A Single Oxidosqualene Cyclase Produces the Seco-Triterpenoid α-Onocerin

Abstract

8,14-seco-Triterpenoids are characterized by their unusual open C-ring. Their distribution in nature is rare and scattered in taxonomically unrelated plants. The 8,14-seco-triterpenoid α-onocerin is only known from the evolutionarily distant clubmoss genus Lycopodium and the leguminous genus Ononis, which makes the biosynthesis of this seco-triterpenoid intriguing from an evolutionary standpoint. In our experiments with Ononis spinosa, α-onocerin was detected only in the roots. Through transcriptome analysis of the roots, an oxidosqualene cyclase, OsONS1, was identified that produces α-onocerin from squalene-2,3;22,23-dioxide when transiently expressed in Nicotiana bethamiana In contrast, in Lycopodium clavatum, two sequential cyclases, LcLCC and LcLCD, are required to produce α-onocerin in the N. benthamiana transient expression system. Expression of OsONS1 in the lanosterol synthase knockout yeast strain GIL77, which accumulates squalene-2,3;22,23-dioxide, verified the α-onocerin production. A phylogenetic analysis predicts that OsONS1 branches off from specific lupeol synthases and does not group with the known L. clavatum α-onocerin cyclases. Both the biochemical and phylogenetic analyses of OsONS1 suggest convergent evolution of the α-onocerin pathways. When OsONS1 was coexpressed in N. benthamiana leaves with either of the two O. spinosa squalene epoxidases, OsSQE1 or OsSQE2, α-onocerin production was boosted, most likely because the epoxidases produce higher amounts of squalene-2,3;22,23-dioxide. Fluorescence lifetime imaging microscopy analysis demonstrated specific protein-protein interactions between OsONS1 and both O. spinosa squalene epoxidases. Coexpression of OsONS1 with the two OsSQEs suggests that OsSQE2 is the preferred partner of OsONS1 in planta. Our results provide an example of the convergent evolution of plant specialized metabolism.

Figure 1.

α-Onocerin content varies throughout plant growth and across the root length. A, Time course of α-onocerin accumulation in root and aerial tissues (fresh weight) of O. spinosa plants grown in either soil or a hydroponics (Hpn) system. B, Extracted ion chromatograms (480–482 mass-to-charge ratio) of O. spinosa root sections, stem, and leaf tissues. C, Mass spectrum of derivatized α-onocerin standard. D, Representative mass spectrum of an α-onocerin peak in derivatized extracts from root sections. Values represent means ± se from three biological replicates for each time point; letters indicate significant differences determined by ANOVA/posthoc Tukey’s honestly significant difference (HSD) test at P < 0.05.

Figure 2.

Phylogenetic analysis suggests that the α-onocerin pathway genes in O. spinosa and L. clavatum are distantly related and arise from convergent evolution. A, Maximum-likelihood tree constructed on deduced amino acid sequences of eight SQEs aligned by ClustalW spanning 496 positions using the MEGA6 program. The statistical significance of each node was tested by the bootstrap method using 1,000 iterations. Representative names are as follows: OsSQE1, O. spinosa squalene epoxidase1; MtSQE1, M. truncatula mRNA for squalene monooxygenase1; AtSQE1, Arabidopsis squalene epoxidase1; PpSQE, Physcomitrella patens squalene epoxidase. B, Maximum-likelihood tree constructed on deduced amino acid sequences of 24 OSCs with known product profiles aligned by ClustalW spanning 730 positions using the MEGA6 program. The statistical significance of each node was tested by the bootstrap method using 1,000 iterations. Boldface names represent genes from O. spinosa and L. clavatum. Representative names are as follows: OsONS1, O. spinosa onocerin synthase1; OsBAS, O. spinosa putative β-amyrin synthase; OsCAS, O. spinosa putative cycloartenol synthase; OsLAS, O. spinosa putative lanosterol synthase; PsCASPEA, P. sativum cycloartenol synthase; PsOSCPSY, P. sativum β-amyrin synthase; PsOSCPSM, P. sativum mixed amyrin synthase; MtBAS, M. truncatula β-amyrin synthase; AtLUP1, Arabidopsis lupeol synthase1; AtPEN6, Arabidopsis seco-β-amyrin synthase; AtMRN1, Arabidopsis marneral synthase; AtBAS, Arabidopsis β-amyrin synthase; AtCAS1, Arabidopsis cycloartenol synthase; AtLAS1, Arabidopsis lanosterol synthase; PgPNA, Panax ginseng dammarenediol II synthase; LcLCA, L. clavatum cycloartenol synthase; LcLCC, L. clavatum pre-α-onocerin synthase; LcLCD, L. clavatum onocerin synthase; OeOEW, Olea europaea lupeol synthase; GgLUS1, G. glabra lupeol synthase; LjOSC3, Lotus japonicus lupeol synthase; LjOSC7, L. japonicus lanosterol synthase; CpCPQ, Cucurbita pepo cucurbitadienol synthase. GenBank accession numbers for each nucleotide sequence of OSCs and SQEs are given in “Materials and Methods.”

Figure 3.

α-Onocerin biosynthesis in O. spinosa is biochemically different from that of L. clavatum. A, α-Onocerin content of N. benthamiana leaves transiently transformed with OSCs and SQEs of O. spinosa. B, α-Onocerin content of N. benthamiana leaves transiently transformed with α-onocerin synthases of L. clavatum coexpressed with SQEs of O. spinosa. C, Total ion chromatogram showing the production of α-onocerin in yeast strain GIL77 transformed with OsONS1 of O. spinosa. Values represent means ± se from three biological replicates; letters indicate significant differences determined by ANOVA/posthoc Tukey’s HSD test at P < 0.05.

Figure 4.

Docking simulations of the OsONS1 homology model shows that pre-α-onocerin can dock from both ends to the catalytic Asp residue, Asp-482. Color coding is as follows: pink, carbon atoms of pre-α-onocerin; green, carbon atoms in amino acid residues of the OsONS1 homology model; red, oxygen atoms; blue, nitrogen atoms; white, hydrogen atoms; yellow, sulfur atoms. A, Epoxide end of pre-α-onocerin forming a hydrogen bond with catalytic Asp-482 in the active site of the OsONS1 homology model. B, Hydroxyl group from the cyclized end of pre-α-onocerin forming a hydrogen bond with catalytic Asp-482 in the active site of the OsONS1 homology model. C, Steps of the α-onocerin biosynthetic pathway. D, Demonstrated cyclization reaction by rabbit microsomes. Compound 1, 3-((3E,7E)-3,7-Dimethyl-10-((8aS)-2,5,5,8a-tetramethyl-3,4,4a,5,6,7,8,8a-octahydronaphthalen-1-yl)deca-3,7-dien-1-yl)-2,2-dimethyloxirane; compound 2, 8,14-seco-gammacera-7,14-diene-3-ol.

Figure 5.

OsONS1 localizes in the cytoplasm while OsSQEs are restricted to the ER membrane, as visualized by confocal microscopy of N. benthamiana epidermal leaves. Confocal laser scanning microscopy images were collected 3 d after agroinfiltration of N. benthamiana leaves. Representative confocal images of the nucleus (A–F) and parietal cell space (G–L) of leaves expressing eGFP-tagged target proteins are shown. Soluble enzymes may permeate through the nuclear pores and be found inside the nucleus (A–C) and fill out the cytoplasm around the cortical ER (G–I). ER proteins are restrained to nuclear membranes (D–F) and to a characteristic ER membrane network (J–L). Due to turgescent vacuole pushing the cytoplasm and ER near the plasma membrane, a well-defined ER network and diffused cytoplasmic patterns may be visualized when observing the cortical space in epidermal cells of N. benthamiana leaves. Bars = 10 μm.

Figure 6.

OsONS1 interacts with both OsSQE1 and OsSQE2. A, FLIM values for OsONS1 tagged with eGFP on the C terminus coexpressed with several mRFP1-fused constructs in transiently transformed N. benthamiana leaves. B, FLIM values for OsONS1 tagged with eGFP on the N terminus coexpressed with several mRFP1-tagged constructs in transiently transformed N. benthamiana leaves. C, α-Onocerin content of N. benthamiana leaves transiently transformed with OsONS1-eGFP coexpressed with several mRFP1-tagged constructs. D, α-Onocerin content of N. benthamiana leaves transiently transformed with eGFP-OsONS1 coexpressed with several mRFP1-tagged constructs. For A and B, letters indicate significant differences determined by ANOVA/posthoc Scheffe’s method at P < 0.05. For C and D, values represent means ± se from three biological replicates; letters indicate significant differences determined by ANOVA/posthoc Tukey’s HSD test at P < 0.05.

Figure 7.

OsSQE2 and OsONSs coexpress across root sections and leaves of O. spinosa. A, Absolute quantification of OsSQE1 transcripts in different tissues of O. spinosa. B, Absolute quantification of OsSQE2 transcripts in different tissues of O. spinosa. C, Absolute quantification of OsONS transcripts in different tissues of O. spinosa. qRT-PCR was performed on RNA extracted from the pooled tissues of five plants. Values represent means ± se from three different poolings per tissue; letters indicate significant differences determined by ANOVA/posthoc Tukey’s HSD test at P < 0.05.