Transcriptional control of anthocyanin biosynthetic genes in extreme phenotypes for berry pigmentation of naturally occurring grapevines

Abstract

Background: Fruit coloration of red-skinned grapevines is mainly due to anthocyanin pigments. We analysed a panel of nine cultivars that included extreme phenotypes for berry colour, ranging from green (absence of anthocyanins) to red, purple, violet and blue. Expression of six genes of the anthocyanin pathway coding for flavanone-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), flavonoid 3',5'-hydroxylase (F3'5'H), UDP-glucose:flavonoid-3-O-glucosyltransferase (UFGT), glutathione-S-transferase (GST), O-methyltransferase (OMT) and four transcription factors (MybA, MybB, MybC, MybD) was analysed by quantitative RT-PCR at four developmental stages from before the onset of ripening until full maturity and compared to anthocyanin metabolites.

Results: Total anthocyanin content at full maturity correlated well with the cumulative expression of F3H, UFGT and GST throughout ripening. Transcripts of the last two genes were absent in the green-skinned cultivar 'Sauvignonasse', also known as 'Tocai friulano', and were at least 10-fold less abundant in pale red cultivars, such as 'Pinot gris' and 'Gewürztraminer', compared to fully coloured cultivars. Predominance of tri-hydroxylated anthocyanins (delphinidin, petunidin and malvidin) in cultivars bearing dark berries with violet and blue hue was associated with higher ratios of F3'5'H/F3'H transcription, compared to red-skinned cultivars. Higher levels of OMT transcripts were observed in berries of cultivars that accumulated methoxylated forms of anthocyanins more abundantly than non-methoxylated forms.

Conclusion: Colour variation of the grape berry conforms to a peculiar pattern of genotype-specific expression of the whole set of anthocyanin genes in a direct transcript-metabolite-phenotype relationship. Cumulative mRNA levels of the structural genes and their relative abundance throughout ripening explained per se the final phenotype for anthocyanin content, anthocyanin composition, colour intensity and colour hue of grapes at berry maturity.

Figure 1

Anthocyanin concentration and gene expression in berry skin. (A) Total anthocyanins in nine cultivars. Concentration is expressed as mg g^-1 of skin of malvidin equivalents; (B) UFGT and (C) GST gene expression in four cultivars with no, low, medium and high amount of total anthocyanins at four ripening stages. At mid-véraison green berries were analysed separately from red berries on the same cluster; (D, E, F) linear regression between cumulative transcription of three genes (F3H, UFGT and GST) throughout ripening (calculated as the area below the expression curve) and final anthocyanin content.

Figure 2

Anthocyanin hydroxylation and expression of flavonoid 3',5'-hydroxylases. (A) Percentage of tri-hydroxylated anthocyanins among 3-monoglucoside anthocyanins; (B) evolution of anthocyanin composition from the onset of coloration to full maturity; (C) F3'5'H gene expression in four cultivars with no, low, medium and high percentage of tri-hydroxylated anthocyanins at four ripening stages. At mid-véraison green berries were analysed separately from red berries on the same cluster; (D) pattern of F3'5'H gene expression normalised to the expression level of the anthocyanin biosynthetic gene UFGT (F3'5'H /UFGT); (E) linear regression between the cumulative F3'5'H /UFGT ratio and the final percentage of tri-hydroxylated anthocyanins; (F) pattern of F3'5'H gene expression normalised to the expression level of F3'H (F3'5'H /F3'H).

Figure 3

Colour evolution of berry skin in nine cultivars. Colour was reproduced by an image editing software (CorelDraw) using mean L, a, b values averaged on 70 berries at all ripening stages investigated in this study. At mid-véraison green berries were analysed separately from red berries on the same cluster.

Figure 4

Anthocyanin methoxylation and expression of O-methyltransferase. (A) Percentage of methoxylated anthocyanins (peonidin, petunidin and malvidin) among 3-monoglucoside anthocyanins; (B) OMT gene expression in four reference cultivars at four ripening stages. At mid-véraison green berries were analysed separately from red berries on the same cluster; (C) pattern of OMT gene expression normalised to the expression level of the anthocyanin biosynthetic gene UFGT (OMT /UFGT) in all cultivars.