1 Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT, UK
2 Max-Planck Institut für Züchtungsforschung,
Carl-von-Linné-Weg 10, 50829 Köln, Germany
Author for correspondence (e-mail:
b.h.davies{at}leeds.ac.uk)
Accepted 18 November 2003
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SUMMARY |
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Key words: Antirrhinum, Arabidopsis, Boundaries, Meristems, Transcription factors
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Introduction |
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Another phenotypic feature of nam and the cuc mutants is
an inability to form a SAM during normal development. In nam mutants,
the Petunia apical meristem fails to form in most seedlings, but a
variable proportion eventually manage to produce an escape shoot with a
functional SAM (Souer et al.,
1996). Double and triple mutants involving cuc2 and
cuc1, and/or cuc3, also lack a functional SAM
(Vroemen et al., 2003
). In the
case of cuc1 cuc2 double mutants, shoots bearing a functional SAM
have been regenerated from double mutant calli
(Aida et al., 1997
). SHOOT
MERISTEMLESS (STM), which encodes a homeodomain-containing
factor, is one of the key determinants of the Arabidopsis SAM
(Long et al., 1996
). As
STM is not expressed in cuc1 cuc2 double mutant embryos, it
is possible that CUC1 and CUC2 exert their influence on SAM
formation by promoting the expression of STM
(Aida et al., 1999
). Indeed,
CUC1 and CUC2 can even promote adventitious shoot formation
on calli in an STM-dependent manner
(Daimon et al., 2003
). It has
been suggested that a boundary region of reduced cell division is required for
the establishment of a SAM, and that the CUC and STM genes
participate in the formation of such a niche of cells from which meristems can
be formed (Vroemen et al.,
2003
).
Neither the nam nor the cuc1 cuc2 mutants affect the
boundaries of vegetative or inflorescence lateral organs outside the flower,
raising the possibility that other genes are involved in the establishment of
these boundaries (Souer et al.,
1996). Here, we describe cupuliformis (cup)
(Stubbe, 1966
), a classical
Antirrhinum mutant that shows extensive fusion of all lateral organs
and a transitory defect in SAM initiation. We show that CUP encodes a
NAC-domain transcription factor, related to NAM and the CUCs. In contrast to
nam and cuc1 cuc2, cup mutants strongly affect all lateral
organ boundaries, suggesting a greater redundancy of function for the boundary
factors in Petunia and Arabidopsis. Furthermore, we
demonstrate a direct interaction between CUP and a TCP-domain factor, TIC,
which suggests a model for the establishment of organ boundaries in
plants.
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Materials and methods |
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cDNA isolation
The full-length CUP cDNA was isolated by screening an
Antirrhinum cDNA library using a mixed PCR probe generated by
amplifying the conserved Arabidopsis NAC domain region by RT-PCR,
using primers 5'-TCCCACGGACGAAGAGCTCATCAC-3' and
5'-TGGAGCTTCTTGAGATGAAATGGTAAG-3'. The Genomewalker kit (Clontech)
was used to determine the sequence within the 5'-untranslated region of
TIC. Primer TIC1 (5'-GGAAGGATCCGAAGGAGGCGATGAT-3'), derived from
this region, together with primer TIC-2
(5'-ATAAGAATGCGGCCGCTCAAGAATGGTGACTTGTG-3'), derived from the
3' sequence of the truncated TIC clone obtained in the
two-hybrid screen, was used to obtain a full-length TIC transcript by
RT-PCR. Two independently amplified PCR products were sequenced.
Yeast two-hybrid analysis
The full-length CUP gene, lacking the ATG start codon, was cloned
into the DNA-binding-domain vector pGBT9 (Clontech) and transformed into yeast
strain HF7c (Feilotter et al.,
1994), to form the bait strain. The screen was carried out using a
random-primed, whole-plant Antirrhinum cDNA library, as previously
described (Davies et al.,
1996
). Positive colonies appearing within 10 days of the screen
were confirmed by growth in the presence of 20 mM 3-amino-1,2,4-triazole
(3AT). Plasmid DNA was recovered from the positive yeast colonies by
transformation into E. coli, and interactions were verified by
re-transformation of bait and prey into the yeast strains HF7c and SFY526
(Bartel et al., 1993
), as
previously described (Davies et al.,
1996
).
In vitro GST-pulldown assays
A full-length GST-CUP fusion was produced by cloning a CUP PCR
fragment, amplified using primers that introduce BamHI and
NotI sites, into pGEX-4T-1 (Amersham Pharmacia Biotech) to form
pGSTCUP. TIC was also cloned into pGEX-4T-1, between the same
restriction sites, to form pGST-TIC. pGEX-4T-1, pGST-CUP and pGST-TIC were
used to transform E. coli BL21 (DE3) cells (Stratagene). Proteins
were produced and GST pulldowns carried out essentially as described
(Causier et al., 2003), with
the exception that following lysis the pellets were re-suspended into a
solution of 1.5% N-Lauroylsarcosine, 25 mM Triethanolamine, 1 mM EDTA (pH
8.0), and incubated for 10 minutes at 4°C followed by a further
centrifugation to remove cell debris.
Expression analysis
Seedlings and inflorescences were embedded, sectioned and hybridised as
described (Zachgo et al.,
2000). A DIG-labelled antisense RNA probe was transcribed from a
600 bp PCR fragment containing the 3' region of CUP, outside the
conserved NAC-domain. A sense control probe was also tested and no signal
obtained (not shown).
RT-PCR analysis was carried out using first strand cDNA generated from 0.5 µg DNase treated total RNA, using oligo dT and Omniscript RT (Qiagen) according to the manufacturer's instructions. PCR was performed with RT reaction corresponding to 0.0125 µg total RNA using Expand High Fidelity PCR System (Roche) with the following oligos:
TICSMA, 5'-gaattcccgggggaaggaggcgatgatcat-3';
TICBAM3, 5'-cgggatcctcaagaatggtgacttgt-3';
CUPBAM5, 5'-ggaaggatccgagaattacaattgctac-3';
CUPRI3, 5'-ggaagaattcctagtaaccccaaatacg-3';
AMEF1, 5'-tgagaccaccaagtactactg-3'; and
AMEF2, 5'-caacattgtcaccgggaagag-3'.
All primers were confirmed to be specific for the gene to be amplified.
Northern blots were performed as previously described
(Sommer et al., 1990).
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Results |
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cup differs from similar mutants in other species
The cup phenotype can be characterised as a failure to form a SAM
or axillary meristems and an inability to define lateral organ boundaries.
There are clear differences between the phenotypes of cup mutants and
organ boundary mutants in other species
(Souer et al., 1996;
Aida et al., 1997
;
Takada et al., 2001
). The
Petunia nam mutant has fused cotyledons and the SAM is absent. In
contrast to cup, most nam seedlings die, but some continue
to develop by forming an escape shoot. Unlike cup escape shoots,
those nam escape shoots which go on to produce a mature plant, show
normal vegetative and inflorescence development. The Arabidopsis cuc1
cuc2 double mutant also has fused cotyledons. In this case, the
individual single mutants look normal, except for a tendency towards partial
fusion of the cotyledons. cuc1 cuc2 double mutants do not form escape
shoots naturally, but shoots can be induced by regeneration from mutant calli.
As with nam escape shoots, cuc1 cuc2 shoots do not show the
dramatic organ fusion and fasciation effects observed in cup escape
shoots. Flowers produced from regenerated cuc1 cuc2 plants show
fusion of sepals and stamens, petal loss and female sterility. Like
cup flowers, cuc1 cuc2 flowers show a reduction in ovule
number (Ishida et al., 2000
).
nam flowers are male and female sterile, contain an extra whorl of
petals that become fused to the stamens, and have abnormal gynoecia with
aberrant placental and ovule development. In summary, the embryonic and floral
phenotypes of cuc1 cuc2, nam and cup are similar, but only
cup mutants show the striking vegetative defects, which include
repetitive generation of cup-tipped shoots from the hypocotyl, extensive
fusion of leaves, loss of axillary meristems and fasciation.
CUP encodes a NAC-domain gene
NAM, CUC1, CUC2 and CUC3 all encode NAC-domain proteins
(Aida et al., 1997). NAC-domain
proteins form a large family of transcription factors, with more than 100
members in Arabidopsis. The similarity between embryonic and floral
aspects of the cup, cuc1 cuc2 and nam phenotypes raised the
possibility that the cup phenotype resulted from a defect in a
related NAC-domain gene. To test this hypothesis, 25 Antirrhinum
NAC-domain genes were isolated from a cDNA library using a mixture of
heterologous NAC-domain genes as a probe (Accession Numbers: AJ568261-AJ568281
and AJ568337-AJ568340). The closest match to NAM and CUC2
was tentatively named CUP (Fig.
3A), and was used as a probe on Southern blots from segregating
cup populations. Both alleles of cup showed co-segregating
polymorphisms, suggesting that they were caused by transposon insertions. PCR
analysis with gene specific and generic transposon primers identified
insertions of transposons in both alleles
(Fig. 3B). Molecular and
genetic analysis confirmed that escape shoots formed in each allele are not
revertants. In the absence of reversion of either allele, co-segregation
analysis was extended to 73 plants in three segregating populations. In all
cases, the transposon insertion showed absolute cosegregation with the mutant
phenotype. Northern blot analysis confirmed that the CUP gene is not
expressed in either cup allele
(Fig. 3C). Taken together, the
similarity of CUP to NAM and the CUC factors, the presence of transposon
insertions in both alleles of cup, the absolute co-segregation of the
insertions with the mutant phenotype and the lack of CUP expression
in the homozygous cup mutants indicates that loss of CUP
expression is responsible for the phenotypes observed in the cup
mutants.
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CUP interacts with a TCP-domain factor
Three different species, Petunia, Arabidopsis and
Antirrhinum have now been shown to use related NAC-domain genes to
establish the boundaries between lateral organs. In an attempt to understand
how expression of these genes, in cells destined not to develop into organs,
could result in the formation of discrete boundaries, a yeast two-hybrid
screen was performed using CUP as bait. Eight putative CUP interactors were
isolated from the 5x106 colonies screened. DNA sequence
analysis showed that six of these corresponded to known yeast two-hybrid
false-positive sequences, leaving two candidate interactors. One of these
cDNAs was unable to encode a protein because of stop codons in all reading
frames and the other was found to encode a truncated member of the PCF
subclade of the TCP family of transcription factors
(Cubas et al., 1999), which we
designated TIC (TCP-Interacting with CUP). A cDNA corresponding to
full-length TIC was subsequently isolated (Accession Number:
AJ580844). Full-length TIC encodes a 398 amino acid protein with a
characteristic TCP-domain between residues 76-130. The interaction between CUP
and full-length TIC was confirmed by GST pulldown
(Fig. 5A). Interaction between
CUP and TIC was detectable when either partner was expressed as a GST fusion.
In addition, interaction was also detected between labelled TIC and the
TIC-GST fusion protein, suggesting that TIC is also capable of interacting
with itself. Homodimerisation and heterodimerisation between TCP-domain
factors has previously been reported
(Kosugi and Ohashi, 2002
).
TIC expression appears to be too low to be detected by in situ
hybridisation, as no signal could be detected. However, RT-PCR analysis
confirmed that TIC expression overlaps with that of CUP
(Fig. 5B).
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Discussion |
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It appears that the determination of lateral organ boundaries in plants is
controlled by a class of plant-specific transcription factors: the NAC-domain
factors. Although NAC-domain genes form a large gene family, little is known
about their functions. The results presented here support the idea that
members of this gene family are functionally redundant. The Antirrhinum
cup mutant affects lateral organ boundaries throughout the plant,
suggesting that the lack of vegetative and inflorescence phenotypes seen in
nam and cuc1 cuc2 are due to redundancy within this family.
Redundancy of gene function has been previously postulated for NAM,
both to explain the discrepancy between its expression pattern and sites of
phenotypic abnormality (Souer et al.,
1996), and because co-suppression experiments, in which several
NAC-domain genes are silenced, result in a lack of lateral meristems
(Souer et al., 1998
).
Redundancy is also apparent amongst the Arabidopsis CUC genes, but
even in cuc1 cuc2 double mutants vegetative and inflorescence
development is unaffected (Aida et al.,
1997
). Preliminary phenotypic observations have been reported to
suggest that cuc3 plays a role in the establishment of boundaries in
adult plants (Vroemen et al.,
2003
). It therefore appears that less redundancy exists for this
function in Antirrhinum, where the loss of a single gene affects all
lateral organ boundaries. Our low stringency heterologous screen for
NAM/CUC related genes in Antirrhinum identified 25
different NAC-domain containing genes, but only CUP belonged to the
NAM/CUC subclade of this large gene family. This further
supports the view that less redundancy exists for this function in
Antirrhinum than in Arabidopsis. However, as lateral
meristems can occasionally form in cup mutants, and fusion is not
observed between all lateral organs, it remains possible that another function
is also involved in specifying lateral organ boundaries. This could be a
member of the NAC-domain family or an unrelated factor.
A model for boundary formation in plants
The finding that CUP interacts with a TCP-domain factor suggests a model
for the establishment of lateral organ boundaries. The best characterised
members of the TCP-domain factor family are CYCLOIDEA (CYC) and TEOSINTE
BRANCHED 1 (TB1) (Luo et al.,
1996; Doebley et al.,
1997
). A common feature of both these TCP-domain factors is their
role in the prevention of organ outgrowth
(Cubas et al., 1999
).
CYC acts early in flower development to reduce the growth rate in the
dorsal part of the floral meristem. Later, CYC activity prevents the
growth of the dorsal stamen. Similarly in maize, TB1 activity
prevents the outgrowth of lateral branches. Another Antirrhinum
TCP-domain gene, CINCINNATA (CIN), provides further evidence
for the connection between this gene family and the regulation of cell
division (Nath et al., 2003
).
The leaves of cin mutants are wrinkled rather than flat because of a
failure to restrict cell division in the leaf margins. During the development
of leaves in wild-type plants, a front of cell cycle arrest moves
progressively from the tip of the leaf towards the base. In cin
mutants this cell cycle arrest front moves more slowly, resulting in excessive
cell division before eventual arrest. The expression pattern of CIN
in developing leaves suggests that CIN might act to sensitise cells
to the arrest signal (Nath et al.,
2003
). Thus, mutational studies in different species have
identified TCP-domain genes as negative regulators of growth.
Although the mechanism by which these TCP-factors might control cell
division is not yet understood, the other founder members of this family,
PCF1 and PCF2, could provide a clue. PCF1 and
PCF2 were identified in a screen for factors which bind to sites in
the promoter of the rice proliferating cell nuclear antigen (PCNA)
gene, which itself has a role in DNA-synthesis and cell cycle control
(Kosugi and Ohashi, 1997).
More recently an Arabidopsis TCP-domain protein, At-TCP20, was shown
to bind to a motif in the promoter of Arabidopsis PCNA, and in the
promoters of other genes regulated during the G1-S phase transition
(Trémousaygue et al.,
2003
). All three of these TCP-domain factors, which belong to the
same PCF subclade of TCP-domain genes as TIC, are suggested to activate
expression in meristematic cells, but it is also possible that interaction
with other factors could result in repression (or lack of activation) of
target genes. This leads us to postulate a model whereby the NAC-domain factor
CUP, expressed in specific boundary domains surrounding all lateral organs,
interacts with the more widely expressed TCP-domain factor TIC, resulting in
either repression, or failure of activation, of genes which promote cell
division. As a consequence of this interaction, cell division is reduced
between developing meristems and primordia, resulting in the formation of
discrete lateral organs with defined boundaries.
The interaction between CUP and the TCP-domain factor TIC also provides a
possible explanation for the radialisation seen in cup flowers.
Radialisation of the flower is found in cyc and dichotoma
(dich) mutants (Luo et al.,
1999), which result from defects in TCP-domain encoding genes. The
radialisation found in cup mutants could be explained in a variety of
ways, including the direct interaction of CUP and CYC, and/or DICH, or an
alteration in the stoichiometry of interactions within the TCP family
following the removal of a TCP-factor interactor. Alternatively, radialisation
might be a consequence of the loss of organ boundaries and breakdown in
organisation of the floral meristem. Preliminary yeast two-hybrid experiments
suggest that CUP is indeed capable of interacting with CYC (J.L. and B.D.,
unpublished). As we have discovered a direct protein-protein interaction
between members of the NAC-domain and TCP-domain families it is possible that
other members of these families also interact.
The NAC-domain genes NAM, CUC1, CUC2, CUC3 and CUP act to
establish boundaries between lateral organs. The extreme phenotype observed in
cup mutants supports the idea that further redundancy will be
discovered amongst these genes in Petunia and Arabidopsis,
and suggests that members of this gene family regulate the separation of all
lateral organs in plants. The interaction between NAC-domain factors and
TCP-factors provides a model to explain how boundaries might be established,
but this is unlikely to be the whole picture. Several other genes, such as
LATERAL ORGAN BOUNDARIES (LOB), are also expressed at
boundaries (Shuai et al.,
2002). Further work is required to assess the contribution of all
these genes to the establishment of organs boundaries and meristem
formation.
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ACKNOWLEDGMENTS |
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Footnotes |
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