Patterns of cell division and expansion in developing petals of Petunia hybrida

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1 Sex Plant Reprod (2002) 15: DOI /s ORIGINAL ARTICLE Lara Reale Andrea Porceddu Luisa Lanfaloni Chiaraluce Moretti Sara Zenoni Mario Pezzotti Bruno Romano Francesco Ferranti Patterns of cell division and expansion in developing petals of Petunia hybrida Received: 17 May 2002 / Accepted: 19 July 2002 / Published online: 11 September 2002 Springer-Verlag 2002 Abstract The definition of the patterns of cell division and expansion in plant development is of fundamental importance in understanding the mechanics of morphogenesis. By studying cell division and expansion patterns, we have assembled a developmental map of Petunia hybrida petals. Cycling cells were labelled with in situ markers of the cell cycle, whereas cell expansion was followed by assessing cell size in representative regions of developing petals. The outlined cell division and expansion patterns were related to organ asymmetry. Initially, cell divisions are uniformly distributed throughout the petal and decline gradually, starting from the basal part, to form a striking gradient of acropetal polarity. Cell areas, in contrast, increased first in the basal portion and then gradually towards the petal tip. This growth strategy highlighted a cell size control model based on cell-cycle departure time. The dorso-ventral asymmetry can be explained in terms of differential regulation of cell expansion. Cells of the abaxial epidermis enlarged earlier to a higher final extent than those of the adaxial L. Reale C. Moretti B. Romano F. Ferranti Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali, Università degli Studi di Perugia, Borgo XX Giugno 74, Perugia, Italy A. Porceddu ( ) Istituto per il Miglioramento Genetico delle Piante da Orto e da Fiore (CNR), via Università 133 Portici (NA), Perugia, Italy L. Lanfaloni Dipartimento di Biologia Cellulare e Molecolare, Università degli Studi di Perugia, Via Elce di Sotto, Perugia, Italy S. Zenoni M. Pezzotti Dipartimento Scientifico e Tecnologico, Università degli Studi di Verona, Strada Le Grazie 7, Verona, Italy Present address: A. Porceddu, Istituto per il Miglioramento Genetico delle Piante Foraggere (CNR), via Madonna Alta 130, Perugia, Italy A.Porceddu@irmgpf.pg.cnr.it Tel.: , Fax: epidermis. Epidermal appendage differentiation contributed to the remaining asymmetry. On the whole our study provides a sound basis for mutant analyses and to investigate the impact of specific (environmental) factors on petal growth. Keywords Cell division Histone H4 Mitotic cyclin Petal development Petunia hybrida Introduction Development in plants is achieved mainly during postembryogenesis, according to programs that coordinate diverse processes, ranging from cell division and enlargement to differentiation. Shoots and their attendant structures such as leaves and flowers differentiate from a shoot apical meristem (SAM), which is composed of three clonally distinct layers of cells. As organ formation requires the coordinated proliferation of cells in all three layers, the rate and pattern of cell division in each of the layers must be controlled in order to establish the size and shape of these organs. Controls, ensuring that cell division and expansion in the different layers, are mutually coordinated are indispensable for this process (Meyerowitz 1997). The existence of structural similarities between organs, such as the proximodistal and dorsoventral asymmetries, suggests that at least some of these developmental programs are shared between different organs. The role of cell division and expansion on the morphogenesis of vegetative organs has long been a subject of study. Patterns of cell cycling related to morphogenesis of the leaf blade, tissue proliferation and differentiation of specific cell types occur along a prominent longitudinal gradient. In Arabidopsis leaves, for example, dividing cells are initially spread throughout the leaf, but gradually become restricted to proximal portions of the blade and to the petiole, forming a typical basipetal gradient (Donnelly et al. 1999). Although the frequency of cell cycling varies for each specific layer, this gradient is

2 124 seen in every cell layer (Donnelly et al. 1999). Despite the fact that basipetal development is common to leaves of most species, cases of acropetal development have also been described (Hofer et al. 1997; Donnelly et al. 1999). Compared with vegetative organs, the shape and size of floral organs in a plant is highly invariable (Kotilainen et al. 1999), a fact that is advantageous for studying the basis of organogenesis using molecular and genetic approaches. Petals are structurally similar to leaves and their development from the floral meristem has often been compared to that of leaves. As in foliage leaves, petals are initiated by periclinal divisions of cells located more or less deeply beneath, or in, the protoderm (Fahn 1990). Petal axis formation requires the activity of initial apical and sub-apical cells, while lateral growth occurs due to the activity of marginal and sub-marginal initial cells (Martin and Gerats 1993). Further growth is due to the presence of circles of marginal meristematic cells and of a certain number of cell divisions that occur after the separation of the cells from the marginal meristem (Martin and Gerats 1993). At the end of petal development, two parts are generally distinguishable, namely the limb and the tube (Drews et al. 1992). Different types of epidermal appendages are found in petals. Conical-papillate petal cells appear to share parts of a common developmental program with the trichomes in tobacco and Antirrhinum, since their formation is correlated with the expression of the MIXTA gene (Glover 2000). We traced the patterns of cell cycling in developing Petunia hybrida line W115 petals by following the expression of mitotic B1-type cyclin (Porceddu et al. 1999) and histone H4 by in situ hybridisation on longitudinal petal sections. The expression of these two genes provides reliable markers for G2/M and S phase of the cell cycle, respectively (Fobert et al. 1994; Gaudin et al. 2000). The extent of cell expansion was followed both spatially and temporally by assessing cell size in regions of petals at different developmental stages. The assembled picture represents a developmental map of the pattern of cell division and expansion and so, over time, of the shape and size of Petunia petals. The study provides a baseline for mutant characterisation as well as giving information that should lead to a better understanding of the role played by cell division and expansion in the morphogenesis of a flattened organ with a determinate pattern of development. Material and methods Plant materials P. hybrida plants (line W115) were grown under greenhouse conditions. Flowers were classified into 14 developmental stages according to petal length (Table 1). Table 1 Developmental stages In situ hybridisation Digoxigenin-labelled sense and antisense probes were generated from a full-length PethyCycB1 1 cdna clone using a gene-specific primer together with a T3 or T7 primer. Transcriptions were performed with T3 or T7 RNA polymerase. The PetH4 1 cdna clone was amplified using Sp6, T7 and specific primers and transcribed with Sp6 or T7 RNA polymerase. Tissue preparation and hybridisation conditions were as described by Cañas et al. (1994) with several modifications. The flower buds were infiltrated under vacuum with FAA medium (50 ml 100% ethanol, 5 ml acetic acid, 10 ml 37% formaldehyde and 35 ml distilled water) and the sections were deparaffinized by rinsing in Histo-clear (Polymed, Milan; cat. no. HS200). Light microscopy Corollas were dissected from flower buds at each of 14 developmental stages and sectioned. Samples were fixed in glutaraldehyde (5% v/v) overnight at room temperature and post-fixed in osmium tetroxide (1% w/v) for 4 h (both in M cacodylate buffer, ph 7.2). After dehydration with a graded ethanol series and propylene oxide, the samples were embedded in resin (Epon, DDSA and MNA mixture) (Loreto et al. 2001). Transverse and longitudinal sections (1.5 µm thick) from each sample were stained with toluidine blue and mounted in Eukitt for light microscopy observation. Photomicrographs were taken using a Leica DMR HC photomicroscope. Quantitative observations At each developmental stage, starting from stage 3, the petal was divided into the limb, the pigmented portion of the petal, and the tube, which is the unpigmented portion surrounding the pistil and stamens. Both limb and tube were subdivided into proximal and distal parts. The portion along the margin and the portion far from the margin were distinguished in the limb, while in the tube the upper and lower halves were distinguished. The lengths of the two orthogonal axes, one of which was parallel to the proximo-distal petal axis, were measured in each portion. Petals were peeled to cut out the epidermis before measuring and the adaxial and abaxial epidermal parts were analysed by light microscopy. The image analysis software LEICA QWIN was used for measuring cell length, width, and surface of these portions.

3 Scanning electron microscopy For scanning electron microscopy (SEM), petal portions were fixed for 3 h at room temperature in 5% glutaraldehyde/0.07 M sodium cacodylate buffer, post-fixed for 1 h in buffered 1% OsO 4, and dehydrated in a graded ethanol series. After critical point drying in liquid CO 2, samples were mounted on aluminium stubs, sputter coated with gold, and viewed with a Cambridge Stereoscan 90B. Results P. hybrida petals have proximo-distal and dorso-ventral asymmetry The 14 stages cover the developmental range from petal specification to mature petal. Development of P. hybrida petals was studied at each of the 14 stages, from a length of 1 mm to that at anthesis of about 70 mm (Table 1). This developmental range includes the events occurring after petal identity specification. The histological structure of the organ already appears to be completely defined at stage 1 (Fig. 1A). Two regions were cyto/histologically recognisable early in development (stages 1 2): the apical region, composed of meristematic cells (Fig. 1B) and the basal region, composed of cells already undergoing differentiation (Fig. 1C). This acropetal morphogenesis is responsible for asymmetry along the proximo-distal axis of the organ, although exceptions are found locally. This is illustrated by trichome development in the basal portion. While multicellular uniseriate trichomes first appear in the basal portion, macroscopically respecting the acropetal gradient, young trichomes reiteratively differentiate between them as to partition the space. Thus, alternate regions of fully developed and young trichomes are formed. An example of this situation is shown in Fig. 1D. The proximo-distal asymmetry becomes phenotypically evident at stage 6. The basal portion region (tube) displays sepaloid characteristics, while in the apical region (limb) pigmentation has already started (Table 1, Fig. 1E). Macroscopically, the two epidermis (abaxial and adaxial) diverge, giving dorso-ventral asymmetry to the organ. The abaxial epidermis of the limb is composed of larger, densely vacuolated cells, while the adaxial epidermis harbours smaller cells without vacuoles (Fig. 1F). In the tube, both epidermis are composed of elongated cells disposed in vertical columns. The type and frequency of epidermal appendages leads to further asymmetry. In the abaxial epidermis the number of epidermal appendages, i.e. multicellular uniseriate trichomes, discriminates the tube from the limb being higher in the tube (Fig. 1G, H). Cells in the adaxial epidermis of the limb have strong, convex walls that give rise to coneshaped papillae (Fig. 1I). Short pointed hairs predominate in the adaxial epidermis of the tube (Fig. 1J). There is a clear proximo-distal asymmetry in the mesophyll beginning at stage 5. By stage 5, the cells of the basal region have elongated and are arranged in ranks (Fig. 1K) while those of the apical region are isodiametric (Fig. 1L). At stage 7, the cells of the limb assume an irregular shape having large lobes and are separated by intercellular spaces (Fig. 1M). Patterns of cell division as revealed by two in situ markers 125 Patterns of cell division were studied by using two in situ markers. Transverse sections of petals at the 14 developmental stages were hybridised with digoxigeninlabelled antisense RNA probes derived from either PethyCycB1-1, a mitotic P. hybrida cyclin expressed mainly in flowers (Porceddu et al. 1999), or PetH4 1, a P. hybrida histone H4 gene. B1 type cyclins are expressed preferentially during the G2/M transition and have been widely used as an in situ marker of cell division (Fobert et al. 1994) while H4 is expressed during S phase and has been used as an in situ marker of cells undergoing DNA replication (Gaudin et al. 2000). At high magnification, hybridisation signals appeared as purple patchy spots. Increased magnification showed that single cells were labelled. Using sense RNA probes, individual cells were not labelled. Signals were observed in all portions of the petals during initial developmental stages. There was little difference in the number of labelled cells in the epidermis and the mesophyll (Fig. 2A). However, as development proceeded (stages 4 5) the pattern of expression of PethyCycB1 1 discriminated both the parts and the histological organisation of the organ. Generally, the number of labelled cells increased towards the petal tip, delineating a steep acropetal gradient (Fig. 2B). However, clusters of labelled cells were still present in the basal portions at the procambial strands level. The acropetal Fig. 1 A Longitudinal semi-thin section of flower bud at stage 1, from right: sepal, petal, anther. B, C Magnification of A: apical portion of petal composed of meristematic cells (B) and basal portion with the number of mesophyll layers already defined (C). D Scanning electron micrograph: abaxial epidermis of middle portion of petal at stage 9 with young trichomes growing between older ones. E Stereomicrograph of flower bud at stage 6. F Transverse semi-thin section of petal at stage 6; abaxial epidermal cells are more vacuolated and bigger than the adaxial epidermal cells. G Scanning electron micrograph of abaxial epidermis of basal portion of petal (tube) at stage 9; trichomes are numerous, regularly distributed and at different developmental stages. H Scanning electron micrograph of abaxial epidermis of apical portion of petal (limb) at stage 9; trichomes are rare. I Transverse semi-thin section of petal at anthesis; papillae have formed in the adaxial epidermis. J Scanning electron micrograph of adaxial epidermis of basal portion of petal; short pointed hairs are seen in all cells. K Tangential semi-thin section of basal portion of petal at stage 5; rectangular mesophyll cells organised in parallel lines are seen on the right, adaxial epidermal cells on the left. L Petal at stage 5: tangential semi-thin section of apical portion, with randomly distributed isodiametric mesophyll cells. M Transverse semi-thin section of apical portion of petal at stage 7; intercellular spaces are starting to appear in the mesophyll. Bars 40 µm. a Anther, ab abaxial epidermis, ad adaxial epidermis, m mesophyll, p petal, pa papilla, s sepal, t trichome, vb vascular bundle

4 126 Fig. 1 (Legend see page 125)

5 127 Fig. 2A F In situ hybridisation of longitudinal sections of flower at different stages of development with RNA probes from Pethy- CycB1 1. Sections were probed with digoxigenin-labelled antisense RNA and viewed under a bright field that gives a purpleblue label. A Flower at first stages (1 2). At this stage, signals are detected in all petal portions. B Petal at stage 4: signals are numerous in the apical portion and rare in the basal portion. C, D Apical (C) and basal (D) portions of petal at stage 7: signals are only detected in the two epidermis and vascular bundle of the apical portion (C); the signals are absent in the basal portion (D). E Apical portion of petal at stage 8, the density of spots becomes higher in the adaxial epidermis. F Folded portion of limb at stage 8: the signals are present, only in the two epidermis and in the vascular bundle. Bars 80 µm. a Anther, ab abaxial epidermis, ad adaxial epidermis, m mesophyll, o ovary, p petal, vb vascular bundle

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7 gradient of cycling cells became even more pronounced during later developmental stages. For example, at stage 7 the signals were almost exclusively confined to the apical region (Fig. 2C, D). At the petal tip the epidermis showed the highest spot density; in contrast, spots were practically absent in the mesophyll except for some areas still visible around the vascular bundle (Fig. 2C). At later developmental stages, labelled cells were scored only in the epidermis and not in the mesophyll. Cell division is required in the petal epidermis during these developmental stages not only to ensure petal growth, but also for epidermal appendage formation. Consequently, some of the spaced signals detected along the epidermis might have indicated cells that were dividing periclinally to give rise to trichomes. The precocious decrease in cycling cells in the mesophyll layers was associated with cell enlargement and differentiation. While the density of spots in the adaxial and abaxial epidermis was comparable during initial developmental stages, it became higher in the adaxial epidermis at later stages (Fig. 2E). The number of labelled cells decreased gradually from stages 7 8 to anthesis. At stage 8, labelled cells were detected only in the folded part of the limb (Fig. 2F). This gradual decrease continued until stage 11, at which point cycling cells were no longer present in any portions of the petal (data not shown). The expression of a histone H4 was examined in order to confirm that PethyCycB1 1 expression labelled most of the cycling cells in the petal. As expression of this histone is strictly associated with the S phase of the cell cycle, it is considered a reliable in situ marker for cycling cells (Fobert et al. 1994; De Veylder et al. 1999; Gaudin et al. 2000). The temporal and spatial histone H4 expression pattern resembled that of PethyCycB1 1. At the first developmental stages (1 3) signals were observed in every portion of the petal (Fig. 3A, B), while from stage 4 5 they were detected only in the limb (Fig. 3C, D). In this portion, in subsequent stages, expression of PetH4 1 became higher in the outer and inner epidermis than in the mesophyll. At stage 8, labelled cells were detected only in the inner or adaxial epidermis (Fig. 3E, F). Just before anthesis no signals are detectable either in the limb or in the tube (data not shown). The only difference between the hybridisation patterns of PethyCycB1 1 and PetH4 1 was the number of labelled cells was greater with the PetH4 1 probe (Fig. 3). Fig. 3A F In situ hybridisation of longitudinal sections of flowers at different stages of development with RNA probes from the PetH4 1 gene. A, B Apical (A) and basal (B) portion of petal at first stages (1 2); at this stage, signals are detected in all petal portions. Bars 100 µm. C, D Apical (C) and basal (D) portion of petal at stage 4. The signals are more numerous in the apical than in the basal portion. Bars 50 µm. E Apical portion of petal at stage 8. The signals are present only in the two epidermis and in the vascular bundle. Bar 100 µm. F Apical portion of petal at stage 8, more signals are detected in the adaxial epidermis. Bar 50 µm. a Anther, ab abaxial epidermis, ad adaxial epidermis, m mesophyll, p petal, vb vascular bundle Temporal and spatial patterns of cell enlargement in the two petal epidermis 129 Patterns of cell enlargement were examined by measuring the size of epidermal cells of petals starting from stage 3 to anthesis. Because of the extreme asymmetry of petals, four regions were measured in each epidermis: two in the tube and the other two in the limb. The position of these regions partitioned the entire petal length at early developmental stages (3 5), the first being at the petal margin. After stage 6, when flower pigmentation starts, the transition from tube to limb was chosen as the ideal border between the apical and basal portion and the position of this region was selected for the partitioning of each portion. The extraordinary difference documented in the patterns of cell division in the tube was reflected in differences in cell expansion. Cell size decreased gradually from the base to the margin, along a longitudinal gradient complementary to that of cell division. Tube epidermal cells started to increase in size from stages 6 7 (Fig. 4C, D, G, H); in the apical part significant size variations were observed only from stages 9 10 (Fig. 4A, B, E, F), when we observed a further rapid increase in size also in the tube. The cell size increase observed in all portions of petals at stage 10 was justified by the limb unfolding. The extent of cell expansion also differed in the two epidermis. Except for epidermal cells in the basal portion of the tube, enlargement was always greater in the abaxial than in the adaxial epidermis. Cell enlargement may contribute in different ways to organ shape and size determination, depending on whether or not the increased cell size occurs along preferential directions. To ascertain whether the enlargement of epidermal cells was directionally oriented, the length of the cell axes parallel and perpendicular to the petal longitudinal axes was measured. The direction of growth was clearly oriented in epidermal cells of the tube. Cell size increased exclusively along the axis parallel to the proximo-distal petal axis. No increase was seen in the transverse axis (Fig. 4C, D, G, H) and, in contrast, there was an increase of limb cells along both directions (Fig. 4A, B, E, F). These growth modalities govern the shape of the epidermal cell in the two regions. They are highly elongated in the basal region, but nearly isodiametric in the apical region. Discussion The uniformity of floral organ shape and size within species compared to the extraordinary variability across species suggests that these traits are determined by internal developmental controls. However, very little information is available about the nature of these controls. It remains unclear how a particular organ establishes its final cell number or how the regional pattern of cell division and expansion is achieved. We investigated the patterns of cell division and expansion giving rise to shape and size

8 130 Fig. 4A H Morphometric measurements of epidermal cells made at different developmental stages and in different portions of the petal. Drawings show how the petal was divided into the adaxial (light grey) and abaxial (dark grey) epidermis. Data obtained for each portion were organized in the corresponding graph. In each graph, length, width and surface of epidermal cells are represented by dot and dash ( ), dotted ( ) and continuous ( ) lines, respectively. The right y-axes show the µm 2, while the left y-axes show the µm; the x-axes show the number of developmental stage to P. hybrida petals. The stages covered a developmental range from 1 mm to about 70 mm at full anthesis. Because the cell layers were already established at the first stage, organ growth refers to increase of the organ length and width. Variations in cell size reflect complementarity between cell division and expansion patterns During the first stages, petal growth was sustained by cell divisions uniformly distributed in the apical and basal parts. However, this uniformity was already disrupted by stage 2 when a longitudinal gradient of acropetal po-

9 larity was established. The pattern of tissue organisation was discriminated by the density of the dividing cells and was generally higher in the epidermal layer compared to the mesophyll. Variations in cell size occurred along a gradient of opposite polarity with respect to cell division and were first observed in cells of the basal portion, followed by gradual variations in other portions of the petal proceeding towards the tip. On the basis of these findings, we propose that the spatial regulation of the rate of cell production, rather than region specific size controls, is the primary factor in determining the cell size patterns of P. hybrida petals. The rate of cell production has two distinct components, namely the number of dividing cells and their rate of division (Baskin 2000). Thus, variations in the rate of cell production may be ascribed to changes in one or both of these components. Establishing which of the components is responsible requires kinetic measurements, which is plagued with difficulties in a floral organ having a marginal meristem. The finding that both cyclin and histone are distributed along an acropetal gradient indicates that cell divisions initially decreased in the basal portion, where cell expansion increased. This suggests that variations in the number of dividing cells are predominantly responsible for changes in the cell production rate. A similar situation has been described in developing sunflower leaves, where the rapid increase in epidermis cell area is a consequence of the decline in the number of dividing cells (Granier and Tardieu 1998). It would be interesting to correlate this variation with the abundance of a regulatory factor responsible for cell division competence. A likely candidate for this regulation was recently described in Arabidopsis thaliana. Through gain and loss of function experiments, Mizukami and Fischer (2000) have shown that AINTEGUMENTA regulates organ size by alteration of cell number. Ant-1 organs (loss of function) were smaller and had fewer, oversized cells, whereas 35S:ANT organs (gain of function) are larger and had more cells. The authors hypothesised that loss of AINTEGUMENTA expression causes a premature termination of cell division and extends the period of cell expansion resulting in larger than normal cells. Mesophyll cells in the basal portion of P. hybrida petals may behave in a similar manner in that they stop cell division early and expand for longer periods before reaching a larger size when mature. The size difference between epidermal and mesophyll cells in the basal part offers another interesting example of the inter-tissue coordination of cell production rate. The initial decrease in the rate of production of mesophyll cells is compensated by a high production rate of epidermal cells. This ensures the generation of new cells that counterbalance longitudinal growth of the mesophyll layers. This kind of compensation has been already described in mosaics. When the epidermis of an otherwise diploid thorn apple is made polyploid, growth and development occur normally despite greatly enlarged epidermal cells. In such plants, cell proliferation in the epidermis is markedly reduced so that the area of the epidermis matches that of underlying cell layers (Day and Lawrence 2000). Cell shape variations reflect the differential regulation of cell expansion polarity The contribution of cell enlargement to organ shape may depend on whether or not growth occurs along preferential directions. The epidermal and mesophyll cells of the tube grow anisotropically, whereas those of the limb grow isotropically. These growth patterns account for the observation that epidermal cells are highly elongated in the basal region and nearly isodiametric in the apical region. Both hormones and light have been implicated in the determination of the extent and direction of cell expansion. Auxin, gibberellins and brassinosteroids promote preferential cell growth along the longitudinal axis, whereas ethylene and cytokinins induce expansion along the transverse axis (Shibaoka and Nagai 1994; Fukazawa et al. 2000). Gibberellins are produced mainly in developing anthers and are translocated to corollas, where they stimulate elongation and pigmentation. It has been hypothesised that, in P. hybrida petals, gibberellins induce the transcription of genes involved in the control of anthocyanin biosynthesis (Weiss et al. 1990, 1995; Ben-Nissan and Weiss 1996). Our data suggest that this function could be spatially uncoupled from the induction of anisotropic elongation, since only cells of the unpigmented part of the petal underwent anisotropic elongation. Gibberellins may co-operate with a factor that is differentially expressed in the tube and limb. Flower opening has often been associated with a transitory increase in the content of gibberellins. We provide evidence that there is a transient increase in the cell elongation rate that corresponds with flower opening. Although we did not measure gibberellin content in P. hybrida petals, the accelerated elongation we noted may not have been independent of the accumulation of gibberellins at opening stages. In this regard, it would be interesting to correlate petal opening with events occurring in anthers. Epidermal appendage formation contributes to organ asymmetry 131 The longitudinal morphogenetic gradient observed during petal development is locally challenged by the differentiation of epidermal appendages. The picture of trichome development is worth noting in this regard. New trichomes reiteratively differentiated at constant distances from older ones. This striking regularity leads to the conclusion that at least two concurrent controls are supervising the plane of cell division in the abaxial epidermis. The first ensures that anticlinal division of epidermal cells compensates for longitudinal growth of mesophyll layers. The second inhibits periclinal divisions giv-

10 132 ing rise to trichome formation until an appropriate spacing of already developed trichomes is attained. This regular spacing of trichomes has already been described in other systems. In A. thaliana leaves, for example, the average distance between developing trichomes is about three cells. The minimum distance between developing trichomes is established at the time of their initiation by the action of a local activator. Such an activator must be controlled by a positive feedback loop and by an inhibitor acting on the surrounding tissue. This in turn prevents neighbouring cells from undergoing a comparable development (Larkin et al. 1996; Glover 2000). We present a morphological description of petal development in P. hybrida, forming a sound basis for a more in-depth analysis of the mechanisms and functions governing shape in floral organs. Acknowledgement This study was in part supported by a grant from the Italian Ministry of University and Scientific Research, Project: Caratterizzazione di geni per la fertilità nelle piante superiori. References Baskin TI (2000) On the constancy of cell division rate in root meristem. Plant Mol Biol 43: Ben-Nissan G, Weiss D (1996) The Petunia homologue of tomato gast1: transcript accumulation coincides with giberellininduced corolla cell elongation. Plant Mol Biol 32: Cañas LA, Busscher M, Angenent GC, Beltrán JP, van Tunen AJ (1994) Nuclear localization of the petunia MADS box protein FBP1. Plant J 6: Day SJ, Lawrence PA (2000) Measuring dimensions: the regulation of size and shape. Development 127: De Veylder L, de Almeida Engler J, Burssens S, Manevski A, Lescure B, Van Montagu M, Engler G, Inzé D (1999) A new D-type cyclin of Arabidopsis thaliana expressed during lateral root primordia formation. Planta 208: Donnelly PM, Bonetta M, Tsukaya H, Dengler RE, Dengler NG (1999) Cell cycling and cell enlargement in developing leaves of Arabidopsis. Dev Biol 215: Drews GN, Beals TP, Bui AQ, Goldberg RB (1992) Regional and cell-specific gene expression patterns during petal development. Plant Cell 4: Fahn A (1990) Plant anatomy, 4th edn. Butterworth-Heinemann, Oxford Fobert PR, Coen ES, Murphy GJP, Doonan JP (1994) Patterns of cell division revealed by transcriptional regulation of genes during the cell cycle in plants. EMBO J 13: Fukazawa J, Sakai T, Ishida S, Yamaguchi I, Kaniya Y, Takahashi Y (2000) Repression of shoot growth, a bzip transcriptional activator, regulates cell elongation by controlling the level of gibberellins. Plant Cell 12: Gaudin V, Lunness PA, Fobert PR, Towers M, Riou-Khamlichi C, Murray JA, Coen E, Doonan JH (2000) The expression of D- cyclin genes defines distinct developmental zones in snapdragon apical meristems and is locally regulated by the Cycloidea gene. Plant Physiol 122: Glover JB (2000) Differentiation in plant epidermal cells. J Exp Bot 51: Granier C, Tardieu F (1998) Spatial and temporal analyses of expansion and cell cycle in sunflower leaves. Plant Physiol 116: Hofer J, Turne L, Hellens R, Ambrose M, Matthews P, Michael A, Ellis N (1997) UNIFOLIATA regulates leaf and flower morphogenesis in pea. Curr Biol 7: Kotilainen M, Helariutta Y, Mehto M, Pöllänen E, Albert VA, Elomaa P, Teeri TH (1999) GEG participates in the regulation of cell and organ shape during corolla and carpel development in Gerbera hybrida. Plant Cell 11: Larkin JC, Young N, Prigge M, Marks D (1996) The control of trichome spacing and number in Arabidopsis. Development 122: Loreto F, Mannozzi M, Maris C, Nascetti P, Ferranti F, Pasqualini S (2001) Ozone quenching properties of isoprene and its antioxidant role in leaves. Plant Physiol 126: Martin C, Gerats A (1993) Control of pigment biosynthesis genes during petal development. Plant Cell 2: Meyerowitz EM (1997) Genetic control of cell division patterns in developing plants. Cell 88: Mizukami Y, Fischer RL (2000) Plant organ size control: AINTEGUMENTA regulates growth and cell numbers during organogenesis. Proc Natl Acad Sci USA 97: Porceddu A, Reale L, Lanfaloni L, Moretti C, Sorbolini S, Tedeschini E, Ferranti F, Pezzotti M (1999) Cloning and expression analysis of a Petunia hybrida flower specific mitotic-like cyclin. FEBS Lett 462: Shibaoka H, Nagai R (1994) The plant cytoskeleton. Curr Opin Cell Biol 6:10 15 Weiss D, van Tunen AJ, Halevy AH, Mol JNN, Gerats AGM (1990) Stamens and gibberellic acid in the regulation of flavonoid gene expression in the corolla of Petunia hybrida. Plant Physiol 94: Weiss D, van der Luit A, Knegt E, Vermeer E, Mol JNN, Kootes JM (1995) Identification of endogenous gibberellins in Petunia flowers. Plant Physiol 107: