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.2024 Apr 17;24(1):294.
doi: 10.1186/s12870-024-04955-3.

A novel O-methyltransferase Cp4MP-OMT catalyses the final step in the biosynthesis of the volatile 1,4-dimethoxybenzene in pumpkin (Cucurbita pepo) flowers

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A novel O-methyltransferase Cp4MP-OMT catalyses the final step in the biosynthesis of the volatile 1,4-dimethoxybenzene in pumpkin (Cucurbita pepo) flowers

Marion Christine Hoepflinger et al. BMC Plant Biol..

Abstract

Background: Floral scents play a crucial role in attracting insect pollinators. Among the compounds attractive to pollinators is 1,4-dimethoxybenzene (1,4-DMB). It is a significant contributor to the scent profile of plants from various genera, including economically important Cucurbita species. Despite its importance, the biosynthetic pathway for the formation of 1,4-DMB was not elucidated so far.

Results: In this study we showed the catalysis of 1,4-DMB in the presence of 4-methoxyphenol (4-MP) by protein extract from Styrian oil pumpkin (Cucurbita pepo) flowers. Based on this finding, we identified a novel O-methyltransferase gene, Cp4MP-OMT, whose expression is highly upregulated in the volatile-producing tissue of pumpkin flowers when compared to vegetative tissues. OMT activity was verified by purified recombinant Cp4MP-OMT, illustrating its ability to catalyse the methylation of 4-MP to 1,4-DMB in the presence of cofactor SAM (S-(5'-adenosyl)-L-methionine).

Conclusions: Cp4MP-OMT is a novel O-methyltransferase from C. pepo, responsible for the final step in the biosynthesis of the floral scent compound 1,4-DMB. Considering the significance of 1,4-DMB in attracting insects for pollination and in the further course fruit formation, enhanced understanding of its biosynthetic pathways holds great promise for both ecological insights and advancements in plant breeding initiatives.

Keywords: Cucurbita pepo 4-methoxyphenol-O-methyltransferase (Cp4MP-OMT); O-methyltransferase (OMT); 1,4-dimethoxybenzene (1,4-DMB); 4-methoxyphenol (4-MP); Flower scent; Insect pollination; Styrian oil pumpkin.

© 2024. The Author(s).

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Predicted formation of 1,4-dimethoxybenzene in Styrian oil pumpkin flowers. Pictorial representation showing the emission of 1,4-dimethoxybenzene from a Styrian oil pumpkin flower(A) and the proposed enzymatic conversion of 4-methoxyphenol andS-(5′-adenosyl)-L-methionine (SAM) to 1,4-dimethoxybenzene by anO-methyl transferase(B)
Fig. 2
Fig. 2
Catalysis of 4-MP to 1,4-DMB. Representative GC chromatograms showing conversion of 4-MP to 1,4-DMB as compared to the substrates HYQ and 4-HBA(A); boxplots showing the amount of 1,4-DMB produced in the reactions(B). Different letters in the box plot indicate significant differences (4-MP,n = 7; HYQ,n = 5; 4-HBA,n = 6; Kruskal-Wallis and Dunn’s tests) at a level ofp < 0.05
Fig. 3
Fig. 3
Production of 1,4-DMB from Styrian oil pumpkin floral protein extract. Representative GC chromatograms showing conversion of 4-MP to 1,4-DMB from Styrian oil pumpkin floral OMT crude protein extract as compared to the control samples(A); boxplots showing the amount of 1,4-DMB (in ng) produced in the reactions(B). Different letters in the box plot indicate the significant differences (reaction 1,n = 7; reaction 2,n = 6; reaction 3,n = 4; reaction 4,n = 5; Kruskal-Wallis-test and Dunn’s test) at a level ofp < 0.05
Fig. 4
Fig. 4
Phylogenetic tree of methyltransferases acting on OH-groups of aromatic ring structures. A prototypic reaction is shown for each branch. The enzymes were annotated as 4-methoxyphenol-O-methyltransferase (4MP-OMT); resveratrol-MT (RMT); guaiacol-MT (GMT); catechol-MT (COMT), caffeic-caffeoyl-CoA O-MT (CCOMT); The Accessions: Cp4MP-OMTCucurbita pepo (this paper, highlighted in blue; OR913727); 4MP-OMTCucurbita maxima (XP_022986324); 4MP-OMTCucurbita moschata (XP_022944128.1);Rhaphiolepis (LC127201);Rhododendron XP_058210860;Camptotheca (AWH62807);Arabidopsis (AAL07162);Camelina (XP_010448306);Thlaspi (CAH2077901);Rauvolfia (KX687823);Catharantus (KAI5664645); human (AAA68927);Xenopus (NP_001016357); tobacco (X71430); potato (KAH0753093); tomato (NP_001306101);Silene GMT1 (sequence from [42])
Fig. 5
Fig. 5
Location of domains and regions in different OMTs. Six protein sequences that are representative for the different branches of the phylogenetic alignment shown in Fig. 4 were analysed using InterPro. The following sequences were used: pumpkin Cp4MP-OMT (this paper),Rhaphiolepis EjCCOMT (LC127201),Silene SIGMT1 (from [42]), tomato StCOMT (NP_001306101),Arabidopsis AtCCOMT (AAL07162), human HsCOMT (AAA68927). The following families, domains and regions were detected and are highlighted in different colours: protein families: SAM-dependent O-methyltransferase class I-type (PROSITE PS51682), SAM-dependent O-methyltransferase class II-type (PROSITE PS51683), caffeic acid O-methyltransferase-like (InterPro IPR016461); domains: plant methyltransferase dimerization domain (IPR012967), O-methyltransferase domain (IPR001077); regions: Coniferyl alcohol 9-OMT (FUNFAM 1.10.10.10:FF:000213), Caffeic acid OMTs (FUNFAM 1.10.10.10:FF:000357 and 3.40.50.150:FF:000061), Caffeoyl-CoA OMT1 homology (FUNFAM 3.40.50.150:FF:000147), Catechol OMT1-related (FUNFAM 3.40.50.150:FF:000054)
Fig. 6
Fig. 6
Cp4MP-OMT gene expression levels in flower and leaf tissues. Expression levels ofCp4MP-OMT (means ± sd) were calculated relative to expression of actin (housekeeping gene), which was set to 1 (n = 3). Differences between mean values in flower (1,4-DMB producing) and leaf (non-1,4-DMB producing tendril) tissues are significant (unpaired t-test, asterisks (**) indicate difference at a level ofp < 0.01)
Fig. 7
Fig. 7
Expression of His-tagged Cp4MP-OMT inNicotiana benthamiana.A SDS-PAGE andB western blot analyses of crude extract ofNicotiana benthamiana leaf tissue with (+) and without (-) recombinantly expressed His-tagged Cp4MP-OMT. Tobacco leaves were transfected with an expression vector either including the sequence for Cp4MP-OMT (+) or without it (-).C SDS-PAGE andD western blot analyses of His-tagged Cp4MP-OMT purification from crude extract. Lanes are indicated as follows: supernatant (sup), column flow through (ft), wash step (w), and elution (elu). The marker is a molecular mass marker (in kDa). Cp4MP-OMT bands were visualized using an antibody against His(6)-tag (arrows)
Fig. 8
Fig. 8
Production of 1,4-DMB from recombinant Cp4MP-OMT. Representative GC chromatograms showing conversion of 4-MP to 1,4-DMB by Cp4MP-OMT as compared to control(A); box plots showing the amount of 1,4-DMB (in ng) produced in the reactions(B). Asterisks (**) inB indicate significant difference (Cp4MP-OMT,n = 8; control,n = 5; Mann-WhitneyU test) at a level ofp < 0.005. InA, the peak at 18.545 min in 2 is a compound (contaminant) other than 1,4-DMB
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