# FhMYB108 Regulates the Expression of Linalool Synthase Gene in _Freesia hybrida_ and _Arabidopsis_
## 3 Results
### The Expression of the AtTPS14 Gene by FhMYB108 and AtMYC2 in Arabidopsis  
In _A. thaliana_, in addition to MYB21 and MYB24, which have been experimentally shown to modulate terpene metabolism, an additional bHLH-type transcription factor (AtMYC2) has also been identified as a regulator of terpene biosynthesis through the jasmonic acid (JA) pathway based on research reports [39]. The transcription factor AtMYC2 plays a pivotal role in the regulation of sesquiterpenoid biosynthesis, exhibiting high expression levels specifically in _Arabidopsis_ flowers and demonstrating reported interactions with MYB21. The results reported herein demonstrate that _Arabidopsis thaliana_ flower volatiles primarily comprise sesquiterpene carryophyllene (>90%) with minimal levels of linalool, suggesting that AtMYC2 may not play a significant role in promoting linalool accumulation. Based on these results, we can make the following bold assumptions: (1) AtMYC2 is incapable of activating the expression of linalool synthase gene AtTPS14; (2) In _Arabidopsis thaliana_, it is plausible that AtMYC2 interacts with FhMYB108 to suppress the activation of FhMYB108 on AtTPS14. The absence of any discernible impact on linalool content resulting from _FhMYB108_ overexpression can plausibly be attributed to the involvement of AtMYC2. By conducting transcription factor activation experiments, we observed a strong binding affinity of AtMYC2 towards the promoters of _TPS11_ and _TPS21_, while no discernible binding capability was demonstrated towards the promoters of _TPS14_ (Figure 6A). In contrast, the transient expression of _AtMYC2_ in protoplasts resulted in a significant upregulation of _TPS11_ and _TPS21_, while no alteration was observed for the _AtTPS14_ gene, suggesting that AtMYC2 lacks the ability to positively regulate linalool synthase gene expression (Figure 6B).  
Additionally, the interaction between FhMYB108 and AtMYC2 was validated through a double fluorescence complementation assay in this study. The results demonstrated that the fluorescence signal was exclusively observed upon the co-transfection of FHMYB108-GFPN and ATMYC2-GFPC, thereby indicating a potential interaction between FhMYB108 and AtMYC2. Furthermore, FhMYB108 and AtMYC2 were individually or co-transferred with At\({}_{\text{Pro}}\)_TPS14_: GUS into the protoplasts of _A. thaliana_ leaves to quantify alterations in binding affinity. The findings illustrate a pronounced transcriptional activation by FhMYB108 on the _TPS14_ promoter, while AtMYC2 exhibits an inability to activate the transcription of the _TPS14_ promoter. Moreover, the co-expression of AtMYC2 and FhMYB108 significantly diminishes the transcriptional activation capacity of FhMYB108.  
Figure 5: _TPS14_ expression and linalool release in _FhMYB108_ overexpressed _A. thaliana_. (**A**) The quantification of the expression level of _AtTPS14_ upon the overexpression of _FhMYB108_ in _A. thaliana_. (**B**) Linalool content in overexpressed seedlings. In (**A**,**B**), data are shown as the mean \(\pm\) SD of three biological replicates. Differences were assessed using a two-tailed \(t\)-test (ns, \(p\) > 0.05) in (**A**).