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Terpene trilactones (TTLs) are one of the main active ingredients of Ginkgo biloba. Owing to TTL’s unique chemical structure, it is difficult to increase TTL content through chemical and biological methods. Studying its regulatory mechanism is important in the G. biloba industry. The effect of exogenous methyl jasmonate (MeJA) on the physiological and molecular mechanism of TTL biosynthesis was studied. These results showed that MeJA treatment could improve the TTL contents, soluble sugar, starch, soluble protein, endogenous hormones (ZT, GA3, IAA, and ABA), antioxidant enzymes (catalase, peroxidase, and superoxide dismutase), and the efficiency of photosynthesis in G. biloba leaves. A total of 100 differentially expressed genes (DEGs) were identified between the control group and MeJA treatment through RNA-seq analysis. The results indicated that exogenous MeJA treatment upregulated the expression levels of the following genes: BMY (beta-amylase) in the starch and sucrose metabolic pathway; PEX7 (peroxin-7) in the peroxisome pathway; psbA (photosystem II reaction center D1 protein), psbC (photosystem II CP43 chlorophyll apoprotein), psaA (photosystem I P700 chlorophyll a apoprotein A1), and petF (photosynthetic electron transport ferredoxin) in the photosynthesis pathway; and CYP450 (Gb-16765) (cytochromeP450). Exogenous MeJA treatment can promote physiological indexes (photosynthetic efficiency, starch, sucrose, antioxidant enzyme activities, etc.) and then regulating differential genes, thus controlling the synthesis of TTLs.

As unique secondary metabolites of Ginkgo biloba L. and relics of Ginkgoaceae, terpene trilactones (TTLs) possess great medicinal value. In this study, we investigated the effects of ultraviolet (UV) irradiation, cold stress (4 °C), and exogenous hormone treatment on TTLs and key genes expression in ginkgo leaves in annual potted ginkgo seedlings subjected to different stress treatments. The processing conditions were as follows: UV treatment (1500 μJ/m2) for 0, 8, 16, 24, and 48 h; cold stress at 4 °C, 400 µmol photons m−2 s−1; 100 μM abscisic acid (ABA); 100 μM methyl jasmonate (MeJA); 10 mM salicylic acid (SA); and 10 mM ethephon (ETH) treatments for 0, 2, 4, 6, and 8 days. Results of evaporative light-scattering detector–high-performance liquid chromatography showed that UV, cold stress, ABA, SA, MeJA, and ETH treatments increased the TTL content at various levels and up to 21.9%. The relationships among the expression levels of the key enzyme genes GbDXS, GbGGPPS, GbLPS, and GbMVD in the TTL biosynthesis pathway were also analyzed. Changes in the TTL content and expression levels of the key genes were significantly and positively correlated with TTL content. UV, cold stress, ABA, SA, MeJA, and ETH were found to increase the TTL content by upregulating the expression of the key genes. This study aimed to reveal the molecular mechanism through which TTLs are synthesized. The results can also provide theoretical guidance for the application of treatment methods to increase TTL content.

Terpene trilactones (TTLs) are the main secondary metabolites in Ginkgo biloba L. with efficacious pharmacological activity. Jasmonate ZIM-domain (JAZ) protein is a key regulatory factor of the JA signaling pathway, which regulates the biosynthesis of secondary metabolites such as terpenes, alkaloids, and flavonoids. In this study, GbJAZ01~GbJAZ11 were identified from the genome data in G. biloba, which contained TIFY-, Jas-, and weakly conserved NT-domains, and the promoters in most of them contained light, hormone, and stress-responsive elements. Phylogenetic analysis divided all JAZ proteins of Arabidopsis thaliana, Oryza sativa, Picea sitchensis, Taxus chinensis, and G. biloba into nine groups, in which GbJAZs belong to Group VI-IX. GbJAZs have similar functional motifs to A. thaliana and O. sativa, but also contain three specific motifs of gymnosperms, indicating that, although gymnosperms and angiosperms have some conservative structures and functions, their evolutionary processes are independent. Expression pattern analysis showed that the expression levels of GbJAZs were significantly up-regulated by MeJA, but the change pattern and amplitude were different, indicating that the function of GbJAZs in response to a JA signal may be different. After ABA and SA treatment, the expression of GbJAZs was up-regulated or inhibited in varying degrees, and different GbJAZs may be involved in the synergistic or antagonistic effects between JA and other hormone signals. The MeJA significantly increased the content of TTLs in G. biloba leaves, which were significantly positively correlated with the expression levels of GbJAZ01, 02, 07, and 11, and negatively correlated with the expression of GbJAZ04. They may play an important role in JA signaling pathways and the interactions between JA and other hormone signals, and participate in the regulation of the biosynthesis of TTLs. Our results provide a reference for the discovery that GbJAZs are involved in JA signaling pathways, and lay a theoretical foundation for analyzing JA signaling pathways to regulate the synthesis of secondary metabolites.

The WD40 transcription factor family is a superfamily found in eukaryotes and implicated in regulating growth and development. In this study, 167 WD40 family genes are identified in the Ginkgo biloba genome. They are divided into 5 clusters and 16 subfamilies based on the difference analysis of a phylogenetic tree and domain structures. The distribution of WD40 genes in chromosomes, gene structures, and motifs is analyzed. Promoter analysis shows that five GbWD40 gene promoters contain the MYB binding site participating in the regulation of flavonoid metabolism, suggesting that these five genes may participate in the regulation of flavonoid synthesis in G. biloba. The correlation analysis is carried out based on FPKM value of WD40 gene and flavonoid content in 8 tissues of G. biloba. Six GbWD40 genes that may participate in flavonoid metabolism are screened. The biological functions of the WD40 family genes in G. biloba are systematically analyzed, providing a foundation for further elucidating their regulatory mechanisms. A number of WD40 candidate genes involved in the biosynthesis and metabolism of G. biloba also predicted. This study presents an important basis and direction for conducting further research on the regulatory network of flavonoid synthesis and metabolism.

Salicylic acid (SA) is an important endogenous signaling molecule that can not only induce disease resistance but also improve the accumulation of secondary metabolites in plants. Terpene trilactones (TTLs) are unique bioactive components of Ginkgo biloba L. and it can antagonize platelet activating factors. In this study, SA treatment could increase TTL content as well as increase several other physiological indicators related to growth, photosynthetic parameters, starch and sucrose content, antioxidant enzyme activity, and endogenous hormone content in G. biloba. Furthermore, comparative transcriptome analysis between the SA-treated and control was used to reveal the molecular regulation of SA on TTL biosynthesis. RNA-Seq analysis identified 249 differentially expressed genes (DEGs) between the SA treatment and CK group. Up-regulation of three DEGs (BMY1, BMY2, and AMY) related to the starch and sucrose metabolism pathway accelerated the oxidative decomposition of sugar to release energy for biosynthesis. Two up-regulated (PetF and Lhcb1) and two down-regulated (Lhcb2-1 and Lhcb2-2) genes involved in the photosynthesis pathway may contribute to TTL biosynthesis. SA-regulated genes may alter antioxidant enzyme activity and endogenous hormone content levels to facilitate TTL biosynthesis. Among these DEGs, candidate structural genes (HMGR, CYP450, CPR, and TPS) and transcription factors (MYB and WRKY) played key roles in SA-promoted TTL accumulation in G. biloba. Quantitative real-time PCR analysis verified the expression patterns of all of the selected DEGs. The results provide novel insights into the transcriptional regulatory mechanism of the promotive effect of SA on TTL biosynthesis in G. biloba.