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Functional characterization of an anthocyanidin reductase gene from the fibers of upland cotton (Gossypium hirsutum)

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Metabolic profiling, gene cloning, enzymatic analysis, ectopic expression, and gene silencing experiments demonstrate that the anthocyanidin reductase (ANR) pathway is involved in the biosynthesis of proanthocyanidins in upland cotton.

Proanthocyanidins (PAs) are oligomeric or polymeric flavan-3-ols, however, the biosynthetic pathway of PAs in cotton remains to be elucidated. Here, we report on an anthocyanidin reductase (ANR) gene from cotton fibers and the ANR pathway of PAs. Phytochemical analysis demonstrated that leaves, stems, roots, and early developing fibers produced PAs and their monomers, including (−)-epicatechin, (−)-catechin, (−)-epigallocatechin, and (−)-gallocatechin. Crude PA extractions from different tissues were boiled in Butanol:HCl. Cyanidin, delphinidin, and pelargonidin were produced, indicating that cotton PAs include diverse extension unit structures. An ANR cDNA homolog (named GhANR1) was cloned from developing fibers. The open reading frame, composed of 1,011 bp nucleotides, was expressed in E. coli to obtain a recombinant protein. In the presence of NADPH, the recombinant enzyme catalyzed cyanidin, delphinidin, and pelargonidin to (−)-epicatechin and (−)-catechin, (−)-epigallocatechin and (−)-gallocatechin, and (−)-epiafzelechin and (−)-afzelechin, respectively. The ectopic expression of GhANR11 in an Arabidopsis ban mutant allowed for the reconstruction of the ANR pathway and PA biosynthesis in the seed coat. Virus-induced gene silencing (VIGS) of GhANR11 led to a significant increase in anthocyanins and a decrease in the PAs, (−)-epicatechin, and (−)-catechin in the stems and leaves of VIGS-infected plants. Taken together, these data demonstrate that the ANR pathway contributes to the biosynthesis of flavan-3-ols and PAs in cotton.

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Acknowledgments

This research was financially supported by “the National State Laboratory of China”. This project was also supported by “Bai Ren Ji Hua, Hunan Province”.

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Authors and Affiliations

  1. The State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

    Yue Zhu, Haiyun Wang, Guixian Xia & Jiahe Wu

  2. Hunan Provincial Key Laboratory of Plant Resources Conservation and Utilization, College of Biology and Environmental Sciences, Jishou University, No.120 Ren Min Nan Lu, Jishou, 416000, Hunan, People’s Republic of China

    Yue Zhu, Qingzhong Peng & Yuntao Tang

  3. Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, 27695, USA

    De-Yu Xie

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  1. Yue Zhu
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  2. Haiyun Wang
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Correspondence to Jiahe Wu or De-Yu Xie.

Electronic supplementary material

S-Fig. 1 HPLC profiles of flavan-3-ols in crude extracts from different tissues. CA, (-)- catechin; EP, (-)- epicatechin; GC, (-)-gallocatechin; EGC, (-)-epicatechin.

S-Fig. 2 Amino acid sequence alignment from 18 ANR homologs. The Rossmann dinucleotide (NADPH/NADH) binding domain highlighted as “#####” has the sequence G-G-S-G-F-V-A. Abbreviations: LuANR, Lotus uliginosus; GmANR, Glycine max, MtANR, Medicago truncatula; CsANR, Camellia sinensis; DkANR, Diospyros kaki; VvANR, Vitis vinifera; MdANR, Malus x domestica; PcANR, Pyrus communis; PaANR, Prunus avium; TcANR, Theobroma cacao; FaANR, Fragaria x ananassa; AtANR, Arabidopsis thaliana; BdANR, Brachypodium distachyon.

S-Fig. 3 An unrooted phylogenetic tree containing 18 ANR homologs. This unrooted tree was developed using the deduced amino acid sequences of 18 ANR homologs. Abbreviations: LuANR, Lotus uliginosus; GmANR, Glycine max, MtANR, Medicago truncatula; CsANR, Camellia sinensis; DkANR, Diospyros kaki; VvANR, Vitis vinifera; MdANR, Malus x domestica; PcANR, Pyrus communis; PaANR, Prunus avium; TcANR, Theobroma cacao; FaANR, Fragaria x ananassa; AtANR, Arabiopsis thaliana; BdANR, Brachypodium distachyon.

S-Fig. 4 Comparison of HPLC profiles of flavan-3-ols from leaf and stem tissues from VIGS-infected and wild-type plants. CA:(-)-catechin, EP:(-)-epicatechin, GC: (-)-gallocatechin, EGC:(-)-epicatechin.

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Zhu, Y., Wang, H., Peng, Q. et al. Functional characterization of an anthocyanidin reductase gene from the fibers of upland cotton (Gossypium hirsutum). Planta 241, 1075–1089 (2015). https://doi.org/10.1007/s00425-014-2238-4

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