1 Department of Bioscience, Faculty of Applied Bioscience, Tokyo University of
Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan
2 Department of Biological Sciences, Graduate School of Science, The University
of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
3 Molecular Membrane Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama
351-0198, Japan
4 Botanical Gardens, Graduate School of Science, The University of Tokyo, 3-7-1
Hakusan, Bunkyo-ku, Tokyo 112-0001, Japan
5 Plant Science Center, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama-shi,
Kanagawa 230-0045, Japan
Author for correspondence (e-mail:
fukuda{at}biol.s.u-tokyo.ac.jp)
Accepted 27 January 2005
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SUMMARY |
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Key words: Arabidopsis, Mutant, VAN3, Vein, Vascular tissue, ARFGAP
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Introduction |
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Auxin has been nominated as a key molecule in the basic mechanism
controlling venation. A large number of studies indicate that polar auxin
transport plays a crucial role in continuous vascular pattern formation
(Nelson and Dengler, 1997;
Berleth et al., 2000
;
Sachs, 2000
;
Aloni, 2001
;
Dengler, 2001
; Tuner and
Sieburth, 2002; Ye, 2002
). The
administration of chemicals that specifically inhibit polar auxin transport
resulted in the formation of local aggregates of vascular cells in the
marginal regions of newly developing leaves (Mattson et al., 1999;
Sieburth, 1999
). However, such
inhibitors were less effective in preventing vascular differentiation from the
existing procambium. Therefore, these inhibitors seem to affect venation by
disrupting the development of procambial patterns. The EMB30/GN gene
encodes a guanine nucleotide exchange factor (GEF) on adenosine diphosphate
(ADP)-ribosylation factor-GTPase
(ARFGEF), which is responsible for the targeted recycling of PIN1
putative auxin efflux carrier. Consequently, the EMB30/GN gene is
required for the maintenance of polar auxin transport
(Shevell et al., 1994
;
Busch et al., 1996
;
Steinmann et al., 1999
;
Geldner et al., 2003
), and
mutations in this gene cause irregular and discontinuous venation, with the
formation of clustered or scattered tracheary elements
(Mayer et al., 1991
;
Mayer et al., 1993
;
Koizumi et al., 2000
).
The importance of auxin in the generation of venation has also been
demonstrated in auxin-response mutants. Arabidopsis mutants defective
in perceiving auxin, such as auxin resistant 6 [axr6
(Hobbie et al., 2000;
Hellmann et al., 2003
)] and
bodenlos (Hamann et al.,
1999
; Hamann et al.,
2002
), exhibit severely reduced vascular networks. In the
monopteros (mp) mutant, which is defective in an
auxin-response transcription factor (IAA24/ARF5), marginal leaf veins are
missing or interrupted and the capacity for polar auxin transport is reduced
(Mayer et al., 1991
;
Berleth and Jurgens, 1993
;
Przemeck et al., 1996
;
Mattsson et al., 2003
).
Recently, using auxin-inducible promoters fused to the ß-glucuronidase
(GUS) reporter gene, three laboratories have visualized auxin
response patterns. Results suggest the preferential accumulation of auxin in
the pre-procambial cells of young leaves
(Avsian-Kretchmer et al., 2002
;
Aloni et al., 2003
;
Mattsson et al., 2003
). This
is additional evidence for the involvement of auxin in the generation of
venation.
Based on physiological analyses of experimentally induced vascular
differentiation, Sachs (Sachs,
1991) proposed the `auxin signal flow canalization hypothesis',
which presents the following scenario for the spatial regulation of vascular
differentiation. Auxin flow, starting initially with diffusion, induces the
formation of the polar auxin transport cell system. This, in turn, promotes
auxin transport and leads to canalization of the auxin flow along a narrow
file of cells. This continuous file of cells differentiates into a strand of
procambial cells, and eventually into vascular cells. The auxin canalization
hypothesis is consistent with the aforementioned data relating auxin and
venation, and is in good agreement with currently accumulating data on PIN
proteins (Benková et al.,
2003
). Hence, the auxin canalization hypothesis might provide a
theoretical framework with which to understand the basic mechanism of venation
generation.
However, at the molecular level, the auxin canalization hypothesis contains many unresolved problems. How are the sources and sinks of auxin that are necessary for the initial flow of auxin located in specific positions? How does auxin flow rearrange the auxin polar transport system to be canalized? How does the canalized auxin flow induce procambial and vascular differentiation? The possibility also remains that some unknown mechanisms act co-operatively with the auxin canalization mechanism to generate venation.
According to the assumptions of the auxin canalization hypothesis, a
continuous flow of auxin is a prerequisite for vascular patterning. Therefore,
the overall architecture of the vascular pattern is expected to be more
sensitive to genetic lesions than is vascular continuity. A number of
Arabidopsis mutants, including lop1/tornado1
(Carland and McHale, 1996),
vascular network defective1 to 6 [van1-6
(Koizumi et al., 2000
)],
scarface, [sfc (Deyholos
et al., 2000
)] and cotyledon vein pattern 1 and
2 [cvp1, cvp2 (Carland
et al., 1999
; Carland et al.,
2002
)], have discontinuous secondary vascular strands in their
cotyledons and leaves. Interestingly and unexpectedly, in most of these
mutants, although the vein networks are fragmented, the overall architecture
is normal. The high frequency of venation mutants of this type cannot be
explained simply by the auxin canalization hypothesis.
To elucidate the molecular basis of the spatial regulation of leaf vascular development, we have identified the causal gene of the van3 mutant, which of the six van mutants shows the most specific and restricted effect on the continuity of procambial cells. The VAN3 gene encodes a unique type of ARFguanosine triphosphatase (GTPase)-activating protein (GAP), which is located in the trans-Golgi network (TGN). Phenotypic analysis of the van3 mutant suggests that the VAN3 ARFGAP may play an important role in the vesicle transport responsible for the auxin signaling that is required for vascular differentiation.
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Materials and methods |
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Double mutant analyses
To generate double mutants of van3 with the pin1-3,
emb30-7/gn or mpT370, plants heterozygous for
van3 were crossed with plants heterozygous for pin1-3,
emb30-7/gn or mpT370. Double mutants were identified
within F2 families that segregated for each single mutant, and were
distinguished by the presence of the distinct morphological features
characteristic of each parental mutant phenotype. Furthermore, the genotypes
of van3 emb30-7/gn and van3 mp double mutants were confirmed
by cleaved amplified polymorphic sequences (CAPS; data not shown). In the
double mutant combinations of van3 with pin1, and
van3 with emb30-7/gn, mutants from each combination
segregated at ratios of about 9:3:3:1 (WT:van3:pin1:van3
pin1 = 348:112:105:30, 2 0.500, <P<0.750;
WT:van3:emb30-7/gn:van3 emb30-7/gn = 339:89:93:30,
2 0.050, <P<0.100;
WT:van3:mp:van3 mp = 236:76:90:28,
2 0.143, <P<0.504).
Chemicals
Naphthalene acetic acid (NAA; Sigma-Aldrich),
N-1-naphthylphthalamic acid (NPA; Tokyo Kasei Kogyo, Tokyo, Japan),
and brefeldin A (BFA; Sigma-Aldrich) were used as 100 mM stock solutions in
dimethylsulfoxide (DMSO). These chemicals were added to the autoclaved
medium.
Histochemical staining for GUS
For the analysis of DR5::GUS expression in van3 mutants,
the van3 mutation was introduced into DR5::GUS transgenic
plants by crossing. For histochemical analysis, GUS staining was performed as
described by Koizumi et al. (Koizumi et
al., 2000), except that samples were incubated in the GUS
substrate solution for 2 hours. Fixed samples were dehydrated through a graded
ethanol series and embedded in Technovit 7100 resin (Heraeus Kulzer, Germany).
Sections (6 µm) were cut with a microtome and observed under a light
microscope equipped with Nomarski optics. The density of GUS-positive spots
was measured using first-node leaves of 7-day-old seedlings. Leaves of about
the same length (850-1,100 µm) were used. Spots and leaf area measurements
were made after the specimens were photographed. For the analysis of the auxin
response, cotyledons of 7-day-old seedlings and first-node leaves of
11-day-old plants were excised at the center. They were then incubated in 1 ml
of liquid GM containing NAA for 6 hours, with subsequent histochemical
detection.
RTPCR analysis
Total RNA was isolated as described previously
(Sawa et al., 2002), and
RTPCR analysis to quantify the expression of auxin-inducible genes was
performed according to the instructions for the Ready-To-Go RTRCR Beads
(Amersham Pharmacia Biotech), using a set of primers specific to the
VAN3 gene: 5'-GCTCCTCTCACATACAAATT-3' (forward), and
5'-GCTTTCTGGACAGAGAAATAGC-3' (reverse). To detect the levels of
control transcripts, we used ACT2 primers
5'-CTTCCTTGACTGCTTCTC-3' (forward) and
5'-TCATCGTCACCACCTTCA-3' (reverse).
Positional cloning of VAN3
The VAN3 locus was mapped between the RCI1B and nga151 markers on
chromosome 5 (Koizumi et al.,
2000). A number of new simple sequence length polymorphism (SSLP)
and CAPS markers between RCI1B and nga151 markers were developed from data
obtained from the TAIR database and Cereon Genomics (data not shown). In the
F2 generation produced from crosses between van3
heterozygotes (Ler) and Columbia, recombinants between the
VAN3 locus and the new SSLP and CAPS loci were scored. From 1034
chromosomes, 784 were analyzed and the VAN3 locus was identified in
the 89 kb region between the T31B5c and T22N19c markers. This corresponds to
two adjoining bacterial artificial chromosome (BAC) clones including 22
putative genes in the interval between the T31B5c and T22N19c markers
(Arabidopsis Genome Initiative). These were PCR-amplified from the
Ler strain and van3, and completely sequenced using the
BigDye Terminator Cycle Sequencing Kit on an ABI PRISM 370 Genetic Analyzer.
Among these putative genes, only the T31B5.120 (At5g13300) gene
contained a mutation in a putative exon. An 8.6 kb XbaI-SpeI
genomic DNA fragment that included the 1.1 kb upstream region of this gene and
the 1.1 kb downstream region (position 51627-60181 of T31B5 BAC) was cloned
into the vector pGreen 0179. The clone was introduced into Agrobacterium
tumefaciens strain C58 and transformed into plants carrying the
heterozygous van3 mutation (van3-1/VAN3) using the
floral dip method. After hygromycin selection, T2 seeds were
collected from individual T1 plants and T2 lines were
constructed. All T2 line seeds were grown with hygromycin and the
segregation of resistance was examined. In the T2 line, plants
presumed to carry single copies of T-DNA and a heterozygous van3
mutation segregated at ratios close to 15:1 (WT: van3 = 197:10,
2 0.250, <P<0.500). These results led us to
conclude that the VAN3 gene corresponds to T31B5.120.
Subcellular localization of VAN3
Full-length VAN3 cDNA was isolated by RTPCR from the A.
thaliana Columbia ecotype, and an XhoI/NcoI restriction
site was introduced at both ends. The fragment was translationally fused to
the N terminus of Venus yellow fluorescent protein. The chimeric gene was
subcloned under the control of the cauliflower mosaic virus 35S promoter and
the Nos terminator. 35S::ARA7GFP
(Ueda et al., 2001),
35S::ARA6GFP (Ueda et al.,
2001
), 35S::HDELGFP
(Takeuchi et al., 2000
),
35S::SYP31 (Takeuchi et al.,
2002
), 35S::VAMP727GFP, and 35S::SYP41GFP were used
as intracellular markers of early endosomes, late endosomes, ER, cis-Golgi,
early endosomes and TNG, respectively. Double transient expression of
35::VAN3Venus and of intracellular markers in the protoplasts of
cultured Arabidopsis cells were analyzed as described by Ueda et al.
(Ueda et al., 2001
).
Protoplasts from gnom mutant cells were prepared as described by
Geldner et al. (Geldner et al.,
2003
). Fluorescence was observed by confocal laser microscopy
(LSM510 META, Carl Zeiss).
ARFGAP assays
Myristoylated yeast ARF1p (myrARF1p) was purified from
Escherichia coli co-transfected with expression vectors for ARF1p and
yeast N-myristoyltransferase, as described previously
(Randazzo et al., 1994;
Randazzo et al., 1995
).
ARFGAP activity was determined by an in vitro assay that measured a
single round of GTP hydrolysis in recombinant myrARF1p
(Makler et al., 1995
;
Huber et al., 2001
;
Huber et al., 2002
).
MyrARFlp (5 µM) was first loaded with 5 µM
[
-32P]GTP in ARF-loading buffer [25 mM Hepes (pH 7.5), 1 mM
dithiothreitol, 2 mM ethylenediaminetetraacetic acid (EDTA), 1 mM
MgCl2, 10 mM ATP, 3 mM dimyristoyl phosphatidylcholine and 0.1%
sodium cholate]. The reaction was stopped with the addition of
MgCl2 at a final concentration of 3 mM, and a GAP assay was
performed at 30°C in 50 mM Hepes (pH 7.5), 4 mM MgCl2, 10 mM
ATP, and recombinant VAN3. The reaction was initiated with the addition of 1
µM [
-32P]GTP-loaded ARF1p, and stopped with the addition
of 250 mM EDTA. The medium was then placed on ice. Nucleotides were separated
by thin-layer chromatography on poly(ethyleneimine)-cellulose sheets developed
with 1 M LiCl and 1 M HCOOH. The sheets were dried, autoradiographed on an
imaging plate, and quantitatively analyzed with a BioImaging Analyzer (BAS
2500, Fuji Photo Film). In the absence of ARF1p, VAN3 showed no GTP hydrolysis
activity (data not shown).
Fat western blotting
Phospholipids (Sigma-Aldrich) were prepared in chloroform as stock
solutions at concentrations of 1 mg/ml. Solutions (10 µl) containing 0.5 or
1 µg of lipid were spotted individually onto nitrocellulose. The membrane
and lipids were dried at room temperature for 1 hour, and the nitrocellulose
was incubated with 3% (w/v) fatty-acid-free bovine serum albumin (isolated by
cold ethanol precipitation; SigmaAldrich A-6003) in Tris-buffered
salineTween 20 (TBST) solution [10 mM Tris (pH 8.0), 140 mM NaCl, 0.1%
(v/v) Tween 20] for 1 hour. The membrane was then placed in a solution
containing GST-tagged recombinant type V VAN3 fusion protein diluted in TBST
(0.5 µg/ml) and incubated at 4°C overnight with shaking. The
nitrocellulose was then washed with TBST three times for 10 minutes each and
incubated with anti-VAN3 antibody diluted 1:2000 in TBST for 1 hour at room
temperature. The membrane was then washed three times for 10 minutes each in
TBST at room temperature and incubated for 1 hour at room temperature with
goat anti-rabbit-IgG antibody conjugated with horseradish peroxidase, diluted
1:10000 in TBST. The nitrocellulose was washed again three times in TBST for
10 minutes each and incubated for 5 minutes in a 1:1 mixture of peroxidase
substrate and luminol/enhancer (Pierce) for subsequent chemiluminescence
detection. The nitrocellulose was exposed to Hyperfilm ECL (Amersham Pharmacia
Biotech) for 0.5-1 minute. The anti-VAN3 antibody was raised against a
synthetic peptide encoded between the BAR and PH domains, EKMQEYKRQVDRESR,
injected into a rabbit (Sawady Technology, Tokyo, Japan).
Yeast two-hybrid analysis
A two-hybrid analysis was performed using the Matchmaker Two-Hybrid System
3 (Clonetech); pGADT7 was used for GAL4 AD and pGBKT7 was used for GAL4
DNA-BD. A standard complete yeast extract-peptone-dextrose medium was used for
cell growth, and synthetic dextrose medium was used as the selective medium to
which tryptophan, leucine, adenine and histidine were added as needed to final
concentrations of 200 mg/l, 1 g/l, 200 mg/l and 200 mg/l, respectively.
Protein-protein interactions were detected by yeast (strain AH109) viability
on agar plates without adenine or histidine. Full-length VAN3 cDNA
and seven types of truncated VAN3 cDNAs
(Fig. 5D) were amplified using
PCR, confirmed by sequencing, and cloned into pGADT7 and/or pGBKT7, as shown
in Fig. 5D.
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Results |
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About half the emb30-7 seedlings produced fused cotyledons similar
to those of the pin1-3 mutant, and the emb30-7 cotyledons
had irregularly concentrated vascular tissues
(Fig. 1G)
(Koizumi et al., 2000). In the
van3 emb30-7 double mutant, about half the seedlings produced fused
cotyledons that contained a single midvein and fragmented lateral veins
(Fig. 1H). Lateral veins of the
van3 emb30-7 cotyledons were more fragmented than those of
emb30-7, and no concentrated vascular tissues were observed. The
architecture of the venation was similar to that of the van3 mutant.
Rosette leaves of the emb30-7 mutant have concentrated vascular
tissues with an increased number of trachery elements (TEs). This phenotype
was also observed in rosette leaves treated with an auxin transport inhibitor
(Fig. 1I,
Fig. 2A-D). The van3
emb30-7 double mutant produced rosette leaves similar to those of the
van3 mutant (Fig.
1D,J), suggesting that the concentrated vascular pattern induced
in the emb30 mutant is suppressed by the van3 mutation.
|
VAN3 and the polar auxin transport system do not act in the same pathway in vascular pattern formation
To further investigate the relationship between VAN3 function and polar
auxin transport in vein pattern formation, we treated the van3 plants with the
auxin transport inhibitor, N-1-naphthylphthalamic acid (NPA). In the
first-node leaves of wild-type plants, vascular differentiation was enhanced
along the entire lamina margin, and the marginal vascular tissues were
connected to the central vascular tissues with an increased number of
non-branched vascular tissues (Fig.
2A-D). These effects depended on NPA concentration
(Fig. 2A-D) (Mattsson et al., 1999;
Mattsson et al., 2003
;
Sieburth, 1999
). In the
first-node leaves of the van3 mutant grown with NPA, vascular formation was
enhanced and the vasculature was fragmented. However, the overall pattern was
the same as that of wild-type plants treated with NPA
(Fig. 2E-H, most obvious in F).
Similar effects were observed in the van3 leaves treated with
2,3,5-triiodobenzoic acid (TIBA) (data not shown). These results suggest that
the effects of the auxin transport inhibitor and the VAN3 mutation are
additive in the formation of the venation pattern in the Arabidopsis leaf.
Thereafter, VAN3 probably acts independently of polar auxin transport in
vascular pattern formation, although we cannot exclude the possibility that
VAN3 functions in the polar auxin transport system.
Brefeldin A (BFA) prevents the polarized transport of PIN1 protein to the
plasma membrane by inhibiting the activation of GNOM ARFGEF
(Geldner et al., 2001). In
wild-type plants grown with BFA, the size of the first-node leaf was reduced,
and the vein pattern was simplified in a BFA-concentration-dependent manner
(Fig. 2I-L). TEs were also
excessively differentiated in the upper part of the leaf margin, but not in
the central region treated with BFA (Fig.
2K,L). After treatment with 20 µM BFA, tertiary veins
occasionally developed discontinuously
(Fig. 2L). In the van3
mutant treated with BFA, secondary and tertiary veins were missing and excess
vascular formation seemed to be suppressed
(Fig. 2M-P).
Minor veins are discontinuously formed with auxin accumulation, and the auxin response is reduced in the van3 mutant
To understand the effect of the VAN3 mutation on auxin
distribution, we examined the expression pattern of the DR5::GUS
construct as a marker of auxin accumulation. In the first-node leaves of
wild-type seedlings, GUS staining was observed as a dotted pattern in the
hydathodes and developing minor veins (Fig.
3A-E). Each dot represented a cell or a few cells, and their
shapes varied from round and oval to elongated
(Fig. 3E). In the van3
mutant, the GUS staining pattern in developing leaves was similar to that of
the wild-type (Fig. 3F-J).
However, the number of GUS-expressing spots was significantly reduced [wild
type: 46.7±3.8/mm2 of leaf (n=24); van3:
5.8±2.3/mm2 of leaf (n=18); values represent means
± s.e.m.]. These results suggest a reduction in the number of
auxin-accumulating cells and/or a reduction in auxin sensitivity.
|
VAN3 encodes an ARFGAP
To gain further insight into the molecular nature of the VAN3
gene, we isolated it using a positional cloning method. The VAN3
locus was mapped to chromosome 5, in the 89 kb region between molecular
markers T31B5c and T22N19c (Fig.
4A). We sequenced the genomic DNA of the Ler strain and
the van3-1 mutant spanning 22 annotated open reading frames (ORFs)
identified in this region, and found a point mutation only in ORF T31B5.120
(At5g13300). An 8.6 kb wild-type genomic fragment that includes 1.1
kb upstream from the putative transcription start site and 1.1 kb downstream
from the putative transcription termination site of this ORF complemented the
discontinuous vascular phenotype of the van3 mutant (detailed in
Materials and methods). We identified ORF At5g13300 as the
VAN3 gene.
|
The BAR domain is expected to mediate proteinprotein interactions
(Navarro et al., 1997),
whereas the PH domain is known to mediate protein-lipid interactions
(Harlan et al., 1994
). The
ARFGAP domain contains a consensus zinc finger motif and functions in
the stimulation of GTP hydrolysis
(Cukierman et al., 1995
). ANK
repeats are involved in protein-protein interactions and associate to form a
higher-order structure. A homology search using the DNA Data Bank of Japan
revealed significant sequence similarity between the putative VAN3 protein and
the human ARFGAPs with coiled-coil domains,
ANK repeats and PH domains, ACAP1 (32% identical) and
ACAP2 (33% identical) (Jackson et al.,
2000
). VAN3 has the same domain structure as ACAP1 and ACAP2
(Fig. 4B). The ARFGAP
domain is highly conserved between VAN3 and the ACAPs
(Fig. 4C), suggesting that the
VAN3 protein may function as an ARFGAP. Many genes showing significant
sequence similarities to the VAN3 gene were found in Arabidopsis
thaliana, Oryza sativa, Anopheles gambiae, Mus musculus, Fugu rubripes,
Drosophila melanogaster, Dictyostelium discoideum and Caenorhabditis
elegans. In the Arabidopsis genome, three genes, At5g61980,
At1g10870 and At1g60860, show significant sequence similarities
to the VAN3 gene. The deduced amino acid sequences of these homologs
are 47-62% identical to that of VAN3, and the domain structures of these
proteins are the same as that of VAN3 (Fig.
4B). In particular, sequences of the BAR domains are strongly
conserved between VAN3 and these predicted proteins
(Fig. 4C).
VAN3 expression is induced by auxin and is self-regulated
Next, we examined the effects of auxin on VAN3 expression in the
cotyledons. We used RTPCR because VAN3 and VAN3
homologs have high sequence similarity and probes specific to the 5'-
and 3'-UTR regions of these genes did not give clear signals on northern
analysis. The intensity of the PCR band corresponding to the VAN3
gene increased significantly with auxin treatment of wild-type cotyledons
(Fig. 4D). In contrast,
VAN3 gene expression level seems to be reduced by auxin treatment in
the van3 mutant (Fig.
4D). This indicates that VAN3 expression is upregulated
by auxin, and its auxin-dependent induction may be positively regulated by
VAN3 itself.
VAN3 protein has ARFGAP activity
We examined the ARFGAP activity of the VAN3 protein using an in
vitro ARFGAP assay. A glutathione S-transferase (GST) fusion protein
and type V VAN3 protein that lacked the BAR domain
(Fig. 5D) were expressed in
E. coli and purified. The hydrolysis of GTP on recombinant yeast
Arf1p was measured with or without recombinant VAN3 protein. VAN3 protein
induced GTP hydrolysis on yeast Arf1p in a concentration- and
incubation-time-dependent manner (Fig.
5A,B). These results indicate that VAN3 protein functions as an
ARFGAP that regulates ARF cycling between the active ARFGTP form
and the inactive ARFguanosine diphosphate (GDP) form in the vesicle
transport pathway.
Recombinant VAN3 protein binds to PI-4-P
The PH domain is found in a wide variety of signaling proteins and binds to
phosphoinositides (Harlan et al.,
1994). Furthermore, human ACAPs, which have significant sequence
similarity to VAN3, are known to bind the lipid phosphatidylinositol
4,5-bisphosphate [PtdIns(4,5)P2]. To determine whether the
VAN3 protein binds to a lipid, we purified recombinant type V VAN3 protein
(Fig. 5D) using affinity
chromatography (data not shown). The ability of the recombinant VAN3 protein
to bind different phospholipids was examined using fat western blotting,
developed by Stevenson et al. (Stevenson
et al., 1998
). Fig.
5C shows the results of fat western blotting probed with
recombinant VAN3 protein. VAN3 bound to phosphatidylinositol (PtdIns),
phosphatidylinositol 4-monophosphate (PtdIns4P) and
PtdIns(4,5)P2, but not to phosphatidic acid (PA),
phosphatidylcholine (PC), or phosphatidylethanolamine (PE). VAN3 bound to
PtdIns4P with higher affinity than to PtdIns or
PtdIns(4,5)P2. Recombinant GST protein did not bind to any
of the lipids tested (data not shown).
VAN3 forms a homodimer
The BAR domain is known to mediate protein-protein interactions
(Navarro et al., 1997). To
determine whether the VAN3 protein forms a homodimer through the BAR domain,
we performed yeast two-hybrid analyses. Eight types of VAN3 cDNA
fragments were translationally fused to the GAL4 activation domain (AD) and/or
GAL4 DNA-binding domain (DNA-BD) (Fig.
5D). Types I-VII were composed of the BAR domain (amino acids
17-221); the PH domain (amino acids 221-441); the GAP and C-terminal domains
(amino acids 439-827); the BAR and PH domains (amino acids 17-431); the PH-C
terminal domain (amino acids 221-827); the GAP domain (439-706); and the
truncated BAR domain (1-85), respectively. As shown in
Fig. 5D, when the intact BAR
domain was used in the yeast two-hybrid analysis, protein-protein interactions
were detected. This indicates that the BAR domain is required and is
sufficient for the formation of the VAN3 homodimer.
VAN3 localizes to a subpopulation of the TGN
ARFGAP is a key component in vesicle formation for membrane
transport, so VAN3 protein is expected to locate to an organelle involved in
the secretory system. Furthermore, human ACAPs are located in endosomes. GNOM
ARFGEF, which is involved in vascular formation, is also considered to
function in endosomes. However, VAN3 and ACAPs have different binding
affinities for lipids, and VAN3 and GNOM seem to act differently in vascular
pattern formation and have different effects on auxin signaling. Therefore, we
identified the subcellular location of VAN3 to better understand its function.
VENUS (Nagai et al.,
2002)-tagged VAN3 and green fluorescent protein
(GFP)-tagged subcellular marker genes were co-introduced into
Arabidopsis suspension-cultured cells
(Fig. 6A), and their
subcellular locations were observed with a confocal laser scanning microscope.
The location of VAN3Venus did not overlap with that of the endoplasmic
reticulum (ER) marker HDELGFP
(Takeuchi et al., 2000
) or the
Golgi body marker SYP31GFP that marks the cis faces of Golgi
stacks (Takeuchi et al.,
2002
). Furthermore, it also did not colocalize with the endosome
marker, ARA6GFP (Fig.
6B-D), ARA7GFP and Vamp727GFP (data not shown)
(Ueda et al., 2001
;
Ueda et al., 2004
). Nor did it
colocalize with the lipophilic endocytic tracer FM4-64 (data not shown). We
also examined the localization of VAN3Venus in gnom
suspension-cultured cells because cultured gnom cells contain
abnormally enlarged endosomes that mediate the endosomeplasma membrane
recycling of PIN1 (Geldner et al.,
2003
). The structure of the organelle in which the
VAN3Venus protein was located was completely different from the
enlarged organelle that was stained with the endosome marker ARA7GFP in
cultured gnom cells (Ueda et al.,
2001
; Geldner et al.,
2003
) (Fig. 6E,F).
This indicates that VAN3 is not located in the endosomes in which GNOM
functions. However, VAN3Venus-positive compartments overlapped with
those of the TGN marker, SYP41GFP
(Bassham et al., 2000
;
Uemura et al., 2004
)
(Fig. 6G-I). These results
indicate that the VAN3 protein is located in the TGN. Interestingly, not all
the TGN was positive for VAN3Venus. This unique localization pattern
suggests that the TGN is not uniform, but is functionally differentiated in
plant cells.
|
![]() |
Discussion |
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---|
Recombinant VAN3 protein showed ARFGTPase-stimulating activity on
yeast Arf1p, demonstrating that VAN3 can function as an ARFGAP
(Fig. 5A,B). The kind of ARF(s)
activity that is regulated by VAN3 has yet to be identified. The six mammalian
ARFs have been grouped into three classes: class I (ARF13), class II
(ARF4 and ARF5) and the most distinctive group, class III (ARF6)
(Moss and Vaughan, 1995;
Jürgens and Geldner,
2002
). ACAPs regulate ARF6-dependent membrane trafficking in
animals (Jackson et al.,
2000
). In the Arabidopsis genome, six of nine putative
ARF genes encode class I ARF proteins with 98100% amino acid identity.
The other three putative Arabidopsis ARFs diverge from animal and
yeast ARFs and are difficult to classify into known groups. No clear
evolutionarily conserved homolog of ARF6 is found in the Arabidopsis
genome. Therefore, it would be interesting to determine which
Arabidopsis ARF is the real substrate of VAN3. These results may cast
new light on the function of ARFGAPs. It is reported that the GAP
activity of AZAP-type ARFGAPs is controlled by phospholipids
(Brown et al., 1998
;
Jackson et al., 2000
;
Kam et al., 2000
). Each AZAP
subfamily has a distinct phosphoinositide dependence. The GAP activity of
ASAPs appears to be specifically stimulated by PA and
PtdIns(4,5)P2. ARAPs are regulated by
PtdIns(3,4,5)P3, and ACAPs by
PtdIns(3,5)P2 and PtdIns(4,5)P2
(Randazzo and Hirsch, 2004
).
In contrast, whereas VAN3 shows an obvious affinity for PtdIns4P and
weak binding to PtdIns and PtdIns(4,5)P2, it shows no
binding to phosphatidic acid (PA) (Fig.
5C). Although we have not yet determined the phospholipid
dependency of the ARFGAP activity of VAN3, this finding suggests
differences in the phospholipid-dependent regulatory mechanism of
ARFGAP activity in animals and plants. These different lipid
dependencies may also be responsible for the subcellular localization of
AZAP-type ARFGAPs. The PH or Phox homology (PX) domains can contribute
to targeting a protein to a specific membrane compartment
(Peter et al., 2004
). Recent
results suggest that the BAR domain is responsible for dimerization, membrane
binding and a curvature-sensing module
(Lee and Schekman, 2004
;
Peter et al., 2004
).
Interestingly, there are also reports that the BAR domain can form
heterodimers in vivo and in vitro (Navarro
et al., 1997
; Wigge et al.,
1997
; Colwii et al., 1999). These results remind us that VAN3 may
form not only homodimers, but also heterodimers with its homologs to
cooperatively regulate the transportation of a cargo protein that regulates
vascular continuity.
In animals, an ACAP member, ARF6GAP, localized to focal adhesions
and recycling endosomes (Jackson et al.,
2000). However, the VAN3 protein does not appear to function in
endosomal trafficking because VAN3 co-localizes with the TGN marker SYP41
(Fig. 6GI) and not with
the endosomal marker ARA6GFP (Fig.
6D) or the endocytic tracer FM (data not shown). This suggests
that VAN3 functions in membrane trafficking at the TGN. This result was
supported with transgenic plants, in which VAN3::VAN3-VENUS
complemented the van3 mutant phenotypes (data not shown). Different
phospholipid dependencies may contribute to the different subcellular
locations of VAN3 and ACAPs. These results imply that VAN3 is a plant-specific
ACAP-type ARFGAP that functions in transporting cargo proteins involved
in distinct cellular events in plants.
Interestingly, VAN3 did not co-localize with all the cellular structures stained with the TGN marker SYP41 (Fig. 6I). This suggests the existence of functionally different TGNs. It is generally believed that the TGN has a uniform function as a sorting center where trafficking proteins are directed to the plasma membrane, endosomes and prevacuolar compartments. However, our observations suggest that the TGN may be differentiated, and therefore that the final target of the cargos may already be selected before they are delivered to the TGN (Fig. 7). This idea is consistent with the specific and restricted effects of VAN3 on vascular continuity. VAN3 homologs and/or other ARFGAPs might be candidate regulators of alternative, or more general, TGN transporting pathways.
|
Expression of the DR5::GUS construct was not induced by the application of auxin to van3 leaves (Fig. 3K-V). This implies that the VAN3 protein functions in auxin signaling. Because VAN3 expression was upregulated by the application of auxin, there may be positive feedback between VAN3 expression and auxin signaling. Furthermore, the enhanced mp phenotype (Fig. 1L) and reduced MP expression level in the van3 mutants (data not shown) suggest that VAN3 may regulate auxin signaling upstream from MP.
The gnom mutants show a concentrated venation pattern
(Fig. 1G). GNOM
encodes an ARFGEF that is believed to regulate the subcellular
localization of PIN1 and to contribute to polar auxin transport
(Geldner et al., 2003).
Therefore, the concentrated venation in the gnom mutants may result
from highly accumulated auxin in the leaf resulting from the aberrant
localization of PIN1. BFA, which represses the GNOM function, induced
concentrated venation, especially at the leaf margins, as occurs in the
gnom mutants (Fig.
2IL). The van3 mutation partially suppressed the
concentrated venation pattern in both the gnom and BFA-treated leaves
(Fig. 1I,J,
Fig. 2I-P). This observation
may be explained as follows. The reduced auxin signaling caused by the
van3 mutation may suppress the overproduction of vascular tissues
caused by excess auxin accumulation in gnom leaves or BFA-treated
leaves. How does VAN3 regulate auxin signaling? VAN3 may be responsible for
the transportation of the components of intracellular auxin signaling, such as
receptors or signal transduction intermediates, from the TGN. Consequently,
the loss of function of VAN3 results in reduced auxin sensitivity.
DR5::GUS expression is often used as a marker of auxin accumulation
(Sabatini et al., 1999
;
Friml et al., 2003
; Mattson et
al., 2003), but more correctly shows auxin reactivity. The reduced sensitivity
of auxin in the van3 mutant is consistent with this hypothesis.
However, we cannot exclude the possibility that VAN3 is involved in the
trafficking of a secretory protein(s) that functions in the intercellular
signaling necessary for the continuous formation of procambial cells. Xylogen
is an arabinogalactan protein that is secreted from procambium cells to
neighboring cells, inducing them to differentiate into vascular tissue, and
its mutants show a disconnected vascular pattern
(Motose et al., 2004
).
Therefore, xylogen might be a good candidate cargo protein of VAN3-related
vesicles.
Discontinuous venation pattern in van3 mutants
We must distinguish between the discontinuous formation of procambium and
that of mature xylem cells. Although the maturation process of a xylem strand
is known to occur sometimes discontinuously from the continuously formed
procambium in leaves (Esau,
1965; Aloni, 2001
;
Pyo et al., 2004
), it is still
unclear whether the procambium is formed discontinuously in normal leaves. In
the van3 mutant, fragmented venation is caused by the discontinuous
formation of the procambium (Koizumi et
al., 2000
). What is the mechanism underlying this discontinuity?
Aloni et al. (Aloni et al.,
2003
) showed that during the development of the leaf primordium,
there are orderly shifts in the sites of DR5::GUS expression. This
progress from the elongation tip, continues downward along the expanding blade
margins, and ends at the central regions of the lamina. In the lamina, as we
demonstrated here, DR5::GUS expression occurs in small round, oval or
elongated cells that are distributed separately from each other, and in some
cases are attached to form a short column
(Fig. 3E). These
DR5::GUS-expressing cells appear to be procambial initials or
procambial cells. In more mature leaves, DR5::GUS expression is
observed in elongated procambial cells in veins
(Aloni et al., 2003
). These
results suggest the following scenario for the continuous formation of
procambial strands. The precursors of procambial cells are formed separately
with a high level of auxin, and then differentiate into procambial cells. The
procambial cells then induce neighboring parenchyma cells to differentiate
into procambial cells. The resultant short columns of procambial cells become
attached and form a continuous strand of procambial cells.
DR5::GUS-expressing cells in the van3 leaves were
distributed at a lower density than those in wild-type leaves
(Fig. 3A-J). In van3
leaves, the application of auxin did not enhance DR5::GUS expression
(Fig. 3K-V). This suggests that
the van3 mutation reduces auxin sensitivity in leaves. Auxin
signaling mutants, such as axr6 and mp, also show
discontinuous venation patterns (Berleth
and Jürgens, 1993
; Hobbie
et al., 2000
). Because auxin induces the differentiation of
procambial cells/procambial initials from parenchymal cells
(Fukuda, 2004
), these results
imply that the reduced sensitivity of auxin in van3 leaves causes a
decrease in the number of procambial initials that are differentiated from
parenchymal cells. As a result, the increased distance between each procambial
initial in the van3 leaves may prevent them from connecting to one
another, thus forming a discontinuous vascular network.
The vesicle transport system appears to play an important role in the development and environmental responses of plants. In this study, we have shown for the first time a novel AZAP-type ARFGAP that functions in pattern formation in the plant vascular network. The location of the protein in the TGN and its role in auxin signaling provide new insight into the vesicular transport involved in vascular pattern formation. Identification of the components of the VAN3-related vesicle transport system, especially the cargo protein, is the next crucial problem to be solved.
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ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
These authors contributed equally to this work
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