A cool temperature-induced ubiquitination-controlled transcription factor promotes starch degradation and ripening in kiwifruit.

Ripening of kiwifruit (Actinidia spp.) is highly sensitive to ethylene, but reliance on exogenous ethylene often results in over-softening, greatly reducing shelf life. Here, we discovered a pathway induced by cool temperature (CT; 5°C-10°C) that directly orchestrates starch-to-sugar conversion in kiwifruit under conditions in which ethylene perception is inhibited by 1-methylcyclopropene. Through transcriptomic and metabolomic profiling, we identified AcBAM3.3, a β-amylase gene that is specifically induced by CT but not by ambient temperature. A CT-inducible ERF transcription factor, AcCTS1 (CT-specific factor 1), was found to directly bind the promoters of AcBAM3.3 and AcBAM3.5 and activate their transcription, as confirmed by dual-luciferase, electrophoretic mobility shift, and yeast one-hybrid assays. We also identified an E3 ubiquitin ligase, AcPUB11, which targets AcCTS1 for 26S proteasomal degradation, repressing starch degradation at room temperature. Under CT, reduced AcPUB11 abundance allows for AcCTS1 accumulation, driving AcBAM3.3 and AcBAM3.5 expression and promoting ripening. Functional characterization via overexpression, RNAi, and CRISPR-Cas9 in both callus and fruit confirmed the AcPUB11-AcCTS1-AcBAM3s module as the central regulator of CT-induced starch metabolism. Our findings define a ubiquitination-controlled transcriptional regulatory module that mediates fruit adaptation to cool environments, providing a mechanistic foundation for temperature-controlled starch degradation during ripening.