BMC Genomics 2006, 7:12 http://www.biomedcentral.com/1471-2164/7/12 EdFxiifgfpeurreresensti8otnissoufesstructural genes of the flavonoid pathway in Expression of structural genes of the flavonoid path- way in different tissues. Expression profiles of flavonoid hydroxylase genes were analysed alongside nine major struc- tural genes of the flavonoid pathway in the following tissues: 1, apex and apical leaflet (pale green); 2, apex and apical leaf- lets (reddish pigmented shoot tips); 3, fully expanded leaves; 4, immature flowers; 5, rootlets; 6, seeds; 7, berry flesh; 8, berry skin (pre-véraison, green); 9, berry skin (post-véraison, red). The actin gene was used as a constitutive gene for the normalisation of cDNA samples. hydroxylases into the UV-protecting flavonols. Inner tis- sues (seeds and berry flesh) and underground organs (rootlets) did not exhibit detectable levels of Fls1. Expression profiling of flavonoid hydroxylase genes in skin of ripening berries The time-course of expression of flavonoid hydroxylase genes in the red-skinned cultivar 'Merlot' revealed that gene VvF3'H-1 is expressed throughout ripening. High transcript levels were detected from the first sampling date at the stage when catechins are rapidly synthesised and peak at 5.6 mg g-1 of berry skin (Figure 9). VvF3'H-1 expression decreased 8–10 weeks after blooming, at the ripening stage when most genes committed to the flavo- noid pathway have been reported to level off [1]. VvF3'H- 1 transcripts increased soon after, at the onset of véraison (August 12th), which were at that point in time 2-fold higher in red berries than in green berries collected from the same bunch. VvF3'H-1 expression lasted till harvest, with a secondary peak of expression one week before har- vest, concurrently at the peak of anthocyanin content. The gene VvF3'5'H-1 was weakly expressed during the ear- liest ripening stages. At the onset of véraison, transcripts of VvF3'5'H-1 were poorly detectable in green berries, while they increased 50-fold in berries that had already turned red within the same bunch. The peak of VvF3'5'H-1 expression corresponded to August 24th, at the stage when all berries had turned red. As described for VvF3'H-1, a weaker peak of expression was found one week before harvest. Expression of VvF3'5'H-2 was checked by semi- quantitative PCR because the different isoforms of this gene were distinguishable by size (Figure 10). Transcripts of the isoforms VvF3'5'H-2-long and VvF3'5'H-2-medium did appear at the onset of véraison and lasted till full rip- ening, whilst the isogene VvF3'5'H-2-short was constitu- tively expressed throughout the ripening. Accumulation, composition of anthocyanins and colour evolution in berry skin during the progress of ripening Biometric and analytical parameters have been used to follow the progress of ripening (Figure 9). The onset of ripening began on August 3rd when sugars started to accu- mulate and acidity commenced to decline. The onset of véraison (August 12th) was considered as the time when 10% of the berries had turned red (H° = 102.98, C* = 18.27). Bunches went through this phase of colour transi- tion until August 24th when all berries had turned red (H° = 20.46, C* = 7.43). Anthocyanins have been detected by HPLC analysis since the onset of véraison on August 12th (0.4 mg g-1). The accumulation of anthocyanins lasted until September 20th, one week before harvest (19.8 mg g- 1), slightly decreasing at harvest (17.1 mg g-1). At the earliest stage of the véraison, cyanidin-based anthocyanins (cyanidin and peonidin) were roughly as abundant as delphinidin-based anthocyanins (delphini- din, malvidin and petunidin) (42% versus 58%). Then, on August 24th the percentage of delphinidin-based anthocyanin (79%) peaked simultaneously with the high- est level of expression of VvF3'5'H-1. The fraction of tri- substituted anthocyanins slightly decreased in the follow- ing weeks (77–78%), then remaining substantially unchanged until harvest (75%). Berry skin colour moder- ately varied from H° = 331.55, C* = 3.83 to H° = 306, C* = 2.59 from September 2nd to September 28th. Anthocy- anin composition in berry skin changed over the period of Page 12 of 17 (page number not for citation purposes)