Plant cell walls primarily comprise lignin, which performs functions of mechanical support, water transport, and stress responses. Lignin biosynthesis pathway proceeds through metabolic grid featuring complexity and diversity in enzymatic reaction. Cinnamate-4-hydroxylase (C4H, EC 1.14.13.11) is the gene encoding enzyme that catalyzes the second step of phenylpropanoid pathway responsible for biosynthesis of lignin. A full-length cDNA of C4H (designated as GbC4H), which spanned 1816-bp with a 1518-bp open reading frame encoding a 505-amino-acid protein, was cloned from Ginkgo biloba. A GbC4H genomic DNA fragment, spanning 3249-bp, was cloned and found to contain two exons and one intron. GbC4H protein showed high similarities with other plant C4Hs to include conserved domains of cytochrome P450 family. GT-1, W-box, and Myb/Myc recognition sites involved in stress response were detected in a 1265-bp upstream promoter region of GbC4H. Phylogenetic analysis suggested the common evolutionary ancestor shared by plant C4Hs including the gymnosperm enzyme. pET-28a-GbC4H plasmid was constructed and expressed in Escherichia coli strain BL21. Enzymatic assay revealed that recombinant GbC4H protein catalyzes conversion of trans-cinnamic acid to p-coumaric acid. Expression analyses in different organs showed high expression of GbC4H in stems and roots, whereas low expressions was found in fruits, carpopodium, and petioles. Further analysis indicated linear correlation of lignin contents with transcript levels of GbC4H among different tissues. GbC4H transcription was increased by treatments with UV-B, cold, salicylic acid, and abscisic acid, indicating the possible role of GbC4H in response to stresses and hormonal signal. Understanding of GbC4H function could benefit molecular breeding and reinforcement of defense mechanisms in Ginkgo.
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