Vanilla

Morphology, Anatomy, and Histology of Mature Vanilla Bean

Part of the information presented below is largely taken from the following references: De Lanessan, 1886; Villiers et al., 1909; Swamy, 1947; Roux, 1954; Odoux et al., 2003; and French, 2005.

The mature green vanilla bean has a roughly triangular transverse section with a central cavity containing numerous black seeds (Figure 10.4). From an anatomical and histological viewpoint, from the outer to the inner part of the fruit we find the epicarp, the mesocarp, and the endocarp (Figure 10.5d).

FIGURE 10.4 Cross section of mature vanilla bean.

FIGURE 10.5 (See color insert following page 136.) From the flower to the mature bean. Cross sections (3 μm) of a vanilla bean embedded in Technovit 7100 resin, at different stages after staining with periodic acid-Schiff (PAS)–Naphthol Blue Black: (a) 9 days after pollination (dap); (b) 14 dap; (c) 60 dap; (d) 8 months after pollination. (Data from Odoux et al., 2003. Annals of Botany 92: 437–444.) The walls and the storage sugars are stained in pink; the proteins in blue. en, endocarp; ep, epicarp; fu, funicle; me, mesocarp; pl, placenta; s, seed; vb, vascular bundle.

The epicarp is made up of a layer of contiguous cells, which vary in length, are polygonal in shape, and run parallel to the long axis of the bean. These thick-walled cells, which are stained in intense pink by Schiff reagent (Figure 10.5d) due to their biochemical composition (cellulose, hemicelluloses, and pectic substances), differentiate into a thick cuticle on the outer part of the fruit. The layer of the cells that forms the epicarp provides a protective layer for the bean.

The mesocarp makes up the majority of the fruit’s volume (Figure 10.5d). It consists of parenchyma cells. It is composed of vacuolated cells that increase in size from the epicarp or endocarp toward the central part of the mesocarp, where their size may reach 300 μm. This considerable increase in size appears to be accompanied by an increase in ploidy through endoreplication (S. Brown, pers. comm.).

The mesocarp is vascularized. There are three groups of three triangularly arranged vascular bundles (Figure 10.5d). They mark the center of each carpel and, from an evolutionary viewpoint, represent the main vascular bundle of the macro-phylla. Among these triangularly arranged groups of bundles, we note the presence of three additional vascular bundles located at the center of the mesocarp, midway between the epicarp and the endocarp. The vascular bundles of the bean are of the closed collateral type, as with most monocotyledons.

Inside the mesocarp, across two of the three groups of three vascular bundles, a radial layer of specialized cells can be observed on cross histological bean sections (Figure 10.5a). The cells that make up this layer are aligned along the ray of the bean and contain carbohydrate reserves in the form of starch granules. These two layers that radiate out from the inner to the outer part of the mesocarp mark the location of the two future dehiscence lines (Figure 10.5a).

The endocarp is made up of one or two layers of small cells that cover the inside of the fruit’s cavity (Figure 10.5c).

At the center of the carpellary leaf are found specialized cells, the papillae. The papillae are intensely stained by Schiff reagent (Figure 10.5d). In agreement with the previous observations (De Lanessan, 1886), the papillae are also intensely stained by Nile Red (Figure 10.6), indicating the presence of high concentration of storage lipids. Some proteinaceous material, stained greenish-blue by Naphthol Blue Black, can be seen in the papillae and in the central cavity in the immediate vicinity of the apical ends of the papillae (Figures 10.5c and d).

FIGURE 10.6 (See color insert following page 136.) Visualization of lipid storage in the papillae of a mature vanilla bean after Nile Red staining (imaging with confocal microscope Zeiss 510 Meta, laser 488 nm and 405 nm, yellow: Nile Red staining, blue: autofluorescence of walls and papillae).

Each side of the pod bears a placenta composed of four to five layers of parenchyma cells and covered by an epidermis. The placenta is divided into two longitudinal placental laminae-bearing funicles, made up of three or four layers of cells (Figure 10.5b), to which seeds are attached. On cross-sectioning, each pair of placental laminae appears as finger-shaped lobes bent inside the central cavity (Figure 10.5d). The cells of the epidermis of the placental laminae contain numerous lipid storage vesicles, as in the papillae.

At maturity, the cavity of the fruit contains numerous small seeds (0.2 μm on average) that are oblong in shape with a dark-colored integument. Each seed is attached to a long, narrow funicle. The seeds are held in mucilage that was found to be essentially polysaccharidic in nature (Odoux and Brillouet, 2009); remnants of this mucilage stained in light pink are shown in Figure 10.5d around the holes previously occupied by the seeds.

Vanilla Bean Ontogenesis: From the Flower to the Mature Pod

The vanilla flowers, in groups of 10 or 15, form small bunches at the leaf axil. White, greenish, or pale yellow in color, they have the typical structure of orchid flowers, the most evolved of all the flowers in the plant kingdom. The perianth of these flowers is made up of three sepals and three petals. The lowest petal of the flower, the lip, is usually large, and is spurred. Under the perianth is a very long ovary, which ends with a short pedicel attaching the flower to the inflorescence axis (Figure 10.2). Before pollination, the vanilla ovary is far from being fully developed. After pollination—natural pollinators are not very well known (Lubinsky et al., 2006)—the perianth withers and falls off (Figure 10.2), while the wall of the inferior ovary progressively evolves to form the fruit’s pericarp (capsule), and the ovules inside the cavity of the ovary develop into seeds. The first sign of the ovary developing into a fruit is a considerable and rapid increase in its size. From an anatomical and histocytological viewpoint, the most spectacular change concerns the inner part of the ovary. After fertilization, a polarized elongation of the endocarp cells toward the cavity of the ovary can be observed. These cells will develop into secretory trichomes, the papillae. At 9 or 14 days after pollination, the papillae remain undifferentiated (Figures 10.5a and b). At this stage, the upper part of the central cavity of the pollinated ovary contains a tissue composed of degenerative cells deeply stained in pink by Schiff reagent (Figure 10.5b). This tissue could correspond to the tissues termed “transmitting tissues” by Arber (1937), a parenchyma that provides a nutrient substrate, which aids the pollen tube to grow through the style and inside the ovary cavity (Figures 10.5a and b).

The differentiation zone of the papillae is not continuous; it is located at the center of the carpel leaf, in the pericarp zone situated under the three triangularly arranged vascular bundles (Figure 10.5b). Two months after pollination, the papilla cells begin their elongation and differentiation. At this stage, their length can reach 20 μm (Figure 10.5c). At maturity, the papillae are around 200 μm in length (Fig ure 10.5d).

Under ultraviolet light, the papillae autofluoresce in white (Figure 10.7a). The cell wall of the mature papillae, which thickens in an uneven manner, undergoes lysis in its distal extremity, facilitating the secretion of its content into the cavity of the capsule (Figure 10.5d).

FIGURE 10.7 Fresh cross sections (100 μm) of mature vanilla bean (8 months after pollination) observed with epifluorescence microscope (Leica DM6000, filter A: 340–380 nm excitation, 425–800 emission). (a) a general view of placenta and papillae; (b) magnification of funicle, seeds, and “matrix.” en, Endocarp; fu, funicle; pl, placenta; vb, vascular bundle.

A white fluorescent substance (Figures 10.7b and 10.8) can also be observed, which surrounds the seeds and partially fills the bean cavity. This substance—called the “matrix” by French (2005)—does not appear to be of cellular origin (i.e., the extremity of funicles), but rather appears to be amorphous and different from the polysaccharidic mucilage.

FIGURE 10.8 Longitudinal view of a mature bean opened in one of the three corners (in the papillae area) with a razor blade and observed with stereomicroscope Zeiss Lumar V12 [white fluorescence of walls (in the mesocarp), papillae and “matrix” = autofluorescence through UV excitation].

The placentas (especially the placental lamina extremity and funicles) also autofluoresce (golden yellow fluorescence, Figures 10.7a and b).

In the mesocarp, white fluorescent globules can be observed in the cells, which may correspond to polyphenols. The lignified tissues of the vascular bundles (xylem and sclerenchyma fibers) emit a bluish autofluorescence linked to the lignin contained in their walls (Figure 10.7a).

Ovule Ontogenesis: From the Ovule to the Seed

In vanilla plants, there is a considerable interval between microsporogenesis, which produces the pollen, and macrosporogenesis, which results in the embryo sac. Surprisingly, the differentiation of the ovules at the top of the placentas mainly occurs after pollination. At two days after pollination, the ovules remain undifferentiated (Figure 10.9a). The placental laminae branch out into a large number of funicles that constitute the placentas (Figure 10.9a). They are made up of four layers of cells that gradually vacuolate from the base (the placental lamina side) to the top. The end of each placenta contains a pool of meristematic cells (actively dividing cells that have dense cytoplasm with a centrally positioned nucleus) (Figure 10.9a). It is this mer-istematic end that assures the growth of the placenta, and later the shift of the ovules into the terminal position. This is clear at 15 days after pollination (Figure 10.9b). The outer integument and the inner integument of the ovule differentiate almost simultaneously with the individualization of the spore mother cell, which will undergo meiosis (Figure 10.9b). The outer integument is made up of four layers of cells and up to eight at its base. This characteristic differs from the other orchids, whose inner integument is made up of a single layer of cells. The inner integument and the outer integument of the ovule are not fused and there is a gap between the two.

The nucellus shows a considerable development in relation to other orchids; it persists in the form of several cells at the base of the seeds when they are disseminated.

Most commonly in orchids, endosperm development is limited to 10 nuclei on an average (up to a maximum of 12). This endosperm is rapidly digested, from the first divisions of the zygote. Unlike the endosperm, the nucellus persists until the embryo development ends, particularly at the chalaza, when the seed is mature. The zygote is surrounded by a thickened outer cell wall and has a central star-shaped nucleus with a single nucleolus stained in black by Naphthol Blue Black (Figure 10.9c). The embryo, which ceases to develop very early on (just after the globular stage but before the torpedo stage), accumulates lipid and protein reserves in the form of aleurone grains that are intensely stained black by Naphthol Blue Black (Figure 10.9d).

FIGURE 10.9 From the ovule to the seed: Cross sections (3 μm) of vanilla beans at different stages of development, embedded in Technovit 7100 resin after staining with PAS–Naphthol Blue Black. (a) 2 dap; (b) 15 dap; (c) 20 dap; (d) 200 dap. The walls and the storage sugars are stained in pink, the proteins in blue. fu: Funicle; pl: placenta.

The vanilla seed coat is the result of the evolution of the outer integument and the inner integument of the ovule. The outermost layer of cells in the outer integument of the ovule becomes sclerous (Figure 10.9d). After fertilization, these cells undergo a polarized elongation following a perpendicular axis at the surface of the ovule. They accumulate brown compounds along their wall, which gradually make them opaque and give the seed coat a reticulated ornamentation. This change differs from those generally observed in other orchids, whose seed coats are finer and translucent.

As noted in the introduction, one of the most important characteristics of V. planifolia is that its glucosidase activity and its glucosylated precursor content (especially its glucovanillin content) are all exceptionally high. Inasmuch as the aromatic quality of the vanilla is closely linked to the hydrolysis of these glucosylated precursors by the β-glucosidase(s) present in the bean (see also Chapters 11 and 12), various researches have been carried out to determine their accumulation and biosynthesis sites in the fruit.

Tissue and Cellular Localization of Glucosidase Activity and Glucovanillin in the Mature Vanilla Bean

Finally, very little research has been conducted to try to identify the parts of the fruit that contain the glucoside precursors of the aroma components and the glucosidase activity.

However, for a long time, De Lanessan (1886) had implicitly suggested the hypothesis that the aroma precursors and the glucosidase activity occur in the central placental region, because he observed that only this part of the fruit had a characteristic smell after the bean had been cut in fine longitudinal slices from the external part toward the internal part.

However, Arana (1943) and Jones and Vicente (1949) found that most of the gluco-vanillin (60–80%) was present in the fleshy part of the bean (external mesocarp), and the rest was present in the internal placenta, whereas the enzymatic activity occurs exclusively in the external part of the bean (Arana, 1943) (Figure 10.10a). Arana concludes that the glucovanillin in the internal part of the fruit has to spread to the external part where the enzyme is found or vice versa, in order to be hydrolyzed during vanilla curing or when the fruit matures on the vine. This hypothesis has been maintained by all the authors who have published work on vanilla during the past 60 years.

FIGURE 10.10 Tissue localization of glucovanillin and β-glucosidase activity in vanilla bean according to (a) Arana (1943) and Jones and Vicente (1949), (b) Odoux et al. (2003), (c) Joel et al. (2003) and Havkin-Frenkel et al. (2005). (Data from Odoux, E., Fruits. 61, 171–184, 2006.)

Odoux et al. (2003), on the other hand, showed that glucovanillin was found only in the internal part of the fruit and that it is mainly present in the placentas and, to a lesser extent, in the papillae (Figure 10.10b). In a more detailed study, Odoux and Brillouet (2009) found that, given the mass ratios of the different tissues and of their respective glucovanillin contents, 92.2% of glucovanillin was found in the placentas, compared to 7% in the papillae and 0.8% in the mesocarp. They found no glucovanillin in the intralocular space around the seeds, except as traces (which could be artifacts).

These authors (Odoux et al., 2003) found that glucosidase activity was much higher in the placentas than in the mesocarps or the papillae (expressed as total activity per mass unit of fresh tissue); the distribution of β-glucosidase activity expressed as a percentage of the maximum value was found as follows (Odoux and Havkin-Frenkel, 2005): 11% in the mesocarp, 100% in the placentas, and 20% in the papillae (Figure 10.10b); in other words, there is a near-perfect superposition between the distribution of glucovanillin and the enzymatic activity. As a result, the enzyme and the glucovanillin do not need to spread in the fruit tissues for hydrolysis to occur (see also Chapter 11).

Other research studies (Joel et al., 2003; Havkin-Frenkel et al., 2005) confirmed that glucovanillin was present in the white, inner part of the fruit (placentas and papillae). They also suggested the presence of glucovanillin in the intralocular space (Figure 10.10c), a result obtained by staining with catechin-HCl, after its biosynthe-sis (see below) and excretion by the papillae.

However, Havkin-Frenkel et al. (2005) found a decreasing gradient of enzymatic activity (expressed as specific activity) from the external part toward the internal placental region. The distribution of β-glucosidase activity expressed as a percentage of the maximum value was found as follows (Odoux and Havkin-Frenkel, 2005): 100% in the green outer fruit tissue, 43% in the placental tissue, and 15% in the hair cells (Figure 10.10c). Havkin-Frenkel et al. (2005), whose results are diametrically opposed to those of Arana (1943), also conclude that glucovanillin or the enzyme must spread through the bean tissues for its complete hydrolysis.

It is important to note that the results obtained by Havkin-Frenkel et al. (2005) and Odoux et al. (2003), concerning tissue localization of enzymatic activity, are not necessarily contradictory. Indeed, the specific activity is the ratio between the total activity and the protein content, and this protein content is much higher in the placentas than in the mesocarps (Odoux and Brillouet, 2009). In such a study, expressing enzymatic activity as specific activity—as Havkin-Frenkel et al. (2005) did—is not useful and may lead to incorrect interpretations.

At cellular level, the glucosidase activity is located in the cytoplasm or the apo-plasm (Figure 10.11). However, it is neither vacuolar nor parietal (Odoux et al., 2003). Glucovanillin was not positively present, but different considerations (concentration commonly around 300 mM and volume ratios of cellular compartments) suggest that it may be present in the vacuole (Figure 10.11), which is the preferred compartment for storing secondary metabolites (Boudet et al., 1984; Wink, 1997; Beckman, 2000; Bartholomew et al., 2002). It can also be present in the extracellular region around the seeds, as suggested by Joel et al. (2003), but the results obtained by Odoux and Brillouet (2009) contradict this hypothesis.

FIGURE 10.11 Cellular localization of glucovanillin and β-glucosidase activity according to Odoux et al. (2003).

Despite considerable controversy and confusion, the localization of glucovanillin and glucosidase activity at the tissue level has now been clarified. It remains to be determined whether glucovanillin is present in the intralocular space around the seeds, which may be important in confirming a possible tissue specialization in the biosynthesis of glucovanillin (see below).

At cellular and subcellular levels, the localization of β-glucosidase can be clarified using techniques such as immunolocalization; similarly, the cellular localization of glucovanillin remains to be determined.

Accumulation of Glucovanillin and β-Glucosidase During Vanilla Bean Development

Another area where very little research has been carried out is the evolution of glucovanillin and of β-glucosidase activity during bean development, with even less research been done on their evolution by tissue type.

The only point of agreement that has emerged from the various research studies on the evolution of glucovanillin (Ranadive et al., 1983; Sagrero-Nieves and Schwartz, 1988; Kanisawa et al., 1994; Brodélius, 1994; Havkin-Frenkel et al., 1999) is that the accumulation of vanillin or glucovanillin in the fruit starts from the 15th week after pollination and continues until around the 30th week.

However, the form in which vanillin is accumulated (free or glucosyl form) may lead to confusion. Ranadive et al. (1983) and Sagrero-Nieves and Schwartz (1988) show the evolution of free vanillin without prior hydrolysis; Ranadive’s results even show that free vanillin represents between 50% and 90% of the potential total vanillin. Brodélius (1994) considers that most of the vanillin is in glucosyl form with the free form not exceeding 15% of the potential total amount. Kanisawa et al. (1994) do not report the presence of vanillin in its free form during the development of the green fruit, and Havkin-Frenkel et al. (1999) indicate that vanillin is only accumulated in its glucosyl form. The latter point is confirmed by Leong (1991), who does not find the free form in the green beans. Arana (1943) had already found that vanillin is present almost exclusively in glucosyl form. Except in certain exceptional cases, our own analyses have always shown that in mature green fruits, the glucosyl form is predominant (at around 95% of the total), if we prevent any accidental hydrolysis during glucovanillin extraction (e.g., by conducting extraction in pure methanol at −18°C). However, a more timely study (unpublished results) on the evolution of glucovanillin during the development of the fruit showed that for beans around 3, 5, 7, and 9 months of development after pollination, the percentage of free vanillin in relation to the total (glucosyl plus free forms) was 33, 6, 1.5, and 0.2%, respectively. It would be interesting to get a confirmation of this evolution, which raises questions on the role of glucosylation of vanillin in the vanilla bean.

According to different researchers who monitored the evolution of glucosidase activity during bean development on the vine (Wild-Altamirano, 1969, Ranadive et al., 1983; Kanisawa et al., 1994), it would appear that this activity is measurable at all stages of fruit growth. However, the enzyme activity increases considerably between the third and the fourth month after pollination, reaching a maximum at around the fifth month. Therefore, the evolution of β-glucosidase activity during bean growth is on par with that of glucovanillin. The assays (unpublished results) conducted by the authors for glucosidase activity in fruits harvested during February 2005 in Madagascar at a developmental stage estimated at less than two months after pollination (highly asymmetrical fruit shape with a floral part that is far more rounded than the peduncular part) showed a glucosidase activity of around 650 nkatal/g of fresh weight for the floral part, compared to 230 nkatal/g of fresh weight for the peduncular part. These activities were already high and suggested an activity gradient in phase with the fruit development. For fruits from the same batch that had reached full size, but had not developed for more than 5 months after pollination, the glucosi-dase activities for the floral parts and peduncles were almost identical, at around 1100 nkatal/g of fresh weight, or the mean value obtained for fruits at the usual harvesting time (see below).

Glucovanillin contents obtained for the green fruit after eight months of development differ greatly from one research study to the other. If we convert the different values given in the literature into grams of glucovanillin per 100 g of dry weight, they range between 2% (Sagrero-Nieves and Schwartz, 1988) and 12% (Havkin-Frenkel et al., 1999). Further research studies have confirmed that mature green beans could comprise of glucovanillin around 10–15% of the dry weight (Ansaldi et al., 1988; Leong, 1991; Brunerie, 1993; Odoux, 2000; Havkin-Frenkel et al., 2005). Determination of glucovanillin from 70 green beans of seven different batches during the year 2000 in Madagascar (unpublished results) showed that glucovanillin contents could vary from 1.5% to 12% of dry weight depending on the fruit, with the majority of individual beans presenting a glucovanillin content of around 10%. Other determinations conducted in 2006 on batches from Papua New Guinea even showed maximum glucovanillin contents of more than 20% of dry weight for certain fruits, confirming the extreme variability that may exist in the glucovanillin content of V. planifolia fruits.

For β-glucosidase activities, it is impossible to compare the values given in the bibliography because of the means of expression (units) used, the protocols for obtaining enzyme extracts, the nature of the buffers used (pH, ionic strength, etc.), the molarity of the substrate (usually pNPG), and so on. Our own experience in this field has shown that in mature green beans with a physiologically healthy appearance and with a standardized, accurate protocol (Odoux, 2004), this activity could also vary considerably. Based on around 100 fruits from the Madagascan harvest in 2000 (unpublished results), the β-glucosidase activities ranged from around 100 to 2000 nkatal/g of fresh weight, with the majority of individual beans presenting an activity of around 1000 nkatal/g of fresh weight.

More systematic studies on the evolution of glucovanillin (and other glucosides) and of glucosidase activity during bean development are essential in order to confirm or refute the research already published. In the case of the aroma components, very strict analytical protocols must be established in order to remove any ambiguity regarding the form in which they are present at the different stages of development; it would also be useful to standardize the assays for glucosidase activity, for which the results can almost never be compared from one study to another.

These evolutions should also be measured by tissue type; in the case of gluco-sides, this could make it possible to obtain additional information on the biosynthetic pathways and sites for these components (see the following section).

Biosynthetic Site and Pathway for Glucovanillin

In his work on biosynthesis of vanillin, Lecomte (1901, 1913) concluded that it involved “coniferoside” (coniferin) that produced coniferyl alcohol through enzymatic hydrolysis, which was then turned into vanillin through the action of an “ oxidase.” Goris, who isolated “vanilloside” (glucovanillin) in 1924, suggested that a second possible pathway consisted of imagining the action of the “oxidase” before that of the “hydrolase” (Figure 10.12). Unable to isolate either the “coniferoside” or the coniferyl alcohol, he finally concluded that these hypotheses were unconfirmed (Goris, 1947). However, they were later resumed by Anwar (1963).

FIGURE 10.12 Biosynthetic pathway of vanillin proposed by Lecomte (1901, 1913) and Goris (1947).

It is now accepted that vanillin is a product of the biosynthetic pathway of shikimic acid, via phenylalanine, which leads to the phenylpropane compounds through enzymatic deamination, and primarily to cinnamic acid (Figure 10.13). Successive enzymatic hydroxylations and methylations then lead to the formation of p-hydroxycinnamic acids and, notably, coumaric, caffeic, ferulic, and sinapic acids.

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.