An EjSPT–EjTCP4– EjRR8 Regulatory Module Links Cytokinin Homeostasis and Cell Proliferation to Fruit Growth in Loquat

Abstract

Fruit size is a key determinant of crop yield and commercial value. Here, we investigated fruit development in two loquat cultivars with contrasting fruit sizes. Through hormone quantification, cytological analyses, and transcriptome profiling, we found that cytokinin-related changes were closely associated with loquat fruit-size variation, and further identified EjRR8 as a key regulatory gene. Gain- and loss-of-function analyses indicated that EjRR8 modulates cytokinin accumulation and cell proliferation, thereby contributing to fruit growth. Furthermore, we identified EjSPT and EjTCP4 as upstream transcription factors of EjRR8. Yeast one-hybrid, Dual-Luciferase, and EMSA assays showed that both factors directly bind to the EjRR8 promoter, while functional validation demonstrated that they positively affect cytokinin accumulation. Notably, EjSPT and EjTCP4 physically interacted and synergistically activated EjRR8 expression, forming a regulatory module that links transcriptional control with cytokinin-related responses and cell proliferation. These findings reveal an EjSPT–EjTCP4–EjRR8 module associated with cytokinin accumulation, cell division and fruit-size variation. This study provides a molecular framework for breeding loquat cultivars with larger fruits and offers broader insights into the regulation of fruit development in woody crops.