A 7-deoxyloganetic acid glucosyltransferase contributes a key step in secologanin biosynthesis in Madagascar periwinkle

Abstract

Iridoids form a broad and versatile class of biologically active molecules found in thousands of plant species. In addition to the many hundreds of iridoids occurring in plants, some iridoids, such as secologanin, serve as key building blocks in the biosynthesis of thousands of monoterpene indole alkaloids (MIAs) and many quinoline alkaloids. This study describes the molecular cloning and functional characterization of three iridoid glucosyltransfeases (UDP-sugar glycosyltransferase6 [UGT6], UGT7, and UGT8) from Madagascar periwinkle (Catharanthus roseus) with remarkably different catalytic efficiencies. Biochemical analyses reveal that UGT8 possessed a high catalytic efficiency toward its exclusive iridoid substrate, 7-deoxyloganetic acid, making it better suited for the biosynthesis of iridoids in periwinkle than the other two iridoid glucosyltransfeases. The role of UGT8 in the fourth to last step in secologanin biosynthesis was confirmed by virus-induced gene silencing in periwinkle plants, which reduced expression of this gene and resulted in a large decline in secologanin and MIA accumulation within silenced plants. Localization studies of UGT8 using a carborundum abrasion method for RNA extraction show that its expression occurs preferentially within periwinkle leaves rather than in epidermal cells, and in situ hybridization studies confirm that UGT8 is preferentially expressed in internal phloem associated parenchyma cells of periwinkle species.

Figure 1.

Possible Secologanin Biosynthesis Pathways. Intermediates in the secologanin biosynthesis pathway: 1, geraniol; 2, iridotrial; 3, 7-deoxyloganetic acid; 4, 7-deoxyloganic acid; 5, loganic acid; 6, loganin; 7, secologanin; 8, 7-deoxyloganetin; 9, 7-deoxyloganin; and 10, loganetin. Enzymes involved in Secologanin biosynthesis: A, Geraniol 10-hydroxylase; B, 10-hydroxygeraniol oxidoreductase; C, iridoid synthase/monoterpene cyclase; D, iridodial oxidoreductase; E, 7-deoxyloganetic acid glucosyltransferase; F, 7-deoxyloganic acid hydroxylase; G, loganic acid methyltransferase; H, SLS; I, 7-deoxyloganic acid methyltransferase; J, 7-deoxyloganetic acid methyltransferase; K, 7-deoxyloganetin glucosyltransferase; L, 7-deoxyloganin hydroxylase; M, 7-deoxyloganetin hydroxylase; N, Loganetin glucosyltransferase. The genes that have been cloned and functionally characterized are in italics in this list and include UGT8 described in this study.

Figure 2.

Differential Conversions of 7-Deoxyloganetic Acid and 7-Deoxyloganetin by Recombinant UGT6, UGT7, and UGT8 to 7-Deoxyloganic Acid and 7-Deoxyloganin. Iridoid substrates (1 mM) were incubated with each rUGT in the presence of 5 mM UDP-Glc for 2 h at 30°C, and the reaction mixture was subjected to HPLC analysis as described in Methods. The arrowheads indicate the respective reaction products (7-deoxyloganic acid or 7-deoxyloganin) being made when recombinant enzymes were incubated with their respective iridoid substrates.

Figure 3.

Differential Expression of UGT8 Correlates with That of the Last Two Steps in Secologanin Biosynthesis, along with Iridoid and MIA Metabolite Profiles in Periwinkle Plant Organs. Relative gene expression of UGT6, UGT7, UGT8, LAMT, and SLS was determined by quantitative RT-PCR analyses performed on total RNA extracted from periwinkle leaf pairs 1 to 5, from open flowers and flower buds, and from root tissues. Each point represents the mean of relative transcript abundance to RPPOC (gene encoding 60S acidic ribosomal protein P0-C) ± sd from at least triplicate measurements of biological and technical replicates. Metabolite (catharanthine, vindoline, and secologanin) levels are plotted as mg/gram fresh weight (FW) and as mg/organ. Each box and bar represents an average value and a se, respectively, from four different plant samples.

Figure 4.

Localization of UGT8 Transcripts in IPAP Cells of Young Developing Leaves of Periwinkle. (A) Relative expression of UGT6, UGT7, UGT8, LaMT, and SLS in relation to RPP0C reference gene in leaf epidermis enriched transcript extracted by carborundum abrasion compared with those found in whole-leaf extracts. Each point represents the mean of relative transcript abundance to RPPOC ± sd from at least triplicate measurements of biological and technical replicates. (B) to (E) Localization by in situ hybridization of UGT8 mRNA in young developing leaves of C. longifolius. Serial longitudinal 10-µm sections made from young leaves (10 to 15 mm long) were hybridized with UGT8-antisense ([B] and [D]) and UGT8-sense ([C] and [E]) probes. Bars = 500 µm in (B) and (C) and 50 µm in (D) and (E). [See online article for color version of this figure.]

Figure 5.

Downregulation of UGT8, LAMT, and SLS Affects the Accumulation of Iridoids and MIAs in Periwinkle. (A) Silencing of UGT8, LAMT, and SLS was conducted by monitoring the iridoid metabolite profiles by UPLC-MS in silenced plants (UGT8-vigs, LAMT-vigs, and SLS-vigs) compared with the profiles obtained with plants treated with EV controls. UPLC-MS analysis of iridoid profiles were detected at A₂₄₀ and by RTs related to iridoid standards: deoxyloganetic acid (RT = 4.68 min; m/z = 197), loganic acid (RT = 1.90 min; m/z = 377), loganin (RT = 3.1 min; m/z =391), and secologanin (RT = 3.88 min; m/z = 389). (B) Silencing of UGT8, LAMT, and SLS was measured by monitoring relative transcript abundance of each iridoid pathway gene by quantitative RT-PCR. Differences in transcript levels for each silenced gene were measured relative to those obtained in EV and mock treatments and are represented as mean ± se. Gene-specific primers for each UGT8, LAMT, and SLS were used for comparison of transcript abundance between EV and for each VIGS treatment. The data represent measurements performed with six biological replicates (with three technical replicates per biological replicate) of mock, EV, UGT8-vigs, LAMT-vigs, and SLS-vigs treatments. (C) and (D) Measurements of iridoids (loganic acid, loganin, and secologanin) (C) and MIAs (catharanthine and vindoline) (D) in untreated (wild type [WT]), EV, mock, UGT8-vigs, LAMT-vigs, and SLS-vigs treated periwinkle plants were performed with the same six biological replicates used for transcript analysis in (B). Significant differences were considered with *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test for the transcript analysis and metabolite contents of EV-infected plants and in each of the silenced lines. fw, fresh weight.

Figure 6.

Spatial Model of Iridoid Biosynthesis and Translocation in Periwinkle Leaves. The MEP pathway and iridoid biosynthesis to 7-deoxyloganic acid occurs in IPAP cells, while the terminal LAMT and SLS reactions occur in the leaf epidermis. The model shows 7-deoxyloganic acid hydroxylase in leaf epidermis, but this reaction and its location still remain to be elucidated. The model shows that MIA assembly from secologanin and tryptamine also takes place in leaf epidermal cells. Solid lines represent a single enzymatic step, whereas double arrows indicate the involvement of multiple enzyme steps. The question mark indicates the putative transport system of 7-deoxyloganic acid from IPAP cells to leaf epidermal cells. [See online article for color version of this figure.]