BACKGROUND
fleshy fruits are highly prized for nutritional content, flavor, fragrance, and appearance.
while most fruits are eaten when ripe, a subset, including many that for culinary purposes are viewed as vegetables, are consumed immature.
cucumbers , which are used as fresh product and processed into pickles, are typically harvested at the middle or end of the exponential growth phase, 1– <dig> weeks post-pollination, and approximately 2– <dig> weeks prior to fruit maturation.
early fruit development is typified by phases of cell division and expansion  <cit> .
in cucumber fruit, which develop from an enlarged inferior ovary, cell division occurs most rapidly prior to anthesis and then continues more slowly in the first 0– <dig> days post anthesis  <cit> .
this phase largely overlaps with the period of highest respiration  <cit> .
fruit elongation begins almost immediately after pollination, with the most rapid increase occurring approximately 4– <dig> days post pollination   <cit> .
the rapid increase in cell size mirrors the rapid increase in fruit length, with obvious increase in vacuolization of mesocarp cells, and thickening in epidermal cell walls occurring between  <dig> and  <dig> dpp  <cit> .
cell division and expansion are largely completed by 12– <dig> dpp, with some variation depending on cultivar and season  <cit> .
in addition to cell division and expansion, early development also includes specialized tissue and organ development and interaction with the abiotic and biotic environment.
for example, developing cucumber fruit exhibit a distinct change in susceptibility to the soil-borne, oomycete pathogen, phytophthora capsici; young fruit are highly susceptible, while older fruit are resistant  <cit> .
there is a sharp transition in susceptibility that occurs at approximately 10– <dig> dpp coinciding with the end of the period of rapid fruit elongation.
this age-related resistance suggests additional kinds of developmental changes occurring in the young cucumber fruit.
although a limited number of studies have examined gene expression during early fruit development, a picture reflecting cell division and expansion is beginning to emerge based on transcriptomic studies of apple, cucumber, grape, tomato and watermelon.
among the enriched categories associated with tomato fruit set, were genes associated with protein biosynthesis, histones, nucleosome and chromosome assembly and cell cycle, suggesting a profile reflective of active cell division  <cit> .
in contrast, various water, sugar and organic acid transport-associated genes were under-represented, but then increased with the transition from cell division to cell expansion.
highly expressed categories of genes expressed in expanding cucumber, as well as apple, grape, tomato, melon and watermelon fruits, included cytoskeleton and cell wall modifying genes such as tubulins, expansins, endo- <dig> -b-glucanase, beta glucosidases, pectate lyases, and pectin methylesterases, and transport associated genes such as aquaporins, vacuolar h+atpases, and phloem-associated proteins  <cit> .
the most highly represented transcripts in rapidly expanding cucumber fruit  also were strongly enriched for defense related homologs including, lipid, latex, and defense-related genes, e.g., chitinase, thionin, hevein, snakin, peroxidase, catalase, thioredoxin, and dehydrins  <cit> .
the early stages of fruit development, including fruit set and exponential growth, are clearly essential for all fruits.
however, despite their importance as determinants of fruit size and yield, there has been little detailed analysis of this phase of development.
most studies to date, including recent transcriptomic studies, have focused on late development, or a broad range of developmental stages, with only a single snapshot during early development eg.,  <cit> .
in this study we combined morphological characterization with transcriptome analysis to provide new insight into important early fruit developmental stages and processes.
our observations, performed at five time points during the period from fruit set through the end of exponential fruit growth, indicate that this is a dynamic period of cucumber fruit development involving an array of internal and external morphological, physiological, and transcriptomic changes that act in concert with phases of active cell division, expansion, and response to the environment.
relative to anthesis and early fruit set, the period of peak- and late-exponential growth includes a large portion of highly represented transcripts, either of unknown function, or without homologs in arabidopsis, suggesting unique factors contributing to the rapid growth phase in cucurbits.
the end of exponential growth was marked by a shift in transcriptome profile characterized by abiotic and biotic stress related genes and significant enrichment for transcription factor gene homologs associated with stress response and development, suggesting that the interval between expansive growth and ripening may include a programmed transition toward enhanced defense.
RESULTS
morphological changes during early cucumber fruit development
young vlaspik cucumber fruit followed a highly reproducible progression of growth and development including visible external and internal morphological changes.
increase in size occurred rapidly after fertilization with most rapid growth occurring between  <dig> and  <dig> dpp .
after approximately  <dig> dpp, fruit size remained largely constant until fruit maturation at approximately  <dig> dpp.
at  <dig> dpp , deep ridges along the length of fruit covered the surface of the fruit.
densely spaced spines were randomly scattered relative to the ridges .
in contrast to ridges, which were most prominent at anthesis, warts, which are typically are formed at the base of spines, were diminutive at  <dig> dpp.
they rapidly developed to become highly prominent at  <dig> dpp but then flattened out with further fruit expansion.
both ridges and warts were nearly absent by  <dig> dpp.
the spines followed a maturation process culminating in abscission.
at  <dig> dpp spine color was translucent light green.
at approximately  <dig> dpp they started to senesce, turning yellow, then white at 12– <dig> dpp.
by  <dig> dpp many had abscised from the fruit surface.
at anthesis, the exocarp was dark green.
dark green/light green stripes and specks on the surface of the fruit began to emerge around  <dig> dpp.
the fruit surface at anthesis also has a dull appearance due to ‘bloom’ , a fine white powder primarily composed of silica oxide   <cit> .
the bloom disappeared first from the peduncle end around  <dig> dpp, then the blossom end by  <dig> dpp; by  <dig> dpp, it had disappeared completely, leaving a shiny fruit surface.
the cuticle layer showed increased thickness with age.
after  <dig> and  <dig> dpp it stained more darkly with sudan iv, indicating increased cutin or wax content that appeared to penetrate between the pallisade cells in the epidermal layer .
with respect to internal fruit morphology, both placenta and pericarp rapidly expanded from 4– <dig> dpp.
the rate and amount of expansion was very similar for both tissues .
the mesocarp was initially green at  <dig> and  <dig> dpp, but became progressively lighter with age.
increase in mesocarp cell size is accompanied bv increased vacuolization between  <dig> and  <dig> dpp  <cit> .
the placenta tissue became gelatinous between  <dig> and  <dig> dpp and hardening of seed coats occurred between  <dig> and  <dig> dpp .
<dig> pyrosequencing data
<dig> pyrosequencing analysis of cdna libraries prepared from pericarp rna samples of fruit harvested  <dig>   <dig>   <dig>   <dig>  and  <dig> days post-pollination provided  <dig>  million reads .
the resulting data were assembled into  <dig>  contigs with a mean length of  <dig> base pairs .
all transcripts were mapped to the assembled cucumber genome of huang et al.
<cit> , although in some cases more than one transcript mapped to the same location.
the number of the reads per contig ranged from  <dig> to more than  <dig>  with a mean of  <dig> reads per contig and median of  <dig> reads/contig.
assembed contig length increased steadily with the number of ests/contig, until approximately  <dig> reads/contig where it leveled off with an average length of approximately  <dig> bp .
similarly, frequency of identification of homologs in arabidopsis increased with number of ests/contig, leveling off at approximately 90% with approximately  <dig> reads/contig .
gene ontology  assignment to those contigs with putative homologs in arabidopsis showed a similar distribution of gene functions as are present in the full arabidopsis genome , suggesting broad representation of the genome .
approximately half of the contigs with ≥ <dig> reads but without homologs in arabidopsis had putative homologs in other species.
the final portion, approximately 5% of the total , either did not have any identified homologs in the current ncbi nr database, or only had putative homologs in cucurbit species, suggesting that these transcripts may be unique to cucumber or cucurbits relative to the plant species sequenced to date.
these potentially cucurbit-unique transcripts included  <dig> very highly expressed contigs, represented by at least  <dig> ests .
eighteen had putative functional assignments, eight of which were known cucurbit specific phloem-related proteins, such as phloem lectins and phloem proteins .
changes in transcript abundance during early fruit growth
based on the observed relationship between ests/contig, contig length, and putative homologs in arabidopsis , subsequent bioinformatic analyses were performed on contigs represented by at least  <dig> ests.
the distribution of contigs represented by at least  <dig> ests that did not have putative homologs outside of cucurbit species was not evenly distributed across fruit age .
the  <dig>   <dig>  and  <dig> dpp libraries contained nearly twice as many contigs without identified homologs in arabidopsis as was observed for the  <dig> and  <dig> dpp libraries.
of the  <dig> very highly abundant transcripts without known homologs outside of cucurbits, only three were not observed in the  <dig>   <dig> or  <dig> dpp samples.
in contrast,  <dig> of the cucurbit specific transcripts did not appear in  <dig> or  <dig> dpp samples.
to validate usefulness of the  <dig> sequence data for analysis of transcript abundance, a set of fourteen genes representing different levels of est representation/contig across the different fruit ages were selected for quantitative real time -pcr analysis .
these included genes such as cyclin-dependent kinase b2; <dig> with high transcript levels early in development  or expansin a <dig> with higher transcript levels at 8– <dig> dpp.
comparison of transcript level at a given age relative to baseline expression at 0dpp  showed good correspondence between values obtained by  <dig> sequencing and qrt-pcr .
there was also good correspondence between the qrt-pcr results obtained from two different growth experiments in the greenhouse , indicating biological reproducibility of patterns of gene expression across fruit ages, and validity of the use of frequency of est representation in the  <dig> library as a measure of level of gene expression.
principal component analysis  was performed on transcript levels among the libraries from the five fruit ages .
the first two components, which accounted for nearly 90% of the variation, separated the fruit ages into three groups,  <dig> and  <dig> dpp,  <dig> dpp, and  <dig> and  <dig> dpp.
examination of fruit growth rate indicated that these age groups correspond with cell division/pre-exponential growth, peak exponential expansion, and late/post-exponential expansion stages of growth, respectively .
comparison of the transcripts present in each of the age groups showed that the great majority were detected in all three age groups.
the fewest unique transcripts were present in the  <dig> dpp sample, consistent with a developmental gradient of transcription moving from 0– <dig> to  <dig> to 12– <dig> dpp.
both the pca and venn diagram  show the least commonality between the 0– <dig> and 12– <dig> dpp age groups.
the most highly represented contigs in each age group  exhibited markedly different profiles of putative gene function.
among those in common to all three groups were housekeeping genes including numerous ribosomal protein genes, and several tubulins, actins, and redox-related genes , as well as several with unknown function or no identifiable homolog in arabidopsis.
examination of the transcripts that were very highly represented in only one age group , showed that 0– <dig> dpp was the only one to include histone genes.
this observation is consistent with the high level of dna replication occurring in young, dividing cells.
similarly, ribosomal protein genes were among the most highly represented transcripts at 0– <dig> dpp  but minimally present in the  <dig> or 12– <dig> dpp ages .
the 12– <dig> dpp group was marked by numerous abiotic stress related genes.
strikingly, genes with unknown function or without arabidopsis homologs, dominated the group at  <dig> dpp, accounting for more than half of the contigs .
the exponential growth stage of tomato also was associated with a larger proportion of ests with unknown function relative to other ages  <cit> .
fewer genes with unknown function or without arabidopsis homologs occurred in the 12– <dig> dpp group  and only  <dig> member of the 0-4dpp group had no assigned putative function or was without a homolog in arabidopsis.
to identify less highly represented genes that were strongly enriched at a specific age group, contigs were normalized for portion of reads observed at different time points.
if transcription levels were constant during development, 20% of the transcript reads would be observed at each of the five sample ages .
overall distribution of portion of transcripts observed at a given age followed this expectation for the transcriptome set, with a mean value of  <dig> %,  <dig> %, and  <dig> %, respectively for 0+ <dig>   <dig>  and 12+16dpp age groups .
the tails of the distribution  were examined for genes for which transcript levels were strongly enriched in a specific age group .
this resulted in three non-overlapping sets of genes .
there also was minimal overlap with the genes listed in table  <dig>  the genes listed in table  <dig> had an average of  <dig> ests/contig whereas the mean number of ests/contig for the genes identified in this manner was  <dig>  as was seen for the most highly represented group of genes, there was uneven distribution of genes without homologs in arabidopsis or with unknown function; those accounted for  <dig> % for 0+ <dig> dpp enriched transcripts, but for  <dig> % and  <dig> % for 8dpp and 12+ <dig> dpp enriched transcripts, respectively.
fruit set/pre-exponential growth
functional enrichment analysis of those transcripts with age-group enriched transcript levels indicated that the 0– <dig> dpp age group had significantly increased representation of genes associated with cell organization and biogenesis, and dna or rna metabolism, that subsided with age .
in addition to histone genes, which were also among the most highly abundant transcripts for this age group , numerous putative cell cycle genes, cyclin- and cyclin dependent kinase-related gene family members, exhibited greater than 90% of transcript reads at 0– <dig> dpp .
extensive protein interaction and gene expression data from arabidopsis have allowed for the development of a picture of the cyclin interactome, including characterization of complexes associated with different cell phases  <cit> .
cyclin related genes strongly enriched at 0– <dig> dpp in the cucumber fruit transcriptome, such as putative homologs of cdkb1; <dig>  cdkb2; <dig>  cycb1:2; cycd3; <dig>  cycd3; <dig>  cycd5; <dig>  were among those associated with the mitosis and post-mitosis  phases in the arabidopsis interactome.
elevated expression of several of these genes was also observed during fruit set in pollinated vs. unpollinated apple and cucumber flowers  <cit> .
in contrast, the homolog of cdka; <dig> , which was uniformly represented in the young cucumber fruit transcriptome, was associated with cyclin complexes throughout the arabidopsis cell cycle.
the categories of plastid and chloroplast also were significantly enriched in the 0– <dig> dpp group, then declined with age.
this is consistent with the decrease in chlorophyll observed after 4dpp; chlorophyll content per gram fresh weight peaked at  <dig> dpp, and then decreased until  <dig> dpp .
the assembled contigs included  <dig> transcripts whose homologs in arabidopsis had annotations including one or more of the following terms: chlorophyll, chloroplast, photosystem, or thylakoid .
overall patterns of transcript abundance for these genes paralleled chlorophyll content in the developing fruit .
k-means cluster analysis allowed for further identification of transcripts showing progressive patterns of representation with fruit age .
the chloroplast and other photosynthesis related genes described above, along with homologs of at least  <dig> additional chloroplast located proteins and enzymes predominated in the  <dig> or 4+ <dig> clusters, but were minimally observed in the  <dig>  0+ <dig>  or  <dig> dpp clusters, and did not appear in the later clusters .
exponential growth
the group of genes with peak abundance at the  <dig> dpp, exponential growth stage, included cytoskeleton, cell wall, and water and carbohydrate transport genes.
tubulins, actin-related proteins, extensins, expansins, cellulose synthases, pectinase modifying enzymes, aquaporins, vacuolar h+atpases, and phloem filament and lectin proteins, were among those strongly represented, as has been observed for other rapidly growing fleshy fruits such as tomato, apple, grape, and watermelon  <cit> .
the major latex protein related genes also exhibited peak levels at  <dig> dpp, including two extremely highly transcribed genes that together accounted for more than  <dig>  reads .
putative homologs of vacuolar atp synthase subunits b, d, h and p <dig>  showed coordinate transcript abundance, with comparable levels increasing steadily until  <dig> dpp, and then gradually declining two very highly represented homologs of the vacuolar aquaporin gene , gamma tip tonoplast intrinsic protein, also peaked at 4-8dpp .
all of the cucurbit specific phloem proteins listed in table  <dig> and the four putative homologs of the arabidopsis phloem protein  a <dig> family members observed in the data set peaked somewhat later, at 8– <dig> dpp with minimal transcript levels at  <dig> and  <dig> dpp .
cucurbits are characterized by a unique and functionally divergent network of extrafascicular phloem external to the vascular bundles  <cit> .
the highly expressed proteinaceous phloem filaments, comprised of the cucurbit-specific pp <dig> proteins, and the more widely distributed pp <dig> phloem lectin proteins  <cit> , were found to be primarily associated with the extrafasicular phloem  <cit> .
strong expression of phloem protein genes during rapid growth has been observed in other studies, including pp <dig> expression in green stage watermelon fruit  <cit> .
specific expression of pp <dig>  was observed in young pumpkin  hypocotyls, peaking at  <dig> days after germination in concert with the period of peak growth and vascular differentiation  <cit> .
in contrast, cucumber homologs of the atpp2-b family had a nearly inverted pattern of transcript levels relative to pp2-a genes, peaking at  <dig> dpp, and dropping during exponential growth, suggesting possible functional divergence .
the period of rapid fruit enlargement was also associated with marked changes in fruit surface, including an increase in cuticle thickness as is typically observed during rapid plant growth  <cit> , and loss of the silica oxide powder based ‘bloom’.
the homolog of the cucurbita moschata silicon transporter  showed age specific transcript abundance peaking at  <dig> dpp then dropping sharply, coinciding with the time of bloom loss from the middle of the fruit .
among the genes identified in other systems to be associated with cuticle biosynthesis are the extracellular gdsl motif lipase/hydrolase proteins and lipid transfer proteins, which have been implicated in lipid transport to extracellular surfaces  <cit> .
the cucumber fruit transcriptome set included eleven gdsl motif lipase/hydrolase protein family members that were represented by at least  <dig> ests, including five with more than  <dig> ests.
the majority showed peak levels at  <dig> or 12– <dig> dpp, with virtually no measured reads until either  <dig> or  <dig> dpp .
twelve lipid transfer protein  family members with greater than  <dig> ests/contig also were observed in the transcriptome data set, including four with greater than  <dig> ests.
as for the gdsl motif lipase/hydrolase protein genes, the majority of the lipid transfer proteins were most highly represented from 8– <dig> dpp; transcript levels of one gene peaked at 4– <dig> dpp .
a homolog of the transcription factor gene shine <dig> , which is associated with cuticle production in arabidopsis   <cit>  also exhibited peak transcript abundance at  <dig> dpp.
additionally, transcript levels of two cyctochrome p <dig> family members  that have been associated with cutin biosynthesis  <cit> ; and two putative beta amyrin synthases, enzymes which have been associated with cuticular wax synthesis in tomato  <cit> , also peaked at 8dpp .
in contrast, two putative gdsl family members and one lipid transfer protein with moderate transcript levels   were observed almost exclusively at  <dig> dpp, suggesting possible floral, rather than fruit, expression .
late/post exponential growth
stress-related genes  were over-represented at all stages, but considerably more so at 12– <dig> dpp than at the younger ages of 0– <dig> and  <dig> dpp .
the 12+ <dig> dpp age group had the highest representation of abiotic and biotic stress related genes, including a variety of heat shock, redox, biotic defense and ethylene-related transcripts .
of the  <dig> genes in this group,  <dig> have high homology with genes associated with plant stress, including at least  <dig> transcription stress-related factors such as wrky <dig> activator of sa-dependent defense; radical induced cell death; ethylene response, salt stress, and heat shock transcription factors .
overall, the group of genes with peak abundance at 12+ <dig> dpp was significantly enriched for transcription factor genes   accounting for 16% of the top  <dig> % set.
this may be contrasted with the total cucumber fruit transcriptome data set where transcription and transcription factor activity related genes were represented at a normalized frequency of  <dig>  relative to occurrence in the arabidopsis genome.
transcription factors in the top  <dig> % of 0+ <dig> and  <dig> dpp groups also were represented at a comparable frequency to the arabidopsis genome, accounting for  <dig> % and  <dig> % of the gene list, respectively.
in addition to the stress related transcription factors with specific representation at 12– <dig> dpp, several putative transcription factor homologs were annotated to be associated with development [e.g., embryo sac development , morphogenesis , and cell expansion  .
furthermore, transcripts of other genes with homologs that have been implicated in development related processes are specifically observed at 12– <dig> days, such as putative homologs of tctp ; btb and taz domainprotein 1; calcium-binding ef hand family protein; seed development related ; and bax inhibitor  <dig>
CONCLUSIONS
examination of early cucumber fruit growth from the period of pollination and initial fruit set through the end of the exponential growth phase shows a dynamic series of physiological and morphological changes .
transcriptomic analysis of the predominant genes represented in the different age groups as identified either by total number of reads , portion of transcript reads observed at that age, or genes grouped by k-means cluster analysis, told a story aligned with the sequential stages of development.
transcript representation in the youngest ages, 0– <dig> dpp, was uniquely characterized by genes associated with cell division, cell organization and biogenesis.
at  <dig> dpp, transcription of the cell cycle genes was declining, while chloroplast, photosynthesis, and chloroplast-localized genes were peaking.
transcripts highly abundant during the exponential growth phase, 4– <dig> dpp, included extensive representation of genes associated with cell structure such as cytoskeleton, vacuoles and cell walls, along with surface lipid metabolism related genes, in concert with the period of greatest increase in cuticle thickness.
a second shift in the transcriptome profile was observed at 12– <dig> dpp with significant enrichment of abiotic and biotic stress related genes and stress-related and developmental transcription factor gene homologs.
the enriched representation of numerous transcription factors relative to earlier ages suggests a programmatic change away from fruit growth, toward defense, and ultimately fruit maturation.
this is also the time period where we have observed transition of cucumber fruit from susceptibility to resistance to p. capsici <cit> .
classically, fleshy fruit development is described to consist of three stages post pollination: cell division, cell expansion, and ripening  <cit> .
these results suggest that the interval between expansive growth and ripening may include further developmental differentiation; an emphasis on defense would be consistent with the role of fruit in protecting the developing seeds during embryo maturation prior to facilitating seed dispersal.
finally, approximately 5% of the contigs represented by ≥ <dig> reads either did not have identified putative homologs, or did not have homologs outside of cucurbits suggesting potentially unique genes specific to cucumber or cucurbits.
the observation that these genes, as well as genes with homologs but with no annotated function, rarely occurred in the 0– <dig> dpp group, suggests commonality among processes associated with early fruit set and cell division and/or greater knowledge about the fruit set stage.
the predominance of transcripts without non-cucurbit homologs or with unknown predicted functions during the peak exponential growth stage may reflect fewer studies to date about this phase of growth, or unique adaptations of cucurbits to allow for extreme fruit growth rates associated with these species.
collectively, the transcriptomic information provided by the young cucumber fruit samples coupled with morphological analyses provide an informative picture of early fruit development characterized by phases of active cell division, fruit expansion including novel or uncharacterized genes, and response to the environment, as summarized in figure  <dig>  the progressive modules of transcript abundance tell a story of cell division, development of photosynthetic capacity, cell expansion and fruit growth, phloem activity, protection of the fruit surface, and finally transition away from fruit growth toward defense and maturation.

 fleshy fruits are highly prized for nutritional content, flavor, fragrance, and appearance. while most fruits are eaten when ripe, a subset, including many that for culinary purposes are viewed as vegetables, are consumed immature. cucumbers, which are used as fresh product and processed into pickles, are typically harvested at the middle or end of the exponential growth phase, 1– weeks post-pollination, and approximately 2– weeks prior to fruit maturation. early fruit development is typified by phases of cell division and expansion. in cucumber fruit, which develop from an enlarged inferior ovary, cell division occurs most rapidly prior to anthesis and then continues more slowly in the first 0– days post anthesis. this phase largely overlaps with the period of highest respiration. fruit elongation begins almost immediately after pollination, with the most rapid increase occurring approximately 4– days post pollination. the rapid increase in cell size mirrors the rapid increase in fruit length, with obvious increase in vacuolization of mesocarp cells, and thickening in epidermal cell walls occurring between and dpp. cell division and expansion are largely completed by 12– dpp, with some variation depending on cultivar and season. in addition to cell division and expansion, early development also includes specialized tissue and organ development and interaction with the abiotic and biotic environment. for example, developing cucumber fruit exhibit a distinct change in susceptibility to the soil-borne, oomycete pathogen, phytophthora capsici; young fruit are highly susceptible, while older fruit are resistant. there is a sharp transition in susceptibility that occurs at approximately 10– dpp coinciding with the end of the period of rapid fruit elongation. this age-related resistance suggests additional kinds of developmental changes occurring in the young cucumber fruit. although a limited number of studies have examined gene expression during early fruit development, a picture reflecting cell division and expansion is beginning to emerge based on transcriptomic studies of apple, cucumber, grape, tomato and watermelon. among the enriched categories associated with tomato fruit set, were genes associated with protein biosynthesis, histones, nucleosome and chromosome assembly and cell cycle, suggesting a profile reflective of active cell division. in contrast, various water, sugar and organic acid transport-associated genes were under-represented, but then increased with the transition from cell division to cell expansion. highly expressed categories of genes expressed in expanding cucumber, as well as apple, grape, tomato, melon and watermelon fruits, included cytoskeleton and cell wall modifying genes such as tubulins, expansins, endo- -b-glucanase, beta glucosidases, pectate lyases, and pectin methylesterases, and transport associated genes such as aquaporins, vacuolar h+atpases, and phloem-associated proteins. the most highly represented transcripts in rapidly expanding cucumber fruit also were strongly enriched for defense related homologs including, lipid, latex, and defense-related genes, e.g., chitinase, thionin, hevein, snakin, peroxidase, catalase, thioredoxin, and dehydrins. the early stages of fruit development, including fruit set and exponential growth, are clearly essential for all fruits. however, despite their importance as determinants of fruit size and yield, there has been little detailed analysis of this phase of development. most studies to date, including recent transcriptomic studies, have focused on late development, or a broad range of developmental stages, with only a single snapshot during early development eg.,. in this study we combined morphological characterization with transcriptome analysis to provide new insight into important early fruit developmental stages and processes. our observations, performed at five time points during the period from fruit set through the end of exponential fruit growth, indicate that this is a dynamic period of cucumber fruit development involving an array of internal and external morphological, physiological, and transcriptomic changes that act in concert with phases of active cell division, expansion, and response to the environment. relative to anthesis and early fruit set, the period of peak- and late-exponential growth includes a large portion of highly represented transcripts, either of unknown function, or without homologs in arabidopsis, suggesting unique factors contributing to the rapid growth phase in cucurbits. the end of exponential growth was marked by a shift in transcriptome profile characterized by abiotic and biotic stress related genes and significant enrichment for transcription factor gene homologs associated with stress response and development, suggesting that the interval between expansive growth and ripening may include a programmed transition toward enhanced defense.  morphological changes during early cucumber fruit development young vlaspik cucumber fruit followed a highly reproducible progression of growth and development including visible external and internal morphological changes. increase in size occurred rapidly after fertilization with most rapid growth occurring between and dpp. after approximately dpp, fruit size remained largely constant until fruit maturation at approximately dpp. at dpp, deep ridges along the length of fruit covered the surface of the fruit. densely spaced spines were randomly scattered relative to the ridges. in contrast to ridges, which were most prominent at anthesis, warts, which are typically are formed at the base of spines, were diminutive at dpp. they rapidly developed to become highly prominent at dpp but then flattened out with further fruit expansion. both ridges and warts were nearly absent by dpp. the spines followed a maturation process culminating in abscission. at dpp spine color was translucent light green. at approximately dpp they started to senesce, turning yellow, then white at 12– dpp. by dpp many had abscised from the fruit surface. at anthesis, the exocarp was dark green. dark green/light green stripes and specks on the surface of the fruit began to emerge around dpp. the fruit surface at anthesis also has a dull appearance due to ‘bloom’, a fine white powder primarily composed of silica oxide. the bloom disappeared first from the peduncle end around dpp, then the blossom end by dpp; by dpp, it had disappeared completely, leaving a shiny fruit surface. the cuticle layer showed increased thickness with age. after and dpp it stained more darkly with sudan iv, indicating increased cutin or wax content that appeared to penetrate between the pallisade cells in the epidermal layer. with respect to internal fruit morphology, both placenta and pericarp rapidly expanded from 4– dpp. the rate and amount of expansion was very similar for both tissues. the mesocarp was initially green at and dpp, but became progressively lighter with age. increase in mesocarp cell size is accompanied bv increased vacuolization between and dpp. the placenta tissue became gelatinous between and dpp and hardening of seed coats occurred between and dpp. pyrosequencing data pyrosequencing analysis of cdna libraries prepared from pericarp rna samples of fruit harvested and days post-pollination provided million reads. the resulting data were assembled into contigs with a mean length of base pairs. all transcripts were mapped to the assembled cucumber genome of huang et al., although in some cases more than one transcript mapped to the same location. the number of the reads per contig ranged from to more than with a mean of reads per contig and median of reads/contig. assembed contig length increased steadily with the number of ests/contig, until approximately reads/contig where it leveled off with an average length of approximately bp. similarly, frequency of identification of homologs in arabidopsis increased with number of ests/contig, leveling off at approximately 90% with approximately reads/contig. gene ontology assignment to those contigs with putative homologs in arabidopsis showed a similar distribution of gene functions as are present in the full arabidopsis genome, suggesting broad representation of the genome. approximately half of the contigs with ≥ reads but without homologs in arabidopsis had putative homologs in other species. the final portion, approximately 5% of the total, either did not have any identified homologs in the current ncbi nr database, or only had putative homologs in cucurbit species, suggesting that these transcripts may be unique to cucumber or cucurbits relative to the plant species sequenced to date. these potentially cucurbit-unique transcripts included very highly expressed contigs, represented by at least ests. eighteen had putative functional assignments, eight of which were known cucurbit specific phloem-related proteins, such as phloem lectins and phloem proteins. changes in transcript abundance during early fruit growth based on the observed relationship between ests/contig, contig length, and putative homologs in arabidopsis, subsequent bioinformatic analyses were performed on contigs represented by at least ests. the distribution of contigs represented by at least ests that did not have putative homologs outside of cucurbit species was not evenly distributed across fruit age. the and dpp libraries contained nearly twice as many contigs without identified homologs in arabidopsis as was observed for the and dpp libraries. of the very highly abundant transcripts without known homologs outside of cucurbits, only three were not observed in the or dpp samples. in contrast, of the cucurbit specific transcripts did not appear in or dpp samples. to validate usefulness of the sequence data for analysis of transcript abundance, a set of fourteen genes representing different levels of est representation/contig across the different fruit ages were selected for quantitative real time -pcr analysis. these included genes such as cyclin-dependent kinase b2; with high transcript levels early in development or expansin a with higher transcript levels at 8– dpp. comparison of transcript level at a given age relative to baseline expression at 0dpp showed good correspondence between values obtained by sequencing and qrt-pcr. there was also good correspondence between the qrt-pcr results obtained from two different growth experiments in the greenhouse, indicating biological reproducibility of patterns of gene expression across fruit ages, and validity of the use of frequency of est representation in the library as a measure of level of gene expression. principal component analysis was performed on transcript levels among the libraries from the five fruit ages. the first two components, which accounted for nearly 90% of the variation, separated the fruit ages into three groups, and dpp, dpp, and and dpp. examination of fruit growth rate indicated that these age groups correspond with cell division/pre-exponential growth, peak exponential expansion, and late/post-exponential expansion stages of growth, respectively. comparison of the transcripts present in each of the age groups showed that the great majority were detected in all three age groups. the fewest unique transcripts were present in the dpp sample, consistent with a developmental gradient of transcription moving from 0– to to 12– dpp. both the pca and venn diagram show the least commonality between the 0– and 12– dpp age groups. the most highly represented contigs in each age group exhibited markedly different profiles of putative gene function. among those in common to all three groups were housekeeping genes including numerous ribosomal protein genes, and several tubulins, actins, and redox-related genes, as well as several with unknown function or no identifiable homolog in arabidopsis. examination of the transcripts that were very highly represented in only one age group, showed that 0– dpp was the only one to include histone genes. this observation is consistent with the high level of dna replication occurring in young, dividing cells. similarly, ribosomal protein genes were among the most highly represented transcripts at 0– dpp but minimally present in the or 12– dpp ages. the 12– dpp group was marked by numerous abiotic stress related genes. strikingly, genes with unknown function or without arabidopsis homologs, dominated the group at dpp, accounting for more than half of the contigs. the exponential growth stage of tomato also was associated with a larger proportion of ests with unknown function relative to other ages. fewer genes with unknown function or without arabidopsis homologs occurred in the 12– dpp group and only member of the 0-4dpp group had no assigned putative function or was without a homolog in arabidopsis. to identify less highly represented genes that were strongly enriched at a specific age group, contigs were normalized for portion of reads observed at different time points. if transcription levels were constant during development, 20% of the transcript reads would be observed at each of the five sample ages. overall distribution of portion of transcripts observed at a given age followed this expectation for the transcriptome set, with a mean value of %, %, and %, respectively for 0+ and 12+16dpp age groups. the tails of the distribution were examined for genes for which transcript levels were strongly enriched in a specific age group. this resulted in three non-overlapping sets of genes. there also was minimal overlap with the genes listed in table the genes listed in table had an average of ests/contig whereas the mean number of ests/contig for the genes identified in this manner was as was seen for the most highly represented group of genes, there was uneven distribution of genes without homologs in arabidopsis or with unknown function; those accounted for % for 0+ dpp enriched transcripts, but for % and % for 8dpp and 12+ dpp enriched transcripts, respectively. fruit set/pre-exponential growth functional enrichment analysis of those transcripts with age-group enriched transcript levels indicated that the 0– dpp age group had significantly increased representation of genes associated with cell organization and biogenesis, and dna or rna metabolism, that subsided with age. in addition to histone genes, which were also among the most highly abundant transcripts for this age group, numerous putative cell cycle genes, cyclin- and cyclin dependent kinase-related gene family members, exhibited greater than 90% of transcript reads at 0– dpp. extensive protein interaction and gene expression data from arabidopsis have allowed for the development of a picture of the cyclin interactome, including characterization of complexes associated with different cell phases. cyclin related genes strongly enriched at 0– dpp in the cucumber fruit transcriptome, such as putative homologs of cdkb1; cdkb2; cycb1:2; cycd3; cycd3; cycd5; were among those associated with the mitosis and post-mitosis phases in the arabidopsis interactome. elevated expression of several of these genes was also observed during fruit set in pollinated vs. unpollinated apple and cucumber flowers. in contrast, the homolog of cdka;, which was uniformly represented in the young cucumber fruit transcriptome, was associated with cyclin complexes throughout the arabidopsis cell cycle. the categories of plastid and chloroplast also were significantly enriched in the 0– dpp group, then declined with age. this is consistent with the decrease in chlorophyll observed after 4dpp; chlorophyll content per gram fresh weight peaked at dpp, and then decreased until dpp. the assembled contigs included transcripts whose homologs in arabidopsis had annotations including one or more of the following terms: chlorophyll, chloroplast, photosystem, or thylakoid. overall patterns of transcript abundance for these genes paralleled chlorophyll content in the developing fruit. k-means cluster analysis allowed for further identification of transcripts showing progressive patterns of representation with fruit age. the chloroplast and other photosynthesis related genes described above, along with homologs of at least additional chloroplast located proteins and enzymes predominated in the or 4+ clusters, but were minimally observed in the 0+ or dpp clusters, and did not appear in the later clusters. exponential growth the group of genes with peak abundance at the dpp, exponential growth stage, included cytoskeleton, cell wall, and water and carbohydrate transport genes. tubulins, actin-related proteins, extensins, expansins, cellulose synthases, pectinase modifying enzymes, aquaporins, vacuolar h+atpases, and phloem filament and lectin proteins, were among those strongly represented, as has been observed for other rapidly growing fleshy fruits such as tomato, apple, grape, and watermelon. the major latex protein related genes also exhibited peak levels at dpp, including two extremely highly transcribed genes that together accounted for more than reads. putative homologs of vacuolar atp synthase subunits b, d, h and p showed coordinate transcript abundance, with comparable levels increasing steadily until dpp, and then gradually declining two very highly represented homologs of the vacuolar aquaporin gene, gamma tip tonoplast intrinsic protein, also peaked at 4-8dpp. all of the cucurbit specific phloem proteins listed in table and the four putative homologs of the arabidopsis phloem protein a family members observed in the data set peaked somewhat later, at 8– dpp with minimal transcript levels at and dpp. cucurbits are characterized by a unique and functionally divergent network of extrafascicular phloem external to the vascular bundles. the highly expressed proteinaceous phloem filaments, comprised of the cucurbit-specific pp proteins, and the more widely distributed pp phloem lectin proteins, were found to be primarily associated with the extrafasicular phloem. strong expression of phloem protein genes during rapid growth has been observed in other studies, including pp expression in green stage watermelon fruit. specific expression of pp was observed in young pumpkin hypocotyls, peaking at days after germination in concert with the period of peak growth and vascular differentiation. in contrast, cucumber homologs of the atpp2-b family had a nearly inverted pattern of transcript levels relative to pp2-a genes, peaking at dpp, and dropping during exponential growth, suggesting possible functional divergence. the period of rapid fruit enlargement was also associated with marked changes in fruit surface, including an increase in cuticle thickness as is typically observed during rapid plant growth, and loss of the silica oxide powder based ‘bloom’. the homolog of the cucurbita moschata silicon transporter showed age specific transcript abundance peaking at dpp then dropping sharply, coinciding with the time of bloom loss from the middle of the fruit. among the genes identified in other systems to be associated with cuticle biosynthesis are the extracellular gdsl motif lipase/hydrolase proteins and lipid transfer proteins, which have been implicated in lipid transport to extracellular surfaces. the cucumber fruit transcriptome set included eleven gdsl motif lipase/hydrolase protein family members that were represented by at least ests, including five with more than ests. the majority showed peak levels at or 12– dpp, with virtually no measured reads until either or dpp. twelve lipid transfer protein family members with greater than ests/contig also were observed in the transcriptome data set, including four with greater than ests. as for the gdsl motif lipase/hydrolase protein genes, the majority of the lipid transfer proteins were most highly represented from 8– dpp; transcript levels of one gene peaked at 4– dpp. a homolog of the transcription factor gene shine, which is associated with cuticle production in arabidopsis also exhibited peak transcript abundance at dpp. additionally, transcript levels of two cyctochrome p family members that have been associated with cutin biosynthesis; and two putative beta amyrin synthases, enzymes which have been associated with cuticular wax synthesis in tomato, also peaked at 8dpp. in contrast, two putative gdsl family members and one lipid transfer protein with moderate transcript levels were observed almost exclusively at dpp, suggesting possible floral, rather than fruit, expression. late/post exponential growth stress-related genes were over-represented at all stages, but considerably more so at 12– dpp than at the younger ages of 0– and dpp. the 12+ dpp age group had the highest representation of abiotic and biotic stress related genes, including a variety of heat shock, redox, biotic defense and ethylene-related transcripts. of the genes in this group, have high homology with genes associated with plant stress, including at least transcription stress-related factors such as wrky activator of sa-dependent defense; radical induced cell death; ethylene response, salt stress, and heat shock transcription factors. overall, the group of genes with peak abundance at 12+ dpp was significantly enriched for transcription factor genes accounting for 16% of the top % set. this may be contrasted with the total cucumber fruit transcriptome data set where transcription and transcription factor activity related genes were represented at a normalized frequency of relative to occurrence in the arabidopsis genome. transcription factors in the top % of 0+ and dpp groups also were represented at a comparable frequency to the arabidopsis genome, accounting for % and % of the gene list, respectively. in addition to the stress related transcription factors with specific representation at 12– dpp, several putative transcription factor homologs were annotated to be associated with development [e.g., embryo sac development, morphogenesis, and cell expansion. furthermore, transcripts of other genes with homologs that have been implicated in development related processes are specifically observed at 12– days, such as putative homologs of tctp; btb and taz domainprotein 1; calcium-binding ef hand family protein; seed development related; and bax inhibitor  examination of early cucumber fruit growth from the period of pollination and initial fruit set through the end of the exponential growth phase shows a dynamic series of physiological and morphological changes. transcriptomic analysis of the predominant genes represented in the different age groups as identified either by total number of reads, portion of transcript reads observed at that age, or genes grouped by k-means cluster analysis, told a story aligned with the sequential stages of development. transcript representation in the youngest ages, 0– dpp, was uniquely characterized by genes associated with cell division, cell organization and biogenesis. at dpp, transcription of the cell cycle genes was declining, while chloroplast, photosynthesis, and chloroplast-localized genes were peaking. transcripts highly abundant during the exponential growth phase, 4– dpp, included extensive representation of genes associated with cell structure such as cytoskeleton, vacuoles and cell walls, along with surface lipid metabolism related genes, in concert with the period of greatest increase in cuticle thickness. a second shift in the transcriptome profile was observed at 12– dpp with significant enrichment of abiotic and biotic stress related genes and stress-related and developmental transcription factor gene homologs. the enriched representation of numerous transcription factors relative to earlier ages suggests a programmatic change away from fruit growth, toward defense, and ultimately fruit maturation. this is also the time period where we have observed transition of cucumber fruit from susceptibility to resistance to p. capsici. classically, fleshy fruit development is described to consist of three stages post pollination: cell division, cell expansion, and ripening. these results suggest that the interval between expansive growth and ripening may include further developmental differentiation; an emphasis on defense would be consistent with the role of fruit in protecting the developing seeds during embryo maturation prior to facilitating seed dispersal. finally, approximately 5% of the contigs represented by ≥ reads either did not have identified putative homologs, or did not have homologs outside of cucurbits suggesting potentially unique genes specific to cucumber or cucurbits. the observation that these genes, as well as genes with homologs but with no annotated function, rarely occurred in the 0– dpp group, suggests commonality among processes associated with early fruit set and cell division and/or greater knowledge about the fruit set stage. the predominance of transcripts without non-cucurbit homologs or with unknown predicted functions during the peak exponential growth stage may reflect fewer studies to date about this phase of growth, or unique adaptations of cucurbits to allow for extreme fruit growth rates associated with these species. collectively, the transcriptomic information provided by the young cucumber fruit samples coupled with morphological analyses provide an informative picture of early fruit development characterized by phases of active cell division, fruit expansion including novel or uncharacterized genes, and response to the environment, as summarized in figure the progressive modules of transcript abundance tell a story of cell division, development of photosynthetic capacity, cell expansion and fruit growth, phloem activity, protection of the fruit surface, and finally transition away from fruit growth toward defense and maturation. 
progressive waves of transcript abundance were associated with cell division, development of photosynthetic capacity, cell expansion and fruit growth, phloem activity, protection of the fruit surface, and finally transition away from fruit growth toward a stage of enhanced stress responses. the transcriptome data coupled with morphological analyses provide an informative picture of early fruit development. these results suggest that the interval between expansive growth and ripening includes further developmental differentiation with an emphasis on defense. principal component analysis separated the fruit ages into three groups corresponding with cell division/pre-exponential growth ), peak exponential expansion, and late/post-exponential expansion stages of growth. all contigs were mapped to the cucumber genome. the group of genes with peak transcript levels at 8dpp included cytoskeleton, cell wall, lipid metabolism and phloem related proteins. early stages of fruit development from initial set through exponential growth are critical determinants of size and yield, however, there has been little detailed analysis of this phase of development. a second shift in transcript profile was observed at 12-16dpp, which was characterized by abiotic and biotic stress related genes and significant enrichment for transcription factor gene homologs, including many associated with stress response and development. cucumis sativusexponential fruit growthfruit maturationfruit setfruit surfacegene expression the increased transcript levels of cucurbit-specific genes during the exponential growth stage may indicate unique factors contributing to rapid growth in cucurbits. this group was also dominated by genes with unknown function or without known homologs outside of cucurbits. in this study we combined morphological analysis with pyrosequencing to study transcript level changes occurring in young cucumber fruit at five ages from anthesis through the end of exponential growth.
 early stages of fruit development from initial set through exponential growth are critical determinants of size and yield, however, there has been little detailed analysis of this phase of development. in this study we combined morphological analysis with pyrosequencing to study transcript level changes occurring in young cucumber fruit at five ages from anthesis through the end of exponential growth.  the fruit samples produced million ests which were assembled into contigs with a mean length of base pairs and a mean of reads per contig. all contigs were mapped to the cucumber genome. principal component analysis separated the fruit ages into three groups corresponding with cell division/pre-exponential growth ), peak exponential expansion, and late/post-exponential expansion stages of growth. transcripts predominantly expressed at and dpp included homologs of histones, cyclins, and plastid and photosynthesis related genes. the group of genes with peak transcript levels at 8dpp included cytoskeleton, cell wall, lipid metabolism and phloem related proteins. this group was also dominated by genes with unknown function or without known homologs outside of cucurbits. a second shift in transcript profile was observed at 12-16dpp, which was characterized by abiotic and biotic stress related genes and significant enrichment for transcription factor gene homologs, including many associated with stress response and development.  the transcriptome data coupled with morphological analyses provide an informative picture of early fruit development. progressive waves of transcript abundance were associated with cell division, development of photosynthetic capacity, cell expansion and fruit growth, phloem activity, protection of the fruit surface, and finally transition away from fruit growth toward a stage of enhanced stress responses. these results suggest that the interval between expansive growth and ripening includes further developmental differentiation with an emphasis on defense. the increased transcript levels of cucurbit-specific genes during the exponential growth stage may indicate unique factors contributing to rapid growth in cucurbits. cucumis sativusexponential fruit growthfruit maturationfruit setfruit surfacegene expression 