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Molecular cloning and functional characterization of a cinnamate 4-hydroxylase-encoding gene from Camptotheca acuminata

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Abstract

Cinnamate 4-hydroxylase (C4H) catalyzes the regioselective para-hydroxylation of trans-cinnamic acid to form p-coumaric acid, the biosynthetic precursor of phenylpropanoid-based polymers. These biopolymers play an essential role in plant structure construction, development, and defense. Herein the open reading frame of CaC4H2 was cloned from Camptotheca acuminata, a deciduous camptothecin-producing tree native to China. CaC4H2 showed 94 % amino acid residues identity with those of reported CaC4H, which suggested that CaC4H2 is an isoform of C4Hs presented in C. acuminata. The intact CaC4H2 was overexpressed in Escherichia coli with its functional reaction partner cytochrome P450 reductase, CamCPR, which transfers electrons from NADPH to CaC4H2 to support the catalytic hydroxylation activity of CaC4H2. Upon incubating trans-cinnamic acid with the recombinant CaC4H2 and tCamCPR, the formation of p-coumaric acid was confirmed by the HPLC–DAD and UPLC-DAD-ESIMS analyses, which indicated the catalytic hydroxylation activity of CaC4H2. Quantitative real-time PCR analyses showed that CaC4H2 was expressed in all tissues of C. acuminata seedlings, which is consistent with the well-known conclusion that the C4H-catalyzed hydroxylation reaction is a key step within the biosynthetic pathway of phenylpropanoids. The functional characterization of CaC4H2 will be useful for molecular breeding and sustainable utilization and protection of the camptothecin-producing plant.

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Acknowledgments

This work was supported in part by the Science and Technology Project for Outstanding Youths in Life Science (KSCX2-EW-Q-6) from the Chinese Academy of Sciences, the Applied and Basic Research Program of Sichuan Province (2015JY0058), and the 21172216 project from the National Natural Science Foundation of China.

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Authors and Affiliations

  1. Center for Natural Products Research, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, 610041, People’s Republic of China

    Wei Li, Lixia Yang, Liangzhen Jiang, Guolin Zhang & Yinggang Luo

  2. State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, 200032, People’s Republic of China

    Yinggang Luo

  3. University of Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China

    Wei Li & Lixia Yang

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  1. Wei Li
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  2. Lixia Yang
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Correspondence to Yinggang Luo.

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Communicated by H. Peng.

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Li, W., Yang, L., Jiang, L. et al. Molecular cloning and functional characterization of a cinnamate 4-hydroxylase-encoding gene from Camptotheca acuminata . Acta Physiol Plant 38, 256 (2016). https://doi.org/10.1007/s11738-016-2275-7

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  • DOI: https://doi.org/10.1007/s11738-016-2275-7

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