At some point between the 7th and 4th millennia BC in the Caucasus region (spanning modern day Iran, Turkey and Georgia), our ancestors unknowingly began the process of grapevine selection that would lead to the eventual domestication of this crop species (Terral et al., 2010). During the domestication process; selection was based on observable phenotypes within the naturally occurring (“wild”) populations, and potentially included: size/yield, survival/resistance to stresses (biotic and/or abiotic), flavour/aroma (taste), colour, and any combination of a number of quality-associated attributes. The >6 000 years of selection have culminated in the diversity of germplasm we currently have for grapevines, estimated at between 5 000 and 8 000 accessions (Moreno-Sanz et al., 2008). If only grape berry colour is considered as a trait; the degree of diversity present within cultivated grapevines is visually evident with a spectrum of shades ranging from white and greys, through pinks and reds to black. In the current genomic era, these germplasm collections, and specifically the genetic basis (mutations) of these varietal differences, provides valuable tools for plant breeders to improve grapevines for a variety of prospective targets.

Terpenes are the collective term for a large group of chemical compounds that all share a common C5-precursor, isopentenyl diphosphate, yet perform diverse biological functions that range from light-harvesting for photosynthesis to herbivory defence. Some terpenes are essential for plants (e.g. the carotenoids in light-harvesting and photoprotection, and the phytohormones in growth and development); whereas others are referred to as secondary metabolites and are involved in “specialised metabolism” and are not essential for the plant, but typically provide some or other survival and/or fitness benefit.

Due to their characteristic properties, volatile terpenes have a long association with humans as flavours, fragrances, dyes, cosmetics, insecticides and numerous therapeutic applications. The presence of volatile C10-monoterpenes, C13-norisoprenoids and C15-sesquiterpenes are responsible for the characteristic flavours, aromas and scents associated with a number of plant species. These compounds are dominant in many plants, e.g. lemons (limonene), eucalyptus (eucalyptol), pine (pinene), mint (menthol), geranium (geraniol) or roses (ß-damascenone). Specifically in grapes and wine, these volatile monoterpenes contribute predominantly to the floral aromas (e.g. geranium, rose, lily, lavender and citrus blossom). Terpenes can be present in the free (volatile) form or stored as non-volatile, water-soluble glycosidically (sugar) bound conjugates. Monoterpenes are prevalent in the varietal character of the Muscats (e.g. Muscat d’Alexandrie and Morio Muscat), but can also contribute to that of Weisser Riesling, Bukettraube and Gewürztraminer. Sesquiterpenes are less common with rotundone being a well-known example with a very low odour threshold (16 ng/ℓ in wine), and is responsible for the peppery aroma found in, e.g. Shiraz grapes and wine (and pepper). Wine is, however, a complex blend of thousands of metabolites that can originate from the grape (e.g. sugars, organic acids and phenols) or as products of the winemaking process (via the fermentation and/or bioconversions during fermentation and/or ageing). The ultimate wine flavour and aroma are therefore determined by the complex assortment of literally hundreds of metabolites with both the absolute concentration and/or ratios of specific metabolites playing a role in the ultimate perceived flavour and aroma.

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FIGURE 1. Discovery and analytical pipeline for the study of terpene synthases and the volatile terpenes in grapevine. FIGURE 2. Presence and potential biological activity of terpenes from grape flowers. (A) Vanillin-stained terpenes in grapevine flowers (stained coloured bands indicative of terpenic compounds); (B) DPPH assay for antioxidant activity (pale yellow zones indicative of anti-oxidant activity). (1) Authentic standard of the sesquiterpenoid valencene; (2) Terpene extracts from Shiraz; and (3) Sauvignon Blanc flowers.

 

The most abundant terpenes in Muscat and other aromatic wines are linalool, geraniol, nerol, a-terpineol and hotrienol (Marais, 1983). The aroma thresholds of these compounds are low (ng/ℓ to μg/ℓ range), and their abundance can vary with several orders of magnitude. From a wine quality perspective the volatile terpenes are important contributors to the overall bouquet of wines, but the actual biological function of these compounds in grapevines is poorly understood. Numerous studies in other plant species have demonstrated that volatile organic compounds (e.g. terpenes and green leaf volatiles (i.e. C6-hexanols) play an important role in plant defence. Volatiles can perform various functions in defence, e.g. they can directly act as repellents to insects and other herbivores, or indirectly as attractants to predators of herbivores, or act as volatile signals where they prime defence responses against pathogens in adjacent leaves and neighbouring plants.

The completion of a grapevine (cv. Pinot noir) genome sequence provided researchers with a molecular snapshot of the full genetic potential of grapevine. The grapevine genome represented the fourth plant-, and the first woody fruit-bearing plant genome sequenced, highlighting the importance of the grapevine to many of the world’s economies. Numerous interesting findings have been reported in the literature that fall outside of the scope of this article, but with specific reference to terpenes it was found that grapevines possess one of the largest terpene synthase (TPS) families of all the sequenced plant genomes (second only to that of Eucalyptus): ~89 genomic loci were identified that encoded for putative TPSs (where a TPS is the enzyme that catalyses the conversion of a prenyl phosphate precursor (i.e. geranyl diphosphate and farnesyl diphosphate) to a mono- or sesquiterpene volatile product, respectively. Functional characterisation of a number of the identified TPSs and their volatile products highlighted the complexity: a single TPS enzyme could potentially form multiple volatile terpenes, and the volatile terpenes produced are subsequently susceptible to further rearrangements (both enzymatic and non-enzymatic) to produce numerous potential terpenic products (from a single precursor).

With its large over-represented TPS family; it is surprising that relatively little is still known on the role of terpenes in grapevines. A number of grapevine studies have, however, contributed to our current understanding. A study by Martin et al. (2009) showed that sesquiterpene synthesis was predominantly localised to the anthers and pollen in Cabernet Sauvignon flowers and emission peaked before bloom. This production of volatiles in the male flower parts, and the timing (before bloom) is biologically unique for floral scent production: most plant species form volatile terpenes during bloom and then typically not in the male flower parts. Volatiles can act as attractants for pollinators and complex mixtures have co-evolved to attract specific agents. The domesticated grapevine, unlike its cross-pollinating wild relatives, is self-pollinating and therefore does not require pollinators for fertilisation. Tasin et al. (2006) showed that the female grapevine moth (Lobesia botrana) was attracted by a ratio-specific blend of three terpenoids: β-caryophyllene, β-farnesene and dimethyl-nonatriene (DMNT) for oviposition (egg-laying). The authors speculated that the ratio was required for discrimination between host and non-host plants, which is essential for maintenance of associations between insects and suitable plant hosts. It has also been demonstrated that methyl jasmonate elicits the production of monoterpenes and sesquiterpenes in grapevine leaves (Hampel et al., 2005) and in grape cell cultures (D’Onofrio et al., 2009). Jasmonates are well-studied elicitors of the defence response in plants. A study by Lawo et al. (2011) on the volatiles emitted by rootstock Teleki 5C (a Hungarian interspecific cross of Vitis vinifera x V. riparia) roots following phylloxera (Daktulospaira vitifaliae Fitch) infection; showed that the infested roots released significantly more volatiles than the uninfested roots. Interestingly, 25% of the volatile metabolites significantly upregulated in the infested roots were terpenes. Interestingly, ß-caryophyllene (a sesquiterpene) was exclusively formed in the infested roots and was not detected in the control roots. Teleki 5C has moderate resistance to nematode feeding, and good resistance to phylloxera. From the preceding it is clear that terpenes occur in complex, structurally diverse mixtures and fulfil various ecological roles.

With ~89 potential grapevine TPSs, it becomes particularly challenging to correlate a specific TPS-encoding gene (or genomic locus) to the production of a specific volatile terpene.

Our research focusses on a number of fundamental biological questions:

  • Where and when are TPSs formed
  • Which TPS(s) produce which volatile terpene(s)
  • How do grapevine cultivars differ in their TPSs and volatile terpene profiles; and
  • Why does a grapevine produce volatile terpenes (biological activity/significance)

These questions are addressed by isolating and characterising candidate TPSs in an optimised heterologous yeast system. Yeast do not naturally produce any endogenous terpenes and, therefore, provide a suitable null background for these studies. Heterologous expression of a TPS in yeast results in the de novo production of volatile terpenes that can be analysed by gas chromatography (GC) mass spectra (MS). The volatile terpene(s) produced are used to assign a function(s) to the isolated TPS and, by extension, the respective genomic locus. This locus is then a potential functional marker for future breeding/selection. The volatile terpene(s) produced by the respective TPS are further analysed to elucidate their possible activity (e.g. against reactive oxygen species (ROS), pathogen) in order to determine the in planta function of the volatile terpenes.

Using the described pipeline (Figure 1), three TPS-encoding genes (VvTPS3, VvTPS12 and VvTPS15) have been isolated from grapevine cDNA, and successfully expressed in yeast. The recombinant yeasts were shown to produce predominantly sabinene (TPS3), Germacrene D (TPS12) and eucalyptol (TPS15), respectively. Sabinene is a monoterpene (C10H16) and is one of the compounds that contribute to the spiciness of, e.g. black pepper; Germacrene D is a sesquiterpene (C15H24) with antimicrobial and anti-insecticidal activity, and eucalyptol (1,8-cineole) is a monoterpene (C10H18O) and its scent is described as fresh/camphor-like. These volatile terpenes formed are currently being tested for their biological activity (Figure 2). Furthermore, additional TPSs are being identified and isolated from a number of commercially relevant wine grape cultivars for functional analysis. Sequence data and the functional characterisation provide potential varietal-specific differences that contribute to the typicity of grapevine cultivars. Linking TPS sequences and expression of specific TPS-encoding genes to the production of volatile terpenes in different grapevine organs (spatial and/or temporal) or in response to viticultural manipulations is required to understand, and ultimately exploit, these versatile biological compounds.

Summary

The terpenic composition of grape berries contributes to the varietal character of the grape and subsequent wine. The biosynthesis of terpenes is catalysed by terpene synthase enzymes. The recent grapevine genome sequence revealed a large (>89) terpene synthases family, making the study of this family particularly challenging. Understanding the basis of the varietal aromas of cultivars is still an active field of study, as is the impact of production practices in modulating the aromatic potential of the berries. Although the functioning of the enzymes are understood mechanistically, the relationship between the enzymes (presence and activity) and the origin (and fate) of terpenes in the complex grape matrix is still unknown. The availability of the grapevine genome sequence and the molecular tools developed in grapevine studies (i.e. genomics, transcriptomics and metabolomics) make it possible to identify, isolate and study individual terpene synthase-encoding genes. A specific terpene synthase encoding gene can then be linked to the production of a specific terpenic product and the derived functional information, together with gene expression data, can be used to evaluate the effect of, e.g. a stress and/or viticultural treatment to the metabolism of terpenes in grapes to understand, and ultimately exploit, these versatile biological compounds.

– For more information, contact Philip Young at pryoung@sun.ac.za.

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