Physiological, transcriptomic, and metabolomic insights into divergent responses to gibberellin and paclobutrazol in walnut (Juglans regia) leaves.

Gibberellins (GA) and paclobutrazol (PP) are plant growth regulators with contrasting effects on plant development, but their regulatory associations in perennial tree crops like walnut (Juglans regia) remain incompletely understood. In this study, 'Lvling' walnut trees were foliar-sprayed with 50 mg L-1 GA and 1000 mg·L-1 PP at 30 days after flowering.Physiological, transcriptomic, and metabolomic analyses were then performed to clarify the mechanisms by which these two growth regulators affect the growth and development of walnut leaves. GA treatment decreased abscisic acid (ABA) and indole-3-acetic acid (IAA) and increased zeatin riboside, isopentenyladenine, chlorophyll, soluble sugars, and proteins; by contrast, PP increased ABA and IAA but reduced chlorophyll, soluble sugars, and starch, thereby delaying growth. Transcriptome analysis showed that GA upregulated GA signalling and starch metabolism genes (GID1, SLY1, AGPase, SUS), while PP activated ABA signalling (NCED3, ABF2) and stress-resistance genes (LOX2, PR1). Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that GA mainly influenced carotenoid and starch-sucrose metabolism, whereas PP regulated α-linolenic acid metabolism and anthocyanin biosynthesis. Metabolomics further revealed that GA promoted the accumulation of flavonoids and jasmonic acid, while PP increased the abundance of osmotic adjustment compounds such as proline and glutathione. Overall, these results suggest that GA and PP induce divergent regulatory patterns in walnut leaves, with GA favoring growth-related hormone signaling and carbon metabolism, and PP favoring stress- and defense-related metabolic reprogramming. This study provides candidate genes and metabolic pathways for optimizing the application of growth regulators in walnut cultivation.