Eugenol (4-Allyl-2-methoxyphenol) is an important class of benzene ring/phenylpropionic acid volatiles widely present in plant flowers and mature fruits, and has various biological effects and health care functions. Eugenol can attract biological pollination and spread seeds, defend herbivores, and can also be used as a protective agent to prevent the invasion of parasitic organisms such as pathogens; Eugenol also has antioxidant, antibacterial, anti-insect and flavoring functions, and has received close attention from researchers. . At the same time, eugenol is a key component of strawberry fruit aroma, but its content in octoploid cultivated strawberry fruit is significantly lower than that in diploid wild-type strawberry. To date, the biosynthetic pathway of eugenol has been largely understood in plants, and most of the identified phenylpropanoid pathway regulators (such as the R2R3-MYB transcription factors FaMYB10 and FaEOBII, the DNA-binding zinc finger transcription factor FaDOF2) have been validated in strawberries The ripening stage plays a role later in fruit development, while eugenol content is higher in strawberry fruits at the large green fruit stage, but it is unclear how eugenol accumulation is transcriptionally regulated at this stage.
In strawberries, the eugenol synthase-encoding genes FaEGS1 and FaEGS2 and the cinnamyl alcohol dehydrogenase-encoding gene FaCAD1 are essential for eugenol production. FaEGS1 and FaEGS2 were mainly expressed in strawberry green fruit and ripe fruit, respectively. In this study, in order to identify the transcription factors involved in the regulation of FaEGS1, yeast single-hybrid screening was performed using the FaEGS1 promoter as bait, and a R2R3-MYB transcription factor FaMYB63 was further studied in the screening results. FaMYB63 was localized to the nucleus, and RT-qPCR analysis showed that FaMYB63 expression was tissue- and stage-specific, consistent with eugenol content during strawberry fruit development, and was inhibited by abscisic acid (ABA) and activated by auxin (IAA). FaMYB63 overexpression and RNAi-mediated silencing resulted in significant changes in the transcript levels of the eugenol synthesis genes FaEGS1, FaEGS2 and FaCAD1, which in turn affected eugenol accumulation. EMSA, yeast monohybrid, GUS activity and dual luciferase activity assays showed that the transcription of FaEOBII and FaMYB10 were also regulated by FaMYB63, but not vice versa. Taken together, these results provide new insights into the regulatory network of the strawberry eugenol synthesis pathway.
