Independent evolution of plant natural products: Formation of benzoxazinoids in Consolida orientalis (Ranunculaceae)

Abstract

Benzoxazinoids (BXDs) are important defense compounds produced by a number of species from different, evolutionarily unrelated plant families. While BXD biosynthesis has been extensively studied in the grasses (monocots) and core eudicots, the mechanism of BXD synthesis in the basal eudicots is still unclear. We used an integrated metabolomics and transcriptomics approach to elucidate the BXD pathway in Consolida orientalis, a Ranunculaceae species known to produce the BXD DIBOA-Glc. Overexpression of candidate genes in Nicotiana benthamiana identified a flavin-dependent monooxygenase (CoBX2-3) and two cytochrome P450 enzymes (CoBX4 and CoBX5) that catalyze the oxidation steps that transform indole into DIBOA. Co-expression of CoBx2-3, CoBx4, and CoBx5 with the previously described indole synthase gene CoBx1 and the UDP-glucosyltransferase gene CoBx8 in N. benthamiana resulted in the reconstitution of a fully active BXD pathway. The fact that CoBX2-3, CoBX4, and CoBX5 are not phylogenetically related to their counterparts in the grasses and core eudicots suggests independent evolution of benzoxazinoid biosynthesis in these three angiosperm lineages.

Keywords: DIBOA-Glc; benzoxazinoids; biosynthesis; defense compounds; evolution; independent evolution; pathway; plant biochemistry; plant defense; secondary metabolism.

Figure 1

Occurrence of benzoxazinoids in the angiosperms and metabolic profiling of Consolida species. A, BXDs have been reported in the core eudicots, basal eudicots, and monocots. Plant families containing BXD-producing species are colored. The tree has been readapted from Kew gardens Tree of Life (32). B, biosynthetic pathway leading to the formation of BXDs in plants. Ranunculaceae, Apocynaceae, Plantaginaceae, and Lamiaceae produce DIBOA-Glc, while Poaceae and Acanthaceae can further convert DIBOA-Glc into the methoxylated derivative DIMBOA-Glc. C, Consolida orientalis but not the closely related C. regalis accumulates DIBOA-Glc. 100 ± 5 mg of plant tissue were extracted with methanol and analyzed through liquid chromatography-time of flight mass spectrometry (LC-qTOF-MS). Extracted ion chromatograms (EIC) are shown. D, accumulation of BXDs in different organs of C. orientalis. 100 ± 5 mg of leaf tissue were extracted with methanol and analyzed through LC-qTOF-MS. EIC are shown. Means and SD are displayed (n = 3 biological replicates, independent plants). Columns labeled with different letters represent statistically significant differences for each compound among tissues (p < 0.05, one-way ANOVA with Tukey’s correction for multiple comparisons). Detailed statistical values for each comparison are reported in Supplemental Dataset 1.

Figure 2

Expression of previously identified CoBx1 and CoBx8 in Nicotiana benthamiana. Activity of CoBX1 (A) and CoBX8 (B) in N. benthamiana in combination with BXD biosynthetic genes from maize. 100 ± 5 mg of leaf tissue was extracted with methanol and analyzed using LC-qTOF-MS. Means and SD are shown (n = 3 biological replicates, independent plants). Unpaired t test was used to evaluate differences among samples, (A) p = 0.0062, t = 5.261, df = 4; (B) p = 0.004, t = 5.955, df = 4.

Figure 3

Relative expression of the 20 C. orientalis transcripts best correlating with CoBx1. Values from 0 (lowest) to 100 (highest) show the relative expression of each gene among the conditions tested. Genes with RPKM > 20 and Pearson co-expression values with CoBx1 > 0.7 were selected. The values reported are the average of three biological replicates (independent plants). Enzyme abbreviations: indole-3-glycerol-phosphate lyase (IGL), cytochrome P450 (CYP), flavin-containing monooxygenase (FMO), UDP-glucosyltransferase (UGT).

Figure 4

The identified CoBX enzymes allow the biosynthesis of 3HI2O-Glc, HBOA-Glc, and DIBOA-Glc in N. benthamiana. A, CoBX2-3, CoBX4, and CoBX5 were transiently co-expressed with ZmBx1 + ZmBx8, ZmBx1, 2, 3 + ZmBx8, and ZmBx1, 2, 3, 4 + ZmBx8, respectively. Leaf material was extracted with methanol and extracts were analyzed using LC-qTOF-MS. Accumulation of BXDs was confirmed with authentic standards for HBOA-Glc and DIBOA-Glc. No authentic standard was available for 3HI2O-Glc, therefore we co-expressed ZmBx1, 2, 3, + ZmBx8 in N. benthamiana to generate this compound. Extracted ion chromatograms (EIC) are shown. B, the FMO CoBX2-3 independently evolved in the Ranunculaceae, Acanthaceae, and Lamiaceae. Amino acid sequences were aligned with WebPrank and a maximum likelihood tree was inferred using iQTree. Bootstrap values above 70% are displayed. FASTA sequences used for the construction of phylogenetic trees are provided in Supplemental Dataset 2. C, Consolida orientalis BXD pathway reconstitution. Transient expression of all C. orientalis Bx genes in N. benthamiana. 100 ± 5 mg of N. benthamiana leaf tissue was extracted with methanol. Extracts were analyzed using LC-qTOF-MS. Means and SD are shown (n = 3 biological replicates, independent plants). Unpaired t test was used to evaluate the effect of the addition of each CoBx gene. 3HI2O-Glc: p < 0.0001, t = 26.12, df = 4; HBOA-Glc: p < 0.0001, t = 26.60, df = 4; DIBOA-Glc: p = 0.0003, t = 12.19, df = 4.

Figure 5

CoBX4 is evolutionarily related to CYP82C and CYP82D enzymes that hydroxylate coumarins, flavonoids, or indole-like scaffolds.A, amino acid sequences were aligned with WebPrank and a maximum likelihood tree was inferred using iQTree. Bootstrap values above 60% are displayed. FASTA sequences used for the construction of phylogenetic trees are provided in Supplemental Dataset 2. B, examples for reactions catalyzed by CYP82C and CYP82D enzymes acting on substrates structurally related to benzoxazinoids.

Figure 6

Independent evolution of the BXD pathway in the angiosperms. Characterized enzymes and respective enzyme classes are indicated in boxes colored according to the plant lineage in which the enzymes were identified. As: Aphelandra squarrosa, Co: Consolida orientalis, Lg: Lamium galeobdolon, Sd: Scoparia dulcis, TSB-like: pseudoenzyme involved in indole biosynthesis, Wr: Wrightia religiosa, Zm: Zea mays.