A coumarin‐specific prenyltransferase catalyzes the crucial biosynthetic reaction for furanocoumarin formation in parsley

Abstract

SUMMARYFuranocoumarins constitute a sub-family of coumarin compounds with important defense properties against pathogens and insects, as well as allelopathic functions in plants. Furanocoumarins are divided into two sub-groups according to the alignment of the furan ring with the lactone structure: linear psoralen and angular angelicin derivatives. Determination of furanocoumarin type is based on the prenylation position of the common precursor of all furanocoumarins, umbelliferone, at C6 or C8, which gives rise to the psoralen or angelicin derivatives, respectively. Here, we identified a membrane-bound prenyltransferase PcPT from parsley (Petroselinum crispum), and characterized the properties of the gene product. PcPT expression in various parsley tissues is increased by UV irradiation, with a concomitant increase in furanocoumarin production. This enzyme has strict substrate specificity towards umbelliferone and dimethylallyl diphosphate, and a strong preference for the C6 position of the prenylated product (demethylsuberosin), leading to linear furanocoumarins. The C8-prenylated derivative (osthenol) is also formed, but to a much lesser extent. The PcPT protein is targeted to the plastids in planta. Introduction of this PcPT into the coumarin-producing plant Ruta graveolens showed increased consumption of endogenous umbelliferone. Expression of PcPT and a 4-coumaroyl CoA 2'-hydroxylase gene in Nicotiana benthamiana, which does not produce furanocoumarins, resulted in formation of demethylsuberosin, indicating that furanocoumarin production may be reconstructed by a metabolic engineering approach. The results demonstrate that a single prenyltransferase, such as PcPT, opens the pathway to linear furanocoumarins in parsley, but may also catalyze the synthesis of osthenol, the first intermediate committed to the angular furanocoumarin pathway, in other plants.

Molecular Cloning and Characterization of a Geranyl Diphosphate-Specific Aromatic Prenyltransferase from Lemon

“…able to maintain the normal level of final products. A similar result was also observed in our earlier study using PcPT (Karamat et al, 2014).…”

“…iPSORT predicted that ClPT1 has a transit peptide at its N terminus, and PSORT found a putative cleavage site between G36 and G37 (Supplemental Fig. S1A Collakova and DellaPenna, 2001;Savidge et al, 2002;Yazaki et al, 2002;Ohara et al, 2006;Sadre et al, 2006;Sasaki et al, 2008;Tsurumaru et al, 2010;Yang et al, 2011;Karamat et al, 2014). ClPT1 could not be grouped into any previously identified clade, including membrane-bound PTs ( Fig.…”

“…In the lemon flavedo, 8-geranyltransferase activity for umbelliferone was also detected (Munakata et al, 2012). To date, only one gene encoding these enzymes has been described; this gene, which encodes a parsley PT (PcPT), was very recently isolated (Karamat et al, 2014).…”

“…Since then, genes encoding various flavonoid PTs have been identified in Leguminosae (Akashi et al, 2009;Sasaki et al, 2011;Shen et al, 2012). Although other prenylated aromatic compounds, including coumarins, xanthons, phenylpropanoids, and phloroglucinols, have been isolated from many plant species, no gene encoding a PT for those aromatics has been isolated, except for the gene encoding a phloroglucinol-specific enzyme (HlPT1) from hops (Humulus lupulus) and a the recently isolated coumarin dimethylallyltransferase from parsley (Tsurumaru et al, 2010Karamat et al, 2014). These isolated plant aromatic PTs show strong preference for dimethylallyl diphosphate (DMAPP) as the prenyl donor substrate, although in nature, many geranylated phenolics and less farnesylated phenolics have been described.…”

Biphenyl 4-Hydroxylases Involved in Aucuparin Biosynthesis in Rowan and Apple Are Cytochrome P450 736A Proteins

“…Infiltration with A. tumefaciens containing the empty vector served as a negative control. After infiltration (48 h), a portion of the infiltrated leaf was examined with a confocal microscope to ensure transient expression, while the remaining part of the leaf was used to prepare microsomes, essentially as described by Karamat et al (2014). The microsomal protein (20 mg) was incubated with 10 mM 3-hydroxy-5-methoxybiphenyl in a final B4H assay volume of 200 mL and adjusted with 100 mM potassium phosphate buffer, pH 7.5.…”