Integrated Multi-Omics Analysis Elucidates Hormonal and Transcriptional Reprogramming During Walnut Bud Break and Identifies JrGAI as a Negative Regulator

Background/Objectives: The release from bud dormancy and subsequent break are critical processes for perennial woody plants to acclimatize to seasonal variations. This study aimed to elucidate the hormonal and transcriptional mechanisms governing walnut bud development. Methods: We utilized the early-fruiting, thin-shelled, and stress-tolerant cultivar ‘Jinboxiang 1’, collecting bud samples at four distinct developmental stages: Dormancy, Bud swelling, Bud burst, and Leaf unfolding. By integrating hormone metabolome and transcriptome analyses with weighted gene co-expression network analysis (WGCNA), key regulatory elements were systematically identified. Results: A total of seventy-seven hormones and related metabolites spanning eight classes were identified, with cytokinins and auxins being predominant. Transcriptomic profiling revealed 2935, 1963, and 3519 differentially expressed genes across the three successive transitions, respectively. Early bud swelling was linked to pathways involving hormone signaling, MAPK cascades, hypoxia responses, and defense mechanisms. Bud burst was associated with processes related to cutin, suberin, wax, fatty acids, carbohydrates, and secondary metabolism, whereas leaf unfolding predominantly involved photosynthetic activity and photosystem assembly. A core set of 276 genes was enriched in pathways related to jasmonate metabolism, ethylene signaling, α-linolenic acid metabolism, and MAPK signaling. WGCNA revealed 16 co-expression modules, among which the blue module was progressively activated during break and harbored JrGAI, a gene encoding a DELLA protein and a known repressor of gibberellin signaling. Heterologous overexpression of JrGAI in poplar reduced plant height and stem diameter and delayed bud break after dormancy release, supporting JrGAI as a candidate DELLA-associated negative regulator of bud development in walnut. Conclusions: These results reveal a stage-specific regulatory network underlying walnut bud development and demonstrate that JrGAI functions as a DELLA-associated negative regulator of both vegetative growth and bud break in walnut.