Time-Series Metabolome and Transcriptome Analyses Reveal the Genetic Basis of Vanillin Biosynthesis in Vanilla

Abstract

Vanillin, the principal aromatic compound in vanilla, is primarily derived from mature pods of vanilla (Vanilla planifolia Andrews). Although the biosynthetic pathway of vanillin has been progressively elucidated, the specific key enzymes and transcription factors (TFs) governing vanillin biosynthesis require further comprehensive investigation via combining transcriptomic and metabolomic analysis. For this study, V. planifolia (higher vanillin producer) and V. imperialis (lower vanillin producer) were selected. Time-series metabolomics analysis revealed 160-220 days after pollination (DAPs) as the critical phase for vanillin biosynthesis. Combined time-series transcriptome analysis revealed 984 upregulated differentially expressed genes (DEGs) in key periods, 2058 genes with temporal expression, and 4326 module genes through weighted gene co-expression network analysis (WGCNA), revealing six major classes of TFs: No Apical Meristem (NAC), Myb, WRKY, FLOWERING PROMOTING FACTOR 1-like (FPFL), DOF, and PLATZ. These TFs display strong regulatory relationships with the expression of key enzymatic genes, including P450s, COMT, and 4CL. The NAC TF family emerged as central regulators in this network, with NAC-2 (HPP92_014056) and NAC-3 (HPP92_012558) identified as key hub genes within the vanillin biosynthetic gene co-expression network. The findings of this study provide a theoretical foundation and potential target genes for enhancing vanillin production through genetic and metabolic engineering approaches, offering new opportunities for sustainable development in the vanilla industry and related applications.

Keywords: Vanilla planifolia Andrews; combined analysis; transcriptome and metabolome; vanillin.

Figure 1

Metabolic and transcriptional profiling during vanilla (Vanilla spp.) growth and development. (A) Fluctuations in glucovanillin accumulation patterns across developmental stages. (B) Expression profiles of vanillin synthase gene VpVAN (HPP92_026221), with distinct expression patterns observed between low-vanillin V. imperialis (V) and high-vanillin V. planifolia (X) accessions.

Figure 2

Differential gene identification and analysis. (A) Differentially expressed genes were identified among six pairwise comparisons and overall (B). (C) Venn diagram illustrates overlaps of upregulated differentially expressed genes across six comparison groups. (D) GO enrichment analysis was performed on 984 commonly upregulated differentially expressed genes. V: low-vanillin V. imperialis, X: high-vanillin V. planifolia.

Figure 3

Expression patterns of metabolome (A) and transcriptome (B) in vanilla fruits and across six developmental stages, with different groups representing distinct expression trends; dark thick lines represent average expression profiles of all genes in each cluster.

Figure 4

Co-expression network analysis of vanillin biosynthesis genes. (A) Hierarchical clustering trees with different topological overlaps of expressed genes. (B) Heat maps of correlations between modules and metabolites associated with vanillin synthesis. Different colors represent different modules (7 in total); numbers in grid represent Pearson correlations between modules and traits, ranging from −1 (blue) to 1 (red). Color gradient indicates strength of correlations, with darker blue signifying stronger negative relationships and deeper red representing more significant positive associations. (C) Relationship between gene significance (GS) and gene and module membership (MM) in MEgreen module. (D) Top 10 genes with highest Maximal Clique Centrality (MCC) scores were identified as hub genes. Black pentagrams indicate genes encoding NAC transcription factors.

Figure 5

Integrated analysis. (A) The Venn diagram displays the overlapping upregulated differentially expressed genes among six comparison groups, Cluster 9, and the vanillin-related WGCNA module. (B) The red pentagrams denote the highest-scoring hub genes in the co-expression network, while the black pentagrams indicate the second-highest-scoring hub genes, one of which also emerged as the top-scoring hub gene in the WGCNA. Only Pearson’s r ≥ 0.6 are displayed.