Anthocyanins and terpenoids are secondary metabolites with well-documented health benefits. Isopentenyl transferases (IPTs) are key enzymes in cytokinin (CK) biosynthesis. While ADP/ATP-type IPTs and their associated trans-zeatin (tZ)-CKs and iP-CKs are considered to play regulatory roles in growth and development, as well as stress acclimation in plants, tRNA-type IPTs and cis-zeatin CKs (cZ-CKs), which may serve housekeeping functions, remain less studied. In this study, the tRNA-type IPT gene FveIPT2 was overexpressed in woodland strawberries (Fragaria vesca). Overexpression had minimal impact on plant growth and CK levels but resulted in transgenic fruits exhibiting a significant increase in total phenolic, flavonoid, and anthocyanin contents, indicating enhanced fruit quality. Metabolite profiling revealed substantial increases in nine specific anthocyanins and 24 out of 47 detected terpenoids in the transgenic fruits. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis confirmed the upregulation of genes involved in anthocyanin and terpenoid biosynthesis and transport. These findings demonstrate that while tRNA-type IPTs may primarily play housekeeping roles, FveIPT2 overexpression can significantly enhance fruit quality by boosting terpenoid and anthocyanin accumulation, highlighting the unexpected potential of these genes to improve the nutritional value of edible fruits.

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