The R2R3-MYB transcription factor FaMYB63 participates in regulation of eugenol production in strawberry

FaMYB63 functions as a crucial regulator to control biosynthetic genes of the phenylpropanoid pathway and eugenol accumulation in strawberry fruit. The biosynthetic pathway of volatile phenylpropanoids, including 4-allyl-2-methoxyphenol (eugenol), has been investigated in petunia (Petunia hybrida). However, the regulatory network for eugenol accumulation in strawberry (Fragaria x ananassa Duch.) fruit remains unclear. Here, an R2R3-type MYB transcription factor (TF; FaMYB63) was isolated from strawberry by yeast one-hybrid (Y1H) screening using the promoter of the FaEGS1 (eugenol synthase 1 [EGS 1]) gene, which encodes the enzyme responsible for the last step in eugenol biosynthesis. FaMYB63 is phylogenetically distinct from other R2R3-MYB TFs, including FaEOBCYRILLIC CAPITAL LETTER BYELORUSSIAN-UKRAINIAN ICYRILLIC CAPITAL LETTER BYELORUSSIAN-UKRAINIAN I (EMISSION OF BENZENOID II [EOBII]), which also participates in regulating eugenol biosynthesis in strawberry receptacles. Reverse transcription quantitative PCR (RT-qPCR) assays showed that the expression of FaMYB63 was tissue-specific and consistent with eugenol content through strawberry fruit development, was repressed by abscisic acid, and was activated by auxins (indole-3-acetic acid). Overexpression and RNA interference-mediated silencing of FaMYB63 resulted in marked changes in the transcript levels of the biosynthetic genes FaEGS1, FaEGS2, and FaCAD1 (cinnamyl alcohol dehydrogenase 1 [CAD1]) and, thereby, the accumulation of eugenol. Electrophoretic mobility shift, Y1H, GUS activity, and dual-luciferase activity assays demonstrated that the transcript levels of FaEOBCYRILLIC CAPITAL LETTER BYELORUSSIAN-UKRAINIAN ICYRILLIC CAPITAL LETTER BYELORUSSIAN-UKRAINIAN I and FaMYB10 were regulated by FaMYB63, but not the other way around. Together, these results demonstrate that FaMYB63 directly activates FaEGS1, FaEGS2, FaCAD1, FaEOBCYRILLIC CAPITAL LETTER BYELORUSSIAN-UKRAINIAN ICYRILLIC CAPITAL LETTER BYELORUSSIAN-UKRAINIAN I, and FaMYB10 to induce eugenol biosynthesis during strawberry fruit development. These findings deepen the understanding of the regulatory network that influences eugenol metabolism in an edible fruit crop.