Characterization of Main Responsive Genes Reveals Their Regulatory Network Attended by Multi-Biological Metabolic Pathways in Paclobutrazol (PAC)-Modulated Grape Seed Development (GSD) at the Stone-Hardening Stage.

Paclobutrazol (PAC) is a significant inhibitor of gibberellin biosynthesis that profoundly influences grape seed development (GSD) through the modulation of key molecular pathways. Here, we identified 6659 differentially expressed genes (DEGs) in GSD under PAC treatment, with 3601 up-regulated and 3058 down-regulated. An analysis of hormone-associated DEGs revealed that auxin-related genes (16) were the most up-regulated, followed by genes associated with brassinosteroid and ABA. In contrast, cytokinin- and gibberellin-related genes exhibited a suppressive response. PAC treatment also triggered extensive reprogramming of metabolic pathways, including 44 genes involved in starch and sucrose metabolism (24 up-regulated, 20 down-regulated), 101 cell wall-related genes (53 up-regulated, 48 down-regulated), and 110 transcription factors (77 up-regulated, 33 down-regulated). A cis-element analysis of the promoters of 76 hormone-responsive genes identified 14 types of hormone-responsive cis-elements, with ABRE being the most prevalent. Genes responsible for inactivating active hormones, such as ABA-, and , were also identified. Concurrently, PAC negatively regulated hormone-active genes, including , , and , leading to reduced levels of these hormones. PAC modulates GSD by mediating the dynamic balance of multi-hormone accumulations. Furthermore, development-related cis-elements such as the AACA-motif, AAGAA-motif, AC-I, AC-II, O2-site, as-1, CAT-box, CCAAT-box, circadian, GCN4-motif, RY-element, HD-Zip 1, HD-Zip 3, MSA-like, MYB-like sequence, MYB-binding site, and MYB recognition site, were found in key DEGs involved in starch and sucrose metabolism, cell wall remodeling, and epigenetic regulation. This indicates that these pathways are responsive to PAC modulation during GSD. Finally, we developed a comprehensive regulatory network to illustrate the PAC-mediated pathways involved in GSD. This network integrates multi-hormonal signaling, cell wall remodeling, epigenetic regulation, and transcription factors, highlighting PAC's pivotal role in GSD. Our findings provide new insights into the complex mechanisms underlying PAC's effects on grapevine development.