# Impact of Environmental Factors on Stilbene Biosynthesis
## 2 Biosynthesis of Stilbenes and Stilbenoids
### Methylation  
Methylation of stilbene hydroxyl side groups leads to the formation of methoxystilbenes (Figure 6). Among the most known methoxystilbenes, there is pinosylvin monomethyl ether (3-hydroxy-5-methoxystilbene) found in several _Pinus_ spp. [113; 114] and _Alnus_ spp. [115], and pterostilbene (3,5-dimethoxy-4'-hydroxystilbene), biosynthesised by red sandalwood (_Pterocarpus santalinus_ [PERSON]) [116], Indian Kino (_Pterocarpus marsupium_ [PERSON].) [117], _Vaccinium_ spp. berries [118], and, at low levels, in grapevine leaves and berries [119; 120; 121; 122; 123; 124; 125; 126; 127; 128; 129]. Methylation of stilbenes, as well as of several other thousands of plant secondary metabolites, is catalyzed by \(S\)-adenosyl-L-methionine (SAM)-dependent \(O\)-methyltransferaseases (OMTs; EC 2.1.1). Methylation of hydroxyl groups alters the solubility and reactivity of stilbenes and can therefore affect their biological activity. For instance, pterostilbene has been shown to be 5-10 times more active than non-methylated resveratrolol inhibiting the germination of downy mildew (_Plasmopara viticola_) sporangia and grey mold (_Botrytis cinerea_) conidia [120]. Pinosylvin monomethyl ether has been reported to have significantly lower antifungal and antibacterial activity than pinosylvin [121], although it has shown greater activity against some brown-rot fungi [122]. Investigations on the relationship between chemical structure and biological activity revealed increased cytotoxicity and anticancer activity associated with resveratrolol methylation [123; 124]. The substitution of hydroxy with methoxy groups enhances the lipophilicity of pterostilbene over resveratrolol, which results in high bioavailability. This difference in pharmacokinetics might explain the higher bioactivity of pterostilbene over its parental compound resveratrolol. Methylated resveratrolol derivatives have consequently become attractive target compounds for both biproduction and metabolic engineering [125; 126]. Several plant OMTs have been characterized in the last decades. However, the majority of them have been found to be involved in the methylation of aromatic hydroxyl groups of different compounds such as benzylisoquinoline alkaloids [127], phenylpropanoids [128], and flavonoids [129], while only a few stilbene-specific OMTs have been reported to date. _V. vinifera_ resveratrol OMT (VvROMT) was shown to specifically catalyze the methylation of resveratrol and pinosylvin (3,5-dihydroxycystislene) at the C-3 or C-5 positions [130]. VvROMT was shown to convert resveratrol to pterostilbene both in vitro and _in planta_. The transient co-expression of _VvROMT_ and _VvSTS_ in tobacco resulted in the accumulation of pterostilbene. _VvROMT_ gene expression in grapevine leaves was induced by different stresses, including _P. viticola_ infection and UV radiation, accordingly with the role of pterostilbene in chemical plant defense [130]. _S. bicolor_ resveratrol OMT (SbOMT1) has been shown to catalyze the 4\({}^{\prime}\)-O-methylation of resveratrol both in vitro and _in planta_[125; 131]. In 2019, [PERSON] and colleagues [132] isolated and characterized a putative aromatic \(O\)-methyltransferase gene (_AcOMT_) in _Acorus calamus_ (Araceae) using RNA-seq analysis. Recombinant AcOMT1 expressed in _Escherichia coli_ showed high 4\({}^{\prime}\)-\(O\)-methylation activity toward resveratrol and its derivative, isarbonotigenin (3,4\({}^{\prime}\),5-trihydroxy-3\({}^{\prime}\)-methoxystilbene). In Scots pine, pinosylvin can be methylated by a pinosylvin O-methyltransferasearse (PsPMT1) to pinosylvin monomethyl ether, following ozone or fungal elicitation treatment [133]. However, it should be noted that PsPMT1 showed a relatively broad substrate specificity, methylating several compounds such as stilbene aglycones, flavonoids, and hydroxycinnamic acids, many of these even more efficiently than pinosylvin. In 2017, [PERSON] and co-workers [134] subsequently isolated and characterized an \(O\)-methyltransferasearse from _P.