Abstract
BACKGROUND: Tree rejuvenation is the transition from adult state to juvenile state. Successive grafting can result in tree rejuvenation. Flavonoids constitute a prominent class of secondary metabolites that play critical roles in regulating cellular physiology, signaling, and the transduction between plant and environment interactions. Meanwhile, endogenous phytohormones are instrumental in restoring the juvenile features of trees. However, little is known about the flavonoid and hormone biosynthesis in rejuvenated Ginkgo biloba L. RESULTS: Here we rejuvenated the surviving 4,000-year-old Ginkgo through successive grafting. The results revealed leaf cleavage, leaf width, leaf length, and leaf area increased significantly in S(1) (first-step graft). Additionally, the total flavonoid content of S(2) (second-step graft) was the highest. Indole acetic acid (IAA), gibberellins (GA), and zeatin-riboside (ZR) increased, and abscisic acid (ABA) decreased in each successive generation. Transcriptome analysis of gene expression fragments per kilobase of exon model per million mapped fragments (FPKM) revealed 1756-5689 significantly differentially expressed genes (DEGs) that clustered into nine distinct expression modules. The weighted gene co-expression network analysis (WGCNA) revealed bisque4 and palevioletred3 had the highest correlation coefficient with the phenotype and physiology of rejuvenated Ginkgo and identified only eight genes were annotated in top hub ten. The expression profiles corresponding DEGs in flavonoid biosynthesis (25 DEGs) and plant hormone signal transduction (36 DEGs) were revealed. Most of these DEGs were up-regulated from S(1) to S(2), while down-regulated in S(3) (third-step graft). Small RNA sequencing revealed 3281 miRNAs including 621 novel miRNAs. Combined analysis both of transcriptome and small RNA, miR395 was the core miRNA and targeted to iron-containing protein (FAO1) and pentatricopeptide repeat-containing protein At1g11290 (PCMP-H40). CONCLUSIONS: The comprehensive analysis of the RNA-seq, small RNA, and physiological data in this study provided candidate genes and clarified the regulatory mechanism of successive grafting in rejuvenated Ginkgo, suggesting that the number of successive generations of grafted rejuvenation should not exceed the third generation (S₃).