mediated plant transformation.
Trends Plant Sci. 7,193
-195.[CrossRef][Medline]
Katekar, G. F. and Geissler, A. E. (1977).
Auxin Transport Inhibitors: III. Chemical Requirements of a Class of Auxin
Transport Inhibitors. Plant Physiol.
60,826
-829.
Kleine-Vehn, J., Dhonukshe, P., Swarup, R., Bennett, M. and
Friml, J. (2006). Subcellular trafficking of the Arabidopsis
auxin influx carrier AUX1 uses a novel pathway distinct from PIN1.
Plant Cell 18,3171
-3181.
Kramer, E. M. and Bennett, M. J. (2006). Auxin
transport: a field in flux. Trends Plant Sci.
11,382
-386.[CrossRef][Medline]
Lauber, M. H., Waizenegger, I., Steinmann, T., Schwarz, H.,
Mayer, U., Hwang, I., Lukowitz, W. and Jurgens, G. (1997).
The Arabidopsis KNOLLE protein is a cytokinesis-specific syntaxin.
J. Cell Biol. 139,1485
-1493.
Lawton, M. A., Yamamoto, R. T., Hanks, S. K. and Lamb, C. J.
(1989). Molecular cloning of plant transcripts encoding protein
kinase homologs. Proc. Natl. Acad. Sci. USA
86,3140
-3144.
Luschnig, C., Gaxiola, R. A., Grisafi, P. and Fink, G. R.
(1998). EIR1, a root-specific protein involved in auxin
transport, is required for gravitropism in Arabidopsis thaliana.
Genes Dev. 12,2175
-2187.
Marchant, A., Kargul, J., May, S. T., Muller, P., Delbarre, A.,
Perrot-Rechenmann, C. and Bennett, M. J. (1999). AUX1
regulates root gravitropism in Arabidopsis by facilitating auxin uptake within
root apical tissues. EMBO J.
18,2066
-2073.[CrossRef][Medline]
Marchant, A., Bhalerao, R., Casimiro, I., Eklof, J., Casero, P.
J., Bennett, M. and Sandberg, G. (2002). AUX1 promotes
lateral root formation by facilitating indole-3-acetic acid distribution
between sink and source tissues in the Arabidopsis seedling. Plant
Cell 14,589
-597.
Michniewicz, M., Zago, M. K., Abas, L., Weijers, D.,
Schweighofer, A., Meskiene, I., Heisler, M. G., Ohno, C., Zhang, J., Huang, F.
et al. (2007). Antagonistic regulation of PIN phosphorylation
by PP2A and PINOID directs auxin flux. Cell
130,1044
-1056.[CrossRef][Medline]
Muller, A., Guan, C., Galweiler, L., Tanzler, P., Huijser, P.,
Marchant, A., Parry, G., Bennett, M., Wisman, E. and Palme, K.
(1998). AtPIN2 defines a locus of Arabidopsis for root
gravitropism control. EMBO J.
17,6903
-6911.[CrossRef][Medline]
Okada, K., Ueda, J., Komaki, M. K., Bell, C. J. and Shimura,
Y. (1991). Requirement of the auxin polar transport system in
early stages of Arabidopsis floral bud formation. Plant
Cell 3,677
-684.
Paciorek, T., Zazimalova, E., Ruthardt, N., Petrasek, J.,
Stierhof, Y. D., Kleine-Vehn, J., Morris, D. A., Emans, N., Jurgens, G.,
Geldner, N. et al. (2005). Auxin inhibits endocytosis and
promotes its own efflux from cells. Nature
435,1251
-1256.[CrossRef][Medline]
Paponov, I. A., Teale, W. D., Trebar, M., Blilou, I. and Palme,
K. (2005). The PIN auxin efflux facilitators: evolutionary
and functional perspectives. Trends Plant Sci.
10,170
-177.[CrossRef][Medline]
Petrasek, J., Mravec, J., Bouchard, R., Blakeslee, J. J., Abas,
M., Seifertova, D., Wisniewska, J., Tadele, Z., Kubes, M., Covanova, M. et
al. (2006). PIN proteins perform a rate-limiting function in
cellular auxin efflux. Science
312,914
-918.
Sabatini, S., Beis, D., Wolkenfelt, H., Murfett, J., Guilfoyle,
T., Malamy, J., Benfey, P., Leyser, O., Bechtold, N., Weisbeek, P. et al.
(1999). An auxin-dependent distal organizer of pattern and
polarity in the Arabidopsis root. Cell
99,463
-472.[CrossRef][Medline]
Sakai, T., Kagawa, T., Kasahara, M., Swartz, T. E., Christie, J.
M., Briggs, W. R., Wada, M. and Okada, K. (2001). Arabidopsis
nph1 and npl1: blue light receptors that mediate both phototropism and
chloroplast relocation. Proc. Natl. Acad. Sci. USA
98,6969
-6974.
Santner, A. A. and Watson, J. C. (2006). The
WAG1 and WAG2 protein kinases negatively regulate root waving in Arabidopsis.
Plant J. 45,752
-764.[CrossRef][Medline]
Sauer, M., Balla, J., Luschnig, C., Wisniewska, J., Reinohl, V.,
Friml, J. and Benkova, E. (2006). Canalization of auxin flow
by Aux/IAA-ARF-dependent feedback regulation of PIN polarity. Genes
Dev. 20,2902
-2911.
Sawano, A. and Miyawaki, A. (2000). Directed
evolution of green fluorescent protein by a new versatile PCR strategy for
site-directed and semi-random mutagenesis. Nucleic Acids
Res. 28,E78
.[CrossRef][Medline]
Scarpella, E., Marcos, D., Friml, J. and Berleth, T.
(2006). Control of leaf vascular patterning by polar auxin
transport. Genes Dev.
20,1015
-1027.
Swarup, K., Benkova, E., Swarup, R., Casimiro, I., Peret, B.,
Yang, Y., Parry, G., Nielsen, E., De Smet, I., Vanneste, S. et al.
(2008). The auxin influx carrier LAX3 promotes lateral root
emergence. Nat. Cell Biol.
10,946
-954.[CrossRef][Medline]
Swarup, R., Friml, J., Marchant, A., Ljung, K., Sandberg, G.,
Palme, K. and Bennett, M. (2001). Localization of the auxin
permease AUX1 suggests two functionally distinct hormone transport pathways
operate in the Arabidopsis root apex. Genes Dev.
15,2648
-2653.
Teale, W. D., Paponov, I. A. and Palme, K.
(2006). Auxin in action: signalling, transport and the control of
plant growth and development. Nat. Rev. Mol. Cell
Biol. 7,847
-859.[CrossRef][Medline]
Ulmasov, T., Murfett, J., Hagen, G. and Guilfoyle, T. J.
(1997). Aux/IAA proteins repress expression of reporter genes
containing natural and highly active synthetic auxin response elements.
Plant Cell 9,1963
-1971.[Abstract]
Vieten, A., Vanneste, S., Wisniewska, J., Benkova, E.,
Benjamins, R., Beeckman, T., Luschnig, C. and Friml, J.
(2005). Functional redundancy of PIN proteins is accompanied by
auxin-dependent cross-regulation of PIN expression.
Development 132,4521
-4531.
Vieten, A., Sauer, M., Brewer, P. B. and Friml, J.
(2007). Molecular and cellular aspects of
auxin-transport-mediated development. Trends Plant
Sci. 12,160
-168.[CrossRef][Medline]
Willige, B. C., Ghosh, S., Nill, C., Zourelidou, M., Dohmann, E.
M., Maier, A. and Schwechheimer, C. (2007). The DELLA domain
of GA INSENSITIVE mediates the interaction with the GA INSENSITIVE DWARF1A
gibberellin receptor of Arabidopsis. Plant Cell
19,1209
-1220.
Wisniewska, J., Xu, J., Seifertova, D., Brewer, P. B., Ruzicka,
K., Blilou, I., Rouquie, D., Benkova, E., Scheres, B. and Friml, J.
(2006). Polar PIN localization directs auxin flow in plants.
Science 312,883
.
Yang, Y., Hammes, U. Z., Taylor, C. G., Schachtman, D. P. and
Nielsen, E. (2006). High-affinity auxin transport by the AUX1
influx carrier protein. Curr. Biol.
16,1123
-1127.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in Development:
This article has been cited by other articles:
|
|
 |
|
  J. Petrasek and J. Friml Auxin transport routes in plant development Development, August 15, 2009; 136(16): 2675 - 2688. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Sukumar, K. S. Edwards, A. Rahman, A. DeLong, and G. K. Muday PINOID Kinase Regulates Root Gravitropism through Modulation of PIN2-Dependent Basipetal Auxin Transport in Arabidopsis Plant Physiology, June 1, 2009; 150(2): 722 - 735. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
