An Alternative Pathway for Formononetin Biosynthesis in Pueraria lobata

Abstract

The O-methylation is an important tailing process in Pueraria lobata isoflavone metabolism, but the molecular mechanism governing it remains not elucidated. This manuscript describes the mining of key O-methyltransferases (OMTs) involved in the process. Using our previously constructed P. lobata transcriptome, the OMT candidates were searched, extensively analyzed, and their functions were investigated by expression in yeast, Escherichia coli, or Glycine max hairy roots. Here, we report the identification of the key OMT gene responsible for formononetin production in P. lobata (designated as PlOMT9). PlOMT9 primarily functions as an isoflavone-specific 4′-O-methyltransferase, although it shows high sequence identities with isoflavone 7-O-methyltransferases. Moreover, unlike the previously reported OMTs that catalyze the 4′-O-methylation for formononetin biosynthesis at the isoflavanone stage, PlOMT9 performs this modifying step at the isoflavone level, using daidzein rather than 2,7,4′-trihydroxy-isoflavanone as the substrate. Gene expression analyses and metabolite profiling supported its proposed roles in P. lobata. Using the system of transgenic G. max hairy roots, the role of PlOMT9 in the biosynthesis of formononetin was further demonstrated in vivo.

Global Transcriptome Analyses Reveal Differentially Expressed Genes of Six Organs and Putative Genes Involved in (Iso)flavonoid Biosynthesis in Belamcanda chinensis

“…The most common sites of methylation of isoflavonoids in legumes are the C4′- and 7-positions. To date, C3′-, 4′-, 5-, or 7- O- methylation activity of IOMTs has been characterized in several legumes ( Khouri et al, 1988 ; He et al, 1998 ; He and Dixon, 2000 ; Liu and Dixon, 2001 ; Akashi et al, 2003 ; Deavours et al, 2006 ; Li et al, 2016a , b ; Mochida et al, 2017 ). No isoflavone-6- O- methyltransferase has yet been found so far.…”

Comparative Genomics of Spatholobus suberectus and Insight Into Flavonoid Biosynthesis

“…In order to detect the candidate genes in the flavonoid biosynthesis pathways in the Leguminosae species, including S. suberectus, G. max, L. japonicus, G. uralensis, C. arietinum. The similarity calculated by BLASTP searching (e-value <= 1e−5) with known members from the model species G. max and A. thaliana and other reported plants, we have searched 15 genes involved in flavonoid synthesis in the above genome, including phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H, a CYP450 gene) and 4-coumarate CoA ligase (4CL), the first three enzymes in phenylpropanoid pathway (Saito et al, 2013), and others enzymes in this pathway including chalcone synthase (CHS), chalcone isomerase (CHI) (Saslowsky and Winkel-Shirley, 2001), isoflavone synthase (IFS, a CYP450 gene), 2-hydroxyisoflavanone dehydratase (HID) (Jung et al, 2000;Shimamura et al, 2007), flavanone-3-hydroxylase (F3H, a 2-OGD gene) (Pelletier and Shirley, 1996), flavanone-3'hydroxylase (F3'H, a CYP450 gene) (Schoenbohm et al, 2000), dihydroflavonol 4-reductase (DFR) (Shirley et al, 1992), anthocyanidin synthase (ANS, alias LDOX, a 2-OGD gene) (Bowerman et al, 2012), anthocyanidin reductase (ANR) (Devic et al, 1999), flavonol synthase (FLS, a 2-OGD gene) (Pelletier et al, 1997), leucoanthocyanidin reductase (LAR) (Tanner et al, 2003), O-methyltransferase (OMT) (Hashim et al, 1990;Dhaubhadel et al, 2003;Li et al, 2016), and chalcone reductase (CHR, alias PKR, also named PKR) (Shimada et al, 2005). Because the homology of different genes is different, we choose different Identity thresholds.…”

Peer Review #1 of "Transcriptome sequencing and analysis reveals the molecular response to selenium stimuli in Pueraria lobata (willd.) Ohwi (v0.2)"

“…The isoflavone-related genes were further mined to determine whether the biosynthesis of the isoflavone in P. lobata was affected by sodium selenite treatment. The biosynthesis of isoflavones were divided into three stages: the phenylpropane pathway, the flavonoids pathway, and the isoflavone pathway He et al, 2011;Li et al, 2016a;Li et al, 2016b;Wang et al, 2016;Wang et al, 2017). Five PALs, three C4Hs, and five 4CLs were found in the PeerJ reviewing PDF | (2019:09:41362:2:0:NEW 3 Feb 2020)…”