1 Institute of Cell and Molecular Biology, University of Edinburgh, King's
Buildings, Mayfield Road, Edinburgh EH9 3JR, UK
2 Section of Plant Biology, University of California Davis, 1 Shields Avenue,
Davis, CA 95616, USA
Author for correspondence (e-mail:
andrew.hudson{at}ed.ac.uk)
Accepted 1 April 2004
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SUMMARY |
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Key words: Antirrhinum majus, GRAMINIFOLIA, PROLONGATA, YABBY, Leaf asymmetry
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Introduction |
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In Arabidopsis, the related HD-ZIP genes
PHABULOSA (PHB), PHAVOLUTA (PHV) and
REVOLUTA (REV) specify adaxial leaf identity. The activity
of these genes normally becomes restricted to the adaxial domain of newly
initiated leaf primordia (McConnell et
al., 2001; Otsuga et al.,
2001
). The characterisation of gain-of-function PHB and
PHV alleles, which result in their ectopic abaxial expression and
adaxial fate, have suggested two mechanisms that might normally limit PHB and
PHV activity to the adaxial domain of developing organs. One proposes that the
HD-ZIP proteins are activated by binding a ligand adaxially, promoting their
own expression so that gain-of-function mutations, which affect the potential
ligand binding site, render the proteins constitutively active
(McConnell et al., 2001
). The
hypothetical ligand may come from the centre of the SAM, because leaf initials
surgically isolated from the SAM fail to form adaxial cell types
(Sussex, 1955
). The second
explanation is that a short microRNA (miRNA) complementary to wild-type RNA
from the HD-ZIP loci causes degradation of PHB and
PHV, and possibly REV, transcripts in the abaxial leaf
domain (Emery et al., 2003
;
Reinhart et al., 2002
;
Rhoades et al., 2002
).
Consistent with this model is the finding that these miRNAs accumulate in the
abaxial domain of Arabidopsis and maize lateral organs
(Juarez et al., 2004
;
Kidner and Martienssen, 2004
).
Because transcripts from the gain-of-function alleles no longer match the
miRNA perfectly and are resistant to degradation
(Tang et al., 2003
), they
might persist in the abaxial domain to specify ectopic adaxial fate. This
second model does not exclude the possibility that the HD-ZIP proteins are
also activated by a ligand. However, these models make different assumptions
about how organ asymmetry is first specified. Adaxial HD-ZIP activation by a
ligand from the centre of the SAM could constitute the first step in organ
polarisation, whereas inactivation by the abaxially localised miRNA implies
that the organ is already polarised or that the miRNA is itself the polarising
signal.
KANADI (KAN) genes, which are both necessary and
sufficient for abaxial fate in Arabidopsis leaves
(Eshed et al., 1999;
Eshed et al., 2001
;
Kerstetter et al., 2001
), are
needed to limit HD-ZIP gene expression to an adaxial domain
(Eshed et al., 2001
). Because
loss of PHB, PHV and REV activity has a similar effect to ectopic adaxial
expression of KAN, it has been suggested that HD-ZIPs act at
least partly by restricting the domain in which KAN genes promote
abaxial fate (Eshed et al.,
2001
; Emery et al.,
2003
). However, it is currently unclear how asymmetric expression
of HD-ZIP and KAN genes is first established, and the extent
to which each gene family acts by repressing the other.
Another family of transcription factor genes - the YABBY
(YAB) genes - are implicated in abaxial organ fate because their
expression is restricted abaxially in organ primordia and yab
mutations disrupt development of abaxial cell types
(Sawa et al., 1999;
Siegfried et al., 1999
;
Villanueva et al., 1999
).
Reduced activity of the two YAB genes FILAMENTOUS FLOWER
(FIL) and YAB3 results in a partial loss of abaxial cell
identity but not its replacement by adaxial identity, as in kan
mutants (Eshed et al., 2001
;
Siegfried et al., 1999
).
Similarly, ectopic YAB expression is not sufficient to confer abaxial
fate on all cells and the ability of ectopic KAN expression to
abaxialise cells is not dependent on FIL or YAB3 activity. The role of
YAB genes in organ asymmetry is therefore enigmatic.
Although most flowering plants, in common with Arabidopsis, have
asymmetric lateral organs the extent to which their regulatory mechanisms are
conserved remains largely untested. In the distantly related eudicot,
Antirrhinum majus, the MYB gene PHANTASTICA
(PHAN) acts redundantly with other factors that are sensitive to cold
to promote adaxial identity - loss of PHAN activity causes
abaxialisation of organs and loss of lateral growth
(Waites and Hudson, 1995;
Waites et al., 1998
). Reduced
activity of the Arabidopsis PHAN orthologue, ASYMMETRIC
LEAVES1 (AS1), has a lesser effect on ad-abaxial organ asymmetry
(Byrne et al., 2000
;
Xu et al., 2003
) and it is
unclear whether the different developmental requirements for PHAN and
AS1 reflect divergence in the functions of genes that they regulate.
The only known target of PHAN - the homeobox gene, HIRZINA,
which it represses in leaves and petals - does not cause asymmetry defects
when mis-expressed (Golz et al.,
2002
). It is therefore not obvious whether PHAN might
regulate the orthologues of genes that control organ asymmetry in
Arabidopsis.
To further understand the control of organ asymmetry and growth we have analysed the roles of the paralogous Antirrhinum YAB genes, GRAMINIFOLIA (GRAM) and PROLONGATA (PROL). GRAM expression becomes confined to an abaxial domain at the margins of leaf primordia, where it promotes lateral growth and abaxial identity. However, the role of GRAM in promoting abaxial identity is redundant if adaxial fate is not specified, suggesting that GRAM acts to exclude adaxial identity - a role supported by ectopic abaxial expression of a PHB homologue in gram mutant leaves. Although expression of both GRAM and PROL is confined to an abaxial domain by PHAN activity, GRAM acts redundantly with PHAN and with PROL to promote adaxial organ identity non cell-autonomously. GRAM expression in only the abaxial epidermal cell layer of organ primordia is sufficient to confer normal identity and growth to more adaxial cells, indicating that GRAM promotes abaxial fate non cell-autonomously. The contrasting roles of GRAM in promotion and repression of adaxial fate might serve to define and reinforce an ad-abaxial boundary required for continued leaf growth.
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Materials and methods |
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Periclinal chimeras for an olive (oli) mutation, which
prevents accumulation of chlorophyll at higher light intensities
(Hudson et al., 1993), were
generated from the unstable oli-605 allele in gram-1 and
GRAM+ backgrounds. Plants were grown at 15°C for 10
days to induce excision of the Tam3 transposon from oli-605,
then maintained at 25°C, to inhibit further transposition, in a light
intensity of
200 µmol/m2/second, to distinguish
OLI+ revertant and oli mutant tissue. Chlorophyll
was identified in hand-cut sections by epifluorescence at 365 nm excitation.
Periclinal chimeras were maintained and propagated vegetatively from
cuttings.
phan gram double mutants were obtained in the F2 of either phan-249 x gram-1 (in the Sippe 50 genetic background), or phan-607 x gram-3 (in the JI.75 background). About 6% of F2 progeny showed an enhanced mutant phenotype that included lack of an embryonic apical meristem. These plants were confirmed as gram phan double mutants by Southern hybridisation. gram prol and phan prol double mutants were obtained in the F2 of gram-1 x prol-1 and phan-249 x prol-1, respectively, and their genotypes confirmed by PCR.
Molecular biology
cDNA clones of AmYAB2 (AY451398), AmFIL (GRAM,
AY451396) and AmYAB5 (PROL; AY451397) were obtained by
low-stringency screening of an Antirrhinum inflorescence cDNA library
with the Arabidopsis INNER NO OUTER gene
(Villanueva et al., 1999).
Additional cDNAs from these genes and two additional paralogues,
AmCRC (AY451399) and AmINO (AY451400) were kindly provided
by Zsuzsanna Schwarz-Sommer (MPIZ, Germany). The introns of AMYAB3
and AMYAB5 were identified by PCR amplification of genomic DNA.
Sequence phylogenies were reconstructed from inferred full-length amino acid
sequences using CLUSTAL and PAUP software.
Both gram-1 and gram-3 gave rise to a low frequency of
wild-type progeny and gram-3 produced wild-type branches, consistent
with both mutations being caused by unstable transposons. Transposons were
identified by PCR with transposon- and GRAM-specific primers. Primers
to a sequence conserved in CACTA transposons were used with
AMYAB5-specific primers to screen DNA from a collection of mutants
maintained at IPK, Gatersleben. These detected a CACTA insertion within the
first intron of AmYAB5 in the inbred line, MAM265, which had slightly
larger leaves than the wild-type lines, JI.75 and Sippe 50. In an
F2 of MAM265 x JI.75 (n=94) leaf size showed
continuous variation and Student's t-tests detected no significant
differences in leaf length or width between amyab5/amyab5,
amyab5/+ and homozygous wild-type siblings (P>0.20 in all
pair-wise comparisons). This suggested that the amyab5 allele did not
condition a mutant phenotype and that the phenotype of MAM265 was consistent
with a different genetic background to JI.75 or Sippe 50. The amyab5
allele, however, segregated with an enhanced gram mutant phenotype in
6% of the F2 progeny of MAM265 x gram-1. These
plants (n=23) were confirmed as amyab5 gram-1 double mutants
by PCR genotyping, while all 18 tested gram mutant siblings carried
at least one wild-type AmYAB5 allele, indicating enhancement of the
gram phenotype by amyab5, or a very closely linked gene. Two
amyab5 gram double mutants produced branches with a gram
single mutant phenotype. PCR analysis confirmed that these branches carried
revertant AmYAB5 alleles with sequence footprints characteristic of
CACTA transposon excision; strongly suggesting that enhancement of the
gram phenotype was due to the amyab5 mutation. In the
absence of other detectable mutations in MAM265, which had originally been
proposed to carry the prolongata (prol) mutation, the
amyab5 allele was named prol-1.
Three PHB homologues, most similar to PHB, REV and ATHB8/ATH-15, respectively, were obtained by probing an Antirrhinum cDNA library with a PHB cDNA. The most PHB-like gene (AmPHB; AY451395) encoded a protein with 84% identical amino acids to PHB in a 230 amino acid region spanning the START domain.
Microscopy
Epidermal impressions were made in Loctite Superglue on a microscope slide
and examined with phase contrast optics. Histological sections (5 µM) were
made from material embedded in JB-4 resin and stained with Toluidine Blue
(Ruzin, 1999). Scanning
electron microscopy and in situ hybridisation were performed as described
previously (Golz et al., 2002
)
The digoxigenin-labelled probes GRAM-long and PROL were
transcribed from near full-length cDNA clones and the GRAM 3'
probe from the final three exons downstream of the Tam3 insertion in
gram-3. Antisense AmPHB probes were transcribed from a 700
bp cDNA that spanned the region encoding the START domain.
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Results |
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Wild-type leaves differ in size according to the node at which they are produced, reaching their maximum mature length and width at nodes 3 or 4 (Fig. 1A). All leaves of gram-1 and gram-2 mutants were consistently half the width of wild-type ones (Fig. 1A,B) and also shorter than wild type up to node 3, after which they were similar in length.
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Leaves of gram mutants have regions of darker green tissue, characteristic of the adaxial side of the leaf, at the abaxial margins (Fig. 1B). In section, elongated palisade cells extend around the edge of the leaf into the abaxial margin (Fig. 1F), making the lamina thicker towards its edge. This phenotype suggested that GRAM is needed for abaxial cell identity at the leaf margin and that adaxial identity occurs in its absence. Similarly, epidermal cells with adaxial characters were found in the abaxial margins and the cells normally associated with the leaf edge extended further into the abaxial epidermis (Fig. 1D), suggesting that GRAM also promotes abaxial identity in epidermal cells. Epidermal marginal cells, which normally form at the leaf edge overlying the junction between spongy and palisade mesophyll, were absent from the displaced adaxial-abaxial boundary in gram leaves (Fig. 1B,D).
Loss of GRAM activity also caused adaxial mesophyll cells away from the
leaf margin to partly resemble abaxial spongy mesophyll in shape and spacing
(Fig. 1F). This suggested that
GRAM is not only needed for abaxial identity at leaf margins but to
promote adaxial identity elsewhere in the leaf. More severe loss of adaxial
cell identity was observed occasionally in needle-like leaves produced by
gram-3 mutants (Fig.
1G), which contained a central vein in which xylem was surrounded
by phloem (Fig. 1H,I). Because
phloem develops abaxial to xylem in the wild-type leaf, the needle-like leaves
appeared to have lost adaxial, and gained abaxial, identity. gram
mutant petals, like leaves, were smaller than wild-type ones and free for more
of their length (Fig. 2A,E),
suggesting that GRAM is also needed for petal growth. Where petals
remained united, pronounced furrows developed in their adaxial (inner) sides
flanked by ridges (arrowheads in Fig.
2F,G). Cells within the furrow had ectopic abaxial identity, as
seen by their darker red pigmentation and lack of yellow hairs. Similarly, the
ridges flanking each furrow contained a radially symmetric vein with an
abaxialised arrangement of cell types (compare
Fig. 2B-D with
Fig. 2F-H), suggesting that
GRAM is needed for adaxial identity at petal margins. gram
mutants also showed reduced growth of the style and occasional homeotic
conversions of floral organ identity
(Navarro et al., 2004).
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In Antirrhinum, in common with most dicots, the SAM consists of three cell populations - a single layer of protoderm cells (L1), a single layer of sub-epidermal cells (L2) and a core of L3 cells. The fates of cells derived from the L2 layer were followed in GRAM+ and gram mutant leaves using stable periclinal chimeras in which L2 was marked by an olive mutation that reduces chlorophyll content (see Materials and methods). Towards the midrib of GRAM+ leaves, L2 contributed one layer of yellow adaxial palisade cells and one abaxial layer of yellow spongy mesophyll cells covering a core of L3-derived green cells (Fig. 3A). The medial part of the leaf therefore appeared green. Nearer the leaf edges, all internal cells were derived from L2 and therefore the margins appeared yellow. The proportion of the blade with internal L2-derived cells varied from about one-third to two-thirds of the leaf width (Fig. 3A). The boundary between green (L3-derived) and yellow (L2-derived) tissue did not correspond to any structural feature and its position varied in different leaves or in opposite halves of the same leaf. In contrast, L3 contributed most of the internal cells in a gram mutant leaf (often more cells than in GRAM+) and the position of the boundary between yellow and green tissue was more consistent (Fig. 3B). This suggested firstly, that GRAM promotes cell divisions in the margins of leaf primordia, where internal tissues are derived entirely from L2, and secondly that L3 contributes more cells to the gram mutant leaf than to the wild-type leaf, perhaps in compensation for reduced marginal growth.
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GRAM encodes YABBY activity
Both gram-1 and gram-3 showed the genetic instability
characteristic of transposon-induced mutations. Because GRAM was
needed to promote abaxial organ identity and a similar role had been
attributed to members of the YAB gene family in Arabidopsis
(Siegfried et al., 1999), we
tested whether GRAM might encode YAB activity. Five Antirrhinum
YAB genes were identified as cDNAs, each encoding a protein with the
N-terminal zinc finger and C-terminal HMG-like YAB domain characteristic of
the family (Fig. 4B).
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Tam3 was lost from gram-3 in five independent GRAM+ revertants. None carried sequence footprints, often associated with Tam3 excision, presumably because footprints would disrupt the highly conserved YAB domain. A single reversion of gram-1 to wild-type involved the loss of Tam2 together with 85 bp of flanking intron sequence. These results confirmed that AmFIL corresponded to the GRAM locus.
A transposon insertion in the AmYAB5 gene was also identified in
an inbred line carrying the classic mutation, prolongata-1
(prol-1; Fig. 4B)
(Stubbe, 1966), but
conditioned no mutant phenotype in an otherwise wild-type genetic background
(see Materials and methods).
GRAM and PROL are expressed abaxially in developing lateral organs
GRAM RNA expression, revealed by in situ hybridisation, was
similar in all lateral organs. It was detected first in incipient primordia
within the SAM or floral meristem (stage P0 in leaves) and abaxially in newly
initiated (early P1) primordia (Fig.
5A,B). It then became restricted mainly to the abaxial margins of
growing primordia from about stage P2 (Fig.
5B). This later pattern of expression was consistent with the
proposed role of GRAM in promoting abaxial cell fate and growth in
leaf margins. PROL RNA was always less abundant than GRAM
(Fig. 5D,E), but like
GRAM it was expressed abaxially from stage P1. Later expression,
unlike GRAM, was detected predominantly in provascular cells and to a
lesser extent in the mesophyll cells in the centre of each primordium.
PROL RNA was not detectable in the prol-1 mutant by in situ
hybridisation or RT-PCR (data not shown).
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Loss of PHANTASTICA (PHAN) activity has the opposite effect to
gram of allowing leaf cells in adaxial positions to assume abaxial
fates (Waites and Hudson,
1995). The degree to which phan mutant leaves are
abaxialised increases with decreasing temperature: at 25°C all leaves are
mosaics of adaxial and ectopic abaxial tissue
(Fig. 6C), at 20°C leaves
at higher nodes on the plant are needle-like and consist only of abaxial cell
types (Fig. 6A) while at
15°C phan mutants are unable to maintain a functional SAM.
|
To determine whether GRAM and PROL function redundantly to promote abaxial identity, plants were generated carrying both gram and prol-1 mutations. Surprisingly prol-1 enhanced the gram mutant phenotype in the same way as phan mutations (Fig. 6N-S). Initially all gram prol seedlings lacked a SAM, however shoots eventually formed from adventitious meristems at the root-hypocotyl junction (Fig. 6N). All of the leaves that formed on these shoots were radially symmetrical and had hairs that were specific to the abaxial surface of wild-type leaves (Fig. 6O-Q). Lack of adaxial cell types was confirmed by histology (Fig. 6R) and by the ubiquitous expression of gram mutant transcript and complete absence of AmPHB expression (Fig. 6S,T). These results suggests that PROL promotes adaxial organ fate redundantly with GRAM and that it is not required, alone or redundantly with GRAM, for abaxial fate when adaxial fate is not specified. Unlike gram mutations, prol-1 did not modify the phan mutant phenotype (data not shown).
Neither GRAM nor PROL are needed for abaxial cell fate in
the absence of adaxial identity. The role of GRAM in promoting
abaxial organ fate might therefore be to repress adaxial identity. To test
this we examined its interaction with AmPHB, an Antirrhinum
homologue of PHB, which is necessary and sufficient for adaxial fate
in Arabidopsis leaves (McConnell
et al., 2001). Sense RNA from AmPHB, like its
Arabidopsis homologue, was expressed in the wild-type SAM, uniformly
in newly initiated leaf primordia and adaxially from late stage P1
(Fig. 5G,H). In contrast,
AmPHB expression was not adaxially restricted in P2 and P3 primordia
of gram mutants and was particularly abundant at their margins
(Fig. 5I). This expression
pattern was therefore consistent with GRAM acting to repress
AmPHB expression and adaxial identity from at least stage P2.
GRAM acts non cell-autonomously
GRAM and PROL are expressed abaxially but required, non
cell-autonomously, to promote the identity of adaxial cells. To test whether
GRAM can also affect abaxial fate non cell-autonomously, we exploited
the ability of the gram-3 mutation to give rise to clones of
wild-type cells following transposon excision. Plants homozygous for the
unstable gram-3 allele occasionally produced branches with a
wild-type phenotype. In most cases, the flowers on these branches gave rise to
75% wild-type progeny on self-pollination, suggesting that the
subepidermal (L2) layer of the SAM, from which gametes are derived, carried a
revertant GRAM+ allele. One phenotypically wild-type
branch, however, produced only gram mutant progeny, suggesting that
it was a periclinal chimera carrying a revertant GRAM+
allele in either the L1 or L3 layers of the SAM. These possibilities were
tested by in situ hybridisation with a probe that could detect wild-type
GRAM RNA but not the transcripts produced from gram-3, which
terminate within the transposon insertion
(Fig. 7A-D). In the chimeric
wild-type branch, the downstream probe detected high levels of GRAM
transcripts only in L1 cells within the normal, abaxial domain of
GRAM expression (Fig.
7F), indicating that this branch had normal GRAM activity in
epidermal cells but not in sub-epidermal, L2-derived cells. Consistent with
this, a wild-type GRAM+ allele could be amplified from the revertant
branch, but not from gram mutant branches of the same plant. These
findings indicated that GRAM activity in abaxial epidermal cells is sufficient
for normal identity and proliferation of more adaxial cells, presumably via an
intercellular signal.
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Discussion |
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Loss of GRAM activity allows cells in abaxial marginal positions to assume
adaxial identities. Because the leaves of plants lacking GRAM activity in a
phan or prol mutant background retain abaxial identity,
GRAM does not appear to be necessary for abaxial fate per se, but
rather to exclude adaxial identity from the abaxial leaf margins. This does
not exclude the possibility that other genes might specify abaxial identity
independently of GRAM. Obvious candidates include other members of
the YAB gene family, at least two of which - PROL and
AmYAB2 - are expressed abaxially in developing leaves of wild-type
and abaxialised phan gram mutants (data not shown). PROL,
however, is not needed for abaxial identity, alone or in combination with
GRAM, because abaxial identity is retained in leaves lacking activity
of both GRAM and PROL or PHAN and PROL. In
Arabidopsis, members of the KAN family are also required for
abaxial fate (Eshed et al.,
2001; Kerstetter et al.,
2001
). Two KAN genes are known to be expressed in
Antirrhinum leaves (J.F.G., unpublished) and might therefore specify
abaxial fates in the absence of GRAM and PROL activity.
Activity of the HD-ZIP proteins PHB, PHV and REV is sufficient to confer
adaxial identity in Arabidopsis leaves
(Emery et al., 2003;
McConnell et al., 2001
) and
restriction of their activity to an adaxial domain is considered to be an
early step in elaboration of organ asymmetry. In Antirrhinum leaves,
GRAM is needed to restrict expression of AmPHB, a
PHB homologue, to an adaxial domain of organ primordia, consistent
with GRAM acting to repress HD-ZIP-dependent adaxial fate. It is,
however, unclear whether GRAM is needed to set up the domain of
HD-ZIP expression or to maintain it. It is also unclear whether
abaxial expression of GRAM is established in response to adaxial
HD-ZIP activity.
The role of GRAM in repressing adaxial fate differs from that
proposed for the homologous Arabidopsis genes, FIL and
YAB3. Reduced activity of both Arabidopsis genes has a
similar effect to gram mutations on leaf growth
(Kumaran et al., 2002;
Siegfried et al., 1999
),
suggesting that FIL and YAB3 together provide a
GRAM-like function. However fill yab3 mutants have less
severe polarity defects involving only a partial loss of abaxial cell
characters but no clear gain of adaxial identity. In this respect the effects
of gram mutations are more similar to loss of both KAN1 and
KAN2 activity in Arabidopsis, which is also accompanied by
ectopic HD-ZIP expression, as in gram
(Eshed et al., 2001
). The
different requirements for GRAM compared to FIL and
YAB3 might reflect divergence in the function of the YAB
gene family in the two species (e.g. from GRAM having assumed or
retained KAN-like functions). Alternatively, they might result from
different degrees of functional overlap between YAB genes within each
species. These possibilities might be tested with additional yab
loss-of-function mutations in both species. In the case of
Antirrhinum, reduced activity of PROL, for which no
orthologous Arabidopsis mutant has been reported, has no
developmental effects when GRAM is active, suggesting that
PROL is redundant. Because reduced activity of both GRAM and
PROL results in loss of adaxial identity, it does not reveal whether
PROL and GRAM might function redundantly to repress adaxial
identity.
In addition to promoting abaxial fate by repression of adaxial identity,
GRAM and PROL together promote adaxial identity. This role
is apparent in the loss of adaxial cell characters from gram single
mutant leaves and the complete replacement of adaxial by abaxial tissues in
gram prol double mutant leaves. In gram prol, the polarity
defect is also accompanied by reduced SAM activity, as seen in other mutants
with abaxialised leaves (e.g. Eshed et al.,
2001; Waites and Hudson,
2001
). Although GRAM is expressed ectopically in the
abaxialised leaves of phan mutants, gram mutations also
enhance the abaxialised organ phenotype of phan mutants in a similar
way to the handlebars (hb) mutation
(Waites and Hudson, 2001
) and,
like hb, remove its sensitivity to cold. This is consistent with
HB and GRAM acting in a cold-sensitive pathway that promotes
adaxial identity redundantly with PHAN. However, the relationship
between these genes is likely to be more complex because hb gram
double mutants (not shown) resemble hb phan, gram phan and gram
prol mutants, whereas the prol mutation enhances the phenotype
of gram, but not phan mutants.
The finding that the KNOX gene, HIRZ, is expressed
ectopically in abaxialised leaves of phan mutants has lead to the
suggestion that KNOX expression might cause polarity defects (e.g.
Tsianstis et al., 1999). In Arabidopsis, FIL and YAB3 have
also been found necessary to prevent KNOX expression in leaves
(Kumaran et al., 2002),
suggesting that GRAM might have a similar role in KNOX
repression and that the enhanced mutant phenotype of the phan gram
mutant might reflect increased KNOX mis-expression. Two observations,
however, argue against this. Firstly, ectopic KNOX expression could
not be detected in gram single mutant leaves and secondly, a
Hirz gain-of-function mutation that causes ectopic HIRZ
expression, as in phan mutants, failed to cause leaf polarity defects
in a GRAM+ background or to enhance the polarity defects
of gram mutants (data not shown)
(Golz et al., 2002
).
Because both GRAM and PROL show abaxially restricted
expression but promote adaxial identity, they appear to be necessary for a non
cell-autonomous signal from the abaxial to adaxial domain. A further non
cell-autonomous role of GRAM was revealed by a periclinal chimera in
which GRAM expression only in the most abaxial cell layer (L1) of the
primordium was sufficient for normal development of leaves and flowers. The
simplest explanation for both these non cell-autonomous effects is that they
involve the same intercellular signalling mechanism. Because GRAM protein is
absent from the adaxial region of developing leaves
(Navarro et al., 2004), it is
likely to regulate production of a downstream signal in abaxial cells, rather
than to acts as a signal itself.
Loss of adaxial identity is also observed in plants lacking activity of
GRAM and STYLOSA (STY)
(Navarro et al., 2004),
suggesting that STY is also required for the adaxial promoting
signal. GRAM and STY proteins interact physically and are co-expressed only in
abaxial cells of early organ primordia, suggesting that STY and GRAM together
regulate the signalling mechanism from early in organ development.
GRAM has opposite roles in the two parts of the leaf - repression of adaxial identity in the abaxial domain and promotion of adaxial identity. This seems unlikely to result from differences in the concentration of a signalling molecule, because the boundary of GRAM expression can be shifted abaxially to the junction between L1-L2 cell layers in a periclinal chimera without causing a shift in the boundary between development of adaxial and abaxial tissues.
Although the adaxial-promoting and adaxial-repressing roles of
GRAM might appear paradoxical, similar phenomena appear to be common
in other signal-response systems. For example, the Decapentaplegic signalling
protein is secreted by the most dorsal cells of the Drosophila embryo
and promotes expression of the Zerknült (Zen)
transcription factor, which confers amniosera fate, in a dorsal domain
(Ray et al., 1991). It also
induces more ventral expression of Brinker (Br), which
represses Zen transcription cell-autonomously
(Jazwinska et al., 1999
). The
interaction of Br with Zen is necessary to refine the dorsoventral boundary of
Zen expression (Muller et al.,
2003
). In an analogous way the opposite effects of GRAM
might serve to refine the boundary between adaxial and abaxial cells of organ
primordia preventing the specification of intermediate cell identities. It
might also serve to maintain the boundary, which is proposed to be necessary
for lateral growth. This view of GRAM function is consistent with the
observed loss of ad-abaxial distinction at the margins of gram leaves
and the loss of lateral growth in this region. Use of CYCLIN D3a
expression as a marker for cell division, suggested that lateral growth of
primordia was not affected until relatively late in development, consistent
with a requirement for GRAM to maintain an ad-abaxial boundary. Both adaxial
asymmetry and growth are maintained in the medial parts of the leaf, perhaps
because of the activity of other genes (e.g. additional YAB family
members). A requirement for GRAM to maintain a growth-promoting
ad-abaxial boundary is also consistent with the lack of ectopic growth at the
ectopic boundary between adaxial and abaxial cell types in the ventral margin
of gram mutant leaves.
Evidence for ab-adaxial signalling in leaves has also been provided by
Arabidopsis plants with reduced activity of the abaxially expressed
KAN1 gene. kan1 mutants show abaxial defects, but also
dosage-dependent reductions in adaxial trichome density
(Kerstetter et al., 2001).
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Max-Planck-Institut für Züchtungsforschung,
Carl-von-Linné-Weg 10, 50829 Köln, Germany
Present address: Department of Plant Biology, The University of Georgia,
Athens, GA 30602-7271, USA
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