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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- O -β-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, Nm AMT6 almost exclusively yielded petunidin 3- O -glucoside rather than malvidin 3- O -glucoside. This specificity is consistent with the anthocyanin composition of Nemophila petals.

plant biotechnology

Molecular cloning of flavonoid biosynthetic genes and biochemical characterization of anthocyanin &lt;i&gt;O&lt;/i&gt;-methyltransferase of &lt;i&gt;Nemophila menziesii&lt;/i&gt; Hook. and Arn.