Integration of tomato reproductive developmental landmarks and expression profiles, and the effect of SUN on fruit shape

Initiation of floral organ primordia

The first landmark represented inflorescence formation and flower initiation (Table 1). The transition to flowering and inflorescence formation in LA1589 has been described previously [36]. Briefly, transition to flowering commenced with the termination of the vegetative meristem into an inflorescence meristem. Floral initiation occurred through the apparent bifurcation of the inflorescence meristem resulting in bud number 1 (Fig. 2A and 2B). The flatter inflorescence meristem continued its indeterminate growth pattern, while the more domed meristem developed into a flower (Fig. 2A and 2B). Following flower initiation, the emergence of the sepal primordia around the perimeter of the floral apex of bud number 2 marked the second landmark (Fig. 2A). The five tomato sepals initiated in a helical pattern of 144° (Fig. 2C). The sepals continued to grow and covered the floral meristem approximately 4 days after floral initiation (Fig. 2D and 2E). At the time of sepal enclosure, petal primordia started to arise, representing landmark 3. Following petal primordia emergence, stamen primordia emerged in alternate positions to the petals (Fig. 2F and 2G), at approximately 5 days after floral initiation, representing landmark 4. Sepals and petals continued to elongate while carpel primordia began to emerge in the floral center (Fig. 2G), marking landmark 5, which occurred approximately 6 days after floral bud initiation. The carpel walls or valves continued to enlarge, while the central part comprising the septum and the central column formed congenitally with the carpel walls, revealing the formation of the two locular cavities of wild type tomato ovary (Fig. 2H). The carpel walls elongated slightly faster than the central column revealing the locular cavity prior to ovary enclosure and initiation of the style, which occurred 8 days post bud initiation (Fig. 2I and 2J).

Flower Development Landmarks; Buzgo et al. (2004)	Days after flower initiation in tomato	Perianth organs	Reproductive organs
			Ovary and ovule development	Stamen and pollen development
(1) Inflorescence formation and flower initiation	1	Flattened inflorescence apex becomes dome-shaped.
(2) Initiation of outermost perianth organs	2	Emergence of sepal primordia in a helical pattern.
(3) Initiation of inner perianth organs.	4	Simultaneous emergence of petal primordia in alternating positions to the sepals. Sepals overlay the floral meristem
(4) Stamen initiation	5	Sepals and petals elongate.		Simultaneous initiation of stamen primordia.
(5) Carpel initiation	6	Petals start curling over the stamens	Carpel primordia arise.
	7		Central column that will form the locular cavities arise.	Stamen filament start developing and two anther lobes become visible.
(6) Microsporangia initiation	8		Central column continues to elongate. Carpels fuse at the apex of the ovary. Style initiation. Initiation of placental development.	Primary pariety cells develop into endothecium, middle layers and tapetum. Sporogenous layers visible.
(7) Ovule initiation	9		Ovule primordia begin to emergence from the placenta.	The two lobes of the anther and the locule are distinguishable, microsporocyte and tapetal cells are distinguishable. Binucleate tapetal cells.
(8) Male meiosis	10			Microsporogenesis. Microsporocytes or microspore mother cells undergo meiosis I and II and forming tetrads.
(9) Female meiosis	11		Megasporogenesis. Megaspore mother cell (meiocyte or megasporocyte) is visible. Meiosis I. The nucellus is small resulting in a tenui-nucellate ovule.
	12	Petals grow to the top of sepals	The single integument begins to grow over the nucellus resulting in unitegmic ovules.	Callose wall surrounding the tetrads degrades releasing the microspores. Tapetum starts degenerating.
	13	Petals emerge from the sepals.	Micropyle development.	Free microspores are being incased in a thick polysaccharide wall; tapetum degenerated.
	14	Onset of sepal opening	Megagametogenesis and development of the embryo sac.	Microspores come vacuolated, and begins asymmetric mitosis
	15			Bi-cellular pollen grain.
	16		Ovule development nears completion.	The vegetative cell and generative cell are well distinguishable
(10) Anthesis	19	Petal opening

The timing of the landmarks described by Buzgo et al (2004) in S. pimpinellifolium accession LA1589 floral development.

Reproductive organ formation

Male reproductive development initiated with microsporangia development, which represented landmark 6, and occurred approximately eight days after floral bud initiation (Table 1 and Fig. 3A). The primary sporogenous layers were visible at this stage (Fig. 3A). Nine days after floral bud formation, the tapetal cells were binucleate, and the developing microsporocytes were also visible (Fig. 3B and 3C). At 10 days after floral bud initiation, microsporocytes or pollen mother cells were undergoing meiosis (Fig. 3D), marking landmark 8. A callose wall surrounded the four haploid nuclei of the tetrads (Fig. 3E). One day later, the callose walls began to degrade and the microspores were being released (Fig. 3F). At 13 days after floral bud initiation, the tapetum was degenerating; and the microspores were single and encapsulated in a thick wall (Fig. 3G and 3H). One day later, the microspores became vacuolated (Fig. 3I) and underwent one asymmetric mitosis. Fifteen days after floral bud initiation, the microspores were bi-cellular (Fig. 3J) and a day later, the generative and vegetative cells were clearly distinguishable within the developing pollen (Fig. 3K). At day 17 after floral bud initiation, the generative cell displayed the characteristic crescent shaped nucleus (Fig. 3L and 3M). The second mitosis of the generative cell did not occur until after pollination.

Female reproductive development initiated with the development of the ovules and represented landmark 7 (Fig. 4A). Approximately 9 days after floral bud initiation, the style and the ovary were nearly equal in length, and ovule primordia were emerging on the placental tissues (Fig. 4A). Ovules were clearly visible one day later (Fig. 4B). Two days after ovule primordia initiation and 11 days after floral bud initiation, a single integument started to envelope the single cell layered nucellus and the developing megasporocyte, resulting in a unitegmic tenui-nucleate ovule representing landmark 9 (Fig. 4C). Apparently the megasporocyte underwent the first meiotic division at this stage (Fig. 4D). A day later, the single integument at the base of the nucellus was clearly visible, while the megasporocyte is undergoing the second meiotic division, representing the first stage of megagametogenesis (Fig. 4E). Fourteen days after floral bud initiation, the integument enveloped the nucellus completely and the micropyle was well defined. The embryo sac development was taking place as evidenced by concentrated dark staining at the micropyle end. The presence of the megaspore at the chalaza end of the ovule indicated the development of the egg apparatus (Fig. 4F).

Fertilization and fruit set

Anthesis or flower opening was the final floral landmark as well as the first fruit landmark (Table 1 and 2). At the time of anthesis, the anther lobes dehisced to release the pollen, which after landing on the receptive stigma, germinated. Pollen tubes had grown close to the base of the style 6 hours after pollination, and reached the ovules approximately 2 hours later (Fig. 5A and 5B). Ten to 12 hours after pollination, the pollen tubes had released their content resulting in fertilization of the ovules (Fig. 5C) and representing fruit development landmark 2 (Table 2). Senescence of floral organs, namely petal, stamens and style is associated with successful fertilization and was visible approximately two days after anthesis as shown in Fig. 1B.

Fruit Development Landmarks	Days post anthesis	Descriptions of fruit development in tomato
		Fruit growth (Gillaspy et al1993)	Embryo/seed development
(1) Anthesis	0	Mature ovary, phase I.	Mature gametes. Pollen is shed, which will land on the stigma and germinate. Pollen tubes growth through the style.
(2) Fertilization	1–2	End of phase I, beginning of phase II.	Fusion of sperm and egg nuclei.
(3) 4–16 Cell Stage Embryo	3–6	Phase II and III, cell division and elongation stage.	First embryo divisions.
(4) Globular Stage Embryo	6–10	Phase III, cell expansion stage.	Globular embryo.
(5) Heart Stage Embryo	10–12	Phase III, cell expansion stage.	Heart Stage embryo lasts approximately one day and occurs 10–12 dpa.
(6) Torpedo Stage Embryo	13–16	Phase III, continued fruit enlargement.	Torpedo Stage embryo lasts approximately one day and occurs 13–16 dpa.
(7) Coiled Stage Embryo	20	Phase III, continued fruit enlargement.	Cotyledon expansion and curl as they elongate. Embryo appears physically mature, but the seed is not yet viable.
	20–28		Seed maturation period
(8) Seed germination	29–31	The fruit has reached the mature green stage. Fruit becomes sensitive to ethylene.	Seeds are becoming viable for germination.
(9) Fruit ripening	33–40	Ripening starts at the onset of the breaker stage. Changes in pigmentation are visible.	After ripening of seed.
(10) Ripe Fruit	40	Red ripe stage of tomato.

Timing of the fruit landmarks in S. pimpinellifolium LA1589.

Development of the pericarp after pollination

Following fertilization, tomato fruit growth consists of cell division and cell expansion [19]. We analyzed the growth of the pericarp following pollination to establish the timing of cell division and cell elongation in the developing LA1589 pericarp. Pericarp width doubled from anthesis to 2 days post anthesis (dpa), and then further doubled at 5 and 10 dpa, respectively (Fig. 6). Cell number across the pericarp increased from 10 at anthesis to 17 at 2 dpa, and reached the final number of 19–21 at 5 dpa (Fig. 6F), implying that cell division ended at or before that time. Mesocarp cell expansion started as early as 2 dpa (Fig. 6B). These results indicated that cell division and expansion occurred concurrently in the pericarp of the early developing fruit. Note the presence of the cuticle layer and starch granules in the epicarp and mesocarp respectively, of 10 dpa fruit (Fig. 6D).

Seed development

As indicated above, cell division overlapped with cell elongation during the early stages of fruit development. Moreover, the cell division stage was short, ending before 5 dpa in LA1589, whereas the cell elongation stage spanned fruit development from 2 dpa until mature green stage. Thus, these two fruit developmental stages, which correspond to tomato development phases II and III, provided limited guides for referencing. To develop additional landmarks for the developmental stages of tomato fruit growth, we analyzed morphological changes in embryo development, which occur concomitantly with fruit growth in most angiosperm plant species.

We propose the third fruit developmental landmark as the stage of 4–16 celled embryo, which occurred approximately 4 dpa (Fig. 7A and 7B). The fourth landmark was represented by the globular embryo stage at 6 to 10 dpa (Fig. 7C and 7D). Heart shape embryo was the fifth landmark and occurred between 10 and 12 dpa (Fig. 7E and 7F) highlighting the beginning of cotyledon growth. The 13–16 dpa embryo was torpedo shape, marking the sixth landmark (Fig. 7G and 7H). After the sixth landmark, the cotyledons grew into a coil and reached the seventh landmark approximately at 20 dpa. At this stage, the embryo approached its final size, but the seed was not yet viable for germination (Fig. 7I and 7J). The eighth fruit developmental landmark was reached when the seeds harvested from the maturing fruit were viable for germination. Seed were collected from maturing fruit starting at 26 dpa until 33 dpa. Up until 29 dpa, there was little or no seed germination (Fig. 8). However, at 30 dpa, the germination rate increased dramatically thus reaching landmark eight. At 32 dpa, nearly 100% of the seeds germinated.

Fruit ripening

Tomato fruit ripening stages consist of mature green, breaker and red ripe [19, 23]. At the mature green stage, ethylene treatment will result in a rapid reddening of the fruit [23, 37–39]. We measured ethylene sensitivity in half of the harvested fruits while determining the germination ability of the seed in the other half that were collected at selected times (see above). Ethylene sensitivity was achieved over a short period of up to two days, and coincided with the time when the seed became viable for germination (Fig. 8). Forty percent of fruit had responded to ethylene at 30 dpa when 43% of the seeds were viable for germination. Fruit younger than 29 dpa did not respond to ethylene treatment (Fig. 8). The ninth landmark is the onset of fruit ripening, coinciding with the breaker stage when color began to change at approximately 32 dpa. This stage is followed by the tenth and final landmark of ripe fruit.

Gene expression profiles of floral and fruit development

Expression of organ identity and patterning genes

Of the genes representing the developmental processes, key floral and fruit patterning genes were examined for their expression profiles during reproductive development (see Additional file 2, Fig. 9). Genes orthologous or homologous to the Arabidopsis ABCE genes required for floral organ identity have been identified in tomato [41, 42]. On our array, the tomato floral organ identity genes differentially expressed at the three stages include B class genes TAP3 (TC116723) [26], TPI (TC117703) and SlMBP1/LePI-B (TC119919) [42], C class gene TAG1 (TC124766) [43], and E class gene TM29 [44]. The tomato ortholog TC121763 of Arabidopsis NAP that is directly activated by B class gene APETALA3 and PISTILLATA in Arabidopsis [45] was also differentially expressed. All the above-mentioned genes showed higher expression in floral buds and/or anthesis-stage flowers (see Additional file 2), in agreement with their previously identified expression patterns. Another tomato B class gene TM6 (TC117238) was not differentially expressed, likely due to its more ubiquitous expression in floral organs [26]. While there is no clear ortholog of Arabidopsis A class genes in tomato [42], the closest related AP1 gene, MADS-MC (TC118643) [46], showed no expression changes in the three developmental stages. Many of the organ identity genes encode MADS box proteins of MIKC-type, and in vitro interaction analysis of twenty-two tomato MADS box proteins show modified as well as novel interaction patterns that had evolved for the family members in this species [47].

In addition to these organ identity genes, other genes play key roles in patterning of the fruit. In Arabidopsis, these include the apical-basal patterning genes: ETTIN (ETT) [48], LEUNIG (LUG) [49], TOUSLED (TSL) [50], STYLISH (STY1 and STY2) [51], SPATULA (SPT) [52], NO TRANSMITTING TRACT (NTT) [53], and HECATE (HEC1, HEC2 and HEC3) [54], involved in basal valve growth, carpel and septum fusion, elongation of apical tissues, and style and transmitting tract formation, respectively. There are also genes patterning valve and valve margin of the fruit along the medio-lateral axis, including SHATTERPROOF (SHP) [55], ALCATRAZ (ALC) [56], INDEHISCENCE (IND) [57], REPLUMLESS (RPL) [58], and FRUITFULL (FUL) [59]. The Arabidopsis gene SEEDSTICK (STK) is required for ovule identity and patterning as well as seed disposal [60], and ERECTA (ER) regulates fruit shape by controlling cell expansion and cell division [61]. JAGGED (JAG) acts redundantly with the polarity genes FILAMOUS FLOWER (FIL) and YABBY3 (YAB3) to activate FUL and SHP [10]. Additional polarity genes required for proper patterning and establishment of organ boundaries are CRABS CLAW (CRC) [62], KANADI (KAN1 and KAN2) [63], GYMNOS (GYM) [64], PHAVOLUTA (PHV) and PHABULOSA (PHB) [65]. Tomato genes homologous to Arabidopsis patterning genes FIL (TC126122), FUL (TC125305 and TC126125), CRC (TC125410), ER (TC121018, TC122809 and TC123029), PHB (TC130887), and SPT (TC126307) were more abundantly expressed in tomato flower buds compared to the other tissues. The tomato SHP homolog TC118705 showed higher expression in anthesis-stage flowers and fruits at 5 dpa than in floral buds. The STK homolog in tomato TAGL11 (TC119398), which is expressed in the inner integument of the ovules and the endothelium in developing seeds [41], was expressed higher in fruits at 5 dpa compared to other time points (see Additional file 2), suggesting that it may also play a role in tomato fruit development. Tomato genes with high similarity to Arabidopsis fruit patterning genes ETT, GYM, KAN2, LUG, PHV, RPL, HEC1, STY1 and TSL were not differentially expressed between the three stages, whereas no tomato homologs for JAG, NTT, ALC, IND, YAB3, STY2 were included on our array. Further, the hierarchical clustering of all the 122 differentially expressed developmental processes genes revealed that flower bud and 5 dpa fruit shared expression profiles of the same developmental genes, whereas anthesis-stage flower showed a distinctive profile (Fig. 9, see Additional file 2), which is in agreement with results from other gene profiling studies in Arabidopsis [66–68].

Expression of phytohormone-related genes

Phytohormones play essential roles in many aspects of plant development. Among the three developmental time points, 79 phytohormone-related genes were differentially expressed (see Additional file 3). Of these genes, 30 were involved in auxin conjugation, transport or signaling. Most of the auxin-related genes (22 of 30) were either up- or down-regulated in 5 dpa fruit (Fig. 10, see Additional file 3). Moreover, most of the genes with similarity to GH3 involved in IAA conjugation were repressed after pollination, whereas three auxin response factor genes TC118569 (ARF4), TC122720 (ARF8), and TC122700 (ARF9), were expressed at the lowest level in anthesis stage flowers. Further, transcripts of three auxin transporter genes, TC127164, TC123055 and TC120936, homologous to AUX1, PIN4 and an auxin efflux carrier family protein, respectively, were less abundant in 5 dpa fruit (Fig. 10, see Additional file 3). Several genes involved in biosynthesis of tryptophan (TC119571, TC121695, TC125473, TC127841, TC129375, and TC130235), a precursor of IAA, were not developmentally regulated in this study, neither was the ortholog of Arabidopsis auxin receptor TRANSPORT INHIBITOR RESPONSE1 (TIR1, TC121284) [69]. The ortholog of ALDEHYDE OXIDASE 1 (AAO1, TC117167) involved in auxin biosynthesis [70], was expressed at higher level in anthesis flower. This may imply that many components in auxin pathway are channeled to the increasing demand for auxin-dependent programs to fulfill rapid fruit growth after pollination.

Some GA-related genes were also differentially expressed in the three developmental stages. Transcript levels of the tomato ortholog TC124105 of AtKAO2 that catalyzes the conversion of ent-kaurenoic acid to GA₁₂ in gibberellin biosynthesis pathway [71], was more abundant in 5 dpa fruit compared to other stages. In contrast, the expression of SlGA2ox2 (TC127124), involved in catabolism of GA [72], was lower in the developing fruits than in flower buds at 10 days preanthesis and anthesis-stage flowers. Interestingly, transcripts of three tomato homologs TC118018, TC121133 and TC124715 of Arabidopsis GA receptors GA INSENTIVE DWARF1B and C (GID1B and GID1C) [73], were less abundant in 5 dpa fruit. This suggests that although GA levels may increase in 5 dpa fruit as a result of increased biosynthesis and reduced catabolism, the sensitivity to the hormone may decrease as a result of reduced expression of the receptor. GA biosynthesis genes of the GA 20-oxidase and GA 3-oxidase families were either not differentially expressed (SlGA20ox-3, SlGA3ox-2) or not included on the array (SlGA20ox-1, -2 and SlGA3ox-1, -3). Most of the seven GA responsive genes were not differentially expressed following pollination with the exception of tomato gene TC126562 encoding GASA/GAST/Snakin family protein that was upregulated after anthesis (Fig. 10, see Additional file 3).

Transcripts of all the eight brassinosteroid-related genes were more abundant in 5 dpa fruit, whereas the majority of jasmonate- and ethylene-related genes were less abundant in 5 dpa fruit (see Additional file 3). Expression of genes involved in ABA biosynthesis and response like were also lower in 5 dpa fruits. The putative ortholog of Arabidopsis gene CYP707A3 (TC129465), encoding the major ABA 8'-hydroxylase involved in ABA catabolism [74], is expressed at higher level in 5 dpa fruit compared to the other stages, suggesting that the ABA levels are reduced during the early fruit growth.

Fruit shape changes in LA1589 NILs differing at sun

We used the floral and fruit developmental landmarks described above to determine when SUN affects tomato fruit shape. SUN controls fruit elongation and its high expression results in oval shaped fruit [32]. We analyzed the changes in fruit shape from anthesis onward in NILs in LA1589 background differing at the sun locus because at anthesis the ovary shape is only marginally different (Fig. 11). The LA1589pp has round fruit and carries the wild type allele, while LA1589ee carries the Sun1642 allele of sun resulting in an elongated fruit (Fig. 11A). The difference in fruit shape between the two NILs became apparent immediately following fertilization and was most pronounced between 6 and 10 dpa coinciding with the globular embryo stage of fruit landmark 4. At the end of the sixth fruit landmark, representing the seed torpedo stage, the fruit shape index of the LA1589ee NIL started to decrease. After the landmark of seed germination corresponding to mature green stage, the fruit shape index remained constant. LA1589pp fruit showed a decrease in fruit shape index from > 1 at anthesis to < 1 at 5 dpa (Fig. 11A). We examined SUN expression in the developing fruits of the LA1589 NILs starting from anthesis-stage ovaries until ripe fruit. In LA1589ee, SUN was expressed at a high level until fruit landmark 7 coinciding with coiled embryo and seed maturation stage at 20 dpa (Fig. 11B). A detailed investigation of its expression immediately before and after anthesis showed that SUN transcript levels increased from 2 days prior to anthesis to 2 dpa and thus showed a similar kinetics to that of the changes in fruit shape (Fig. 11B).

Gene expression profiles associated with SUN

SUN has been hypothesized to affect fruit shape by altering hormone levels such as auxin [32]. However, several auxin biosynthesis genes, including ALDEHYDE OXIDASE 1 (AAO1) and most genes encoding tryptophan biosynthesis enzymes that were present on the array, were not changed in the NILs. Gibberellins (GA) also play important roles in cell division and elongation [75, 76]. Similarly, none of the GA biosynthesis genes on the array were differentially expressed. We also performed Northern blots on GA biosynthesis genes that were not on the array and found that none were differentially expressed in the NILs either (data not shown). This implied that SUN is not directly involved in regulating auxin and GA levels.

Plant materials

Seeds of S. pimpinellifolium accession LA1589 were obtained from the C.M. Rick Tomato Genetics Resource Center, Davis, California, USA. Nearly Isogenic Lines (NILs) that differ at sun locus were resulted from the high-resolution recombinant screens conducted to fine map the locus [84]. After multiple backcrosses and molecular marker analysis, we estimated that the introgression of the Sun1642 allele in the LA1589 background is less then 100 kb with very few, if any, other regions of the genome harboring the Sun1642 allele. Plants were grown under standard conditions with supplemental lighting in the greenhouse.

Timing of flower opening on individual inflorescences

Eighty three inflorescences from four independent experiments were tagged before flower opening. Anthesis was recorded each day at the same time, and two flowers that opened on the same day were recorded as 0 days between flowerings.

Seed viability determination

Seeds were extracted from the fruit harvested on tagged inflorescences that were hand pollinated to ensure uniform fruit set. The dates of pollination were recorded and the fruits were harvested based on days after anthesis. Seeds were extracted and incubated for 20 min in 25% HCl to remove the gelatinous layer surrounding the seed, rinsed with distilled water and germinated for one week in the dark at 30°C on moist Whatman paper.

Ethylene sensitivity of developing fruit

Tagged flowers were hand pollinated and the dates were recorded. Fifteen to 20 fruit from mature green to breaker (26–33 dpa) were treated for 16 hours in a sealed chamber with 10 μl/L ethylene and the color changes were monitored two days later. Color for each fruit was recorded into different categories (green, color turning, orange, yellow and red) before and after ethylene treatment, and ethylene sensitivity was expressed by fruits with changed colors in total fruits assayed.

Timing of fertilization

Flowers were emasculated one day prior to anthesis and hand-pollinated the next day. Pistils were collected at 6, 8, 10, 12 and 24 hours after pollination. Dissected pistils were fixed in 3:1 95% ethanol:glacial acetic acid overnight at room temperature. Samples were subsequently softened for 24 hours in 10 N NaOH, rinsed five times in ddH₂O and stained using 0.1% aniline blue (aniline blue fluorochrome, Biosupplies Australia) in 0.1 M potassium phosphate buffer pH8.0 for 4 hours in the dark. Samples were mounted in 30% glycerol and viewed on a Leica DM IRB epifluorescence microscope using the UV filter set (Chroma filter A, BP340-380, LP425).

Fruit shape changes during development

Data were collected from five individual NIL plants per genotype homozygous for sun. For ovary and developing fruits from anthesis to 34 dpa, developing fruit were cut in half longitudinally and images were obtained using camera connected to dissection microscope (0–7 dpa) or using scanner (fruit older than 7 dpa). Shape index (length divided by width) were obtained with ImageJ http://rsb.info.nih.gov/ij/ on images taken. Each time point has at least three ovaries or fruits from each individual plant.

Scanning electron microscopy of floral development

Flowers were processed in its entirety or partially dissected under the dissecting microscope prior to fixation. Samples were infiltrated and fixed with 3% gluteraldehyde, 2% paraformaldehyde in 0.1 M potassium phosphate buffer pH7.4 for two hours at room temperature and then over night at 4°C. After 3 washes with ddH₂O samples were post fixed with 1% osmium tetroxide, washed 3 times with ddH₂O and dehydrated following a graded ethanol series (once for 25%, 50%, 70%, 90%, twice 100%). Critically point dried (Samdri-790, Tousimis Research Corporation) samples were mounted on aluminum stubs, and sputter-coated with platinum (Polaron). When necessary, flower buds were further dissected after platinum coating. Samples were viewed and images recorded with a Hitachi 3500N scanning electron microscope under high vacuum.

Light microscopy

Flower and fruit samples were infiltrated and fixed in 3% glutaraldehyde, 4% paraformaldehyde, 0.05% Triton X-100 in 0.1 M potassium phosphate buffer at pH 7.2 for two hours at room temperature and then over night at 4°C. After three washes with potassium phosphate buffer, samples were processed for embedding into London Resin White (LRW) (EMS) or paraffin (PolyFin, Polyscience).

For LRW embedding, samples were dehydrated in a graded ethanol series (25%, 50%, 70%, twice 90%), infiltrated with a graded resin and 90% ethanol series (1:3, 1:1, 3:1, twice 100% resin), embedded in airtight gelatine capsules (EMS) and polymerized overnight at 60°C. Five μm thick sections were collected on glass slides and stained with 0.1% sodium bicarbonate, 0.5% toluidine blue, in 25% EtOH before light microscopy observation.

For paraffin embedding, samples were dehydrated in a graded ethanol series (50%, 80%, 90% twice 100%), and subsequently infiltrated, first in a graded ethanol/tertiary butyl alcohol (TBA) series at room temperature (2:1, 1:1, 1:2, twice 100% TBA), and then in a graded TBA/paraffin series (1:3, 1:1, 3:1, twice 100% paraffin) at 56°C and embedded in paraffin. 6–10 μm sections were collected on silane treated glass slides (Polyscience). Deparaffinized sections were stained 10 minutes with 10 mg/ml safranin O in 50% ethanol, and 10 seconds with 5 mg/ml astra blue containing 20 mg/ml tartaric acid following Jensen procedure [85]. Sections were observed on the Leica DM IRB light microscope (Leica Microsystems, Wetzlar Germany) and images were captured using the MagnaFire model S99802 digital camera (Optronics, Goleta, CA).

For fluorescent microscopy, sections were deparaffinized, blocked with 10 mM potassium phosphate buffer (pH7.4), 150 mM NaCl (PBS) containing 10 mM NaAzide, 0.2%BSA, 1% normal goat serum for 30 minutes. Tubulin was detected using a 1/500 dilution of the mouse anti-tubulin monoclonal IgG1 (Molecular Probes) as primary antibody, and AlexaFluor488 sheep anti-mouse secondary antibody (Invitrogen, USA). Antibody incubations were performed in incubation buffer (PBS containing 10 mM NaAzide, 0.2%BSA) at room temperature for 4 hours for the primary antibody, and 2 hours for the secondary antibody. After each incubation, the sections were washed five times with PBS. Cell nuclei were counterstained for 8 minutes with 0.25 mM SytoxOrange (Invitrogen, USA). Sections were then mounted with GelMount (Biomedia) and observed on a Leica TCS-NT confocal microscope.

Additional developing embryos were visualized using differential interference contrast microscopy. Samples were fixed in 10:3:1 ethanol, glacial acetic acid, chloroform mixture. Tissue was rinsed in 90% ethanol twice, and then cleared in modified Hoyer's solution consisting of 60 ml of distilled water, 7.5 g arabic gum, 100 g chloral hydrate, 5 ml of glycerin. Samples were mounted in 70% glycerol, smashed using the cover slip and viewed with a Nomarski objective or phase contrast using the Leica DM IRB light microscope.

Pericarp cell number and size measurements

Fruits were harvested at 0, 2, 5, and 10 dpa. Prior to fixation, fruit of 5 and 10 dpa were cut longitudinally and perpendicular to the septum, while fruit of 0 and 2 dpa were fixed as a whole. The fixed tissues were embedded into London Resin White as described above. Sections were collected from 6 and 20 samples per time point. Pericarp consists of epicarp (the single outermost cell layer), endocarp (the single innermost cell layer) and mesocarp comprising of cells in-between epicarp and endocarp. Cell lengths of epicarp and endocarp were determined by averaged lengths of 5–10 cells along. The length of the mesocarp was measured in the middle region of the mesocarp sampling 5–10 cells. Cell volume was calculated based on formula V = L*W*((L+W)/2), where V represents cell volume, L = cell length, W = cell width.

Microarray analysis

The tomato microarray was custom-designed oligoarray manufactured by Nimblegen (Nimblegen Inc. USA) based on TIGR tomato Tentative Contigs sequences (release 9, http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=tomato). It consists of 15270 TCs corresponding to 7600 different clusters (transcripts) and each TC was represented by 12 pairs of perfect and mismatch probes of 24-mers.

Total RNA for microarray analysis were extracted from 10-day preanthesis flower bud and anthesis flower and fruits at 5 dpa using Trizol reagent (Invitrogen Inc. USA). Before RNA extraction, tissues harvested at 7-day interval from five plants were pooled for each genotype. Three biological replicates were conducted with three sets of LA1589 sun NILs growing during different time periods resulting in 3 time points × 2 genotypes × 3 replicates = 18 array hybridizations. Microarray hybridizations, image scanning and data extracting were performed by Nimblegen Inc. Background correction and data normalization were performed by Robust Microarray Analysis (RMA, [86]) in Bioconductor. Differentially expressed genes (DEs) among the three stages were selected by multiple testing package multtest [40] of R http://www.r-project.org using the RMA expression values. To update the gene description and annotation, sequences of the differentially expressed genes were BLASTed against Arabidopsis protein database (version 7 released on July 24, 2007 by TAIR, http://www.arabidopsis.org.) using blastx. Description of proteins encoded by some differentially expressed genes with low homology (p < 1e-10) to Arabidopsis proteins was assigned with the annotation of the newest TC (release version 11 by TIGR) or those with best hit in NCBI database http://www.ncbi.nlm.nih.gov. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus [87] and are accessible through GEO Series accession number GSE15453 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE15453.

Northern blot

RNA was isolated from the whole fruit or flower using Trizol reagent (Invitrogen Inc. USA) (for ovary and fruits of 20 dpa or younger), or the hot borate method (for fruits of 25, 30, and 34 dpa old) [88]. For Northern blot, 10 μg of the total RNA of each sample was separated in 1.2% Agarose gel in 1XMOPS buffer with formaldehyde, transferred onto Hybond N membrane (Amersham Biosciences) and hybridized at 42°C in formamide-containing hybridization buffer with radiolabeled gene-specific probes sequentially after previous probes were stripped.