Handbook of Vanilla Science and Technology

In V. planifolia, at least five different OMTs have been characterized. From the tissue culture a cDNA encoding a multifunctional OMT was isolated and functionally characterized. Studies on its substrate specificity showed that it preferentially methylated the typical caffeic acid O-methyltransferase (COMT) substrates, caffeoylaldehyde and 5-hydroxyco-niferylaldehyde, but also had activity with 3,4-dihydroxybenzaldehyde, the proposed immediate vanillin precursor (Pak et al. 2004). Its presence was found in leaves, stems, and roots but not in pods of V. planifolia. Although the enzyme was active on 3,4-dihydroxybenzaldehyde, its tissue location and substrate specificity indicated it was unlikely to be involved in vanillin biosynthesis and is most likely a COMT involved in lignin biosynthesis. Based on the relative activities of this enzyme, it is most probably connected with methylation of the 3-OH of caffeoylaldehyde and 5-OH of 5-hydroxyco-niferylaldehyde during S-lignin formation (Dixon et al. 2001; Parvathi et al. 2001). Thus, in pods there must be another distinct 3,4-dihydroxybenzaldehyde O-methyltransferase (DOMT) responsible for vanillin biosynthesis. Strong DOMT activity was found in extracts of the inner part of pod at 5 to 11 months post-pollination (Pak et al. 2004), which is the pod developmental period when glucovanillin accumulation begins (Havkin-Frenkel et al. 1999).

In cell suspension culture of V. planifolia, two kinetin-inducible COMTs have been described. One was caffeic acid 4-O-methyltransferase, apparently connected with vanillic acid biosynthesis in cell suspension culture in the pathway proposed by Funk and Brode-lius (1990). The other kinetin-inducible enzyme was a typical caffeic acid 3-O-methyl-transferase connected to lignin biosynthesis (Xue and Brodelius 1998).

Two other O-methyltransferases from V. planifolia, Van OMT-2, and Van OMT-3, which have novel substrate preferences, have also been described (Li et al. 2006). These enzyme are capable of efficient methylation of the outer hydroxyl group in substrates having a 1,2,3-trihydroxybenzene structure, such as in methylgallate or in the B ring of the flavonol myricetin. Although the DNA sequences of Van OMT-2 and Van Omt-3 are 52% identical with the V. planifolia COMT described above (Pak et al. 2004), their activity with the usual COMT substrates was negligible. Based on phylogenetic analysis it was proposed that these enzymes evolved from the V. planifolia COMT (Li et al. 2006). The role of these enzymes in V. planifolia requires further elucidation.

17.5 BENZYL ALCOHOL DEHYDROGENASE (BAD)

4-Hydroxybenzylalcohol is one of the most abundant potential vanillin precursors found in green vanilla beans (Kanisawa et al. 1994). In V. planifolia embryo culture, it accumulates up to 3% of dry weight (Havkin-Frenkel etal. 1996). It was suggested that this alcohol is formed from 4-hydroxybenzaldehyde as a result of aromatic alcohol NADPH-dependent dehydrogenase activity (BAD). Although this enzyme in V. planifolia was not sufficiently investigated, there is data indicating its high activity in vanilla embryo culture. The enzyme was also found capable of fast and efficient reduction of vanillin to vanillyl alcohol (Havkin-Frenkel and Podstolski, unpublished).

17.6 GLYCOSYLTRANSFERASES (GTS)

Many secondary metabolites including derivatives of benzoate, salicylate, phenylpropene, coumarins, flavonoids, anthocyanidins, terpenoids, and alkaloids occur in plant tissues as glucosides (Vogt and Jones 1997). Some of the presumed intermediates of the vanillin biosynthetic pathway downstream of 4-coumaric acid, such as 4-hydroxybenzaldehyde, 4-hydroxybenzyl alcohol, vanillin, vanillic acid, vanillyl alcohol, bis[4-(β-D-glucopyrano-syloxy)-benzyl]-2-isopropyltartrate and bis[4-(β-D-glucopyranosyloxy)-benzyl]-2-(2-butyl) tartrate (in Figure 17.1 referred to as “tartrate esters”) have been identified in extracts of green vanilla beans (Kanisawa et al. 1994; Dignum et al. 2004). Due to their more hydrophilic nature, glucosides are as a rule more soluble in water in comparison to their aglycones, the non-sugar partners. Higher solubility of glucosides allows for their higher concentration in cell storage compartments, usually vacuoles. Their high solubility may also facilitate transport of these metabolites between cell compartments and plant tissues and organs. As discussed below, vanillin accumulates in the green beans as a glucoside.