LAUSR

Summary Potassium (K+), an abundant cation in plant cells, is important in fruit development and plant resistance. However, how cellular K+ is directed by potassium channels in fruit development and quality formation of strawberry (Fragaria × ananassa) is not yet fully clear. Here, a two‐pore K+ (TPK) channel gene in strawberry, FaTPK1, was cloned using reverse transcription–PCR. A green fluorescent protein subcellular localization analysis showed that FaTPK1 localized in the vacuole membrane. A transcription analysis indicated that the mRNA expression level of FaTPK1 increased rapidly and was maintained at a high level in ripened fruit, which was coupled with the fruit's red colour development, suggesting that FaTPK1 is related to fruit quality formation. The down‐ and up‐regulation of the FaTPK1 mRNA expression levels using RNA interference and overexpression, respectively, inhibited and promoted fruit ripening, respectively, as demonstrated by consistent changes in firmness and the contents of soluble sugars, anthocyanin and abscisic acid, as well as the transcript levels of ripening‐regulated genes PG1 (polygalacturonase), GAL6 (beta‐galactosidase), XYL2 (D‐xylulose reductase), SUT1 (sucrose transporter), CHS (chalcone synthase) and CHI (chalcone flavanone isomerase). Additionally, the regulatory changes influenced fruit resistance to Botrytis cinerea. An isothermal calorimetry analysis showed that the Escherichia coli‐expressed FaTPK1 recombinant protein could bind K+ with a binding constant of 2.1 × 10–3 m −1 and a dissociation constant of 476 μm. Thus, the strawberry TPK1 is a ubiquitously expressed, tonoplast‐localized two‐pore potassium channel that plays important roles in fruit ripening and quality formation.