Ginkgo biloba, usually referred to as a “living fossil,” is widely planted in many countries because of its medicinal value and beautiful appearance. Owing to ginkgo’s high resistance to drought stress, ginkgo seedlings can even survive withholding water for several days without exhibiting leaf wilting and desiccation. To assess the physiological and transcriptomic mechanisms involved in the drought stress and re-watering responses of Ginkgo biloba, ginkgo seedlings were subjected to drought treatment for 15 d (D_15 d) and 22 d (D_22 d) until they had severely wilted, followed by re-watering for 3 d (D_Re3 d) to restore normal growth. Variations in physiological characteristics (relative water content, malondialdehyde (MDA) content, stomatal aperture, and antioxidant enzyme activity) during drought and re-watering were assessed. In total, 1692, 2031, and 1038 differentially expressed genes (DEGs) were upregulated, while 1691, 2820, and 1910 were downregulated in D_15 d, D_22 d, and D_Re3 d, respectively, relative to the control. Three pathways, namely, plant hormone signal transduction, plant–pathogen interaction, and the plant MAPK signaling pathway, were enriched during drought stress and re-watering. The DEGs involved in plant hormone signal transduction pathways (those of IAA, CTK, GA, ABA, ETH, BR, SA, and JA) and the major differentially expressed transcription factors (TFs; MYB, bHLH, AP2/ERF, NAC, WRKY, and bZIP) were identified. Quantitative real-time PCR revealed six TFs as positive or negative regulators of drought stress response. These phenotype-related physiological characteristics, DEGs, pathways, and TFs provide valuable insights into the drought stress and re-watering responses in G. biloba.

B-box (BBX) transcription factors, which are specific to the plant kingdom, play a crucial role in regulating light-dependent growth, development, secondary metabolite biosynthesis, and the response to biotic and abiotic stresses. Despite their significance, there has been a lack of systematic investigation into the BBX gene family in Ginkgo biloba. In the present study, we identified nine BBX genes within the G. biloba reference genome, distributed across seven chromosomes, and classified them into four groups based on their phylogenetic relationships with the BBX gene families of Arabidopsis thaliana. Our analysis of gene structure, conserved domains, and motifs suggests that GbBBXs exhibit a high degree of conservation throughout evolutionary history. Additionally, synteny analysis revealed that dispersed duplication events have contributed to the expansion of the BBX gene family in G. biloba. An examination of cis-regulatory elements indicated that numerous GbBBX genes contain motifs associated with light, hormones, and stress, suggesting their potential roles in responding to these signals and environmental adaptation. Expression profiles obtained from RNA-Seq data and quantitative Real-Time PCR (qRT-PCR) analyses of GbBBX genes across various organs, hormone treatments, and leaves with differing flavonoid content, as well as during both short-term and long-term water stress, demonstrated their potential roles in flavonoid regulation and responses to hormones and water stress. Subcellular localization studies indicated that the proteins GbBBX5, GbBBX7, GbBBX8, and GbBBX9 are localized within the nucleus. This study is the first thorough analysis of the BBX gene family in G. biloba, providing a valuable foundation for further understanding their evolutionary context and functional roles in flavonoid regulation and responses to water stress.

SQUAMOSA promoter-binding protein-like (SPL) transcription factors specific to plants are vital for regulating growth, development, secondary metabolite biosynthesis, and responses to both biotic and abiotic stresses. Despite their importance, no systematic investigations or identifications of the SPL gene family in Ginkgo biloba have been conducted. In this study, we identified 13 SPL genes within the Ginkgo biloba reference genome, spanning seven chromosomes, and categorized these genes into six groups based on their phylogenetic relationships with Arabidopsis thaliana SPL gene families. Our analysis of gene structure, conserved domains, motifs, and miR156 target predictions indicates that GbSPLs are highly conserved across evolutionary timelines. Furthermore, synteny analysis highlighted that dispersed duplication events have expanded the SPL gene family in Ginkgo biloba. Examination of the cis-regulatory elements revealed that many GbSPL genes possess motifs associated with light, hormones, and stress, implying their involvement in flavonoid biosynthesis and adaptation to environmental conditions. RNA-Seq and qRT-PCR expression profiles of GbSPL genes across various tissues and low- and high-flavonoid leaves and during both short-term and long-term water stress illustrated their roles in flavonoid biosynthesis and responses to water stress. Subcellular localization experiments showed that GbSPL2 and GbSPL11 proteins are situated within the nucleus. Our research offers the first systematic characterization of the SPL gene family in Ginkgo biloba, establishing a valuable foundation for understanding their evolutionary background and functional roles in flavonoid biosynthesis and water stress response.

To assess the regulatory mechanisms involved in the transcriptomic response of Ginkgo biloba to water shock and rehydration, ginkgo seedlings were subjected to dehydration for 0, 3, 6, 12, and 24 h, followed by rehydration for 12 h (Re12 h). A total of 1388, 1802, 2267, 2667, and 3352 genes were upregulated, whereas 1604, 1839, 1934, 2435, and 3035 genes were downregulated, at 3, 6, 12, 24, and Re12 h, respectively, compared to 0 h. Two KEGG pathways—the plant pathogen interaction pathway and mitogen-activated protein kinase (MAPK) signaling pathway—were enriched under water shock but not under rehydration. Moreover, plant hormone signal transduction was enriched under both water shock and rehydration. Differentially expressed genes (DEGs) involved in the ABA signaling pathway (PYR/PYLs, PP2Cs, and SnRK2s) and major differentially expressed transcription factors (MYB, bHLH, AP2/ERF, NAC, WRKY, and bZIP TFs) were identified. qRT-PCR analysis further revealed GbWRKY3 as a negative regulator of the water shock response in G. biloba. The subcellular localization results revealed GbWRKY3 as a nuclear protein. These phenotype-related DEGs, pathways, and TFs provide valuable insight into the water shock and rehydration response in G. biloba.