Integration of tomato reproductive developmental landmarks and expression profiles, and the effect of SUN on fruit shape
- Han Xiao†1,
- Cheryll Radovich†1,
- Nicholas Welty†1,
- Jason Hsu3,
- Dongmei Li3,
- Tea Meulia2 and
- Esther van der Knaap1Email author
© Xiao et al; licensee BioMed Central Ltd. 2009
Received: 30 December 2008
Accepted: 07 May 2009
Published: 07 May 2009
Universally accepted landmark stages are necessary to highlight key events in plant reproductive development and to facilitate comparisons among species. Domestication and selection of tomato resulted in many varieties that differ in fruit shape and size. This diversity is useful to unravel underlying molecular and developmental mechanisms that control organ morphology and patterning. The tomato fruit shape gene SUN controls fruit elongation. The most dramatic effect of SUN on fruit shape occurs after pollination and fertilization although a detailed investigation into the timing of the fruit shape change as well as gene expression profiles during critical developmental stages has not been conducted.
We provide a description of floral and fruit development in a red-fruited closely related wild relative of tomato, Solanum pimpinellifolium accession LA1589. We use established and propose new floral and fruit landmarks to present a framework for tomato developmental studies. In addition, gene expression profiles of three key stages in floral and fruit development are presented, namely floral buds 10 days before anthesis (floral landmark 7), anthesis-stage flowers (floral landmark 10 and fruit landmark 1), and 5 days post anthesis fruit (fruit landmark 3). To demonstrate the utility of the landmarks, we characterize the tomato shape gene SUN in fruit development. SUN controls fruit shape predominantly after fertilization and its effect reaches a maximum at 8 days post-anthesis coinciding with fruit landmark 4 representing the globular embryo stage of seed development. The expression profiles of the NILs that differ at sun show that only 34 genes were differentially expressed and most of them at a less than 2-fold difference.
The landmarks for flower and fruit development in tomato were outlined and integrated with the effect of SUN on fruit shape. Although we did not identify many genes differentially expressed in the NILs that differ at the sun locus, higher or lower transcript levels for many genes involved in phytohormone biosynthesis or signaling as well as organ identity and patterning of tomato fruit were found between developmental time points.
Plants display a diverse array of shapes, sizes and categories of fruit. Within the Solanaceae family fruit categories range from capsules, drupes, pyrenes, berries, to several other types of non-capsular dehiscent fruits . Within one species such as tomato (Solanum lycopersicum L.), fruit morphology varies dramatically among cultivated accessions. The dramatic diversity in tomato fruit shape and size is due to domestication and continued selection for its fruit characters [2, 3]. Fruit formation starts with the development of the floral meristem. Within the floral meristem, the expression of organ identity genes gives rise to the four whorls namely the sepals, petals, stamen and gynoecium. The coordinate spatial and temporal expression of several classes of homeotic genes specifies the identity of floral organs [4–7]. A class genes control sepal identity, A and B class genes specify the identity of petals, B and C genes define stamen identity, and C genes control carpel identity. The E class genes act redundantly in specifying the identity of floral whorls in combinations with the A, B and C genes [5–7].
After organ specification within the floral meristem, a complex growth patterning is observed in the fourth floral whorl comprising the gynoecium, which will become the fruit after fertilization of the ovules. Along the apical-basal axis, the developing tissue types of the gynoecium are the stigma, style, ovary and gynophore, whereas along the mediolateral axis of the ovary the valves or pericarp, septum or columella, placenta and ovules are formed. In fruit such as that of Arabidopsis, the gynoecium also includes two dehiscence-related tissues, replum and valve margin [8, 9]. Combined with the organ and tissue identity genes, patterning is controlled by the expression of genes determining organ polarity . A critical stage of fruit patterning occurs at fertilization which, when successful, results in seed formation. Fruit of most species will abort if there is none or limited fertilization and seed set. Phytohormones, particularly auxin and gibberellins (GA), play critical roles in fruit set and early growth triggered by pollination and fertilization. Auxin and GA can also induce parthenocarpic fruits by triggering pollination-independent fruit growth in several species including tomato [11–15].
Descriptions of flower and fruit developmental stages have been established for several species. The stages have been used to interpret gene function, and to determine the spatial and temporal expression of genes involved in organ identity and patterning. In addition, detailed descriptions of developmental stages are needed for comparative analyses to unravel genetic and molecular mechanisms that give rise to floral and fruit diversity. Ideally, these stages should describe key developmental events that are shared among flowering plant species, so that the landmarks could be compared and queried across databases using key morphological developmental features. Buzgo et al (2004) compared three distant angiosperm species and proposed ten floral landmark stages. These landmarks comprise "inflorescence formation and flower initiation", "sepal initiation", "petal initiation", "stamen initiation", "carpel initiation", "microsporangia formation", "ovule initiation", "male meiosis", "female meiosis", and "anthesis" , which have been adopted in studies of several other species [17, 18]. However, key fruit landmark stages that are applicable across species have not been described to date. For example, whereas Arabidopsis fruit development is described in eight stages, tomato fruit development is described in four [19, 20]. Phase I of tomato fruit development comprises ovary development ending with fertilization. Phase II describes early fruit growth following fertilization and spans cell division and early embryo development. Phase III spans cell expansion and embryo maturation. The final phase IV is the ripening phase . Both cell division and elongation occur concomitantly in the different parts of the tomato fruit, thus these two phases are not well separated during growth of the organ [21, 22]. More importantly, the stages described for Arabidopsis and tomato detail species-specific events that are not applicable across species. Therefore, the establishment of universally applied fruit developmental landmarks would allow comparative analysis of data obtained from different species.
Tomato, classified as a berry fruit, represents an excellent model for floral and fruit development and is used extensively in comparative studies within the Solanaceae family [2, 3, 19, 23]. Whereas some information is known about the regulation of organ identity and specification [24–29], information about fruit patterning in Solanaceous species is rather limited. Varieties that differ in fruit morphology offer an important resource to further our understanding on its patterning. Fruit size and shape of tomato are controlled by major and minor QTL loci [2, 3, 30]. For some of these major QTL, the underlying genes are known. SUN and OVATE control fruit elongation and therefore affect patterning along the apical-basal axis [31, 32]. FW2.2 and FAS control fruit mass via increases of the placenta area and locule number, respectively, and thus affect patterning along the medio-lateral axis [33, 34]. SUN encodes a member of the IQD protein family . The founding member of the IQD protein family AtIQD1 is localized in the nucleus and its overexpression leads to increases in glucosinolate production in Arabidopsis . The high expression of SUN in tomato leads to elongated fruit, whichis hypothesized to control increases in secondary metabolites and/or hormone levels. In the near-isogenic lines (NILs) that differ at SUN, the most significant fruit shape changes occur after anthesis during fruit set . However a detailed developmental time-course describing fruit shape changes that would aid in understanding the mechanism by which SUN acts has not been described. Moreover, an evaluation of flower and fruit expression profiles in the S. pimpinellifolium LA1589 background has not been performed to date.
In this study, we adopt the floral landmarks established previously , and also propose new landmarks of fruit development that are applicable across angiosperm plant species. These landmarks are superimposed onto the fruit shape changes controlled by SUN and combined with gene expression profiles of floral buds 10 days prior to anthesis, anthesis-stage flowers and fruit 5 days post pollination.
Initiation of floral organ primordia
Flower developmental landmarks.
Flower Development Landmarks; Buzgo et al. (2004)
Days after flower initiation in tomato
Ovary and ovule development
Stamen and pollen development
(1) Inflorescence formation and flower initiation
Flattened inflorescence apex becomes dome-shaped.
(2) Initiation of outermost perianth organs
Emergence of sepal primordia in a helical pattern.
(3) Initiation of inner perianth organs.
Simultaneous emergence of petal primordia in alternating positions to the sepals. Sepals overlay the floral meristem
(4) Stamen initiation
Sepals and petals elongate.
Simultaneous initiation of stamen primordia.
(5) Carpel initiation
Petals start curling over the stamens
Carpel primordia arise.
Central column that will form the locular cavities arise.
Stamen filament start developing and two anther lobes become visible.
(6) Microsporangia initiation
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
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
Microsporogenesis. Microsporocytes or microspore mother cells undergo meiosis I and II and forming tetrads.
(9) Female meiosis
Megasporogenesis. Megaspore mother cell (meiocyte or megasporocyte) is visible. Meiosis I. The nucellus is small resulting in a tenui-nucellate ovule.
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.
Petals emerge from the sepals.
Free microspores are being incased in a thick polysaccharide wall; tapetum degenerated.
Onset of sepal opening
Megagametogenesis and development of the embryo sac.
Microspores come vacuolated, and begins asymmetric mitosis
Bi-cellular pollen grain.
Ovule development nears completion.
The vegetative cell and generative cell are well distinguishable
Reproductive organ formation
Fertilization and fruit set
Fruit developmental landmarks.
Fruit Development Landmarks
Days post anthesis
Descriptions of fruit development in tomato
Fruit growth (Gillaspy et al1993)
Mature ovary, phase I.
Mature gametes. Pollen is shed, which will land on the stigma and germinate. Pollen tubes growth through the style.
End of phase I, beginning of phase II.
Fusion of sperm and egg nuclei.
(3) 4–16 Cell Stage Embryo
Phase II and III, cell division and elongation stage.
First embryo divisions.
(4) Globular Stage Embryo
Phase III, cell expansion stage.
(5) Heart Stage Embryo
Phase III, cell expansion stage.
Heart Stage embryo lasts approximately one day and occurs 10–12 dpa.
(6) Torpedo Stage Embryo
Phase III, continued fruit enlargement.
Torpedo Stage embryo lasts approximately one day and occurs 13–16 dpa.
(7) Coiled Stage Embryo
Phase III, continued fruit enlargement.
Cotyledon expansion and curl as they elongate. Embryo appears physically mature, but the seed is not yet viable.
Seed maturation period
(8) Seed germination
The fruit has reached the mature green stage. Fruit becomes sensitive to ethylene.
Seeds are becoming viable for germination.
(9) Fruit ripening
Ripening starts at the onset of the breaker stage. Changes in pigmentation are visible.
After ripening of seed.
(10) Ripe Fruit
Red ripe stage of tomato.
Development of the pericarp after pollination
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.
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
Functional classification of differentially expressed genes during flower and early fruit development
10 days preanthesis
5 DPA fruit
Cell cycle and Cell wall
Electron transport or energy pathway
Metabolism and other cellular processes
Regulation of transcription
Response to stimuli
Expression of organ identity and patterning genes
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) , LEUNIG (LUG) , TOUSLED (TSL) , STYLISH (STY1 and STY2) , SPATULA (SPT) , NO TRANSMITTING TRACT (NTT) , and HECATE (HEC1, HEC2 and HEC3) , 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) , ALCATRAZ (ALC) , INDEHISCENCE (IND) , REPLUMLESS (RPL) , and FRUITFULL (FUL) . The Arabidopsis gene SEEDSTICK (STK) is required for ovule identity and patterning as well as seed disposal , and ERECTA (ER) regulates fruit shape by controlling cell expansion and cell division . JAGGED (JAG) acts redundantly with the polarity genes FILAMOUS FLOWER (FIL) and YABBY3 (YAB3) to activate FUL and SHP . Additional polarity genes required for proper patterning and establishment of organ boundaries are CRABS CLAW (CRC) , KANADI (KAN1 and KAN2) , GYMNOS (GYM) , PHAVOLUTA (PHV) and PHABULOSA (PHB) . 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 , 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
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 , was more abundant in 5 dpa fruit compared to other stages. In contrast, the expression of SlGA2ox2 (TC127124), involved in catabolism of GA , 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) , 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 , 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
Gene expression profiles associated with SUN
Differentially expressed genes in LA1589 sun NILs
Pyridoxal 5'-phosphate-dependant histidine decarboxylase
Harpin-induced protein 1 (Hin1) (AT5G11890).
Putative GPI protein (At5g53870)
Weakly similar to potato resistance gene cluster AF265664.
Auxin-responsive family protein (AT3G25290)
Plant thionin family protein (AT1G12663)
Plastocyanin-like domain-containing protein (AT5G53870)
DNAJ-LIKE 20 (At4g13830)
Universal stress protein (USP) family protein (At3g62550)
Thiamine biosynthesis family protein/thiC family protein (AT2G29630)
Unknown protein (AT4G32480)
alternative oxidase 2 (AT5G64210)
60S ribosomal protein L6 (RPL6A) (AT1G18540)
MEE59 (maternal effect embryo arrest 59) (AT4g37300)
ALPHA-CRYSTALLIN DOMAIN 31.2 (At1g06460 mRNA)
Single-stranded DNA binding protein precursor (AT2G37220)
HEPTAHELICAL TRANSMEMBRANE PROTEIN1 (AT5g20270)
Phi-1. Arabidopsis thaliana phosphate-responsive protein (EXO)
Pectin methylesterase inhibitor isoform (AT5G62360)
Auxin/aluminum-responsive protein (AT4G27450)
Sulfate transporter (AT3G51895)
proteinase inhibitor isoform
Gty37 protein; putative cell wall protein (AT2G20870)
2OG-Fe(II) oxygenase family (AT2G36690)
THI1 protein (AT5G54770)
Late embryogenesis abundant protein
MEE59 (maternal effect embryo arrest 59) (AT4g37300)
SUN has been hypothesized to affect fruit shape by altering hormone levels such as auxin . 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.
The formation of the flower and fruit can be described by a series of landmarks that coincide with key development events. Floral landmarks described by Buzgo et al. (2004) and fruit landmarks proposed herein provide the framework for comparative analyses of floral and fruit development among angiosperm species. Moreover, understanding the common mechanisms of reproductive development also provides the basis from which to dissect the differences observed among species and the evolution of fruit form .
For tomato, S. pimpinellifolium accession LA1589 is an excellent model for flower and fruit development because of its predictable growth pattern, large numbers of flowers per inflorescence and inflorescences per plant. Previous studies in cherry tomato (S. lycopersicum var. cerasiforme) described flower development in 20 stages from sepal initiation to anthesis and established the correlation between major cellular events in reproductive organs with perianth markers . The main floral developmental events we described for LA1589 are in agreement with those observations in cherry tomato, although we started floral development with inflorescence formation and floral initiation rather than sepal initiation. Inflorescence formation and floral initiation is a major event in floral development, and the critical transformation from vegetative meristem to floral meristem is tightly regulated by floral meristem identity genes, such as LEAFY and APETALA1 [79, 80]. Therefore, floral landmark 1 will be of great interest in dissecting functions and expression patterns of floral meristem identity genes in tomato as well as genes that play a role in fruit size and shape. Previous fruit development of cultivated tomato has been divided into phases based on cell division activities . We observed a very short duration of cell division in the pericarp of LA1589 fruit (less than 5 dpa), in contrast to ~7 to 10 dpa in cultivated tomato . Embryogenesis and seed formation in many flowering plants occur concomitantly with fruit development, therefore we described the ontogeny of the fruit following key events in embryogenesis and seed formation. Thus, herein we provide a complete set of consensus landmarks for flower and fruit stages starting from floral initiation until fruit ripening. These landmarks highlight major events in reproductive development and serve as a guide in floral and fruit developmental research. The use of common terminology will make data and information from different species queryable, while also facilitates comparative analysis across species.
Recently, a genome-wide analysis of the transcriptional changes induced by pollination and GA application of ovaries was performed . A comparison between ours and the previously published study showed that some phytohormone related genes were shared in the two studies. Four auxin-related genes, encoding GH3.3 (TC118161), auxin responsive family protein (TC130798), amino acid permease (TC122973) and auxin efflux carrier family protein (TC120936), shared the same expression patterns between the two experiments. However, none of the GA-related genes were shared in the two studies. Abscisic acid (ABA) and ethylene may also play roles in fruit set and fruit growth post pollination as genes involved in biosynthesis and signaling of these phytohormones were differentially expressed after pollination . Similar to the Vriezen et al study (2008), several ACC synthase genes were differentially expressed and all the ethylene biosynthesis genes were less abundant in 5 dpa fruits, suggesting reduced levels of this hormone after pollination. The expression of ABA biosynthesis genes, such as neoxantin synthase (NSY) and 9-cis-epoxycarotenoid dioxygenase (LeNCED), is reduced in fruits post pollination . Similarly, in our study zeaxanthin epoxidase (ZEP/ABA1) was less abundant in 5 dpa fruit compared to flower. In both studies, an ABA 8'-hydroxylase gene (cytochrome P450 family member) involved in ABA catabolism , was more abundant in fruits post pollination. This suggests that ABA, like ethylene, is in low demand during fruit set and early growth. Recently, Galpaz et al (2008) determined that tomato high-pigment 3 (hp3) mutant with a mutation in the ZEP gene produces a higher level of fruit lycopene linked to increased plastid number as a result of ABA deficiency . Because the hp3 mutant makes smaller fruit , certain amounts of ABA may be required for fruit growth after anthesis.
Transcriptional profiles of other classes of genes were also similar between the previously published study  and ours. More than half (13 of 22) of cell cycle-related genes and half (13) of the cell wall-related genes were shared between the two studies (see Additional file 5) . Two cyclin genes TC120949 and TC128804, showing highest similarities to Arabidopsis CYCLIN D3;1 (CYCD3;1) and CYCLIN B1;4 (CYCB1;4), were induced by pollination, but not by GA treatment based on previous observations . However, their higher expression before and after anthesis in our experiments suggests that the two genes are not only inducible by pollination but also involved in pre-anthesis activation of cell division possibly in response to other hormone cues such as cytokinin. In Arabidopsis, CYCD3;1 responds to cytokinin to activate cell division at the G1-S cell cycle phase .
After establishing the morphological landmarks for flower and fruit development in tomato, we superimposed the effect of SUN on fruit formation. SUN controls fruit shape after anthesis . From the landmark fertilization to the landmark globular embryo stage, the fruit shape index dramatically increased in the accession that expresses SUN to a high level (Fig. 11). The coincidence between the dynamics of fruit shape index mediated by SUN and fruit growth suggests that SUN mainly acts in fast growing tissues, which is further supported by high expression of SUN in the oval shaped fruits during early fruit growth. Although we hypothesized that SUN may indirectly affect hormone or secondary metabolite levels and as such altering organ shape , the identified differentially expressed genes did not support that notion. Moreover, the very low number of differentially expressed genes was surprising considering that the expression of SUN was quite high in the lines carrying oval-shaped fruit at the time points sampled.
Following the universal landmarks proposed by Buzgo et al (2004), we outlined flower and fruit developmental landmarks in tomato. Transcriptional profiles of flower and developing fruit at three main stages have been integrated with their corresponding landmarks, which will be useful for identifying important regulatory components responsible for key developmental processes. We identified genes encoding patterning, phytohormone and cell cycle-related proteins modulated during flower and early fruit development, which will provide basis for further studies on tomato fruit growth. The usefulness of the landmarks was demonstrated by examining the fruit shape changes mediated by SUN.
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 . 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.
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 . 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.
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, ) in Bioconductor. Differentially expressed genes (DEs) among the three stages were selected by multiple testing package multtest  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  and are accessible through GEO Series accession number GSE15453 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE15453.
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) . 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.
The authors thank J. Moyseenko and L. Duncan for plant care and the Molecular and Cellular Imaging Center staff for technical help. We thank Drs. S. Hogenhout and S. Kamoun for collaborations on the Nimblegen microarray experiments. We also thank Dr. M. Buzgo for advice, and Drs JC Jang and M. Jones for critical reading of this manuscript. This work was funded by National Science Foundation grants DBI 0227541 to EVDK.
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