Furanones and pyrones. Furanones and pyrones (Figure 4) are important fruit constituents or have been isolated from the bark and leaves of several tree species (Schwab and Roscher, 1997). Although hexoses and pentoses are the primary photosynthetic products and serve as excellent flavor precursors in the Maillard reaction, only a limited number of natural volatiles originate directly from carbohydrates without prior degradation of the carbon skeleton. Such compounds include the furanones and pyrones (Bood and Zabetakis, 2002).
Figure 4. Carbohydrate-derived flavor molecules, including 4-hydroxy-2,5-dimethyl-3(2H)-furanone (furaneol), 2,5-dimethyl-4-methoxy-3(2H)-furanone (methoxyfuraneol), 4-hydroxy-5-methyl-3(2H)-furanone (norfuraneol), 2-ethyl-4-hydroxy-5-methyl-3(2H)-furanone (homofuraneol), 4-hydroxy-2-methylene-5-methyl-3(2H)-furanone (HMMF) and 3-hydroxy-2-methyl-4H-pyran-4-on (maltol).
Download figure to PowerPoint
Substituted 4-hydroxy-3(2H)-furanones and the pyrone maltol constitute an uncommon group of flavor molecules with exceptional low odor thresholds. Furanones have been detected in a few plant species in which they are emitted only by the fruits. Maltol has been isolated from the bark and leaves of Larix deciduas, Evodiopanax innovans, Cercidiphyllum japonicum and four kinds of Pinaceae plants (Tiefel and Berger, 1993). Incorporation experiments using labeled precursors revealed that d-fructose-1,6-diphosphate is an efficient biogenetic precursor of furaneol. In strawberry (Fragaria × ananassa) and tomato (Solanum lycopersicum), the hexose diphosphate is converted by an as yet unknown enzyme to 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone, which serves as the substrate for an enone oxidoreductase recently isolated from ripe fruit (Klein et al., 2007; Raab et al., 2006). A highly similar sequence was identified in an EST collection for pineapple (Ananas comosus), another species which produces furaneol in its fruits. In strawberry, furaneol is further metabolized by an O-methyltransferase (FaOMT) to methoxyfuraneol (Wein et al., 2002). An ortho-diphenolic structure was identified as a common structural feature of the accepted substrates, and is also present in the dienolic tautomer of furaneol. Genetic transformation of strawberry with the FaOMT sequence in the antisense orientation, under the control of a constitutive promoter, resulted in a near total loss of methoxyfuraneol, demonstrating the in vivo methylation of furaneol by FaOMT (Lunkenbein et al., 2006). However, the reduced level of methoxyfuraneol was only perceived by one third of the volunteer panelists, consistent with results obtained by aroma extract dilution assays. Norfuraneol and homofuraneol have been identified in tomato and melon fruits, respectively, but their biogenetic pathways and that of maltol remain unknown (Schwab and Roscher, 1997). However, studies in tomato and yeast have identified phosphorylated carbohydrates as potential precursors of the furanones (Hauck et al., 2003; Sasaki et al., 1991).
The furanones are mutagenic to bacteria and cause DNA damage in laboratory tests. However, they are also very effective anti-carcinogenic agents in the diets of animals, and their antioxidant activity is comparable to that of ascorbic acid (Slaughter, 1999). Norfuraneol has been identified as a male pheromone in the cockroach Eurycolis florionda (Walker), and furaneol deters fungal growth. Furaneol is also one of the key flavor compounds in the attractive aroma of fruits (Farine et al., 1994). It has been proposed that the evolved biological function of the furanones is to act as inter-organism signal molecules in various systems. The 4-hydroxy-3(2H)-furanones associated with fruit aromas act to attract animals to the fruit, which ensures seed dispersal. In the case of humans, the coincidental chemical synthesis of these compounds in foods during preparation results in these foods appearing particularly attractive through transferred operation of the original signaling mechanisms (Slaughter, 1999).
Terpenoid pathway. Terpenoids are enzymatically synthesized de novo from acetyl CoA and pyruvate provided by the carbohydrate pools in plastids and the cytoplasm. Although fatty acid oxidation is one of the major pathways producing acetyl CoA, this process probably does not contribute to the formation of terpenoids as it takes place in peroxisomes. Terpenoids constitute one of the most diverse families of natural products, with over 40 000 different structures of terpenoids discovered so far. Many of the terpenoids produced are non-volatile and are involved in important plant processes such as membrane structure (sterols), photosynthesis (chlorophyll side chains, carotenoids), redox chemistry (quinones) and growth regulation (gibberellins, abscisic acid, brassinosteroids) (Croteau et al., 2000). The volatile terpenoids – hemiterpenoids (C5), monoterpenoids (C10), sesquiterpenoids (C15) and some diterpenoids (C20) – are involved in interactions between plants and insect herbivores or pollinators and are also implicated in general defense or stress responses (Dudareva et al., 2004; Pichersky and Gershenzon, 2002; Pichersky et al., 2006). Terpenoids, mainly the C10 and C15 members of this family, were found to affect the flavor profiles of most fruits and the scent of flowers at varying levels (Figure 5). Citrus fruit aroma consists mostly of mono- and sesquiterpenes, which accumulate in specialized oil glands in the flavedo (external part of the peel) and oil bodies in the juice sacs. The monoterpene R-limonene normally accounts for over 90% of the essential oils of the citrus fruit (Weiss, 1997). The sesquiterpenes valencene and α- and β-sinensal, although present in minor quantities in oranges, play an important role in the overall flavor and aroma of orange fruit (Maccarone et al., 1998; Vora et al., 1983; Weiss, 1997). Nootkatone, a putative derivative of valencene, is a small fraction of the essential oils, but has a dominant role in the flavor and aroma of grapefruit (MacLeod and Buigues, 1964; Shaw and Wilson, 1981), while the monoterpene S-linalool was found to be an important general strawberry aroma compound (Aharoni et al., 2004; Larsen and Poll, 1992) and is found in many other fruits including peaches, guavas, nectarines, papayas, mangoes, passion fruits, tomatoes, litchi, oranges, prickly pears and koubos (Baldwin et al., 2000; Bernreuther and Schreier, 1991; Flath and Takahashi, 1978; Idstein et al., 1985; Ninio et al., 2003; Ong and Acree, 1998; Visai and Vanoli, 1997). The combination of the monoterpenes geraniol, citronellol and rose oxide is a key component of the characteristic aroma of aromatic muscat grapes as well as the special scent of roses (Bayrak, 1994; Dunphy and Allcock, 1972; Luan et al., 2005).
Terpenoids are also the primary constituents of the essential oils of many types of herbs. The peltate glandular trichomes of peppermint produce copious amounts of a commercially valuable, menthol-rich essential oil, composed primarily of p-menthane monoterpenes (Turner and Croteau, 2004). The glandular trichomes of sweet basil (Ocimum basilicum) are rich in phenylpropenes as well as monoterpenes and sesquiterpenes (Iijima et al., 2004a). Lemon-scented herbs of various plant families, such as lemon basil (Ocimum × citratus, Lamiaceae), lemongrass (Cymbopogon citratus, Poaceae) and lemon verbena (Aloysia citriodora, Verbenaceae), accumulate citral, a mixture of the cis–trans isomeric monoterpene aldehydes neral and geranial (Lewinsohn et al., 1998; Iijima et al., 2004a,b; Gil et al., 2007). Therefore, many terpenoids are commercially important and are widely used as flavoring agents, perfumes, insecticides, anti-microbial agents and important raw material for the manufacture of vitamins and other key chemicals. Many terpenoids have medicinal properties; consequently they are of interest to the pharmaceutical industry as anti-retroviral agents or anti-malarial compounds (Modzelewska et al., 2005). As a result, modulation of terpenoid biosynthesis in medicinal and aromatic plants has received much interest (Gómez-Galera et al., 2007; Mahmoud and Croteau, 2001, 2002; Mahmoud et al., 2004; Muñoz-Bertomeu et al., 2006, 2007; Tadmor and Lewinsohn, 2007). Synthetic variations and derivatives of natural terpenes and terpenoids also greatly expand the variety of aromas used in perfumery and flavors used in food additives.
Despite their diversity, all terpenoids derive from the common building units isopentenyl diphosphate (IDP) and its isomer dimethylallyl diphosphate (DMADP) (Croteau and Karp, 1991; Croteau et al., 2000; McGarvey and Croteau, 1995). In plants, both IDP and DMADP are synthesized via two parallel pathways, the mevalonate (MVA) pathway, which is active in the cytosol, and the methylerythritol 4-phosphate (MEP) pathway, which is active in the plastids (Lichtenthaler, 1999; Rodriguez-Concepción and Boronat, 2002; Rohdich et al., 2002; Rohmer, 2003). It is generally recognized that the cytosolic pathway is responsible for the synthesis of sesquiterpenes, phytosterols and ubiquinone, whereas monoterpenes, gibberellins, abscisic acid, carotenoids and the prenyl moiety of chlorophylls, plastoquinone and tocopherol are produced in plastids (Lichtenthaler, 1999; Rodriguez-Concepción and Boronat, 2002; Rohdich et al., 2002; Rohmer, 2003), but indications of cross-talk between the plastidic and cytosolic pathways have been found in tobacco, Arabidopsis and snapdragon petals (Aharoni et al., 2004; Dudareva et al., 2005; Ohara et al., 2003). The direct precursors of terpenoids, linear geranyl diphosphate (GDP, C10), farnesyl diphosphate (FDP, C15) and geranylgeranyl diphosphate (GGDP, C20), are produced by the activities of three prenyl transferases. Terpene synthases are the primary enzymes responsible for catalyzing the formation of hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15) or diterpenes (C20) from the substrates DMADP, GDP, FDP or GGDP, respectively.
Prenyl transferases catalyze the addition of IDP units to prenyl diphosphates with allylic double bonds to the diphosphate moiety. Most of the prenyl transferases accept DMADP as the initial substrate, but they also bind GDP or FDP depending on the particular prenyltransferase (Greenhagen and Chappell, 2001; Tarshis et al., 1994, 1996; Withers and Keasling, 2007). The availability of GDP and FDP are often the key factor in the production of monoterpenes and sesquiterpenes in plants. This problem was elegantly overcome in metabolic engineering experiments by the co-expression of GDP and FDP synthases with appropriate monoterpene and sesquiterpene synthases over-expressed in tobacco (Wu et al., 2006). This strategy, together with targeting of the over-expression to the plastid compartment, resulted in increased synthesis of the sesquiterpenes amorpha-4,11-diene and patchoulol and the monoterpene S-limonene (Wu et al., 2006).
The third phase of terpene volatile biosynthesis involves conversion of the various prenyl diphosphates DMADP, GDP, FDP and GGDP to hemiterpenes, monoterpenes, sesquiterpenes and diterpenes, respectively, by the large family of the terpene synthases. Triterpenes (and sterols) and tetraterpenes (such as carotenoids) are derived from the condensation of two molecules of FDP or GGDP, respectively. Plant hemiterpene, monoterpene, sesquiterpene and diterpene synthases are evolutionarily related to each other and are structurally distinct from triterpene or tetraterpene synthases. Much of the progress achieved in recent years in terpenoid metabolism is described elsewhere in this issue (Bohlmann and Keeling, 2008). Many terpene synthases have been isolated and characterized from various plant species (Bohlmann et al., 1998; Tholl, 2006).
While many terpene volatiles are direct products of terpene synthases, many others are formed through transformation of the initial products by oxidation, dehydrogenation, acylation and other reactions (Croteau and Karp, 1991; Croteau et al., 2000; Dudareva et al., 2004; Pichersky et al., 2006). For example, (−)-(1R,2S,5R)-menthol, the principal monoterpene of commercial peppermint essential oil and the component responsible for the familiar cooling sensation of peppermint and its products, is formed by eight enzymatic steps involving monoterpene synthases, isomerases and reductases (Ringer et al., 2005; Turner and Croteau, 2004). The biosynthesis starts with the formation of 4S-limonene from GPP and ends with the reduction of (−)-menthone to (−)-menthol. Mentha arvensis is the primary species of mint used to make natural menthol crystals and natural menthol flakes. As with many widely used aroma chemicals, the annual demand for menthol of 6300 tonnes greatly exceeds the supply from natural sources.
Metabolic engineering of the terpenoid pathway is a constantly improving tool, used for the fundamental study of terpenoid biosynthesis (Lücker et al., 2001, 2004; Ohara et al., 2003). In addition, this tool is being used more and more for the understanding of chemical diversity in crops (Köllner et al., 2004; Portnoy et al., 2008), as well as improvement of traits in crops such as disease and pest resistance (Kappers et al., 2005; Schnee et al., 2006; Wu et al., 2006), enhanced and altered aroma formation (Lavy et al., 2002; Lewinsohn et al., 2001; Mahmoud and Croteau, 2001) and production of medicinal compounds (Wu et al., 2006). Most of the recent progress in this field has been summarized by Lücker et al. (2007). A recent example in which flavor engineering was detected by non-trained test panelists involved ectopic expression of the lemon basil geraniol synthase gene under the control of the fruit ripening-specific tomato polygalacturonase promoter (Davidovich-Rikanati et al., 2007). This caused diversion of the plastidial terpenoid pathway for production of lycopene to the accumulation of high levels of geraniol and about ten novel geraniol derivatives, and had a profound impact on tomato flavor and aroma, as evaluated organoleptically.
Apocarotenoid formation. Carotenoids are tetraterpenoid pigments that accumulate in the plastids of leaves, flowers and fruits, where they contribute to the red, orange and yellow coloration. In addition to their roles in plants as photosynthetic accessory pigments and colorants, carotenoids are also precursors of apocarotenoids (also called norisoprenes) such as the phytohormone abscisic acid, the visual and signaling molecules retinal and retinoic acid, and aromatic volatiles such as β-ionone. Evidence, based on comparative genetics, has indicated that carotenoid pigmentation patterns have profound effects on the apocarotenoid and monoterpene aroma volatile compositions of tomato and watermelon fruits (Lewinsohn et al., 2005a,b). This work indicated that the various flavors and aromas of otherwise similar fruit of different colors have a real chemical basis and are not solely due to psychological preconception. Indeed, enzymes capable of cleaving carotenoids at specific sites were found to be involved in the synthesis of a number of apocarotenoids. Carotenoid cleavage dioxygenases (CCDs) catalyze the oxidative cleavage of carotenoids, resulting in production of apocarotenoids (Schmidt et al., 2006). CCDs often exhibit substrate promiscuity, which probably contributes to the diversity of apocarotenoids found in nature. Apocarotenoids are commonly found in the flowers, fruits, and leaves of many plants (Winterhalter and Rouseff, 2002), and possess flavor aroma properties together with low aroma thresholds. They are found among the potent flavor compounds in wines and contribute to floral and fruity attributes (Winterhalter and Schreier, 1994). Therefore, they have been subject to extensive research in recent years with regard to their structure and flavor potential (Winterhalter and Rouseff, 2002). The synthesis of β-ionone, geranyl acetone and 6-methyl-5-hepten-2-one in tomato fruits increases 10–20-fold during fruit ripening, and these compounds were produced by the activity of the genes LeCCD1A and LeCCD1B that were isolated from tomato fruits (Simkin et al., 2004). In tomato fruit, β-ionone is present at very low concentrations (4 nl l−1), but due to its low odor threshold (0.007 nl l−1) is the second most important volatile contributing to fruit flavor (Baldwin et al., 2000). Silencing of LeCCD1A and LeCCD1B resulted in a significant decrease in the β-ionone content of ripe fruits, implying a role for these genes in C13 norisoprenoid synthesis in vivo (Simkin et al., 2004). Reduction of Petunia hybrida CCD1 transcript levels in transgenic plants led to a 58–76% decrease in β-ionone synthesis in the corollas of selected petunia lines, indicating a significant role for this enzyme in volatile synthesis (Simkin et al., 2004). Also, a potential CCD gene was identified among a Vitis vinifera L. EST collection, and recombinant expression of VvCCD1 confirmed that the gene encodes a functional CCD that cleaves zeaxanthin symmetrically yielding 3-hydroxy-β-ionone and a C14 dialdehyde (Mathieu et al., 2005). CCDs were also found to be involved in the formation of important aroma compounds in melon (Cucumis melo) (Figure 6). The product of the CmCCD1 gene, whose expression is up-regulated upon fruit development, was shown to cleave carotenoids, generating geranylacetone from phytoene, pseudoionone from lycopene, β-ionone from β-carotene, and α-ionone and pseudoionone from δ-carotene (Ibdah et al., 2006).
Figure 6. Carotenoids and their degradation products. Carotenoid substrates (left) are oxidatively cleaved to yield the apocarotenoid derivatives (right).
Download figure to PowerPoint