This study aimed to investigate the mechanism underlying primary bud necrosis (PBN) in grapevines. PBN is a physiological disorder that significantly reduces grape yields. The four varieties, ‘Shine Muscat’, ‘Summer Black’, ‘Ruby Seedless’, and ‘Hutai 8’, were investigated and found to exhibit differences in PBN, which was positively correlated with the speed and extent of inflorescence differentiation. Among them, ‘Summer Black’ was most susceptible to PBN. Treatment with gibberellin acid 3 (GA₃) notably accelerated and exacerbated PBN in ‘Summer Black’, whereas the endogenous gibberellin (GA) inhibitor chlorocholine chloride (CCC) delayed or prevented PBN onset. Histological observations of dormant bud tissues revealed PBN progression in stages, starting with the expansion of cells in the necrosis zone (NZ), followed by cell wall irregularities and collapse, buckling cell layer formation, and subsequent cell separation. In the water control group, NZ mainly occurred in the bud scale layer. However, by the second week after GA₃ treatment, primary buds visibly elongated, and NZ was formed at multiple locations along the primary buds. Transcriptomic analyses revealed significant regulation of stress-related genes, including reactive oxygen species (ROS) and heat-shock proteins (HSPs), following GA₃ treatment. Genes related to jasmonic acid (JA) biosynthesis and signaling pathways were upregulated after week 2, whereas CCC treatment led to the downregulation of these genes. Furthermore, genes associated with cations such as calcium, iron, and copper showed significant changes across all transcriptome samples. Genes associated with the degradation of cell membranes and cell walls were upregulated in samples treated with GA₃ and water control. Overall, these findings suggested that GA₃ promoted PBN by enhancing JA synthesis and modulating the cell necrosis pathway via JA signaling. This process involved ROS accumulation and activation of cation pathways, leading to endomembrane and cell wall degradation, cell rupture, and, ultimately, PBN development.

Southern China has high temperatures and receives concentrated rainfall; therefore, the two-crop-a-year cultivation system has been applied to grape production so as to resolve the problem of relative seasonal surplus of grape yield. However, a common issue associated with this technique is the tendency of the second season fruits to be smaller than the first season fruits. We here used the first and second season fruits of ‘Summer Black’ at different ripening stages as research materials. Phenotypic and histological analyses revealed fewer cell number occurring between 7 and 14 days post anthesis (DPA) in the second season fruits, which ultimately resulted in a smaller fruit size compared with the first season fruits. To unravel the mechanism underlying this phenomenon, first and second season fruits of four time periods (7, 14, 21, and 28 DPA) were selected for RNA-seq analysis. This analysis identified 10431 differentially expressed genes (DEGs). These DEGs were classified into 9 clusters through GO and KEGG enrichment analyses. Then the time-ordered gene co-expression network (TO-GCN) analysis with the breadth-first search algorithm showed that DEGs in the GCN were divided into 8 levels. The DEGs of early berry development (L1–L3) were enriched in heat stress- and cell division-related pathways. The field investigation of effective accumulated temperature confirmed that the growth and development of the second season fruits were subjected to high temperature stress during 7–14 DPA. Moreover, based on the results of interactive analysis of TO-GCN and transcriptional regulation prediction of L1–L3 genes, we constructed a unique hierarchical regulatory network for the heat stress regulation of berry size. The expression level of 5 candidate genes was verified through qRT-PCR. Vitvi10g00469 (HSFB2A), Vitvi16g00982 (HSFB2A), Vitvi02g00387 (HSFB2B), Vitvi15g01542 (NTL9), and Vitvi06g00592 (DIVARICATA) were upregulated in 7–14 DPA, whereas Vitvi18g00777 (HSFB4) was downregulated in 7 DPA. These results suggest that during intense cell division, heat stress might act as a major factor causing a reduction in cell number, thereby ultimately resulting in the smaller size of the second season fruits.