## 3 Results
### Discernment of Cis-Acting Elements in the Promoter Region of DcTPSb1  
The promoter region of _DcTPSb1_ contains a wide variety of cis-acting elements commonly found in promoter sequences. These cis-acting elements play important roles in modulating the biological function of genes. According to their functions related to stress, phytohormones, plant growth and development, these elements are often categorized into three categories in Figure 6. Within subgroup related to plant growth and development (21/49), a total of nine types of cis-elements involved in endosperm expression (AAGAA-motif), shoot expression (As-1 element), as well as meristem expression of root and apical (A-box, CAT-box), light responsiveness (ATCT-motif, GA-motif, G-Box, MRE, and TCT-motif) were observed, with the TCT-motif being the most prevalent at 24%. In the stress responsiveness category (9/49), diverse elements linked to stress (STRE, 33%), anaerobic induction (ARE, 11%), and wounding (W box, WRE3, and WUN-motif, each at 22%, 22%, and 11%, respectively) responsiveness were identified. The majority of cis-elements (19/49) fell under the phytohormone responsiveness category, responding to abscisic acid (ABRE), MeJA (MYC, CGTCA-motif, and TGACG-motif), and gibberellin (P-box and TATC-box). Particularly notable was the prevalence of MeJA-responsiveness elements, comprising 68% of the hormone-related cis-elements. Based on these findings, it is suggested that _DcTPSb1_ could potentially be regulated by MeJA or light, inducing or repressing its expression.  
Figure 5: Functional characterization of DcTPSb1. (**a**) Purification of DcTPSb1 recombinant protein. M, marker; S, supernatant; R, rinse solution. The red frame indicates purified target protein; Eluted protein represents GST-DcTPSb1 protein with 89.2 kDa. (**b**) Gas chromatograms of three monoter-penes generated by DcTPSb1 when GPP (Geranyl pyrophosphate) was used as a substrate. Control added high-temperature inactivated DcTPSb1 protein, in which no products were generated.  
Analysis of Volatile Metabolite Differences Between Tobacco Overexpression DcTPSb1 and Wild-Type Using GC-MS  
Compared to the wild type, there were no floral phenotypic changes in the transgenic tobacco plants. Subjecting blooming flowers from empty and overexpressed plants to GC-MS analyses revealed the presence of terpenoid VOCs in _N. benthamiana_, accounting for 21.07% of the total VOCs (Figure 7a), a notably higher percentage than other compound types, including 166 terpenoid varieties. The substance with significant changes is shown in Figure 7b. Among the significantly altered metabolites in overexpressed tobacco, terpen were the most diverse and varied. DcTPSb1, members of the TPS-b subfamily known as monoterpene synthases, likely played a crucial role in regulating the changes in the monoterpene level. Figure 7c displays the top 10 upregulated and top 10 downregulated substances. Noteworthy changes were observed in the relative content of three monoterrenes-\(\alpha\)-Pinene, (1S)-(-)-alpha-Pinene, and (1R)-(+)-\(\alpha\)-Pinene-with significantly increased levels among the differential metabolites.  
Figure 6: The distributions, categories, and numbers of twenty cis-acting elements in 2000 bp upstream region of the initiation codon (ATG) of _DcTPSb1_ in _D. chrysotoxum_. The above figure represents a wide distribution throughout the entire region of 20 cis-acting elements (represented by 20 types of blocks, respectively) distributed throughout the entire region with three categories in three colors. The following figure shows 20 types of cis-acting components belonging to three categories, represented by three colors. The detailed distribution of motifs is shown in Table S4.