Molecular cloning of flavonoid biosynthetic genes and biochemical characterization of anthocyanin <i>O</i>-methyltransferase of <i>Nemophila menziesii</i> Hook. and Arn.

Abstract

Blue flower color of Nemophila menziesii Hook. and Arn. is derived from a metalloanthocyanin, nemophilin, which comprises petunidin-3-O-[6-O-(trans-p-coumaroyl)-β-glucoside]-5-O-[6-O-(malonyl)-β-glucoside], apigenin-7-Oβ-glucoside-4′-O-(6-O-malonyl)-β-glucoside, and Mg 2+ and Fe 3+ ions. The flavonoid biosynthetic pathway of nemophilin has not yet been characterized. RNA-Seq analysis of the petals yielded 61,491 contigs. These were searched using BLAST against petunia or torenia flavonoid biosynthetic proteins, which identified 11 putative full-length protein sequences belonging to the flavonoid biosynthetic pathway. RT-PCR using primers designed on the basis of these sequences yielded 14 sequences. Spatio-temporal transcriptome analysis indicated that genes involved in the early part of the pathway are strongly expressed during early-petal development and that those in the late part at late-flower opening stages, but they are rarely expressed in leaves. Flavanone 3-hydroxylase and flavonoid 3′,5′-hydroxylase cDNAs were successfully expressed in yeast to confirm their activities. Recombinant anthocyanin O-methyltransferase cDNA (NmAMT6) produced using Escherichia coli was subjected to biochemical characterization. Km of NmAMT6 toward delphinidin 3-O-glucoside was 22 µM, which is comparable with Km values of anthocyanin O-methyltransferases from other plants. With delphinidin 3-O-glucoside as substrate, NmAMT6 almost exclusively yielded petunidin 3-O-glucoside rather than malvidin 3-O-glucoside. This specificity is consistent with the anthocyanin composition of Nemophila petals.

Genome-wide identification of the AOMT gene family in wax apple and functional characterization of SsAOMTs to anthocyanin methylation

“…During fruit ripening period, SsAOMT5 was upregulated continuously in both Tub and Daye cultivars and got the peak at 50 days after blossom (T5); especially, the expression of SsAOMT5 in Daye cultivar increased sharply and was extremely significantly higher than that in Tub cultivar at T5 stage. This was different to the results of pomegranate, in which PgOMT04 and PgOMT09 in peel were always kept with a high expression throughout fruit development ( Zhang et al., 2021 ), and also different to the finding that NmAMT in petals of N. menziesii significantly expressed only at the early stages of flower development ( Okitsu et al., 2018 ). This discrepancy was most likely related to species.…”

Functional Analysis of a Methyltransferase Involved in Anthocyanin Biosynthesis from Blueberries (Vaccinium corymbosum)

“…Enzyme activity assay was conducted based on previous studies with some modifications. , Five flavonols (kaempferol, quercetin, morin, myricetin and dihydromyricetin), three anthocyanidins (cyanidin, delphinidin and malvidin), two flavonol glycosides (quercetrin and myricetrin) and three anthocyanins (cyanidin 3- O -glucoside, delphinidin 3- O -glucoside, petunidin 3- O -glucoside), along with methyl group donor SAM were selected to evaluate the substrate selectivity of VcOMT. The enzyme activity assay mixture (total volume was 100 μL) contained 0.1 M Tris–HCl, pH 7.0, 10 mM SAM, 100 μg VcOMT and 10 mM substrate.…”