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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.

Ginkgo biloba, which contains flavonoids as bioactive components, is widely used in traditional Chinese medicine. Increasing the flavonoid production of medicinal plants through genetic engineering generally focuses on the key genes involved in flavonoid biosynthesis. However, the molecular mechanisms underlying such biosynthesis are not yet well understood. To understand these mechanisms, a combination of second-generation sequencing (SGS) and single-molecule real-time (SMRT) sequencing was applied to G. biloba. Eight tissues were sampled for SMRT sequencing to generate a high-quality, full-length transcriptome database. From 23.36 Gb clean reads, 12,954 alternative polyadenylation events, 12,290 alternative splicing events, 929 fusion transcripts, 2,286 novel transcripts, and 1,270 lncRNAs were predicted by removing redundant reads. Further studies reveal that 7 AS, 5 lncRNA, and 6 fusion gene events were identified in flavonoid biosynthesis. A total of 12 gene modules were revealed to be involved in flavonoid metabolism structural genes and transcription factors by constructing co-expression networks. Weighted gene coexpression network analysis (WGCNA) analysis reveals that some hub genes operate during the biosynthesis by identifying transcription factors (TFs) and structure genes. Seven key hub genes were also identified by analyzing the correlation between gene expression level and flavonoids content. The results highlight the importance of SMRT sequencing of the full-length transcriptome in improving genome annotation and elucidating the gene regulation of flavonoid biosynthesis in G. biloba by providing a comprehensive set of reference transcripts.