# Mechanistic Insights into the Bornyl Diphosphate Synthase from _Lavandula angustifolia_
## 3 Results
### Spatial and Temporal Regulation Patterns of LaBPPS Gene  
To assess _LaBPPS_ expression patterns across different tissues and time points, real-time quantitative polymerase chain reaction (RT-qPCR) was conducted. The results revealed markedly higher transcript levels in flowers compared to leaves, stems, and roots, with peak expression occurring at 8:00 (Figure 7). At this time point, flowers exhibited the highest expression levels (152.9-fold), substantially surpassing levels in leaves (2.9-fold), stems (1.3-fold), and roots (1.1-fold). On the other hand, temporal profiling in flowers showed maximal expression at 8:00 (152.9-fold), followed by 14:00 (8.5-fold), while 20:00 and 2:00 displayed lower expression (2.3-fold and 1.7-fold, respectively) (Figure 7). These results identify flowers as the dominant site of _LaBPPS_ activity. The observed tissue-specific and \begin{table}
\begin{tabular}{c c c} \hline \hline
**Constructs** & \(K_{m}\) **(μM)** & \(K_{cat}\) **(min\({}^{-1}\))** \\ \hline WT (wild-type) & 29.37 \(\pm\) 1.21 & 6.23 \(\pm\) 0.47 \\ \(\Delta\)1–58 & 28.12 \(\pm\) 1.75 & 6.78 \(\pm\) 0.35 \\ \(\Delta\)1–74 & 41.72 \(\pm\) 2.15 & 3.19 \(\pm\) 0.12 \\ \(\Delta\)59–74 & 38.49 \(\pm\) 2.58 & 3.37 \(\pm\) 0.42 \\ \(\Delta\)578–602 & 14.83 \(\pm\) 1.09 & 11.71 \(\pm\) 0.86 \\ \hline \hline \end{tabular} Note: Kinetic parameters were determined using Hanes–Woolf plots.  
\end{table} 
Table 4: Kinetic parameters of different LaBPPS constructs. Figure 6: **N- and C-terminus regulated the activity of LaBPPS. ** (**a**) The regions spanning residues 1–58, 59–74, and 578–602 are colored in orange, magenta, and green, respectively. (**b**) Deletion of region 1–58 (A1–58) did not affect the activity, which remained identical to that of the wild-type (WT) protein. In contrast, deletions of region 1–74 or 59–74 (\(\Delta\)1–74 or \(\Delta\)59–74) resulted in a significant reduction in the activity compared to the WT protein. However, deletion of 578–602 (\(\Delta\)578–602) led to a substantial increase in the activity. The activity of WT LaBPPS was set to 100%. diurnal expression patterns suggest spatially and temporally regulated terpene synthesis, indicating transcriptional control over terpenoid biosynthesis. To further investigate the spatiotemporal dynamics of gene expression, we analyzed _LaBPPS_-derived metabolites using gas chromatography-mass spectrometry (GC-MS). We found that the highest metabolite accumulation was detected in the flower at 8:00 among these conditions (Table 5). The highest yield of metabolites was observed in the flower compared to the corresponding yields from the other tissues (leaf, stem and root) (Table 5). Notably, metabolite production at 8:00 significantly surpassed yields assessed at other time points (2:00, 14:00, and 20:00) (Table 5). This pattern aligns with the transcriptional activity revealed by the RT-qPCR results, confirming coordinated expression and metabolic output of the _LaBPPS_ gene. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Tissues** & **Time Points** & **Metabolite (Quantity \#g/g Dry Tissue)** \\ \hline \multirow{4}{*}{Flower} & 2:00 & 1.29 \(\pm\) 0.56 \\  & 8:00 & 239.53 \(\pm\) 6.47 \\  & 14:00 & 13.27 \(\pm\) 1.14 \\  & 20:00 & 3.47 \(\pm\) 0.83 \\ \hline \multirow{4}{*}{Leaf} & 2:00 & 0.38 \(\pm\) 0.02 \\  & 8:00 & 8.49 \(\pm\) 0.92 \\ \cline{1-1}  & 14:00 & 1.95 \(\pm\) 0.73 \\ \cline{1-1}  & 20:00 & 0.09 \(\pm\) 0.01 \\ \hline \multirow{4}{*}{Stem} & 2:00 & 0.11 \(\pm\) 0.06 \\  & 8:00 & 0.95 \(\pm\) 0.06 \\ \cline{1-1}  & 14:00 & 0.25 \(\pm\) 0.03 \\ \cline{1-1}  & 20:00 & 0.13 \(\pm\) 0.09 \\ \hline \multirow{4}{*}{Root} & 2:00 & 0.08 \(\pm\) 0.02 \\ \cline{1-1}  & 8:00 & 0.38 \(\pm\) 0.01 \\ \cline{1-1}  & 14:00 & 0.06 \(\pm\) 0.01 \\ \cline{1-1}  & 20:00 & 0.05 \(\pm\) 0.02 \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Analysis of metabolite derived from LaBPPS. Figure 7: _LaBPPS gene expression profiles in different tissues (flower, leaf, stem, and root) during a 24 h day/night cycle.