FaMYB63 localization and expression and eugenol content in different stages and under different treatments. A, Subcellular localization of CaMV35S:FaMYB63–GFP fusions in transiently transformed N. benthamiana leaves. All experiments were assayed 72 h after infiltration. The same N. benthamiana leaves were stained with DAPI to show the locations of the nuclei. Bars = 20 μm. B, Analysis by RT-qPCR of FaMYB63 transcript levels (left axis) and the eugenol content (right axis) in fruit receptacles at different developmental stages of Fragaria × ananassa cv. ‘Benihoppe’. C, Analysis by RT-qPCR of FaMYB63 transcript levels in achenes at different developmental stages of F. × ananassa cv. ‘Benihoppe’. G1, small green fruit stage; G2, large green fruit stage; G3, green-white fruit stage; W, white fruit stage; T, red-turning fruit stage; R, red-ripening fruit stage; OR, over-ripening fruit stage. Relative expression values were relative to receptacles at the G1 stage in all cases, which was assigned an arbitrary value equal to one. In (D) and (E), achenes were removed from developing fruits in the G2 stage of F. × ananassa cv. ‘Benihoppe’ for analysis of (D) FaMYB63 expression by RT-qPCR (left axis) and eugenol content (right axis) and (E) IAA content. Control + G2, control, large green fruit receptacle; G2-A + L, G2 fruit receptacle without achenes for 5 d covered by a lanolin paste; G2-A + IAA + L, G2 fruit receptacle without achenes for 5 d treated with the synthetic auxin IAA (1 mM) in lanoline paste. F and G, Analysis by RT-qPCR of FaMYB63 and FaNCED1 transcript levels (in bars) and content of eugenol (in lines) and (H and I) ABA content. F and H, Control, green-white fruits were injected with sterile water; NDGA, green-white fruits were injected with 100-μM NDGA. G and I, Control, fruit pedicels were immersed in MS medium containing sucrose; Water Stress, fruit pedicels were exposed to air without MS medium. Values are the mean ± sd of three biological replicates. The different letters above the columns in (B) and (C) indicate significant differences at the 5% level (P < 0.05, Duncan’s test). Statistical significance with respect to the reference sample (control) was determined by the Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001.