Transcriptome analyses of early cucumber fruit growth identifies distinct gene modules associated with phases of development

Abstract

ABBACKGROUND: 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 454 pyrosequencing to study transcript level changes occurring in young cucumber fruit at five ages from anthesis through the end of exponential growth.

Results: The fruit samples produced 1.13 million ESTs which were assembled into 27,859 contigs with a mean length of 834 base pairs and a mean of 67 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 (0 and 4 days post pollination (dpp)), peak exponential expansion (8dpp), and late/post-exponential expansion stages of growth (12 and 16 dpp). Transcripts predominantly expressed at 0 and 4 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.

Conclusions: 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.

Figure 1

Growth and development of cucumber fruits. (A) Increase in fruit length and diameter as a function of days post pollination (dpp). (B) Changes in fruit surface during fruit growth including spine maturation and abscission, development and subsidence of warts, presence of ‘bloom’ and loss of chlorophyll. (C) Micrographs showing changes in fruit surface with age (magnification 200x; staining was with Sudan IV). (D) Thickness of fruit pericarp and placenta. (E) Cross section of developing cucumber fruit at 0, 4, 8, 12 and 16 dpp

Figure 2

Comparison of transcripts expressed in the cucumber fruit libraries. (A) Portion of contigs represented by at least 30 ESTs in a given fruit age library [0, 4, 8, 12, or 16 days post pollination (dpp)] that did not have putative homologs in Arabidopsis or other sequences present in the NCBI nr database. (B) Principal component analysis of transcripts expressed at the five different fruit ages. (C) Relationship between ages grouped by principal component analysis and fruit growth. (D) Venn diagrams showing genes commonly expressed among the three age groups

Figure 3

Portion of gene expression observed in each age group and biological enrichment analysis. (A) Distribution of the portion of gene expression observed at each age group for all contigs with ≥30 reads. Shading represents those contigs most strongly expressed for each of the age groups (top 2.5%). (B) Biological enrichment analysis of contigs with age group-enriched expression as identified in (A). Functional distribution, normalized frequency, and bootstrap standard deviation (SD) of contigs with putative Arabidopsis homologs was determined using the categories classification from the Classification SuperViewer from Bio-Array Resource for Arabidopsis Functional Genomics for Gene Ontology [25]. Shading indicates those categories that are significantly enriched (P < 0.05)

Figure 4

Functional groups of genes showing age-specific expression. (A) Expression of cyclin related genes relative to fruit age plotted as percent total expression for that transcript observed at each age [putative homologs of cyclins B1;2 (At5g06150), B1;4 (At2g26760); D1;1 (At1g70210);, D3;1 (At4g34160), D3;3 (At3g50070), D5;1 (At4g37630), and cyclin dependent kinases (CDK), CDKB1;2 (At2g38620), CDKB2;2 (At1g20930), CDKD1;3 (At1g18040), CDKE;1 (At5g63610); CKS1 (At2g27960)]. (B) GDSL-motif lipase/hydrolase family protein genes (putative homologs of At1g09390, At1g56670, At2g04570, At2g42990, At3g16370, At3g48460, At5g03610, At5g14450, At5g33370, At5g62930) and transcription factor SHINE1 (At 1 g15360; dotted line). (C,) Lipid transfer protein (LTP) family protein genes (putative homologs of At1g48750, At1g62510, At2g10940, At2g45180, At5g01870, At5g64080). (D) Phloem proteins. Solid lines indicate cucurbit specific phloem proteins as listed in Table 1. Dotted lines indicate putative homologs to Arabidopsis phloem proteins (ATPP) 2-A genes (two for ATPP2-A1, one for ATPP2-A9, and ATPP2-A13); dashed lines are putative homologs of ATPP2-B genes (ATPP-B10 and ATPP-B12). (E) Transcription factors showing preferential expression at 12 +16 dpp (putative homologs of At1g27730, At1g50640, At2g17040, At2g26150, At2g40140, 2 g46240, At3g15210, At3g15510, At3g16770, AT3g56400, At4g11660, At4g16780, At4g39250, At5g25560, At5g51190)

Figure 5

Chlorophyll content and expression of chlorophyll and chloroplast-related transcripts in relationship to fruit age. (A) Chlorophyll content. (B) Expression of chlorophyll and chloroplast-related transcripts in relationship to fruit age. Gene expression is plotted as percent of total expression observed for that transcript at each age. The heavy white line represents average percent gene expression at each age for the 91 genes (Supplemental file 2) with homologs in Arabidopsis annotated to be associated with chlorophyll or chloroplasts

Figure 6

Profiles of cucumber fruit transcripts showing age-specific expression patterns as determined by K-means analysis. Analyses were performed using Cluster 3.0 software [25]

Figure 7

Schematic representation of morphological and gene expression changes occurring during early cucumber fruit development. Gene expression data refer to periods of peak expression for indicated gene categories. Data for respiration, cell division, and susceptibility to P. capsici are from Marcelis and Hofman-Eijer [4], Colle et al. [7], and Gevens et al. [9], respectively