<i>FaLWD1-like</i> enhances <i>FaMYB10</i> -driven transcriptional activation of <i>FaF3'H</i> and mediates cyanidin-specific accumulation in pink/white strawberry

Abstract Background and Aims Anthocyanins, vital health-beneficial plant pigments, are regulated by structural genes and MYB-bHLH-WD40 complexes. Cultivated strawberry (Fragaria × ananassa) displays diverse fruit colors, with its anthocyanin profile mainly comprising nearly 70% pelargonidin and 30% cyanidin. Variations in this ratio lead to distinct fruit coloration, yet the regulatory mechanisms governing this proportion remain largely unknown. Here, via systematic analysis of anthocyanin metabolic patterns in five strawberry cultivars with contrasting fruit colors, we identified a molecular module controlling cyanidin biosynthesis in strawberry. Methods This study used five commercially cultivated strawberry cultivars with gradient fruit skin colors ('Zhenhongmeiling', 'Benihoppe', 'Kaorino', 'Fenyu No.2', 'Mengzhiying') as materials. We analyzed the anthocyanin metabolic regulatory network during fruit skin development via high-performance liquid chromatography (HPLC), RNA-seq, yeast hybridization and transient overexpression assays. Key Results The results showed that pelargonidin-based anthocyanins dominated (54%–68%) in the red cultivars ('Zhenhongmeiling', 'Benihoppe', 'Kaorino'), while cyanidin-based anthocyanins were the main pigments (72%–85%) in pink ('Fenyu No.2') and white ('Mengzhiying') cultivars. RNA-seq and functional validation revealed that the low expression of most flavonoid structural genes reduced anthocyanin diversity and total content in pink/white strawberries, and weak FaLWD1-like expression decreased the transcriptional activation of FaMYB10 on target structural genes. Further verification confirmed that FaLWD1-like drastically elevated the activation of FaMYB10 on the FaF3'H promoter by 10.85-fold relative to the 35S control. Additionally, co-overexpression of FaLWD1-like and FaMYB10 increased cyanidin 3-O-glucoside content by 28%–29% in pink/white strawberries compared with FaMYB10 single overexpression. Conclusions This study uncovers a novel mechanism where the FaLWD1-like-FaMYB10 module regulates cyanidin synthesis in strawberry, providing a novel target for the improvement of strawberry fruit coloration.