## 4 Discussion
### Correlation of the Relative Expression Level of VvOMTs in Berry Skins with IBMP Biosynthesis  
The research on the biosynthesis of MPs in grapes is still insufficient knowledge, but the final step involving the HPs being methylated into the MPs had been confirmed [24; 25; 26]; especially, the four VvOMTs involved in it had been identified in wine grapes [27; 28; 29]. At present, in all relative studies about the IBMP accumulation, the VvOMTs expression pattern is almost the only way to interpret the results from the mechanistic level [40; 41].  
In the grape berries, 72% of the IBMP is located in the berry skins [42], so the relative expression level of VvOMTs in grape berry skins were monitored during the experiment period in two consecutive years (2020 and 2021) in this study.  
In 2021, the relative expression level of _VvOMT1_ was synchronized with the IBMP accumulation in grape, but in 2020, the maximum level of IBMP accumulation was 8 days earlier than the maximum _VvOMT1_ relative expression. After DCIP incorporation, the relative expression level of _VvOMT1_ in berry skin was significantly up-regulated at 121 and 128 DAA in 2020, but showed no significant difference from the Control and Mock treatments in 2021.  
The expression pattern of _VvOMT3_ in berry skin was synchronized with IBMP accumulation in grape in two consecutive years, and the peak relative expression level of _VvOMT3_ was almost synchronized with the maximum IBMP level. After DCIP incorporation, the relative expression level of _VvOMT3_ in berry skins was remarkably increased at 67 DAA in 2020 and was significantly up-regulated from 69 to 85 DAA in 2021, which was 16-49 days earlier than the significant increase of IBMP accumulation in grape berries.  
These results suggested that both the _VvOMT1_ and _VvOMT3_ play a significant role in regulating the IBMP accumulation; the IBMP content in grape berries was positively correlated with the up-regulated expression of _VvOMT1_[27; 28], but in particular, the _VvOMT3_ expression pattern was almost synchronized with the IBMP accumulation, which plays a major role in MPs biosynthesis in grape berries due to the fact that it had a high affinity for the HP precursors of MPs [29; 41].  
The relative expression levels of _VvOMT2_ and _VvOMT4_ did not show a positive correlation with IBMP accumulation in grape berries during the experimental period over the two years. This may be because _VvOMT2_ favors the methylation of 3-isopropyl-2-hydroxypyrazine (IPHP) to 3-isopropyl-2-methoxypyrazine (IPMP) in grape berries [27]. The _VvOMT4_ might have little relation to IBMP accumulation in grape, which might be why the expression level of _VvOMT4_ always had been ignored in other studies about MPs' accumulation.  
Only a few studies have focused on the metabolic pathways of MPs' biosynthesis. Interpreting the mechanism of relevant MPs study findings only from the VvOMTs expression pattern, which is involved in the final step of MPs' biosynthesis pathway, has certain limitations. More research on MPs' metabolic pathway is needed to further explore.  
On the whole, the results drawn from this study will provide reference for further understanding and much deeper research on IBMP biosynthesis.