The Intracellular Localization of the Vanillin Biosynthetic Machinery in Pods of Vanilla planifolia

Abstract

Vanillin is the most important flavor compound in the vanilla pod. Vanilla planifolia vanillin synthase (VpVAN) catalyzes the conversion of ferulic acid and ferulic acid glucoside into vanillin and vanillin glucoside, respectively. Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) of vanilla pod sections demonstrates that vanillin glucoside is preferentially localized within the mesocarp and placental laminae whereas vanillin is preferentially localized within the mesocarp. VpVAN is present as the mature form (25 kDa) but, depending on the tissue and isolation procedure, small amounts of the immature unprocessed form (40 kDa) and putative oligomers (50, 75 and 100 kDa) may be observed by immunoblotting using an antibody specific to the C-terminal sequence of VpVAN. The VpVAN protein is localized within chloroplasts and re-differentiated chloroplasts termed phenyloplasts, as monitored during the process of pod development. Isolated chloroplasts were shown to convert [14C]phenylalanine and [14C]cinnamic acid into [14C]vanillin glucoside, indicating that the entire vanillin de novo biosynthetic machinery converting phenylalanine to vanillin glucoside is present in the chloroplast.

Fig. 1

Transverse section of a 6-month-old vanilla pod, with arrows pointing to the different tissues present.

Fig. 2

Images obtained by DESI-MSI of longitudinal (left column) and cross- (right column) sections of a 6-month-old vanilla pod. (A and B) Photo of vanilla pod tissue obtained immediately following longitudinal and cross cryosectioning, respectively. (C and D) Vanillin distribution (m/z 153). (E and F) Vanillin glucoside distribution (m/z 337). (G and H) Sucrose distribution (m/z 381). (I and J) Colored overlay of vanillin (red), vanillin glucoside (green) and sucrose (blue) distributions.

Fig. 3

Western blot analysis of the presence of mature and immature forms of VpVAN in protein extracts. of vanilla pods and in N. benthamiana leaves following transient expression of VpVAN. Lane A: pre-stained Bio-Rad protein ladder. Lane B: unstained Bio-Rad protein ladder. Lane C: protein extract from N. benthamiana leaves following transient expression of VpVAN probed with an antibody specific to the C-terminal sequence of VpVAN. Lane D: protein extract of a 7-month-old vanilla pod probed with an antibody specific to the C-terminal sequence of VpVAN. A 10 μg aliquot of protein were applied to each lane.

Fig. 4

Mature VpVAN homodimer and putative oligomers detected in the crude V. planifolia extracts as demonstrated by SDS–PAGE followed by Western blot analysis. Lane A: crude protein extract from a 7-month-old vanilla pod from V. planifolia. Lane B: pre-stained Bio-Rad protein ladder. Lane C: unstained Bio-Rad protein ladder. 1, VpVAN mature protein (25 kDa); 2, VpVAN immature protein (39 kDa); 3, putative mature VpVAN homodimer (50 kDa); 4, putative mature VpVAN trimer (75 kDa); 5, putative mature VpVAN oligomers (100 kDa). The proteins present in a 7-month-old vanilla pod were separated by SDS–PAGE (12% Criterion™ TGX Stain-Free Precast Gels) and visualized using the ChemiDoc MP Imaging System (Bio-Rad). The major protein components present in the pod extracts are marked with * (red) while VpVAN and putative oligomers are marked with * (yellow). Western blot analysis was conducted using a C-terminal sequence-specific antibody against VpVAN in a 1 : 50 dilution. At this extremely high antibody concentration, the primary antibody demonstrated specificity towards VpVAN. No cross-reactions were observed with the major protein components present in the extract.

Fig. 5

The ratio between monomeric and putative oligomeric forms of VpVAN as analyzed by treatments with reductant and oxidant as montiored by SDS–PAGE followed by Western blot analysis. Crude protein extracts from 8-month-old pods were analyzed. Protein bands recognized by the VpVAN antibody are marked with * (yellow). Lane A: pre-stained Bio-Rad protein ladder. Lane B: treatment with 50 mM TCEP for 15 min. Lane C: treatment with 50 mM TCEP for 15 min followed by 50 mM potasium fericyanide for 30 min. Lane D: unstained Bio-Rad protein ladder.

Fig. 6

Immunohistochemical localization of VpVAN in cytoplasmic organelles in a 7-month-old V. planifolia pod as observed by confocal microscopy. (A) Transverse section of a 7-month-old V. planifolia pod providing an overview of VpVAN localization in the mesocarp. (B) Close-up of a single mesocarp cell showing the intracellular localization of VpVAN in plastids in the cytoplasm. (C) The same section as shown in (B) observed with translucent light. Images were obtained from transverse section of a 7-month-old V. planifolia pod using the VpVAN C-terminal antibody and a goat anti-rabbit antibody labeled with FITC. The images were recorded using a Leica SPII confocal scanning microscope. Arrows indicate the position of selected cytoplasmic plastids harboring VpVAN. Abbreviations: epi, epicarp; v, vacuole.

Fig. 7

Immunolocalization of VpVAN to chloroplasts and phenyloplasts in 7-month-old V. planifolia pods using an antibody specific to the C-terminal sequence of VpVAN. (A–F) Data obtained from two transverse sections of two different pods are shown; V. planifolia pod 1 (A–C) and V. planifolia pod 2 (D–F). (A, D and G) Visualization of the examined transverse vanilla pod sections using light microscopy documenting the localization of chloroplasts. Chloroplasts are identified based on their Chl content. Selected chloroplasts are marked with black arrows. (B and E) Immunodetection of VpVAN in chloroplasts and phenyloplasts by FITC and fluorescence microscopy. The chloroplasts and phenyloplasts are stained green due to the antibody reaction. Selected phenyloplasts and chloroplasts are marked with white stars and white arrows, respectively. (C and F) Localization of VpVAN in chloroplasts (white arrows) and phenyloplasts (white stars) as monitored by fluorescence microscopy using a filter setting enabling simultaneous detection of FITC and Chl fluorescence. Chl fluorescence appears in red and demonstrates the localization of chloroplasts. Phenyloplasts that are totally free of Chl appear in green on (C) and (F) while those plastids retaining a small amount of Chl due to incomplete re-differentiation appear in yellow in (C) and (F). (G, H and I) Control panels; vanilla pod sections probed with pre-immune serum. No cross-reactions were observed. Chloroplasts: black arrows (A, D and G) and white arrows (B, C, E, F, H and I). Phenyloplasts: white stars. The same selected set of chloroplasts and phenyloplasts is labeled on the different panels to facilitate interpretation of the nature of the observed structures. Scale bars correspond to 100 μm. epi, epicarp.

Fig. 8

Intact chloroplasts isolated from 8-month-old V. planifolia pods synthesize vanillin de novo. (A) The purity and integrity of the isolated chloroplasts as monitored by light microscopy. (B) Vanillin biosynthetic activity in isolated chloroplasts as monitored by administration of [¹⁴C]phenylalanine (lane 1B) and [¹⁴C-cinnamic acid (lane 2B) as substrates. An extract from the inner part of a V. planifolia pod disc incubated with [¹⁴C]vanillin was applied (lane 3B) to visualize the migration position of [¹⁴C]vanillin glucoside.