FIGURE 10.13 Presumed biosynthetic pathway of the phenylpropane compounds via shikimic acid and l-phenylalanine. (Data from Odoux, E., Fruits, 61, 171–184, 2006.)
Most of the research published in an attempt to clarify the subsequent stages of the biosynthetic pathway of vanillin was conducted using cell cultures and showed that different pathways could be activated, depending on the experimental conditions (reviewed by Dignum et al., 2001; Walton et al., 2003). It is therefore difficult to draw any definitive conclusions and even more difficult to attempt to extrapolate the results obtained in these conditions to the plant.
FIGURE 10.14 Biosynthetic pathway of vanillin proposed by Zenk (1965) and Negishi et al. (2009).
What emerges from the research conducted on the fruits, of which there is very little information (Zenk, 1965; Kanisawa, 1993; Kanisawa et al., 1994; Negishi et al., 2009), is that two biosynthetic pathways are suggested:
• The first one suggests that the direct precursor of vanillin is ferulic acid (C6–C3 compound), which means that the C3 side chain of the molecule is later shortened to give vanillin (C6–C1 compound) (Figure 10.14). This is the argument put forward by Zenk (1965) and confirmed by the recent work of Negishi et al. (2009). In both cases, the results were obtained after incorporating 14C-labeled molecules on green vanilla discs and monitoring their conversion.
The results obtained by Negishi et al. (2009) also suggest that biosynthesis does not cause glucosylated intermediate compounds to intervene; vanillin is therefore synthesized in the aglycon form, and then glucosylated once produced.
• The second one suggests that shortening of part C3 occurs higher up at the \level of 4-coumaric acid (C6–C3 compound) to give 4-hydroxybenzalde-hyde (C6–C1 compound), which is then hydroxylated and methylated to give vanillin (Figure 10.15). This argument is favored by Kanisawa et al. (1994)—although he also suggests a pathway via diglucosides and does not rule out the possibility of ferulic acid as a precursor—based on the different compounds identified in the fruit at different stages of development. This is also the argument defended by the team at Rutgers University, Princeton, USA, who purified a 4-hydroxybenzaldehyde synthase (4HBS) (from cell cultures) and a methyltransferase (DOMT) (from the bean) that can catalyze, respectively, the conversion of 4-coumaric acid into 4-hydroxybenzal-dehyde and 3,4-dihydroxybenzaldehyde (protocatechuic aldehyde) into vanillin (Podstolski et al., 2002; Pak et al., 2004). The existence of an enzyme able to hydroxylate 4-hydroxybenzaldehyde into 3,4-dihydroxy-benzaldehyde remains to be proven, preferably in the fruit. The results obtained by Negishi et al. (2009) do not show any conversion of 4-hydroxy-benzaldehyde into vanillin.
FIGURE 10.15 Biosynthetic pathway of vanillin proposed by Kanisawa et al. (1994) and Podstolski et al. (2002). In the pathway suggested by Kanisawa et al. (1994), the compounds involved are glucosylated from 4-coumaric acid (not shown in the figure).
In the pathway suggested by Kanisawa et al. (1994), glucosylation takes place as soon as 4-coumaric acid appears; the subsequent reactions would therefore involve glucosylated intermediates up to glucovanillin.
As discussed in the previous sections, glucovanillin is present in fully mature fruits mainly in the placentas and, to a lesser extent, in the papillae. According to Joel et al. (2003), papillae are the site of biosynthesis of glucovanillin in the bean, based on the presence of 4-hydroxybenzaldehyde synthase (4HBS) in the cytoplasm of the cells. The presence of 4HBS was revealed by immunolocalization, but the research note that supported the presentation of the results gives no details of the methodology used. Glucovanillin (or precursors of this molecule) is then secreted by these papillae in the extracellular space around the seeds. French (2005) believes that the presence of other enzymes involved in the biosynthesis of glucovanillin must also be shown in order to confirm this hypothesis.
Furthermore, if papillae were the only site of biosynthesis of glucovanillin, this would raise the question of how it is transported to the placentas (Odoux and Brillouet, 2009), which are the preferred sites of accumulation.
A great deal of research remains to be carried out to clarify the biosynthetic pathway(s) of glucovanillin in the vanilla bean, and also to determine the tissue(s) involved.
References
Ansaldi, G., Gil, G., and Le Petit, J. 1988. Procédé d'obtention d'arôme naturel de vanille par traitement des gousses de vanille verte et arôme obtenu. Brevet, INPI. No. 2, 634 979, p. 7.
Anwar, M.H. 1963. Paper chromatography of monohydroxyphenols in vanilla extract. Analytical Chemistry 35:1974–1976.
Arana, F.E. 1943. Action of a β-glucosidase in the curing of vanilla. Food Research. 8:343–351.
Arana, F.E. 1944. Vanilla curing and its chemistry. Federal Experiment Station of the US Department of Agriculture, Mayaguez, Puerto Rico, Bulletin No. 42, 17pp.
Arber, A. 1937. The interpretation of the flower: A study of some aspects of morphological thought. Biology Review 12:157–184.Balls, A.K. and Arana, F.E. 1941. The curing of vanilla. Industrial and Engineering Chemistry 33(8):1073–1075.
Bartholomew, D.M., Van Dyk, D.E., Cindy Lau, S.-M., O’keefe, D.P., Rea, P.A., and Viitanen, P.V. 2002. Alternate energy-dependent pathways for the vacuolar uptake of glucose and glutathione conjugates. Plant Physiology 130:1562–1572.
Beckman, C.H. 2000. Phenolic-storing cells: Keys to programmed cell death and periderm formation in wilt disease resistance and in general defence responses in plants? Physiological and Molecular Plant Pathology 57:101–110.
Boudet, A.M., Alibert, A., and Marigo, G. 1984. Vacuoles and tonoplast in the regulation of cellular metabolism. In: A.M. Boudet, A. Alibert, G. Marigo, and P.J. Lea, eds. Membranes and Compartmentation in the Regulation of Plant Functions. Clarendon Press, Oxford, 29–47.
Brodélius, P.E. 1994. Phenylpropanoid metabolism in suspension cultures of Vanilla planifolia Andr. V. High performance liquid chromatographic analysis of phenolic glycosides and aglycones in developing fruits. Phytochemical Analysis 5:27–31.
Brunerie P.M. 1993. Procédé d’obtention d’arôme naturel de vanille par traitement enzyma-tique des gousses de vanille verte et arôme obtenu. International Patent Application, 1993, No PCT/FR92/00837, 11pp.
Cameron, K. and Chase, M. 2000. Nuclear 18S rDNA sequences of Orchidacea confirm the subfamilial status and circumscription of Vanilloideae. In: K.L. Wilson and D.A. Morrison, eds. Monocots—Systematics and Evolution, Vol. 1 of Proceedings of the Second International Conference on the Comparative Biology of the Monocots. CSIRO, Melbourne, 457–464.
Childers, N.F. and Cibes, H.R. 1948. Vanilla culture in Puerto Rico. Federal Experiment Station of the US Department of Agriculture, Mayaguez, Puerto Rico, Circular No. 28, 94pp.
De Lanessan, J.L. 1886. Vanille. In: J.L. De Lanessan, ed. Les plantes utiles des colonies fran-çaises. Imprimerie Nationale, Paris, 62–74.
Dignum, M.J.W., Kerler, J., and Verpoorte, R. 2001. Vanilla production: Technological, chemical and biosynthetic aspects. Food Reviews International 17:199–219.