Tubby-like proteins (TLPs) play important roles in plant growth and development and in responses to abiotic stress. However, TLPs in strawberry remain poorly studied. In this study, eight TLPs were identified in woodland strawberry (Fragaria vesca subspecies vesca ‘Ruegen’). Protein structure analysis revealed that the structure of FvTLPs is highly conserved, but evolutionary and gene structure analyses revealed that the evolutionary pattern of FvTLP family members differs from that of their orthologous genes in Arabidopsis, poplar, and apple. Subcellular localization assays revealed that FvTLPs were localized to the nucleus and plasma membrane. FvTLPs showed no transcriptional activity. Yeast two-hybrid assays revealed that FvTLPs interact with specific FvSKP1s. The expression patterns of FvTLPs in different tissues and under various abiotic stresses (salt, drought, cold, and heat) and hormone treatments (ABA (abscisic acid) and MeJA (methyl jasmonate)) were determined. The expression patterns of FvTLPs indicated that they play a role in regulating growth and development and responses to abiotic stress in F. vesca. The GUS (beta-glucuronidase) activity of FvTLP1pro::GUS plants in GUS activity assays increased under salt and drought stress and abscisic acid treatment. The results of this study provide new insights into the molecular mechanisms underlying the functions of TLPs.

Growing market demand exists for strawberry, with nutrient-rich and health-promoting properties, beyond mere taste and flavor. Genetic biofortification is a powerful strategy to enhance nutrient metabolites in strawberry. Both GABA and organic acids contribute to human health by supporting nervous system relaxation and enhancing metabolic and digestive functions, respectively. However, the regulatory mechanisms underlying their accumulation remain poorly understood. In this study, we analyzed the accumulation patterns of GABA and organic acids across four fruit developmental stages in two representative cultivars, ‘Monterey’ and ‘Benihoppe’. Ripening ‘Benihoppe’ fruits accumulated higher levels of GABA and citric acid, whereas ‘Monterey’ fruits contained more malic acid. Integrated transcriptome analysis identified key structural genes and transcription factors (TFs) involved in GABA biosynthesis. Notably, functional characterization revealed that FaGAD4 significantly promotes GABA accumulation and simultaneously enhances the content of anthocyanin and ascorbic acid (AsA). Overall, this study provides novel insights into the regulatory mechanisms of GABA accumulation in strawberry fruit and identifies FaGAD4 and potential TFs as valuable genetic targets for molecular breeding.