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Physical Interaction of Floral Organs Controls
Petal Morphogenesis in Arabidopsis1[W][OA]
Seiji Takeda, Akira Iwasaki, Noritaka Matsumoto, Tomohiro Uemura, Kiyoshi Tatematsu, and
Kiyotaka Okada*
Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606–8502, Japan (S.T., A.I., N.M.,
K.O.); Laboratory of Plant Organ Development, National Institute for Basic Biology, Okazaki, Aichi 444–8585,
Japan (A.I., K.T., K.O.); Graduate School of Life and Environmental Sciences, Kyoto Prefectural University and
Kyoto Prefectural Institute of Agricultural Biotechnology, Seika, Kyoto 619–0244, Japan (S.T.); and Department
of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113–0033, Japan (T.U.)
Flowering plants bear beautiful flowers to attract pollinators. Petals are the most variable organs in flowering plants, with their
color, fragrance, and shape. In Arabidopsis (Arabidopsis thaliana), petal primordia arise at a similar time to stamen primordia and
elongate at later stages through the narrow space between anthers and sepals. Although many of the genes involved in
regulating petal identity and primordia growth are known, the molecular mechanism for the later elongation process remains
unknown. We found a mutant, folded petals1 (fop1), in which normal petal development is inhibited during their growth through
the narrow space between sepals and anthers, resulting in formation of folded petals at maturation. During elongation, the fop1
petals contact the sepal surface at several sites. The conical-shaped petal epidermal cells are flattened in the fop1 mutant, as if
they had been pressed from the top. Surgical or genetic removal of sepals in young buds restores the regular growth of petals,
suggesting that narrow space within a bud is the cause of petal folding in the fop1 mutant. FOP1 encodes a member of the
bifunctional wax ester synthase/diacylglycerol acyltransferase family, WSD11, which is expressed in elongating petals and
localized to the plasma membrane. These results suggest that the FOP1/WSD11 products synthesized in the petal epidermis
may act as a lubricant, enabling uninhibited growth of the petals as they extend between the sepals and the anthers.
Floral organs usually develop sequentially from the
outermost whorl toward the inner, in the order of sepals, petals, stamens, and carpels. Sepals arise first and
form a tight external covering, which functions as a
barrier to protect the developing internal floral organs
from physical or biological attacks from the outside.
Petals initiate when sepals cover the flower bud and
grow rapidly at a later stage. Petal development can be
divided into several stages, each of which has been
well described at a molecular level in Arabidopsis
(Arabidopsis thaliana; Smyth et al., 1990; Irish, 2008).
Petal and stamen primordia arise simultaneously at
developmental stage 5. Stamens grow faster than
petals until stage 8, and the anthers fill the upper
1
This work was supported by a Grant-in-Aid for Creative Scientific Research (no. 19GS0315 to K.O.), a Grant-in-Aid for Scientific
Research on Priority Areas (no. 19060004 to K.O.), the Grant for the
Biodiversity Research of the 21st Century Centers Of Excellence
(grant no. A14 to K.O. and S.T.), and a Grant-in-Aid for Scientific
Research (C; grant no. 22570042 to S.T.).
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Seiji Takeda ([email protected]).
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internal space created by the protective dome-like closed
sepals. After stage 9, petal growth is accelerated, and
petals elongate through a narrow space generated by the
sepals and anthers.
Floral organ identity is established in a concentric
pattern by MADS and APETALA2 (AP2)/ETHYLENE
RESPONSE FACTOR (ERF) transcription factors, which
is described by the floral ABCE or quartet model
(Theissen and Saedler, 2001). Petal identity is fixed in
the second whorl by the combined function of class
A, B, and E genes (Bowman et al., 1989; Weigel and
Meyerowitz, 1994; Krizek and Fletcher, 2005). Suppression of AGAMOUS activity in the perianth whorls
is important for petal growth, and this process is controlled by AP2, AINTEGUMENTA, LEUNIG, SEUSS,
RABBIT EARS, ROXY1, and STERILE APETALA (Liu
and Meyerowitz, 1995; Byzova et al., 1999; Conner and
Liu, 2000; Krizek et al., 2000, 2006; Franks et al., 2002;
Sridhar et al., 2004; Xing et al., 2005; Grigorova et al.,
2011). Petal primordia arise at four loci in the second
whorl, and this positioning is established independently
of the process that determines organ identity (Griffith
et al., 1999; Brewer et al., 2004; Takeda et al., 2004; Xing
et al., 2005; Lampugnani et al., 2013). After initiation,
the growth of petals depends on the activity of cell division and expansion along the proximal-distal axis,
which is partly regulated by JAGGED (Dinneny et al.,
2004; Ohno et al., 2004). Final petal size is determined by
the balance of cell proliferation and expansion (Mizukami
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Physical Interaction Controls Petal Morphogenesis
and Fischer, 2000; Szécsi et al., 2006). The coordinated
growth of petals and other floral organs leads to flower
opening, which is regulated by auxin, jasmonic acids, and
transcription factors such as MYB21, MYB24, AUXIN
RESPONSE FACTOR6 (ARF6), ARF8, and BIGPETALp
(Brioudes et al., 2009; Tabata et al., 2010; Varaud et al.,
2011; Reeves et al., 2012). Most of the known genes involved in petal development encode transcription factors
and act in the early developmental processes; however,
regulators involved in later stages of the petal elongation
process remain unidentified.
We isolated a mutant of Arabidopsis, folded petals
1 (fop1), in which petals are folded at the mature stage,
but whose overall size and shape are normal. We
found that the petals become stuck in the narrow space
between the sepals and stamens in the bud, causing
the petal to fold at flower opening, and that this defect
is rescued by removal of sepals. Characterization and
expression analysis of FOP1 suggests that FOP1 products, synthesized in the petal epidermis, play a lubricant
role during petal elongation.
RESULTS
fop1 Petals Do Not Elongate Normally through the
Narrow Space between Sepals and Anthers
The fop1-1 mutant was identified from a screen of
floral organ-defective mutants in Arabidopsis. Petals
of wild-type flowers were straight or slightly curved
outwards (Fig. 1A), whereas the mutant petals were
folded twice in the shape of a letter N (Fig. 1B), with an
outward fold in the medial region and an inward fold
in the distal portion. When fully expanded, the size,
shape, color, and vascular pattern of a mature petal of
the fop1-1 mutant were almost the same as those of the
wild type, except for its folded pattern (Supplemental
Fig. S1, A, B, O, and P), suggesting that the fop1 mutation is responsible for the physical process of growth
rather than cell proliferation or expansion. This is
supported by the expression pattern of a cell proliferation marker gene, HISTONE4, in petals, which
was not significantly different from the wild type
(Supplemental Fig. S1, C–J; Krizek, 1999; Gaudin et al.,
2000; Dinneny et al., 2004). These data suggest that the
overall petal shape and growth are not altered in fop11. We examined the phenotype of two transfer DNA
(T-DNA) insertion mutants for the FOP1 gene after
gene identification (Supplemental Fig. S3, see below)
and found that they showed a similar phenotype to
fop1-1 (Fig. 1, C and D). On the basis of this phenotypic
and genetic similarity, we used fop1-1 for further
analysis.
Morphological changes in developing petals were
examined by sectioning of the bud at several developmental stages. Petal primordia of the fop1-1 mutant
formed at stage 5 grew normally up to stage 9, when
the petal tip reached the base of an anther (Fig. 1, E, F,
K, and L). At stage 10, when wild-type petals elongate
through the space between a sepal and an anther (Fig.
1G), the mutant petals did not show smooth elongation, becoming stuck between an anther and a sepal
(Fig. 1M). Dissection of unopened buds revealed that
when the tip of wild-type petals reached the top of the
long stamens (Fig. 1H), the mutant petals failed to
undergo straight elongation (Fig. 1N). The degree of
folding increased at points where the mutant petal was
stuck between the anther and sepal (Fig. 1, O and P),
whereas wild-type petals remained straight (Fig. 1I),
even though their middle part could contact the anther
Figure 1. fop1 petal phenotype. Flowers of the
wild type (A), fop1-1 (B), fop1-2 (C), and fop1-3
(D). Arrowheads (B–D) show folded petals. E to P,
Histological analysis of wild-type (E–J) and fop11 (K–P) flowers. E and K, Stage 7. F and L, Stage 9.
G and M, Stage 10. H to J and N to P, Stage 12. I,
J, O, and P, High-magnification images of the
squares in H and N, respectively. Arrowhead in O
shows the contacting petal tip to sepal. se, Sepal;
p, petal; st, stamen; ca, carpel. Bars = 50 mm.
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Takeda et al.
and sepal (Fig. 1J). These observations suggest that the
FOP1 gene is involved in the smooth elongation of
petals as they grow though the narrow space in a floral
bud.
fop1 Petals Make Contact with the Sepal Surface
Next, we examined the contact region between
petals and sepals using scanning electron microscopy
to identify where the folding starts in the mutant. In
the wild type, at the stage where the petals are equal in
height to the anthers, no direct contact with the sepals
was observed (Fig. 2, A–C). At a similar stage in fop1-1,
the petal tip made contact with the sepal surface, and
the middle part of the petal touched the sepal surface
at several sites (Fig. 2, D–F). When wild-type petals
grow over the top of anthers, epicuticular nanoridges
started to deposit on the surface of the petal epidermis
(Fig. 2, G–I). At this stage in fop1-1, petals were seen to
have started folding (Fig. 2J), with the petal tip being in
contact with the sepal surface (Fig. 2K) and contacted
sepals at several sites (Fig. 2L). Before flower opening,
epidermal cells of wild-type petals are covered with
epicuticular nanoridges and become conical in shape
(Fig. 2, M–O). At this stage, petal folding of fop1-1 was
apparent (Fig. 2P), the petal apex turned outward (Fig.
2Q), and the surface of the conical epidermal cells were
flattened, as if they had been pressed and rubbed from
above (Fig. 2R). The flattened cells were frequent on
the abaxial surface in the distal part of a folded petal
facing a sepal (Supplemental Fig. S1L). Similar flattip cells were also found in wild-type petals, but
the number of such cells was comparatively few
(Supplemental Fig. S1K). Cuticle formation is not altered in fop1 because petal cells had epicuticular
nanoridges, although they were affected after its formation. The surface of epidermal cells of anthers or
sepals was not altered in the mutant (data not shown).
Taken together, the flattened surface of the epidermal
cells in fop1-1 mutant petals could be the result of
strong pressure and friction between the petals and
sepals.
Sepal Removal Restores the Straight Growth of fop1 Petals
These data indicate that the petals on the fop1 mutant do not easily extend through the space between
the sepals and the anthers. To confirm this further, we
examined whether regular petal elongation is restored
in open buds where sepals do not form a tight covering so that the physical contact between petals and
sepals or anthers would not be strong. First, we removed one or two sepals from a fop1-1 mutant bud
before petal folding started and let the buds grow.
After 3 d, the petals grew flat and did not show the
folding phenotype (n = 19; Fig. 3, A and B;
Supplemental Fig. S2). Sepal removal also restored
petal growth in individuals whose petals had already
Figure 2. Scanning electron microscopy images of floral organ surface. A to
C, G to I, and M to O, Wild type. D to F, J to L, and P to R, fop1-1. A to F,
Stage 10. G to L, Stage 11. M to R, Stage 12. B and C, Higher magnification
of A, showing a space between petal and sepal (arrows). E and F, Higher
magnification of D, showing direct contact between petal and sepal (arrows). H and I, Higher magnification of G, showing straight petal elongation
(G) and abaxial epidermis of petals (I) starting the deposition of the epicuticular nanoridges on their surface. K and L, Higher magnification of J,
showing direct contact of petal and sepal (arrows). N and O, Petal surface of
the same flower as M. Abaxial epidermal cells of petals deposit nanoridges
on cell surface. Q, Higher magnification of P, showing the petal edge turning
outward. R, Higher magnification of petals in Q, showing the trace of friction (arrows). The front side sepal is removed in A, D, G, J, M, and P to show
the inside of flowers. sep, Sepal; pet, petal; ant, anther. Bars = 200 mm (D, G,
J, M, and P), 100 mm (A and H), 50 mm (N), 20 mm (Q), 10 mm (B, E, I, O,
and R), 5 mm (C, K, and L), and 3 mm (F).
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Physical Interaction Controls Petal Morphogenesis
started to fold (n = 5; Fig. 3, C and D; Supplemental
Fig. S2). The epidermal cells were flattened in the fop11 mutant, especially in the marginal regions of the
folding site (Fig. 3E), although the petals adjacent to
removed sepals had much less flattened cells (Fig. 3F),
suggesting that sepal removal reduces the physical
contact of petals and restores the straight growth.
Next, we tested the petal phenotype of a double
mutant carrying both fop1 and pressed flower (prs). The
prs mutant is defective in sepal formation and lacks
two sepals in the lateral positions, thus forming an
open floral bud (Fig. 3G; Matsumoto and Okada,
2001). As we expected, petals of the fop1-1 prs-1 double
mutant did not fold; instead, they elongated normally
(Fig. 3H). Next, we examined the structure of the second whorl organ in the ap3-5 fop1-1 double mutant. In
ap3-5, which is one of the mutants of homeotic transition of floral organs, petals and stamens are converted to sepals and carpels, respectively (Fig. 3I;
Bowman et al., 1989). The sepaloid second whorl organs in the ap3-5 fop1-1 double mutant did not show
the folded phenotype (Fig. 3J). Rescue of the mutant
phenotype may be due to the change of the floral organ identity or to the increased space provided by the
Figure 3. Surgical and genetic removal of sepals restores straight
growth of petals. A, Flower bud of fop1-1 at stage 11. Sepal on the
abaxial side is removed. Petals are shown with arrows. B, The same
flower with A after 3 d, showing the side of the sepal removal. C, A
sepal-removed flower at the stage when petals have already folded
(arrows). D, The same flower with C after 3 d, with the straight growth
of petals. E, Scanning electron microscopy image of petal epidermis in
fop1-1. Surface of epidermal cells, especially those at the marginal
region, are flattened. F, Scanning electron microscopy image of petal
epidermis in fop1-1, where a sepal has been removed and petals grow
straight. G, prs-1 flower. H, fop1-1 prs-1 double mutant flower. Petals
of the double mutant are not folded. I, ap3-5 flower. J, ap3-5 fop11 double mutant flower. Bars = 500 mm (A–D) and 50 mm (E and F).
loss of anthers. Together, elimination of the tightness
in a floral bud restored the regular petal elongation,
suggesting that physical contact of floral organs is the
cause of the folded petals in the mutant.
FOP1 Encodes WSD11, a Member of the WAX
SYNTHASE/DIACYLGLYCEROL
ACYLTRANSFERASE Family
We mapped the FOP1 gene on chromosome 5 and
identified a mutation in At5g53390 (Supplemental Fig.
S3A). We found that G at nucleotide 820 of the coding
sequence was substituted with A in fop1-1, replacing
Gly with Arg in the mutant protein (Supplemental Fig.
S3B). We examined the border sequences of three TDNA insertion lines found in the SIGnAL database
(http://signal.salk.edu/cgi-bin/tdnaexpress) within
or near the gene (Supplemental Fig. S3B; Alonso et al.,
2003) and confirmed that the three lines had a small
deletion at the T-DNA insertion sites (Supplemental
Fig. S3B). Two of them, SALK_093133 (named fop1-2)
and SALK_137481 (fop1-3), showed a similar phenotype to fop1-1 (Fig. 1, B–D). The SALK_149804 line, in
which T-DNA was inserted at 31 bp upstream from the
ATG initiation codon, was indistinguishable from the
wild type and did not show the petal-folding phenotype.
To confirm whether At5g53390 is FOP1, a genomic
fragment comprising the 2.2-kb promoter, 2.2-kb
open reading frame, and 1.6-kb 39 region was transformed into the fop1-1 mutant. We obtained 80 independent primary transgenic plants carrying the FOP1
genomic fragment, and all lines complemented the
petal defects, indicating that At5g53390 is FOP1.
We cloned a full-length FOP1 complementary DNA
(cDNA) using a RACE strategy and identified the region covering the 48-bp 59 untranslated region, 217-bp 39
untranslated region, and seven exons (Supplemental Fig.
S3B).
FOP1 corresponds to WSD11, a member of the
bifunctional WAX SYNTHASE/DIACYLGLYCEROL
ACYLTRANSFERASE (WS/DGAT) family (Kalscheuer
and Steinbüchel, 2003; Li et al., 2008). Ten genes encode
similar proteins in Arabidopsis, and a gene located next
to FOP1 on chromosome 5, At5g53380, is the closest
homolog of the same family (Supplemental Fig. S3A;
Li et al., 2008). The amino acid sequence of FOP1
showed 17.3% identity with that of Acinetobacter spp.
WS/DGAT (GenBank accession no. AAO17391; Kalscheuer
and Steinbüchel, 2003). Alignment of the FOP1,
At5g53380, and Acinetobacter spp. WS/DGAT proteins
revealed three domains of high homology, and the first
domain (amino acids 106–155 of FOP1) includes a
putative active-site HHxLxDGxS box, which is conserved among Mycobacterium spp. and plant WS/
DGAT proteins (Supplemental Fig. S3C; Kalscheuer
and Steinbüchel, 2003; Li et al., 2008), suggesting that
FOP1 is involved in the synthesis of wax esters and
triacylglycerols.
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Takeda et al.
FOP1 Is Expressed in the Epidermis of Growing Petals
We investigated the spatiotemporal expression pattern of FOP1 during flower development. Reverse
transcription (RT)-PCR analysis revealed that FOP1
was preferentially expressed in inflorescences, including young floral buds, and in open flowers (Fig. 4A).
We performed in situ hybridization with FOP1 probes,
but no signals were obtained, probably due to low
expression levels. We then generated transgenic plants
carrying FOP1p:GUS, in which the GUS gene was
expressed under the control of the 2.2-kb FOP1 promoter. Out of 29 independent transgenic lines obtained
by screening, 26 lines showed GUS signals. In floral
buds at stage 2, when sepal primordia initiated, GUS
signal was detected in the floral meristem and sepal
primordia (Fig. 4B). The GUS expression was high in
petal primordia at stage 9, with weak expression in the
other floral organs (Fig. 4C). At stage 11, FOP1 is
expressed in the marginal region of petals and in
ovules (Fig. 4D). We also generated a translational
fusion line by transforming the FOP1p:FOP1-GFP
transgene into the fop1-1 mutant. The petal phenotype
of the mutant was rescued, indicating that the fusion
protein was functional in plants (Fig. 4F). GFP signal
was detected at the margin of elongating petals, when
they grew through the space between anthers and sepals (Fig. 4E), and the expression expanded widely in
the distal part of petals (Fig. 4F). FOP1 had lower expression in sepals and anthers than in petals, indicating that function of FOP1 in petals is required for
straight elongation. The FOP1-GFP fusion protein was
localized at the periphery of petal epidermal cells (Fig.
4G). We further examined the cellular localization of
FOP1 protein by expressing the GFP-FOP1 fusion gene
transiently in suspension-cultured cells of Arabidopsis
(Fig. 4H). The GFP signal was detected in the periphery of the cell, which overlapped with the FM4-64
signal (Fig. 4, I and J), indicating that FOP1 is localized to the plasma membrane. Together, these data
suggest that FOP1 is involved in wax ester synthesis at
the plasma membrane in the petal epidermis.
DISCUSSION
Figure 4. FOP1 is expressed in elongating petals. A, RT-PCR analysis
of FOP1 transcripts. FOP1 is expressed in inflorescences, including
young floral buds, and in open flowers. ACT8 was used as a control. B
to D, GUS expression in inflorescences and flowers carrying the
FOP1p:GUS transgene. B, GUS is expressed strongly in floral meristem
and sepal primordia. C, Strong GUS expression in petal primordia of a
stage 9 flower (arrowheads). D, GUS expression is observed at petal
margin (arrowheads) and ovules in a stage 11 flower. E to G, GFP
fluorescence in fop1-1 flowers of plants carrying the FOP1p:FOP1GFP transgene. The fusion protein is expressed preferentially at the
margin of petals of a stage 10 flower (E) and in the apical part of petals
of a stage 12 flower (F). G, A merged image of bright-field and GFP
images of petal epidermal cells. The FOP1-GFP fusion protein is localized at the cell periphery. H to J, Suspension cell expressing GFPFOP1 transiently. H, Bright-field image. I, GFP image. J, Merged image
of the GFP and FM4-64 images, showing that GFP-FOP1 is localized to
plasma membrane. Note that the upper cell, which is supposed not to
carry the construct, shows only FM4-64 signal. IM, Inflorescence
meristem; FM, floral meristem; se, sepal; st, stamen; ov, ovule; ca,
carpel. Bars = 50 mm (B and C), 500 mm (D), and 10 mm (H–J).
Study of the fop1 mutant directed attention to an
aspect of petal growth in a floral bud, namely to how
petals elongate through the narrow space between
sepals and anthers. Figure 5A describes the schematic
model highlighting the differences in the petal elongation process between wild type and fop1. Growth of
petal primordia is normal up to stage 9; however, after
stage 9, the petal tip becomes stuck between sepals and
anthers in the mutant, while in the wild type, petals
elongate straight. Our data suggest that the products
of FOP1 act as a lubricant on the petal surface, enabling petals to elongate smoothly through the narrow
space in a floral bud. It is also possible that the FOP1
products are involved in making cuticle rigid, so that
in the fop1 mutant, the petal surface intensity is lower
and easy to get rubbed off, enhancing the petal folding.
FOP1 encodes a homolog of the WS/DGAT enzymes, suggesting that biosynthesis of wax esters
and/or triacylglycerols mediated by FOP1 is required
for the smooth petal elongation. Several mutants defective in wax biosynthesis or secretion show a similar
petal-folding phenotype, and their petals are sensitive
to dye immersion due to the lower repellency of the
petal surface (Panikashvili et al., 2009; Li-Beisson et al.,
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Physical Interaction Controls Petal Morphogenesis
Figure 5. Schematic model of the FOP1 function.
A, Model explaining the process of petal folding
in fop1 floral buds at stage 8 (left), stage 9 (center), and stage 12 (right). Red represents petals.
Left and right halves show the petal structure in
the wild type and fop1, respectively. The blue
area represents the region of contact between
petals and sepals. Arrows indicate the direction of
petal elongation. B, Model explaining the process
of petal surface modification. During petal elongation between sepal and anther, FOP1 products
may be secreted to the surface, enabling smooth
elongation through the narrow space. Two ATPbinding cassette transporters, ABCG11 and ABCG13,
may be involved in a similar process (center). Before
maturation, epicuticular nanoridges deposit the outermost surface of petal epidermis, mediated by
GPAT6, CYP77A6, DCR, and PEC1/ABCG32, preventing organ fusion and protecting petals from water
loss and pathogen attack. WT, Wild type.
2009). We immersed the fop1-1 petals in the dye toluidine blue (Tanaka et al., 2004), but they were not
stained (Supplemental Fig. S1, M and N). We also
examined the difference of the hexane-soluble surface
contents of floral buds between wild type and fop1-1,
but found no significant change (Supplemental Fig.
S4). Arabidopsis WSD1, a homolog of FOP1, has high
wax synthase activity and lower but significant DGAT
activity (Li et al., 2008), suggesting that FOP1 has the
same activities as WSD1. Identification of substrates
and products of FOP1 will elucidate the role of petal
surface components in petal elongation.
The petal surface, as with the entire aerial plant
body, is covered by cuticle (Pollard et al., 2008;
Samuels et al., 2008; Domínguez et al., 2011). Cuticle
mainly consists of cutin and cuticular wax. Cutin is an
insoluble lipid polymer, consisting of aliphatics (C16
and C18 fatty acids), aromatics, and glycerols, and
covers the external surface of the cell wall. The outer
layer of the cutin is covered with cuticular wax, a
complex of C20 to C60 aliphatics, aldehydes, ketones,
and wax esters, coating the outermost surface of the
plant body (Pollard et al., 2008). A series of Arabidopsis mutants defective in cuticle synthesis and secretion show the biological roles of cuticles as a barrier
to biotic or abiotic stresses, osmotic stress, water loss,
and damage from UV radiation, and in preventing the
fusion of leaves and floral organs (Yephremov et al.,
1999; Pruitt et al., 2000; Krolikowski et al., 2003; Aharoni
et al., 2004; Kurdyukov et al., 2006; Bessire et al., 2007;
Shi et al., 2011; Wang et al., 2011). Some mutants are
known to be involved in petal morphogenesis: Lack of
nanoridges of petals, due to a mutation in DEFECTIVE
IN CUTICULAR RIDGES (DCR, encoding a BAHD acyltransferase), CYP77A6 (cytochrome P450 family),
GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE6
(GPAT6), or PERMEABLE CUTICLE1 (PEC1), result in
increased permeability of petals to a dye and cause
organ fusion (Li-Beisson et al., 2009; Panikashvili
et al., 2009; Bessire et al., 2011). Compared with these
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Takeda et al.
mutants, fop1 petals form nanoridges on the petal
epidermis, and the degree of organ fusion is, if any,
only subtle (Fig. 2). Because the petal folding starts
before the nanoridges deposit on the surface of petal
epidermis, we propose that FOP1 is involved in the
synthesis of wax-related products in the petal epidermis before nanoridge deposition (Fig. 5B). Similar
petal-folding defects are found in mutants of the
DESPERADO/AtWBC11/ABCG11 and ABCG13 genes,
both of which encode members of ATP-binding cassette transporters (Panikashvili et al., 2007, 2011),
suggesting that they are involved in a similar process
to that of FOP1 (Fig. 5B).
Is the petal elongation mechanism conserved among
flowering plants? A similar petal-folding phenotype is
also known in a breed of Japanese morning glory (Ipomoea nil). Corollas of the crepe (cp) mutant fold twice
like those of fop1, forming an additional tube-like
structure surrounding the stamens and carpels
(Supplemental Fig. S5; Miyake and Imai, 1927). Similar
to our results, the removal of calyx restored the formation of funnel-shaped corollas (Nishino and Gotoh,
2002). These characters suggest that the cp phenotype
might be caused by a similar mechanism to that of fop1
petals. Molecular identification of the CP gene will
provide an answer.
CONCLUSION
Here, we propose a mechanism for the smooth
elongation of petals. After primordia initiation, petals
elongate through the narrow space generated by anthers
and sepals. FOP1/WSD11 is expressed in elongating
petals and may catalyze the wax ester biosynthesis at
the epidermal plasma membrane. The FOP1 products
may act as a lubricant that enables petals to grow
straight in a narrow space in a bud. Identifying the
substrates and products of FOP1/WSD11 is the next
challenge in the effort to understanding the relationship between the cell surface components and petal
morphogenesis.
formaldehyde, 5% [v/v] acetic acid), dehydrated in an ethanol series, and
embedded in Technovit 7100 resin (Heraeus Kulzer). Sections of 5-mm thickness were stained with 0.1% (w/v) toluidine blue. For petal clarification,
flowers were fixed in acetic acid and ethanol mixture (1:9) and cleared in
clearing solution (40 g chloral hydrate, 10 mL glycerol, 5 mL distilled water).
The length and width of cleared petals were measured using the Image ProPlus 5.0 software. For whole-mount toluidine blue staining, petals were
treated with 0.05% (w/v) toluidine blue for 2 min and washed in distilled
water twice (Tanaka et al., 2004). Samples were visualized under an Axiophot
2 microscope (Carl Zeiss).
Mapping and Cloning of FOP1
F2 plants generated by crossing fop1-1 with the Columbia ecotype were
used for mapping. Information about the RPS4NT, nga129, JV61/62, and
EG7F2 markers was obtained from The Arabidopsis Information Resource
(http://www.arabidopsis.org). Other markers were generated as sequence
markers based on the polymorphisms revealed by genome sequencing. The
sequences of the oligonucleotides used in mapping and gene cloning are listed
in Supplemental Table S1. cDNA cloning was performed by both 59 RACE and
39 RACE using the SMART RACE cDNA Amplification Kit (Clontech). Sequencing was performed using the ABI BigDye Terminator Cycle Sequencing
Ready Reaction Kit and an ABI Prism 3100 Genetic Analyzer (Applied Biosystems).
Complementation Test
The genomic fragment including the 2.2-kb promoter, 2.2-kb open reading
frame, and 1.6-kb 39 regions of FOP1 was cloned as follows. Fragments amplified by PCR using the 53390R2 and FOP3R primers were digested with SpeI
and HindIII, and cloned into pBluescriptII SK1 (Stratagene) to generate
pFg3SK. The promoter region was amplified using the FOP5SalIF and
FOPpXbaIR primers, digested by SalI and XbaI, and cloned into pBluescriptII
SK1 to generate pFpSK. The HindIII fragment from pFpSK was subcloned
into pFg3SK to generate pFgfSK, and the KpnI-SacI fragment of pFgfSK was
subcloned to pPZP211 (Hajdukiewicz et al., 1994) and pPZP211NP (a gift from
T. Nishimura, Nagoya University) to generate pFgf21135 and pFgf211NP,
respectively. These constructs were introduced into the fop1-1 mutant by a
vacuum infiltration procedure with the Agrobacterium tumefaciens strain
C58C1. Transgenic plants were screened on an agar medium containing 30 mg
mL-1 kanamycin and 100 mg mL-1 carbenicillin. Sequences of oligonucleotide
primers used for cloning are listed in Supplemental Table S1.
RNA Isolation and RT-PCR
Total RNA was isolated with the Isogen reagent (Nippon Gene) or RNeasy
Plant Mini Kit (Qiagen). One microgram of total RNA was reverse transcribed
with the SuperScript II reverse transcription kit (Invitrogen). The oligonucleotide primers used for RT-PCR are as follows: FOP1, 53390F1a and 53390R1,
and ACTIN8, ACT8F and ACT8R. The sequences of the oligonucleotide
primers are listed in Supplemental Table S1.
MATERIALS AND METHODS
mRNA in Situ Hybridization
Plant Growth Conditions
mRNA in situ hybridization was performed as previously described
(Matsumoto and Okada, 2001). The HISTONE4 probe was prepared as previously described (Dinneny et al., 2004).
The Wassilewskija and Columbia ecotypes of Arabidopsis (Arabidopsis
thaliana) were used as the wild type. The fop1-1 mutant was isolated from an
M2 population of long hypocotyl5 (Wassilewskija ecotype background; Oyama
et al., 1997) mutagenized by ethyl methanesulfonate. T-DNA insertion mutants were obtained from the SIGnAL Web site (http://signal.salk.edu) and
the Arabidopsis Biological Resource Center (http://abrc.osu.edu). Seeds were
sown on the surface of vermiculite in small pots and incubated for 1 week at
4°C. Plants were grown under continuous white light at 22°C to 24°C.
Histology and Microscopes
For scanning electron microscopy, samples were prepared as previously
described (Matsumoto and Okada, 2001) and observed with S-3200N (Hitachi)
and JSM-5800 (JEOL) microscopes. For histological analysis, inflorescences
were fixed in formaldehyde-acetic acid (50% [v/v] ethanol, 3.7% [v/v]
Histochemical Analysis of FOP1
The HindIII fragment from pFpSK was subcloned into pBI101 (Clontech) to
generate pFpGUSBI. pFpGUSBI was transformed into Columbia plants and
screened as described above. Samples were incubated in a staining buffer (100
mM NaPO4, pH 7.0, 10 mM EDTA, pH 8.0, 5 mM potassium ferricyanide, 5 mM
potassium ferrocyanide, 0.1% Triton X-100 (w/v), and 0.5 mg mL–1 5-bromo-4chloro-3-indolyl-b-glucuronic acid) at 37°C for 6 h. The stained samples were
treated with 70% ethanol for 10 min at room temperature, 100% ethanol for 30
min at 37°C, and 70% ethanol for 10 min at room temperature, and then
cleared in clearing solution. Samples were visualized under a Leica M420
(Leica Microsystems) or an Axiophot 2 microscope with a differential interference contrast filter. For transient expression in suspension cells, the FOP1
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
Physical Interaction Controls Petal Morphogenesis
cDNA was amplified by PCR with FOP1FSacI2nt and FOP1RKpnI primers
(Supplemental Table S1), digested with SacI and KpnI, and cloned into 35SGFP-NOS/pUC18 plasmid. The generated 35S:GFP:FOP1c construct was
transformed into Arabidopsis suspension-cultured cells by polyethylene glycolbased method as has been previously described (Uemura et al., 2004). The fluorescent images were taken using a LSM710 confocal microscope (Carl Zeiss).
Generation of FOP1p:FOP1-GFP
The G3GFP and FOP1 cDNAs were cloned into pPZP211 and pPZP211NP
to generate pFcG21135 and pFcG211NP, respectively. The HindIII fragment
from pFpSK was subcloned into pFcG21135 and pFcG211NP to generate
pFpFcG21135 and pFpFcG211NP, respectively. Transformation and screening
were performed as described above.
Gas Chromatography
Surface fatty acids were extracted from flowers of 6-week-old plants with
hexane. C19COOH (Fluka) and N,O-bis(trimethylsilyl)trifluoroactamide with
1% trimethylchlorosilane (Sigma) were added as internal controls. The solution was incubated at 80°C for 30 min and concentrated and analyzed with a
GC-14A gas chromatograph (Shimadzu) with the use of a DB-1 column (J&W
Scientific). The measurement conditions were 170°C to 265°C with a 2°C increase per minute, then to 310°C with a 4°C increase per minute, and hold for
10 min (alcohol-insoluble residue: 0.5 kg cm–2; hydrogen: 0.5 kg cm–2; carrier
[P1]: 1 kg cm–2; carrier [P2]: 2 kg cm–2 [carrier: He]).
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession numbers FOP1/At5g53390 and NM-124718.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Characterization of the fop1-1 petal.
Supplemental Figure S2. Sepal removal experiment in fop1-1.
Supplemental Figure S3. Molecular characterization of FOP1.
Supplemental Figure S4. GC analysis of floral buds.
Supplemental Figure S5. Petal phenotype of crepe mutant in Japanese
morning glory.
Supplemental Table S1. Oligonucleotide primers used in this work.
ACKNOWLEDGMENTS
We thank Eisho Nishino (Chiba University, Japan), Eiji Nitasaka (Kyushu
University, Japan), Noriyoshi Yagi (Kyoto University, Japan), Mitsuhiro Aida
(Nara Institute of Science and Technology, Japan), Koichi Toyokura (National
Institute for Basic Biology), Maki Kondo (National Institute for Basic Biology),
Mikio Nishimura (National Institute for Basic Biology), the Model Plant
Research Facility (National Institute for Basic Biology), and the Functional
Genomics Facility (National Institute for Basic Biology) for their help with this
work; Rebecca Horn (John Innes Centre) for critical reading of the paper; and
the Salk Institute and the Arabidopsis Biological Resource Center for providing T-DNA insertion mutants.
Received November 29, 2012; accepted January 9, 2013; published January 11,
2013.
LITERATURE CITED
Aharoni A, Dixit S, Jetter R, Thoenes E, van Arkel G, Pereira A (2004) The
SHINE clade of AP2 domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance when
overexpressed in Arabidopsis. Plant Cell 16: 2463–2480
Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P,
Stevenson DK, Zimmerman J, Barajas P, Cheuk R, et al (2003)
Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science
301: 653–657
Bessire M, Borel S, Fabre G, Carraça L, Efremova N, Yephremov A, Cao Y,
Jetter R, Jacquat AC, Métraux JP, et al (2011) A member of the PLEIOTROPIC DRUG RESISTANCE family of ATP binding cassette transporters is required for the formation of a functional cuticle in Arabidopsis.
Plant Cell 23: 1958–1970
Bessire M, Chassot C, Jacquat AC, Humphry M, Borel S, Petétot JM,
Métraux JP, Nawrath C (2007) A permeable cuticle in Arabidopsis leads
to a strong resistance to Botrytis cinerea. EMBO J 26: 2158–2168
Bowman JL, Smyth DR, Meyerowitz EM (1989) Genes directing flower
development in Arabidopsis. Plant Cell 1: 37–52
Brewer PB, Howles PA, Dorian K, Griffith ME, Ishida T, Kaplan-Levy
RN, Kilinc A, Smyth DR (2004) PETAL LOSS, a trihelix transcription
factor gene, regulates perianth architecture in the Arabidopsis flower.
Development 131: 4035–4045
Brioudes F, Joly C, Szécsi J, Varaud E, Leroux J, Bellvert F, Bertrand C,
Bendahmane M (2009) Jasmonate controls late development stages of
petal growth in Arabidopsis thaliana. Plant J 60: 1070–1080
Byzova MV, Franken J, Aarts MG, de Almeida-Engler J, Engler G,
Mariani C, Van Lookeren Campagne MM, Angenent GC (1999) Arabidopsis STERILE APETALA, a multifunctional gene regulating inflorescence,
flower, and ovule development. Genes Dev 13: 1002–1014
Conner J, Liu Z (2000) LEUNIG, a putative transcriptional corepressor that
regulates AGAMOUS expression during flower development. Proc Natl
Acad Sci USA 97: 12902–12907
Dinneny JR, Yadegari R, Fischer RL, Yanofsky MF, Weigel D (2004) The
role of JAGGED in shaping lateral organs. Development 131: 1101–1110
Domínguez E, Heredia-Guerrero JA, Heredia A (2011) The biophysical
design of plant cuticles: an overview. New Phytol 189: 938–949
Franks RG, Wang C, Levin JZ, Liu Z (2002) SEUSS, a member of a novel
family of plant regulatory proteins, represses floral homeotic gene expression with LEUNIG. Development 129: 253–263
Gaudin V, Lunness PA, Fobert PR, Towers M, Riou-Khamlichi C, Murray
JA, Coen E, Doonan JH (2000) The expression of D-cyclin genes defines
distinct developmental zones in snapdragon apical meristems and is
locally regulated by the Cycloidea gene. Plant Physiol 122: 1137–1148
Griffith ME, da Silva Conceição A, Smyth DR (1999) PETAL LOSS gene
regulates initiation and orientation of second whorl organs in the Arabidopsis flower. Development 126: 5635–5644
Grigorova B, Mara C, Hollender C, Sijacic P, Chen X, Liu Z (2011) LEUNIG and SEUSS co-repressors regulate miR172 expression in Arabidopsis flowers. Development 138: 2451–2456
Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP family
of Agrobacterium binary vectors for plant transformation. Plant Mol
Biol 25: 989–994
Irish VF (2008) The Arabidopsis petal: a model for plant organogenesis.
Trends Plant Sci 13: 430–436
Kalscheuer R, Steinbüchel A (2003) A novel bifunctional wax ester
synthase/acyl-CoA:diacylglycerol acyltransferase mediates wax ester
and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1. J
Biol Chem 278: 8075–8082
Krizek BA (1999) Ectopic expression of AINTEGUMENTA in Arabidopsis
plants results in increased growth of floral organs. Dev Genet 25: 224–236
Krizek BA, Fletcher JC (2005) Molecular mechanisms of flower development: an armchair guide. Nat Rev Genet 6: 688–698
Krizek BA, Lewis MW, Fletcher JC (2006) RABBIT EARS is a second-whorl
repressor of AGAMOUS that maintains spatial boundaries in Arabidopsis flowers. Plant J 45: 369–383
Krizek BA, Prost V, Macias A (2000) AINTEGUMENTA promotes petal
identity and acts as a negative regulator of AGAMOUS. Plant Cell 12:
1357–1366
Krolikowski KA, Victor JL, Wagler TN, Lolle SJ, Pruitt RE (2003) Isolation
and characterization of the Arabidopsis organ fusion gene HOTHEAD.
Plant J 35: 501–511
Kurdyukov S, Faust A, Nawrath C, Bär S, Voisin D, Efremova N, Franke
R, Schreiber L, Saedler H, Métraux JP, et al (2006) The epidermisspecific extracellular BODYGUARD controls cuticle development and
morphogenesis in Arabidopsis. Plant Cell 18: 321–339
Lampugnani ER, Kilinc A, Smyth DR (2013) Auxin controls petal initiation
in Arabidopsis. Development 140: 185–194
Li F, Wu X, Lam P, Bird D, Zheng H, Samuels L, Jetter R, Kunst L (2008)
Identification of the wax ester synthase/acyl-coenzyme A:diacylglycerol
acyltransferase WSD1 required for stem wax ester biosynthesis in Arabidopsis. Plant Physiol 148: 97–107
Plant Physiol. Vol. 161, 2013
1249
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.
Takeda et al.
Li-Beisson Y, Pollard M, Sauveplane V, Pinot F, Ohlrogge J, Beisson F
(2009) Nanoridges that characterize the surface morphology of flowers
require the synthesis of cutin polyester. Proc Natl Acad Sci USA 106:
22008–22013
Liu Z, Meyerowitz EM (1995) LEUNIG regulates AGAMOUS expression in
Arabidopsis flowers. Development 121: 975–991
Matsumoto N, Okada K (2001) A homeobox gene, PRESSED FLOWER,
regulates lateral axis-dependent development of Arabidopsis flowers.
Genes Dev 15: 3355–3364
Miyake K, Imai Y (1927) On the double flowers of the Japanese morning
glory. J Genet 19: 97–130
Mizukami Y, Fischer RL (2000) Plant organ size control: AINTEGUMENTA regulates growth and cell numbers during organogenesis. Proc
Natl Acad Sci USA 97: 942–947
Nishino E, Gotoh S (2002) Reversed corolla tube formation in crepe mutant
of Japanese morning glory. J Plant Res (Suppl) 115: 134
Ohno CK, Reddy GV, Heisler MG, Meyerowitz EM (2004) The Arabidopsis JAGGED gene encodes a zinc finger protein that promotes leaf
tissue development. Development 131: 1111–1122
Oyama T, Shimura Y, Okada K (1997) The Arabidopsis HY5 gene encodes
a bZIP protein that regulates stimulus-induced development of root and
hypocotyl. Genes Dev 11: 2983–2995
Panikashvili D, Savaldi-Goldstein S, Mandel T, Yifhar T, Franke RB,
Höfer R, Schreiber L, Chory J, Aharoni A (2007) The Arabidopsis
DESPERADO/AtWBC11 transporter is required for cutin and wax secretion. Plant Physiol 145: 1345–1360
Panikashvili D, Shi JX, Schreiber L, Aharoni A (2009) The Arabidopsis
DCR encoding a soluble BAHD acyltransferase is required for cutin
polyester formation and seed hydration properties. Plant Physiol 151:
1773–1789
Panikashvili D, Shi JX, Schreiber L, Aharoni A (2011) The Arabidopsis
ABCG13 transporter is required for flower cuticle secretion and patterning of the petal epidermis. New Phytol 190: 113–124
Pollard M, Beisson F, Li Y, Ohlrogge JB (2008) Building lipid barriers:
biosynthesis of cutin and suberin. Trends Plant Sci 13: 236–246
Pruitt RE, Vielle-Calzada JP, Ploense SE, Grossniklaus U, Lolle SJ (2000)
FIDDLEHEAD, a gene required to suppress epidermal cell interactions
in Arabidopsis, encodes a putative lipid biosynthetic enzyme. Proc Natl
Acad Sci USA 97: 1311–1316
Reeves PH, Ellis CM, Ploense SE, Wu MF, Yadav V, Tholl D, Chételat A,
Haupt I, Kennerley BJ, Hodgens C, et al (2012) A regulatory network
for coordinated flower maturation. PLoS Genet 8: e1002506
Samuels L, Kunst L, Jetter R (2008) Sealing plant surfaces: cuticular wax
formation by epidermal cells. Annu Rev Plant Biol 59: 683–707
Shi JX, Malitsky S, De Oliveira S, Branigan C, Franke RB, Schreiber L,
Aharoni A (2011) SHINE transcription factors act redundantly to pattern the archetypal surface of Arabidopsis flower organs. PLoS Genet 7:
e1001388
Smyth DR, Bowman JL, Meyerowitz EM (1990) Early flower development
in Arabidopsis. Plant Cell 2: 755–767
Sridhar VV, Surendrarao A, Gonzalez D, Conlan RS, Liu Z (2004) Transcriptional repression of target genes by LEUNIG and SEUSS, two interacting regulatory proteins for Arabidopsis flower development. Proc
Natl Acad Sci USA 101: 11494–11499
Szécsi J, Joly C, Bordji K, Varaud E, Cock JM, Dumas C, Bendahmane M
(2006) BIGPETALp, a bHLH transcription factor is involved in the
control of Arabidopsis petal size. EMBO J 25: 3912–3920
Tabata R, Ikezaki M, Fujibe T, Aida M, Tian CE, Ueno Y, Yamamoto KT,
Machida Y, Nakamura K, Ishiguro S (2010) Arabidopsis auxin response
factor6 and 8 regulate jasmonic acid biosynthesis and floral organ development via repression of class 1 KNOX genes. Plant Cell Physiol 51: 164–175
Takeda S, Matsumoto N, Okada K (2004) RABBIT EARS, encoding a
SUPERMAN-like zinc finger protein, regulates petal development in
Arabidopsis thaliana. Development 131: 425–434
Tanaka T, Tanaka H, Machida C, Watanabe M, Machida Y (2004) A new
method for rapid visualization of defects in leaf cuticle reveals five intrinsic patterns of surface defects in Arabidopsis. Plant J 37: 139–146
Theissen G, Saedler H (2001) Plant biology. Floral quartets. Nature 409:
469–471
Uemura T, Ueda T, Ohniwa RL, Nakano A, Takeyasu K, Sato MH (2004)
Systematic analysis of SNARE molecules in Arabidopsis: dissection of
the post-Golgi network in plant cells. Cell Struct Funct 29: 49–65
Varaud E, Brioudes F, Szécsi J, Leroux J, Brown S, Perrot-Rechenmann C,
Bendahmane M (2011) AUXIN RESPONSE FACTOR8 regulates Arabidopsis petal growth by interacting with the bHLH transcription factor
BIGPETALp. Plant Cell 23: 973–983
Wang ZY, Xiong L, Li W, Zhu JK, Zhu J (2011) The plant cuticle is required
for osmotic stress regulation of abscisic acid biosynthesis and osmotic
stress tolerance in Arabidopsis. Plant Cell 23: 1971–1984
Weigel D, Meyerowitz EM (1994) The ABCs of floral homeotic genes. Cell
78: 203–209
Xing S, Rosso MG, Zachgo S (2005) ROXY1, a member of the plant glutaredoxin family, is required for petal development in Arabidopsis
thaliana. Development 132: 1555–1565
Yephremov A, Wisman E, Huijser P, Huijser C, Wellesen K, Saedler H
(1999) Characterization of the FIDDLEHEAD gene of Arabidopsis reveals
a link between adhesion response and cell differentiation in the epidermis. Plant Cell 11: 2187–2201
1250
Plant Physiol. Vol. 161, 2013
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.