## 2 Results and Discussion
### Effect of TDZ on Gene Expression of Berries  
Monoterpenes were the most abundant class of compounds detected in Shine Muscat berries. In order to characterize the monoterenoid metabolic pathway at the gene expression level, we performed targeted transcript analyses by RT-qPCR. The monoterenoid metabolism pathway has been previously characterized in different grape species. Critical steps for monoterpene biosynthesis have been revealed within the MEP pathway [38], and as a result, we focused our studies on the genes within this pathway, in addition to the relevant and functionally characterized TPS genes that act downstream of the MEP pathway [39]. Specifically, we selected the 1-deoxyxyulose-5-phosphate synthase (_VvDXS1_ and _VvDXS3_), 1-deoxy-p-xylylose-5-phosphate reductoisomerase (_VvDXR_) and (_E_)-4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (_VvHDR_) genes due to their considerations as the key enzymes responsible for controlling the flux of the MEP pathway [40]. In addition, we also selected the _VvPNGer_ and _VvPNIinNer1_ genes, which were confirmed as the highest expressed _TPS_ genes leading to the biosynthesis of monoterpenes such as linalool and geraniol. During the two consecutive years of investigation, the expression profiles of most of these selected genes showed a decrease after the treatment with TDZ compared with the control. From Figures 3 and 4 (2018 and 2019, respectively), the _VvDXS1_ transcript abundance was significantly lower in the TDZ-treated berries on 28 August (harvest stage), which represented the time point where berries were at a commercially relevant maturity level. On the other hand, no significant differences in the _VvDXS1_ expression were observed prior to the mature berries. The _VvDXS3_ expression was significantly lower in the TDZ-treated samples during two developmental stages (i.e., veraison and maturity stages). These results are in good accordance with two separate reports which identified an association between _VvDXS1_ and _VvDXS3_ and the content of monoterpenoids in _Vitis_ spp. [41, 42]. The _VvDXR_ and _VvHDR_ genes showed an obvious reduction in transcript abundance after the first application of TDZ and also throughout the last three time points of the sample collection--including the pre-veraison, veraison and commercially mature stages. Additionally, _VvHDR_ overall was the highest expressed MEP pathway gene. In \"Gewurztraminer\" berries, the accumulation of the _VvHDR_ transcript exhibited a good correlation with the accumulation of terpen [43]. However, in Riesling berries, light treatment did not have any effect on the _VvHDR_ expression, and the accumulation of the _VvHDR_ transcript was not significantly correlated with the accumulation of linalool or other monoterpenes [44]. In our study, a 5-fold increase in expression was observed for the _VvDXS3_ gene during fruit development, making it the most responsive gene affected by the TDZ treatment observed  
Figure 2: Effect of TDZ on grape terpene synthase (TPS) activity in 2018 and 2019 with the progression of berry development. Mean \(\pm\) Standard error (\(n=3\)). Different letters indicate significant differences ([PERSON]’s multiple range test, \(p\leq 0.05\)). under our experimental conditions. These data are in agreement with the hypothesis that _DXS_ is the first-rate limiting enzyme for the biosynthesis of plasidial monoterpenes [45]. The initial substrates for the biosynthesis of monoterpencids are C5-unit isopentenyl pyrophosphate and its isomer dimethylallyl pyrophosphate. These substrates are converted to GPP by geranyl diphosphate synthases (GPPS). Terpene synthases are subsequently responsible for catalyzing the formation of monoterpenes from GPP. To provide an additional characterization of this biosynthetic pathway, two _TPS_ genes involved in linalool and geraniol formation were specifically selected for transcript analyses [37; 46; 47], revealing nearly identical patterns of gene expression.