Sex Plant Reprod (2003) 15:311–320 DOI 10.1007/s00497-003-0167-7
ORIGINAL ARTICLE
Vladimir Brukhin · Michel Hernould · Nathalie Gonzalez · Christian Chevalier · Armand Mouras
Flower development schedule in tomato Lycopersicon esculentum cv. sweet cherry Received: 3 September 2002 / Accepted: 3 February 2003 / Published online: 15 March 2003 Springer-Verlag 2003
Abstract The ontogeny of tomato (Lycopersicon esculentum cv. sweet cherry) flowers was subdivided into 20 stages using a series of landmark events. Stamen primordia emergence and carpel initiation occur at stage 4; archesporial and parietal tissue differentiate at stage 6 and meiosis in anthers begins at stage 9. Subepidermal meristematic ovule primordia are formed on the placenta at stage 9; megasporogenesis begins at stage 11–12 and embryo sac differentiation and ovule curvature take place at stage 14, once the pollen is maturing. We established a correlation between the characteristic cellular events in carpels and stamens and morphological markers of the perianth. The model of tomato flower development schedule was then used to analyse the spatial, temporal and tissue-specific expression of gene(s) involved in the regulation of floral organ development. As an example, the expression pattern of ORFX, a gene controlling cell size in tomato fruits, shows that expression starts very early during the ontogeny of reproductive organs. Keywords Lycopersicon esculentum · Flower development · Microsporogenesis · Megasporogenesis · Histological analysis
V. Brukhin ()) · M. Hernould · N. Gonzalez · C. Chevalier · A. Mouras UMR 0619 Physiologie Biotechnologie Vgtales, INRA-Universits Bordeaux 1 et 2; Institut Biologie Vgtale Molculaire, INRA-CR Bordeaux, BP 81, 33883 Villenave d’Ornon Cedex, France e-mail:
[email protected] Tel.: +41-1-6348265 Fax: +41-1-6348204 Present address: V. Brukhin, Institute of Plant Biology, University of Zurich, Zollikerstrasse 107, 8008 Zurich, Switzerland e-mail:
[email protected] Tel.: +41-1-6348265 Fax: +41-1-6348204
Introduction Research in plant developmental biology has progressed very rapidly in the past decade due to the application of molecular biology techniques, the isolation and characterisation of various developmental mutants, and the cloning of genes specifically involved in different stages of plant development. It has been established that floral organ development is controlled by the differential expression of tissue- and organ-specific genes (Meyerowitz 1998; Theissen et al. 2000). Such genes may be involved in organ/tissue differentiation by triggering the regulatory cascade of key genes controlling the identity of floral organs, i.e. the “ABC” model of floral development (Coen and Meyerowitz 1991; Weigel and Meyerowitz 1994; Ng and Yanofsky 2000). However, a prerequisite to understanding the mechanisms of flower development is its dissection into distinct stages. Initially, this requires morphological and cytological definition of developmental stages at high resolution from initiation of floral meristem to floral maturity, upon which a subsequent systematic genetic and molecular analysis may be based. Chronological ranking of these stages and linking them to the size of the flower meristem is also required in order to study temporal changes in flower development. Such a morphological description has been carried out for widely used model plants e.g. Nicotiana tabacum (Koltunow et al. 1990), Arabidopsis thaliana (Smyth et al. 1990; Schneitz et al. 1995), and Silene latifolia (Farbos et al. 1997). However, despite many years of intensive genetic selection, we still lack information about many aspects of tomato (Lycopersicon esculentum) flower development, i.e. how it is coordinated with embryonic or seed development, how development of gynoecium and androecium are correlated with each other and with other flower structures and, finally, what the tomato flower development schedule is. Previous analyses of very early stages of tomato flower development, performed by light- and scanning electron microscopy (SEM), showed that flower organs in the four whorls are produced in a serial sequence (sepals, petals,
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stamens and carpels, respectively), that sepal primordia within whorl 1 (stage 1) arise independently according to a spiral sequence, and that in whorl 2 (stage 2), whorl 3 (stage 3–4) and whorl 4 (stage 5), the organs arise simultaneously. During the formation of each whorl, the meristem increases in size in order to produce the set of cells required to generate the primordia of the next whorl (Sawhney and Greyson 1972; Chandra Sekar and Sawhney 1984; Nester and Zeevaart 1988; Rasmussen and Green 1993). During the process of microsporogenesis in tomato anthers, there is a correlation between the cytological process and the bud length (Sawhney and Bhadula 1988). Comprehensive data on pollen mother cell (PMC) differentiation during the meiotic prophase in tomato show tapetal cell and pollen development from tetrad stage to pollen maturation at anthesis (Polowick and Sawhney 1992, 1993a, 1993b, 1993c). Although studies describing ultrastructural changes during microsporogenesis are available, they are not precisely linked with events in gynoecium and flower development. In addition, data on the differentiation of sporogenous cells and ovule differentiation in carpels remain scarce. Moreover, the timing of meiosis in both the male and female floral organ during their development is not well known. It is now admitted that size and cell number in vegetative and floral organs are determined early in shoot apical meristems (Meyerowitz 1997). In addition, fruit size in tomato is controlled by a quantitative trait locus, fw2.2, containing the gene ORFX, which is expressed in early flower buds (as small as 3–5 mm in length) (Frary et al. 2000). This latter study suggested that ORFX may be a negative regulator of cell division and concluded that allelic variation at the fw2.2 locus modulated fruit size, at least in part, by controlling carpel cell number before anthesis. In this paper we provide a comprehensive description of tomato flower development. The analysis focuses mainly on L. esculentum cv. sweet cherry, since this cultivar is frequently used as a model plant for tomato flower and fruit development. Using our cytological observations and data on tomato flower development reported previously, we have been able to identify critical stages at which major histogenetic events in floral meristem and flower development take place. We also show that the flower development schedule can be used to analyse the expression pattern of any gene(s) acting in tomato flowers, as illustrated by the expression pattern of ORFX during flower ontogeny.
Materials and methods Material Seeds of L. esculentum cv. sweet cherry, WVA 106 were germinated in peat pellets and seedlings with three to four leaves were transferred into 15 cm plastic pots containing loam:sand:peat (1:1:1). The plants were grown in a growth chamber under a thermoperiod of 26/20C (day/night) and a photoperiod of 14/10 h
(light/dark) with light intensity of approximately 5,000 Lx. Plants were supplied every week with a commercial fertiliser. Inflorescence and flower sampling were performed on 2-month-old plants after the production of three inflorescences. Methods Histological analysis For histological analysis, flower buds of 0.5–10 mm in length were fixed in FAA solution (4% paraformaldehyde, 50% ethanol, 5% acetic acid in 1 PBS), placed under vacuum for 10 min and incubated overnight at 4C, dehydrated in alcohol and embedded in paraffin (Paraplast plus; Sigma). At least ten buds were sampled and checked for each stage of development. Histological preparations were performed according to Gabe (1968). For histological analysis, 8 mm-thick sections were stained with 0.05% toluidine blue. For meiosis analysis in anther and ovule, 3 mm thin sections were stained with diaminophenylindole (DAPI) (1 mg/ml). Slides were observed under a microscope (Zeiss-Axioplan). Scanning electron microscopy Samples were fixed in 69% acetone/29% H2O/2% glutaraldehyde (100%) for 12 h, washed, dehydrated with acetone and transferred into 100% acetone. Samples were then critical point dried, mounted and sputtered with gold. Some samples were post-fixed with OsO4. Specimens were examined by SEM (Hitachi S-4000) at an accelerating voltage of 20 kV. Images were saved as digital files. In situ hybridisation In order to use ORFX as a probe, a set of PCR primers spanning nucleotides 56–76 and 259–277 of the ORFX sequence (accession number AF261774) was used to amplify the appropriate fragment of tomato cDNA. The DNA fragment was inserted into the plasmid pBluescribe. Sense and antisense digoxygenin (DIG)-labelled riboprobes were generated by run-off transcription using T7 and SP6 RNA polymerases according to the manufacturer’s protocol (Roche Diagnostics, Meylan, France). For in situ hybridisation, tomato flower buds were sampled and processed as described by Bereterbide et al. (2002).
Results Tomato flower development Flower induction and development Tomato belongs to the Solanaceae family. It is a dayneutral plant featuring a sympodial growth habit of the main shoot and axillary branches. After producing eight to ten leaves, the vegetative meristem transformed into the inflorescence meristem. The next vegetative apex was formed by the lateral bud in the axil of the leaf closest to the apical meristem. Later, the growth of this meristem pushed the inflorescence meristem to the side. The inflorescence meristem ultimately appeared as a lateral monochasial inflorescence, which is a scorpioid uniparous cyme of six to ten flowers (Fig. 1). Flowers contained five sepals, alternating with five petals, five anti-sepalous stamens forming a cone around the style (lateral cohesion of anthers by the interweaving of hairs) and two fused
313 Fig. 1 Lycopersicon esculentum (cherry tomato) flower development. Cyme inflorescences of scorpioid uniparous type: first (a) and second inflorescences (b) with flower buds ranging from stage 6 to 20. c Diagram illustrating flower development stages from stage 6 (1 mm in length) up to 20 (10 mm). For stages 1–5: see Fig. 2. Bars 5 mm
carpels forming a style. The flower was partly gamosepalous and gamopetalous. In mature tomato flowers, petals and stamens were longer than the style, and the anthers formed a tube around the style. Tomato flower development (flowers ranking from 1 to 10 mm in length) is presented in Fig. 1. The developmental process has been subdivided into 20 stages. The following sections detail the comparative development of flower organs in relation to bud size and histology of organs (Table 1; Figs. 1, 2, 3, 4). Early stages of flower development The very early stages of flower development (ranging from 200 to 1,000 mm in length) have already been described (Sawhney and Greyson 1972; Chandra Sekar and Sawhney 1984; Rasmussen and Green 1993) and the data reported here confirm these observations. They can be summarised as follows: the first flower of the inflorescence was formed from the flattened inflorescence apex and the next flower initiated from the base of the first flower apex. Stage 1 corresponded to the emergence of sepal primordia (Fig. 2a); stage 2 to petal primordia alternating with sepals (Fig. 2b); stage 3 to stamen primordia bulging (Fig. 2c); stage 4 to the folding of the remaining meristem edges indicating carpel initiation (Fig. 2d); stage 5 to the appearance of distinct ovary sites in the carpel region (Fig. 2e) and stage 6 to stamens showing the characteristic bi-lobed shape and the beginning of ovary closure (Fig. 2f).
Calyx and corolla development. From stage 6 onwards (flower bud of approximately 1 mm in length), the sepals and petals continued to elongate and the petals approached the top of sepals at stage 9 (Figs. 1c, 2g). The onset of calyx opening was at stage 12, when the bud had reached a size of 5 mm in length (Figs. 1c, 3d). At stage 13, the corolla emerged from the calyx (buds of 5–6 mm in length). The sepals became completely separated at the top of the calyx at stage 14 (Figs. 1c, 3g) and the corolla tube bulged above the calyx at stage 15, but the petals were still closed and green-yellowish. At stage 18, in 9-mm long buds, the sepals reached a maximum size when the corolla limb began to open (Figs. 1c, 4d) and the petals turned yellow. At stage 20, the corolla limb was fully expanded (Fig. 1c) and the yellow petals were of maximum size (approximately 10 mm) Stamen development The initiation of stamen primordia occurred early in stage 3 (Fig. 2c) in 0.5-mm long flowers and stamen primordia became delineated at stage 4 (Rasmussen and Green 1993 and this work) (Fig. 2d). At stage 5, anthers and filaments were initiated (Fig. 2e) and at stage 6 the stamens achieved a bi-lobed configuration with archesporial cell complex beginning to be distinguishable beneath the epidermis (Fig. 2f). Archesporial cells gave rise to the inward sporogenous and outward parietal cells, the latter went on to initiate the tapetum. The sporogenous tissue was distinct from tapetal and anther wall. The anther was tetrasporangiate. At stage 7, the microsporocytes displayed a large nucleus, dense cytoplasm and were
Initiation of sepal primordia Initiation of petal primordia alternating with sepals Sepal and petal primordia emerge
1 2 3
4 5 6
7
8
9
10 11
12
13
14
15
16
17 18
19
20
0.3–0.4 0.5
0.6 0.8 1
1.5
2
3
3.5 4
5
6
6.5
7
8
8.5 9
9.5
10
Corolla limb open or halfway open; anthers visible; sepals: maximum size =6 mm Flower open, corolla limb fully expanded and bright yellow
Corolla emerges from calyx Sepals: 5.4 mm, petals: 5.8 mm Sepals completely separated, corolla bulges at tip of calyx Closed corolla tube bulge above calyx, green-yellowish petals Corolla elongating; petals green-yellowish and slightly open sepals: 5.8 mm, petals: 8 mm Corolla tube bulge enlarging, petal tips yellow Corolla limb beginning to open, petals yellow
Sepals: 4 mm Petals: 3.0 mm Calyx opens slightly at top of bud; sepals: 5.0 mm, petals: 4.5 mm
Petals approaching top of sepals; sepals: 3 mm
Sepals: 1.5 mm
Sepals and petals: elongation and vascularisation Sepals: 1.0 mm
Perianth: morphological markers
Stage
Bud length (mm)
Mature ovules
Carpel: thickness of ovary wall 7–9 rows of cells; ovule: micropyle formation Style elongation Stigma reaches the top of anthers; ovules: fully differentiated anatropous ovules
Ovule curvature
Integument grows around nucellus
Carpels: placental column formation, carpel wall vascularisation; emergence of style; ovule: subepidermic meristematic dome Ovule: megaspore cell differentiation; Style: transmission tract differentiation; vascularisation of central septa Ovule: callose deposition on MMC Ovule: beginning of meiosis in some ovules; initiation of integument Carpel: carpel wall with 6–7 rows of cells; Ovule: majority of ovules in meiosis; vascularisation of funiculus, initiation of curvature Ovule: embryo sac differentiation
Fusion of carpels
Undulation of meristem: carpel initiation Carpel locules appear as faint cavities Carpels growing up; primordium of placenta
Folding of carpel meristem ridge
Reproductive Meristem dome
Carpel
Anthers dehiscence; mature pollen release
Stamen: maximum size =7.0 mm; anther: pollen mitosis Anther: two-celled pollen Pollen: differentiation of vegetative and generative nuclei Anther: lipid bodies accumulation in pollen
Anther: vacuolated microspores, pollen sculpturing, resorption of connective tissue Anther: complete degeneration of tapetum
Anther: tapetum degeneration
Anther: free irregularly shaped microspores
Anther: tetrad of microspores Anther: resorption of callose
Anther: binucleate tapetum; PMC meiosis initiation
Stamen primordia bulges at the periphery of the meristem Stamen: delineation of primordia Stamen: anther differentiation Anther: characteristic bi-lobed shape; parietal and archesporial tissue Stamen: vascularisation of filament; anther: differentiation of PMC and tapetum Anther: callose deposition around PMC
Stamen
Reproductive organs: morphological and major marker events
Table 1 Comparative development of flower organs in relation to bud size and histology of organs. MMC Megaspore mother cells, PMC pollen mother cells
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315 Fig. 2a–l Main features of flower development during stages 1–9. a Stage 1: successive emergence of sepal (S) primordia. b Stage 2: petal (P) primordia alternate with sepals (removed). c Stage 3: stamen (St) primordia bulge. d Stage 4: folding of the remaining meristem edges (arrow) indicating carpel (C) initiation. e Stage 5: emergence of carpels and appearance of distinct ovary cavities. f Stage 6: sepals grow up while petals bend down to cover reproductive organs. Stamens show the characteristic bi-lobed shape (inset: transverse section), and differentiate archesporial tissue. Carpels grow up but are still unfused (arrow) and the position of the placenta (pl) is determined. g Stage 8: carpels have just fused (arrow). h Magnification of the ovary showing carpel wall (cw) and the emergence of ovule primordia (op) from the placenta (pl). i Magnification of anther showing callose deposition around pollen mother cells (PMC) and binucleate tapetum (t, arrow). ex Exothecium. j Stage 9: style elongation and differentiation of transmitting tract (arrow). k Magnification of the ovary showing ovules (ov) differentiating. l Magnification of anther showing PMC in meiosis and the binucleate (arrow) tapetum (t). a–c, e Scanning electron micrographs (SEM); d, f, g, j toluidine blue-stained histological sections; h, i, k, l DAPI-stained histological sections. Bars a, b, 60 mm; c 90 mm; d, e 100 mm; f 500 mm; g, j 250 mm; h, i, k, l 25 mm
surrounded by a cell layer (the tapetum), which started to become binucleate at stage 8 (Fig. 2i). At the premeiotic stage, the potentially sporogenous cells were angular in outline. As prophase I progressed, the PMC became less angular and callose deposition gave them a dark boundary, which could be observed at stage 8 (Fig. 2i), just prior to initiation of meiosis at stage 9 (Fig. 2l). Simultaneous cytokinesis in megaspore mother cells (MMC) followed meiosis. Microspore tetrads were tetrahedral and encased
in callose (Fig. 3c). At stages 10–11 (buds 4 mm in length) meiosis was underway and the microspores were released from the tetrads. Upon release (stage 12), the microspores were irregularly shaped. Many of them still retained the pointed shape on the inner surfaces originally oriented towards the centre of the tetrad. From stages 12 (Fig. 3f) to 15, the microspores differentiated into pollen grains. Pollen sculpturing started at stage 14. Conspicuous features of the microspores at this stage were large
316 Fig. 3a–i Main features of flower development during stages 10–14. a Stage 10. b Magnification of the ovary showing ovules; inset differentiating megaspore mother cell (MMC) (arrow). c Magnification of anther showing tetrad of microspores (arrow). t Binucleate inner tapetum. d Stage 12. e Magnification of the ovary showing initiation of ovule curvature (ov) and meiosis of megaspore (arrow in inset). f Magnification of anther showing free microspores (ms). t Tapetum. g Stage 14. h Magnification of the ovary showing ovules with developed integument and embryo sac (es) differentiating (inset integument grows around nucellus). i Magnification of anther showing free microspores within anther locule where tapetum is degenerating (arrow). a, d, e, g, h Toluidine blue-stained histological sections; b, c, f, i DAPIstained histological sections; h inset SEM. Bars a, d, g 500 mm; b, c, e, f, h, i 50 mm
vacuoles in the cytoplasm and a near spherical shape (Fig. 3i). Following the vacuolation of the microspores at stage 16, pollen entered mitosis (Fig. 4c) and became bicellular with small, irregularly shaped, vacuoles and distinct vegetative and generative cells (Fig. 4f). As the microsporangia matured, reabsorption of connective tissue and the beginning of tapetum degeneration in anthers (Fig. 3i) were observed. From stage 16 onwards, the stamens no longer elongated, complete tapetum degeneration occurred and dark inclusions, corresponding to lipid bodies, were observed in mature pollen (stage 20, Fig. 4i). Anther dehiscence occurred at stage 19 and the corolla limb was fully expanded at stage 20.
Carpel development After the emergence of sepal, petal and stamen primordia (stage 3; Fig. 2c), the folding of the carpel meristem ridge on the reproductive meristem dome was observed. The first undulation of the meristem, indicative of carpel initiation, occurred at stage 4 (Fig. 2d) and the primordium of the placenta emerged at stage 5 (Fig. 2e). At stages 6–7, the carpels continued growing and initiated vascularisation (Fig. 2f). Apical fusion of carpels and emergence of the central column and style occurred at stages 7–8 (Fig. 2g). At stage 8, ovule primordia were initiated by periclinal divisions of the subepidermal
317 Fig. 4a–i Main features of flower development during stages 16–20. a Stage 16. b Magnification of the ovary showing differentiating ovules and micropyle formation (arrow). c Magnification of anther showing microspores mitosis (arrow). d Stage 18. e Magnification of the ovary showing characteristic anatropous ovule. int Integument, es embryo sac, f funiculus, arrow micropyle. f Magnification of maturing pollen with vegetative and generative nuclei (arrows). g Ovary at stage 20. h Distribution of mature anatropous ovules within the ovary (carpel wall removed). i Mature pollen containing lipid bodies (arrow). a, d, g Toluidine blue-stained histological sections; b, c, e, f DAPI-stained histological sections; h SEM. Bars a, d, g 500 mm; b, c, e, f, i 50 mm; h 200 mm
meristematic cells perpendicular to placenta (Fig. 2h). By this time, the petals had approached the tops of the sepals in the perianth (Fig. 2j). The young ovules subsequently appeared in rows of small, relatively tightly packed, bulges along the placenta (Fig. 2k). Originally, cells within the epidermal and subepidermal zones of the placenta had been of the same size (Fig. 2h). The first changes appeared when archesporial cells become visible. Megasporocytes were easily recognised by their increased size, large nuclei and their location one cell layer beneath the nucellus (Fig. 3b, and inset). The megasporocytes differentiated at stage 10 and callose deposition was observed around MMC. The integument was initiated at stage 11–12 at the base of ovules and was observed as growing bulges (Fig. 3e). Meiosis of megasporocytes took place at stage 11–12 in buds of 4–5 mm in length (Fig. 3e, inset), although meiosis in ovules is not as perfectly
synchronised as it is in anthers, i.e. sporogenous complexes in the same placenta were found to be at diverse developmental stages, ranging from just after the final mitotic division involved in megasporocyte formation to late prophase I. A single megaspore, usually the one above the linear tetrad of megaspores, was located just beneath the epidermal layer of the nucellus, and developed into a Polygonum-type embryo sac. At the same time, curvature of ovules and vascularisation of central septa and funiculus occurred (Fig. 3g, h). Megagametogenesis and integument development were observed to be concomitant events. The integument continued to grow around the nucellus (Fig. 3h, inset) and enveloped it completely (Fig. 3h). Despite its haploid state, the functional megaspore nucleus was larger than nuclei in the surrounding diploid sporophytic tissue. The embryo sac developed as the functional megaspore underwent
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mitosis at stage 14. Embryo sac formation (Fig. 3h) was an asynchronous process that could last from stage 14 until stage 18. We observed ovules forming the micropyle (Fig. 4b) and the presence of differently evolved embryo sacs on the same placenta. During ovule development, the growing embryo sac displaced the nucellar tissue at the micropylar and chalazal ends. After the third mitotic division of the functional megaspore and subsequent cellularisation, the embryo sac consisted of seven cells and eight nuclei. The mature tomato ovule was anatropous, unitegmic and tenuinucellar (Fig. 4e) as in most representatives of the Solanaceae (Davis 1963). The style reached its maximum size (~7 mm) and ceased growth at stage 18 (Fig. 4d). At stage 20, the corolla limb expanded (Fig. 1c), exposing the staminal cone covering the gynoecium, which contained fully mature ovules (Fig. 4h). Expression pattern of ORFX, used as a molecular marker for tomato flower development In order to analyse the spatial, temporal and tissuespecific expression of ORFX, an in situ hybridisation procedure was applied to sections of tomato vegetative meristem and flowers. By using antisense (Fig. 5, left panel) and sense (control) (Fig. 5, right panel) probes, the ORFX expression pattern can be summarised as follows: transcripts were detected in the vegetative meristem and the adaxial side of leaf primordia (Fig. 5a). During the early stages of flower development (stage 1–6), transcripts were detected in the whole flower meristem and particularly in emerging primordia, with the exception of the sepals (Fig. 5c). From stages 7 to 10, characterised by the appearance of archesporial cells and microsporogenesis, transcripts were present at a high level in stamens (mainly in anther wall and locules but not in filaments) and carpels (mainly in differentiating ovules and transmitting tract, Fig. 5e). The faint staining of petals indicated a very low level of transcript. From stage 15, transcripts were present only in ovules at the level of the integument in contact with the remaining nucellus. No transcript accumulation was visible either in the nucellus or in the embryo sac (Fig. 5g). Control experiments performed with DIG-labelled sense probes did not show any hybridisation signal (Fig. 5b, d, f, h).
Discussion Fig. 5a–h Detection of mRNA hybridising with an ORFX probe in developing flowers of L. esculentum cv. sweet cherry. Left Antisense probe, right sense probe. Longitudinal sections of vegetative meristem and flower bud at stage 1 (a, b), and flower buds at stage 3–4 (c, d), 9 (e, f) and 15–16 (g, h). Hybridisation signal appears as dark staining
This paper describes the morphological characteristics of flower development in L. esculentum cv. sweet cherry, from the appearance of the reproductive meristem bulge to the mature flower (i.e. just before pollination and fertilisation). Stages 1–6 have already been defined and described elsewhere (Sawhney and Greyson 1972; Chandra Sekar and Sawhney 1984; Rasmussen and Green 1993). Our observations regarding the main events during early flower development in tomato are in agreement with previous reports. Furthermore, we provide new informa-
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tion, based on morphological landmark events combined with histological data, regarding the later stages of tomato flower development (stages 6–20). In particular we present data concerning the timing of meiosis in male (stage 9) and female (stage 11–12) organs. We also define the period of differentiation of sporogenous and tapetal tissue to be at stage 6–7, the meiosis process from stage 8 to 10, the release of microspores from tetrads in the anthers at stage 12 and the period of embryo sac differentiation in ovules from stage 14 to 18. An important aspect is the gap between microsporogenesis and megasporogenesis. Our observations confirm data showing that, in the tomato gynoecium, the archesporial cells enlarge and acquire the function of megasporocytes, while the tetrads of microspores are formed in the anthers (Mazzucato et al. 1998). The existence of such a detailed staging system is of great importance since it provides a better understanding of the relative timing of the morphological events that accompany tomato flower development. As illustrated by the expression pattern of ORFX, a gene involved in the control of cell number and development, the tomato flower development schedule provides a basis for the analysis of genes involved in flower development and fruit set. The criteria for classifying the different stages of flower development are based on the ontogeny of male and female reproductive organs. In our description of flower development, we establish a correlation between the main cellular events occurring in carpels and stamens, and the morphological markers of the perianth: initiation of meiosis in PMC and protruding of ovules from the placenta occurs when the petals are approaching the tops of the sepals; free microspores are released from the tetrads when the calyx opens slightly at the top of the bud; onset of embryo sac differentiation coincides with the bulging of the corolla tube at the tip of the calyx and complete separation of the sepals at the top of the calyx; pollen mitosis takes place when the petals are turning yellow and are slightly open. Flower development was also analysed in other commercial large fruit-bearing tomato cultivars, i.e. L. esculentum cv. Palmiro and L. esculentum cv. Elsa Craig. Although not performed in as much detail as for cv. sweet cherry, these studies reported the same characteristic landmarks. For example, timing of microsporogenesis corresponded to ovule formation (stage 9–10); megasporogenesis, paralleling microspore release from callose wall, occurred in flowers where petals emerged from the calix (stage 12); microspore mitosis and ovule curvature/ maturation occurred in flower buds in which the petals were still closed and turning yellowish (stage 16). The main difference between sweet cherry and other cultivars is the length of flowers: for example, stage 9 (microsporogenesis) occurred in 3-mm long sweet cherry flowers while in other cultivars the flowers were 5 mm long at this stage; stage 16 occurred in 8-mm long sweet cherry flowers versus 10–11 mm flowers in others cultivars. Thus, the correlation between cellular events and mor-
phological markers remains the same in all the tomato cultivars investigated. In conclusion, we have constructed a detailed reference model of tomato flower development for the later study of spatial, temporal, and tissue-specific expression of genes involved in flower development. Acknowledgements This work was supported by the Ministre de l’Enseignement Suprieur et de la Recherche (MESR) and by the Region Aquitaine. V.B. was supported by a postdoctoral fellowship from NATO and the “Ministre de l’Education Nationale de la Recherche et de la Technologie”. N.G. was supported by a grant from the Ministre de l’Education Nationale de la Recherche et de la Technologie (MENRT). We are indebted to Jean-Philipe Bru (Universit Victor Segalen Bordeaux 2, France) and Urs Jauch (Institut of Plant Biology, University of Zrich, Switzerland) for technical assistance. We thank Dr. Mark Curtis for critical reading of this manuscript.
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