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.

Dynamic metabolic solutions to the sessile life style of plants

“…For comparison the solubility of vanillin in water is 30 mg ml –1 136. These observations support a function of NADES as a potential native solvent inside the phenyloplasts enabling accumulation of vanillin glucoside at 4 M (corresponding to ∼1800 mg ml –1 ) concentrations without precipitation or crystallization in fresh vanilla pods 117,121. Interestingly, the extractability of vanillin from cured vanilla pods using NADES was further improved by addition of 25–60% water.…”

“…The molecular mechanisms that trigger the re-differentiation from chloroplast to phenyloplasts are currently unknown. The vanilla pod is the prime plant source of vanillin and the site of vanillin glucoside biosynthesis and storage 118–121. Recently, it was demonstrated that vanillin is biosynthesized in the chloroplasts and phenyloplasts by the enzyme Vp VAN catalyzing the conversion of ferulic acid and its glucoside into vanillin and vanillin glucoside, respectively (Fig.…”

“…4). 121 Toxic vanillin is stored as its non-toxic glucoside 122. Using multiple cell imaging approaches, it was discovered that vanillin glucoside was progressively accumulated in the inner volume of the phenyloplasts ultimately filling the entire phenyloplast at pod maturity 117.…”

Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana

“…Wang et al 2016). Production of halogenated indican (Fräbel et al 2018) and vanillin (Gallage et al 2018) in N. benthmaiana also benefited from chloroplast localisation. In contrast, a recent report found that production of diterpenoids (typically synthesised in the chloroplast) was dramatically enhanced by co-opting the cytosolic mevalonate pathway to produce GGPP rather than the chloroplast MEP pathway (De La Peña and Sattely 2021).…”

Characterization of the UDP-glycosyltransferase UGT72 Family in Poplar and Identification of Genes Involved in the Glycosylation of Monolignols

“…Monolignol homeostasis may therefore rely on the activity of UGTs which colocalize with monolignol enzymes to directly glycosylate some of the intermediate products of this metabolic pathway to circumvent potential toxicity. Such a mechanism has been suggested for a vanillin-specific UGT in order to maintain a content at sub-toxic levels in the cell [ 63 ]. Assembly and disassembly of a given metabolon, for instance, an UGT coupled to a P450 protein, would provide additional flexibility in specialized metabolite biosynthesis.…”