DEVELOPMENTAL
BIOLOGY
152,383-392
(1992)
Fiddlehead: An Arabidopsis Mutant Constitutively Expressing an Organ Fusion Program That Involves Interactions between Epidermal Cells SUSANJ. LOLLE,~ ALICE Y. CHEUNG,AND IAN M. SUSSEX’ Department
of Biology, P.O. Box 6666, Yale University, Accepted April
New Haven, Connecticut
06511
l-6, 1992
In most circumstances plant epidermal cells do not respond to surface contact with adjacent plant parts. We have identified and characterized a mutant of Arabidopsis thaliana, designated jiddlehead, where lateral appendages of the shoot fuse with one another. While fusion between floral organs is most frequent, leaf fusions also occur. Using scanning and transmission electron microscopy, we show that adhesion takes place between epidermal cells and does not involve cytoplasmic union. We also show that the frequency of organ fusion is dictated by organ proximity. In wildtype Arabidopsis, postgential fusion takes place exclusively in the gynoecium, whereas in theJddlehead mutant, this program becomes expressed constitutively. The existence of such a mutant demonstrates that postgenital fusion is a genetically o I992 Academic press, he. distinct program superimposed upon other aspects of gynoecial development in Arubidopsis. INTRODUCTION
The shoot apical meristem of dicotyledonous plants is organized into distinct layers. The outermost layer, designated Ll, gives rise to the epidermis covering the entire shoot while the inner layers produce the rest of the shoot (Dermen and Stewart, 1973; Jegla and Sussex, 1989; Poethig and Sussex, 1985a,b; Satina, 1944; Satina and Blakeslee, 1941,1943; Satina et al, 1940; Stewart and Burk, 1970; Stewart and Dermen, 1975). The Ll or epidermal lineage is the most stable of the meristematic cell populations and its derivatives serve many important functions during the growth and development of the plant. In addition to regulating gas exchange and providing a barrier against desiccation and pathogen infiltration, the epidermis ensures the structural and functional integrity of the plant. If the surfaces of two organs grow into contact, epidermal cells typically do not respond and even upon wounding and grafting make little or no contribution to regenerated tissues (Moore, 1984; Walker and Bruck, 1985). In developing flowers, and particularly during formation of the female reproductive organ (the gynoecium), however, some epiderma1 cells participate in cell wall fusions. Upon contact, cells adhere but do so without undergoing cytoplasmic union (Cusick, 1966). Such late ontogenetic or postgenital fusion represents an exceptional case during normal development of cell-cell interaction between epidermal cells. 1 To whom correspondence should be addressed. ’ Current address: Department of Plant Biology, University fornia, Berkeley, CA 94720.
of Cali-
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Late ontogenetic fusion has been extensively studied in Catharanthus roseus in which two carpel primordia fuse during gynoecial development. Early in development, the surfaces of two originally separate carpel primordia grow into contact and fuse. Epidermal cells along the resulting suture undergo rapid and substantial changes in cytoplasmic density, cell shape, and cell division planes, redifferentiating into parenchyma cells (Verbeke and Walker, 1985, 1986; Walker, 19’75a,b,c; Walker, 1978a,b). Both cellular recognition and communication are involved in this process. Not all contacting epidermal cells undergo dedifferentiation, however, and appear to do so only if organ fusion occurs early in ontogeny (Walker, 1975a). Water-soluble morphogenetic factors mediate all of these changes but only carpel epiderma1 cells are responsive under the described experimental conditions (Siegel and Verbeke, 1989). In wildtype (wt) Arabidopsis plants ontogenetic fusion is limited to the formation of the ovary septum and the transmitting tissues of the gynoecium (Okada ef al., 1989). With this exception, no other instance of organ fusion occurs in Arabidopsis during either normal vegetative or floral development. We have isolated a mutant of Arabidopsis thaliana, designated fiddlehead vdh), which overcomes the normal restrictions placed on organ fusion during ontogeny such that vegetative and floral organs fuse postgenitally. We have named the mutant fiddlehead because adhering floral buds cause apical curling of inflorescences, resulting in structures reminiscent of fern fiddleheads. In an effort to elucidate the nature of the organ fusions that occur in fdh plants, we have studied floral development and analyzed contacting tissues using scanning (SEM) and transmission 0012-1606/92 $5.00 Copyright All rights
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
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(TEM) electron microscopy. The morphological evidence presented here reveals that organ fusion in fdh plants occurs when epidermal cells contact along their periclinal walls. Epidermal cells adhere but do not undergo cytoplasmic union, conforming to the classical definition of ontogenetic or postgenital fusion (Cusick, 1966). In addition to epidermal cell adhesion, organ fusion is accompanied by striking changes in epidermal cell surface morphology, indicating that morphogenetic factors are produced.
VOLUME152,1992 TABLE 1 LINKAGE ANALYSISOFfdh MUTATION Number of individuals with indicated phenotype
Genotype (Chromosome no.) up1 (1) us (2) up9 (3) wz (4) pi (5)
MATERIALS
AND METHODS
Mutant Isolation and Plant Growth Ethyl methane sulfonate mutagenized M2 bulked seed of the Landsberg erecta ecotype was purchased from Guhy Nurseries (Tucson AZ). A total of 16,000M2 plants were screened, and selfed progeny were collected from a subset of individuals. Fiddlehead was isolated from one such family. Plants were grown under either a long (16 hr day/8 hr night) or a short day (8 hr light/l6 hr night) regime with a mix of fluorescent and incandescent lights (175 pmole . m-’ * set-’ at pot level). Growth room temperatures averaged 20°C (+2”C). Seedswere planted in a 9:3:1 vermiculite:soil:sand mixture and watered once daily with a nutrient solution containing 6 g/gallon ‘7-6-19All Purpose Hyponex (Hyponex Co., Copley, OH) and 0.4 g/gallon 15-16-1’7Peat Lite Special (Peters Co., Allentown, PA). Genetic crosses were performed manually using plants heterozygous for the fdh mutation. Marker lines were obtained from Dr. J. Haley (Harvard University). Microscopy Tissue was collected from plants and fixed in either FAA (3.7% formaldehyde, 50% ethanol, 5% acetic acid) or a mixture of 4% glutaraldehyde-2% paraformaldehyde in Sorensen’s buffer (0.05 M pH 7.0) for 2 hr. Samples fixed in FAA were stored at 4°C in 70% ethanol and either prepared for SEM or dehydrated to 100% butanol and embedded in paraffin. Samples for SEM were dehydrated to 100% ethanol and critical point dried in liquid CO,. Dried tissue was mounted on SEM stubs and sputter coated with gold-paladium using an SPI sputter coater. Specimens were examined in an ISI SS40 scanning electron microscope with an accelerating voltage of 5-10 kV. Glutaradehyde-fixed tissue was washed in buffer, postfixed in 2% osmium tetroxide (0.05 M Sorensen’s buffer, pH 7.0), and dehydrated through a graded ethanol series. Samples were embedded in Spurr’s epoxy resin (Spurr, 1969; Electron Microscope Sciences). For
wt
.fdh
197 639 293
244
216 242
Double mutant
Marker
39 90 71 66
7
43 214
4” 36 22 25
110 83 82
Expected 17.8 68.8 33.0 24.5
25.9
Note. Segregation data for F, progeny of crosses between fdh (as male parent) and the chromosome marker lines listed in column 1. a From the calculated number of expected double mutants listed in the last column only segregation frequencies of fdh with as indicate genetic linkage.
light microscopy, Spurr’s-embedded material was cut to approximately 1 pm, and for TEM to 600-800 A using a Sorvall ultramicrotome (MT-2). Samples for TEM were stained with 3% uranyl acetate for 10 min and 0.3% lead citrate for 5 min and examined in a Phillips 300 TEM at 80 kV. RESULTS
The fiddlehead Phenotype Is Due to Mutation
a
Single Recessive
The segregation frequencies of the fdh mutation with known phenotypic markers are summarized in Table 1. The data indicate that the fdh phenotype results from a single recessive nuclear mutation which maps to chromosome 2. Apl fdh double mutants have a reduced incidence of organ fusion (see below) and as a result, the number of double mutants identified was artificially low. Fusion Can Occur between All Lateral Organs of the Shoot In the fdh mutant fusions occur predominantly between organs within single floral buds as well as between adjacent buds within a given inflorescence. Newly initiated floral buds become trapped within the inflorescence by surrounding older buds while internode elongation and pedicel elongation continue (Fig. 1). Hand dissection of individual fdh flowers reveals that all four organ whorls participate in fusion events and do so in a variety of combinations. Sepals, for example, fuse to one another and to petals while stamens adhere to all whorl organs within the flower including neighboring stamens. At anthesis, petals and stamens remain seques-
385
FIG. 1. Inflorescences of wildtype (A) andjiddkeheud (B-D) plants. (A) A 6-week-old wt Arcrbidopsis plant showing the inflorescence bolt and apex (open arrow). At anthesis, flowers spontaneously self-pollinate, giving rise to the seed-containing siliques (si). (B-D) In,fdh plants, fusion between sepals (s) of emerging flower buds (fb) and continued pedicel (pd) and internode elongation eventually result in curling of the entire inflorescence, producing structures reminiscent of fern fiddleheads. Elongation growth results in the emergence of pistils (p) from within the fused sepals but without pollination taking place. Afdh inflorescence (open arrows) at (B) 6 weeks and at (C) 14 weeks. (D) A scanning electron micrograph showing a,fdh inflorescence at 10 weeks postgermination. A, B, and C are the same magnification. Scale bar in A = mm. Bar in D = Wm.
tered within the fused calyx of individual floral buds. Continued growth and elongation of the gynoecium, which is constrained only by limited fusion to anthers and apically to sepals, eventually result in pistil emergence (Fig. 1D). At maturity,f% flowers have correct organ numbers occupying correct whorl positions. The ovary, style, and stigma are structurally normal and fdh flowers produce mature pollen and ovules. Stamens elongate but remain physically restricted from contact with the receptive stigmatic surface, and flowers do not spontaneously self-pollinate.
Early floral development in fdh plants is indistinguishable from wt as revealed by both light microscopy (data not shown) and SEM. As in wt, fdh floral organs are initiated as discrete primordia (Fig. 2). Only after organs have grown into contact does development diverge from that observed in wt. Shortly after sepals enclose the flower bud, small epidermal cell clusters can be seen between closely appressed neighboring buds and at sepal margins (Fig. 2E). Eventually sutures form between sepals within a whorl and between the sepals of adjacent contacting floral buds. As floral development
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FIG.2. Scanning electron micrographs showing wt (A to C) and early,fih (D to F) floral development. (A) Arubidopsis flowers arise in a helical array from the inflorescence meristem. The apical cluster contains flower buds at various stages of maturation. Just prior to anthesis, sepals (s) open exposing four petals (pe), six stamens (a), and the central gynoecium or pistil (p). (B) Early in development maturing inner whorl organs are completely enclosed by four overlapping sepals (arrow shows discrete sepal margin). (C) During gynoecial development the inner walls of the tubular primordium grow into contact, as shown here, and fuse giving rise to the ovary septum and the transmitting tissues, At maturity, the primordium will have closed apically and ovules will have formed along the margins of the fused septum. (D) Early floral development in fdh is indistinguishable from wt. Sepal primordia (sp) are initiated as discrete organs that eventually enclose the floral meristem. (E and F) Infdh, fusion commences after sepals grow into contact. Neighboring buds are united by cell clusters bridging the abaxial sepal surfaces (E) eventually forming a near continuous suture (arrows in F). Scale bars indicate magnification in micrometers. proceeds, growth causes shearing or tearing of epiderma1 layers and disfiguration of some organs. Petals are most severely affected. On average petals expand to only 25% of the size reached in wt flowers at maturity. Rosette and cauline leaves also participate in fusions (Fig. 3A). These fusions can involve one or more leaves. As is true for floral organs, fusion can occur along the margins or on the adaxial or abaxial surface. Although the majority of fdh plants do achieve a relatively normal
vegetative architecture, at a low frequency leaf fusions completely block organ expansion, leading to termination of growth and eventual plant death. In most of these instances, inflorescence stems will have fused to neighboring leaves. Arabidopsis plants switch from vegetative to floral development within approximately 7-10 days if grown under 16 hr photoperiods (Miksche and Brown, 1965). If light exposure is limited to 8 hr photoperiods the switch
387
FIG. 3. Scanning electron micrographs showing leaf fusions in,fak (A) and floral organ fusion in the inHorcscence of an clplfilh double mutant (B). (A) Fusion within and between rosette leaves occurs at variable but lower frequency than fusion between floral organs. Two individual leaves which have fused with each other along their margins (arrows) are shown nest to a single leaf which is fusing to itself (open arrow) ipt, petiole). (B) In trp~,fdl) double mutants fusion patterns diverge from those usually observed in the single mutant. In the tcpl genetic background the physical proximity of the floral whorls is altered such that individual flowers as well as the inflorcsccnce achieve a more open configuration. Bract-like organs (b) often remain free from one another although fusion can still occur between these organs and the inner stamens and pistil (p). Stamen filaments elongate despite anther (a) fusion to the pistil (p) (st. stipulcs). Scale bars indicate magnification in micrometers.
to flowering is significantly delayed and vegetative growth is prolonged for months (Besnard-Wibaut, 1981; Miksche and Brown, 19651. To test whether organ fusion in .fdh plants can occur independent of floral morphogenesis, a segregating population of 50 plants was maintained under short day growth conditions for 2 months. Following the 2-month period, plants were shifted to 16 hr photoperiods so as to induce flowering, score for floral fusion, and to confirm the.fdh phenotype. Only 2 of 12 jidh, plants were identified which had not fused vegetatively under the short day conditions.
The differing frequencies with which fusion events are observed in the .fdh mutant suggest that physical proximity is an important determinant of fusion. In any given.fdh mutant plant some floral organs always participate in fusions while vegetative organs fuse at variable but lower frequency. The incidence of leaf fusion increases from a few percent injiih plants grown singly to 50% in plants grown under crowded conditions. This dependence of leaf fusion on crowding and the consistently
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high fusion frequencies in flowers indicate that organs with greater spatial separation are less likely to participate in a fusion event. Thus, leaves that are arranged in a spiral phyllotaxy (the spatial arrangement of organs on the plant axis) fuse infrequently whereas floral organs, which are arranged in whorls and experience no separation by internodal elongation, often fuse. If the spatial arrangement of organs does influence the relative incidence of fusion, altering this arrangement should also change the pattern of fusions observed. Floral internodes which normally do not elongate between the medial and the lateral first whorl organs do so in the apetelal (apl) floral mutant (Irish and Sussex, 1990). In aplfdh double mutants, contrary to the situation in the fdh single mutant where sepals fuse to form a calyx-like tube, the bract-like first whorl organs typically remain free from one another and more often adhere to other organs within the flower or the inflorescence (Fig. 3B). Flowers in the double mutant acquire an open configuration, revealing fused internal floral organs not normally visible in flowers of the single fdh mutant. In general, changes in floral architecture in these double mutant plants are less striking than those in flowers of the single fdh mutant. Double mutant plants were difficult to distinguish from apl floral mutants in instances where only a few organ fusions had occurred. Although all lateral organs of the shoot are competent to fuse in homozygous fdh plants, fusion between two contacting sibling plants has not been observed under our experimental conditions. Similarly, fdh plants do not adhere to nonliving surfaces. @gan Fusion Occurs without
Cytoplasmic
Union
Analysis of adhering tissues in the fdh mutant by light microscopy (data not shown) and TEM reveals a continuity of intact epidermal cells between the participating organs and indicates that fusion occurs without cytoplasmic union (Fig. 4). At all junctions analyzed, whether involving floral or vegetative fusions, the contacting epidermal cells form a distinct cellular bilayer. Subepidermal cells are not affected. Terminally differentiated epidermal cell types such as guard cell pairs can be distinguished within fusion sutures. Epidermal cells from the same as well as different organ types and of different developmental ages fuse. As shown in Fig. 4C, epidermal cells from a sepal are fused to the epidermis of a differentiating petal primordium. TEM analyses of fusion sutures reveal the presence of an electron-dense material deposited between the cell walls of the adherent epidermal cells (Fig. 4). This material accumulates as tissues mature (Fig. 4B). No intercellular connections (plasmodesmata) have been ob-
V0~~~~152,1992
served between fused cells at any stage during fusion ontogeny, although such connections are evident in the anticlinal walls of epidermal cells (Fig. 4C). Initially, an electron-dense cuticle is present on the outer cell wall surface of adherent epidermal cells (Fig. 4D) which is less readily detected as participating tissues mature (Fig. 4B). Organ Fusion Induces Morphological Surface
Changes of the Cell
Epidermal cells display a cell surface topology which is characteristic for a given organ type when viewed under the SEM. Infdh, epidermal cells are indistinguishable from wt at all stages of development in regions where organ surfaces are not in contact. Along fusion sutures, however, fdh epidermal cells lose their organspecific surface topology (Fig. 5). Cells become smooth and lose their characteristic surface ridging. Such surface changes are evident at fusion junctions on any organ and, with the exception of sepal margins, affect only one or two cell rows back from the suture. Although the surface morphology of contacting epidermal cells is altered, organ identity is not. At junctions where adjacent sepals have not completely fused and sepal margins are in contact with the underlying petal epidermis, surface changes are strikingly polarized. As shown in Fig. 5D, only the one row of petal cells next to the sepal margin differs from the neighboring petal cells while at least five rows of sepal epidermal cells have undergone changes in surface topology. Identical patterns of surface change are also seen at fusion junctions between first and second whorl organs in double mutants homozygous for fdh and either the pistillata (pi) (data not shown) or the apetela3 (ap3) floral homeotic mutations (Fig. 5E). In both the pi and the ap3 mutants, petals are transformed to sepal-like organs (Bowman et al., 1989). In the ap3fdh double mutant (Fig. 5E), cells on the surface of the sepal-like second whorl organ which are in contact with the overlapping first whorl sepal margin show a clearly defined front of surface change identical to that seen on petal cells in the single mutant (Fig. 5D). Organ Fusion Occurs after Primordial
Initiation
Several lines of evidence show that organ fusion takes place soon after primordial initiation. As previously demonstrated, organ primordia are not fused when initiated (see Fig. 2) but become fused before expansion growth ceases.The limited expansion achieved by petals in fdh flowers indicates that these organs fuse to neighboring sepals prior to or by stage 10 in floral development, as defined by Smyth et al. (1990). Similarly, in the apl fdh double mutant, anthers fuse prior to stamen
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FIG. 4. Transmission electron micrographs of fused epidermal cells infdk flowers at various stages of development. (A) Overlapping margins of two fused sepals from a single flower bud shown in cross-section. Epidermal cells have fused forming a suture (arrows) along the adhering periclinal cell walls. No intervening mesophpll cells separate epidermal cells at margins of individual sepals potentiating contact between internal periclinal epidermal cell walls. (B) Cross-section of two fused sepal epidermal cells from an older flower bud showing intact periclinal cell walls (cw) at high magnification. An electron-dense matrix has accumulated between the adherent walls (material between arrows) (cy, cytoplasm). (C) Epidermal cells on the abaxial surface of a petal (pe) fused to adjacent sepal cells shown in cross-section. B matrix of electron-dense material is deposited between the fused cell walls (arrows). Plasmodesmata occur in the anticlinal walls of adjacent epidermal cells (open arrows) but are not present in the periclinal walls of epidermal cells that have fused during ontogeny. (D) Adhering sepal (s) and anther epidcrmal cells shown in cross-section. When cells fuse a darkly staining cuticular surface layer can be distinguished initially (paired arrows) which becomes less well defined at later stages in development (see B) (c, chloroplast). Scale bars indicate magnification in micrometers.
filament elongation (see Fig. 3) since filaments protrude outward from the main axis. Since these fused anthers also show evidence of shearing, anther to pistil fusion occurs prior to pistil elongation. Furthermore, limited attempts to force fusion by physically appressing two mature rosette leaves from an individual plant have not resulted in organ adhesion. DISCUSSION
We have identified a single gene mutation that can eliminate both the spatial and the developmental restrictions placed on organ fusion in Arabidopsis. Our findings provide genetic evidence for the existence of a distinct developmental pathway modulating interactions between epidermal cells in Arabidopsis Mutation
of the FDH gene alters epidermal competence to adhere such that shoot epidermal cells become responsive to contact. This competence is acquired independent of flowering. Organ fusions are most common in flowers and impair reproductive function completely. We know of only one other mutant in plants where abnormal organ adhesion has been described (Hake et al., 1989). This maize mutant has been only partially characterized, however, and details of the ontogeny and mechanism of adhesion have not been determined. Based on our morphological analyses and on the apparent specificity of the fusion response, we propose thatfdh plants undergo true ontogenetic fusion (Cusick, 1966). As defined by Cusick (1966), fusion occurs after organ initiation and growth, is epidermis-specific, and may require some recognition process. Organ fusion in
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FIG.5. Scanning electron micrographs showing epidermal cell surface morphology of first and second whorl organs in wt (A and B) andfiih, (C to E) flowers. (A) Sepal epidermal cells on the abaxial surface of a wt flower. With the exception of the interposed guard cells (gc), the elongate sepal epidermal cells display an elaborate and characteristic cuticular relief. (B) Abaxial petal cells are easily identified by their cuboidal shape and extensive cuticular surface striations. (C) At fusion junctions between adjacent sepals within a whorl, morphological changes occur on the surfaces of fused cells symmetrically about the suture (arrow). Both cell surface characteristics and cell definition are lost. Guard cells (gc) remain distinguishable but like the surrounding sepal epidermal cells lose cell definition. (D) Incomplete fusion between adjacent sepals inf’ih flowers can result in protrusion of the underlying petal tissue. At junctions where sepal-petal fusion has occurred, such as the one shown here, surface changes are strikingly polarized with loss of cuticular topology clearly evident in a single row of petal (pe) ceils (arrows) and in five rows of sepal (s) cells. In some cases, there is a sharp front of surface change affecting only one-half of each of the individual petal cells. (E) In apSfdh double mutants, similar patterns of surface change are observed. The epidermal cells on the subjacent sepal-like whorl (‘pe’) show an identical front of cell surface change (arrows). As is the case in the single mutant, epidermal cells show a directional loss of surface topology. Scale bars indicate magnification in micrometers.
fdh is mediated by adhesion of the epidermal cell walls, does not involve cytoplasmic union, and does not alter organ identity. Fdh plants do not adhere to nonliving surfaces. Fusion as it occurs in,fdh, therefore, is distinct from a wound or grafting response where adhesion of subepidermal cells can occur to nonliving surfaces (Moore, 1984; Moore and Walker, 1981a,b). TEM analysis of fusion junctions reveals the presence of an electron-dense material between fused epidermal cells as well as the absence of plasmodesmata. The frequencies with which fusion takes place between different organs on fdh plants suggest that physi-
cal proximity determines whether two organs will fuse. Chance of contact and incidence of fusion increase with decreasing space. Although we have observed every permutation of organ to organ fusion in the fdh shoot, fusions occur preferentially between floral organs. In apl fdh double mutants where internode elongation occurs between first whorl organs, the incidence of fusion between these bract-like organs is reduced. A similarly reduced incidence of fusion is seen between the spirally arranged rosette leaves. Therefore, we suggest that all organs of the fdh shoot are competent to respond to surface contact with adjacent plant parts during some pe-
LOLLE,CHEUNG,AND&JSSEX
,frlh: A Gene Regultrtin,u Ory~crtl Fusiorl
riod in their development. Physical contact must be achieved for organ fusion to occur. The ubiquity of sepal-petal fusions seen in fdh flowers suggests that the window of fusion competence may be fairly broad. Although petal primordia are initiated immediately after sepal primordia, they are the last floral organs to mature (Smyth et al., 1990). The limited expansion of fused petals infdh flowers suggests that fusion is complete prior to the period where petals experience the greatest expansion growth. It is not clear, however, whether both tissues participate equally and reciprocally in the fusion process. Furthermore, our attempts to force fusion between mature leaves offdh mutant plants have failed, corroborating that fusion competence is restricted temporally and probably is limited to actively maturing organs. In C. roseus, where ontogenetic fusion has been most extensively studied, there is convincing evidence that cell fusion is mediated by water-soluble morphogenetic factors (Siegel and Verbeke, 1989). Are morphogenetic factors produced by fdh cells? On organs that have undergone fusion, cell surface topology is altered such that the cell surface becomes smooth and cell definition is lost at the fusion suture, as seen by SEM. Across heterogeneous organ sutures, such as sepal-petal junctions, fused cell rows show a polarization in these surface changes consistent with the presence of a diffusible morphogenetic front. These findings indicate that contacting epidermal cells produce and respond to diffusible morphogenetic factors. As was demonstrated in C. roseus (Siegel and Verbeke, 1989; Walker, 1975c), only a few cell rows are affected by these factors. What role, if any, these factors play in the fusion process is unclear but it will be of great interest to determine whether they share any functional similarity to those described by Siegel and Verbeke (1989). Due to the sequestration of the contacting cells within the gynoecium in C. roseus, it would be difficult to determine whether surfaces changes such as those described in thefdh mutant accompany the other events detailed ultrastructurally. Transverse sections show that intervening mesophyll cells are often completely absent at sepal margins, facilitating contact between 5 to 10 rows of upper and lower surface epidermal cells along their internal periclinal walls. This number of epidermal cells is exactly coincident with the number of cells that undergo changes in surface topology and cell definition at many of the sepal margins in fdh flowers. The discrete enhancement of this cell surface phenotype along the margins indicates that contact between periclinal cell walls induces the cell surface changes and that contact with either periclinal wall of the epidermal cell (either the external or the internal facing wall) may be sufficient to induce the observed changes. Since epidermal cells distal to the su-
391
tures remain unaffected, lateral contact between epidermal cells (contact between the anticlinal walls) does not induce these changes. Our morphological data indicate that ontogenetic differences between fusing organs infdh plants have some influence on the morphological response of cells along fusion sutures. At sepal-sepal junctions, cell modification is symmetrical about the fusion suture. At sepalpetal junctions, however, cell surface changes are polarized and show a sharp front of change. Although such modifications may be a feature peculiar to petal epiderma1 cells and transformed second whorl organs may behave like petal cells in this regard (since identical gradations of change are seen on sepal-like second whorl organs in the apSfiIh double mutant), it is equally plausible that ontogenetic differences are important determinants of the cell response. In C. I’ose?:us,cells fusing early in carpel ontogeny experience a full spectrum of developmental changes culminating in cell redifferentiation, whereas epidermal cell identity is maintained in regions where cells grow into contact later in development (Walker, 1975a). In C. roseus, the fusing primordia appear to be homologous and are initiated synchronously (Boke, 1949), whereas organs that fuse in jdh plants are often initiated at different times and mature asynchronously. In ,fdh plants, fusion occurs between heterogenous epidermal populations and occurs with less stringent temporal coordination. As such, epiderma1 cells may be incapable of extensive dedifferentiation if they grow into contact late during ontogeny and the variability of response may reflect a diminution in the ability of cells to communicate and interpret morphogenetic signals. Our data demonstrate that organ fusion can be expressed ectopically during Arubidopsis development and does not in and of itself stimulate differentiation of carpel-specific structures such as ovules. Many of the homeotic floral mutants of Arabidopsis form ovules on transformed organs that have not previously participated in a fusion event (Bowman et al., 1989; Haughn and Somerville, 1988; Irish and Sussex, 1990). Similarly, studies on gynoecial development in C. I’ose?Is demonstrate that physically preventing primordial fusion does not block gynoecial maturation (Walker, 1978a). Furthermore, phylogenetic data suggest that intercapel fusion resulted from the superimposition of a distinct epidermal adhesion program upon carpel development (Takhtajan, 1991). It has been suggested that the physical union of carpel primordia enhances reproductive titness by promoting higher seed set (Carr and Carr, 1961). Fusion makes ovules sequestered on any constituent carpel accessible to fertilization irrespective of which carpel the incoming pollen was associated with initially.
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Based on the evidence presented here, we propose that the FDHgene functions to maintain epidermal cells in a distinct developmental state. As in any physical phenomenon there is first a susception phase, in this case physical contact. Second, there is a perception phase where the physical signal is transformed to a physiological response. Third, the physiological response is manifested at the cell wall interfaces. This suggests thatfdh cell walls are altered such that the response to this sequence is organ fusion. The regulating mechanism may be direct, involving protein binding to a gene promoter for example, or indirect, such as occurs in multimeric protein complex formation. We suggest that this developmental pathway is normally expressed only in the developing carpels and, in wt Arabidopsis therefore, the FDH gene is subject to developmental regulation in the carpels. We thank Barry Piekos for instruction in the use of and assistance with the scanning and transmission electron microscopes. We thank Mary Helen Goldsmith, Carolyn Slayman, and Clifford Slayman for their support and interest throughout the course of this work. We also thank Graeme Berlyn, Marc Kirschner, Timothy Nelson, David Taylor, Hong Wang, and Hen-ming Wu for valuable discussion and critical reading of the manuscript. This research was supported by a McKnight Foundation Award to Yale University. S.J.L. was supported by a McKnight Foundation Postdoctoral Fellowship. REFERENCES Besnard-Wibaut, C. (1981). Effectiveness of gibberellins and 6-benzyladenine on flowering of A rubidopsis thaliunu. Physiol. Plant. 53, 205-221. Boke, N. H. (1949). Development of the stamens and carpels in Vi~u Roseu L. Am. J. Bof. 36, 535-54’7. Bowman, J. L., Smyth, D. R., and Meyerowitz, E. M. (1989). Genes directing flower development in Arubidopsis. Plnxt Cell 1, 37-52. Carr, S. G. M., and Carr, D. J. (1961). The functional significance of syncarpy. Phytomorpholgyy 11,249-256. Cusick, F. (1966). On phylogenetic and ontogenetic fusions. 1n “Trends in Plant Morphogenesis” (E. G. Cutter, Ed), pp. 171-183. Longmans, Green. Dermen, H., and Stewart, R. N. (19’73). Ontogenetic study of floral organs of peach (Prunus persicu) utilizing cytochimeral plants. Am. J. Bot. 60, 283-291. Hake, S., Walko, R., Lane, B., and Lowe, B. (1989). Transposon mutagenesis in developmental biology: Methodology and results. Curr. Topics Plunt Biochem. Phgsiol. 8, 237-250. Haughn, G. W., and Somerville, C. R. (1988). Genetic control of morphogenesis in Arubidopsis. Den Genet. 9, 73-89. Irish, V. F., and Sussex, I. M. (1990). Function of the upetalnl gene during Arnbidopais floral development. Pltrnt Cell 2, 741-753. Jegla, D. E., and Sussex, I. M. (1989). Cell lineage patterns in the shoot meristem of the sunflower embryo in the dry seed. DPU. Biol. 131, 215-225. Miksche, J. P., and Brown, J. A. M. (1965). Development of vegetative and floral meristems of Arubidopsis thliam~ Am. J. Bof. 52, 533537.
Moore, R. (1984). Cellular interactions during the formation of approach grafts in Sedum tdephoidrs (Crassulaceae). Gun. .I. Bot. 62, 2476-2484.
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Moore, R., and Walker, D. B. (1981a). Studies on vegetative compatibility-incompatability in higher plants: I. A structural study of a com(Crassulaceae). Am. J. Bat. patible autograft in Se&urn telephoides 68,820-830.
Moore, R., and Walker, D. B. (1981b). Studies on vegetative compatibility-incompatibility in higher plants: II. A structural study of an incompatible heterograft between Sedum telephoides (Crassulaceae) and So2anunz pe?lrLelli (Solanaceae). Am. .I Bot. 68, 831-842. Okada, K., Komaki, M. K., and Shimura, Y. (1989). Mutational analysis of pistil structure and development in Arabidopsis. Cell Difleer. Dev. 28,27-38.
Poethig, R. S., and Sussex, I. M. (1985a). The developmental morphology and growth dynamics of the tobacco leaf. Planta 165,158-169. Poethig, R. S., and Sussex, I. M. (1985b). The cellular parameters of leaf development in tobacco: A clonal analysis. Plunta 165,170-184. Satina, S. (1944). Periclinal chimeras in Dutura in relation to development and structure (A) of the style and stigma (B) of calyx and corolla. Ant. J. Bof. 31,493-502. Satina, S., and Blakeslee, A. F. (1941). Periclinal chimeras in Dutura sfrumoniwm in relation to development of leaf and flower. Am. J. Bof. 28, 862-871. Satina, S., and Blakeslee, A. F. (1943). Periclinal chimeras in Datura in relation to the development of the carpel. Am. J. Bot. 30,453-462. Satina, S., Blakeslee, A. F., and Avery, A. G. (1940). Demonstration of the three germ layers in the shoot apex of Dufuru by means of induced polyploidy in periclinal chimeras. Am. J. Bot. 27, 895-905. Siegel, B. A., and Verbeke, J. A. (1989). Diffusible factors essential for epidermal cell redifferentiation in Cufharunthw roseus. Science 244, 580-582.
Smyth, D. R., Bowman, J. L., and Meyerowitz, E. M. (1990). Early flower development in Ara,bidopsis. Plant Cell 2, 755-767. Spurr, A. R. (1969). A low viscosity epoxy resin embedding medium for Res. 26,31-43. electron microscopy. J Ulfrusfrucf. Stewart, R.N., and Burk, L. G. (1970). Independence of tissues derived from apical layers in ontogeny of the tobacco leaf and ovary. Am. J. Bof. 57, 1010-1016. Stewart, R. N., and Dermen, H. (1975). Flexibility in ontogeny as shown by the contribution of the shoot apical layers to leaves in periclinal chimeras. Am. J Bat. 62, 935-947. Takhtajan, A. (1991). “Evolutionary Trends in Flowering Plants.” Columbia Univ. Press, New York. Verbeke, J. A., and Walker, D. B. (1985). Rate of induced cellular dedifferentiation in Catharunthus roxp%cs.Am. J. Bot. 72, 1314-1317. Verbeke, J. A., and Walker, D. B. (1986). Morphogenetic factors controlling differentiation and dedifferentiation of epidermal cells in the gynoecium of Cufhuruntk~~s rose~s. Pluntu 168, 43-49. Walker, D. B. (1975a). Postgenital carpel fusion in Cuthurunthus rose~i.s (Apocynaceae). I. Light and scanning electron microscopic study of gynoecial ontogeny. Am. J. But. 62,457-467. Walker, D. B. (1975b). Postgenital carpel fusion in Cafhuronthw roSBZLS. II. Fine structure of the epidermis before fusion. Protoplusmu 86,29-41.
Walker, D. B. (1975c). Postgenital carpel fusion in Cutharaxthus TOSPZCS. III. Fine structure of the epidermis during and after fusion. Profoplasmu
86, 43-63.
Walker, D. B. (1978a). Morphogenetic factors controlling differentiation and dedifferentiation of epidermal cells in the gynoecium of C’ufharx~72tlcu.s ~OSPXS.I. The role of pressure and cell confinement. Pluntcr 142, 181-186. Walker, D. B. (197813). Postgenital carpel fusion in Cutharanfhus roSPUS(Apocynaceae). IV. Significance of the fusion. Am. J. Bof. 65, 119-121. Walker, D. B., and Bruck, D. K. (1985). Incompetence of stem epiderma1 cells to dedifferentiate and graft. Can. J Bat. 63, 2129-2131.
BIOLOGY
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(1992)
Fiddlehead: An Arabidopsis Mutant Constitutively Expressing an Organ Fusion Program That Involves Interactions between Epidermal Cells SUSANJ. LOLLE,~ ALICE Y. CHEUNG,AND IAN M. SUSSEX’ Department
of Biology, P.O. Box 6666, Yale University, Accepted April
New Haven, Connecticut
06511
l-6, 1992
In most circumstances plant epidermal cells do not respond to surface contact with adjacent plant parts. We have identified and characterized a mutant of Arabidopsis thaliana, designated jiddlehead, where lateral appendages of the shoot fuse with one another. While fusion between floral organs is most frequent, leaf fusions also occur. Using scanning and transmission electron microscopy, we show that adhesion takes place between epidermal cells and does not involve cytoplasmic union. We also show that the frequency of organ fusion is dictated by organ proximity. In wildtype Arabidopsis, postgential fusion takes place exclusively in the gynoecium, whereas in theJddlehead mutant, this program becomes expressed constitutively. The existence of such a mutant demonstrates that postgenital fusion is a genetically o I992 Academic press, he. distinct program superimposed upon other aspects of gynoecial development in Arubidopsis. INTRODUCTION
The shoot apical meristem of dicotyledonous plants is organized into distinct layers. The outermost layer, designated Ll, gives rise to the epidermis covering the entire shoot while the inner layers produce the rest of the shoot (Dermen and Stewart, 1973; Jegla and Sussex, 1989; Poethig and Sussex, 1985a,b; Satina, 1944; Satina and Blakeslee, 1941,1943; Satina et al, 1940; Stewart and Burk, 1970; Stewart and Dermen, 1975). The Ll or epidermal lineage is the most stable of the meristematic cell populations and its derivatives serve many important functions during the growth and development of the plant. In addition to regulating gas exchange and providing a barrier against desiccation and pathogen infiltration, the epidermis ensures the structural and functional integrity of the plant. If the surfaces of two organs grow into contact, epidermal cells typically do not respond and even upon wounding and grafting make little or no contribution to regenerated tissues (Moore, 1984; Walker and Bruck, 1985). In developing flowers, and particularly during formation of the female reproductive organ (the gynoecium), however, some epiderma1 cells participate in cell wall fusions. Upon contact, cells adhere but do so without undergoing cytoplasmic union (Cusick, 1966). Such late ontogenetic or postgenital fusion represents an exceptional case during normal development of cell-cell interaction between epidermal cells. 1 To whom correspondence should be addressed. ’ Current address: Department of Plant Biology, University fornia, Berkeley, CA 94720.
of Cali-
383
Late ontogenetic fusion has been extensively studied in Catharanthus roseus in which two carpel primordia fuse during gynoecial development. Early in development, the surfaces of two originally separate carpel primordia grow into contact and fuse. Epidermal cells along the resulting suture undergo rapid and substantial changes in cytoplasmic density, cell shape, and cell division planes, redifferentiating into parenchyma cells (Verbeke and Walker, 1985, 1986; Walker, 19’75a,b,c; Walker, 1978a,b). Both cellular recognition and communication are involved in this process. Not all contacting epidermal cells undergo dedifferentiation, however, and appear to do so only if organ fusion occurs early in ontogeny (Walker, 1975a). Water-soluble morphogenetic factors mediate all of these changes but only carpel epiderma1 cells are responsive under the described experimental conditions (Siegel and Verbeke, 1989). In wildtype (wt) Arabidopsis plants ontogenetic fusion is limited to the formation of the ovary septum and the transmitting tissues of the gynoecium (Okada ef al., 1989). With this exception, no other instance of organ fusion occurs in Arabidopsis during either normal vegetative or floral development. We have isolated a mutant of Arabidopsis thaliana, designated fiddlehead vdh), which overcomes the normal restrictions placed on organ fusion during ontogeny such that vegetative and floral organs fuse postgenitally. We have named the mutant fiddlehead because adhering floral buds cause apical curling of inflorescences, resulting in structures reminiscent of fern fiddleheads. In an effort to elucidate the nature of the organ fusions that occur in fdh plants, we have studied floral development and analyzed contacting tissues using scanning (SEM) and transmission 0012-1606/92 $5.00 Copyright All rights
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
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(TEM) electron microscopy. The morphological evidence presented here reveals that organ fusion in fdh plants occurs when epidermal cells contact along their periclinal walls. Epidermal cells adhere but do not undergo cytoplasmic union, conforming to the classical definition of ontogenetic or postgenital fusion (Cusick, 1966). In addition to epidermal cell adhesion, organ fusion is accompanied by striking changes in epidermal cell surface morphology, indicating that morphogenetic factors are produced.
VOLUME152,1992 TABLE 1 LINKAGE ANALYSISOFfdh MUTATION Number of individuals with indicated phenotype
Genotype (Chromosome no.) up1 (1) us (2) up9 (3) wz (4) pi (5)
MATERIALS
AND METHODS
Mutant Isolation and Plant Growth Ethyl methane sulfonate mutagenized M2 bulked seed of the Landsberg erecta ecotype was purchased from Guhy Nurseries (Tucson AZ). A total of 16,000M2 plants were screened, and selfed progeny were collected from a subset of individuals. Fiddlehead was isolated from one such family. Plants were grown under either a long (16 hr day/8 hr night) or a short day (8 hr light/l6 hr night) regime with a mix of fluorescent and incandescent lights (175 pmole . m-’ * set-’ at pot level). Growth room temperatures averaged 20°C (+2”C). Seedswere planted in a 9:3:1 vermiculite:soil:sand mixture and watered once daily with a nutrient solution containing 6 g/gallon ‘7-6-19All Purpose Hyponex (Hyponex Co., Copley, OH) and 0.4 g/gallon 15-16-1’7Peat Lite Special (Peters Co., Allentown, PA). Genetic crosses were performed manually using plants heterozygous for the fdh mutation. Marker lines were obtained from Dr. J. Haley (Harvard University). Microscopy Tissue was collected from plants and fixed in either FAA (3.7% formaldehyde, 50% ethanol, 5% acetic acid) or a mixture of 4% glutaraldehyde-2% paraformaldehyde in Sorensen’s buffer (0.05 M pH 7.0) for 2 hr. Samples fixed in FAA were stored at 4°C in 70% ethanol and either prepared for SEM or dehydrated to 100% butanol and embedded in paraffin. Samples for SEM were dehydrated to 100% ethanol and critical point dried in liquid CO,. Dried tissue was mounted on SEM stubs and sputter coated with gold-paladium using an SPI sputter coater. Specimens were examined in an ISI SS40 scanning electron microscope with an accelerating voltage of 5-10 kV. Glutaradehyde-fixed tissue was washed in buffer, postfixed in 2% osmium tetroxide (0.05 M Sorensen’s buffer, pH 7.0), and dehydrated through a graded ethanol series. Samples were embedded in Spurr’s epoxy resin (Spurr, 1969; Electron Microscope Sciences). For
wt
.fdh
197 639 293
244
216 242
Double mutant
Marker
39 90 71 66
7
43 214
4” 36 22 25
110 83 82
Expected 17.8 68.8 33.0 24.5
25.9
Note. Segregation data for F, progeny of crosses between fdh (as male parent) and the chromosome marker lines listed in column 1. a From the calculated number of expected double mutants listed in the last column only segregation frequencies of fdh with as indicate genetic linkage.
light microscopy, Spurr’s-embedded material was cut to approximately 1 pm, and for TEM to 600-800 A using a Sorvall ultramicrotome (MT-2). Samples for TEM were stained with 3% uranyl acetate for 10 min and 0.3% lead citrate for 5 min and examined in a Phillips 300 TEM at 80 kV. RESULTS
The fiddlehead Phenotype Is Due to Mutation
a
Single Recessive
The segregation frequencies of the fdh mutation with known phenotypic markers are summarized in Table 1. The data indicate that the fdh phenotype results from a single recessive nuclear mutation which maps to chromosome 2. Apl fdh double mutants have a reduced incidence of organ fusion (see below) and as a result, the number of double mutants identified was artificially low. Fusion Can Occur between All Lateral Organs of the Shoot In the fdh mutant fusions occur predominantly between organs within single floral buds as well as between adjacent buds within a given inflorescence. Newly initiated floral buds become trapped within the inflorescence by surrounding older buds while internode elongation and pedicel elongation continue (Fig. 1). Hand dissection of individual fdh flowers reveals that all four organ whorls participate in fusion events and do so in a variety of combinations. Sepals, for example, fuse to one another and to petals while stamens adhere to all whorl organs within the flower including neighboring stamens. At anthesis, petals and stamens remain seques-
385
FIG. 1. Inflorescences of wildtype (A) andjiddkeheud (B-D) plants. (A) A 6-week-old wt Arcrbidopsis plant showing the inflorescence bolt and apex (open arrow). At anthesis, flowers spontaneously self-pollinate, giving rise to the seed-containing siliques (si). (B-D) In,fdh plants, fusion between sepals (s) of emerging flower buds (fb) and continued pedicel (pd) and internode elongation eventually result in curling of the entire inflorescence, producing structures reminiscent of fern fiddleheads. Elongation growth results in the emergence of pistils (p) from within the fused sepals but without pollination taking place. Afdh inflorescence (open arrows) at (B) 6 weeks and at (C) 14 weeks. (D) A scanning electron micrograph showing a,fdh inflorescence at 10 weeks postgermination. A, B, and C are the same magnification. Scale bar in A = mm. Bar in D = Wm.
tered within the fused calyx of individual floral buds. Continued growth and elongation of the gynoecium, which is constrained only by limited fusion to anthers and apically to sepals, eventually result in pistil emergence (Fig. 1D). At maturity,f% flowers have correct organ numbers occupying correct whorl positions. The ovary, style, and stigma are structurally normal and fdh flowers produce mature pollen and ovules. Stamens elongate but remain physically restricted from contact with the receptive stigmatic surface, and flowers do not spontaneously self-pollinate.
Early floral development in fdh plants is indistinguishable from wt as revealed by both light microscopy (data not shown) and SEM. As in wt, fdh floral organs are initiated as discrete primordia (Fig. 2). Only after organs have grown into contact does development diverge from that observed in wt. Shortly after sepals enclose the flower bud, small epidermal cell clusters can be seen between closely appressed neighboring buds and at sepal margins (Fig. 2E). Eventually sutures form between sepals within a whorl and between the sepals of adjacent contacting floral buds. As floral development
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DEVELOPMENTALBIOLOGYVOLUME152,1992
FIG.2. Scanning electron micrographs showing wt (A to C) and early,fih (D to F) floral development. (A) Arubidopsis flowers arise in a helical array from the inflorescence meristem. The apical cluster contains flower buds at various stages of maturation. Just prior to anthesis, sepals (s) open exposing four petals (pe), six stamens (a), and the central gynoecium or pistil (p). (B) Early in development maturing inner whorl organs are completely enclosed by four overlapping sepals (arrow shows discrete sepal margin). (C) During gynoecial development the inner walls of the tubular primordium grow into contact, as shown here, and fuse giving rise to the ovary septum and the transmitting tissues, At maturity, the primordium will have closed apically and ovules will have formed along the margins of the fused septum. (D) Early floral development in fdh is indistinguishable from wt. Sepal primordia (sp) are initiated as discrete organs that eventually enclose the floral meristem. (E and F) Infdh, fusion commences after sepals grow into contact. Neighboring buds are united by cell clusters bridging the abaxial sepal surfaces (E) eventually forming a near continuous suture (arrows in F). Scale bars indicate magnification in micrometers. proceeds, growth causes shearing or tearing of epiderma1 layers and disfiguration of some organs. Petals are most severely affected. On average petals expand to only 25% of the size reached in wt flowers at maturity. Rosette and cauline leaves also participate in fusions (Fig. 3A). These fusions can involve one or more leaves. As is true for floral organs, fusion can occur along the margins or on the adaxial or abaxial surface. Although the majority of fdh plants do achieve a relatively normal
vegetative architecture, at a low frequency leaf fusions completely block organ expansion, leading to termination of growth and eventual plant death. In most of these instances, inflorescence stems will have fused to neighboring leaves. Arabidopsis plants switch from vegetative to floral development within approximately 7-10 days if grown under 16 hr photoperiods (Miksche and Brown, 1965). If light exposure is limited to 8 hr photoperiods the switch
387
FIG. 3. Scanning electron micrographs showing leaf fusions in,fak (A) and floral organ fusion in the inHorcscence of an clplfilh double mutant (B). (A) Fusion within and between rosette leaves occurs at variable but lower frequency than fusion between floral organs. Two individual leaves which have fused with each other along their margins (arrows) are shown nest to a single leaf which is fusing to itself (open arrow) ipt, petiole). (B) In trp~,fdl) double mutants fusion patterns diverge from those usually observed in the single mutant. In the tcpl genetic background the physical proximity of the floral whorls is altered such that individual flowers as well as the inflorcsccnce achieve a more open configuration. Bract-like organs (b) often remain free from one another although fusion can still occur between these organs and the inner stamens and pistil (p). Stamen filaments elongate despite anther (a) fusion to the pistil (p) (st. stipulcs). Scale bars indicate magnification in micrometers.
to flowering is significantly delayed and vegetative growth is prolonged for months (Besnard-Wibaut, 1981; Miksche and Brown, 19651. To test whether organ fusion in .fdh plants can occur independent of floral morphogenesis, a segregating population of 50 plants was maintained under short day growth conditions for 2 months. Following the 2-month period, plants were shifted to 16 hr photoperiods so as to induce flowering, score for floral fusion, and to confirm the.fdh phenotype. Only 2 of 12 jidh, plants were identified which had not fused vegetatively under the short day conditions.
The differing frequencies with which fusion events are observed in the .fdh mutant suggest that physical proximity is an important determinant of fusion. In any given.fdh mutant plant some floral organs always participate in fusions while vegetative organs fuse at variable but lower frequency. The incidence of leaf fusion increases from a few percent injiih plants grown singly to 50% in plants grown under crowded conditions. This dependence of leaf fusion on crowding and the consistently
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high fusion frequencies in flowers indicate that organs with greater spatial separation are less likely to participate in a fusion event. Thus, leaves that are arranged in a spiral phyllotaxy (the spatial arrangement of organs on the plant axis) fuse infrequently whereas floral organs, which are arranged in whorls and experience no separation by internodal elongation, often fuse. If the spatial arrangement of organs does influence the relative incidence of fusion, altering this arrangement should also change the pattern of fusions observed. Floral internodes which normally do not elongate between the medial and the lateral first whorl organs do so in the apetelal (apl) floral mutant (Irish and Sussex, 1990). In aplfdh double mutants, contrary to the situation in the fdh single mutant where sepals fuse to form a calyx-like tube, the bract-like first whorl organs typically remain free from one another and more often adhere to other organs within the flower or the inflorescence (Fig. 3B). Flowers in the double mutant acquire an open configuration, revealing fused internal floral organs not normally visible in flowers of the single fdh mutant. In general, changes in floral architecture in these double mutant plants are less striking than those in flowers of the single fdh mutant. Double mutant plants were difficult to distinguish from apl floral mutants in instances where only a few organ fusions had occurred. Although all lateral organs of the shoot are competent to fuse in homozygous fdh plants, fusion between two contacting sibling plants has not been observed under our experimental conditions. Similarly, fdh plants do not adhere to nonliving surfaces. @gan Fusion Occurs without
Cytoplasmic
Union
Analysis of adhering tissues in the fdh mutant by light microscopy (data not shown) and TEM reveals a continuity of intact epidermal cells between the participating organs and indicates that fusion occurs without cytoplasmic union (Fig. 4). At all junctions analyzed, whether involving floral or vegetative fusions, the contacting epidermal cells form a distinct cellular bilayer. Subepidermal cells are not affected. Terminally differentiated epidermal cell types such as guard cell pairs can be distinguished within fusion sutures. Epidermal cells from the same as well as different organ types and of different developmental ages fuse. As shown in Fig. 4C, epidermal cells from a sepal are fused to the epidermis of a differentiating petal primordium. TEM analyses of fusion sutures reveal the presence of an electron-dense material deposited between the cell walls of the adherent epidermal cells (Fig. 4). This material accumulates as tissues mature (Fig. 4B). No intercellular connections (plasmodesmata) have been ob-
V0~~~~152,1992
served between fused cells at any stage during fusion ontogeny, although such connections are evident in the anticlinal walls of epidermal cells (Fig. 4C). Initially, an electron-dense cuticle is present on the outer cell wall surface of adherent epidermal cells (Fig. 4D) which is less readily detected as participating tissues mature (Fig. 4B). Organ Fusion Induces Morphological Surface
Changes of the Cell
Epidermal cells display a cell surface topology which is characteristic for a given organ type when viewed under the SEM. Infdh, epidermal cells are indistinguishable from wt at all stages of development in regions where organ surfaces are not in contact. Along fusion sutures, however, fdh epidermal cells lose their organspecific surface topology (Fig. 5). Cells become smooth and lose their characteristic surface ridging. Such surface changes are evident at fusion junctions on any organ and, with the exception of sepal margins, affect only one or two cell rows back from the suture. Although the surface morphology of contacting epidermal cells is altered, organ identity is not. At junctions where adjacent sepals have not completely fused and sepal margins are in contact with the underlying petal epidermis, surface changes are strikingly polarized. As shown in Fig. 5D, only the one row of petal cells next to the sepal margin differs from the neighboring petal cells while at least five rows of sepal epidermal cells have undergone changes in surface topology. Identical patterns of surface change are also seen at fusion junctions between first and second whorl organs in double mutants homozygous for fdh and either the pistillata (pi) (data not shown) or the apetela3 (ap3) floral homeotic mutations (Fig. 5E). In both the pi and the ap3 mutants, petals are transformed to sepal-like organs (Bowman et al., 1989). In the ap3fdh double mutant (Fig. 5E), cells on the surface of the sepal-like second whorl organ which are in contact with the overlapping first whorl sepal margin show a clearly defined front of surface change identical to that seen on petal cells in the single mutant (Fig. 5D). Organ Fusion Occurs after Primordial
Initiation
Several lines of evidence show that organ fusion takes place soon after primordial initiation. As previously demonstrated, organ primordia are not fused when initiated (see Fig. 2) but become fused before expansion growth ceases.The limited expansion achieved by petals in fdh flowers indicates that these organs fuse to neighboring sepals prior to or by stage 10 in floral development, as defined by Smyth et al. (1990). Similarly, in the apl fdh double mutant, anthers fuse prior to stamen
389
FIG. 4. Transmission electron micrographs of fused epidermal cells infdk flowers at various stages of development. (A) Overlapping margins of two fused sepals from a single flower bud shown in cross-section. Epidermal cells have fused forming a suture (arrows) along the adhering periclinal cell walls. No intervening mesophpll cells separate epidermal cells at margins of individual sepals potentiating contact between internal periclinal epidermal cell walls. (B) Cross-section of two fused sepal epidermal cells from an older flower bud showing intact periclinal cell walls (cw) at high magnification. An electron-dense matrix has accumulated between the adherent walls (material between arrows) (cy, cytoplasm). (C) Epidermal cells on the abaxial surface of a petal (pe) fused to adjacent sepal cells shown in cross-section. B matrix of electron-dense material is deposited between the fused cell walls (arrows). Plasmodesmata occur in the anticlinal walls of adjacent epidermal cells (open arrows) but are not present in the periclinal walls of epidermal cells that have fused during ontogeny. (D) Adhering sepal (s) and anther epidcrmal cells shown in cross-section. When cells fuse a darkly staining cuticular surface layer can be distinguished initially (paired arrows) which becomes less well defined at later stages in development (see B) (c, chloroplast). Scale bars indicate magnification in micrometers.
filament elongation (see Fig. 3) since filaments protrude outward from the main axis. Since these fused anthers also show evidence of shearing, anther to pistil fusion occurs prior to pistil elongation. Furthermore, limited attempts to force fusion by physically appressing two mature rosette leaves from an individual plant have not resulted in organ adhesion. DISCUSSION
We have identified a single gene mutation that can eliminate both the spatial and the developmental restrictions placed on organ fusion in Arabidopsis. Our findings provide genetic evidence for the existence of a distinct developmental pathway modulating interactions between epidermal cells in Arabidopsis Mutation
of the FDH gene alters epidermal competence to adhere such that shoot epidermal cells become responsive to contact. This competence is acquired independent of flowering. Organ fusions are most common in flowers and impair reproductive function completely. We know of only one other mutant in plants where abnormal organ adhesion has been described (Hake et al., 1989). This maize mutant has been only partially characterized, however, and details of the ontogeny and mechanism of adhesion have not been determined. Based on our morphological analyses and on the apparent specificity of the fusion response, we propose thatfdh plants undergo true ontogenetic fusion (Cusick, 1966). As defined by Cusick (1966), fusion occurs after organ initiation and growth, is epidermis-specific, and may require some recognition process. Organ fusion in
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DEVELOPMENTALBIOLOGYV0~~~~152.1992
FIG.5. Scanning electron micrographs showing epidermal cell surface morphology of first and second whorl organs in wt (A and B) andfiih, (C to E) flowers. (A) Sepal epidermal cells on the abaxial surface of a wt flower. With the exception of the interposed guard cells (gc), the elongate sepal epidermal cells display an elaborate and characteristic cuticular relief. (B) Abaxial petal cells are easily identified by their cuboidal shape and extensive cuticular surface striations. (C) At fusion junctions between adjacent sepals within a whorl, morphological changes occur on the surfaces of fused cells symmetrically about the suture (arrow). Both cell surface characteristics and cell definition are lost. Guard cells (gc) remain distinguishable but like the surrounding sepal epidermal cells lose cell definition. (D) Incomplete fusion between adjacent sepals inf’ih flowers can result in protrusion of the underlying petal tissue. At junctions where sepal-petal fusion has occurred, such as the one shown here, surface changes are strikingly polarized with loss of cuticular topology clearly evident in a single row of petal (pe) ceils (arrows) and in five rows of sepal (s) cells. In some cases, there is a sharp front of surface change affecting only one-half of each of the individual petal cells. (E) In apSfdh double mutants, similar patterns of surface change are observed. The epidermal cells on the subjacent sepal-like whorl (‘pe’) show an identical front of cell surface change (arrows). As is the case in the single mutant, epidermal cells show a directional loss of surface topology. Scale bars indicate magnification in micrometers.
fdh is mediated by adhesion of the epidermal cell walls, does not involve cytoplasmic union, and does not alter organ identity. Fdh plants do not adhere to nonliving surfaces. Fusion as it occurs in,fdh, therefore, is distinct from a wound or grafting response where adhesion of subepidermal cells can occur to nonliving surfaces (Moore, 1984; Moore and Walker, 1981a,b). TEM analysis of fusion junctions reveals the presence of an electron-dense material between fused epidermal cells as well as the absence of plasmodesmata. The frequencies with which fusion takes place between different organs on fdh plants suggest that physi-
cal proximity determines whether two organs will fuse. Chance of contact and incidence of fusion increase with decreasing space. Although we have observed every permutation of organ to organ fusion in the fdh shoot, fusions occur preferentially between floral organs. In apl fdh double mutants where internode elongation occurs between first whorl organs, the incidence of fusion between these bract-like organs is reduced. A similarly reduced incidence of fusion is seen between the spirally arranged rosette leaves. Therefore, we suggest that all organs of the fdh shoot are competent to respond to surface contact with adjacent plant parts during some pe-
LOLLE,CHEUNG,AND&JSSEX
,frlh: A Gene Regultrtin,u Ory~crtl Fusiorl
riod in their development. Physical contact must be achieved for organ fusion to occur. The ubiquity of sepal-petal fusions seen in fdh flowers suggests that the window of fusion competence may be fairly broad. Although petal primordia are initiated immediately after sepal primordia, they are the last floral organs to mature (Smyth et al., 1990). The limited expansion of fused petals infdh flowers suggests that fusion is complete prior to the period where petals experience the greatest expansion growth. It is not clear, however, whether both tissues participate equally and reciprocally in the fusion process. Furthermore, our attempts to force fusion between mature leaves offdh mutant plants have failed, corroborating that fusion competence is restricted temporally and probably is limited to actively maturing organs. In C. roseus, where ontogenetic fusion has been most extensively studied, there is convincing evidence that cell fusion is mediated by water-soluble morphogenetic factors (Siegel and Verbeke, 1989). Are morphogenetic factors produced by fdh cells? On organs that have undergone fusion, cell surface topology is altered such that the cell surface becomes smooth and cell definition is lost at the fusion suture, as seen by SEM. Across heterogeneous organ sutures, such as sepal-petal junctions, fused cell rows show a polarization in these surface changes consistent with the presence of a diffusible morphogenetic front. These findings indicate that contacting epidermal cells produce and respond to diffusible morphogenetic factors. As was demonstrated in C. roseus (Siegel and Verbeke, 1989; Walker, 1975c), only a few cell rows are affected by these factors. What role, if any, these factors play in the fusion process is unclear but it will be of great interest to determine whether they share any functional similarity to those described by Siegel and Verbeke (1989). Due to the sequestration of the contacting cells within the gynoecium in C. roseus, it would be difficult to determine whether surfaces changes such as those described in thefdh mutant accompany the other events detailed ultrastructurally. Transverse sections show that intervening mesophyll cells are often completely absent at sepal margins, facilitating contact between 5 to 10 rows of upper and lower surface epidermal cells along their internal periclinal walls. This number of epidermal cells is exactly coincident with the number of cells that undergo changes in surface topology and cell definition at many of the sepal margins in fdh flowers. The discrete enhancement of this cell surface phenotype along the margins indicates that contact between periclinal cell walls induces the cell surface changes and that contact with either periclinal wall of the epidermal cell (either the external or the internal facing wall) may be sufficient to induce the observed changes. Since epidermal cells distal to the su-
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tures remain unaffected, lateral contact between epidermal cells (contact between the anticlinal walls) does not induce these changes. Our morphological data indicate that ontogenetic differences between fusing organs infdh plants have some influence on the morphological response of cells along fusion sutures. At sepal-sepal junctions, cell modification is symmetrical about the fusion suture. At sepalpetal junctions, however, cell surface changes are polarized and show a sharp front of change. Although such modifications may be a feature peculiar to petal epiderma1 cells and transformed second whorl organs may behave like petal cells in this regard (since identical gradations of change are seen on sepal-like second whorl organs in the apSfiIh double mutant), it is equally plausible that ontogenetic differences are important determinants of the cell response. In C. I’ose?:us,cells fusing early in carpel ontogeny experience a full spectrum of developmental changes culminating in cell redifferentiation, whereas epidermal cell identity is maintained in regions where cells grow into contact later in development (Walker, 1975a). In C. roseus, the fusing primordia appear to be homologous and are initiated synchronously (Boke, 1949), whereas organs that fuse in jdh plants are often initiated at different times and mature asynchronously. In ,fdh plants, fusion occurs between heterogenous epidermal populations and occurs with less stringent temporal coordination. As such, epiderma1 cells may be incapable of extensive dedifferentiation if they grow into contact late during ontogeny and the variability of response may reflect a diminution in the ability of cells to communicate and interpret morphogenetic signals. Our data demonstrate that organ fusion can be expressed ectopically during Arubidopsis development and does not in and of itself stimulate differentiation of carpel-specific structures such as ovules. Many of the homeotic floral mutants of Arabidopsis form ovules on transformed organs that have not previously participated in a fusion event (Bowman et al., 1989; Haughn and Somerville, 1988; Irish and Sussex, 1990). Similarly, studies on gynoecial development in C. I’ose?Is demonstrate that physically preventing primordial fusion does not block gynoecial maturation (Walker, 1978a). Furthermore, phylogenetic data suggest that intercapel fusion resulted from the superimposition of a distinct epidermal adhesion program upon carpel development (Takhtajan, 1991). It has been suggested that the physical union of carpel primordia enhances reproductive titness by promoting higher seed set (Carr and Carr, 1961). Fusion makes ovules sequestered on any constituent carpel accessible to fertilization irrespective of which carpel the incoming pollen was associated with initially.
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DEVELOPMENTAL BIOLOGY
Based on the evidence presented here, we propose that the FDHgene functions to maintain epidermal cells in a distinct developmental state. As in any physical phenomenon there is first a susception phase, in this case physical contact. Second, there is a perception phase where the physical signal is transformed to a physiological response. Third, the physiological response is manifested at the cell wall interfaces. This suggests thatfdh cell walls are altered such that the response to this sequence is organ fusion. The regulating mechanism may be direct, involving protein binding to a gene promoter for example, or indirect, such as occurs in multimeric protein complex formation. We suggest that this developmental pathway is normally expressed only in the developing carpels and, in wt Arabidopsis therefore, the FDH gene is subject to developmental regulation in the carpels. We thank Barry Piekos for instruction in the use of and assistance with the scanning and transmission electron microscopes. We thank Mary Helen Goldsmith, Carolyn Slayman, and Clifford Slayman for their support and interest throughout the course of this work. We also thank Graeme Berlyn, Marc Kirschner, Timothy Nelson, David Taylor, Hong Wang, and Hen-ming Wu for valuable discussion and critical reading of the manuscript. This research was supported by a McKnight Foundation Award to Yale University. S.J.L. was supported by a McKnight Foundation Postdoctoral Fellowship. REFERENCES Besnard-Wibaut, C. (1981). Effectiveness of gibberellins and 6-benzyladenine on flowering of A rubidopsis thaliunu. Physiol. Plant. 53, 205-221. Boke, N. H. (1949). Development of the stamens and carpels in Vi~u Roseu L. Am. J. Bof. 36, 535-54’7. Bowman, J. L., Smyth, D. R., and Meyerowitz, E. M. (1989). Genes directing flower development in Arubidopsis. Plnxt Cell 1, 37-52. Carr, S. G. M., and Carr, D. J. (1961). The functional significance of syncarpy. Phytomorpholgyy 11,249-256. Cusick, F. (1966). On phylogenetic and ontogenetic fusions. 1n “Trends in Plant Morphogenesis” (E. G. Cutter, Ed), pp. 171-183. Longmans, Green. Dermen, H., and Stewart, R. N. (19’73). Ontogenetic study of floral organs of peach (Prunus persicu) utilizing cytochimeral plants. Am. J. Bot. 60, 283-291. Hake, S., Walko, R., Lane, B., and Lowe, B. (1989). Transposon mutagenesis in developmental biology: Methodology and results. Curr. Topics Plunt Biochem. Phgsiol. 8, 237-250. Haughn, G. W., and Somerville, C. R. (1988). Genetic control of morphogenesis in Arubidopsis. Den Genet. 9, 73-89. Irish, V. F., and Sussex, I. M. (1990). Function of the upetalnl gene during Arnbidopais floral development. Pltrnt Cell 2, 741-753. Jegla, D. E., and Sussex, I. M. (1989). Cell lineage patterns in the shoot meristem of the sunflower embryo in the dry seed. DPU. Biol. 131, 215-225. Miksche, J. P., and Brown, J. A. M. (1965). Development of vegetative and floral meristems of Arubidopsis thliam~ Am. J. Bof. 52, 533537.
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