Convergence and divergence of bitterness biosynthesis and regulation in Cucurbitaceae

Abstract

Differentiation of secondary metabolite profiles in closely related plant species provides clues for unravelling biosynthetic pathways and regulatory circuits, an area that is still underinvestigated. Cucurbitacins, a group of bitter and highly oxygenated tetracyclic triterpenes, are mainly produced by the plant family Cucurbitaceae. These compounds have similar structures, but differ in their antitumour activities and ecophysiological roles. By comparative analyses of the genomes of cucumber, melon and watermelon, we uncovered conserved syntenic loci encoding metabolic genes for distinct cucurbitacins. Characterization of the cytochrome P450s (CYPs) identified from these loci enabled us to unveil a novel multi-oxidation CYP for the tailoring of the cucurbitacin core skeleton as well as two other CYPs responsible for the key structural variations among cucurbitacins C, B and E. We also discovered a syntenic gene cluster of transcription factors that regulates the tissue-specific biosynthesis of cucurbitacins and may confer the loss of bitterness phenotypes associated with convergent domestication of wild cucurbits. This study illustrates the potential to exploit comparative genomics to identify enzymes and transcription factors that control the biosynthesis of structurally related yet unique natural products.

Figure 1. Comparative analysis of cucurbitacin biosynthetic and regulatory genes in cucumber, melon and watermelon

(a) Evolutionary relationship of three cucurbits and structures of CuC, CuB and CuE. mya, million years ago. (b) Distribution of bitter compound and expression profiles of genes in wild or cultivated lines. Genes were renamed according to Table 1. For cucumber, gene expressions were shown by the FPKM value; for melon and watermelon, expressions profiles were determined by qRT-PCR. Relative gene expression values are shown with identical scales (means ± SEM, n=3 biological replicates). The potential biosynthetic enzymes and regulators of CuC, CuB or CuE are indicated in bold.

Figure 2. Elucidation of the catalytic steps invovled in CuB and CuE biosynthesis

(a) GC-MS profiles of the extracts prepared from the yeast that harbored CmBi, ClBi, or empty vector, and an authentic cucurbitadienol standard. EI⁺, electron ionization in positive ion mode; TIC, total ion chromatograms; EIC 134, extracted ion chromatograms of the characteristic fragment ion of cucurbitadienol at a mass/charge ratio (m/z) of 134. (b and c) UPLC-qTOF-MS analysis of the ACT-catalytic reaction products. The sample without CmACT or ClACT protein was served as the negative control. EIC 576.3531 and 574.3374, extracted ion chromatograms of the accurate parent ions at m/z 576.3531 [M+NH]⁺ and 574.3374 [M+NH]⁺, for CuB and CuE, respectively.

Figure 3. Functional elucidations of a multifunctional and a divergent CYPs

(a) UPLC-qTOF-MS analysis of the extracts prepared from the yeast expressing OSC, CPR, and each candidate CYP with APCI in positive ion mode. One expected product (red arrows) is generated by CYP87D20 (Cm890, Cl890A, Cl890B, or Cs890). The structure of this product (right) was elucidated by MS/MS and NMR. EIC 441.3727, extracted ion chromatogram of the accurate parent ion at m/z of 441.3727 [M+H]⁺. (b) UPLC-qTOF-MS analysis of the extracts used in (a) with ESI in positive mode. One expected product (red arrows) is detected. The structure of this product (right) was elucidated by MS/MS and NMR. EIC 457.3676, extracted ion chromatogram of the accurate parent ion at m/z of 457.3676 [M+H]⁺. (c) UPLC-qTOF-MS analysis of the extracts prepared from the yeast accumulating 11-carbonyl-20β-hydroxycucurbitadienol and expressing candidate CYPs with ESI in positive ion mode. One expected product peak (red arrow) is generated by CYP81Q59 (Cm180 or Cl180). The structure of this product (below) was elucidated by MS/MS and NMR. EIC 473.3625, extracted ion chromatogram of the accurate parent ion at m/z of 473.3625 [M+H]⁺.

Figure 4. Characterization of the tissue-specific bitterness regulators from melon or watermelon

(a) Summary of the interactions between the promoters of candidate genes and the regulators from melon or watermelon (see Supplementary Figs 17–20 for further information). Y1H, yeast one-hybrid; Luc, luciferase trans-activation assay. (b) Transient expression of potential regulators in the non-bitter cotyledons of melon or watermelon activated the transcription of OSC and induced generation of bitter chemicals. Gene expression levels and contents of cucurbitacins were determined 5 days after agroinfiltration. Relative values are shown with identical scales (means ± SEM, n=3 biological replicates). INF, sample infiltrated with TF expression construct; CK, sample infiltrated with empty vector. (c) Comparison of the domestication sweep region among the three cucurbits. Numeric pi values are shown in Supplementary Table 1. The fruit-specific regulator genes (indicated in red) in melon or watermelon are also located within a large domestication sweep region. The Y axis represents the distribution of nucleotide diversity. Chr, chromosome.