# New Insights into the Mechanism of Spatiotemporal Scent Accumulation in Orchid Flowers
## 2 Results
### Spatiotemporal Quantification of VOCs in KOVA Flowers  
Seven monoterepenes, nerol, D-limonene, linalool, geraniol, (-)_-trans_-Isopiperitenol, carvone, and citronellol, were identified from the flower tissues, and the total relative contents of monoterepenes gradually increased in each part during flowering (Figure 3A). Among these flower parts, the total relative content of monoterepenes in the hypochile at D8 was the highest, and linalool and geraniol represented the highest proportions of the total monoterepene contents, accounting for \(\sim\)38% and 40%, respectively, which were lower than those in the sepal and petal (Figure 3B). However, the relative contents of nerol, D-limonene, (-)_-trans_-Isopiperitenol, and citronellol, in the total monoterepene content accounted for proportions of \(\sim\)5%, 8%, 2%, and 7%, respectively, in the hypochile at D8, which were higher than those in the other flower parts (Figure 3B). Some monoterepenes, including nerol, D-limonene, (-)_-trans_-Isopiperitenol, and citronellol, were only detected in KOVA flower tissues, while _beta_-Ocimene, _cis_-allo-Ocimene, _cis_-Citral, and citral were also not detected (Figures 2 and 3). Additionally, the relative contents of linalool and geraniol in four flower parts increased from stages D7 to D8 (Figure 3), which was in accordance with the variation tendency of those emitted by KOVA flowers between the two stages (Figure 2). Most of these monoterepenes were present in each part of the KOVA flower at D8, except for carvone, which was only detected in the hypochile at D7 (Figure 4). Additionally, the relative content of each monoterepene was highest in the hypochile. The relative contents of nerol, D-limonene, and (-)_-trans_-Isopiperitenol at stage D8 were notably higher in the hypochile than those in the other parts of the flower (Figure 4). Expression Profiles of the Genes Involved in the MEP Pathway in the KOVA Flower Parts during Floral Development  
Given that monoterepenes are the main contributors to the floral scent of KOVA, we used transcriptome data produced by a previous study [25] to analyze the spatiotemporal expression profiles of the MEP pathway structural genes in the four flower parts during development. Most MEP pathway structural genes were not activated in the KOVA flowers at the hub stages, including D1 (developmental stage 1; i.e., bulb length <2 cm) and D4 (developmental stage 4; i.e., bulb length 4-5 cm), but they were highly expressed at D7 and D8 (Figure 5). Among these MEP pathway structural genes, the expression of _DXS_s encoding the rate-limiting enzyme and _HD_S_s showed strong temporal specificity in that they only had high expression levels in the flowers at D8 (Figure 5), which corresponded to the increased content of monoterepenes in flowers from D7 to D8 (Figures 2 and 3). In addition, the expression profiles of the _MT_Ps encoding the enzyme involved in the last step of monoterepe biosynthesis were clearly spatiotemporal, and they maintained their high levels in the hypochile from D4 to D7 compared with their levels in other parts (Figure 5). These data were consistent with the finding that the contents of monoterepenes in the hypochile were higher than those in the other flower parts (Figures 2 and 4). Figure 3: The types and relative contents of monoterpenes in four parts of the KOVA flower at stages D7 and D8. (**A**) The total relative contents of the monoterpenes in each part of the flower at D7 and D8. The four parts of KOVA flowers at D7 and D8 were sampled and analyzed using headspace GC–MS. The sepal, petal, epichile, and hypochile are abbreviated as Sep, Pet, Epi, and Hyp, respectively. The relative content represents the result calculated by the external standard method based on data produced by GC. The total relative content represents the sum total of all monoterpene relative contents. The data are the mean \(\pm\) SD from three biological replicates.