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An integrated approach to demonstrating the ANR pathway of proanthocyanidin biosynthesis in plants

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Abstract

Proanthocyanidins (PAs) are oligomers or polymers of plant flavan-3-ols and are important to plant adaptation in extreme environmental conditions. The characterization of anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) has demonstrated the different biogenesis of four stereo-configurations of flavan-3-ols. It is important to understand whether ANR and the ANR pathway widely occur in the plant kingdom. Here, we report an integrated approach to demonstrate the ANR pathway in plants. This includes different methods to extract native ANR from different tissues of eight angiosperm plants (Lotus corniculatus, Desmodium uncinatum, Medicago sativa, Hordeum vulgare, Vitis vinifera, Vitis bellula, Parthenocissus heterophylla, and Cerasus serrulata) and one fern plant (Dryopteris pycnopteroides), a general enzymatic analysis approach to demonstrate the ANR activity, high-performance liquid chromatography-based fingerprinting to demonstrate (−)-epicatechin and other flavan-3-ol molecules, and phytochemical analysis of PAs. Results demonstrate that in addition to leaves of M. sativa, tissues of other eight plants contain an active ANR pathway. Particularly, the leaves, flowers and pods of D. uncinatum, which is a model plant to study LAR and the LAR pathways, are demonstrated to express an active ANR pathway. This finding suggests that the ANR pathway involves PA biosynthesis in D. uncinatum. In addition, a sequence BLAST analysis reveals that ANR homologs have been sequenced in plants from both gymnosperms and angiosperms. These data show that the ANR pathway to PA biosynthesis occurs in both seed and seedless vascular plants.

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Abbreviations

ANR:

Anthocyanidin reductase

HPLC:

High-performance liquid chromatography

TLC:

Thin layer chromatography

PA:

Proanthocyanidins

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Acknowledgments

This work is dedicated to Dr. Richard A. Dixon for his 60th birthday celebration. This work was firstly initiated at Noble Foundation and then continued by supports from Bai-Ren-Ji-Hua’s startup of Jishou University, Hunan, P.R. China, and North Carolina State University startup.

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Authors and Affiliations

  1. 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 City, 416000, Hunan Province, People’s Republic of China

    Qing-Zhong Peng, Yue Zhu, Ci Du, Ke-Gang Li & De-Yu Xie

  2. Department of Plant Biology, North Carolina State University, Raleigh, NC, 27695, USA

    Zhong Liu & De-Yu Xie

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  1. Qing-Zhong Peng
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  2. Yue Zhu
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  3. Zhong Liu
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Correspondence to De-Yu Xie.

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S-Fig. 1 DMACA staining of D. uncinatum flowers. a Two examples of flowers treated with buffer without DMACA; b two examples of flowers showing deep blue color after treated 2 min with 0.1 % DMACA (g/ml) in buffer. The blue coloration results from reaction of DMACA and proanthocyanidins.

S-Fig. 2 HPLC analysis of catechin and epicatechin from leaves of Dryopteris pycnopteroides; a an extract of D. pycnopteroides leaves; b authentic standards; abbreviation, CA: catechin, EP: epicatechin).

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Peng, QZ., Zhu, Y., Liu, Z. et al. An integrated approach to demonstrating the ANR pathway of proanthocyanidin biosynthesis in plants. Planta 236, 901–918 (2012). https://doi.org/10.1007/s00425-012-1670-6

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