Antagonistic and Synergistic Roles of Tomato AFP3 Isoforms in Hormonal Regulation and Development

The ABA INSENSITIVE5 BINDING PROTEIN (AFP) family plays a critical role in abscisic acid (ABA) signaling through interaction with the transcription factor ABI5, impacting seed germination and stress responses. Here, we characterize tomato AFP3, which produces two isoforms: a full-length protein and a shorter microProtein (sAFP3) containing only the C-terminal domain. Functional analyses reveal contrasting roles of these isoforms in development; while AFP3 overexpression accelerates shoot growth but impairs seed germination, both afp3 loss-of-function and sAFP3-expressing mutants (afp3-D) exhibit stunted growth and developmental defects. Transcriptome profiling highlights that AFP3 and sAFP3 differentially regulate hormone-related pathways, including salicylic acid, gibberellic acid, and jasmonic acid metabolism. Proteomic interaction studies demonstrate that AFP3 and sAFP3 physically interact, sharing partners involved in hormone signaling. Hormone quantification confirms that AFP3 modulates multiple phytohormones, with elevated ABA in afp3-D mutants and increased gibberellic acid, jasmonic acid, and salicylic acid in both afp3 and afp3-D mutant backgrounds. Moreover, AFP3 controls flower and fruit development, influencing yield and ripening. Together, these findings identify AFP3 as a key integrator of hormonal crosstalk that coordinates growth, development, and stress responses in tomato. The production of dual AFP3 isoforms through alternative transcription, combined with microProtein-mediated dominant-negative regulation, reveals a sophisticated mechanism for dynamically fine-tuning transcriptional networks. This versatile strategy underscores how plants--and potentially other organisms--achieve precise control over complex signaling pathways.