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; pclass="underline" 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.
β-Glucosidase and Glucovanillin Metabolism in the Vanilla Bean
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).