1 Division of Biological Sciences, Graduate School of Science, Hokkaido
University, Sapporo 060-0810, Japan
2 Department of Biological Sciences, Graduate School of Science, The University
of Tokyo, Hongo, Tokyo 113-0033, Japan
* Present address: Department of Botany, Graduate School of Science, Kyoto
University, Kyoto 606-8502, Japan
Author for correspondence (e-mail:
perfect{at}sci.hokudai.ac.jp)
Accepted 31 October 2002
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SUMMARY |
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Key words: Arabidopsis, Epidermis, Homeodomain, L1, MERISTEM LAYER1, PROTODERMAL FACTOR2, Shoot apical meristem
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INTRODUCTION |
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An important issue regarding transcription factors is identification of
their target genes, but information on plant homeodomain target genes is
limited. The Arabidopsis WUSCHEL (WUS) gene encodes a
homeodomain protein belonging to a distinct class and functions in specifying
stem cell identity in shoot and floral meristems
(Mayer et al., 1998). Lohmann
et al. have identified WUS-binding sites in the second intron of the floral
homeotic gene AGAMOUS (AG) and revealed that WUS acts
together with another transcription factor, LEAFY (LFY), as a direct activator
of AG (Lohmann et al.,
2001
). Arabidopsis ATHB-2, which belongs to a subgroup of
the HD-ZIP proteins and plays a role in the shade avoidance response in
photomorphogenesis (Steindler et al.,
1999
), has been shown to bind its own promoter and create a
negative autoregulatory loop (Ohgishi et
al., 2001
). Given the functional significance of individual
members of the homeodomain proteins in plant growth and development, their
target DNA sequences and downstream genes must be investigated further.
Arabidopsis PROTODERMAL FACTOR 1 (PDF1) encodes a
proline-rich cell-wall protein that is expressed exclusively in the L1 of
shoot meristems. By using progressive deletions of a promoter fragment of the
PDF1 gene, we previously showed that a cis-regulatory element named
the L1 box is required for the L1-specific gene expression
(Abe et al., 2001). The L1 box
is well-conserved within the promoter regions of all L1-specific genes
analyzed so far. Furthermore, recombinant ATML1 specifically binds to the L1
box in vitro (Abe et al.,
2001
). Here we report on the characterization of the
PROTODERMAL FACTOR2 (PDF2) gene, which shares the highest
homology with ATML1 in the Arabidopsis genome. PDF2
also shows L1-specific expression. atml1 pdf2 double mutation results
in severe defects in shoot epidermal cell differentiation, which are not
observed in plants carrying mutations of only one of the genes. Our results
suggest that PDF2 and ATML1 play a critical role in
maintaining the L1 cells, possibly by regulating the expression of essential
L1-specific proteins.
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MATERIALS AND METHODS |
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The T-DNA insertion alleles of PDF2 and ATML1 were
isolated by screening a total of 60,480 T-DNA-tagged lines generated at the
University of Wisconsin Knockout Arabidopsis Facility
(Krysan et al., 1999). A
primer specific for the T-DNA left border (LB, 5'-CATTT TATAA TAACG
CTGCG GACAT CTAC-3') was used in tandem with PDF2-specific
primers (PDF2-F, 5'-ATATT GATCA GTGCC TTGAA GGAAA CCAA-3' and
PDF2-R, 5'-CTTGT TACTT GCTCC ACAAG AATCC CATT-3') or
ATML1-specific primers (ML1-F, 5'-TGGGA TATAC AGGCA GAAGA AAATC
GAGA-3' and ML1-R, 5'-ACCTT CTGCA AAAAC ACAAA CCAAA
ACAT-3'). These T-DNA-tagged mutants had been created in the Ws ecotype,
and were backcrossed at least three times into the wild-type Col-0 before
analysis. Primers used for the mutant screen were also employed for PCR-based
genotyping.
Cloning of PDF2
The partial PDF2 cDNA fragment that was originally isolated by
cDNA subtraction (Abe et al.,
1999) was used as a probe to screen an Arabidopsis cDNA
library derived from wild-type inflorescences (kindly provided by Drs J.
Mulligan and R. Davis, Stanford University). Plaque hybridization and
sequencing were performed as described previously
(Abe et al., 1999
). An almost
full-length cDNA fragment was isolated and cloned as an EcoRI
fragment into pBluescript II (Stratagene) to generate pPDF2-02. The 5'
end of the PDF2 transcript was determined by using a 5'-RACE
kit (Takara, Kyoto, Japan). One µg of total RNA from inflorescences was
used as a template for first-strand cDNA synthesis. The full-length
PDF2 cDNA sequence has been deposited with GenBank under the
accession number AB056455.
Expression analyses
Preparation of total RNA and RNA gel blot analyses were performed as
described previously (Abe et al.,
1999). For the PDF2 probe, gel-purified EcoRI
fragment of pPDF2-02 was labeled by the random-priming method. The
ATML1 probe was prepared from an EcoRI-PstI
fragment of the ATML1 cDNA clone
(Abe et al., 2001
). Probes for
PDF1 and EF1
have been described
(Abe et al., 1999
). For reverse
transcription (RT)-PCR, one µg of total RNA from aerial parts of 10-day-old
seedlings was used as a template for first-strand cDNA synthesis with an
oligo(dT) primer. Nucleotide sequences of PCR primers were PDF2-F and PDF2-R
for PDF2, ML1-F and ML1-R for ATML1, PDF1-F (5'-TCCCT
CTGGC TCACA TGGAA-3') and PDF1-R (5'-GTCTC TAACT TGAGG
GGTTG-3') for PDF1, ACRF (5'-TGAAG AACAC AATGC
TCGAG-3') and ACR-R (5'-TATCT CTTCC TCAAG ACTCC-3') for
ACR4 (Tanaka et al.,
2002
), and STM-F (5'-ACAGC ACTTC TTGTC CAATG GCTT-3')
and STM-R (5'-GAAGA CCATA GCTTC CTTGA AAGG-3') for
SHOOTMERISTEMLESS (STM) (Long et
al., 1996
).
In situ hybridization was performed as described previously
(Abe et al., 1999). To prepare
a PDF2 gene-specific riboprobe, the PDF2
5'-untranslated region (UTR) was amplified by PCR using pPDF2-02 as a
template with PDF2 5'-specific primers (PDF2-5'F,
5'-CTGAG TGATC ATAGT CAATC ATCC-3' and PDF2-5'R,
5'-AGTAG TGACT TCGGT ACCTG ACTT-3'), and was cloned as a
BclI-KpnI fragment into pBluescript II. Sense and antisense
probes were generated by using T7 and T3 RNA polymerases with
digoxigenin-11-UTP (Boehringer), respectively.
Gel shift assays
The PDF2 protein-coding sequence was amplified by PCR using
pPDF2-02 as a template with specific primers (PDF2P-F, 5'-GATCA TAGTG
AATTC TCCAT AACAA-3' and PDF2P-R, 5'-GAAAC CATAA CCAAG CTTAA
TCCT-3'). The sequence was cloned as an EcoRI-HindIII
fragment into pMAL-p2X (New England Biolab) to make a maltose-binding protein
(MBP)-PDF2 fusion construct. The fusion protein was produced in E.
coli TB1 and was purified according to the manufacturer's protocol. Gel
retardation assays were performed as described previously
(Abe et al., 2001).
Microscopy
For scanning electron microscopy (SEM), seedlings grown in MS plates were
fixed in FAA (50% ethanol, 5% formaldehyde and 5% acetic acid), dehydrated in
an ethanol series and critical point-dried using liquid CO2. After
coating with gold, samples were viewed using a Hitachi scanning electron
microscope. For light microscopy, tissue samples were fixed in FAA, dehydrated
in an ethanol series and embedded in Technovit 7100 resin (Kulzer GmbH,
Wehrheim, Germany). Sections (10 µm) were stained for 2 minutes in an
aqueous 0.1% Toluidine Blue solution.
Plant transformation
For the 35S::PDF2 sense and antisense constructs, gel-purified
EcoRI fragment of pPDF2-02 was blunt-ended with T4 DNA polymerase and
inserted into a SmaI site of pUC-NOS, a pUC18 derivative containing
an SacI-EcoRI fragment of the nopaline synthase gene
(NOS) terminator from pBI101
(Jefferson et al., 1987). The
resulting clones carrying the PDF2 cDNA in sense or antisense
orientation were used as XbaI-EcoRI fragments to replace the
ß-glucuronidase gene downstream of the cauliflower mosaic virus (CaMV)
35S promoter of the pBI121 Ti-vector (Clontech). The constructs were
introduced into Agrobacterium strain C58C1 by electroporation and
transformed into wild-type plants by the floral dip method
(Clough and Bent, 1998
).
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RESULTS |
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The 5' promoter region of PDF2 contains an L1 box which has
been identified as a cis-regulatory element responsible for L1-specific gene
expression (Abe et al., 2001),
as shown in Fig. 1A. The L1 box
of PDF2 is preceded by a TTAATGG heptamer, a potential target
sequence of WUS (Lohmann et al.,
2001
). The 100-bp region between the L1 box and the predicted
transcription start site does not contain a putative TATA box. A similar
arrangement of such elements is also found in the ATML1 promoter
region (Fig. 1A).
Expression pattern of PDF2
We examined the expression pattern of PDF2 by RNA gel blot
analysis. Total RNA samples prepared from each tissue were probed with a
PDF2-specific probe. PDF2 expression was detected mainly in
flower bud clusters including shoot apices
(Fig. 2A). PDF2 mRNA
was also present in leaves, stems, siliques and 10-day-old seedlings. We
detected only a faint signal of PDF2 expression in root tissue.
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The spatial expression pattern of the PDF2 gene was further
examined by RNA in situ hybridization. PDF2 mRNA was readily detected
in the L1 layer of vegetative shoot meristems and the protoderm of leaf
primordia (Fig. 2B).
L1-layer-specific expression was also found in floral and apical inflorescence
meristems (Fig. 2C). In
developing flowers, PDF2 mRNA was present in protodermal cells of
primordia of all floral organs (Fig.
2D), but later the signal became restricted to the protodermis of
developing ovules (Fig. 2E).
PDF2 expression was evenly distributed in the quadrant-stage embryo
(Fig. 2F), but was confined to
the outermost cell layer in the early globular-stage embryo
(Fig. 2G). These expression
patterns are indistinguishable from those of ATML1
(Lu et al., 1996).
PDF2 binds to the L1 box
Our previous study demonstrated that the recombinant ATML1 protein can bind
to the L1 box within the PDF1 promoter in vitro
(Abe et al., 2001). To
determine whether PDF2 also interacts with the L1 box, we performed gel
retardation assays using the recombinant PDF2 protein. PDF2 was produced as a
fusion protein with a maltose-binding protein (MBP) in E. coli cells,
purified and then tested for its binding ability to the L1 box probe. Complex
formation was observed with the recombinant PDF2 but not with MBP alone
(Fig. 3). Specific interaction
of PDF2 with the authentic L1 box was confirmed with effective competition
with the unlabeled probe and no complex formation with the mutated L1 box
(Fig. 3).
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Isolation of pdf2 and atml1 knockout mutants
To define the role of PDF2 in L1 layer differentiation, we screened a
collection of 60,480 T-DNA tag lines
(Krysan et al., 1999) for
knockout mutants of the PDF2 gene by using a PCR-based screening
strategy, and we identified one allele designated pdf2-1. The
pdf2-1 allele has a T-DNA insertion in the fifth exon of the
PDF2 gene (Fig. 1A) but exhibits no abnormal phenotype with respect to growth and morphology. We
further screened for ATML1 knockout mutants and isolated one allele
designated atml1-1. The atml1-1 allele has a T-DNA insertion
in the ninth exon of the ATML1 gene
(Fig. 1A) but also displays
normal growth and morphology.
We therefore examined the effect of double mutation. Because both loci are located on the same chromosome, we first selected plants that were homozygous for pdf2-1 and heterozygous for atml1-1 in the F2 population of the cross between pdf2-1 and atml1-1 mutants and examined segregation of genotypes in their selfed progeny (Table 1). We found that all plants showing severe defects in cotyledon development were homozygous for both pdf2-1 and atml1-1 (Fig. 4A). The same result was obtained in the progeny of plants that were homozygous for atml1-1 and heterozygous for pdf2-1 (Table 1). Furthermore, in reciprocal crosses of plants that were homozygous for pdf2-1 and heterozygous for atml1-1 with wild-type plants, progeny that was heterozygous for atml1-1 or homozygous for the wild-type ATML1 allele segregated at a ratio of 1:1. The whole progeny was heterozygous for pdf2-1 and showed normal growth. Thus, neither PDF2 nor ATML1 are required for germ cell development. SEM showed shoot apical dome-like structures in pdf2-1 atml1-1 mature embryos (Fig. 4B), but sections revealed an irregular surface of the shoot apex and a lack of distinct cell layers (Fig. 4C). In contrast, the anatomy of the root apical meristem and root growth in pdf2-1 atml1-1 were indistinguishable from the wild type (Fig. 4D and data not shown).
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Although pdf2-1 atml1-1 double mutants failed to survive after
germination under greenhouse conditions, those grown in MS agar supplemented
with 3% sucrose produced leaves that appeared moist, glossy and more pointed
than wild-type leaves (Fig.
5A-C). The surface of these leaves appeared to lack an epidermis
(Fig. 5D). The adaxial and
abaxial surfaces of pdf2-1 atml1-1 leaves consisted of cells that
resembled wild-type palisade and spongy mesophyll, respectively
(Fig. 5E-H). In cross sections
of double mutant leaves, vascular tissue but no epidermal cells were observed
(Fig. 5I). However, we
occasionally observed abnormal clusters of stomatal guard cells on both
adaxial and abaxial leaf surfaces (Fig.
5J). So far, all double mutant plants died without having produced
flowers. We also examined whether the pdf2-1 atml1-1 double mutation
affects transcript levels of L1-specific genes. RT-PCR analysis revealed that
pdf2-1 atml1-1 seedlings accumulated no transcripts of PDF1
and ACR4 (Tanaka et al.,
2002), an Arabidopsis homolog of the maize
CRINKLY4 gene that encodes a receptor protein kinase implicated in
leaf epidermis differentiation (Becraft et
al., 1996
) (Fig.
6).
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PDF2 overexpression delays flowering
To examine the effect of PDF2 overexpression, transgenic
Arabidopsis plants in which the full-length PDF2 cDNA is
transcribed under the control of the CaMV 35S promoter were generated. We
obtained 15 independent lines, and they were classified into two groups based
on their phenotypes, one showing delayed flowering and another showing altered
flower morphology. RNA gel blot analysis using total RNA from flower bud
clusters revealed that the transgenic lines with the late-flowering phenotype
accumulated much higher levels of PDF2 mRNA than did the wild type,
whereas the lines with abnormal flowers accumulated reduced levels of
PDF2 mRNA (Fig. 7).
These results suggest that the late-flowering phenotype is caused by
overexpression of PDF2 and the abnormal flower development is a
consequence of reduced PDF2 expression.
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When grown at 22°C under continuous illumination, the PDF2 overexpression lines produced approximately 12 extra rosette leaves before bolting compared to wild-type plants (Fig. 8A,B). In situ hybridization revealed that PDF2 mRNA was ectopically expressed throughout the shoot apex of these overexpression lines (Fig. 8C,D). However, the PDF2 overexpression had no effect on the mRNA levels of ATML1 and PDF1 (Fig. 7).
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Morphology of plants with reduced PDF2 expression
We also generated plants with the antisense PDF2 construct and
found abnormalities in flower development (not shown) which were almost
identical to those found in plants carrying the 35S::PDF2 construct
but showing reduced PDF2 mRNA accumulation
(Fig. 9). Therefore, the
abnormal flower phenotype observed in these lines with the 35S::PDF2
construct is most probably because of co-suppression of the endogenous gene
with the introduced construct (for a review, see
Depicker and Van Montagu,
1997). Interestingly, these co-suppression and antisense plants
also showed reduction in ATML1 and PDF1 mRNA levels
(Fig. 7 and not shown).
Morphological aberrations were found in sepals and petals. Sepals of these
plants were often fused along their edges toward the base, and petals were
short and narrow (Fig. 9A,B).
Although these flowers did not fully open and usually failed in
self-pollination, we confirmed by enforcing crosses that the fertility was
normal. SEM revealed that no phenotype was manifested at young bud stages
(Fig. 9C,D). Examination of
tissue sections revealed no obvious difference between the cell-layered
structures of shoot apical meristems in the wild type and these transgenic
lines (Fig. 9E,F).
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The epidermal surface morphology of each floral organ was further examined
by using SEM. At the mid-floral stage (stage nine according to Smyth et al.)
(Smyth et al., 1990), petal
epidermal cells of wild-type flowers are rounded and some of them are still
under division (Fig. 9G). At
maturity, the adaxial (interior) epidermal cells become more cone-shaped with
straight cuticular ridges, whereas the abaxial (exterior) epidermal cells
become cobblestone-like in appearance (Fig.
9I,K). Petal epidermal cells of the lines with reduced
PDF2 expression were noticeably large and rugged at stage nine
(Fig. 9H). Later, they became
tubular on both adaxial and abaxial sides
(Fig. 9J,L). In contrast,
wild-type sepals differentiated stomata cells and some extremely elongated
cells in the abaxial epidermis (Fig.
9M). Sepals of the lines with reduced PDF2 expression
also contained stomata cells but fewer elongated cells in the abaxial
epidermis (Fig. 9N). Epidermal
cell morphology in anthers, filaments, carpels and other vegetative organs was
indistinguishable between the wild type and these transgenic lines (not
shown).
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DISCUSSION |
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High similarity of the homeodomain among the members of the HD-GL2 class
raises the possibility that they may share the same L1 box as a target-binding
site and regulate an overlapping set of target genes. Our searches of the
GenBank database revealed that the Arabidopsis genome contains 16
genes of the HD-GL2 class (not shown). Four maize genes of this class are
expressed in distinct regions of the embryonic protoderm during early
development (Ingram et al.,
2000). Functional specificity of these members in recognition of
target genes might be conferred by temporal and spatial expression patterns of
each individual and combinatorial interactions with other transcription
factors of the same or different class to form a transcription complex. The
presence of a ZIP motif indicates potential dimer formation
(Sessa et al., 1993
). In
contrast, many studies on Drosophila homeodomain proteins have
suggested the importance of cofactor interactions in modulating DNA-binding
site specificity, transcriptional activity or both
(Popperl et al., 1995
). The
predicted START domain of PDF2 might also serve as a regulatory domain.
Alterations in the START domain of Arabidopsis PHABULOSA and
PHAVOLUTA, both of which belong to an HD-ZIP class distinct from the HD-GL2
class, may render the proteins constitutively active and cause a dominant
phenotype of abnormal radial patterning in shoots
(McConnell et al., 2001
).
The effect of PDF2 overexpression indicates that PDF2 is
insufficient for ectopically activating PDF1, which contains an L1
box within the promoter region, suggesting the requirement for another
factor(s) for its ectopic expression. It is possible that additional
cis-elements other than the L1 box and their binding factors are involved in
the activation of target genes. Consistent with this, chimeric promoter
constructs consisting of tandemly repeated L1 box-containing fragments (21 bp
x 4) of PDF1 and a minimal 90-bp CaMV 35S promoter with a
reporter gene did not activate the reporter (M. A., unpublished). The
late-flowering phenotype is reminiscent of the one reported for fwa
mutants, in which the FWA gene encoding a homeodomain protein of the
HD-GL2 class is ectopically expressed
(Soppe et al., 2000). In
wild-type plants, FWA is expressed only in developing and germinating
seeds (Soppe et al., 2000
). It
remains unknown whether FWA binds to the L1 box element or not. If so, both
PDF2 and FWA might activate or repress a common target gene(s) to delay
flowering.
PDF2 and ATML1 function in shoot epidermal cell
differentiation
Based on the phenotype, we conclude that the pdf2-1 atml1-1 double
mutant fails to differentiate epidermal cells. Surprisingly, this has little
effect on the development of the mesophyll and the vascular cells, and on the
establishment of dorsiventrality. Furthermore, expression of PDF1 and
ACR4 was found to be downregulated in pdf2-1 atml1-1. These
results suggest that PDF2 and ATML1 activate L1-specific
genes, and consequently serve in the differentiation of epidermal cells from
the L1 of shoot meristems. The common location of PDF2 and
ATML1 in a duplicated block of the Arabidopsis genome,
together with the similarity of the expression pattern and the absence of
abnormal phenotypes in single mutants, suggests that PDF2 and
ATML1 are functionally interchangeable. The double mutant leaves have
clusters of guard cells which are normally differentiated from protodermal
cells with some spacing and mature basipetally
(Pyke et al., 1991;
Larkin et al., 1997
). This
suggests that the competence to form stomatal initials is present in
pdf2-1 atml1-1 plants.
Transgenic lines with reduced PDF2 expression levels displayed the
abnormal flower phenotype. The apparent contradiction between this phenotype
and the absence of the abnormal phenotype in pdf2-1 may be
attributable to the difference in the ATML1 expression level, which
is not affected in pdf2-1 but reduced in the transgenic lines. Given
the fact that PDF2 is not necessarily required for ATML1
expression (Fig. 6), the
reduced ATML1 expression levels found in these transgenic lines may
not be caused by reduced PDF2 expression but by concurrent
co-suppression of PDF2 and ATML1 with the 35S::PDF2
construct because of the high sequence similarity between them. The abnormal
phenotype became manifest preferentially in the epidermis of sepals and
petals, whereas no phenotype was detected in stamens, pistils and other
vegetative organs. The total amount of PDF2 and ATML1 gene
products in the transgenic lines could be still sufficient for normal
development of these organs even though some genes, including PDF1,
would be affected. The critical requirement for the PDF2 or ATML1 function in
sepal and petal epidermal cell differentiation might be in agreement with the
predominant contribution of L1-derived cells in sepals and petals in
Arabidopsis (Jenik and Irish,
2000).
How is the L1 layer established and maintained?
In summary, our results suggest that both PDF2 and ATML1 function in
activating L1 layer-specific genes through interaction with the L1 box and
consequently serve in the differentiation of epidermal cells from the L1 layer
of shoot and floral meristems (Fig.
10A). As is the case with ATML1, the 5' promoter
region of PDF2 contains an L1 box
(Fig. 1A), which may suggest a
positive feedback loop of PDF2 expression. Because PDF2 and
ATML1 are expressed in the quadrant-stage embryo
(Lu et al., 1996;
Sessions et al., 1999
)
(Fig. 2F), such a feedback loop
would seem to necessitate negative regulators that suppress expression in the
basal and inner cells at the 16-cell stage
(Fig. 10B). Although both of
the PDF2 and ATML1 promoter regions contain a potential
target site of WUS (Fig. 1A)
whose expression starts at the four apical inner cells in 16-cell embryos
(Mayer et al., 1998
), it
remains to be determined whether the suppression of the autoregulatory loop of
PDF2 and ATML1 involves WUS-like transcription factors.
Furthermore, the mechanism of the initial onset of PDF2 and
ATML1 expressions before the eight-cell stage remains unknown.
Identification of additional regulatory molecules will be the next important
step to elucidate the mechanisms of the establishment and maintenance of the
L1 layer in higher plants. Phenotypes similar to that of pdf2 atml1
have been reported for gurke
(Torres-Ruiz et al., 1996
) and
tumorous shoot development (Frank
et al., 2002
) mutants of Arabidopsis. Detailed analysis
of these mutated genes may provide additional clues.
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ACKNOWLEDGMENTS |
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