The E3 ligase PpMBR2 targets PpABI5 for degradation and negatively regulates cold tolerance in peach fruit

Abscisic acid (ABA) is known to alleviate chilling injury (CI) of peach fruit, but the molecular mechanisms is poorly understood. Here, a regulatory module that mediates peach fruit CI response at both transcriptional and post-translational levels was identified. ABA induced the gene expression and protein accumulation of abscisic acid-insensitive 5 (PpABI5). In turn, PpABI5 stimulated the expression of genes involved in proline and flavonoid biosynthesis, antioxidant defense, and fatty acid desaturation. Transient and ectopic overexpression of PpABI5 enhanced cold tolerance in peach and tomato fruit, accompanied by elevated proline and flavonoid biosynthesis, enhanced antioxidant capacity, and fatty acid desaturation. PpABI5 interacted with an E3 ligase MED25-binding RING-H2 protein2 (PpMBR2), which ubiquitinated PpABI5 at the specific site of K140, thereby modulated its degradation and attenuated PpABI5-mediated transactivation of target genes. PpMBR2-overexpression aggravated fruit CI, whereas PpMBR2-silencing produced the opposite effect. Furthermore, PpABI5 interacted with an ethylene response factor CRF3 (PpERF-CRF3), and this interaction enhanced their transcriptional regulation of target genes. Collectively, the results reveal a dynamic regulatory module PpMBR2-PpABI5-PpERF-CRF3 that governs cold tolerance in peach fruit by regulating proline and flavonoid biosynthesis, antioxidant defense and fatty acid desaturation. These findings advance our understanding of phytohormone crosstalk and site-specific ubiquitination modification in fruit CI.