Identification of the flavonoid hydroxylases from grapevine and their regulation during fruit development

Abstract

Flavonoids are important secondary metabolites in many fruits, and their hydroxylation pattern determines their color, stability, and antioxidant capacity. Hydroxylation of the B-ring of flavonoids is catalyzed by flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H), and may also require cytochrome b5. We report the identification of genes encoding F3'H, F3'5'H, and a putative cytochrome b5 from grapevine (Vitis vinifera L. cv Shiraz) and their transcriptional regulation in fruit. Functionality of the genes VvF3'H and VvF3'5'H1 was demonstrated by ectopic expression in petunia (Petunia hybrida), which altered flower color and flavonoid composition as expected. VvF3'H was expressed in grapes before flowering, when 3'-hydroxylated flavonols are made, and all three genes were expressed after flowering, when proanthocyanidins (PAs) are synthesized. In berry skin, expression of all three genes was low at the onset of ripening (véraison) but increased after véraison concomitant with the accumulation of 3'- and 3',5'-hydroxylated anthocyanins. VvF3'H and VvCytoB5 were expressed in seeds but not VvF3'5'H1, consistent with the accumulation of 3'-hydroxylated PAs in this tissue. VvCytoB5 expression was correlated with expression of both VvF3'H and VvF3'5'H1 in the different grape tissues. In contrast to red grapes, where VvF3'H, VvF3'5'H1, and VvCytoB5 were highly expressed during ripening, the expression of VvF3'5'H1 and VvCytoB5 in white grapes during ripening was extremely low, suggesting a difference in transcriptional regulation. Our results show that temporal and tissue-specific expression of VvF3'H, VvF3'5'H1, and VvCytoB5 in grapes is coordinated with the accumulation of the respective hydroxylated flavonols and PAs, as well as anthocyanins. Understanding the regulation of flavonoid hydroxylases could be used to modify flavonoid composition of fruits.

Figure 1.

Schematic representation of the flavonoid biosynthetic pathway. Enzymes involved in the pathway shown are CHS, chalcone synthase; CHI, chalcone isomerase; F3′H; F3′5′H; F3H, flavanone-3β-hydroxylase; DFR; LDOX; FLS, flavonol synthase; LAR; ANR; and UFGT. Examples for the different hydroxylation patterns of the flavonoid B-ring are given for naringenin (4′-hydroxylated), eriodictyol (3′,4′-hydroxylated), and pentahydroxyflavone (3′,4′,5′-hydroxylated).

Figure 2.

Phylogenetic tree showing selected flavonoid hydroxylases from GenBank or EMBL database grouping into the CYB75A and CYB75B cytochrome P450 family, respectively. The grapevine hydroxylases VvF3′H (CAI54278) and VvF3′5′H1 (CAI54277) are shown in bold. The ClustalW multiple sequence alignment was formed using the default parameters of the MEGA package (Kumar et al., 2004). The tree was constructed from the ClustalW alignment using the UPGMA method by the MEGA program. The scale bar represents 0.1 substitutions per site, and the numbers next to the nodes are bootstrap values from 100 replicates.

Figure 3.

Functional characterization of VvF3′H and VvF3′5′H1 by their ectopic expression in the petunia ht1 mutant line Skr4 × Sw63. A, Flowers of untransformed control Skr4 × Sw63 (pale lilac) and lines transgenic for VvF3′H (reddish-pink) and VvF3′5′H1 (purple). B, HPLC analysis of anthocyanidins and flavonols from flowers of the control line Skr4 × Sw63, the line H1.2 transgenic for VvF3′H, and O2.2 transgenic for VvF3′5′H1 showing the accumulation of the respective hydroxylated flavonoid. At least three additional independent Skr4 × Sw63 lines transgenic for VvF3′H or VvF3′5′H1 were analyzed and showed a similar phenotype and HPLC profile as the presented lines O2.2 or H1.2, respectively. The anthocyanidins delphinidin and pelargonidin as well as the flavonol myricetin were not detectable in any of the analyzed samples. C, Cyanidin; P, peonidin; M, malvidin; K, kaempferol; Q, quercetin.

Figure 4.

Deduced VvCytoB5 (TC45693) amino acid sequence and comparison with the cytochrome b₅ protein DIF-F (Q9ZSP7) from petunia and other related sequences. A, Sequence comparison of the deduced amino acid sequence of TC45693 from grapevine and the cytochrome b₅ protein DIF-F (Q9ZSP7) from petunia. Identical amino acids are boxed in black, and similar amino acids are boxed in gray. B, Phylogenetic tree constructed with several cytochrome b₅-related sequences recovered by using the deduced amino acid sequence of TC45693 in a BLAST algorithm on the GenBank and EMBL databases. VvCytoB5 (TC45693) is shown in bold, and the tree was constructed as described in Figure 2. The scale bar represents 0.5 substitutions per site, and the numbers next to the nodes are bootstrap values from 100 replicates.

Figure 5.

Gene expression of VvDFR, VvF3′H, VvF3′5′H1, and VvCytoB5 (TC45693) in grapes during the early stages of berry development, in seeds, and in berry skin. Flowering is marked by an arrow and occurred 8 weeks before véraison, which is the onset of ripening in grapes. Transcript levels were determined by real-time PCR and are shown relative to expression of VvUbiquitin1 in each sample. All data are presented as mean of three replicates.

Figure 6.

Gene expression of VvDFR, VvLDOX, VvF3′H, VvUFGT, VvF3′5′H1, and VvCytoB5 (TC45693) was measured in red (dark bars) and white (light bars) grape cultivars by real-time PCR. Gene expression of the indicated genes was analyzed at 2 weeks after berry ripening (véraison) in different red and white grape varieties. Transcript levels were determined by real-time PCR and are shown relative to expression of VvUbiquitin1 in each sample. All data are presented as mean of three replicates.

Figure 7.

Transcript levels of VvMybA, VvUFGT, VvF3′5′H1, and VvCytoB5 in the red grape cultivar Shiraz (dark bars) and the white cultivar Chardonnay (light bars) at early berry development (10–6 weeks before véraison) and 2 weeks after véraison. Gene expressions were determined by real-time PCR and are shown relative to expression of VvUbiquitin1 in each sample. All data are presented as mean of three replicates.