Utilisation of the MVA pathway to produce elevated levels of the sesquiterpene α-copaene in potato tubers
Graphical abstract
Tuber-specific over-expression of a native sesquiterpene synthase results in elevated levels of the aroma volatile α-copaene but does not affect potato tuber flavour.
Highlights
► We describe the first report of metabolic engineering of sesquiterpenes in potato. ► Transgene expression level and α-copaene abundance positively correlate. ► Flavour metabolite correlations with taste panel assessments are reported. ► Sensory analysis suggests that α-copaene is not a major component of potato flavour.
Introduction
Potato tuber flavour is a major consideration in consumer preference but is a difficult trait to assess quantitatively (McGregor, 2007). Increasing our understanding of the molecular basis of this important quality trait will help us to make improvements in potato germplasm. Potato flavour perception is complex and results from the interaction of both volatile and non-volatile compounds (Maga, 1994, Taylor et al., 2007). This is further complicated because storage conditions and cooking treatments and have a major impact on the levels and type of flavour compounds present (Duckham et al., 2001, Duckham et al., 2002, Oruna-Concha et al., 2001, Oruna-Concha et al., 2002). Previous work has shown that the non-volatile compounds including the major umami amino acids, glutamate and aspartate, and the 5-ribonucleotides, GMP and AMP are key determinants of potato flavour (Morris et al., 2007) whereas chemicals representing the so-called sweet, sour, salty and bitter tastes do not provide a cooked potato taste (Solms, 1971, Solms and Wyler, 1979). Other non-volatile compounds e.g., salts, sugars or glycoalkaloids, do not enhance potato flavour although their presence at high levels may decrease palatability (Sinden et al., 1976, Ross et al., 1978).
Volatile compounds produced by raw and cooked potatoes have been studied extensively (Maga, 1994, Taylor et al., 2007). Recently, boiled tuber volatiles produced by Solanum tuberosum group Phureja cultivars were compared with profiles from S. tuberosum group Tuberosum (Winfield et al., 2005, Shepherd et al., 2007a, Shepherd et al., 2007b). Phureja tubers score consistently higher than Tuberosum when assessed by sensory evaluation (Winfield et al., 2005, Shepherd et al., 2007a, Shepherd et al., 2007b). Interestingly, compared with Tuberosum, Phureja tubers produce significantly higher levels (up to 100-fold) of the sesquiterpene (C15) volatile α-copaene (Ducreux et al., 2008), an important aroma compound in several food plants including sweet potato (Cui et al., 2010), lettuce and carrots (Nielsen and Poll, 2007). Microarray analysis of gene expression facilitated the cloning of a potato sesquiterpene synthase gene that encodes an enzyme that catalyses the conversion of farnesyl diphosphate to α-copaene (Ducreux et al., 2008). With the identification of the potato α-copaene synthase gene, it is now possible to manipulate the tuber volatile profile through transgenic modification, providing a means of testing the importance of this volatile metabolite to sensory evaluation. Indeed, previous reports have shown that it is possible to increase the levels of important flavour volatiles in tomato fruit using a transgenic approach by manipulating terpene biosynthesis (Davidovich-Rikanati et al., 2007).
To date, a number of sesquiterpene synthases have been cloned and characterized in plants (Nagegowda, 2010, Nagegowda and Dudareva, 2007). Several examples of metabolic engineering of sesquiterpenes have been reported in plants including Arabidopsis (Aharoni et al., 2003, Kappers et al., 2005), tobacco (Hohn and Ohlrogge, 1991, Wallaart et al., 2001) and tomato (Davidovich-Rikanati et al., 2008). Although engineering of these compounds is achievable there are inherent requirements and limitations to consider (Aharoni et al., 2006) e.g., the limited availability of the cytosolic sesquiterpene precursor farnesyl diphosphate. The aim of this study was to investigate whether potato tubers could be engineered to produce elevated sesquiterpene levels by tuber-specific over-expression of a native α-copaene synthase gene. If so, how does increasing the level of α-copaene in potato influence potato flavour? To this end, flavour metabolites were measured in cooked tuber samples from control and transgenic lines and correlations with taste panel assessments are reported.
Section snippets
Generation and screening of transgenic potato lines with altered α-copaene levels
Primary potato transgenic lines were generated over-expressing the potato α-copaene synthase gene. Expression was driven by the tuber-specific patatin promoter in order to avoid potential effects on development in non-tuber tissues. Developing tubers from 29 independent over-expressing transgenic lines were screened for α-copaene synthase gene expression. Successful, stable over-expression of α-copaene synthase was achieved in the tubers of transgenic lines with increases ranging from 66 to
Concluding remarks
We have shown that it is possible to increase the level of a specific sesquiterpene in transgenic potato tubers by introducing a native α-copaene synthase gene in the sense orientation under the control of a tuber-specific promoter. Levels obtained were in the same order of magnitude as those present in the S. tuberosum group Phureja cultivars previously analysed. To our knowledge, this is the first example of metabolic engineering of sesquiterpene compounds in potato tubers. Although this
Plant material and sampling
S. tuberosum L. (cv. Desiree) plants were grown from seed tubers or in vitro propagated tissue culture plants in 30 cm diameter pots containing standard compost mix. Plants were raised in a glasshouse maintained at a daytime temperature of 20 °C and a nocturnal temperature of 15 °C. Light intensity (photosynthetic photon flux density) ranged from 400 to 1000 μmol m−2 s−1 and the mean day length was 16 h. Uniform sized tubers were selected and formed the basis for all the analysis described in this
Acknowledgements
We are grateful to Dr Sean Conner for carrying out the glycoalkaloid analysis.
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