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Divergent and Overlapping Function of Five 4-Coumarate/Coenzyme A Ligases from Populus tomentosa

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Abstract

4-Coumarate/coenzyme A ligases (4CLs) play key roles in the biosynthesis of plant secondary compounds and act at the divergence point from general phenylpropanoid metabolism to several major branch pathways, such as lignins and flavonoids. In this study, five 4CL genes in Populus tomentosa were cloned and characterized. Quantitative polymerase chain reaction (qPCR) and promoter-β-glucuronidase analysis showed that the expression of Pto4CLs is significantly divergent and certainly overlapping. The five Pto4CL recombinant proteins also have different substrate specificities and catalytic turnover rates. Pto4CL4 has the highest substrate affinity and catalytic turnover rate compared with other Pto4CL proteins. All five Pto4CL recombinant proteins had no detectable activity towards sinapate. Deletion of V338 residues in Pto4CL mutants resulted in activity towards sinapate. The low accumulation of sinapate and high proportion of S unit lignin implies that the ability of 4CL to catalyze the activation of sinapate is not necessary for lignin biosynthesis. Overexpression of the five Pto4CLs in transgenic tobacco using the 35S promoter revealed significantly increased lignin contents and decreased hydroxycinnamate derivative contents in phloem, xylem, root, and leaf tissues. An increased content of naringenin in Pto4CL4 transgenic tobacco roots was also observed. Our results suggest that the substrate specificity and divergent and overlapping expression patterns of Pto4CL isoforms may play important roles in regulating the lignin and flavonoid biosynthesis.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (31170574 and 30671697) and the 111 Project (Project No. B13007).

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Authors and Affiliations

  1. College of Life Sciences and Biotechnology, Beijing Forestry University, 100083, Beijing, People’s Republic of China

    Guodong Rao, Xiang Pan, Yongzhuo Zhang, Shan Cao, Xiangning Jiang & Hai Lu

  2. State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry, Chinese Academy of Forestry, Beijing, People’s Republic of China

    Guodong Rao

  3. Analytical and Testing Center, Beijing Forestry University, Beijing, People’s Republic of China

    Fang Xu

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  1. Guodong Rao
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  2. Xiang Pan
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  3. Fang Xu
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  6. Xiangning Jiang
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Correspondence to Hai Lu.

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Guodong Rao and Xiang Pan contributed equally to this work.

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Rao, G., Pan, X., Xu, F. et al. Divergent and Overlapping Function of Five 4-Coumarate/Coenzyme A Ligases from Populus tomentosa . Plant Mol Biol Rep 33, 841–854 (2015). https://doi.org/10.1007/s11105-014-0803-4

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