Physiological, transcriptomic, and metabolomic analyses reveal the molecular regulatory mechanisms of walnut fruit in response to gibberellin and paclobutrazol

Gibberellins (GAs) and paclobutrazole (PP333) are crucial plant growth regulators, but their molecular mechanisms in walnut fruit development have yet to be elucidated. This study used ‘Lüling’ walnut plants, whose leaves were sprayed with different concentrations of GAs and PP333 during the fruit expansion period. The effects of those two treatments on the endogenous hormones, metabolite accumulation, and gene expression in the fruits were explored through a combined analysis of plant physiology, transcriptomics, and metabolomics. The results showed that the GAS treatment significantly increased the content of gibberellins (with a 321% maximal increase), cytokinins (KT, IP), and auxin (IAA), while reducing the level of abscisic acid (ABA). It also promoted the accumulation of soluble sugars (+ 50%), starch (+ 19.4%), and soluble proteins (+ 18.18%). The transcriptomic analysis revealed that GAS regulated fruit development by activating certain pathways, namely those for ABC transporters, carbon metabolism, and carotenoid biosynthesis. By contrast, PP333 inhibited the GA signaling pathway and downregulated the expression of genes involved in starch metabolism. Further, the WGCNA analysis identified genes related to amino acid synthesis, such as LOC109010970 . According to the transcription factor analysis, both MYB and bHLH families of transcription factors played a central regulatory role in hormone signal transduction. This study uncovered the mechanisms by which GAS and PP333 regulate the process of walnut fruit development through a ‘hormone–metabolism–gene’ network, providing a valuable theoretical basis for the precise application of growth regulators in walnut cultivation.