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.

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.