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-VvPP2CA, IAA-VvGH3.1, and CK-VvARR9-1, were also identified. Concurrently, PAC negatively regulated hormone-active genes, including BR-VvXTH25, SA-VvTGA21-3, and JA-VvTIFY3B, 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.

The seedless trait in grapes primarily arises from pseudo-parthenocarpy, driven by early seed abortion involving programmed cell death (PCD) and autophagy. However, the specific regulatory networks governing these pathways remain poorly defined. To address this, we systematically characterized the PCD-related (vacuolar processing enzyme [VPE] and metacaspase [MC]) and autophagy-related (ATG) gene families in the seedless cultivar ‘Jingkejing’ utilizing comprehensive bioinformatic analyses, spatiotemporal transcriptomic profiling, and transient expression assays. We identified three VPE, six MC, and 33 ATG genes, whose promoters were significantly enriched in hormone-responsive and transcription factor (TF)-binding motifs. Transcriptomic profiling across critical seed abortion stages highlighted VvγVPE and VvMC9 as key differentially expressed genes. Furthermore, protein–protein interaction predictions indicated extensive crosstalk among PCD/ATG proteins, kinases, and ubiquitin system components. Notably, transient expression assays confirmed that two core transcription factors, VvWRKY46 and VvERF71, specifically regulate downstream PCD-related targets, supporting a proposed multi-hormonal and TF-mediated regulatory network during seed abortion. These findings elucidate the molecular mechanisms underlying pseudo-parthenocarpy, providing essential candidate gene resources for the marker-assisted breeding of seedless grapes.