BACKGROUND
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  <cit> .
within one species such as tomato , 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  <cit> .
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  <cit> .
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  <cit> .
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  <cit> .
combined with the organ and tissue identity genes, patterning is controlled by the expression of genes determining organ polarity  <cit> .
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 , 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  <cit> .
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  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"  <cit> , which have been adopted in studies of several other species  <cit> .
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  <cit> .
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  <cit> .
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  <cit> .
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  <cit> .
whereas some information is known about the regulation of organ identity and specification  <cit> , 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  <cit> .
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  <cit> .
fw <dig>  and fas control fruit mass via increases of the placenta area and locule number, respectively, and thus affect patterning along the medio-lateral axis  <cit> .
sun encodes a member of the iqd protein family  <cit> .
the founding member of the iqd protein family atiqd <dig> is localized in the nucleus and its overexpression leads to increases in glucosinolate production in arabidopsis  <cit> .
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  that differ at sun, the most significant fruit shape changes occur after anthesis during fruit set  <cit> .
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 la <dig> background has not been performed to date.
in this study, we adopt the floral landmarks established previously  <cit> , 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  <dig> days prior to anthesis, anthesis-stage flowers and fruit  <dig> days post pollination.
RESULTS
we used s. pimpinellifolium accession la <dig> for the tomato flower and fruit developmental studies due to its indeterminate growth habit and the abundant number of flowers and inflorescences throughout its life cycle.
for example, la <dig> carries on average  <dig> flowers per inflorescence , whereas a typical cultivated variety carries only  <dig> to  <dig> flowers per inflorescence  <cit> .
in addition, flower development is highly regular in the wild relative la <dig> compared to most cultivated types  <cit> .
to time the developmental stages of consecutive buds and then fruits on an inflorescence, we recorded the time of anthesis for each flower in a total of  <dig> inflorescences investigated over four independent experiments.
as shown in figure 1c, the second flower opened 70% of the time one day after the first flower, 29% of the time on the same day as the first flower, and 1% of the time two days after the first flower and so on.
in general, consecutive flower opening occurred at one-day intervals 75% of the time, until the 16th flower on a given inflorescence .
flower buds developed after the 16th on a given inflorescence tended to open more irregularly and often at an interval of 2-days or more.
by inference, this result implied that the first  <dig> floral meristems arose 75% of the time in one-day interval from one another.
therefore, we concluded that until the 16th flower on a given inflorescence, the developing flower and fruit respectively, are staged at close to one-day intervals from one another.
initiation of floral organ primordia
the first landmark represented inflorescence formation and flower initiation .
the transition to flowering and inflorescence formation in la <dig> has been described previously  <cit> .
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  <dig> .
the flatter inflorescence meristem continued its indeterminate growth pattern, while the more domed meristem developed into a flower .
following flower initiation, the emergence of the sepal primordia around the perimeter of the floral apex of bud number  <dig> marked the second landmark .
the five tomato sepals initiated in a helical pattern of 144° .
the sepals continued to grow and covered the floral meristem approximately  <dig> days after floral initiation .
at the time of sepal enclosure, petal primordia started to arise, representing landmark  <dig>  following petal primordia emergence, stamen primordia emerged in alternate positions to the petals , at approximately  <dig> days after floral initiation, representing landmark  <dig>  sepals and petals continued to elongate while carpel primordia began to emerge in the floral center , marking landmark  <dig>  which occurred approximately  <dig> 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 .
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  <dig> days post bud initiation .
the timing of the landmarks described by buzgo et al  in s. pimpinellifolium accession la <dig> floral development.
reproductive organ formation
male reproductive development initiated with microsporangia development, which represented landmark  <dig>  and occurred approximately eight days after floral bud initiation .
the primary sporogenous layers were visible at this stage .
nine days after floral bud formation, the tapetal cells were binucleate, and the developing microsporocytes were also visible .
at  <dig> days after floral bud initiation, microsporocytes or pollen mother cells were undergoing meiosis , marking landmark  <dig>  a callose wall surrounded the four haploid nuclei of the tetrads .
one day later, the callose walls began to degrade and the microspores were being released .
at  <dig> days after floral bud initiation, the tapetum was degenerating; and the microspores were single and encapsulated in a thick wall .
one day later, the microspores became vacuolated  and underwent one asymmetric mitosis.
fifteen days after floral bud initiation, the microspores were bi-cellular  and a day later, the generative and vegetative cells were clearly distinguishable within the developing pollen .
at day  <dig> after floral bud initiation, the generative cell displayed the characteristic crescent shaped nucleus .
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  <dig> .
approximately  <dig> days after floral bud initiation, the style and the ovary were nearly equal in length, and ovule primordia were emerging on the placental tissues .
ovules were clearly visible one day later .
two days after ovule primordia initiation and  <dig> 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  <dig> .
apparently the megasporocyte underwent the first meiotic division at this stage .
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 .
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 .
fertilization and fruit set
anthesis or flower opening was the final floral landmark as well as the first fruit landmark .
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  <dig> hours after pollination, and reached the ovules approximately  <dig> hours later .
ten to  <dig> hours after pollination, the pollen tubes had released their content resulting in fertilization of the ovules  and representing fruit development landmark  <dig> .
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.
timing of the fruit landmarks in s. pimpinellifolium la <dig>
development of the pericarp after pollination
following fertilization, tomato fruit growth consists of cell division and cell expansion  <cit> .
we analyzed the growth of the pericarp following pollination to establish the timing of cell division and cell elongation in the developing la <dig> pericarp.
pericarp width doubled from anthesis to  <dig> days post anthesis , and then further doubled at  <dig> and  <dig> dpa, respectively .
cell number across the pericarp increased from  <dig> at anthesis to  <dig> at  <dig> dpa, and reached the final number of 19– <dig> at  <dig> dpa , implying that cell division ended at or before that time.
mesocarp cell expansion started as early as  <dig> dpa .
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  <dig> dpa fruit .
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  <dig> dpa in la <dig>  whereas the cell elongation stage spanned fruit development from  <dig> 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– <dig> celled embryo, which occurred approximately  <dig> dpa .
the fourth landmark was represented by the globular embryo stage at  <dig> to  <dig> dpa .
heart shape embryo was the fifth landmark and occurred between  <dig> and  <dig> dpa  highlighting the beginning of cotyledon growth.
the 13– <dig> dpa embryo was torpedo shape, marking the sixth landmark .
after the sixth landmark, the cotyledons grew into a coil and reached the seventh landmark approximately at  <dig> dpa.
at this stage, the embryo approached its final size, but the seed was not yet viable for germination .
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  <dig> dpa until  <dig> dpa.
up until  <dig> dpa, there was little or no seed germination .
however, at  <dig> dpa, the germination rate increased dramatically thus reaching landmark eight.
at  <dig> dpa, nearly 100% of the seeds germinated.
fruit ripening
tomato fruit ripening stages consist of mature green, breaker and red ripe  <cit> .
at the mature green stage, ethylene treatment will result in a rapid reddening of the fruit  <cit> .
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 .
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 .
forty percent of fruit had responded to ethylene at  <dig> dpa when 43% of the seeds were viable for germination.
fruit younger than  <dig> dpa did not respond to ethylene treatment .
the ninth landmark is the onset of fruit ripening, coinciding with the breaker stage when color began to change at approximately  <dig> dpa.
this stage is followed by the tenth and final landmark of ripe fruit.
gene expression profiles of floral and fruit development
to obtain a global overview of gene expression in flower and fruit, we compared the profiles between three critical developmental time points.
the first stage was young flower buds at floral landmark  <dig>  representing ovule initiation .
the second stage was the anthesis-stage, representing flower landmark  <dig> and fruit landmark  <dig>  the third and last stage was  <dig> dpa fruits, representing the 4– <dig> cell embryo stage and fruit landmark  <dig>  differentially expressed genes were identified using the resampling-based multiple testing method  <cit> .
without the cutoff of fold-change applied,  <dig> genes with adjusted p <  <dig>  were differentially expressed in at least one of the three stages .
among them,  <dig> genes showed higher expression at anthesis, whereas  <dig> and  <dig> genes showed higher expression in young flower buds and  <dig> dpa fruits, respectively .
functional classification of the differentially expressed genes showed a distinct distribution of genes involved in various biological processes for the three stages investigated.
for example, more genes involved in developmental processes were found in flower buds during ovule initiation and anthesis-stage flowers than in  <dig> dpa fruit.
on the other hand, phytohormone-related genes were predominantly found in anthesis-stage flowers and  <dig> dpa fruits compared to flower buds .
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 .
genes orthologous or homologous to the arabidopsis abce genes required for floral organ identity have been identified in tomato  <cit> .
on our array, the tomato floral organ identity genes differentially expressed at the three stages include b class genes tap <dig>   <cit> , tpi  and slmbp1/lepi-b   <cit> , c class gene tag <dig>   <cit> , and e class gene tm <dig>  <cit> .
the tomato ortholog tc <dig> of arabidopsis nap that is directly activated by b class gene apetala <dig> and pistillata in arabidopsis  <cit>  was also differentially expressed.
all the above-mentioned genes showed higher expression in floral buds and/or anthesis-stage flowers , in agreement with their previously identified expression patterns.
another tomato b class gene tm <dig>  was not differentially expressed, likely due to its more ubiquitous expression in floral organs  <cit> .
while there is no clear ortholog of arabidopsis a class genes in tomato  <cit> , the closest related ap <dig> gene, mads-mc   <cit> , 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  <cit> .
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   <cit> , leunig   <cit> , tousled   <cit> , stylish   <cit> , spatula   <cit> , no transmitting tract   <cit> , and hecate   <cit> , 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   <cit> , alcatraz   <cit> , indehiscence   <cit> , replumless   <cit> , and fruitfull   <cit> .
the arabidopsis gene seedstick  is required for ovule identity and patterning as well as seed disposal  <cit> , and erecta  regulates fruit shape by controlling cell expansion and cell division  <cit> .
jagged  acts redundantly with the polarity genes filamous flower  and yabby <dig>  to activate ful and shp  <cit> .
additional polarity genes required for proper patterning and establishment of organ boundaries are crabs claw   <cit> , kanadi   <cit> , gymnos   <cit> , phavoluta  and phabulosa   <cit> .
tomato genes homologous to arabidopsis patterning genes fil , ful , crc , er , phb , and spt  were more abundantly expressed in tomato flower buds compared to the other tissues.
the tomato shp homolog tc <dig> showed higher expression in anthesis-stage flowers and fruits at  <dig> dpa than in floral buds.
the stk homolog in tomato tagl <dig> , which is expressed in the inner integument of the ovules and the endothelium in developing seeds  <cit> , was expressed higher in fruits at  <dig> dpa compared to other time points , suggesting that it may also play a role in tomato fruit development.
tomato genes with high similarity to arabidopsis fruit patterning genes ett, gym, kan <dig>  lug, phv, rpl, hec <dig>  sty <dig> and tsl were not differentially expressed between the three stages, whereas no tomato homologs for jag, ntt, alc, ind, yab <dig>  sty <dig> were included on our array.
further, the hierarchical clustering of all the  <dig> differentially expressed developmental processes genes revealed that flower bud and  <dig> dpa fruit shared expression profiles of the same developmental genes, whereas anthesis-stage flower showed a distinctive profile , which is in agreement with results from other gene profiling studies in arabidopsis  <cit> .
expression of phytohormone-related genes
phytohormones play essential roles in many aspects of plant development.
among the three developmental time points,  <dig> phytohormone-related genes were differentially expressed .
of these genes,  <dig> were involved in auxin conjugation, transport or signaling.
most of the auxin-related genes  were either up- or down-regulated in  <dig> dpa fruit .
moreover, most of the genes with similarity to gh <dig> involved in iaa conjugation were repressed after pollination, whereas three auxin response factor genes tc <dig> , tc <dig> , and tc <dig> , were expressed at the lowest level in anthesis stage flowers.
further, transcripts of three auxin transporter genes, tc <dig>  tc <dig> and tc <dig>  homologous to aux <dig>  pin <dig> and an auxin efflux carrier family protein, respectively, were less abundant in  <dig> dpa fruit .
several genes involved in biosynthesis of tryptophan , a precursor of iaa, were not developmentally regulated in this study, neither was the ortholog of arabidopsis auxin receptor transport inhibitor response <dig>   <cit> .
the ortholog of aldehyde oxidase  <dig>  involved in auxin biosynthesis  <cit> , 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 tc <dig> of atkao <dig> that catalyzes the conversion of ent-kaurenoic acid to ga <dig> in gibberellin biosynthesis pathway  <cit> , was more abundant in  <dig> dpa fruit compared to other stages.
in contrast, the expression of slga2ox <dig> , involved in catabolism of ga  <cit> , was lower in the developing fruits than in flower buds at  <dig> days preanthesis and anthesis-stage flowers.
interestingly, transcripts of three tomato homologs tc <dig>  tc <dig> and tc <dig> of arabidopsis ga receptors ga insentive dwarf1b and c   <cit> , were less abundant in  <dig> dpa fruit.
this suggests that although ga levels may increase in  <dig> 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  or not included on the array .
most of the seven ga responsive genes were not differentially expressed following pollination with the exception of tomato gene tc <dig> encoding gasa/gast/snakin family protein that was upregulated after anthesis .
transcripts of all the eight brassinosteroid-related genes were more abundant in  <dig> dpa fruit, whereas the majority of jasmonate- and ethylene-related genes were less abundant in  <dig> dpa fruit .
expression of genes involved in aba biosynthesis and response like were also lower in  <dig> dpa fruits.
the putative ortholog of arabidopsis gene cyp707a <dig> , encoding the major aba 8'-hydroxylase involved in aba catabolism  <cit> , is expressed at higher level in  <dig> dpa fruit compared to the other stages, suggesting that the aba levels are reduced during the early fruit growth.
fruit shape changes in la <dig> 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  <cit> .
we analyzed the changes in fruit shape from anthesis onward in nils in la <dig> background differing at the sun locus because at anthesis the ovary shape is only marginally different .
the la1589pp has round fruit and carries the wild type allele, while la1589ee carries the sun <dig> allele of sun resulting in an elongated fruit .
the difference in fruit shape between the two nils became apparent immediately following fertilization and was most pronounced between  <dig> and  <dig> dpa coinciding with the globular embryo stage of fruit landmark  <dig>  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 >  <dig> at anthesis to <  <dig> at  <dig> dpa .
we examined sun expression in the developing fruits of the la <dig> nils starting from anthesis-stage ovaries until ripe fruit.
in la1589ee, sun was expressed at a high level until fruit landmark  <dig> coinciding with coiled embryo and seed maturation stage at  <dig> dpa .
a detailed investigation of its expression immediately before and after anthesis showed that sun transcript levels increased from  <dig> days prior to anthesis to  <dig> dpa and thus showed a similar kinetics to that of the changes in fruit shape .
gene expression profiles associated with sun
to further investigate the effect of sun on tomato fruit shape and to identify genes that may interact with sun in regulating morphology, we compared transcriptional profiles of three floral and fruit developmental stages in the nils in la <dig> background that differ at sun.
the stages selected represented the three important events in flower and early fruit development when sun exhibited the greatest differential gene expression , namely  <dig> days pre-anthesis, anthesis and  <dig> days post-anthesis fruit.
in total, we found  <dig> differentially expressed genes between the nil pairs  .
one of the genes, defl <dig> encoding a member of plant defensins, was differentially expressed at all three time points.
another gene encoding maternal effect embryo arrest  <dig>  was upregulated in oval shaped fruit at two time points.
twenty four genes were differentially expressed only in anthesis-stage flowers and eight genes were differentially expressed only in  <dig> dpa fruit.
the differences in the transcript levels of the  <dig> genes were less than two-fold with the exception of defl <dig>  the latter gene is located very close to sun on chromosome  <dig>  therefore, decreased defl <dig> expression in the nil carrying elongated fruit was likely due to the mutation at the locus and not a consequence of increased expression of sun .
the remaining differentially expressed genes did not fall into known developmental pathways.
note that sun and defl <dig>  which are differentially expressed in these nils  <cit>  were not on the array.
*sun and defl <dig> were not on the nimblegen array; genes with rma expression value smaller than  <dig> were considered to bee too low expressed and removed from the analysis.
sun has been hypothesized to affect fruit shape by altering hormone levels such as auxin  <cit> .
however, several auxin biosynthesis genes, including aldehyde oxidase  <dig>  and most genes encoding tryptophan biosynthesis enzymes that were present on the array, were not changed in the nils.
gibberellins  also play important roles in cell division and elongation  <cit> .
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 .
this implied that sun is not directly involved in regulating auxin and ga levels.
discussion
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.
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  <cit> .
for tomato, s. pimpinellifolium accession la <dig> 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  described flower development in  <dig> stages from sepal initiation to anthesis and established the correlation between major cellular events in reproductive organs with perianth markers  <cit> .
the main floral developmental events we described for la <dig> 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 apetala <dig>  <cit> .
therefore, floral landmark  <dig> 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  <cit> .
we observed a very short duration of cell division in the pericarp of la <dig> fruit , in contrast to ~ <dig> to  <dig> dpa in cultivated tomato  <cit> .
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  <cit> .
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 gh <dig>  , auxin responsive family protein , amino acid permease  and auxin efflux carrier family protein , shared the same expression patterns between the two experiments.
however, none of the ga-related genes were shared in the two studies.
abscisic acid  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  <cit> .
similar to the vriezen et al study , several acc synthase genes were differentially expressed and all the ethylene biosynthesis genes were less abundant in  <dig> dpa fruits, suggesting reduced levels of this hormone after pollination.
the expression of aba biosynthesis genes, such as neoxantin synthase  and 9-cis-epoxycarotenoid dioxygenase , is reduced in fruits post pollination  <cit> .
similarly, in our study zeaxanthin epoxidase  was less abundant in  <dig> dpa fruit compared to flower.
in both studies, an aba 8'-hydroxylase gene  involved in aba catabolism  <cit> , 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  determined that tomato high-pigment  <dig>  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  <cit> .
because the hp <dig> mutant makes smaller fruit  <cit> , 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  <cit>  and ours.
more than half  of cell cycle-related genes and half  of the cell wall-related genes were shared between the two studies   <cit> .
two cyclin genes tc <dig> and tc <dig>  showing highest similarities to arabidopsis cyclin d3; <dig>  and cyclin b1; <dig> , were induced by pollination, but not by ga treatment based on previous observations  <cit> .
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; <dig> responds to cytokinin to activate cell division at the g1-s cell cycle phase  <cit> .
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  <cit> .
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 .
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  <cit> , 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.
CONCLUSIONS
following the universal landmarks proposed by buzgo et al , 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.

 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, 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. 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. 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. 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. 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, 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. 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 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. 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. 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. 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. whereas some information is known about the regulation of organ identity and specification, 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. 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. fw and fas control fruit mass via increases of the placenta area and locule number, respectively, and thus affect patterning along the medio-lateral axis. sun encodes a member of the iqd protein family. the founding member of the iqd protein family atiqd 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 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 la 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 days prior to anthesis, anthesis-stage flowers and fruit days post pollination.  we used s. pimpinellifolium accession la for the tomato flower and fruit developmental studies due to its indeterminate growth habit and the abundant number of flowers and inflorescences throughout its life cycle. for example, la carries on average flowers per inflorescence, whereas a typical cultivated variety carries only to flowers per inflorescence. in addition, flower development is highly regular in the wild relative la compared to most cultivated types. to time the developmental stages of consecutive buds and then fruits on an inflorescence, we recorded the time of anthesis for each flower in a total of inflorescences investigated over four independent experiments. as shown in figure 1c, the second flower opened 70% of the time one day after the first flower, 29% of the time on the same day as the first flower, and 1% of the time two days after the first flower and so on. in general, consecutive flower opening occurred at one-day intervals 75% of the time, until the 16th flower on a given inflorescence. flower buds developed after the 16th on a given inflorescence tended to open more irregularly and often at an interval of 2-days or more. by inference, this result implied that the first floral meristems arose 75% of the time in one-day interval from one another. therefore, we concluded that until the 16th flower on a given inflorescence, the developing flower and fruit respectively, are staged at close to one-day intervals from one another. initiation of floral organ primordia the first landmark represented inflorescence formation and flower initiation. the transition to flowering and inflorescence formation in la has been described previously. 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. the flatter inflorescence meristem continued its indeterminate growth pattern, while the more domed meristem developed into a flower. following flower initiation, the emergence of the sepal primordia around the perimeter of the floral apex of bud number marked the second landmark. the five tomato sepals initiated in a helical pattern of 144°. the sepals continued to grow and covered the floral meristem approximately days after floral initiation. at the time of sepal enclosure, petal primordia started to arise, representing landmark following petal primordia emergence, stamen primordia emerged in alternate positions to the petals, at approximately days after floral initiation, representing landmark sepals and petals continued to elongate while carpel primordia began to emerge in the floral center, marking landmark which occurred approximately 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. 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 days post bud initiation. the timing of the landmarks described by buzgo et al in s. pimpinellifolium accession la floral development. reproductive organ formation male reproductive development initiated with microsporangia development, which represented landmark and occurred approximately eight days after floral bud initiation. the primary sporogenous layers were visible at this stage. nine days after floral bud formation, the tapetal cells were binucleate, and the developing microsporocytes were also visible. at days after floral bud initiation, microsporocytes or pollen mother cells were undergoing meiosis, marking landmark a callose wall surrounded the four haploid nuclei of the tetrads. one day later, the callose walls began to degrade and the microspores were being released. at days after floral bud initiation, the tapetum was degenerating; and the microspores were single and encapsulated in a thick wall. one day later, the microspores became vacuolated and underwent one asymmetric mitosis. fifteen days after floral bud initiation, the microspores were bi-cellular and a day later, the generative and vegetative cells were clearly distinguishable within the developing pollen. at day after floral bud initiation, the generative cell displayed the characteristic crescent shaped nucleus. 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. approximately days after floral bud initiation, the style and the ovary were nearly equal in length, and ovule primordia were emerging on the placental tissues. ovules were clearly visible one day later. two days after ovule primordia initiation and 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. apparently the megasporocyte underwent the first meiotic division at this stage. 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. 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. fertilization and fruit set anthesis or flower opening was the final floral landmark as well as the first fruit landmark. 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 hours after pollination, and reached the ovules approximately hours later. ten to hours after pollination, the pollen tubes had released their content resulting in fertilization of the ovules and representing fruit development landmark. 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. timing of the fruit landmarks in s. pimpinellifolium la development of the pericarp after pollination following fertilization, tomato fruit growth consists of cell division and cell expansion. we analyzed the growth of the pericarp following pollination to establish the timing of cell division and cell elongation in the developing la pericarp. pericarp width doubled from anthesis to days post anthesis, and then further doubled at and dpa, respectively. cell number across the pericarp increased from at anthesis to at dpa, and reached the final number of 19– at dpa, implying that cell division ended at or before that time. mesocarp cell expansion started as early as dpa. 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 dpa fruit. 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 dpa in la whereas the cell elongation stage spanned fruit development from 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– celled embryo, which occurred approximately dpa. the fourth landmark was represented by the globular embryo stage at to dpa. heart shape embryo was the fifth landmark and occurred between and dpa highlighting the beginning of cotyledon growth. the 13– dpa embryo was torpedo shape, marking the sixth landmark. after the sixth landmark, the cotyledons grew into a coil and reached the seventh landmark approximately at dpa. at this stage, the embryo approached its final size, but the seed was not yet viable for germination. 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 dpa until dpa. up until dpa, there was little or no seed germination. however, at dpa, the germination rate increased dramatically thus reaching landmark eight. at dpa, nearly 100% of the seeds germinated. fruit ripening tomato fruit ripening stages consist of mature green, breaker and red ripe. at the mature green stage, ethylene treatment will result in a rapid reddening of the fruit. 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. 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. forty percent of fruit had responded to ethylene at dpa when 43% of the seeds were viable for germination. fruit younger than dpa did not respond to ethylene treatment. the ninth landmark is the onset of fruit ripening, coinciding with the breaker stage when color began to change at approximately dpa. this stage is followed by the tenth and final landmark of ripe fruit. gene expression profiles of floral and fruit development to obtain a global overview of gene expression in flower and fruit, we compared the profiles between three critical developmental time points. the first stage was young flower buds at floral landmark representing ovule initiation. the second stage was the anthesis-stage, representing flower landmark and fruit landmark the third and last stage was dpa fruits, representing the 4– cell embryo stage and fruit landmark differentially expressed genes were identified using the resampling-based multiple testing method. without the cutoff of fold-change applied, genes with adjusted p < were differentially expressed in at least one of the three stages. among them, genes showed higher expression at anthesis, whereas and genes showed higher expression in young flower buds and dpa fruits, respectively. functional classification of the differentially expressed genes showed a distinct distribution of genes involved in various biological processes for the three stages investigated. for example, more genes involved in developmental processes were found in flower buds during ovule initiation and anthesis-stage flowers than in dpa fruit. on the other hand, phytohormone-related genes were predominantly found in anthesis-stage flowers and dpa fruits compared to flower buds. 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. genes orthologous or homologous to the arabidopsis abce genes required for floral organ identity have been identified in tomato. on our array, the tomato floral organ identity genes differentially expressed at the three stages include b class genes tap, tpi and slmbp1/lepi-b, c class gene tag, and e class gene tm. the tomato ortholog tc of arabidopsis nap that is directly activated by b class gene apetala and pistillata in arabidopsis was also differentially expressed. all the above-mentioned genes showed higher expression in floral buds and/or anthesis-stage flowers, in agreement with their previously identified expression patterns. another tomato b class gene tm was not differentially expressed, likely due to its more ubiquitous expression in floral organs. while there is no clear ortholog of arabidopsis a class genes in tomato, the closest related ap gene, mads-mc, 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. 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, leunig, tousled, stylish, spatula, no transmitting tract, and hecate, 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, alcatraz, indehiscence, replumless, and fruitfull. the arabidopsis gene seedstick is required for ovule identity and patterning as well as seed disposal, and erecta regulates fruit shape by controlling cell expansion and cell division. jagged acts redundantly with the polarity genes filamous flower and yabby to activate ful and shp. additional polarity genes required for proper patterning and establishment of organ boundaries are crabs claw, kanadi, gymnos, phavoluta and phabulosa. tomato genes homologous to arabidopsis patterning genes fil, ful, crc, er, phb, and spt were more abundantly expressed in tomato flower buds compared to the other tissues. the tomato shp homolog tc showed higher expression in anthesis-stage flowers and fruits at dpa than in floral buds. the stk homolog in tomato tagl, which is expressed in the inner integument of the ovules and the endothelium in developing seeds, was expressed higher in fruits at dpa compared to other time points, suggesting that it may also play a role in tomato fruit development. tomato genes with high similarity to arabidopsis fruit patterning genes ett, gym, kan lug, phv, rpl, hec sty and tsl were not differentially expressed between the three stages, whereas no tomato homologs for jag, ntt, alc, ind, yab sty were included on our array. further, the hierarchical clustering of all the differentially expressed developmental processes genes revealed that flower bud and dpa fruit shared expression profiles of the same developmental genes, whereas anthesis-stage flower showed a distinctive profile, which is in agreement with results from other gene profiling studies in arabidopsis. expression of phytohormone-related genes phytohormones play essential roles in many aspects of plant development. among the three developmental time points, phytohormone-related genes were differentially expressed. of these genes, were involved in auxin conjugation, transport or signaling. most of the auxin-related genes were either up- or down-regulated in dpa fruit. moreover, most of the genes with similarity to gh involved in iaa conjugation were repressed after pollination, whereas three auxin response factor genes tc, tc, and tc, were expressed at the lowest level in anthesis stage flowers. further, transcripts of three auxin transporter genes, tc tc and tc homologous to aux pin and an auxin efflux carrier family protein, respectively, were less abundant in dpa fruit. several genes involved in biosynthesis of tryptophan, a precursor of iaa, were not developmentally regulated in this study, neither was the ortholog of arabidopsis auxin receptor transport inhibitor response. the ortholog of aldehyde oxidase involved in auxin biosynthesis, 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 tc of atkao that catalyzes the conversion of ent-kaurenoic acid to ga in gibberellin biosynthesis pathway, was more abundant in dpa fruit compared to other stages. in contrast, the expression of slga2ox, involved in catabolism of ga, was lower in the developing fruits than in flower buds at days preanthesis and anthesis-stage flowers. interestingly, transcripts of three tomato homologs tc tc and tc of arabidopsis ga receptors ga insentive dwarf1b and c, were less abundant in dpa fruit. this suggests that although ga levels may increase in 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 or not included on the array. most of the seven ga responsive genes were not differentially expressed following pollination with the exception of tomato gene tc encoding gasa/gast/snakin family protein that was upregulated after anthesis. transcripts of all the eight brassinosteroid-related genes were more abundant in dpa fruit, whereas the majority of jasmonate- and ethylene-related genes were less abundant in dpa fruit. expression of genes involved in aba biosynthesis and response like were also lower in dpa fruits. the putative ortholog of arabidopsis gene cyp707a, encoding the major aba 8'-hydroxylase involved in aba catabolism, is expressed at higher level in dpa fruit compared to the other stages, suggesting that the aba levels are reduced during the early fruit growth. fruit shape changes in la 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. we analyzed the changes in fruit shape from anthesis onward in nils in la background differing at the sun locus because at anthesis the ovary shape is only marginally different. the la1589pp has round fruit and carries the wild type allele, while la1589ee carries the sun allele of sun resulting in an elongated fruit. the difference in fruit shape between the two nils became apparent immediately following fertilization and was most pronounced between and dpa coinciding with the globular embryo stage of fruit landmark 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 > at anthesis to < at dpa. we examined sun expression in the developing fruits of the la nils starting from anthesis-stage ovaries until ripe fruit. in la1589ee, sun was expressed at a high level until fruit landmark coinciding with coiled embryo and seed maturation stage at dpa. a detailed investigation of its expression immediately before and after anthesis showed that sun transcript levels increased from days prior to anthesis to dpa and thus showed a similar kinetics to that of the changes in fruit shape. gene expression profiles associated with sun to further investigate the effect of sun on tomato fruit shape and to identify genes that may interact with sun in regulating morphology, we compared transcriptional profiles of three floral and fruit developmental stages in the nils in la background that differ at sun. the stages selected represented the three important events in flower and early fruit development when sun exhibited the greatest differential gene expression, namely days pre-anthesis, anthesis and days post-anthesis fruit. in total, we found differentially expressed genes between the nil pairs. one of the genes, defl encoding a member of plant defensins, was differentially expressed at all three time points. another gene encoding maternal effect embryo arrest was upregulated in oval shaped fruit at two time points. twenty four genes were differentially expressed only in anthesis-stage flowers and eight genes were differentially expressed only in dpa fruit. the differences in the transcript levels of the genes were less than two-fold with the exception of defl the latter gene is located very close to sun on chromosome therefore, decreased defl expression in the nil carrying elongated fruit was likely due to the mutation at the locus and not a consequence of increased expression of sun. the remaining differentially expressed genes did not fall into known developmental pathways. note that sun and defl which are differentially expressed in these nils were not on the array. *sun and defl were not on the nimblegen array; genes with rma expression value smaller than were considered to bee too low expressed and removed from the analysis. sun has been hypothesized to affect fruit shape by altering hormone levels such as auxin. however, several auxin biosynthesis genes, including aldehyde oxidase and most genes encoding tryptophan biosynthesis enzymes that were present on the array, were not changed in the nils. gibberellins also play important roles in cell division and elongation. 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. this implied that sun is not directly involved in regulating auxin and ga levels. discussion 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. 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 la 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 described flower development in 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 la 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 apetala. therefore, floral landmark 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 la fruit, in contrast to ~ to 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 gh, auxin responsive family protein, amino acid permease and auxin efflux carrier family protein, shared the same expression patterns between the two experiments. however, none of the ga-related genes were shared in the two studies. abscisic acid 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, several acc synthase genes were differentially expressed and all the ethylene biosynthesis genes were less abundant in dpa fruits, suggesting reduced levels of this hormone after pollination. the expression of aba biosynthesis genes, such as neoxantin synthase and 9-cis-epoxycarotenoid dioxygenase, is reduced in fruits post pollination. similarly, in our study zeaxanthin epoxidase was less abundant in dpa fruit compared to flower. in both studies, an aba 8'-hydroxylase gene 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 determined that tomato high-pigment 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 hp 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 of cell cycle-related genes and half of the cell wall-related genes were shared between the two studies. two cyclin genes tc and tc showing highest similarities to arabidopsis cyclin d3; and cyclin b1;, 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; 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. 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, 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. 
in addition, gene expression profiles of three key stages in floral and fruit development are presented, namely floral buds days before anthesis, anthesis-stage flowers, and days post anthesis fruit. the tomato fruit shape gene sun controls fruit elongation. sun controls fruit shape predominantly after fertilization and its effect reaches a maximum at days post-anthesis coinciding with fruit landmark representing the globular embryo stage of seed development. 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. we provide a description of floral and fruit development in a red-fruited closely related wild relative of tomato, solanum pimpinellifolium accession la we use established and propose new floral and fruit landmarks to present a framework for tomato developmental studies. to demonstrate the utility of the landmarks, we characterize the tomato shape gene sun in fruit development. the landmarks for flower and fruit development in tomato were outlined and integrated with the effect of sun on fruit shape. domestication and selection of tomato resulted in many varieties that differ in fruit shape and size. 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. the expression profiles of the nils that differ at sun show that only genes were differentially expressed and most of them at a less than 2-fold difference.
 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 la 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 days before anthesis, anthesis-stage flowers, and days post anthesis fruit. 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 days post-anthesis coinciding with fruit landmark representing the globular embryo stage of seed development. the expression profiles of the nils that differ at sun show that only 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. 