Identification and functional characterization of the dirigent gene family in Phryma leptostachya and the contribution of PlDIR1 in lignan biosynthesis

Abstract

Background Furofuran lignans, the main insecticidal ingredient in Phryma leptostachya, exhibit excellent controlling efficacy against a variety of pests. During the biosynthesis of furofuran lignans, Dirigent proteins (DIRs) are thought to be dominant in the stereoselective coupling of coniferyl alcohol to form ( ±)-pinoresinol. There are DIR family members in almost every vascular plant, but members of DIRs in P. leptostachya are unknown. To identify the PlDIR genes and elucidate their functions in lignan biosynthesis, this study performed transcriptome-wide analysis and characterized the catalytic activity of the PlDIR1 protein. Results Fifteen full-length unique PlDIR genes were identified in P. leptostachya. A phylogenetic analysis of the PlDIRs classified them into four subfamilies (DIR-a, DIR-b/d, DIR-e, and DIR-g), and 12 conserved motifs were found among them. In tissue-specific expression analysis, except for PlDIR7, which displayed the highest transcript abundance in seeds, the other PlDIRs showed preferential expression in roots, leaves, and stems. Furthermore, the treatments with signaling molecules demonstrated that PlDIRs could be significantly induced by methyl jasmonate (MeJA), salicylic acid (SA), and ethylene (ETH), both in the roots and leaves of P. leptostachya. In examining the tertiary structure of the protein and the critical amino acids, it was found that PlDIR1, one of the DIR-a subfamily members, might be involved in the region- and stereo-selectivity of the phenoxy radical. Accordingly, LC–MS/MS analysis demonstrated the catalytic activity of recombinant PlDIR1 protein from Escherichia coli to direct coniferyl alcohol coupling into ( +)-pinoresinol. The active sites and hydrogen bonds of the interaction between PlDIR1 and bis-quinone methide (bisQM), the intermediate in ( +)-pinoresinol formation, were analyzed by molecular docking. As a result, 18 active sites and 4 hydrogen bonds (Asp-42, Ala-113, Leu-138, Arg-143) were discovered in the PlDIR1-bisQM complex. Moreover, correlation analysis indicated that the expression profile of PlDIR1 was closely connected with lignan accumulations after SA treatment. Conclusions The results of this study will provide useful clues for uncovering P. leptostachya's lignan biosynthesis pathway as well as facilitate further studies on the DIR family.

Integrative analysis of the metabolome and transcriptome provides insights into the mechanisms of lignan biosynthesis in Herpetospermum pedunculosum (Cucurbitaceae)

“…In subsequent synthesis, coniferyl alcohol serves as an important starting material for the synthesis of Herpetospermum -specific lignans (including herpetrione, herpetotriol, and herpetin), which is a very complex synthesis process controlled by numerous genes and enzymes. Pinoresinol is an important intermediate in the formation of herpetrione, which is obtained through the oxidation and cyclization of two molecules of coniferyl alcohol in the presence of dirigent protein (DIR) [ 23 ]. Once Pinoresinol is formed, it undergoes cyclization and oxidation with another molecule of coniferyl alcohol to obtain another intermediate.…”

“…Moreover, activation of the phenylpropanoid pathway is also linked to the upregulation of DIR and POD , which controls the initiation of species-specific lignan biosynthesis [ 16 , 42 , 43 ]. Heterologous expression of PlDIR from Phryma leptostachya stereoselectively couples coniferyl alcohol to form pinoresinol [ 23 ]. We found a DEG encoding a similar protein in H. pedunculosum (XP_023512165.1, E-value: 2 × 10 − 111 , percentage identity: 84.49%), which is a candidate for the stereo-configuration of a lignan.…”