Anthocyanins: the secret of Sicilian blood oranges

A flesh-specific oligonucleotide custom array was designed to study gene expression during blood orange ripening. The array included 301 probes derived from a subtracted SSH library, a cDNA-AFLP collection, and a set of regulatory genes from the HarvEST Citrus database. The custom array was hybridised using fruits of Moro, a blood cultivar, and Cadenera, a common one, at three different ripening stages: the immature phase, the halfway point of maturation (corresponding to the start of Moro pigmentation) and the full ripening. Among 301 probes, 27 in total, corresponding to 20 different transcripts, gave indication of differential expression in stage-stage and/or cultivar-cultivar comparisons. Transcripts encoding for anthocyanins biosynthesis represented most of the total over-expressed probes. The remaining differentially expressed transcripts were functionally associated with primary metabolism as flavour biosynthesis, defense and signal transduction. The expressed products indicating differentially expression were confirmed by Real time RT-PCR. The main novelty of the customization of the presented array is the use of expressed sequences specifically derived from blood orange flesh to study fruits of different cultivars in some ripening stages and providing further information about processes related to anthocyanin pigmentation in fruit flesh. To investigate the involvement of a member of the Glutathione S-Transferase gene family in the vacuolarization of anthocyanins, the SemiQ RT-PCR analysis was performed on various tissues (albedo, flavedo, flesh, young and adult leaves, ovary) of Moro and Cadenera, comparing in silico prevision and molecular approaches. Real time RT-PCR was conduced on different pigmented and not pigmented tissues of lemon and sweet oranges, showing the involvement fruit-specific (flesh and rind) of a GST in the anthocyanins pathway.