Gene expression in developing watermelon fruit

Abstract

Background: Cultivated watermelon form large fruits that are highly variable in size, shape, color, and content, yet have extremely narrow genetic diversity. Whereas a plethora of genes involved in cell wall metabolism, ethylene biosynthesis, fruit softening, and secondary metabolism during fruit development and ripening have been identified in other plant species, little is known of the genes involved in these processes in watermelon. A microarray and quantitative Real-Time PCR-based study was conducted in watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus] in order to elucidate the flow of events associated with fruit development and ripening in this species. RNA from three different maturation stages of watermelon fruits, as well as leaf, were collected from field grown plants during three consecutive years, and analyzed for gene expression using high-density photolithography microarrays and quantitative PCR.

Results: High-density photolithography arrays, composed of probes of 832 EST-unigenes from a subtracted, fruit development, cDNA library of watermelon were utilized to examine gene expression at three distinct time-points in watermelon fruit development. Analysis was performed with field-grown fruits over three consecutive growing seasons. Microarray analysis identified three hundred and thirty-five unique ESTs that are differentially regulated by at least two-fold in watermelon fruits during the early, ripening, or mature stage when compared to leaf. Of the 335 ESTs identified, 211 share significant homology with known gene products and 96 had no significant matches with any database accession. Of the modulated watermelon ESTs related to annotated genes, a significant number were found to be associated with or involved in the vascular system, carotenoid biosynthesis, transcriptional regulation, pathogen and stress response, and ethylene biosynthesis. Ethylene bioassays, performed with a closely related watermelon genotype with a similar phenotype, i.e. seeded, bright red flesh, dark green rind, etc., determined that ethylene levels were highest during the green fruit stage followed by a decrease during the white and pink fruit stages. Additionally, quantitative Real-Time PCR was used to validate modulation of 127 ESTs that were differentially expressed in developing and ripening fruits based on array analysis.

Conclusion: This study identified numerous ESTs with putative involvement in the watermelon fruit developmental and ripening process, in particular the involvement of the vascular system and ethylene. The production of ethylene during fruit development in watermelon gives further support to the role of ethylene in fruit development in non-climacteric fruits.

Figure 1

List of 2x-ESTs that show at least two-fold differential expression by microarray analysis. A total of 335 2x-ESTs were identified by microarray analysis that showed at least a two-fold difference in modulation as compared to leaf. These 2x-ESTs were compared to similar sequences in the Entrez database by blastx analysis and the strongest known homology was identified. This figure shows the upper quartile, for the full image please see Additional File 1. The 2x-ESTs induced at least two-fold were highlighted in red whereas 2x-ESTs repressed at least two-fold were highlighted in green. Microarray-determined fold-change is shown for each 2x-EST for green, pink, and red flesh as compared to leaf.

Figure 2

Differential modulation of 127 2x-ESTs was confirmed by Q-PCR using two additional biological replicates. One hundred and twenty seven 2x-ESTs that show differential modulation in the microarray (Biorep 1) were confirmed by Q-PCR (Biorep 2 & Biorep 3). This figure shows the first half of these 2x-ESTs, for the full image please see Additional File 2. The 2x-ESTs induced at least two-fold were highlighted in red whereas 2x-ESTs repressed at least two-fold were highlighted in green. Q-PCR fold inductions are shown for each 2x-EST for green, pink, and red flesh as compared to leaf.

Figure 3

Venn diagram of 176 induced 2x-ESTs. Venn diagram showing the overlapping and stage-specific genes up-regulated in flesh tissue of watermelon fruit at three stages of development; green (12-days after pollination), pink (24-days after pollination) and red (36-days after pollination). Eighty-nine genes were expressed at all stages of development; some genes were stage specific (green 23; pink 10; and red 15) while others were overlapping in two stages (for gene identity see Figure 4 and Additional File 3).

Figure 4

The 2x-ESTs that are differentially modulated at a specific fruit stage compared to leaf as determined by microarray analysis. One hundred and seventy-six 2x-ESTs that exhibit induction in one or more fruit types with a false discovery rate (FDR) of less than 0.05 were identified. This figure shows the first 60 of these 2x-ESTs, for the full image please see Additional File 3.

Figure 5

Ethylene production during fruit development. The watermelon cultivar 'Sugar Baby' was grown and ethylene production was measured during the green, white, pink and red fruit developmental stages.