1 School of Biological Sciences, The University of Manchester, 3.614 Stopford
Building, Oxford Road, Manchester M13 9PT, UK
2 Department of Forest Genetics and Plant Physiology, SLU, Umeå SE-901 83,
Sweden
* Author for correspondence (e-mail: simon.turner{at}man.ac.uk)
Accepted 17 February 2003
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SUMMARY |
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Key words: Arabidopsis, Differentiation, Vascular bundle, Patterning, Auxin
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INTRODUCTION |
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In broad flat organs, such as leaves, the vascular tissue consists of an
interconnected network that forms during the course of leaf development. By
contrast, the pattern of primary vascular development in the stem is laid down
in the region immediately below the apical meristem. The differentiation of
vascular tissue is closely linked to the activity of the meristem. Early
experiments demonstrated that the inductive effects of leaves and organ
primordia on vascular development could be replaced by the application of
auxin (Jacobs, 1952;
Young, 1953
). Many subsequent
experiments have demonstrated the effect of auxin on both the induction and
pattern of vascular tissue (for reviews, see
Bennett et al., 1998
;
Berleth and Mattsson, 2000
;
Ye, 2002
). A large number of
experiments investigating vascular tissue induction by auxin were rationalised
by Sachs (Sachs, 1981
;
Sachs, 1991
), who suggested
that the positive effects of high levels of auxin on the capacity for polar
auxin transport (PAT) generate a positive feedback mechanism leading to the
canalisation of auxin flow. The high levels of auxin in these regions result
in vascular differentiation. Identification of the PIN1 protein has provided
evidence to support the canalisation hypothesis. PIN1 is essential for PAT,
and is normally localised on the basal membrane of the cell
(Galweiler et al., 1998
).
During embryo development, transition from a uniform localisation of PIN to an
orientated localisation occurs along the apical-basal axis, which is
consistent with a positive feedback loop for auxin
(Steinmann et al., 1999
).
Furthermore, disrupted localisation of PIN1 in emb30/gnom
mutants results in a proliferation of vascular tissue that is consistent with
auxin delivery to inappropriate cell types
(Koizumi et al., 2000
;
Steinmann et al., 1999
).
A large number of experiments by Sachs suggested that auxin may also play a
role in the inhibition of vascular bundle development. Pre-existing vascular
bundles with high rates of auxin transport inhibited the development of new
vascular strands in their immediate vicinity
(Sachs, 1981;
Sachs, 1991
). Interestingly,
recent experiments by Reinhardt et al.
(Reinhardt et al., 2000
)
suggest that auxin is involved in both inductive and inhibitory affects in
organ formation. Although auxin is able to induce primordia formation, once
formed, the region adjacent to these primordia becomes insensitive to further
auxin application. The mechanism by which auxin, or some other signalling
molecule from the developing primordia, exerts this inhibitory effect remains
unclear.
Isolation of mutants that contain islands of vascular tissue that are not
connected to the existing vascular tissue are harder to reconcile with the
canalisation hypothesis (Carland et al.,
1999; Deyholos et al.,
2000
; Koizumi et al.,
2000
). Some of these mutants have normal responses to auxin and
may be explained more easily by the diffusion-reaction pre-pattern hypothesis.
Based on computer modelling, the diffusion-reaction hypothesis proposes the
interaction of two diffusible substances with varying diffusion rates that
regulate the autonomous formation of veins in an initially uniform field. One
of these substances promotes the induction of vascular tissue, whereas the
other results in inhibition of vascular development
(Koch and Meinhardt, 1994
).
Although there is a good deal of evidence to support the inductive effects of
auxin on vascular development, there is little evidence on what the inhibitory
element might be.
In wild-type Arabidopsis stems, the pattern of vascular bundles is
very ordered with five to eight bundles developing around the stem, separated
by the interfascicular regions. However, the molecular mechanisms that control
this patterning remain unclear. There are a limited number of mutants
available that show altered vascular patterning in the stem. The
amphivasal vascular bundle (avb) mutant
(Zhong et al., 1999) has an
increased number of vascular bundles in the stem, many of which are abnormally
localised in the pith. The vascular bundles also show an amphivasal pattern
with the phloem surrounded by xylem. Another mutant, acaulis5
(acl5) (Hanzawa et al.,
1997
), has vascular bundles that are surrounded by an abnormally
extended sheath of cells with thickened walls. Although the number of veins in
acl5 plants is similar in wild-type plants, the amount of
differentiated vascular tissue within the vascular bundles appears to be
greater in the mutant.
In this study, we report the cloning of a gene involved in maintaining the ordered patterning of vascular bundles within the stem of Arabidopsis. Mutations in this gene lead to disruptions in the patterning of differentiated vascular tissue in the stem, resulting in a continuous ring-like pattern of xylem and phloem, with very little interfascicular tissue and the loss of defined vascular bundles. Based on the phenotype of the vascular tissue in the stem, this mutant was designated continuous vascular (cov1). COV1 is predicted to be an integral membrane protein that may be involved in the perception or transport of a signalling molecule that negatively regulates the differentiation of vascular tissue in the developing stem of Arabidopsis.
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MATERIALS AND METHODS |
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DNA and RNA isolation
DNA was isolated from an F2-mapping population as described by
Guidet et al. (Guidet et al.,
1991). The testcross population was screened using an alternative
rapid DNA extraction procedure. Small sections (6-9 mm2) of the
cotyledons or first emerging leaves of young seedlings were removed and ground
in 20 µl 0.25 M NaOH. Grinding was performed in 96-well PCR plates using
plastic pipette tip ends sealed by passing them through a flame. Once the
tissue was homogenised, 7 µl of the sample was removed and added to 150
µl 0.3 M Tris-HCl buffer (pH 8.0). Each PCR reaction used 7 µl of this
mix, in a total volume of 20 µl. RNA extraction was performed using an
RNeasy Plant Mini Kit (Qiagen), and RNA was quantified on a BioSpec-1601 E
spectrophotometer (Shimadzu). A full-length cDNA (wild-type Columbia) of
COV1 was obtained from a library generated by Asamizu et al.
(Asamizu et al., 2000
), and
also by RT-PCR analysis from wild-type Ler.
PCR and RT-PCR
PCR was performed in a PTC100 thermal cycler (MJ Research) using Taq
polymerase (Life Technologies). Each reaction was performed in a total volume
of 25 µl, containing 100 ng DNA with 50 mM KCl, 10 mM Tris-HCl (pH 9.0),
0.1% Triton X-100, 200 µM dNTPs (each), 1.5 mM MgCl2, 0.5 units
Taq polymerase and 1 µM each primer. PCR conditions were as follows:
94°C for 2 minutes; 35 cycles of 94°C for 30 seconds, 55°C
(variable between primer pairs) for 30 seconds and 72°C for 60 seconds;
and 2 minutes at 72°C. RT-PCR was performed using a One-Step RT-PCR Kit
(ABgene) in a volume of 50 µl with 1 µg of total RNA. Gene-specific
primers were used for amplification. PCR conditions were as follows: 47°C
for 30 minutes; 94°C for 2 minutes; 35 cycles of 94°C for 20 seconds,
55°C for 30 seconds and 72°C for 90 seconds. The positive control
(MS2) was provided with the kit. All PCR products were run on 1-2% agarose
gels in TBE buffer and visualised using ethidium bromide staining.
DNA sequencing
Templates generated from restriction fragment cloning, PCR and RT-PCR were
gel purified using a High Pure PCR Product Purification Kit (Roche), and
cloned into the vector pGEM-T Easy (Promega) for sequencing. Specific primers
were designed and synthesised (MWG Biotech) for use along with vector specific
T7 and SP6 primers. Plasmid templates were prepared using a QIAprep Spin
Miniprep Kit (Qiagen), and sequencing reations were performed using an ABI
Prism Sequencing Kit (Applied Biosystems). Sequence data were analysed using
the ContigExpress program (InforMax). Sequences used for alignment were
identified from BLAST searches of GenBank. Alignments were carried out using
CLUSTAL W (Thompson et al.,
1994).
Expression analysis
Expression analysis was performed using real-time quantitative PCR (qPCR).
Primers and probes were designed for the various genes using Primer Express
(v1.0) (Applied Biosystems), and probes were labelled with 6-FAM (FAM;
5') and tetramethylrhodamin (TAMRA; 3'). First strand synthesis
was performed in a volume of 20 µl, containing 1 µg total RNA with 500
ng poly-(dT) primer and 100 U reverse transcriptase (Promega), at 42°C for
60 minutes. PCR conditions were as follows: 50°C for 2 minutes; 95°C
for 10 minutes; 40 cycles of 95°C for 15 seconds and 60°C for 60
seconds. PCR reactions were performed in a volume of 25 µl, containing 12.5
µl 2x qPCR Mastermix (Eurogentec), 5 pmol probe and 25 pmol each
primer. Data analysis was carried out using the Sequence Detector (v1.7)
program (Applied Biosystems).
Complementation
DNA fragments containing genes of interest were subcloned into the binary
vector pc2300 (Cambia) for complementation purposes. Complementation was
carried out by transforming plants using Agrobacterium tumefaciens
(GV3101) containing various constructs described by Bent and Clough
(Bent and Clough, 1998).
Transformants were identified by growing seed of the transformed plants on
agar plates containing MS (2.2 g/l) and the appropriate selection antibiotic.
After 2 weeks, the resistant seedlings were transplanted into pots and grown
to maturity to determine their phenotype.
Auxin experiments
Endogenous auxin levels were quantified by gas chromatography-selected ion
monitoring-mass spectrometry with 13C6IAA as an internal standard, as
described by Edlund et al. (Edlund et al.,
1995). To determine response to auxin, seedlings were grown on MS
agar plates containing various levels of 2,4-Dichlorophenoxy-acetic acid
(2,4-D; 0.05, 0.1 and 0.25 µM), in a vertical position in a growth cabinet
under continuous light at 22°C.
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RESULTS |
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Cross-sections of mature 4-week-old inflorescence stems showed that both alleles had a significant defect in the patterning and development of the vascular bundles compared with wild-type Ler (Fig. 1). In wild-type Ler plants, six to eight vascular bundles develop in a very ordered and predictable pattern around the stem, with bundles of phloem and xylem separated by interfascicular regions made up of differentiated fibres. However, in the cov1 mutants, the ordered patterning of vascular bundles is lost and is replaced by an almost continuous ring of both phloem and xylem tissue around the whole stem. This increase in vascular tissue appears to replace the area normally occupied by the interfascicular cells such that, in plants exhibiting a severe mutant phenotype, the interfascicular region was virtually absent or significantly reduced (Fig. 1). This proliferation of vascular tissue is also accompanied by a reduction in stem thickness.
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Mapping the cov1 gene
An initial F2-mapping population of 600 plants was generated
from a cross between cov1-1 and wild-type Columbia plants. The
population was scored for the mutant phenotype, and 120 mutant plants were
selected and screened with polymorphic microsatellite (SSLP) markers spanning
all five chromosomes. Linkage was identified with markers on the long arm of
chromosome 2. Further SSLP markers were generated in this region for more
detailed mapping and the gene was localised to a region of 500 kb.
Polymorphic co-dominant markers located on BACs F27F23 (SSLP33) and F21P24
(SSLP23), which were suitable for rapid PCR screening, were identified that
flanked a region of
2 Mb containing the COV1 gene. These markers
were used to screen a testcross population to identify critical recombinants
in this region. The testcross population was generated by crossing
F1 plants generated from the initial cross with Columbia back to
cov1-1 to generate a population that was 50% mutant and 50% wild type
(heterozygous). Out of 2148 testcross plants screened, 105 showed
recombination between the two flanking markers. These critical recombinants
were used to finely map the gene further using additional SSLP and cleaved
amplified polymorphic sequence (CAPS) markers generated on each of the BACs
spanning this region. Based on the mapping data obtained from these plants,
the gene was placed within a region of 77 kb, spanned by the ends of two
adjacent BACs (T2G17 and F11A3) (Fig.
4).
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A full-length EST cDNA clone of At2g20120 (COV1) from a Colombia
background was obtained (Asamizu et al.,
2000) and the DNA sequence deposited in GenBank (Accession Number
AY170845). RT-PCR was performed using mRNA from wild-type Ler, and
the coding sequence was identical to the cDNA clone from Columbia. Comparison
of the cDNA and genomic sequence revealed that the COV1 gene
comprises seven exons and six introns (Fig.
4). The mutation in cov1-1 is located in the third exon
(C
T) and results in an amino acid change from proline to serine
(P76
S). In cov1-2 the mutation occurred in the
fourth exon (G
A) and results in an amino acid change from glycine to
arginine (G130
R). The full-length protein is predicted to be
268 amino acids in length, although there are two possible in frame start
codons (Fig. 6) and it is
unclear which one is used. The predicted structure of the gene indicates that
the N-terminal end of the protein is cytoplasmic, followed by three membrane
spanning domains with the C-terminal end predicted to be in the wall
(Fig. 4). The mutation in the
cov1-1 allele is located in the first membrane spanning domain,
whereas the mutation in cov1-2 is located intracellularly, just after
the second predicted membrane spanning domain. Based on phenotype, the
mutation in cov1-2 is more severe and causes a greater loss of
function of the protein, as evidenced by the extreme dwarf growth habit and
increased disruption of the vascular patterning in the stem.
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Analysis of protein sequences from other genomes has revealed that there is a homologous gene to COV1 in rice, with the predicted sequence from a cDNA exhibiting 59% identity to COV1 (Fig. 6). COV1 also has clear homologues in a large number of bacterial genomes. Alignment with two of these bacterial sequences, from Mesorhizobium loti and Agrobacterium tumefaciens, is shown in Fig. 6. They both exhibit sequence conservation with COV1 along their entire length. All of the related bacterial genes are annotated as membrane proteins of unknown function. These results indicate that these genes may be involved in cellular mechanisms that are specific to plants and bacteria, and that the COV1 gene may play a common role within these organisms.
Expression of COV1 and LCV1
RT-PCR experiments demonstrate that the COV1 gene is alternatively
spliced in both wild-type Ler plants and the two mutant alleles (data
not shown). Sequence analysis revealed that the alternatively spliced product
was the result of the retention of intron 2, which leads to a premature stop
codon and a truncated protein. Real-time quantitative PCR (qPCR) analysis was
carried out to determine the relative levels of the different transcripts in
wild-type and mutant plants. In wild-type Ler and cov1-2
plants, the amount of alternatively spliced product compared with the
correctly spliced product was 0.95%, but in the cov1-1 mutant plants,
this was increased to 2.01% (data not shown). Because of the high degree of
similarity between COV1 and the three related genes, qPCR was used to
determine the expression levels of the specific genes in various plant
tissues. The results of the qPCR experiments showed that COV1 was
highly expressed in flowers and stems; a reasonable level of expression was
also seen in roots, but less in leaves
(Fig. 7). The expression levels
of LCV1 were also measured and were found to be much lower than those
of COV1.
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To determine whether there was any difference in auxin concentration between wild-type Ler and cov1-1 mutant plants, levels of auxin were measured at various positions along the inflorescence stem. These included the region just below the apical meristem; half way down the stem; at the base of the stem; and in the hypocotyl. The results of the analysis showed that there was no significant difference in auxin concentration in the upper part of the stem (Fig. 8). However, in the base of the stem and in the hypocotyl there was a significant difference: wild-type plants contained almost twice the amount of auxin as did cov1-1 plants.
cov1-1 plants were also transformed with a construct containing
the auxin inducible IAA2 promoter coupled with the ß
glucuronidase (GUS) reporter gene as an indicator of the presence of auxin
(Swarup et al., 2001).
Transformed cov1-1 plants were then crossed with wild-type
Ler plants for comparison, to reduce any variation caused by
positional effects. There was little difference in expression patterns of GUS
between the wild-type or cov1-1-mutant plants in either the seedlings
or developing stems (data not shown), which indicates that there was no
difference in the distribution of auxin in the upper part of the stem. There
was no detectable IAA2-GUS expression at the base of the stem in either
wild-type Ler or cov1-1-transformed plants where the auxin
measurements identified a significant difference between wild-type and mutant
plants.
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DISCUSSION |
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Several of the phenotypic characteristics of the cov1 mutants
indicate that the abnormalities might be caused by a defect in auxin
signalling. The mutant plants have a stunted growth habit and wrinkled leaves
(Fig. 3). Several other mutants
defective in auxin signalling and transport also show a dwarf phenotype with
wrinkled or curled leaves (Ruegger et al.,
1997; Hobbie et al.,
2000
; Liscum and Reed,
2002
). Auxin is known to be involved in controlling cell expansion
and has been shown to be involved in regulating hypocotyl elongation in
light-grown, but not dark-grown, plants
(Jensen et al., 1998
;
Nakazawa et al., 2001
). Given
the importance of auxin in all aspects of vascular development,
cov1-1 was examined for its response to auxin
(Sachs, 1981
;
Sachs, 1991
;
Carland et al., 1999
;
Sieburth, 1999
;
Mattsson et al., 1999
).
Seedlings of cov1 respond to auxin in a similar manner to wild-type
plants. Furthermore, cov1-1:axr1-3 double mutants behave in a manner
similar to the single mutants alone (Fig.
8). Both these lines of evidence suggest that auxin signalling is
not the primary defect in cov1-1. Direct measurement of auxin levels
indicates no difference between mutant and wild type in the upper stem when
the vascular defect in cov1 first becomes apparent. There is a small
(twofold) difference in auxin levels at the base of the stem, but this may be
a consequence of the altered vascular tissue in cov1 plants affecting
PAT. It is unlikely to account for the vascular patterning defect that is
first apparent close to the apex.
According to the canalisation hypothesis
(Sachs, 1981;
Sachs, 1991
), auxin and auxin
transport are responsible for the induction of new vascular tissue, and the
subsequent draining of auxin from surrounding cells prevents the formation of
new vascular strands close to pre-existing vascular bundles. Although there is
a vast body of evidence to support the induction and canalisation of the
vascular bundles by auxin, the basis of the inhibitory affect is less clear.
The cov1 phenotype is hard to reconcile with this model because a
normal pattern of vascular development is observed but apparently fails to
inhibit the formation of additional vascular tissue nearby. The cov1
phenotype is more consistent with the diffusion-reaction hypothesis in which a
second inhibitory component is hypothesised. To date, the nature of this
inhibitor has not been identified, but the cov1 phenotype is
consistent with a plant defective in the synthesis, transport or perception of
such an inhibitor.
Polyamines are also known to affect cell size and play an important role in
internode elongation through cell expansion. The acl5 mutant, which
encodes a spermine synthase, exhibits a short inflorescence stem similar to
cov1-2 (Hanzawa et al.,
1997), and, like the acl5 mutant, the cells in the
inflorescence of cov1-1 plants are much shorter and smaller compared
with wild type (data not shown). The acl5 mutant has much shorter and
wrinkled siliques, which is also a characteristic of the cov1
mutants. acl5 plants show an increase in vascular tissue within the
bundles of the stem, but the spacing and organisation remains relatively
similar to the wild type. Polyamines have been hypothesised either to act
downstream of auxin signalling or to regulate the interaction of different
hormones, such as auxin and gibberellin
(Hanzawa et al., 2000
).
However, the mechanism by which polyamines or their derivatives affect plant
development is currently unknown.
The gene responsible for the cov1 mutation has been cloned by a
map-based cloning strategy. Two distinct lines of evidence indicate that the
defect is caused by a mutation in the predicted gene At2g20120. First, both
mutant alleles can be fully complemented using a fragment containing only this
predicted gene. Second, comparison of the cov1 alleles with the
wild-type sequence revealed single base-pair changes within this gene. The
complementation analysis was complicated by the fact that the cov1-1
mutation could be complemented using a fragment that lacked COV1.
This is explained by fact that COV1 appears to be part of a very
recent tandem duplication event. COV1 is adjacent to a very closely
related gene (termed LCV1) that exhibits a 95% amino acid sequence
identity with COV1. LCV1 has a very short promoter region (500
bp) which it shares with a gene coding for one of the subunits of the 26S
proteasome (Fig. 4).
Presumably, if the gene is inserted into the genome in a position that
increases the expression of LCV1, it is sufficiently similar to
compensate for COV1. Evidence that LCV1 is not responsible
for the cov1 mutation comes from the fact that no mutation was
identified in LCV1 in either allele of cov1 and that pGDP1
was only able to partially complement the cov1-2 allele (68% of
plants). The presence of LCV1 makes it unlikely that either of the
cov1 alleles are functional knockouts, as LCV1 is expressed
at low levels in the plant and was shown to be able to partially complement
the mutation.
Both the vascular defect and the growth defects were complemented by pGDP7,
which indicates that all the phenotypes associated with cov1 were a
consequence of a defect in a single gene. Expression of the cov1 gene
is high in stems, which is consistent with the observed phenotype. The
transcript appears to be alternatively spliced, and a small minority (1%)
of the mRNA retained the second intron. It is unlikely that this change
contributes to the mutant phenotype, because the relative increase is small;
however, the exact role of the alternative splicing in wild-type plants
remains unclear.
COV1 is predicted to be an integral membrane protein of unknown function in plants and a large number of bacteria. The high degree of homology between the genes in plants and bacteria indicates that COV1 may play an important role in some common pathway, possibly in signalling. Interestingly, the bacterial species that contain the more closely related homologues of COV1 are nearly all species that are associated with plants, including Mesorhizobium loti and Agrobacterium tumefaciens. Whether these genes are used in the perception or transport of a signal generated in plants remains to be investigated. The cov1-mutant phenotype is consistent with that of a plant defective in a component that negatively regulates the differentiation of both xylem and phloem in the developing stem. This component has been predicted in a number of models for vascular development. Further analysis should allow us to determine further the function of COV1 and how it regulates plant vascular development.
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ACKNOWLEDGMENTS |
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