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Identification of a novel hedycaryol synthase gene isolated from Camellia brevistyla flowers and floral scent of Camellia cultivars

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Abstract

Main conclusion

A novel terpene synthase ( Tps ) gene isolated from Camellia brevistyla was identified as hedycaryol synthase, which was shown to be expressed specifically in flowers.

Camellia plants are very popular because they bloom in winter when other plants seldom flower. Many ornamental cultivars of Camellia have been bred mainly in Japan, although the fragrance of their flowers has not been studied extensively. We analyzed floral scents of several Camellia cultivars by gas chromatography–mass spectrometry (GC–MS) and found that Camellia brevistyla produced various sesquiterpenes in addition to monoterpenes, whereas Camellia japonica and its cross-lines produced only monoterpenes, including linalool as the main product. From a flower of C. brevistyla, we isolated one cDNA encoding a terpene synthase (TPS) comprised of 554 amino acids, which was phylogenetically positioned to a sole gene clade. The cDNA, designated CbTps1, was expressed in mevalonate-pathway-engineered Escherichia coli, which carried the Streptomyces mevalonate-pathway gene cluster in addition to the acetoacetate-CoA ligase gene. A terpene product was purified from recombinant E. coli cultured with lithium acetoacetate, and analyzed by 1H-nulcear magnetic resonance spectroscopy (1H-NMR) and GC–MS. It was shown that a sesquiterpene hedycaryol was produced, because 1H-NMR signals of the purified product were very broad, and elemol, a thermal rearrangement product from hedycaryol, was identified by GC–MS analysis. Spectroscopic data of elemol were also determined. These results indicated that the CbTps1 gene encodes hedycaryol synthase. Expression analysis of CbTps1 showed that it was expressed specifically in flowers, and hedycaryol is likely to be one of the terpenes that attract insects for pollination of C. brevistyla. A linalool synthase gene, which was isolated from a flower of Camellia saluenensis, is also described.

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Abbreviations

Cm:

Chloramphenicol

DMAPP:

Dimethylallyl diphosphate

FPP:

Farnesyl diphosphate

GC:

Gas chromatography

GC-MS:

Gas chromatography–mass spectrometry

GPP:

Geranyl diphosphate

HPLC:

High-performance liquid chromatography

IPP:

Isopentenyl diphosphate

IPTG:

Isopropyl β-d-thiogalactopyranoside

Km:

Kanamycin

LAA:

Lithium acetoacetate

MEP:

Methylerythritol phosphate

MVA:

Mevalonate

NMR:

Nuclear magnetic resonance spectroscopy

ORF:

Open-reading frame

RACE:

Rapid amplification of cDNA ends

RT:

Reverse transcription

RT-PCR:

Reverse transcription-polymerase chain reaction

SPME:

Solid phase micro-extraction

TPS:

Terpene synthase

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Acknowledgments

We thank Mr. Seizou Matsui, Kanazawa branch of the Japan Camellia Association, for kindly providing the C. brevistyla and other Camellia samples and important information on Camellia plants. We also thank to Dr. Eiji Tanaka, Ishikawa Prefectural University, for advice on manuscript preparation. We are grateful to Takasago International Corporation (Tokyo, Japan) for kindly providing the authentic preparation of elemol. This work was supported by the commission for Development of Artificial Gene Synthesis Technology for Creating Innovative Biomaterial from the Ministry of Economy, Trade and Industry (METI), Japan.

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Authors and Affiliations

  1. Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi, Ishikawa, 921-8836, Japan

    Jun-ichiro Hattan, Chie Tagaki, Fumina Ohno & Norihiko Misawa

  2. Department of Food and Nutrition, Japan Women’s University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo, 112-8681, Japan

    Kazutoshi Shindo, Tomoko Ito, Yurica Shibuya & Arisa Watanabe

  3. Industrial Research Institute of Ishikawa, 2-1 Kuratsuki, Kanazawa, Ishikawa, 920-8203, Japan

    Tetsuya Sasaki

  4. Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan

    Jun Ishii

  5. Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan

    Akihiko Kondo

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  1. Jun-ichiro Hattan
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  2. Kazutoshi Shindo
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  3. Tomoko Ito
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Correspondence to Norihiko Misawa.

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Hattan, Ji., Shindo, K., Ito, T. et al. Identification of a novel hedycaryol synthase gene isolated from Camellia brevistyla flowers and floral scent of Camellia cultivars. Planta 243, 959–972 (2016). https://doi.org/10.1007/s00425-015-2454-6

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