Anthocyanins are the main pigments in ripe strawberry fruits. FaMYB10 and abscisic acid (ABA) are the two major regulators of anthocyanin biosynthesis in the maturation process of strawberry fruits. However, the transcriptional regulation of FaMYB10, ABA biosynthesis, and anthocyanin accumulation in strawberry (Fragaria × ananassa) remain largely unknown. Here, a yeast one-hybrid screen using the FaMYB10 promoter identified a class B MADS-domain transcription factor, FaMADS6 in strawberry. FaMADS6 exhibited high expression at the early developmental stage but was hardly detected during maturation of strawberry fruit, a pattern opposite to accumulation of anthocyanins. Transcriptional upregulation of FaMADS6 repressed anthocyanin accumulation and expression of FaMYB10 and the anthocyanin biosynthetic genes, FaCHS, FaCHI, FaF3H, FaANS, and FaUFGT. In contrast, downregulation of FaMADS6 promoted the expression of FaMYB10 and the anthocyanin biosynthetic genes. The promoters of the anthocyanin biosynthetic genes were not directly bound by FaMADS6, in contrast to FaMYB10. Analysis of the DNA binding sequences of FaMADS6 revealed that it also interacted with the promoters of FaNCED2 and FaPYR1, which are involved in the biosynthesis and perception of ABA. Overexpression of FaMADS6 significantly suppressed FaNCED2 and FaPYR1 and ABA synthesis in transgenic strawberry. Together, our findings suggest that FaMADS6 functions as a suppressor of anthocyanin accumulation by directly downregulating FaMYB10 and ABA production during strawberry fruit maturation.

The study of strawberry gene function has been hindered by the low transformation efficiency and long generation time of transgenic plants. This study aimed to develop and optimize methods for generating strawberry (Fragaria × ananassa Duch. ‘Benihoppe’) plants with transgenic hairy roots. This involved inducing hairy roots on strawberry stolons near new plants, optimizing several parameters that affect the survival rate of stolon hairy roots, and using the new hairy root transgenic system to investigate the nitrate-transport function of FaNRT1.1. In vivo injection (IVI) of Agrobacterium rhizogenes K599 (OD₆₀₀ = 1.0) at sites measuring 0.5–1 cm on the tops of mature stolons (9–12 days old) of ‘Benihoppe’ strawberry resulted in the establishment of strawberries with transgenic hairy roots. The IVI induced epidermal bulges and hairy roots in 50% of the stolons. The epidermal bulges were evident, and callus began to grow 35 days post-injection, while hairy roots began to develop near the injection sites at 40 days and became abundant by 60 days. Specific fluorescence signals were observed in all transgenic hairy roots of 40 new plants. Using ¹⁵N nitrate labeling, we confirmed the nitrate-transport—from roots to shoots—function of FaNRT1.1 in the strawberry plants with transgenic hairy roots. Taken together, sufficient hairy roots can be induced using an efficient transgenic hairy root system, which can be effectively applied to gene function research, such as the analysis of the nitrate-transport activity of FaNRT1.1.