Methylation mediated by an anthocyanin, O-methyltransferase, is involved in purple flower coloration in Paeonia

Abstract

Anthocyanins are major pigments in plants. Methylation plays a role in the diversity and stability of anthocyanins. However, the contribution of anthocyanin methylation to flower coloration is still unclear. We identified two homologous anthocyanin O-methyltransferase (AOMT) genes from purple-flowered (PsAOMT) and red-flowered (PtAOMT) Paeonia plants, and we performed functional analyses of the two genes in vitro and in vivo. The critical amino acids for AOMT catalytic activity were studied by site-directed mutagenesis. We showed that the recombinant proteins, PsAOMT and PtAOMT, had identical substrate preferences towards anthocyanins. The methylation activity of PsAOMT was 60 times higher than that of PtAOMT in vitro. Interestingly, this vast difference in catalytic activity appeared to result from a single amino acid residue substitution at position 87 (arginine to leucine). There were significant differences between the 35S::PsAOMT transgenic tobacco and control flowers in relation to their chromatic parameters, which further confirmed the function of PsAOMT in vivo. The expression levels of the two homologous AOMT genes were consistent with anthocyanin accumulation in petals. We conclude that AOMTs are responsible for the methylation of cyanidin glycosides in Paeonia plants and play an important role in purple coloration in Paeonia spp.

Keywords: Anthocyanin O-methyltransferase; Paeonia; catalytic activity; flavonoids; flower coloration; single amino acid substitution..

Fig. 1.

Biochemical pathway and chemical structural information regarding selected flavonoid compounds in plants. (A) Schematic diagram of the biosynthetic pathways of the major flavonoids (Rausher et al., 2008; Wessinger and Rausher, 2012). Dashed arrows represent the unclear steps. The names and structures of three anthocyanidin compounds are indicated. The enzyme names in black boxes are as follows: CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′,5′-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; FNS, flavones synthase; FLS, flavonol synthase; GT, glycosyltransferase; OMT, O-methyltransferase. (B) Chemical structures of anthocyanins, flavones, and flavonols in Paeonia flower petals (Wang et al., 2001; Li et al., 2009).

Fig. 2.

Flowers from five developmental stages and the anthocyanin accumulation at five developmental stages (S1–S5) from Paeonia suffruticosa cv. ‘Gunpohden’ (A) and P. tenuifolia (B), as well as expression patterns of AOMT transcripts at the corresponding developmental stages and in different tissues, measured by qPCR. The expression values have been normalized against the Actin gene and are expressed as relative abundances.

Fig. 3.

Sequence alignments of PsAOMT and PtAOMT with predicted secondary structural elements. The reference sequences VvAOMT (grapevine, BQ796057), GmAOMT (black soybean, ADX43927) and McPFOMT (ice plant, AY145521) are also included. α-Helices and β-strands are represented as cylinders and arrows, respectively. The residues conserved in all OMTs are shaded. (This figure is available in colour at JXB online.)

Fig. 4.

Phylogenetic tree of selected OMT peptides. OMTs in bold are known to act in the methylation of anthocyanins. OMT names and GenBank accession numbers are as follows: Fuchsia, FMT, HB975539; Vitis vinifera, VvAOMT, BQ796057; Torenia, TMT5, HB975529; Petunia difE, FMT, HB975519; Cyclamen persicum×Cyclamen purpurascens, CkmOMT2, BAK74804; Mesembryanthemum crystallinum, McPFOMT, AY145521; Stellaria longipes, SlCCoAOMT, L22203; Glycine max, GmAOMT, ADX43927; Arabidopsis thaliana, AtCCoAOMT, AAM64800; Zea mays, ZmCCoAOMT, AJ242980; Medicago sativa, MsCCoAOMT, AAC28973; Vitis vinifera, VvCCoAOMT, Z54233; Nicotiana tabacum, NtCCoAOMT, U38612; Populus balsamifera subsp., PtCCoAOMT, AJ224896; Homo sapiens, HOMT, A38459; Medicago sativa, MsIOMT, AAC49927; Catharanthus roseus, CrAOMT, AY127568; Oryza sativa, OsROMT, DQ288259; Thalictrum tuberosum, TtOMT, AF064693; Nicotiana tabacum, NtCOMT, AF484252; Vitis vinifera, VvCOMT, AF239740; Medicago sativa, MsCOMT, M63853; Chrysosplenium americanum, CaCOMT, AAA86982 and CaAOMT, U16794. The number beside the branches represents the bootstrap values based on 1000 replicates using MEGA 5. Bar, nucleotide substitutions per site.

Fig. 5.

Molecular model of the PsAOMT active site. The SAH (S-adenosyl-l-homocysteine), Cy3G5G, and substrate-binding residues are represented as sticks, and labelled in cyan, grey, and white, respectively. The oxygen, nitrogen, and sulfur atoms are labelled in red, blue, and yellow, respectively.

Fig. 6.

Characterization of AOMT activity in vivo using transient expression in strawberry and the stable transformation of tobacco. (A) Anthocyanin profiles of strawberry fruits with transient PBI121 or PAP1 expression, and the co-expression of PAP1/PsAOMT, and PAP1/PtAOMT (4 d of expression). Other anthocyanins are shown in Supplementary Table S5, available at JXB online. (B) Anthocyanins from transgenic tobacco flowers (35S::PsAOMT, 35S::PtAOMT and empty vector control, respectively) were analysed by HPLC-MS.

Fig. 7.

CIELab colour space analysis of transgenic tobacco flower colour. L* represents lightness, from black (0) to white (100); a* represents red (positive) to green (negative); b* represents yellow (positive) to blue (negative); and C* represents the chroma or saturation of the colour. Boxes of each parameter with no common letter in the figure body indicate a significant difference at P<0.05 (n≥9).