Two polyketide synthases are necessary for 4‐hydroxy‐5‐methylcoumarin biosynthesis in Gerbera hybrida

Abstract

Gerbera (Gerbera hybrida) is an economically important ornamental species and a model plant of the Asteraceae family for flower development and secondary metabolism. Gerberin and parasorboside, two bitter tasting glucosidic lactones, are produced in high amounts in nearly all gerbera tissues. Gerbera and its close relatives also produce a rare coumarin, 4-hydroxy-5-methylcoumarin (HMC). Unlike most coumarins, 5-methylcoumarins have been suggested to be derived through the acetate-malonate pathway. All of these polyketide-derived glucosylated molecules are considered to have a role in defense against herbivores and phytopathogens in gerbera. Gerbera expresses three genes encoding 2-pyrone synthases (G2PS1-3). The enzymes are chalcone synthase-like polyketide synthases with altered starter substrate specificity. We have shown previously that G2PS1 is responsible for the synthesis of 4-hydroxy-6-methyl-2-pyrone (triacetolactone), a putative precursor of gerberin and parasorboside. Here we show that polyketide synthases G2PS2 and G2PS3 are necessary for the biosynthesis of HMC in gerbera, and that a reductase enzyme is likely required to complete the pathway to HMC. G2PS2 is expressed in the leaf blade and inflorescences of gerbera, while G2PS3 is strictly root specific. Heterologous expression of G2PS2 or G2PS3 in tobacco leads to the formation of 4,7-dihydroxy-5-methylcoumarin, apparently an unreduced precursor of HMC, while ectopic expression in gerbera leads to HMC formation in tissues where nontransgenic tissue does not express the genes and does not accumulate the compound. Using protein modelling and site-directed mutagenesis we identified the residues I203 and T344 in G2PS2 and G2PS3 to be critical for pentaketide synthase activity.

Molecular and functional characterization of two isoforms of chalcone synthase and their expression analysis in relation to flavonoid constituents in Grewia asiatica L

“…Incidentally, gene duplication occurrence may be analysed as an essential resource for the origin of evolutionary advances [ 51 ]. There is growing evidence that the CHS-like enzymes in the CHS-superfamily evolved from CHS via duplication and subsequent divergence over the course of evolution [ 52 , 53 ]. The CHS and CHS-like genes provide an interesting example of evolutionary asymmetry and evolutionary novelty following gene duplication [ 54 ].…”

The O‐methyltransferase PMT2 mediates methylation of pinosylvin in Scots pine

“…High‐resolution MS analyses interfaced with UPLC were done in the Metabolomics unit at the University of Helsinki, Finland as described by Pietiäinen et al . ().…”

Structure Elucidation of Prenyl- and Geranyl-Substituted Coumarins in Gerbera piloselloides by NMR Spectroscopy, Electronic Circular Dichroism Calculations, and Single Crystal X-ray Crystallography

“…The coumarin skeleton is synthesized from acetyl-CoA and malonyl-CoA through the polyketide pathway in plants [42]. Inspired by the biosynthetic pathway of tetrahydrocannabinolic acid (THCA) in Cannabis sativa [43], the monoterpenoid-coumarins identified in this study are plausibly first added a monoterpenoid chain to C-3 of the coumarin skeleton, catalyzed by prenyl transferase (Pathway 1 in Figure 10).…”

“…The coumarin skeleton is synthesized from acetyl-CoA and malonyl-CoA through the polyketide pathway in plants [42]. Inspired by the biosynthetic pathway of tetrahydrocannabinolic acid (THCA) in Cannabis sativa [43], the monoterpenoid-coumarins identified in this study are plausibly first added a monoterpenoid chain to C-3 of the coumarin skeleton, catalyzed by prenyl Chiral resolution of 23 gave two enantiomers (23a and 23b) in a 1:1 ratio ( Figure S2, Supplementary Material), and comparison of the ECD spectra of 23a and 23b with the ECD spectrum calculated for 23b (Figure 9), allowed assignment of the absolute configuration to be 9R,10R,11R,14S for 23a and 9S,10S,11S,14R for 23b, respectively, with ∆ ESI of 0.88.…”