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First published online 30 August 2006
doi: 10.1242/dev.02554
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1 División de Genética and Instituto de Bioingeniería,
Universidad Miguel Hernández, Campus de Elche, 03202 Elche, Alicante,
Spain.
2 Instituto Nacional de Investigación y Tecnología Agraria y
Alimentaria, Departamento de Biotecnología, Carretera de la
Coruña Km. 7, 28040 Madrid, Spain.
3 División de Genética, Universidad Miguel Hernández,
Campus de San Juan, 03550 Alicante, Spain.
Author for correspondence (e-mail:
jlmicol{at}umh.es)
Accepted 24 July 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Arabidopsis, TIP120, CAND1, Venation pattern formation, Natural variation
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Meinhardt, 1984;
Mitchison, 1980;
Mitchison, 1981), the
mechanism by which such structures are built is still not fully understood at
the genetic level. Considerable experimental evidence suggests that indole
acetic acid (IAA, the main auxin) transport is involved in the patterning of
plant vasculature. The spatial distribution of IAA correlates well with the
sites of vascular differentiation in shoot apices and young leaf primordia
(Avsian-Kretchmer et al.,
2002), and the inhibition of auxin polar transport causes a
variety of developmental defects in leaves, including an abnormal vascular
network (Mattsson et al.,
1999; Sieburth,
1999). These observations support the so-called `canalization
hypothesis', which states that the patterning of plant vascular systems
depends on a positive feedback loop acting on the transport of auxin
(Sachs, 1991a;
Sachs, 1991b).
Mutants with aberrant venation patterns in leaves or cotyledons have been
isolated using Arabidopsis thaliana as a model
(Carland and McHale, 1996;
Carland et al., 1999;
Deyholos et al., 2000;
Koizumi et al., 2000;
Steynen and Schultz, 2003).
Some of the corresponding genes have already been cloned, including
COTYLEDON VASCULAR PATTERN1 (CVP1), which encodes a sterol
methyltransferase involved in the biosynthesis of sterol and brassinosteroid
compounds (Carland et al.,
2002), and CVP2, which encodes an inositol polyphosphate
5' phosphatase and confirms a role for inositol 1,4,5-trisphosphate
(IP3)-mediated signal transduction in vein ontogeny
(Carland and Nelson,
2004).
In a search for naturally occurring variations of the leaf venation pattern
in Arabidopsis, we found that the spontaneous hemivenata-1
(hve-1) allele reduces venation complexity in leaves and cotyledons
(Candela et al., 1999). Here,
we analyze the developmental effects of hve-1 and two other recessive
alleles of HVE, which we positionally cloned and found to encode a
CAND1 protein. Our results demonstrate a role for ubiquitin-mediated auxin
signaling in Arabidopsis vein patterning.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and the
pin1cay (N324) and axr1-12
(Leyser et al., 1993) mutants.
The pin1cay mutant was isolated by G. Röbbelen and
later named cay by Serrano-Cartagena et al.
(Serrano-Cartagena et al.,
1999). Allelism of cay and pin1-1 was suggested
by their similar phenotypes and confirmed in complementation crosses. We found
that pin1cay is a null allele carrying a G-to-A
substitution that produces a truncated protein lacking 489 amino acids.
lop1-65 and cvp2-1
(Carland and McHale, 1996;
Carland et al., 1999) were
kindly provided by Francine Carland; mpT370
(Przemeck et al., 1996) by
Thomas Berleth; and the ATHB-8-GUS
(Baima et al., 1995),
CAND1-GUS (Chuang et al.,
2004) and DR5-GUS
(Ulmasov et al., 1997)
transgenic lines by Simona Baima, William Gray and Thomas Guilfoyle,
respectively. We obtained
mpT370/mpT370;hve-3/hve-3 and
mpT370/mpT370;HVE/- seedlings, all of which
were lethal and failed to develop roots and produce leaves. In our hands, the
induction of adventitious roots by hypocotyl basal bisection
(Przemeck et al., 1996) led to
an extremely low survival rate, which made not possible the isolation of a
significant number of mpT370/mpT370 rosettes
for morphometry of leaf vascular patterns.
Plants were grown on agar medium, at 20±1°C and 60-70% relative
humidity and with 7000 lux of continuous fluorescent light
(Ponce et al., 1998). Crosses
and allelism tests were performed as described previously
(Berná et al., 1999).
ATHB-8-GUS and CAND1-GUS transgenic seeds were
sterilized and kept in water at 4°C in the dark for 4 days to ensure
synchronized germination.
Physiological assays
To study the effects of the synthetic auxin 2,4-dichlorophenoxyacetic acid
(2,4-D), seeds were sown on non-supplemented agar medium and the seedlings
were transferred to supplemented media 4 days later. Root lengths were
measured 4 days after the transfer
(Lincoln et al., 1990). The
gravitropic response of roots was studied on Petri dishes containing 1.5% agar
medium, which were kept vertically in a Conviron TC30 growth chamber for 8
days before being rotated 135° and observed 4 days later.
Characterization of vascular patterns
Histological sections were obtained as described previously
(Serrano-Cartagena et al.,
2000). For venation pattern visualization and micrography, lateral
organs were cleared as described previously
(Candela et al., 1999).
Histochemical visualization of ß-glucuronidase (GUS) activity was
performed as described previously
(Donnelly et al., 1999). We
combined the null hve-3 allele with null alleles of other genes, in
most cases in the same genetic background (Col-0), to obtain double mutants,
the venation of which was compared with those of their siblings. Data
corresponding to leaf lamina area, venation density and number of branching
points/mm2 were collected as described previously
(Candela et al., 1999) and
analyzed using the SPSS for Windows version 10.0.6 statistical software
package (SPSS). Kolmogorov-Smirnov tests with Lilliefors correction were
applied to assess the normality of our data. When the data fitted a normal
distribution, t tests were used to compare mean values. When
normality was not accepted, the non-parametric Mann-Whitney U test
was used instead.
Molecular characterization of the HVE gene
Linkage analysis (Ponce et al.,
1999) was used for the positional cloning of HVE. We have
developed several novel molecular markers that are used for high-resolution
mapping of the HVE gene (Table
1). Two RFLP markers that flank nga1145
(Lister and Dean, 1993) were
converted to PCR-based CAPS markers
(Konieczny and Ausubel, 1993):
MnlI restriction of the ve012 PCR product rendered a single band in
Ei-5 (339 bp) and two in Ws-2 (300 and 39 bp), whereas EcoRI
restriction of the m246 PCR product rendered a 1.6 kb band in Ws-2 and two 0.8
kb bands in Ei-5. In addition, we found a polymorphic (AT)n
microsatellite, which we named T17M13b, based on the available sequence of the
T17M13 BAC clone. Sequencing the candidate region allowed us to identify five
novel single nucleotide polymorphisms (SNP), named T8K22-4, P450, T8K22-7,
T8K22-1 and E2, which were used for mapping the HVE gene. The
synthetic oligonucleotides were purchased from Sigma-Genosys (UK). Genomic DNA
was extracted, PCR-amplified and sequenced as described previously
(Pérez-Pérez et al.,
2004).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This
spontaneous allele strongly reduced venation pattern complexity in vegetative
leaves and cotyledons. We positionally cloned HVE (Fig. 1A) using an F2 mapping population derived from a Ws-2 x hve-1/hve-1 (in an Ei-5 background) cross. After finding linkage between hve-1 and the nga1145 marker, near the upper telomere of chromosome 2, we developed eight molecular markers (Fig. 1A) that were used to screen for recombinants in 1545 F2 plants. Informative recombinants narrowed down the candidate interval to a 61 kb genomic region including 14 annotated genes (Fig. 1A).
To identify HVE among the candidate genes, we studied publicly available T-DNA and transposon alleles. Forty-nine lines carrying insertions within or close to 11 candidate genes (At2g02550-At2g02590, At2g02610-At2g02640, At2g02660 and At2g02670) were searched for phenotypic traits reminiscent of those of hve-1/hve-1 plants. Two of them, N599479 and N610969, carried T-DNA insertions in the At2g02560 gene and displayed a simple leaf vasculature and a bushy inflorescence. Complementation tests confirmed that both lines carried alleles of hve-1. The presence and position of their T-DNA insertions was confirmed by PCR and by sequencing (Table 1). These insertions lie at nucleotides 1195 (N599479) and 5764 (N610969) of the At2g02560 transcription unit (numbering from the ATG; Fig. 1B), as described at http://signal.salk.edu. We named the alleles carried by N599479 and N610969 as hve-2 and hve-3, respectively.
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The HVE gene encodes a CAND1 (TIP120) protein
HVE encodes a predicted protein of 1217 amino acids (see Fig. S1
in the supplementary material) and a molecular weight of 134.6 kDa
(http://mips.gsf.de/cgi-bin/proj/thal/search_gene?code=At2g02560),
closely related to the mammalian TATA-binding protein-interacting protein 120
(TIP120A) (Yogosawa et al.,
1996). TIP120, also known as CAND1 (Cullin-Associated and
Neddylation-Dissociated) (Liu et al.,
2002; Zheng et al.,
2002), regulates the formation of the SCF complexes of ubiquitin
E3 ligases. Although two paralogs have been described in the rat and human
genomes (Aoki et al., 1999),
HVE/CAND1 is a single-copy gene in Arabidopsis.
Since mammalian CUL1 is known to interact with CAND1
(Liu et al., 2002;
Zheng et al., 2002;
Hwang et al., 2003;
Oshikawa et al., 2003), we
generated a recombinant Glutathione-S-transferase-HVE (GST-HVE)
protein that was used in pull-down experiments. CUL1 was translated in vitro
in rabbit reticulocyte lysate in the presence of 35S-Met and then
incubated with GST or GST-HVE. As shown by SDS-PAGE gel autoradiography (see
Fig. S2A in the supplementary material), GST-HVE, but not GST, was able to
interact with CUL1, suggesting that, as their mammalian counterparts, the
HVE/CAND1 protein of Arabidopsis regulates the activity of the SCF
complexes by binding CUL1. While this work was being carried out, other groups
demonstrated that CAND1 and CUL1 physically interact
(Chuang et al., 2004;
Feng et al., 2004).
The morphological phenotype of hve mutants
The hve mutants displayed vegetative leaves of slightly reduced
size (Fig. 2A-D), and a bushy,
extremely branched inflorescence (Fig.
2E-G). Fertility was reduced in hve-1/hve-1 plants and
was very low in hve-2/hve-2 and hve-3/hve-3, which produced
less than 10 seeds per plant. The inflorescence of the hve-1 mutant
was of normal size, whereas those of hve-2 and hve-3 were
dwarf (Fig. 2F,G). Flowering
time and senescence were delayed in all hve mutants. Together with
the molecular characterization of the alleles, these results suggest that
hve-1 is a hypomorphic allele, whereas hve-2 and
hve-3 are null.
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To determine the extent to which HVE is required for the development of the vascular pattern, we cleared cotyledons, vegetative and cauline leaves, sepals and petals of the hve mutants and the Col-0 wild-type accession, which is the genetic background of hve-2 and hve-3 (Fig. 3). The venation patterns of the three hve mutants were similar to one another and clearly distinct from that of Col-0. The cotyledons contained three or four areoles (regions of the lamina completely bordered by veins) in the wild type (Fig. 3A), but only two in the hve mutants, often incompletely closed (Fig. 3H,O,V). The reduction in vein numbers was also apparent in the first two rosette leaves, which had fewer secondary veins than the wild type. No quaternary veins and only a few tertiary veins, which ended blindly within the areoles, were present in mutant leaves, the intramarginal vein of which was occasionally interrupted (Fig. 3B,I,P,W). Similar observations were made in vegetative leaves of the third (Fig. 3C,J,Q,X) and seventh (Fig. 3D,K,R,Y) nodes. The lower structural complexity of the mutant venation patterns was in all cases a consequence of a reduced number of secondary and tertiary veins and the absence or shortening of higher-order veins (quaternary and higher). Cauline leaves, by contrast, were smaller in the hve-2 and hve-3 mutants than in Col-0, but did not show an obvious reduction in vascular density (Fig. 3E,L,S,Z). In wild-type sepals, two secondary veins diverged from the apical end of the primary vein to form two arches (Fig. 3F), whereas a single primary vein and two incomplete secondary veins were observed in the mutants (Fig. 3M,T,AA). Wild-type petals also showed a more elaborate venation pattern than the corresponding organs of the hve mutants (Fig. 3G,N,U,AB).
As seen in paradermal and transverse sections (see Fig. S3 in the supplementary material), the primary vein was thinner in hve-3/hve-3 leaves than in the wild type, but the secondary veins looked normal. The structure of bundle sheath cells, sieve tubes and tracheary elements was also normal in the mutant. No differences with the wild type were found in the cell shape and size of the mutant epidermis, palisade mesophyll and spongy mesophyll. However, the spongy mesophyll had larger air spaces and the palisade mesophyll was partially disorganized in the mutant. A midrib was clearly distinguishable abaxial to the midvein in the wild type, but seemed to be absent in hve-3.
The HVE promoter drives GUS expression in leaf veins
The various organs affected in hve mutants suggested that the
HVE/CAND1 gene is required throughout the life cycle, from
embryogenesis to floral development. To verify this, Col-0 RNA was extracted
from assorted organs and RT-PCR amplified, and HVE was found to be
expressed in every organ studied (see Fig. S2B in the supplementary material).
To further define the postembryonic spatial expression pattern of
HVE/CAND1, we characterized the wild-type expression pattern
(Fig. 4) of a CAND1
promoter-GUS fusion (PETA2-GUS) that includes 2.7 kb of
upstream sequence (Chuang et al.,
2004). Consistent with the pleiotropic phenotype of
loss-of-function hve mutants and with our semi-quantitative RT-PCR
results, the gene was found almost ubiquitously expressed in aerial and
underground organs of 10-day-old plants
(Fig. 4A,B). At this stage, the
gene was widely expressed in rosette leaves, at the highest level in the
vasculature (Fig. 4A). By
contrast, GUS staining was mostly confined to the leaf vasculature in
21-day-old plants, indicating that HVE expression changes dynamically
throughout leaf development (Fig.
4C). For cotyledons and vegetative leaves, the expression in
mesophyll cells was apparently more intense at the beginning of leaf
expansion, disappearing progressively as the leaves grew
(Fig. 4D,E-H,P). Closer
inspection revealed that the gene was expressed before xylem differentiation
in developing veins at the basal actively dividing region of rosette leaves
(Fig. 4R) and, after the
differentiation of tracheary elements, in the living cells of the vascular
bundles (Fig. 4Q). The strong
association of HVE expression and the vascular tissues is consistent
with its role in vascular development (Fig.
4E-K,O-P) and with the defective venation of hve mutants.
By contrast, the reporter was expressed throughout young cauline leaves before
being relegated to their margins, at which time no expression could be
detected in the veins (Fig.
4I). This observation correlates with the absence of vascular
mutant phenotype in the cauline leaves of hve mutants. Similarly, the
expression of the gene changed dynamically in developing flowers, being
general in immature flowers (Fig.
4M) and restricted to the distal tip of sepals, the veins of
petals, and the vasculature, filament and anthers of stamens in mature flowers
(Fig. 4J,K). Expression was
also detected in other organs, including siliques
(Fig. 4L,N), stems
(Fig. 4L), the root meristem
and vascular cylinder (Fig.
4B).
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).
Consistent with a positive role in vascular differentiation, it is induced
during revascularization of injured stems and after the exogenous application
of auxin (Baima et al., 1995),
and its overexpression causes the premature differentiation of procambial
cells into primary xylem (Baima et al.,
2001). We have used an ATHB-8-GUS reporter
(Baima et al., 1995) to trace
back the perturbations caused by hve mutations in venation pattern
formation. The expression of ATHB-8-GUS is restricted to the veins of
wild-type Ws-2 (Baima et al.,
1995) and Col-0 leaves (Fig.
5I-P). Previous results indicated that, at the stage shown in
Fig. 5 (21 days after sowing),
the first leaf is almost fully expanded, whereas the tenth leaf is just
beginning to expand (Candela et al.,
1999;
Pérez-Pérez et al.,
2002). In all these wild-type leaves, the expression of the
reporter uncovered a complex, reticulate pattern consisting of mature and
differentiating vascular strands. By contrast, the expression of the reporter
revealed a much simpler GUS pattern in hve-3/hve-3 leaves than in
wild-type leaves of the same age (compare
Fig. 5A with 5I, 5B with 5J, 5C with 5K,
and 5D with 5L). The GUS pattern was restricted to the fully
differentiated venation pattern of the mutant, which lacked most tertiary and
higher order veins. The absence of GUS staining at the sites where the veins
normally differentiate indicates that ATHB-8-GUS expression is
dependent on the earlier acquisition of vascular fate at specific locations
within the leaf. Identical results were obtained in comparisons of Ws-2 with
hve-1/hve-1 (see Fig. S4 in the supplementary material). The reduction of ATHB-8-GUS expression due to loss of HVE/CAND1 function suggested that HVE/CAND1 is required earlier to determine the sites of ATHB-8 expression, and prompted us to compare the timing of CAND1-GUS (PETA2-GUS) and ATHB-8-GUS expression throughout the development of wild-type first leaf primordia. Their expression was not detected in leaf primordia 2.5-3.0 days after germination (DAG; Fig. 6A,L). The onset of CAND1-GUS was observed in 3.5-DAG primordia (Fig. 6B), when their tips and presumptive midvein regions showed a mild GUS staining, and became stronger 4 or 4.5 DAG in the cells that will differentiate as the midvein (Fig. 6C,D). Later on (5 DAG), when the characteristic cell wall thickenings were apparent in the tracheids of the developing midvein, the promoter turned on in the secondary veins, which were arranged in the form of two loops (Fig. 6E). The veins in these loops displayed differentiated tracheary elements at 5.5 DAG, when two additional loops of secondary veins were developing in the basal region of the lamina. Expression of the transgene at this stage also revealed tertiary veins differentiating within the first two loops (Fig. 6F). GUS staining persisted in mature veins, beyond the procambial stage (Fig. 6F-K), and in young trichomes, and was diffuse in the leaf lamina. The veins of the first leaves of CAND1-GUS plants remained GUS-positive during the studied period, between 3.5 and 21 DAG.
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To ascertain whether the reduced vein number of the hve mutants
results from reduced responsiveness to auxin, we studied the expression of the
DR5-GUS construct (Ulmasov et
al., 1997), which drives GUS expression under the control of auxin
response elements of the synthetic DR5 promoter. Previous studies
have shown that DR5-GUS expression precedes and coincides with the
appearance of procambial strands, and then disappears as the veins mature
(Mattsson et al., 2003). The
DR5-GUS reporter was not expressed in the veins of 21-day-old Col-0
and hve-3/hve-3 leaves from the first, third and seventh nodes (see
Fig. S5 in the supplementary material), although it was in the leaf margin,
the hydathodes and some mesophyll regions. For actively developing leaves of
the 9th and upper nodes, by contrast, the DR5-GUS reporter was
expressed at the tip, hydathodes, and tertiary and higher order veins of the
proximal regions of the lamina in the wild type, but only at the tip in
hve-3/hve-3 leaves.
Physiological assays
We found no differences between the hve mutants and their wild
types with regard to the root gravitropic response and skotomorphogenesis.
Moderate 2,4-D resistance was displayed by these mutants (see Fig. S6 in the
supplementary material), providing further evidence for the involvement of
HVE in auxin responsiveness.
Genetic interactions
A number of genes required for vascular development and auxin transport and
perception have been identified, including the abovementioned CVP1
and CVP2; the recently cloned LOPPED1 (LOP1; also
known as TORNADO1), which encodes a putative leucine-rich repeat
protein of unknown function (Carland and
McHale, 1996; Cnops et al.,
2006); AUXIN RESISTANT1 (AXR1), which encodes a
subunit of the RUB1-activating enzyme that promotes the modification of CUL1
with RUB1 (Lincoln et al.,
1990; Leyser et al.,
1993; del Pozo and Estelle,
1999; del Pozo et al.,
2002); and PINFORMED1 (PIN1), which encodes an
auxin efflux carrier that contributes to the basipetal transport of auxin
(Goto et al., 1987;
Goto et al., 1991;
Gälweiler et al., 1998).
To investigate the functional relationships between HVE and these
genes, we obtained double mutants and quantitatively analyzed their venation
patterns. Leaf area, venation length and number of branching points in the
vascular network were measured and used to calculate vascular density and
number of branching points per surface unit
(Table 2), as described
previously (Candela et al.,
1999).
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Loss of AXR1 function causes auxin insensitivity and a bushy
inflorescence, the latter due to a decrease in the inhibition of apical
dominance mediated by auxin (Estelle and
Somerville, 1987; Lincoln et
al., 1990; Stirnberg et al.,
1999). The phenotypic similarities between hve/hve and
axr1-12/axr1-12 plants, including their bushy inflorescence and low
fertility, point to a functional relationship between HVE and
AXR1. We did not find significant differences in vascular density and
number of branching points per surface unit between axr1-12/axr1-12
and hve-3/hve-3 first leaves
[Table 2; this aspect of the
axr1 phenotype has been reported by Deyholos et al.
(Deyholos et al., 2003)].
However, the leaf epinasty of axr1-12 mutants (see Fig. S7 in the
supplementary material) is not shared by hve mutants. We identified
hve-3/hve-3;axr1-12/axr1-12 double mutants among the F2 progeny of an
HVE/hve-3 x axr1-12/axr1-12 cross
(Fig. 7, see Fig. S7 in the
supplementary material). The hve-3/hve-3;axr1-12/axr1-12 leaves were
epinastic, like those of HVE/-;axr1-12/axr1-12 plants (Fig. S7), and
significantly smaller than those of hve-3/hve-3;AXR1/- individuals
(Table 2). The vascular length
and the number of branching points were similar in
hve-3/hve-3;axr1-12/axr1-12 and hve-3/hve-3;AXR1/- plants,
indicating similar levels of vascular development and the functional
relationship of HVE and AXR1. However, the vascular density
and number of branching points per surface unit were higher in the double
mutant, the leaves of which were smaller than those of the parentals.
We identified hve-3/hve-3;lop1-65/lop1-65 double mutants in F2
families from an HVE/hve-3 x LOP1/lop1-65 cross in a
9:3:3:1 segregation (
2=0.995; P=0.802; the presence
of hve-3 was tested in phenotypically Lop1 and Hve Lop1 plants). The
double mutants showed significantly smaller leaves (see Fig. S7 in the
supplementary material, Table
2), with fewer branching points, fewer branching points per
surface unit, and a shorter vascular network than those of their
hve-3/hve-3;LOP1/- and HVE/-;lop1-65/lop1-65 siblings
(Figs 7, S7 and
Table 2). Their vascular
densities, however, were found to be similar to those of
hve-3/hve-3;LOP1/- plants. Vascular islands were also observed in
some double mutant individuals (Fig.
7).
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We identified hve-3/hve-3;cvp1-3/cvp1-3 double mutants in the F2 of an hve-3/hve-3 x cvp1-3/cvp1-3 cross. The cotyledons of these double mutants displayed an additive phenotype consisting of the simple vascular pattern characteristic of hve-3, combined with the vein disconnections characteristic of cvp1-3. We sequenced CVP1 in these plants to confirm that they were hve-3/hve-3;cvp1-3/cvp1-3. The venation patterns of hve-3/hve-3;cvp1-3/cvp1-3 and hve-3/hve-3;CVP1/- first rosette leaves were not significantly different (Table 2, Fig. 7). By contrast, the venation length, vascular density, number of branching points and the number of branching points per surface area were significantly smaller for HVE/-;cvp1-3/cvp1-3 plants than for their HVE/-;CVP1/- wild-type siblings (Table 2, Fig. 7). This role of CVP1 in the venation patterning of vegetative leaves had remained unnoticed before.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Mullen et al.,
1998; Rutherford et al.,
1998). According to the canalization hypothesis
(Sachs, 1991b), the spacing of
veins is the outcome of a mechanism that requires that auxin exerts a positive
feedback on its own transport. Cells that transport the hormone more
efficiently become prospective vascular bundles after reaching a threshold of
auxin concentration, while the resulting depletion of auxin in neighboring
cells has the same effect as a lateral inhibition mechanism. This model
predicts that decreased auxin sensitivity should result in a sparse vascular
network, such as the one we observed in hve leaves.
Like axr1 mutants, although to a lesser extent, the hve
mutants are insensitive to the synthetic auxin 2,4-D, in agreement with
previous observations (Cheng et al.,
2004; Chuang et al.,
2004; Feng et al.,
2004). By contrast, root gravitropism, skotomorphogenesis and
sensitivity to the auxin transport inhibitor TIBA were normal in
hve/hve plants. Unlike some agravitropic mutants that are insensitive
to exogenous auxin, such as auxin resistant4 (axr4)
(Hobbie and Estelle, 1995) and
auxin-herbicide resistant1 (aux1)
(Yamamoto and Yamamoto, 1998),
hve/hve roots did not display altered gravitropism.
A role for auxin and ubiquitination in venation pattern formation
Following a map-based approach, we cloned HVE and found it to
encode the Arabidopsis ortholog of mammalian CAND1. The important
role of the HVE/CAND1 gene in vascular development has remained
unnoticed so far, even though its cloning and the characterization of several
mutant alleles have been reported by three research groups
(Cheng et al., 2004;
Chuang et al., 2004;
Feng et al., 2004). Human
CAND1 regulates the ubiquitination of target proteins by binding to
unneddylated CULLIN1 (CUL1). CUL1, SKP1 and F-box proteins form SCF complexes
that ligate ubiquitin moieties to proteins that will be degraded by the 26S
proteasome. It has been proposed that the binding of CAND1 to unneddylated
CUL1 hinders the interaction of SKP1 with CUL1 preventing the assembly of the
SCF complex. After neddylation of CUL1, CAND1 is released and the SFC complex
is assembled (Liu et al.,
2002; Zheng et al.,
2002; Hwang et al.,
2003; Oshikawa et al.,
2003).
Ubiquitin-mediated protein degradation is essential for auxin signaling
(Dharmasiri and Estelle,
2004). Auxin triggers the rapid degradation of AUX/IAA
transcriptional repressors through the action of the SCFTIR1
complex (Gray et al., 2001).
AUX/IAA proteins act by sequestering members of the ARF family of
transcription factors (Ulmasov et al.,
1997; Guilfoyle et al.,
1998) that bind auxin response elements (AREs), a conserved
sequence motif found in the promoters of auxin primary responsive genes
(Guilfoyle et al., 1998). The
identification of the F-box protein TIR1 as the auxin receptor thus provides a
simple explanation for the rapid responses to auxin
(Dharmasiri et al., 2005;
Kepinski and Leyser, 2005). In
Arabidopsis, CAND1 binds to unneddylated CUL1, as demonstrated in
vivo (Feng et al., 2004) and
in vitro (Chuang et al., 2004)
(this work), and hence regulates the formation of SCF complexes involving the
F-box proteins UFO, TIR1, COI1 and SLY1.
The fact that not only hve but also axr1 mutants have
impaired leaf venation confirms the role of the ubiquitin pathway in the
patterning of plant vascular tissues. AXR1 encodes a protein similar
to the E1 ubiquitin-activating enzymes and is required for the conjugation of
RUB1 to CUL1 (Leyser et al.,
1993; del Pozo and Estelle,
1999; del Pozo et al.,
2002). As inferred from their similar number of branching points,
the leaves of the hve and axr1 single mutants and hve
axr1 double mutants show similar structural complexity, suggesting that
both genes act in the same developmental pathway. As noticed previously
(Cheng et al., 2004) for other
phenotypic traits, the similar venation defects of axr1 and
hve loss-of-function mutants are paradoxical, as one would expect
them to have opposite phenotypes if the only role of CAND1 is to negatively
regulate the assembly of SCF complexes. The recent finding that the
deneddylation of CUL1 by the COP9 signalosome is enhanced by CAND1
(Min et al., 2005) is equally
paradoxical, as extra neddylation of CUL1 in loss-of-function hve
mutants should also lead to a phenotype opposite to that of axr1
mutants. Alternatively, by sequestering unneddylated CUL1, HVE/CAND1 may cause
that only neddylated CUL1 is incorporated into functional SCF complexes. The
incorporation of unneddylated CUL1 into the SCF complexes may impair their
functionality through a dominant-negative effect, explaining the similar
phenotypes of axr1 and hve mutants, the compromised
functionality of the SCF complexes in cand1-1 mutants
(Feng et al., 2004), and why,
unlike null CUL1 alleles (Shen et
al., 2002), null hve alleles are not lethal.
It has been proposed that the concentration of auxin that leads to the
patterning of the primary and secondary veins is higher than the concentration
required for other veins (Aloni et al.,
2003). If a certain threshold of an auxin signal must be surpassed
for the ground cells to adopt vascular fates, the co-occurrence of decreased
auxin sensitivity and the lower auxin concentration required for the
patterning of higher-order veins may explain why higher-order veins are more
severely affected in hve mutants. We found no expression of the
DR5-GUS reporter in hve-3/hve-3 leaves at the sites where
higher-order veins are normally differentiating in the wild type. This
suggests that loss of HVE function leads to a reduction in GUS
expression driven by the auxin response elements of the synthetic DR5
promoter, and further supports a correlation between an impaired auxin
perception and the failure to form tertiary and higher-order veins.
HVE is required in a general, very early venation patterning mechanism
Based on the correlation between venation density and venation branching
found for different rosette leaves, we previously proposed that a common
patterning mechanism operates in all of them
(Candela et al., 1999). This
hypothesis is reinforced by the simple vascular network of all the rosette
leaves and laminar floral organs of hve mutants. The extent of the
venation abnormalities caused by hve alleles indicates that
HVE is a component of such a common mechanism. Nevertheless, the
apparently normal venation pattern of hve cauline leaves also
indicates the existence of organ-specific elements. The expression of
CAND1-GUS in the hydathodes and margins of fully expanded cauline
leaves suggests additional roles for HVE.
The dwarfism and extremely low fertility caused by hve-2 and
hve-3 suggest that they are null alleles, as also proposed by Feng et
al. (Feng et al., 2004). By
contrast, hve-1 is probably hypomorphic, as its effects on fertility
and overall plant and inflorescence size are not so severe. Mis-splicing of
hve-1 transcripts generates at least two truncated predicted proteins
that may keep some remnant function. Similarly, the phenotype caused by
mis-splicing of another allele, Atcand1-1, is weaker than that of
null alleles (Cheng et al.,
2004). The similar vein pattern phenotypes conferred by
hypomorphic and null alleles of HVE suggest that venation patterning
is more sensitive to HVE loss of function than are plant size and
fertility.
We studied the expression of ATHB-8-GUS, an early marker of
vascular cell identity (Baima et al.,
1995), in hve-1/hve-1 and hve-3/hve-3 plants.
The reporter highlighted a simple vascular pattern with reduced vascular
density and lacking higher order veins. Consistent with their early role, the
expression of both HVE and ATHB-8 was detected before xylem
differentiation in the midvein of the first leaves, and they evolved in a very
similar way. Our results suggest that, at least in higher order veins,
HVE acts to determine the location of new strands. Lack of
HVE expression, however, does not preclude the formation of the
primary vein, and only partially inhibits the formation of the secondary and
tertiary veins. ATHB-8 expression seems to be required for
vein-specific events after vein initiation sites are established, and depends
on prior HVE activity in higher order veins, only partially so in the
case of secondary and tertiary veins, and not at all in the primary vein. As
this conclusion is solely based on the behavior of an ATHB-8-GUS
transgene, further research will be required to shed light on the order of
action of HVE and ATHB-8. Cleared leaves and paradermal and
transverse leaf sections showed no traces of aborted or incompletely developed
vascular strands at the sites where higher-order veins are lacking in the
hve mutants, an observation indicating a perturbation of vascular
initiation rather than final differentiation. The thin primary vein of the
hve-3 mutant is a likely consequence of impaired auxin perception on
the recruitment of provascular cells.
Double mutant analysis suggests that auxin transport and perception act independently to pattern leaf veins
We investigated the functional relationship of HVE with several
genes involved in venation pattern formation or auxin perception or transport,
such as CVP1, CVP2, LOP1 and PIN1. The hve-3
pin1cay double mutant was particularly informative, given that
the simple vascular pattern of hve-3 was affected by the null
pin1cay allele. As PIN1 is an auxin transporter, this
result shows that the minimal vascular pattern of hve leaves still
depends on auxin for its development and that alternative mechanisms of auxin
perception are at work in the mutant. The additive hve-3
pin1cay phenotype was as to be expected if auxin perception
and transport operate independently to pattern the vascular network. As the
architecture of the venation pattern seems to be very sensitive to alterations
in either of these two factors, it follows that they must act coordinately.
Further support for these conclusions came from the application of the auxin
transport inhibitor TIBA to the hve-1 mutant, which caused the same
range of abnormalities as in the wild type.
The phenotypes of hve-3 lop1-65, hve-3 cvp1-3 and hve-3 cvp2-1 double mutants were also considered to be additive, suggesting that additional independent processes are required for vein development. The free-ending higher-order veins of cvp2-1 single mutants indicate a role for CVP2 in the connectivity of vascular strands. The secondary veins of hve-3 cvp2-1 double mutants were often disconnected, suggesting that the secondary veins of hve-3 are equivalent to the higher-order veins of the wild type, as inferred from their similar sensitivity to the loss of CVP2 function. This equivalence suggests a gradual establishment of the venation pattern and may be explained by an impaired auxin sensitivity leading to a premature arrest of the patterning process in the mutant, which is consistent with the mechanism proposed by the canalization hypothesis.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/19/3755/DC1
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ACKNOWLEDGMENTS |
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|
Footnotes |
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Present address: Plant Gene Expression Center, University of California,
Berkeley, Albany, CA 94710, USA
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