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. 2022 Sep;235(5):1884-1899.
doi: 10.1111/nph.18272. Epub 2022 Jun 18.

The tomato cytochrome P450 CYP712G1 catalyses the double oxidation of orobanchol en route to the rhizosphere signalling strigolactone, solanacol

Affiliations

The tomato cytochrome P450 CYP712G1 catalyses the double oxidation of orobanchol en route to the rhizosphere signalling strigolactone, solanacol

Yanting Wang et al. New Phytol. 2022 Sep.

Abstract

Strigolactones (SLs) are rhizosphere signalling molecules and phytohormones. The biosynthetic pathway of SLs in tomato has been partially elucidated, but the structural diversity in tomato SLs predicts that additional biosynthetic steps are required. Here, root RNA-seq data and co-expression analysis were used for SL biosynthetic gene discovery. This strategy resulted in a candidate gene list containing several cytochrome P450s. Heterologous expression in Nicotiana benthamiana and yeast showed that one of these, CYP712G1, can catalyse the double oxidation of orobanchol, resulting in the formation of three didehydro-orobanchol (DDH) isomers. Virus-induced gene silencing and heterologous expression in yeast showed that one of these DDH isomers is converted to solanacol, one of the most abundant SLs in tomato root exudate. Protein modelling and substrate docking analysis suggest that hydroxy-orbanchol is the likely intermediate in the conversion from orobanchol to the DDH isomers. Phylogenetic analysis demonstrated the occurrence of CYP712G1 homologues in the Eudicots only, which fits with the reports on DDH isomers in that clade. Protein modelling and orobanchol docking of the putative tobacco CYP712G1 homologue suggest that it can convert orobanchol to similar DDH isomers as tomato.

Keywords: CYP712G1; orobanchol; oxidation; solanacol; strigolactone biosynthesis; tomato (Solanum lycopersicum).

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Figures

Fig. 1
Fig. 1
The proposed strigolactone (SL) biosynthetic pathway in tomato (Solanum lycopersicum). Elucidated steps are indicated with solid and putative steps with broken arrows. The proposed structures of didehydro‐orobanchol (DDH) isomers are shown in Supporting Information Fig. S8.
Fig. 2
Fig. 2
The discovery of strigolactone (SL) biosynthesis candidate genes in tomato (Solanum lycopersicum). (a) Gene co‐expression network of all genes in the RNA‐seq dataset with four bait genes created with CoExpNetViz and Cytoscape. The default correlation threshold of CoExpNetViz was used (lower percentile rank < 5; upper percentile rank > 95). Genes in group 1 are co‐expressed with SlD27, SlCCD7 and SlCCD8; genes in group 2, are co‐expressed with SlMAX1; genes in group 3 are co‐expressed with SlD27 and SlCCD8; genes in group 4 are co‐expressed with SlCCD8 and SlMAX1; genes in group 5 are co‐expressed with SlCCD8; genes in group 6 are co‐expressed with SlD27, SlCCD8 and SlMAX1; genes in group 7 are co‐expressed with SlD27, SlCCD7, SlCCD8 and SlMAX1; genes in group 8 are co‐expressed with SlCCD7; genes in group 9 are co‐expressed with SlD27 and SlCCD7; genes in group 10 are co‐expressed with SlD27. The 12 P450s we studied in this paper and CYP722C from group 7 were manually moved to a different location on the network canvas (P450 circle on the top right). (b) Venn diagram showing the number of co‐expressed genes with known SL biosynthetic genes (PCC ≥ 0.9) (Oliveros, 2007).
Fig. 3
Fig. 3
Biochemical characterisation of CYP712G1 using transient expression in Nicotiana benthamiana. (a) The reduction of orobanchol (transition [M+H]+ m/z orobanchol_347.2 > 97) in the transient expression assay. (b) The multiple reaction monitoring (MRM) chromatogram of didehydro‐orobanchol (DDH) isomers (transition [M+H]+ m/z 345.16 > 96.96) on enantio‐selective column in transient expression assay. (c) The production of solanacol (transition [M+H]+ m/z 343.16 > 96.97) in the transient expression assay. (d) The MRM chromatogram of DDH isomers (transition [M+H] + m/z 345.16 > 96.96, 345.16 > 175.27 and 345.16 > 203) on enantio‐selective column in tomato root exudate under aeroponic system. EV, empty vector; orobanchol pathway, SlD27 + SlCCD7 + SlCCD8 + Os900 + Os1400; orobanchol + CYP712G1, co‐expression of CYP712G1 with the orobanchol pathway.
Fig. 4
Fig. 4
Expression of strigolactone (SL) biosynthetic genes in roots and SL quantification in the root exudate of virus‐induced gene silencing (VIGS)‐treated tomato plants (cv Moneymaker). (a) The expression of D27, CCD8, MAX1 and CYP712G1, normalised to two reference genes (SGN‐U584254 and SGN‐U563892; Dekkers et al., 2012), in the roots of tomato (cv Moneymaker) 4 wk after infiltration of leaves with tobacco rattle virus (TRV) VIGS constructs (n = 6). (b) Orobanchol level (transition [M+H]+ m/z orobanchol_347.2 > 97) in the root exudate of VIGS‐treated tomato plants. (c) Solanacol level (transition [M+H]+ m/z solanacol_343.16 > 96.97) in the root exudate of VIGS‐treated tomato plants. (d) DDH isomers level (transition [M+H]+ m/z DDH_345.16 > 96.96) in the root exudate of VIGS‐treated tomato plants. (e) Hydroxy‐orobanchol level (transition [M+H]+ m/z hydroxy‐orobanchol_361.16 > 247.05) in the root exudate of VIGS‐treated tomato plants. Root exudates were collected after VIGS infiltration for 4 wk (normalised using root fresh weight, pmol g−1 FW), GUS‐TRV2B is the experimental control and CCD8‐TRV2B is the positive control (n = 6). Error bars represent means ± SEM (significance was determined using Student's t‐test, * and ** = significant at 0.05 and 0.01 levels, respectively; ns, nonsignificant).
Fig. 5
Fig. 5
Multiple reaction monitoring‐liquid chromatography tandem mass spectrometry (MRM‐LC/MS/MS) identification of didehydro‐orobanchol (DDH) isomers and orobanchol stereoisomers of yeast microsome in vitro assay on enantio‐selective column. (a) Structures of orobanchol stereoisomers. (b) Representative chromatogram of the production of DDH isomers (transition [M+H]+ m/z DDH_345.16 > 96.96) in the enzyme assay mixture with yeast microsomes expressing CYP712G1 (empty vector (EV) as control) and two orobanchol isomers after incubation of 3 h as analysed using MRM‐LC–MS (n = 3) on an enantio‐selective column.
Fig. 6
Fig. 6
Docking of potential substrates into tomato CYP712G1. Docking of (a) orobanchol, (b) 2′‐epi‐orobanchol, (c) ent‐orobanchol, (d) ent‐2′‐epi‐orobanchol, (e) 6‐hydroxy‐orobanchol and (f) 7‐hydroxy‐orobanchol into CYP712G1. All substrates are depicted in purple, the heme in brown and protein residues in blue. The distances (in Å) of C7 and C6 in (a) and C7 in (b) to the Fe group of the heme are shown in orange. Other interactions of (a) and (b) to residues in CYP712G1 as returned by the Protein‐Ligand Interaction Profiler (PLIP) tool (Salentin et al., 2015) are shown and the residues are labelled – hydrophobic interactions as dashed grey lines, π‐Stacking as dashed green lines, and hydrogen bonds as dashed blue lines. Both (a) and (b) are involved in hydrophobic interactions with neighbouring phenylalanines and leucines (PHE378, PHE501, LEU249, LEU308). Orobanchol (a) can also form hydrogen bonds with SER124 and TYR118. This TYR118 is also positioned in a way to be stabilised using a π‐stacking interaction between its aromatic ring and that of the orobanchol (represented as a green dashed line between two white spheres at the centres of the aromatic rings). Ent‐orobanchol and ent‐2′‐epi‐orobanchol (d, e) dock in reverse orientation. In (e) and (f) distances of the docked ligand to the two aspartic acids that were mutated are shown if < 5 Å. The binding energies of the ligands for the dockings shown in this figure are provided in Supporting Information Dataset S2.
Fig. 7
Fig. 7
Distribution of the CYP712 gene subfamilies across the Eudicot phylogenetic tree. Asterisks indicate described presence of didehydro‐orobanchol (DDH)isomers. The black and red arrows indicate tomato (Solanum lycopersicum) CYP712G1 and Arabidopsis CYP705 subfamilies, respectively.

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