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Establishment of pomegranate (Punica granatum) hairy root cultures for genetic interrogation of the hydrolyzable tannin biosynthetic pathway

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Abstract

In contrast to the numerous reports on the human therapeutic applications of hydrolyzable tannins (HTs), genes involved in their biosynthesis have not been identified at the molecular level from any plant species. Although we have previously identified candidate HT biosynthetic genes in pomegranate using transcriptomic and bioinformatic analyses, characterization of in planta enzyme function remains a critical step in biochemical pathway elucidation. We here report the establishment of a pomegranate (Punica granatum) hairy root culture system that produces HTs. Agrobacterium rhizogenes strains transformed with a binary vector harboring a yellow fluorescent protein (YFP) gene were used for hairy root induction, allowing visual, non-destructive, detection of transgene incorporation. It also demonstrated that the pomegranate hairy root culture system is suitable for expressing heterologous genes (YFP in this case). Expression of 26 putative UDP-glycosyltransferase (UGT) genes, obtained from a pomegranate fruit peel (a tissue highly abundant in HTs) RNA-Seq library, were verified in wild type and hairy roots. In addition, two candidate UGTs for HT biosynthesis were identified based on HPLC and differential gene expression analyses of various pomegranate tissues. Together with in vitro enzyme activity assays, the hairy root culture system holds great promise for revealing the undivulged HT biosynthetic pathway using pomegranate as a model system.

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Abbreviations

CTAB:

Cetyltrimethylammonium bromide

ESI:

Electrospray ionization

GFP:

Green fluorescent protein

HT:

Hydrolyzable tannin

MS:

Murashige and Skoog

Ri:

Root inducing

RT:

Reverse transcriptase

UGT:

UDP-glycosyltransferase

YFP:

Yellow fluorescent protein

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Acknowledgments

It is an honor to dedicate this paper to Dr. Richard A. Dixon of the Samuel Roberts Noble Foundation on the occasion of his 60th birthday. We would like to thank Dr. John Yoder for providing us with the A. rhizogenes strains and the pHG8-YFP plasmid, David Tricoli for allowing access to the fluorescent stereomicroscope, Mark Silveria and Dr. Oliver Fiehn for LC–MS analysis. This work was supported by the National Science Foundation (MCB1120323) and the UC Davis new faculty startup fund to LT. NNO is supported by NIH Initiative for Maximizing Student Diversity/Development, the NSF Graduate Research Fellowship, the UC Davis Department of Plant Sciences Graduate Research Fellowship and the Henry A. Jastro Research Fellowship.

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Authors and Affiliations

  1. Department of Plant Sciences, Mail Stop 3, University of California, Davis, Davis, CA, USA

    Nadia N. Ono, Pradeepa C. G. Bandaranayake & Li Tian

  2. Department of Crop Science, Faculty of Agriculture, University of Peradeniya, Peradeniya, 20400, Sri Lanka

    Pradeepa C. G. Bandaranayake

Authors
  1. Nadia N. Ono
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  2. Pradeepa C. G. Bandaranayake
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  3. Li Tian
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Corresponding author

Correspondence to Li Tian.

Additional information

N. N. Ono and P. C. G. Bandaranayake contributed equally to this work.

A contribution to the Special Issue on Metabolic Plant Biology.

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425_2012_1706_MOESM1_ESM.docx

Fig. S1 Optimization of pomegranate seed germination. Pomegranate seeds were either abraded with sandpaper (sanded) or approximately 1 mm was cut from the true apex of the seed using a sharp scalpel (cut). The scarified and stratified seeds were sowed on plates, which were incubated at 4 °C for 2–9 days and then transferred to 25oC with a 16-h light period. a 12 days, b 16 days, and c 24 days after sowing (i.e. total incubation time at 4 and 25 °C). Each experiment was repeated three times (20 seeds in each experiment). Results are represented as mean ± SD (n=60) (DOCX 119 kb)

425_2012_1706_MOESM2_ESM.docx

Fig. S2 Root biomass produced from cotyledon, leaf and radicle explants. The transgenic hairy roots were transferred and wounded for four rounds (approximately 3 months), collected and fresh weight measured. Results are represented as mean ± SD (n=9) (DOCX 44 kb)

Table S1 Primer sequences used for amplification of putative UGTs and the actin control. (DOCX 35 kb)

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Ono, N.N., Bandaranayake, P.C.G. & Tian, L. Establishment of pomegranate (Punica granatum) hairy root cultures for genetic interrogation of the hydrolyzable tannin biosynthetic pathway. Planta 236, 931–941 (2012). https://doi.org/10.1007/s00425-012-1706-y

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