Modularity of plant metabolic gene clusters: a trio of linked genes that are collectively required for acylation of triterpenes in oat

Abstract

Operon-like gene clusters are an emerging phenomenon in the field of plant natural products. The genes encoding some of the best-characterized plant secondary metabolite biosynthetic pathways are scattered across plant genomes. However, an increasing number of gene clusters encoding the synthesis of diverse natural products have recently been reported in plant genomes. These clusters have arisen through the neo-functionalization and relocation of existing genes within the genome, and not by horizontal gene transfer from microbes. The reasons for clustering are not yet clear, although this form of gene organization is likely to facilitate co-inheritance and co-regulation. Oats (Avena spp) synthesize antimicrobial triterpenoids (avenacins) that provide protection against disease. The synthesis of these compounds is encoded by a gene cluster. Here we show that a module of three adjacent genes within the wider biosynthetic gene cluster is required for avenacin acylation. Through the characterization of these genes and their encoded proteins we present a model of the subcellular organization of triterpenoid biosynthesis.

Figure 1.

Structures of Oat Root Avenacins. The C-21 acyl group consists of either N-methyl anthranilate or benzoate, depending on the R₁ group. The major avenacin found in oat roots is avenacin A-1, which is acylated with N-methyl anthranilate (Crombie et al., 1984).

Figure 2.

A Gene Encoding a Predicted Methyltransferase Located within the Avenacin Biosynthetic Gene Cluster Is Implicated in Avenacin Synthesis. (A) BAC contig showing the avenacin gene cluster and the location of the Sad9 gene, which is predicted to encode a methyltransferase. Gene names are indicated above and encoded proteins below. (B) Neighbor-joining phylogeny of functionally characterized plant natural product S-adenosyl-Met–dependent methyltransferase enzymes. N-methyltransferase enzymes are indicated with asterisks. The phylogeny includes the sequences used by Liscombe and Facchini, 2007; the MT1 protein (indicated by an arrow); the most similar sequences to As-MT1 encoded by the genomes of Arabidopsis (At4g35160), rice (Oryza sativa; Os9g17560), Brachypodium distachyon (Bd2g19830), and sorghum (Sorghum bicolor; Sb07g024270); and the R. graveolens anthranilate methyltransferase (Rg ANMT; Rohde et al., 2008). Details of these other sequences are provided in Methods, and the full protein sequence alignment can be found in Supplemental Data Set 1 online. Bootstrap values (percentage of 1000 replicates) are shown for key branches. Bar indicates 0.05 substitutions per site. (C) Sad9 (AsMT1) transcript abundance in young leaves, whole roots, root tips (0.5 mm), and roots minus tips, measured by quantitative PCR (transcript abundance expressed relative to elongation factor 1-α); values are means (n = 3 ± se). (D) Immunostaining of root tip sections from 3-d-old wild-type A. strigosa seedlings. Sections were stained with (from left to right) anti-AsMT1 antisera, anti-AsSCPL1 antisera, or 4′,6-diamidino-2-phenylindole (DAPI). A bright-field image and an overlay of the MT1, SCPL1, and DAPI images are also shown. Controls probed with preimmune sera are shown in Supplemental Figure 1 online.

Figure 3.

Detection of MT1, UGT74H5, and SCPL1 in Oat Roots. Immunoblot analysis of soluble protein extracts from 3-d-old A. strigosa seedlings, probed with antisera raised against MT1, UGT74H5, or SCPL1. The bottom panel shows Coomassie blue staining of a replicate gel. Protein extracts were prepared from leaves and roots of 3-d-old A. strigosa wild-type (WT) seedlings and from roots of previously characterized sad1 (mutant line #610) and sad7 (#616) mutants (Papadopoulou et al., 1999; Mugford et al., 2009) and sad9 mutants (#1475, 1310, 961, and 841) (this article). Bands corresponding to the predicted size of the MT1 and UGT74H5 proteins were detected. SCPL1 is posttranslationally cleaved into a large and a small subunit corresponding to the bands detected at 29 and 19 kD and a processing intermediate (33 kD) (Mugford et al., 2009).

Figure 4.

sad9 Mutants Are Blocked in the Methylation of Avenacin Acyl-Groups. (A) Analysis of wild-type and mutant (sad1, sad7, and sad9) root extracts by fluorescence LC-MS. Fluorescence detection shows the absence of avenacin A-1 (A1) in all mutants and the accumulation of DMA in sad9 mutants. N-methyl anthraniloyl-O-Glc (NMA-glc) is not detectable in root extracts from sad9 mutants, but anthraniloyl-O-Glc (Anth-glc) is detected at low levels. Similar metabolite profiles were observed in the other sad9 mutants analyzed (see Supplemental Figure 2 online). Confirmation of the identity of these compounds by mass spectrometry is presented in Supplemental Figure 2 online. LC-MS shows the accumulation of des-acyl avenacin A (DAA; extracted ion chromatogram for mass-to-charge ratio = 983 to 985) in the sad7 mutant, as previously shown by Mugford et al. (2009). AU, arbitrary units. (B) Proposed pathway for acylation of avenacin A-1. N-methyl anthranilate (NMA) is synthesized by the methylation of anthranilate (Anth) by MT1. The glucosyltransferase UGT74H5 glucosylates N-methyl anthranilate to give the activated acyl donor substrate N-methyl anthraniloyl-O-Glc (NMA-Glc). UGT74H5 has a clear preference for N-methyl anthranilate but also has weak activity toward anthranilate to give anthraniloyl-O-Glc (Anth-Glc) (Owatworakit et al., 2012). In wild-type A. strigosa, the SCPL1 acyltransferase uses NMA-Glc as the acyl donor to give avenacin A-1 (A1). In sad9 mutants (lacking MT1), the triterpenoid backbone is acylated with anthranilate instead of N-methyl anthranilate to give DMA.

Figure 5.

sad9 Mutants Have Enhanced Disease Susceptibility. Seedlings were inoculated with the take-all fungus (G. graminis var tritici strain T2) and scored for disease symptoms 3 weeks later as described by Papadopoulou et al. (1999). Dark-brown/black lesions on the roots (arrowed) are typical symptoms of infection. The wild type (WT) (A), sad1 mutant #610 (B), sad7 mutant #616 (C), and sad9 mutant #1475 (D). Images are representative of six to 10 biological replicates across two independent experiments. Bars = 0.25 cm.

Figure 6.

Coexpression of As-MT1 and As-UGT74H5 in Tobacco Leaves Results in the Accumulation and Sequestration of N-Methyl Anthraniloyl-O-Glc. (A) Immunoblot analysis of soluble protein extracts of N. benthamiana leaves probed with antisera raised against MT1, UGT74H5, and SCPL1. The bottom panel shows a replicate gel stained with Coomassie blue. Lanes were loaded with protein extracted from leaves 6 d after infiltration with Agrobacterium tumefaciens strains containing (from left to right) the P19 silencing suppressor alone (P19) or in combination with the CPMV empty vector (EV); vectors containing the coding sequences of MT1, UGT74H5, or SCPL1 alone or coinfiltrated in combination as indicated. (B) Leaves of N. benthamiana photographed under UV illumination 6 d after infiltration with Agrobacterium strains. (C) HPLC analysis with fluorescence detection (excitation at 353 nm; emission at 441 nm) of methanolic extracts from N. benthamiana leaves. Peaks 2 and 3 are N-methyl anthraniloyl-O-Glc and N-methyl anthranilic acid, respectively. Peaks 1 and 4 were found to be derivatives of N-methyl anthraniloyl-O-Glc, which were further modified. Accurate mass determination and fragmentation by MS2 provides putative identification of the additional group on compound 1 (X) as a hexose and on compound 2 (Y) as malate (see Supplemental Figure 6 online). AU, arbitrary units. (D) Confocal microscopy of lower epidermal cells of leaves (from left to right) uninfiltrated or infiltrated with Agrobacterium strains bearing the CPMV empty vector (EV), the CPMV vector carrying the MT1 (Sad9) coding sequence, or MT1 and UGT74H5. Clockwise from top left for each set of four panels: UV fluorescence, chlorophyll autofluorescence, overlay, and transmitted light. Blue fluorescence is detectable across the central body of cells coexpressing MT1 and UGT74H5 (and more weakly, also from cells expressing MT1 alone), consistent with vacuolar compartmentalization of the fluorescent metabolites. The epidermal cells are highly vacuolated; the chlorophyll autofluorescence shows how the chloroplasts are restricted to the periphery of the cells by the enlarged vacuole. Conversely, weak blue fluorescence in the empty vector control leaves is mainly associated with the cell wall; this may be due to a response to Agrobacterium infiltration. Bars = 20 μm.

Figure 7.

Subcellular Localization of As-MT1, As-UGT74H5, and As-SCPL1. (A) to (D) Transmission electron microscopy images from immunogold labeling of ultrathin sections probed with antisera raised against MT1. (A) and (B) Root tip epidermal cells of 3-d-old wild-type (WT) (A) and sad9 (#961) mutant (B) oat seedlings. (C) and (D) Sections from N. benthamiana leaves expressing the CPMV-AsMT1 construct (C) or the CPMV-EV control (D). (E) to (G) Sections probed with antisera raised against UGT74H5. (E) Wild-type oat root tip epidermis. (F) and (G) N. benthamiana leaves expressing CPMV-AsUGT74H5 (F) or CPMV-EV (G). (H) and (I) Sections probed with antisera raised against SCPL1. Wild-type (H) or sad7 (#616) (I) mutant oat root tip epidermis. Gold particle labeling is visible as black dots. Selected subcellular compartments are labeled as follows: C, cytoplasm; V, vacuole; P, plastid; M, mitochondria; and W, cell wall. Images are representative of 10 to 20 biological replicates, and quantification of gold particle densities in different compartments is presented in Supplemental Table 2 online. Bars = 0.5 μm.

Figure 8.

Model of the Subcellular Organization of Avenacin A-1 Biosynthesis in Oat. The acyl group of avenacin A-1 originates from the shikimate pathway in the plastid. Anthranilate (Anth) (which fluoresces purple under UV illumination) is methylated by MT1 (encoded by Sad9) to produce N-methyl anthranilate (NMA) (which fluoresces blue) and then glucosylated by UGT74H5 to give N-methyl anthraniloyl-O-glucose (NMA-Glc). NMA-Glc is transported into the vacuole by an unknown mechanism, where it serves as the acyl donor substrate for SCPL1 (SAD7). The triterpene glycoside is synthesized from the cytoplasmic mevalonate (MVA) pathway products, and the early steps are catalyzed by β-amyrin synthase (SAD1/AsβAS1) and CYP51H10 (SAD2). These steps are likely to be associated with the endoplasmic reticulum (ER) membrane (Wegel et al., 2009). Des-acyl avenacin A is then formed by a series of as yet uncharacterized oxidation and glycosylation steps. The triterpene glycoside is transported into the vacuole, either by a vacuolar membrane transporter or possibly through vesicular trafficking from the endoplasmic reticulum to the vacuole.