Elsevier

Phytochemistry

Volume 65, Issue 19, October 2004, Pages 2649-2659
Phytochemistry

Vitis vinifera terpenoid cyclases: functional identification of two sesquiterpene synthase cDNAs encoding (+)-valencene synthase and (−)-germacrene D synthase and expression of mono- and sesquiterpene synthases in grapevine flowers and berries

https://doi.org/10.1016/j.phytochem.2004.08.017Get rights and content

Abstract

Valencene is a volatile sesquiterpene emitted from flowers of grapevine, Vitis vinifera L. A full-length cDNA from the cultivar Gewürztraminer was functionally expressed in Escherichia coli and found to encode valencene synthase (VvVal). The two major products formed by recombinant VvVal enzyme activity with farnesyl diphosphate (FPP) as substrate are (+)-valencene and (−)-7-epi-α-selinene. Grapevine valencene synthase is closely related to a second sesquiterpene synthase from this species, (−)-germacrene D synthase (VvGerD). VvVal and VvGerD cDNA probes revealed strong signals in Northern hybridizations with RNA isolated from grapevine flower buds. Transcript levels were lower in open pre-anthesis flowers, flowers after anthesis, or at early onset of fruit development. Similar results were obtained using a third probe, (−)-α-terpineol synthase, a monoterpenol synthase. Sesquiterpene synthase and monoterpene synthase transcripts were not detected in the mesocarp and exocarp during early stages of fruit development, but transcripts hybridizing with VvVal appeared during late ripening of the berries. Sesquiterpene synthase transcripts were also detected in young seeds.

Graphical abstract

Terpenoids are important for a variety of quality traits in the grapevine, Vitis vinifera.

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Introduction

Low-molecular-weight terpenoids, including a large array of monoterpenes, sequiterpenes, and norisoprenoids, are commonly found as volatiles emitted from flowers, fruits and leaves of plants. These compounds are widely recognized by humans as important fragrance, flavour and aroma compounds (Schrader and Berger, 2001) and can present desirable quality traits for plant breeding in agriculture, horticulture, and viticulture. The sesquiterpene valencene (1) (Fig. 1) is a major volatile emitted from flowers of white and red varieties of grapevine, Vitis vinifera L. (Buchbauer et al., 1994a, Buchbauer et al., 1994b, Buchbauer et al., 1995). Valencene (1) is also a characteristic fruit flavour and aroma component in some citrus species, and is the likely precursor in the formation of nootkatone (3), a valuable compound associated with grapefruit (Citrus x paradisi) aroma (Sharon-Asa et al., 2003). Other terpenoid volatiles, mainly monoterpene alcohols such as linalool, geraniol, nerol and terpineol, have often been described as flavour and aroma compounds of grapevine berries and wine (e.g. Marais, 1983, Strauss et al., 1986, Wilson et al., 1986, Park and Noble, 1993, Mateo and Jimenez, 2000). Despite the prominence of terpenoid volatiles in grapevine flowers and berries, little is known about the biochemical and molecular regulation of terpenoids in this economically important plant. Recently, Luan and Wüst (2002) showed that the exocarp and mesocarp of grapevine berries contribute to monoterpene formation via the 2C-methyl-d-erythritol 4-phosphate pathway. In earlier work, geranyl diphosphate synthase, a prenyl transferase for the formation of the immediate precursor of monoterpenes, was characterized from grapevine cell cultures (Clastre et al., 1993). Recently, we have initiated a project that is aiming at the discovery and functional characterization of genes of terpenoid formation in grapevine and described the first monoterpene synthase in V. vinifera (Martin and Bohlmann, 2004). Enzymes of the large family of plant terpenoid synthases (TPS) are critical for the generation of structural diversity of monoterpenoids, sesquiterpenoids, and diterpenoids (Bohlmann et al., 1998, Davis and Croteau, 2000). To our knowledge, an enzyme or gene that is specific to the formation of a sesquiterpene volatile in grapevine has not been reported to date. In this paper, we describe a pair of grapevine full-length cDNAs encoding two different sesquiterpene synthases, the multi-product (+)-valencene synthase (VvVal) and a (−)-germacrene D synthase (VvGerD), and expression of TPS transcripts in vegetative tissues and in flower buds, open flowers, and fruits of wine grape cultivars.

Section snippets

Identification of grapevine full-length sesquiterpene synthase cDNAs

Plant genome and EST databases are a valuable resource for the mining of genes of secondary metabolism including genes of terpenoid metabolism (e.g. Lange et al., 2000, Gang et al., 2001, Aubourg et al., 2002, Dudareva et al., 2003). Full-length cDNA sequence databases and full-length cDNA clone collections are a particularly powerful resource for functional genomics (Seki et al., 2002), because they allow for the identification of complete open reading frames (ORFs) for improved gene

Discussion

In this paper we provide a first characterization of sesquiterpene synthase cDNAs in grapevine, V. vinifera, which is economically one of most significant fruit plants worldwide. Terpenoid volatiles are important fruit aroma and flavour components in this species and contribute much to the quality of table grapes and wine (e.g. Marais, 1983, Strauss et al., 1986, Wilson et al., 1986, Park and Noble, 1993, Mateo and Jimenez, 2000). In addition to the many reports on terpenoid profiles of wines

Plant material

Tissues of V. vinifera cv. Chardonnay were harvested from plants grown outside under natural environmental conditions at the horticulture plant growth facilities of the University of British Columbia, Vancouver, BC. Fruits of V. vinifera cv. Gewürztraminer were sampled at vineyards of the Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, in Summerland, Okanagan, BC, Canada. Gewürztraminer fruit samples were harvested in intervals of one week from 23 July 2002 until 8 October

Acknowledgements

This research was supported by grants and infrastructure funds to J.B. from the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Foundation for Innovation (CFI), and the BC Knowledge and Development Funds (BCKDF). We thank Dr. David McArthur, University of British Columbia, Vancouver, for access to Chardonnay plants; Dr. Åsa Lommlé, VitiGen AG Siebeldingen, Germany, for screening of the grapevine full-length cDNA sequence database; Dr. Wilfried König,

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