Physical Interaction of Floral Organs Controls...folded twice in the shape of a letter N (Fig. 1B),...

Physical Interaction of Floral Organs ControlsPetal Morphogenesis in Arabidopsis1[W][OA]

Seiji Takeda, Akira Iwasaki, Noritaka Matsumoto, Tomohiro Uemura, Kiyoshi Tatematsu, andKiyotaka 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 andKyoto Prefectural Institute of Agricultural Biotechnology, Seika, Kyoto 619–0244, Japan (S.T.); and Departmentof 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 theircolor, fragrance, and shape. In Arabidopsis (Arabidopsis thaliana), petal primordia arise at a similar time to stamen primordia andelongate at later stages through the narrow space between anthers and sepals. Although many of the genes involved inregulating petal identity and primordia growth are known, the molecular mechanism for the later elongation process remainsunknown. We found a mutant, folded petals1 (fop1), in which normal petal development is inhibited during their growth throughthe narrow space between sepals and anthers, resulting in formation of folded petals at maturation. During elongation, the fop1petals contact the sepal surface at several sites. The conical-shaped petal epidermal cells are flattened in the fop1 mutant, as ifthey 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 thebifunctional wax ester synthase/diacylglycerol acyltransferase family, WSD11, which is expressed in elongating petals andlocalized to the plasma membrane. These results suggest that the FOP1/WSD11 products synthesized in the petal epidermismay 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 theoutermost whorl toward the inner, in the order of se-pals, petals, stamens, and carpels. Sepals arise first andform a tight external covering, which functions as abarrier to protect the developing internal floral organsfrom physical or biological attacks from the outside.Petals initiate when sepals cover the flower bud andgrow rapidly at a later stage. Petal development can bedivided into several stages, each of which has beenwell described at a molecular level in Arabidopsis(Arabidopsis thaliana; Smyth et al., 1990; Irish, 2008).Petal and stamen primordia arise simultaneously atdevelopmental stage 5. Stamens grow faster thanpetals until stage 8, and the anthers fill the upper

internal space created by the protective dome-like closedsepals. After stage 9, petal growth is accelerated, andpetals elongate through a narrow space generated by thesepals and anthers.

Floral organ identity is established in a concentricpattern by MADS and APETALA2 (AP2)/ETHYLENERESPONSE FACTOR (ERF) transcription factors, whichis described by the floral ABCE or quartet model(Theissen and Saedler, 2001). Petal identity is fixed inthe second whorl by the combined function of classA, B, and E genes (Bowman et al., 1989; Weigel andMeyerowitz, 1994; Krizek and Fletcher, 2005). Sup-pression of AGAMOUS activity in the perianth whorlsis important for petal growth, and this process is con-trolled by AP2, AINTEGUMENTA, LEUNIG, SEUSS,RABBIT EARS, ROXY1, and STERILE APETALA (Liuand Meyerowitz, 1995; Byzova et al., 1999; Conner andLiu, 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 secondwhorl, and this positioning is established independentlyof the process that determines organ identity (Griffithet al., 1999; Brewer et al., 2004; Takeda et al., 2004; Xinget al., 2005; Lampugnani et al., 2013). After initiation,the growth of petals depends on the activity of cell di-vision 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 bythe balance of cell proliferation and expansion (Mizukami

1 This work was supported by a Grant-in-Aid for Creative Scien-tific Research (no. 19GS0315 to K.O.), a Grant-in-Aid for ScientificResearch on Priority Areas (no. 19060004 to K.O.), the Grant for theBiodiversity Research of the 21st Century Centers Of Excellence(grant no. A14 to K.O. and S.T.), and a Grant-in-Aid for ScientificResearch (C; grant no. 22570042 to S.T.).

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Seiji Takeda ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a subscrip-

tion.www.plantphysiol.org/cgi/doi/10.1104/pp.112.212084

1242 Plant Physiology�, March 2013, Vol. 161, pp. 1242–1250, www.plantphysiol.org � 2013 American Society of Plant Biologists. All Rights Reserved.

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and Fischer, 2000; Szécsi et al., 2006). The coordinatedgrowth of petals and other floral organs leads to floweropening, which is regulated by auxin, jasmonic acids, andtranscription factors such as MYB21, MYB24, AUXINRESPONSE 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 in-volved in petal development encode transcription factorsand act in the early developmental processes; however,regulators involved in later stages of the petal elongationprocess 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. Wefound that the petals become stuck in the narrow spacebetween the sepals and stamens in the bud, causingthe petal to fold at flower opening, and that this defectis rescued by removal of sepals. Characterization andexpression analysis of FOP1 suggests that FOP1 prod-ucts, synthesized in the petal epidermis, play a lubricantrole during petal elongation.

RESULTS

fop1 Petals Do Not Elongate Normally through theNarrow Space between Sepals and Anthers

The fop1-1 mutant was identified from a screen offloral organ-defective mutants in Arabidopsis. Petalsof wild-type flowers were straight or slightly curvedoutwards (Fig. 1A), whereas the mutant petals werefolded twice in the shape of a letter N (Fig. 1B), with anoutward fold in the medial region and an inward foldin 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 thewild type, except for its folded pattern (SupplementalFig. S1, A, B, O, and P), suggesting that the fop1 mu-tation is responsible for the physical process of growthrather than cell proliferation or expansion. This issupported by the expression pattern of a cell prolif-eration marker gene, HISTONE4, in petals, whichwas 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 theoverall petal shape and growth are not altered in fop1-1. We examined the phenotype of two transfer DNA(T-DNA) insertion mutants for the FOP1 gene aftergene identification (Supplemental Fig. S3, see below)and found that they showed a similar phenotype tofop1-1 (Fig. 1, C and D). On the basis of this phenotypicand genetic similarity, we used fop1-1 for furtheranalysis.

Morphological changes in developing petals wereexamined by sectioning of the bud at several devel-opmental stages. Petal primordia of the fop1-1 mutantformed at stage 5 grew normally up to stage 9, whenthe petal tip reached the base of an anther (Fig. 1, E, F,K, and L). At stage 10, when wild-type petals elongatethrough the space between a sepal and an anther (Fig.1G), the mutant petals did not show smooth elonga-tion, becoming stuck between an anther and a sepal(Fig. 1M). Dissection of unopened buds revealed thatwhen the tip of wild-type petals reached the top of thelong stamens (Fig. 1H), the mutant petals failed toundergo straight elongation (Fig. 1N). The degree offolding increased at points where the mutant petal wasstuck 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 thewild 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 fop1-1 (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 thesquares in H and N, respectively. Arrowhead in Oshows the contacting petal tip to sepal. se, Sepal;p, petal; st, stamen; ca, carpel. Bars = 50 mm.

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and sepal (Fig. 1J). These observations suggest that theFOP1 gene is involved in the smooth elongation ofpetals as they grow though the narrow space in a floralbud.

fop1 Petals Make Contact with the Sepal Surface

Next, we examined the contact region betweenpetals and sepals using scanning electron microscopyto identify where the folding starts in the mutant. Inthe wild type, at the stage where the petals are equal inheight to the anthers, no direct contact with the sepalswas observed (Fig. 2, A–C). At a similar stage in fop1-1,the petal tip made contact with the sepal surface, andthe middle part of the petal touched the sepal surfaceat several sites (Fig. 2, D–F). When wild-type petalsgrow over the top of anthers, epicuticular nanoridgesstarted to deposit on the surface of the petal epidermis(Fig. 2, G–I). At this stage in fop1-1, petals were seen tohave started folding (Fig. 2J), with the petal tip being incontact with the sepal surface (Fig. 2K) and contactedsepals at several sites (Fig. 2L). Before flower opening,epidermal cells of wild-type petals are covered withepicuticular nanoridges and become conical in shape(Fig. 2, M–O). At this stage, petal folding of fop1-1 wasapparent (Fig. 2P), the petal apex turned outward (Fig.2Q), and the surface of the conical epidermal cells wereflattened, as if they had been pressed and rubbed fromabove (Fig. 2R). The flattened cells were frequent onthe abaxial surface in the distal part of a folded petalfacing a sepal (Supplemental Fig. S1L). Similar flat-tip cells were also found in wild-type petals, butthe number of such cells was comparatively few(Supplemental Fig. S1K). Cuticle formation is not al-tered in fop1 because petal cells had epicuticularnanoridges, although they were affected after its for-mation. The surface of epidermal cells of anthers orsepals was not altered in the mutant (data not shown).Taken together, the flattened surface of the epidermalcells in fop1-1 mutant petals could be the result ofstrong pressure and friction between the petals andsepals.

Sepal Removal Restores the Straight Growth of fop1 Petals

These data indicate that the petals on the fop1 mu-tant do not easily extend through the space betweenthe sepals and the anthers. To confirm this further, weexamined whether regular petal elongation is restoredin open buds where sepals do not form a tight cover-ing so that the physical contact between petals andsepals or anthers would not be strong. First, we re-moved one or two sepals from a fop1-1 mutant budbefore petal folding started and let the buds grow.After 3 d, the petals grew flat and did not show thefolding phenotype (n = 19; Fig. 3, A and B;Supplemental Fig. S2). Sepal removal also restoredpetal growth in individuals whose petals had already

Figure 2. Scanning electronmicroscopy images of floral organ surface. A toC, 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 magnificationof A, showing a space between petal and sepal (arrows). E and F, Highermagnification of D, showing direct contact between petal and sepal (ar-rows). H and I, Higher magnification of G, showing straight petal elongation(G) and abaxial epidermis of petals (I) starting the deposition of the epicu-ticular nanoridges on their surface. K and L, Higher magnification of J,showing direct contact of petal and sepal (arrows). N and O, Petal surface ofthe same flower as M. Abaxial epidermal cells of petals deposit nanoridgeson cell surface. Q, Higher magnification of P, showing the petal edge turningoutward. R, Higher magnification of petals in Q, showing the trace of fric-tion (arrows). The front side sepal is removed in A, D, G, J, M, and P to showthe inside of flowers. sep, Sepal; pet, petal; ant, anther. Bars = 200mm (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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started to fold (n = 5; Fig. 3, C and D; SupplementalFig. S2). The epidermal cells were flattened in the fop1-1 mutant, especially in the marginal regions of thefolding site (Fig. 3E), although the petals adjacent toremoved sepals had much less flattened cells (Fig. 3F),suggesting that sepal removal reduces the physicalcontact of petals and restores the straight growth.Next, we tested the petal phenotype of a double

mutant carrying both fop1 and pressed flower (prs). Theprs mutant is defective in sepal formation and lackstwo sepals in the lateral positions, thus forming anopen floral bud (Fig. 3G; Matsumoto and Okada,2001). As we expected, petals of the fop1-1 prs-1 doublemutant did not fold; instead, they elongated normally(Fig. 3H). Next, we examined the structure of the sec-ond whorl organ in the ap3-5 fop1-1 double mutant. Inap3-5, which is one of the mutants of homeotic tran-sition of floral organs, petals and stamens are con-verted to sepals and carpels, respectively (Fig. 3I;Bowman et al., 1989). The sepaloid second whorl or-gans in the ap3-5 fop1-1 double mutant did not showthe folded phenotype (Fig. 3J). Rescue of the mutantphenotype may be due to the change of the floral or-gan identity or to the increased space provided by the

loss of anthers. Together, elimination of the tightnessin a floral bud restored the regular petal elongation,suggesting that physical contact of floral organs is thecause of the folded petals in the mutant.

FOP1 Encodes WSD11, a Member of the WAXSYNTHASE/DIACYLGLYCEROLACYLTRANSFERASE Family

We mapped the FOP1 gene on chromosome 5 andidentified a mutation in At5g53390 (Supplemental Fig.S3A). We found that G at nucleotide 820 of the codingsequence was substituted with A in fop1-1, replacingGly with Arg in the mutant protein (Supplemental Fig.S3B). We examined the border sequences of three T-DNA insertion lines found in the SIGnAL database(http://signal.salk.edu/cgi-bin/tdnaexpress) withinor near the gene (Supplemental Fig. S3B; Alonso et al.,2003) and confirmed that the three lines had a smalldeletion at the T-DNA insertion sites (SupplementalFig. S3B). Two of them, SALK_093133 (named fop1-2)and SALK_137481 (fop1-3), showed a similar pheno-type to fop1-1 (Fig. 1, B–D). The SALK_149804 line, inwhich T-DNA was inserted at 31 bp upstream from theATG initiation codon, was indistinguishable from thewild type and did not show the petal-folding pheno-type.

To confirm whether At5g53390 is FOP1, a genomicfragment comprising the 2.2-kb promoter, 2.2-kbopen reading frame, and 1.6-kb 39 region was trans-formed into the fop1-1 mutant. We obtained 80 inde-pendent primary transgenic plants carrying the FOP1genomic fragment, and all lines complemented thepetal defects, indicating that At5g53390 is FOP1.We cloned a full-length FOP1 complementary DNA(cDNA) using a RACE strategy and identified the re-gion covering the 48-bp 59 untranslated region, 217-bp 39untranslated region, and seven exons (Supplemental Fig.S3B).

FOP1 corresponds to WSD11, a member of thebifunctional WAX SYNTHASE/DIACYLGLYCEROLACYLTRANSFERASE (WS/DGAT) family (Kalscheuerand Steinbüchel, 2003; Li et al., 2008). Ten genes encodesimilar proteins in Arabidopsis, and a gene located nextto FOP1 on chromosome 5, At5g53380, is the closesthomolog of the same family (Supplemental Fig. S3A;Li et al., 2008). The amino acid sequence of FOP1showed 17.3% identity with that of Acinetobacter spp.WS/DGAT (GenBank accession no. AAO17391; Kalscheuerand Steinbüchel, 2003). Alignment of the FOP1,At5g53380, and Acinetobacter spp. WS/DGAT proteinsrevealed three domains of high homology, and the firstdomain (amino acids 106–155 of FOP1) includes aputative active-site HHxLxDGxS box, which is con-served among Mycobacterium spp. and plant WS/DGAT proteins (Supplemental Fig. S3C; Kalscheuerand Steinbüchel, 2003; Li et al., 2008), suggesting thatFOP1 is involved in the synthesis of wax esters andtriacylglycerols.

Figure 3. Surgical and genetic removal of sepals restores straightgrowth of petals. A, Flower bud of fop1-1 at stage 11. Sepal on theabaxial side is removed. Petals are shown with arrows. B, The sameflower with A after 3 d, showing the side of the sepal removal. C, Asepal-removed flower at the stage when petals have already folded(arrows). D, The same flower with C after 3 d, with the straight growthof petals. E, Scanning electron microscopy image of petal epidermis infop1-1. Surface of epidermal cells, especially those at the marginalregion, are flattened. F, Scanning electron microscopy image of petalepidermis in fop1-1, where a sepal has been removed and petals growstraight. G, prs-1 flower. H, fop1-1 prs-1 double mutant flower. Petalsof the double mutant are not folded. I, ap3-5 flower. J, ap3-5 fop1-1 double mutant flower. Bars = 500 mm (A–D) and 50 mm (E and F).



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FOP1 Is Expressed in the Epidermis of Growing Petals

We investigated the spatiotemporal expression pat-tern of FOP1 during flower development. Reversetranscription (RT)-PCR analysis revealed that FOP1was preferentially expressed in inflorescences, includ-ing 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 lowexpression levels. We then generated transgenic plants

carrying FOP1p:GUS, in which the GUS gene wasexpressed under the control of the 2.2-kb FOP1 pro-moter. Out of 29 independent transgenic lines obtainedby screening, 26 lines showed GUS signals. In floralbuds at stage 2, when sepal primordia initiated, GUSsignal was detected in the floral meristem and sepalprimordia (Fig. 4B). The GUS expression was high inpetal primordia at stage 9, with weak expression in theother floral organs (Fig. 4C). At stage 11, FOP1 isexpressed in the marginal region of petals and inovules (Fig. 4D). We also generated a translationalfusion line by transforming the FOP1p:FOP1-GFPtransgene into the fop1-1 mutant. The petal phenotypeof the mutant was rescued, indicating that the fusionprotein was functional in plants (Fig. 4F). GFP signalwas detected at the margin of elongating petals, whenthey grew through the space between anthers and se-pals (Fig. 4E), and the expression expanded widely inthe distal part of petals (Fig. 4F). FOP1 had lower ex-pression in sepals and anthers than in petals, indicat-ing that function of FOP1 in petals is required forstraight elongation. The FOP1-GFP fusion protein waslocalized at the periphery of petal epidermal cells (Fig.4G). We further examined the cellular localization ofFOP1 protein by expressing the GFP-FOP1 fusion genetransiently in suspension-cultured cells of Arabidopsis(Fig. 4H). The GFP signal was detected in the periph-ery of the cell, which overlapped with the FM4-64signal (Fig. 4, I and J), indicating that FOP1 is local-ized to the plasma membrane. Together, these datasuggest that FOP1 is involved in wax ester synthesis atthe plasma membrane in the petal epidermis.

DISCUSSION

Study of the fop1 mutant directed attention to anaspect of petal growth in a floral bud, namely to howpetals elongate through the narrow space betweensepals and anthers. Figure 5A describes the schematicmodel highlighting the differences in the petal elon-gation process between wild type and fop1. Growth ofpetal primordia is normal up to stage 9; however, afterstage 9, the petal tip becomes stuck between sepals andanthers in the mutant, while in the wild type, petalselongate straight. Our data suggest that the productsof FOP1 act as a lubricant on the petal surface, en-abling petals to elongate smoothly through the narrowspace in a floral bud. It is also possible that the FOP1products are involved in making cuticle rigid, so thatin the fop1 mutant, the petal surface intensity is lowerand easy to get rubbed off, enhancing the petal folding.

FOP1 encodes a homolog of the WS/DGAT en-zymes, suggesting that biosynthesis of wax estersand/or triacylglycerols mediated by FOP1 is requiredfor the smooth petal elongation. Several mutants de-fective in wax biosynthesis or secretion show a similarpetal-folding phenotype, and their petals are sensitiveto dye immersion due to the lower repellency of thepetal surface (Panikashvili et al., 2009; Li-Beisson et al.,

Figure 4. FOP1 is expressed in elongating petals. A, RT-PCR analysisof FOP1 transcripts. FOP1 is expressed in inflorescences, includingyoung floral buds, and in open flowers. ACT8 was used as a control. Bto D, GUS expression in inflorescences and flowers carrying theFOP1p:GUS transgene. B, GUS is expressed strongly in floral meristemand sepal primordia. C, Strong GUS expression in petal primordia of astage 9 flower (arrowheads). D, GUS expression is observed at petalmargin (arrowheads) and ovules in a stage 11 flower. E to G, GFPfluorescence in fop1-1 flowers of plants carrying the FOP1p:FOP1-GFP transgene. The fusion protein is expressed preferentially at themargin of petals of a stage 10 flower (E) and in the apical part of petalsof a stage 12 flower (F). G, A merged image of bright-field and GFPimages of petal epidermal cells. The FOP1-GFP fusion protein is lo-calized at the cell periphery. H to J, Suspension cell expressing GFP-FOP1 transiently. H, Bright-field image. I, GFP image. J, Merged imageof the GFPand FM4-64 images, showing that GFP-FOP1 is localized toplasma membrane. Note that the upper cell, which is supposed not tocarry the construct, shows only FM4-64 signal. IM, Inflorescencemeristem; 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).


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2009). We immersed the fop1-1 petals in the dye tolu-idine blue (Tanaka et al., 2004), but they were notstained (Supplemental Fig. S1, M and N). We alsoexamined the difference of the hexane-soluble surfacecontents of floral buds between wild type and fop1-1,but found no significant change (Supplemental Fig.S4). Arabidopsis WSD1, a homolog of FOP1, has highwax synthase activity and lower but significant DGATactivity (Li et al., 2008), suggesting that FOP1 has thesame activities as WSD1. Identification of substratesand products of FOP1 will elucidate the role of petalsurface 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). Cuticlemainly consists of cutin and cuticular wax. Cutin is aninsoluble lipid polymer, consisting of aliphatics (C16and C18 fatty acids), aromatics, and glycerols, andcovers the external surface of the cell wall. The outerlayer 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 theplant body (Pollard et al., 2008). A series of Arabi-dopsis mutants defective in cuticle synthesis and se-cretion show the biological roles of cuticles as a barrierto biotic or abiotic stresses, osmotic stress, water loss,and damage from UV radiation, and in preventing thefusion of leaves and floral organs (Yephremov et al.,1999; Pruitt et al., 2000; Krolikowski et al., 2003; Aharoniet al., 2004; Kurdyukov et al., 2006; Bessire et al., 2007;Shi et al., 2011; Wang et al., 2011). Some mutants areknown to be involved in petal morphogenesis: Lack ofnanoridges of petals, due to a mutation in DEFECTIVEIN CUTICULAR RIDGES (DCR, encoding a BAHD ac-yltransferase), CYP77A6 (cytochrome P450 family),GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE6(GPAT6), or PERMEABLE CUTICLE1 (PEC1), result inincreased permeability of petals to a dye and causeorgan fusion (Li-Beisson et al., 2009; Panikashviliet al., 2009; Bessire et al., 2011). Compared with these

Figure 5. Schematic model of the FOP1 function.A, Model explaining the process of petal foldingin fop1 floral buds at stage 8 (left), stage 9 (cen-ter), and stage 12 (right). Red represents petals.Left and right halves show the petal structure inthe wild type and fop1, respectively. The bluearea represents the region of contact betweenpetals and sepals. Arrows indicate the direction ofpetal elongation. B, Model explaining the processof petal surface modification. During petal elon-gation between sepal and anther, FOP1 productsmay be secreted to the surface, enabling smoothelongation through the narrow space. Two ATP-binding cassette transporters, ABCG11 and ABCG13,may be involved in a similar process (center). Beforematuration, epicuticular nanoridges deposit the out-ermost surface of petal epidermis, mediated byGPAT6, CYP77A6, DCR, and PEC1/ABCG32, pre-venting organ fusion and protecting petals fromwaterloss and pathogen attack. WT, Wild type.



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mutants, fop1 petals form nanoridges on the petalepidermis, and the degree of organ fusion is, if any,only subtle (Fig. 2). Because the petal folding startsbefore the nanoridges deposit on the surface of petalepidermis, we propose that FOP1 is involved in thesynthesis of wax-related products in the petal epi-dermis before nanoridge deposition (Fig. 5B). Similarpetal-folding defects are found in mutants of theDESPERADO/AtWBC11/ABCG11 and ABCG13 genes,both of which encode members of ATP-binding cas-sette transporters (Panikashvili et al., 2007, 2011),suggesting that they are involved in a similar processto that of FOP1 (Fig. 5B).

Is the petal elongation mechanism conserved amongflowering plants? A similar petal-folding phenotype isalso known in a breed of Japanese morning glory (Ip-omoea nil). Corollas of the crepe (cp) mutant fold twicelike those of fop1, forming an additional tube-likestructure surrounding the stamens and carpels(Supplemental Fig. S5; Miyake and Imai, 1927). Similarto our results, the removal of calyx restored the for-mation of funnel-shaped corollas (Nishino and Gotoh,2002). These characters suggest that the cp phenotypemight be caused by a similar mechanism to that of fop1petals. Molecular identification of the CP gene willprovide an answer.

CONCLUSION

Here, we propose a mechanism for the smoothelongation of petals. After primordia initiation, petalselongate through the narrow space generated by anthersand sepals. FOP1/WSD11 is expressed in elongatingpetals and may catalyze the wax ester biosynthesis atthe epidermal plasma membrane. The FOP1 productsmay act as a lubricant that enables petals to growstraight in a narrow space in a bud. Identifying thesubstrates and products of FOP1/WSD11 is the nextchallenge in the effort to understanding the relation-ship between the cell surface components and petalmorphogenesis.

MATERIALS AND METHODS

Plant Growth Conditions

The Wassilewskija and Columbia ecotypes of Arabidopsis (Arabidopsisthaliana) were used as the wild type. The fop1-1 mutant was isolated from anM2 population of long hypocotyl5 (Wassilewskija ecotype background; Oyamaet al., 1997) mutagenized by ethyl methanesulfonate. T-DNA insertion mu-tants were obtained from the SIGnAL Web site (http://signal.salk.edu) andthe Arabidopsis Biological Resource Center (http://abrc.osu.edu). Seeds weresown on the surface of vermiculite in small pots and incubated for 1 week at4°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 previouslydescribed (Matsumoto and Okada, 2001) and observed with S-3200N (Hitachi)and JSM-5800 (JEOL) microscopes. For histological analysis, inflorescenceswere fixed in formaldehyde-acetic acid (50% [v/v] ethanol, 3.7% [v/v]

formaldehyde, 5% [v/v] acetic acid), dehydrated in an ethanol series, andembedded in Technovit 7100 resin (Heraeus Kulzer). Sections of 5-mm thick-ness 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 inclearing solution (40 g chloral hydrate, 10 mL glycerol, 5 mL distilled water).The length and width of cleared petals were measured using the Image Pro-Plus 5.0 software. For whole-mount toluidine blue staining, petals weretreated with 0.05% (w/v) toluidine blue for 2 min and washed in distilledwater twice (Tanaka et al., 2004). Samples were visualized under an Axiophot2 microscope (Carl Zeiss).

Mapping and Cloning of FOP1

F2 plants generated by crossing fop1-1 with the Columbia ecotype wereused for mapping. Information about the RPS4NT, nga129, JV61/62, andEG7F2 markers was obtained from The Arabidopsis Information Resource(http://www.arabidopsis.org). Other markers were generated as sequencemarkers based on the polymorphisms revealed by genome sequencing. Thesequences of the oligonucleotides used in mapping and gene cloning are listedin Supplemental Table S1. cDNA cloning was performed by both 59 RACE and39 RACE using the SMART RACE cDNA Amplification Kit (Clontech). Se-quencing was performed using the ABI BigDye Terminator Cycle SequencingReady Reaction Kit and an ABI Prism 3100 Genetic Analyzer (Applied Bio-systems).

Complementation Test

The genomic fragment including the 2.2-kb promoter, 2.2-kb open readingframe, and 1.6-kb 39 regions of FOP1 was cloned as follows. Fragments am-plified by PCR using the 53390R2 and FOP3R primers were digested with SpeIand HindIII, and cloned into pBluescriptII SK1 (Stratagene) to generatepFg3SK. The promoter region was amplified using the FOP5SalIF andFOPpXbaIR primers, digested by SalI and XbaI, and cloned into pBluescriptIISK1 to generate pFpSK. The HindIII fragment from pFpSK was subclonedinto pFg3SK to generate pFgfSK, and the KpnI-SacI fragment of pFgfSK wassubcloned to pPZP211 (Hajdukiewicz et al., 1994) and pPZP211NP (a gift fromT. Nishimura, Nagoya University) to generate pFgf21135 and pFgf211NP,respectively. These constructs were introduced into the fop1-1 mutant by avacuum infiltration procedure with the Agrobacterium tumefaciens strainC58C1. Transgenic plants were screened on an agar medium containing 30 mgmL-1 kanamycin and 100 mg mL-1 carbenicillin. Sequences of oligonucleotideprimers 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 RNeasyPlant Mini Kit (Qiagen). One microgram of total RNA was reverse transcribedwith the SuperScript II reverse transcription kit (Invitrogen). The oligonucle-otide primers used for RT-PCR are as follows: FOP1, 53390F1a and 53390R1,and ACTIN8, ACT8F and ACT8R. The sequences of the oligonucleotideprimers are listed in Supplemental Table S1.

mRNA in Situ Hybridization

mRNA in situ hybridization was performed as previously described(Matsumoto and Okada, 2001). The HISTONE4 probe was prepared as pre-viously described (Dinneny et al., 2004).

Histochemical Analysis of FOP1

The HindIII fragment from pFpSK was subcloned into pBI101 (Clontech) togenerate pFpGUSBI. pFpGUSBI was transformed into Columbia plants andscreened as described above. Samples were incubated in a staining buffer (100mM NaPO4, pH 7.0, 10 mM EDTA, pH 8.0, 5 mM potassium ferricyanide, 5 mMpotassium ferrocyanide, 0.1% Triton X-100 (w/v), and 0.5 mg mL–1 5-bromo-4-chloro-3-indolyl-b-glucuronic acid) at 37°C for 6 h. The stained samples weretreated with 70% ethanol for 10 min at room temperature, 100% ethanol for 30min at 37°C, and 70% ethanol for 10 min at room temperature, and thencleared in clearing solution. Samples were visualized under a Leica M420(Leica Microsystems) or an Axiophot 2 microscope with a differential inter-ference contrast filter. For transient expression in suspension cells, the FOP1


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cDNA was amplified by PCR with FOP1FSacI2nt and FOP1RKpnI primers(Supplemental Table S1), digested with SacI and KpnI, and cloned into 35S-GFP-NOS/pUC18 plasmid. The generated 35S:GFP:FOP1c construct wastransformed into Arabidopsis suspension-cultured cells by polyethylene glycol-based method as has been previously described (Uemura et al., 2004). The fluo-rescent 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 pPZP211NPto generate pFcG21135 and pFcG211NP, respectively. The HindIII fragmentfrom pFpSK was subcloned into pFcG21135 and pFcG211NP to generatepFpFcG21135 and pFpFcG211NP, respectively. Transformation and screeningwere performed as described above.

Gas Chromatography

Surface fatty acids were extracted from flowers of 6-week-old plants withhexane. C19COOH (Fluka) and N,O-bis(trimethylsilyl)trifluoroactamide with1% trimethylchlorosilane (Sigma) were added as internal controls. The solu-tion was incubated at 80°C for 30 min and concentrated and analyzed with aGC-14A gas chromatograph (Shimadzu) with the use of a DB-1 column (J&WScientific). The measurement conditions were 170°C to 265°C with a 2°C in-crease per minute, then to 310°C with a 4°C increase per minute, and hold for10 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 datalibraries 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 Japanesemorning glory.

Supplemental Table S1. Oligonucleotide primers used in this work.

ACKNOWLEDGMENTS

We thank Eisho Nishino (Chiba University, Japan), Eiji Nitasaka (KyushuUniversity, Japan), Noriyoshi Yagi (Kyoto University, Japan), Mitsuhiro Aida(Nara Institute of Science and Technology, Japan), Koichi Toyokura (NationalInstitute for Basic Biology), Maki Kondo (National Institute for Basic Biology),Mikio Nishimura (National Institute for Basic Biology), the Model PlantResearch Facility (National Institute for Basic Biology), and the FunctionalGenomics Facility (National Institute for Basic Biology) for their help with thiswork; Rebecca Horn (John Innes Centre) for critical reading of the paper; andthe Salk Institute and the Arabidopsis Biological Resource Center for provid-ing T-DNA insertion mutants.

Received November 29, 2012; accepted January 9, 2013; published January 11,2013.

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