Virus‐induced gene silencing identifies Catharanthus roseus 7‐deoxyloganic acid‐7‐hydroxylase, a step in iridoid and monoterpene indole alkaloid biosynthesis

Abstract

SUMMARYIridoids are a major group of biologically active molecules that are present in thousands of plant species, and one versatile iridoid, secologanin, is a precursor for the assembly of thousands of monoterpenoid indole alkaloids (MIAs) as well as a number of quinoline alkaloids. This study uses bioinformatics to screen large databases of annotated transcripts from various MIA-producing plant species to select candidate genes that may be involved in iridoid biosynthesis. Virus-induced gene silencing of the selected genes combined with metabolite analyses of silenced plants was then used to identify the 7-deoxyloganic acid 7-hydroxylase (CrDL7H) that is involved in the 3rd to last step in secologanin biosynthesis. Silencing of CrDL7H reduced secologanin levels by at least 70%, and increased the levels of 7-deoxyloganic acid to over 4 mg g À1 fresh leaf weight compared to control plants in which this iridoid is not detected. Functional expression of this CrDL7H in yeast confirmed its biochemical activity, and substrate specificity studies showed its preference for 7-deoxyloganic acid over other closely related substrates. Together, these results suggest that hydroxylation precedes carboxy-O-methylation in the secologanin pathway in Catharanthus roseus.

Identification and Characterization of the Iridoid Synthase Involved in Oleuropein Biosynthesis in Olive (Olea europaea) Fruits

“…Identification of Candidate Genes-The amino acid sequences from Madagascar periwinkle (C. roseus) iridoid synthase (ISY), 8-hydroxygeraniol oxidoreductase (8HGO), iridoid oxidase (IO), 7-deoxyloganetic acid-O-glucosyl transferase (7-DLGT), and 7-deoxyloganic acid hydroxylase (7-DLH), recently shown to be involved in monoterpene indole alkaloid biosynthesis (16,17,19,20), were used to search olive-expressed sequence tags by basic local alignment (BLAST) in olive transcriptomic data of olive fruit (33) and other tissues (35). The sequences of candidate genes were assembled in silico using preliminary genomic sequences.…”

“…These candidates were identified in olive transcriptomic data (33,35) by using the local alignment search tool tBLASTn with five C. roseus enzymes involved in iridoid biosynthesis (16,17,19,20) as query sequences. We identified a putative iridoid synthase (OeISY), 8-hydroxygeraniol oxidoreductase (Oe8HGO), iridoid oxidase (OeIO), 7-deoxyloganetic acid-O-glucosyl transferase (Oe7-DLGT1 and Oe7-DLGT2), and 7-deoxyloganic acid hydroxylase-like (Oe7-DLH-like) genes (Table 1 and Figs.…”

A novel cinnamyl alcohol dehydrogenase (CAD)-like reductase contributes to the structural diversity of monoterpenoid indole alkaloids in Rauvolfia

“…Its subsequent ligation to tryptamine yields the universal intermediate, strictosidine, whence all 2500 MIAs known thus far are derived (Stöckigt and Zenk 1977). This ''upstream pathway'' was elucidated in detail on enzymatic and genetic level (GeuFlores et al 2012;Asada et al 2013;Salim et al 2013Salim et al , 2014Miettinen et al 2014) and, with the genetic machinery at hand, heterologous production of the latter plant-specific compound in yeast became feasible (Brown et al 2015). However, the manifold reactions, spearheaded by deglucosylation of strictosidine, providing the abundance of MIA carbon skeletons, are still elusive.…”

Metabolite Diversity in Alkaloid Biosynthesis: A Multilane (Diastereomer) Highway for Camptothecin Synthesis in Camptotheca acuminata

“…The enzyme iridoid synthase catalyzes the subsequent conversion of 8-oxogeranial (1) to iridodial (Geu-Flores et al, 2012), which is oxidized further to 7-deoxyloganetic acid by 7-deoxyloganetic acid synthase (CYP76A26) . 7-Deoxyloganetic acid is glycosylated to 7-deoxyloganic acid (Asada et al, 2013), which undergoes subsequent hydroxylation by CYP72A224 to yield loganic acid, whose carboxyl group is then O-methylated to form loganin (Murata et al, 2008;Salim et al, 2013). Secologanin synthase (CYP72A1) then catalyzes the oxidative ring opening of loganin to secologanin, which contains an aldehyde group suitable for condensation with the amino group of tryptamine (Irmler et al, 2000).…”