Genes involved in the biosynthesis of aroma volatiles and biotechnological applications

13 Genes involved in the biosynthesis of aroma volatiles and biotechnological applications J. C. Pech, A. Latché and B. van der Rest, INRA/INP-ENSAT, UMR 990, Ecole Nationale Supérieure Agronomique de Toulouse, France

13.1 Introduction Aroma volatiles contribute to a large extent to the overall sensory quality of fruit and vegetables. Research during recent decades has been dedicated to the identification of volatile compounds and to the elucidation of some of the biosynthetic routes by either bioconversion or by tracing of precursors (Sanz et al., 1997; D’Auria et al., 2002; Dudareva et al., 2004). Recently, research efforts have been directed to the isolation of the corresponding genes in fruit and vegetables (Aharoni et al., 2000; Yahyaoui et al., 2002; Beekwilder et al., 2004) or in flowers (Shalit et al., 2003; Dudareva and Pichersky, 2006; Pichersky and Dudareva, 2007). Aroma is generally a complex mixture of a wide range of compounds. Each product has a distinctive aroma which is function of the proportion of the key volatiles and the presence or absence of unique components. The most important classes of aromas are: monoterpenes, sesquiterpenes, lipids-, sugars- and amino acid-derived compounds. Therefore, the strategies developed to improve aroma volatiles emitted by fruits and vegetables include a wide range of targets comprising the different metabolic pathways, but also regulatory elements such as hormones and transcription factors, and finally mechanisms involved in the storage or sequestration of volatile precursors, such as glycosylation and storage into the vacuoles (Fig. 13.1). It is noteworthy to mention that the production of aroma volatiles can be unexpectedly affected by engineering other fruit attributes. For

Genes involved in the biosynthesis of aroma volatiles Amino acids

MVA and MEP pathways IPP

DMAPP C5

Decarboxylase Deaminase

Prenyltransferases

Fatty acids Desaturases Unsaturated fatty acids moieties Phospholipases, acyl hydrolase Unsaturated free fatty acids LOX 13-hydroperoxides

Glycosylated compounds Phytoene synth .

HPL

Glucosyltransf.

Aldehydes

Glycosidases

Sugars

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Monoterpene synthases LIS, GS

Carotenoids C40 ADH

Alcohols

Fructose -PP Monoterpenes C10

CCD

Acyl -CoA FaQR

AAT

Cstps1 tps1 FaOMT

DMMF Furaneol

Sesquiterpenes

Ketones Aldehydes Alcohols

Volatile esters

Fig. 13.1 Schematic representation of the steps of aroma volatiles biosynthesis for which genes have been characterized and used as targets for genetic transformation. Genes that have been up- or down-regulated by genetic engineering are under grey background.

instance, down-regulation of polygalacturonase (PG), pectin methyl esterase (PME) and PG+PME in transgenic fruit (Baldwin et al., 2000) that results in lower degradation of pectins, can reduce flavor volatiles by a so far unexplained mechanism. The aims of the chapter are the following: (1) review the information available on genes and gene families identified as participating in the synthesis of different class of aroma volatiles; and (2) describe the potential biotechnological applications for improved aromas in fruit and vegetables and other perspectives.

13.2 Genes involved in the biosynthesis of aroma volatiles Genes involved in the generation of fatty acids derived volatiles Fatty acids, essentially from membrane lipids, are major precursors of volatile compounds participating in the aroma of many fruit and vegetables. Volatile fatty acids derivatives include saturated and unsaturated short-chain alcohols, aldehydes and esters. They are generated by the lipoxygenase (LOX) pathway. Modifications of fatty acids composition Fatty acids in plants are first produced in saturated forms which are thereafter desaturated to mono- and polyunsaturated fatty acids successively by ∆9, ∆12 and ∆15 desaturases that add double bonds at specific positions in the chain. In most plants the level of mono-unsaturated fatty acids is low as compared to saturated and

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poly-unsaturated fatty acids, indicating that the first desaturation by ∆9 desaturase could be a limiting factor of polyunsaturation. Indeed, by expressing a gene encoding a yeast ∆9 desaturase, the levels of 16:1 fatty acids were highly increased in tomato leaves (Wang et al., 1996). The levels of 18:1 and 16:3 fatty acids were slightly increased and the level of 18:3 was significantly decreased. As a consequence, certain volatile compounds derived from 16:1, 16:3 and 18:1 fatty acids were increased to more than 3-fold, such as 1-hydroxy-2-butanone, 1-penten-3-ol, heptanal, 3-hexen-1-ol, 2-octanol, cis-3-hexenal, hexanal and 2-nonenal (Wang et al., 2001). Surprisingly, several C-6 compounds normally derived from 18:3 such as trans-2-hexenal also increased to a similar extent and compounds not known to be derived from fatty acids such as 2-ethyl-furan, 5-ethyl-2-[5H]-furanone, eugenol and 2-ethylthiophene also sharply increased in transgenic leaves. No information is available on the emission of volatile compounds by the fruit. A tomato mutant spr2 was identified as being deficient in jasmonic acid biosynthesis due to considerably lower levels of 18:3 fatty acids than wild type. The mutation corresponded to a gene encoding a chloroplastic ω-3 desaturase now called LeFAD7 (Li et al., 2003). In parallel with the reduction of 18:3 fatty acids, there was a 1.5 to three-fold increase in 18:2 linoleic acid (Cañoles et al., 2006). The production of unsaturated C-6 aldehydes: Z-3-hexenal, Z-3-hexenol, and E-2hexenal and the alcohol Z-3-hexenol derived from 18:3 fatty acids was markedly reduced in leaves and fruit of the mutant line. Conversely, the production of the saturated C-6 hexanal and hexanol were significantly higher in the mutant. Sensory tests indicated that the mutant had lower flavor. It is not known whether the overexpression of the LeFAD7 ω-3 desaturase would increase the flavor of the tomato. Phospholipases Phospholipases are involved in the formation of polyunsaturated free fatty acids that are generally considered as substrates for lipoxygenase (Feussner and Wasternack, 2002). The pathway for the catabolism of phospholipids has been studied in several senescing systems and involves the sequential action of a number of enzymes including phospholipase D (PLD), phosphatidate phosphatase, lipolytic acyl hydrolase and lipoxygenase (Paliyath and Droillard, 1992). Among these, phospholipase D-α (PLD-α) is involved in the mediation of various senescence processes promoted by ethylene or abscissic acid (Bargmann and Munnik, 2006). During tomato fruit development, a PLD-α is induced and its activity peaks at the mature green and turning stages (Pinhero et al., 2003). Tomato antisense lines targeted against PLD-α (Oke et al., 2003) produced fruits that released, after blending, higher quantities of LOX-derived aldehydes, E-pentenal and 2-hexenal. A possible explanation is that low expression of PLD-α delayed the senescence process thus resulting in the preservation of membrane integrity and in providing higher level of precursors for the LOX pathway. However, it cannot be ruled out that other unknown mechanisms associated with the slow-down of senescence be involved.

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Lipoxygenases Lipoxygenases (LOX) catalyze the hydroperoxidation of polyunsaturated fatty acids containing a cis,cis-pentadiene structure. The principal substrates of LOX in plants are linoleic and linolenic acid. There are two classes of LOX: 9-LOX, which generate specifically 9-hydroxyperoxides and 13-LOX which generate specifically 13-hydroxyperoxides. LOX are supposed to be involved in stress responses, wounding, and pathogen attack (Feussner and Wasternack, 2002). But LOX are also involved in the biosynthesis of volatile compounds, such as hexanal, hexenal and hexenol that participate in the aroma of fruit and vegetables. In tomato, five LOX genes (TomloxA, B, C, D, and E) are expressed during fruit ripening (Ferrie et al., 1994; Heitz et al., 1997; Griffths et al., 1999a). TomloxA, B and E are quite similar with 72% to 77% identity at the amino acid level. TomloxC and D, which have a chloroplast targeting signal (Heitz et al., 1997), show 42% and 47% identity to TomloxA, respectively, and 46% identity to each other. The specific role of the five LOX in stress responses, defense or biosynthesis of flavor compounds during fruit ripening has been one of the major questions addressed in recent years through gene silencing. Antisense suppression of TomloxA and TomloxB in tomato fruit has resulted in no significant changes of the fruit flavor volatiles (Griffiths et al., 1999b), while co-suppression of TomloxC strongly affected the production of flavor volatiles (Chen et al., 2004). TomloxC is therefore a good target gene for increasing aroma production through biotechnology in tomato. A homolog of TomloxC, LOX H1, has been down-regulated in potato resulting in the depletion of volatile aliphatic C6 aldehydes formation (Leon et al., 2002). Hydroxyperoxide lyases Hydroxyperoxide lyase (HPL) form very unstable hemiacetals from hydroperoxides (HPO) generated by LOX from polyunsaturated fatty acids leading to the generation of aldehydes and aldehydes enols by spontaneous dissociation. HPL belong to a family of cytochrome P450 proteins (CYP74) that include also allene oxide synthases (AOS) and divinyl ether synthases (DES). HPL can be divided into three subfamilies according to their substrate specificity.

• 13-HPLs show strong preference for 13-HPO (see Fukushige and Hildebrand, •

•

2005a) and cleave the 13-HPO into 12-oxo-(9Z)-dodecenoic acid and C6 aldehydes such as hexanal or (3Z)-hexenal. 9/13-HPLs can act both on 9- and 13-hydroxyperoxides but have often preference for 9-HPO (Matsui et al., 2000; Tijet et al., 2001). Beside cleaving 13-HPLs as indicated above, they can also cleave 9-HPO into 9-oxononanoic acid and C9 aldehydes such as (3Z)-nonenal or (3Z,6E)-nonadienal. The (3Z)aldehydes easily isomerize to their (2E)-enals. A 9-HPL gene has been recently isolated in almonds encoding an HPL with almost exclusive preference for 9-HPO (Mita et al., 2005).

13-HPLs or 9/13 HPLs are considered to be targeted to the chloroplast outer membrane. The 9-HPL protein of almond is associated with lipid bodies (Mita et al.,

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2005). HPLs have differential substrate specificity towards HPO derived fom linolenic acid (HPOT) or linoleic acid (HPOD). For instance, watermelon HPL is three times more active for 13-HPOT than HPOD, guava ten times; green pepper, 12 times; sunflower leaf 16 times; alfafa 1.5 times; sunflower hypocotyl two times (Fukushige and Hildebrand, 2005a). In order to modify the flavor properties of tomato fruits, a cucumber HPL gene which encodes an enzyme having preference for 9-PHOs to form C9-aldehydes hydroxyperoxides has been introduced in tomato plants (Matsui et al., 2001). Despite a high activity of the introduced HPL in leaves and fruit of transgenic tomatoes, little changes have been observed in the composition of volatile shortchain aldehydes and alcohols emitted by the fruit. Such a result was unexpected because tomato fruit have high lipoxygenase activity to form 9-HPO. Possible explanations are that the access of HPL to 9-HPO may not be possible due to compartmentation and/or the affinity of the introduced HPL for 9-HPO is low. The expression of a 9/13 HPL of watermelon having much higher affinity for 13-HPOs (specially those derived from linolenic acid) than for 9-HPOs in tobacco and Arabidopsis plants resulted in enzyme activies that were up to 50 times higher than in wild type plants (Fukushige and Hildebrand, 2005b). However, the effect of the transgene on the emission of volatiles by transgenic leaves has not been quantified in details. The biotechnological relevance of this strategy therefore remains to be proved. The silencing of LOX and HPL has been performed separately in potato plants (Salas et al., 2005). The down-regulation of HPL induced an increase in LOX activity and of the content of most of the C5 volatiles and a decrease of most of the C6 compounds in the leaves. This resulted in an increase in the sweet note and decrease in the green note odor. Suppression of lipoxygenase caused a severe decrease in the amount of volatiles produced by the leaves and in the intensity of their aroma estimated by sensory evaluation. These data open some perspectives, if not the improvement of the existing flavor, at least for the modification of aromas in leafy vegetables by genetic engineering. Alcohol dehydrogenases Alcohol dehydrogenases (ADH) catalyze the reversible conversion of aldehydes to the corresponding alcohols. They have been involved in the response to a wide range of stresses (Chase, 1999). However, ADH genes that are suspected of participation in the production of aromas are expressed in a developmentally regulated manner, particularly during fruit ripening (van der Straeten et al., 1991; Speirs et al., 1998; 2002; Echeverria et al., 2004; Manriquez et al., 2006). In grapes, three ADH genes are expressed during fruit development. VvADH1 and VvADH3 transcripts accumulate transiently in young developing berries, while VvADH2 transcripts strongly increase at the onset of ripening named véraison (Tesnière and Verriès, 2000). Fruit-specific dehydrogenases so far characterized belong to the medium-size zinc-containing class (Chase, 1999). Partial cDNA clones putatively encoding short-chain ADHs have been reported in tomato (Picton et al., 1993) and in pear (Fonseca et al., 2004). In melon, two fruit-specific

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CmADH genes belonging to both the medium- and short-chain types have been isolated. After expression in yeast and purification, it was demonstrated that the two encoded enzymes preferentially work as aldehyde reductases and have specific substrates preferences (Manriquez et al., 2006). In tomato fruit, one of the two ADH genes, LeADH2, participates in the formation of flavor volatiles during fruit ripening. Overexpression of LeADH2 has led to improved flavor of the fruit by increasing the level of alcohols, particularly Z-3-hexenol (Speirs et al., 1998). Fruits overexpressing LeADH2 were identified by a sensory panel as having a more intense ‘ripe fruit’ flavor. Alcohol acyl-transferases Alcohol acyl-transferases (AAT) catalyze the transfer of an acyl-CoA to an alcohol. These enzymes are capable of combining different alcohols and acylCoAs resulting in the synthesis of a wide range of esters accounting for the diversity of esters emitted by the fruit. A number of genes encoding AAT have been isolated and characterized in fruit and vegetables (Aharoni et al., 2000; Beekwilder et al., 2004; Souleyre et al., 2005; Wang and De Luca, 2005; ElSharkawy et al., 2005). In melon, three AAT genes (Cm-AAT1, Cm-AAT3 and Cm-AAT4) have been isolated that are specifically expressed in fruit under the control of the plant hormone ethylene. They have different substrate specificities that contribute to the production of a wide range of esters in the melon (ElSharkawy et al., 2005). Among the three AATs, Cm-AAT1 has the biggest capacity to produce thioesters (Lucchetta et al., 2007) that have considerable sensory importance in cantaloupe melon (Wyllie and Leach, 1990). The importance of AAT substrate specificity has also been outlined in apple (Souleyre et al., 2005) or in grapes where an AAT recently described in Concord grapes is responsible for the distinctive ‘foxy’ aroma (Wang and de Luca, 2005). A large number of acyl-transferase genes are present in plants with around 70 members encountered in Arabidopsis (Pichersky and Gang, 2000). Although performing the same reaction, AAT proteins from different fruit species may be highly divergent. For instance the strawberry AAT and the CmAAT1 of melon have only 22% identity while they have similar preference for substrates (Aharoni et al., 2000; Yahyaoui et al., 2002). Given their importance in aroma accumulation, AAT have also been the target of genetic engineering. In petunia, overexpression of strawberry AAT did not change the volatile emission profiles from flowers and green parts, yet, ester production could be enhanced when coupling the AAT overexpression with precursor feeding such as isoamyl alcohol (Beekwilder et al., 2004). More recently, the RNA interference technique has been used for downregulating AAT in Arabidopsis thaliana (D’Auria et al., 2007) and petunia (Dexter et al., 2007). In Arabidopsis, the target AAT exhibited a substrate preference similar to melon CmAAT1 and strawberry AAT. Its down-regulation resulted in a dramatic reduction in the emission of the green leaf volatile (Z)-3-hexen-1-ylacetate (D’Auria et al., 2007). In petunia, the target AAT was found to control the synthesis of isoeugenol, likely through the esterification of coniferyl alcohol in coniferyl acetate (Dexter et al., 2007).

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13.3 Genes of amino acid metabolism Aldehydes and alcohols derived from the degradation of amino acids constitute a class of highly abundant fruit volatiles. The two compounds, 2-phenylacetaldehyde and 2-phenylethanol and their glycosides are synthesized from phenylalanine. They are abundant in various fruits such as tomato (Baldwin et al., 2000) and some grape varieties (Garcia et al., 2003). Compounds derived from leucine catabolism such as 3-methyl-butanal and 3-methyl-butanol also contribute to the tomato flavour. In addition, alcohols deriving from amino acids can be esterified into compounds having a large impact on fruit odor such as 3-methyl-butyl acetate in banana or ethyl-butanoate and ethyl-hexanoate in strawberries (Perez et al., 1992). Recently, genes encoding enzymes responsible for the decarboxylation of phenylalanine have been identified in tomato (Tieman et al., 2006), petunia and rose (Kaminaga et al., 2006). In both studies, the enzymes described belong to the pyridoxal 5-phosphate dependent amino acid decarboxylases and display subtle differences of sequences and enzymatic properties. The antisense down-regulation of the decarboxylase gene in tomato and petunia led to reduced emission of phenylacetaldehyde and phenylethanol. Conversely, the overexpression in tomato of the amino acid decarboxylase increased up to 10-fold the amount of phenylethanol, phenylacetaldehyde, phenylacetonitrile, and 1-nitro-2-phenylethane released from the transgenic fruits (Tieman et al., 2006). This capacity to modulate the levels of phenylethanol and phenylacetaldehyde is important since these compounds can exert a dual effect: at low concentrations phenylethanol and phenylacetaldehyde are associated with pleasant sweet flowery notes, while at high concentrations the pungent aroma of phenylacetaldehyde has a nauseating and unpleasant odor (Tadmor et al., 2002)

13.4 Genes involved in terpenoid biosynthesis Terpenoids represent a large class of aroma volatiles originating from the condensation of the five carbon precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) generated by the methylerythritol 4-phosphate (MEP) and mevalonate pathways. The condensation is performed by prenyltransferases, such as geranyl diphosphate synthase (GPPS), geranylgeranyl diphospahte synthase (GGPPS) and farnesyl diphosphate synthase (FPPS), to produce prenyl diphosphates. The prenyl diphosphate precursors are then transformed into terpenes, sesquiterpenes and triterpenes by terpene synthases (McGarvey and Croteau, 1995; Tholl, 2006). Many of the terpene volatiles derive directly from the action of terpene synthases, while others are transformed into hydroxylated, dehydrogenated or acteylated compounds. Several reviews have been made directly related to the engineering of plants for terpenoids (McCaskill and Croteau, 1998) and for monoterpenes (Mahmoud and Croteau, 2002). Engineering of the terpene pathway has been initially attempted in plants rich in essential oils such as peppermint.

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13.4.1 Up-regulation of the MEP pathway Increasing the production of monoterpenes through the elevation of IPP and DMAPP was achieved in peppermint by expressing a reductoisomerase of the MEP pathway (Mahmoud and Croteau, 2001). There was a stimulation by 50% of monoterpenes synthesis without any change in the composition of the essential oil. Application to other species of agricultural interest is awaited.

13.4.2 Introduction or up-regulation of monoterpenes synthases Two different monoterpene synthases have been engineered in edible products. First, a Clarkia breweri linalool synthase has been expressed in tomato in a fruitspecific manner (Lewinsohn et al., 2001). It has resulted not only in the production of linalool but also of 8-hydroxy-linalool, probably resulting from the presence in the tomato of a P450 enzyme capable of hydroxylating linalool. More recently, tomato flavour was enriched through heterologous expression of a basil geraniol synthase (Davidovich-Rikanati et al., 2007). Very interestingly, the introduction of geraniol synthase not only resulted in the accumulation of monoterpenes uncommon in tomato fruit (geraniol, citronellol, neric acid, limonene), but the lycopene content was reduced, suggesting that the early plastidial terpenoid pathway was diverted from lycopene accumulation to monoterpene production. Except for these two attempts, most of the studies related to the overexpression of monoterpene synthases have been carried out in flowers or in model plants such as tobacco. Because monoterpene synthesis occurs in the plastids, targeting of gene expression to this organelle has proved to be more efficient than in the cytosol. As an example, transgenic tobacco plants expressing a heterologous limonene synthase produced much more limonene when gene expression was targeted to the plastids than to the cytosol. Expression in the reticulum resulted in the absence of limonene synthesis (Ohara et al., 2003). In some instances, several terpene synthases have been introduced simultaneously, which resulted in the production of new products beside the main and minor compounds resulting from the activity of the three introduced enzymes (Lücker et al., 2004a). Thus, modifying fruit flavor through monoterpene biosynthesis pathways has proven to be very efficient.

13.4.3 Introduction of up-regulation of P450 monoterpene hydroxylase and sesquiterpene synthase genes The introduction of a P450 limonene-3-hydroxylase in a tobacco transgenic line expressing three monoterpene synthases producing limonene, γ-terpinene and (–) β-pinene as the main products resulted in the hydroxylation of (+) limonene into (+) trans-isopiperitenol, an uncommon compound in the plant kingdom (Lücker et al., 2004b). The synthesis of sesquiterpenes has been stimulated by expression of a fungal sesquiterpene cyclase in tobacco (Hohn and Ohlrogge, 1991). A sesquiterpene synthase of citrus involved in the synthesis of the sesquiterpene valencene has been isolated (Sharon-Asa et al., 2003), but biotechnological applications have not been reported.

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13.4.4 Reduction of undesirable monoterpene compounds Reduction of menthofuran, an undesirable monoterpene oil component of peppermint, was achieved by down-regulating the methofuran synthase (Mahmoud and Croteau, 2001). Similar experiments in fruit and vegetables have not been published.

13.4.5 Genes involved in the generation of volatiles from carotenoids Carotenoids are the precursors of C14 and C13 volatile compounds that contribute to flavor and aroma of many fruit, vegetable and flowers, such as β-ionone, geranylacetone (6,10-dimethyl-5,9-undecadien-2-one) and pseudoionone (6,10dimethyl-3,5,9-undecatrien-2-one). Because they originate from coloured carotenoids, a link has long been observed between pigmentation and aroma production in fruit. Lewinsohn et al., (2005) have correlated the pigment content of a number of mutants or genotypes of tomato and watermelon with aroma production. For instance, tomato mutants rich in δ-carotene produce high amounts of α-ionone, while those rich in β-carotene produce more β-ionone. These observations indicate that engineering fruit for increasing the level or for modifying the balance of carotenoids in fruit has probably an effect on the production of aroma volatiles. So far, papers dealing with biotechnology of carotenoids (Rosati et al., 2000; Römer et al., 2000; Fraser et al., 2002) have not taken aroma production into account. The C14 and C13 carotenoid-derived volatile compounds are predicted to derive from carotenoids through an oxidative cleavage mechanisms. In the recent years genes encoding carotenoid cleavage dioxygenase (CCD) have been isolated in plants (Giuliano et al., 2003) that contribute to the generation of apocarotenoids, among which, abscissic acid (Schwartz et al., 1997; Tan et al., 1997), bixin dialdehyde an important food and cosmetic plant pigment (Bouvier et al., 2003a) also known as a contributor to tomato flavor (Baldwin et al., 2000) and crocin, the main pigment of saffron (Bouvier et al., 2003b). Carotenoid dioxygenase genes have been characterized in tomato (Simkin et al., 2004a) and in petunia (Simkin et al., 2004b). In tomato, there are two closely related genes, LeCCD1A and LeCCD1B, the latter being highly expressed during fruit ripening. LeCCD1-supressed tomatoes were generated that show strong reduction of the production of β-ionone, geranylacetone and pseudoionone (Simkin et al., 2004a). In petunia, suppression of PhCCD1 led to a 58% to 76% decrease in β-ionone synthesis (Simkin et al., 2004b). In grapes a VvCCD1 has been isolated and characterized by expression in E. coli (Mathieu et al., 2005). A CmCCD1 gene of melon has been characterized by functional expression in E. coli (Ibdah et al., 2006). It shows up-regulation during fruit ripening. The CmCCD1 gene product cleaves carotenoids at positions 9,10 and 9',10' generating geranylacetone from phytoene, β-ionone from β-carotene, and α-ionone and pseudoionone from δ-carotene. Since the CmCCD1 gene is also expressed in white and pale green melons, despite the lack of (or the low) production of apocarotenoids, it can be concluded that the accumulation of β-ionone is limited by the availability

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of the carotenoid substrate. From a biotechnological point of view the overexpression of these genes in fruit may stimulate the production of aroma volatiles only when the availabilty of substrates is not limiting.

13.5 Genes involved in the generation of aroma volatiles from sugars Furanone structures such as the caramel-like 4-hydroxy-2,5-dimethyl-3(2H)furanone (HDMF or furaneol) and 2,5-dimethyl-4-methoxy-3(2H)-furanone (DMMF) derive from the metabolism of sugars, primarly from D-fructose-1,6diphosphate (Roscher et al., 1998). HDMF is present at high concentration in the aroma of strawberry (up to 55 mg/kg FW) (Larsen and Poll, 1992) and has a low odor threshold (10 ppb) (Schwab and Roscher, 1997). HDMF and DMMF are present in pineapple and a number of different fruit (Schwab and Roscher, 1997). They are not present or undetectable in root, stems, leaves or flowers. A strawberry FaOMT encoding an O-methyltransferase responsible for DMMF biosynthesis has been isolated (Wein et al., 2002). Its expression is up-regulated specifically during fruit ripening and the encoded protein was capable of methylating a number of substrates: catecol, caffeic acid, protocatechuic aldehyde, coffeoyl CoA, and DMF. It is supposed to play a role in lignification of the achenes and vascular bundles and in the synthesis of volatiles such as DMMF. The function of this FaOMT was assessed in planta using overexpression and antisense technology in strawberries (Lunkenbein et al., 2006a). The reduction of FaOMT gene expression altered the HDMF/DMMF ratio, resulting in a near depletion of the DMMF pool, thus confirming the importance of FaOMT in the DMMF formation. In addition, the dual function of this enzyme in the secondary metabolism was also proven since FaOMT down-regulation affected also the concentration of feruloyl glucose, suggesting that it is also involved in the methylation of the caffeoyl group. Recently, an enone oxidoreductase (FaQR) involved in the HDMF formation was isolated from a crude strawberry fruit extract and the corresponding gene has been cloned (Raab et al., 2006). It represents a very promising target for biotechnological engineering.

13.6 Modification of the glycosylated fraction Many aroma precursors are also found as glycosylated compounds. Their release, by enzymatic or chemical hydrolysis, is of particular interest in fruit juice processing and in winemaking (Sarry and Gunata, 2004). The potential of this source of precursor in genetic engineering has not been fully explored. Yet, a first attempt to express an Aspergillus niger β-glucosidase in tobacco leaves had profound effects on the volatile emissions (Wei et al., 2004). Theses effects depended on the subcellular compartment targeted by the heterologous expression, suggesting that the different subcellular fractions indeed contain large pools of glycosylated

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volatiles precursors which can be mobilized by metabolic engineering. In another attempt to engineer fruits flavors, Lunkenbein et al. (2006b) recently identified a UDP-glucose:cinnamate glucosyltransferase responsible for the synthesis of (hydroxyl)cinnamoyl-glucose in strawberries, which is a possible precursor of methyl and ethyl cinnamate. However, the down-regulation of the gene by antisense only decreased the pools of the cinnamoyl- and p-coumaroyl glycosides without any obvious increase or decrease of the derived volatiles. It can also be mentioned that glycosylation can be problematic in the frame of metabolic engineering since the enhanced metabolites can be stored in a glycosylated and non-volatile form. For example, metabolic engineering of terpenoid metabolism in petunia leaves (Lücker et al., 2001) or Arabidopsis flowers (Aharoni et al., 2003) by over-expression of linalool synthase resulted mainly in the accumulation of linalyl-glycosides instead of the free volatile form.

13.7 Regulators controlling aroma biosynthesis: transcription factors and hormones Besides genes encoding enzymes involved in the aromas’ metabolic pathways, genes that regulate the pathways may also represent promising targets for genetic engineering. Two types of regulators may be considered: transcription factors and genes involved in the synthesis or perception of hormones. Although transcription factors have not been targeted for improving aroma volatiles, they have been successfully used to increase fruit contents in antioxidant flavonoids (Bovy et al., 2002; Schijlen et al., 2004). Concerning hormones, plants altered in ethylene synthesis or perception with the aim of extending shelf-life of climacteric fruits, generally displayed a lower production of aroma volatiles (Ayub et al., 1996; Bauchot et al., 1998; Flores et al., 2002; Dandekar et al., 2004; Defilippi et al., 2004; Nuñez-Palenius et al., 2006). Since several hormones influence fruit development and sensory quality (see Klein and Goldschmidt, 2005), they represent a high number of potential targets for genetic engineering.

13.8 Conclusions and perspectives This review shows that many genes participating in the synthesis of aroma volatiles have been isolated in recent years. In many cases functional characterization has been carried out by expression in heterologous model plants or by down-regulation in the original plant. Many studies have been carried out in flowers for which the scent is of the utmost importance and the number of studies that have been dedicated to edible fruit and vegetables are reduced. There is no doubt that the recent development in genomics and proteomics will enhance the number of target genes for genetic manipulation of aroma volatiles biosynthesis. In addition to the genes directly involved in the biosynthesis, it is probable that regulatory genes, such as transcription factors, capable of modulating the aromas’ biosynthetic

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pathways will be discovered. The strategies developed for metabolic engineering should integrate other aspects. For instance, the importance of targeting the expression of the genes to specific organelles or cell compartments has been demonstrated, particularly for monoterpenes. Switching the sub-cellular localization to organelles that are not originally involved in the synthesis of aroma volatiles could result in the synthesis of new volatiles (Kappers et al., 2005). Also, the use of organ-specific promoters is desirable. Overall, the data reported in this review demonstrate the potential of genetic engineering for the improvement of aroma and taste properties of horticultural products. However, most if not all them are related so far to basic studies at the laboratory level. They provide only proof of principle that engineering one of several of these genes could be of practical interest. Field or commercial tests remain to be performed and acceptance by the consumers to be assessed.

13.9 Acknowledgements The authors thank INRA (CEPIA department) and the Midi-Pyrénées Regional Council for financially supporting our own research on genes involved in the synthesis of aroma volatiles thus making possible the writing of this chapter.

13.10 References AHARONI A., KEIZER L.C., BOUWMEESTER H.J., SUN Z., ALVAREZ-HUERTA M., VERHOEVEN H.A., BLAAS J., VAN HOUWELINGEN A.M., DE VOS R.C., VAN DER VOET H., JANSEN R.C., GUIS M., MOL J., DAVIS R.W., SCHENA M., VAN TUNEN A.J. AND O’CONNELL A.P. (2000)

Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays. Plant Cell, 12, 647–662. AHARONI A., GIRI A.P., DEUERLEIN S., GRIEPINK F., DE KOGEL W.J., VERSTAPPEN F.W., VERHOEVEN H.A., JONGSMA M.A., SCHWAB W. AND BOUWMEESTER H.J. (2003) Terpenoid metabolism in wild-type and transgenic Arabidopsis plants. Plant Cell, 15, 2866–2884. AYUB R., GUIS M., AMOR M.B., GILLOT L., ROUSTAN J.-P., LATCHÉ A., BOUZAYEN M. AND PECH J.C. (1996) Expression of ACC oxidase antisense gene inhibits ripening of cantaloupe melon fruits. Nat Biotech, 14, 862–866. BALDWIN E.A., SCOTT J.W., SHEWMAKER C.K. AND SCHUCH W. (2000) Flavor trivia and tomato aromas: biochemistry and possible mechanisms for control of important aroma components. HortScience, 35, 1013–1022. BARGMANN B.O. AND MUNNIK T. (2006) The role of phospholipase D in plant stress responses. Curr Opin Plant Biol, 9, 515–522. BAUCHOT A.D., MOTTRAM D.S., DODSON A.T. AND JOHN P. (1998) Effect of aminocyclopropane1-carboxylic acid oxidase antisense gene on the formation of volatile esters in cantaloupe charentais melon (Cv. Vedrandais). J Agric Food Chem, 46, 4787–4792. BEEKWILDER J., ALVAREZ-HUERTA M., NEEF E., VERSTAPPEN F.W., BOUWMEESTER H.J. AND AHARONI A. (2004) Functional characterization of enzymes forming volatile esters from strawberry and banana. Plant Physiol, 135, 1865–1878. BOUVIER F., DOGBO O. AND CAMARA B. (2003a) Biosynthesis of the food and cosmetic plant pigment bixin (annatto). Science, 300, 2089–2091.

266

Fruit and vegetable flavour

BOUVIER F., SUIRE C., MUTTERER J. AND CAMARA B.

(2003b) Oxidative remodeling of chromoplast carotenoids: identification of the carotenoid dioxygenase CsCCD and CsZCD genes involved in Crocus secondary metabolite biogenesis. Plant Cell, 15, 47–62. BOVY A., DE VOS R., KEMPER M., SCHIJLEN E., ALMENAR PERTEJO M., MUIR S., COLLINS G., ROBINSON S., VERHOEYEN M., HUGHES S., SANTOS-BUELGA C. AND VAN TUNEN A. (2002) High-flavonol tomatoes resulting from the heterologous expression of the maize transcription factor genes LC and C1. Plant Cell, 14, 2509–2526. CAÑOLES M.A., BEAUDRY R.M., LI C. AND HOWE G. (2006) Deficiency of linolenic acid in Lefad7 mutant tomato changes the volatile profile and sensory perception of disrupted leaf and fruit tissue. J Amer Soc Hort Sci, 131, 284–289. CHASE T. (1999) Alcohol dehydrogenases: identification and names for gene families. Plant Mol Biol Rep, 17, 333–350. CHEN G., HACKETT R., WALKER D., TAYLOR A., LIN Z. AND GRIERSON D. (2004) Identification of a specific isoform of tomato lipoxygenase (TomloxC) involved in the generation of fatty acid-derived flavor compounds. Plant Physiol, 136, 2641–2651. DANDEKAR A.M., TEO G., DEFILIPPI B.G., URATSU S.L., PASSEY A.J., KADER A.A., STOW J.R., COLGAN R.J. AND JAMES D.J. (2004) Effect of down-regulation of ethylene biosynthesis on fruit flavor complex in apple fruit. Transgenic Research, 13, 373–384. D’AURIA J.C., CHEN F. AND PICHERSKY E. (2002) Characterization of an acyltransferase capable of synthesizing benzylbenzoate and other volatile esters in flowers and damaged leaves of Clarkia breweri. Plant Physiol, 130, 466–476. D’AURIA, J. C., PICHERSKY, E., SCHAUB, A., HANSEL, A., GERSHENZON, J. (2007) Characterization of a BAHD acyltransferase responsible for producing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabidopsis thaliana. Plant J, 194–207. DAVIDOVICH-RIKANATI, R., SITRIT, Y., TADMOR, Y., IIJIMA, Y., BILENKO, N., BAR, E., CARMONA, B., FALLIK, E., DUDAI, N., SIMON, J. E., PICHERSKY, E., LEWINSOHN, E. (2007) Enrichment of tomato flavor by diversion of the early plastidial terpenoid pathway. Nat Biotech 25, 899–901 DEFILIPPI B.G., DANDEKAR A.M. AND KADER A.A. (2004) Impact of suppression of ethylene action or biosynthesis on flavor metabolites in apple (Malus domestica Borkh) fruits. J Agric Food Chem, 52, 5694–5701. DEXTER, R., QUALLEY, A., KISH, C. M., MA, C. J., KOEDUKA, T., NAGEGOWDA, D. A., DUDAREVA, N., PICHERSKY, E., CLARK, D. (2007) Characterization of a petunia acetyltransferase involved in the biosynthesis of the floral volatile isoeugenol. Plant J, 49, 265–275. DUDAREVA N., PICHERSKY E. AND GERSHENZON J. (2004) Biochemistry of plant volatiles. Plant Physiol, 135, 1893–1902. DUDAREVA N. AND PICHERSKY E. (2006) Floral scent metabolic pathways: their regulation and evolution. In: Biology of Floral Scent (eds N. Dudareva and E. Pichersky), pp. 55– 78. CRC Press, Boca Raton, FL. ECHEVERRIA G., GRAELL J., LOPEZ M.L. AND LARA I. (2004) Volatile production, quality and aroma-related enzyme activities during maturation of ‘Fuji’ apples. Postharvest Biol Technol, 31, 217–227. EL-SHARKAWY I., MANRIQUEZ D., FLORES F.B., REGAD F., BOUZAYEN M., LATCHÉ A. AND PECH J.C. (2005) Functional characterization of a melon alcohol acyl-transferase gene family involved in the biosynthesis of ester volatiles. Identification of the crucial role of a threonine residue for enzyme activity. Plant Mol Biol, 59, 345–362. FERRIE B.J., BEAUDOIN N., BURKHART W., BOWSHER C.G. AND ROTHSTEIN S.J. (1994) The cloning of two tomato lipoxygenase genes and their differential expression during fruit ripening. Plant Physiol, 106, 109–118. FEUSSNER I. AND WASTERNACK C. (2002) The lipoxygenase pathway. Annual Review of Plant Biology, 53, 275–297. FLORES F., EL YAHYAOUI F., DE BILLERBECK G., ROMOJARO F., LATCHÉ A., BOUZAYEN M., PECH J.C. AND AMBID C. (2002) Role of ethylene in the biosynthetic pathway of aliphatic

Genes involved in the biosynthesis of aroma volatiles

267

ester aroma volatiles in Charentais Cantaloupe melons. J Exp Bot, 53, 201–206. FONSECA S., HACKLER L., ZVARA A., FERREIRA S., BALDE A., DUDITS D., PAIS M.S. AND PUSKAS L.G. (2004) Monitoring gene expression along pear fruit development, ripening

and senescence using cDNA microarrays. Plant Science, 167, 457–469. FRASER P.D., RÖMER S., SHIPTON C.A., MILLS P.B., KIANO J.W., MISAWA N., DRAKE R.G., SCHUCH W. AND BRAMLEY P.M. (2002) Evaluation of transgenic tomato plants expressing

an additional phytoene synthase in a fruit-specific manner. Proc Natl Acad Sci USA, 99, 1092–1097. FUKUSHIGE H. AND HILDEBRAND D.F. (2005a) Watermelon (Citrullus lanatus) hydroperoxide lyase greatly increases C6 aldehyde formation in transgenic leaves. J Agric Food Chem, 53, 2046–2051. FUKUSHIGE H. AND HILDEBRAND D.F. (2005b) A simple and efficient system for green note compound biogenesis by use of certain lipoxygenase and hydroperoxide lyase sources. J Agric Food Chem, 53, 6877–6882. GARCIA E., CHACON J.L., MARTINEZ J. AND IZQUIERDO P.M. (2003) Changes in volatile compounds during ripening in grapes of Airén, Macabeo and Chardonnay white varieties grown in La Mancha region (Spain). Food Sci Technol Int, 9, 33–41. GIULIANO G., AL-BABILI S. AND VON LINTIG J. (2003) Carotenoid oxygenases: cleave it or leave it. Trends Plant Sci, 8, 145–149. GRIFFITHS A., BARRY C., ALPUCHE-SOLIS A. AND GRIERSON D. (1999a) Ethylene and developmental signals regulate expression of lipoxygenase genes during tomato fruit ripening. J Exp Bot, 50, 793–798. GRIFFITHS A., PRESTAGE S., LINFORTH R., ZHANG J., TAYLOR A. AND GRIERSON D. (1999b) Fruit-specific lipoxygenase suppression in antisense-transgenic tomatoes. Postharvest Biol Technol, 17, 163–173. HEITZ T., BERGEY D.R. AND RYAN C.A. (1997) A gene encoding a chloroplast-targeted lipoxygenase in tomato leaves is transiently induced by wounding, systemin, and methyl jasmonate. Plant Physiol, 114, 1085–1093. HOHN T.M. AND OHLROGGE J.B. (1991) Expression of a fungal sesquiterpene cyclase gene in transgenic tobacco. Plant Physiol, 97, 460–462. IBDAH M., AZULAY Y., PORTNOY V., WASSERMAN B., BAR E., MEIR A., BURGER Y., HIRSCHBERG J., SCHAFFER A.A., KATZIR N., TADMOR Y. AND LEWINSOHN E. (2006) Functional characterization of CmCCD1, a carotenoid cleavage dioxygenase from melon. Phytochemistry, 67, 1579–1589. KAMINAGA Y., SCHNEPP J., PEEL G., KISH C.M., BEN-NISSAN G., WEISS D., ORLOVA I., LAVIE O., RHODES D., WOOD K., PORTERFIELD D.M., COOPER A.J.L., SCHLOSS J.V., PICHERSKY E., VAINSTEIN A. AND DUDAREVA N. (2006) Plant phenylacetaldehyde synthase is a bifunctional homotetrameric enzyme that catalyzes phenylalanine decarboxylation and oxidation. J Biol Chem, 281, 23357–23366. KAPPERS I.F., AHARONI A., VAN HERPEN T.W., LUCKERHOFF L.L., DICKE M. AND BOUWMEESTER H.J. (2005) Genetic engineering of terpenoid metabolism attracts bodyguards to Arabidopsis. Science, 309, 2070–2072. KLEIN J.D. AND GOLDSCHMIDT E.E. (2005) Hormonal regulation of ripening and senescence phenomena. In: Environmentally Friendly Technologies for Agricultural Produce Quality (ed S. Ben-Yehoshua), pp. 315–331. CRC Press, Boca Raton, FL. LARSEN M. AND POLL L. (1992) Odour thresholds of some important aroma compounds in strawberries. Z Lebensm Unters Forsch, 195, 120–123. LEON J., ROYO J., VANCANNEYT G., SANZ C., SILKOWSKI H., GRIFFITHS G. AND SANCHEZSERRANO J.J. (2002) Lipoxygenase H1 gene silencing reveals a specific role in supplying fatty acid hydroperoxides for aliphatic aldehyde production. J Biol Chem, 277, 416–423. LEWINSOHN E., SCHALECHET F., WILKINSON J., MATSUI K., TADMOR Y., NAM K.H., AMAR O., LASTOCHKIN E., LARKOV O., RAVID U., HIATT W., GEPSTEIN S. AND PICHERSKY E. (2001) Enhanced levels of the aroma and flavor compound S-linalool by metabolic engineering of the terpenoid pathway in tomato fruits. Plant Physiol, 127, 1256–1265.

268

Fruit and vegetable flavour

LEWINSOHN E., SITRIT Y., BAR E., AZULAY Y., MEIR A., ZAMIR D. AND TADMOR Y.

(2005) Carotenoid pigmentation affects the volatile composition of tomato and watermelon fruits, as revealed by comparative genetic analyses. J Agric Food Chem, 53, 3142– 3148. LI C., LIU G., XU C., LEE G.I., BAUER P., LING H.Q., GANAL M.W. AND HOWE G.A. (2003) The tomato suppressor of prosystemin-mediated responses2 gene encodes a fatty acid desaturase required for the biosynthesis of jasmonic acid and the production of a systemic wound signal for defense gene expression. Plant Cell, 15, 1646–1661. LUCCHETTA L., MANRIQUEZ D., EL-SHARKAWY I., FLORES F.B., SANCHEZ-BEL P., ZOUINE M., GINIES C., BOUZAYEN M., ROMBALDI C., PECH J.C. AND LATCHÉ A. (2007) Biochemical and catalytic properties of three recombinant alcohol acyltransferases of melon. Sulfurcontaining ester formation, regulatory role of CoASH in activity and sequence elements conferring substrate preference. J Agric Food Chem, 55, 5213–5220. LÜCKER J., BOUWMEESTER H.J., SCHWAB W., BLAAS J., VAN DER PLAS L.H. AND VERHOEVEN H.A. (2001) Expression of Clarkia S-linalool synthase in transgenic petunia plants results in the accumulation of S-linalyl-beta-D-glucopyranoside. Plant J, 27, 315–324. LÜCKER J., SCHWAB W., VAN HAUTUM B., BLAAS J., VAN DER PLAS L.H., BOUWMEESTER H.J. AND VERHOEVEN H.A. (2004a) Increased and altered fragrance of tobacco plants after metabolic engineering using three monoterpene synthases from lemon. Plant Physiol, 134, 510–519. LÜCKER J., SCHWAB W., FRANSSEN M.C., VAN DER PLAS L.H., BOUWMEESTER H.J. AND VERHOEVEN H.A. (2004b) Metabolic engineering of monoterpene biosynthesis: two-step production of (+)-trans-isopiperitenol by tobacco. Plant J, 39, 135–145. LUNKENBEIN S., SALENTIJN E.M., COINER H.A., BOONE M.J., KRENS F.A. AND SCHWAB W. (2006a) Up- and down-regulation of Fragaria × ananassa O-methyltransferase: impacts on furanone and phenylpropanoid metabolism. J Exp Bot, 57, 2445–2453. LUNKENBEIN S., BELLIDO M., AHARONI A., SALENTIJN E.M., KALDENHOFF R., COINER H.A., MUNOZ-BLANCO J. AND SCHWAB W. (2006b) Cinnamate metabolism in ripening fruit. Characterization of a UDP-glucose:cinnamate glucosyltransferase from strawberry. Plant Physiol, 140, 1047–1058. MAHMOUD S.S. AND CROTEAU R.B. (2001) Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase. Proc Natl Acad Sci USA, 98, 8915–8920. MAHMOUD S.S. AND CROTEAU R.B. (2002) Strategies for transgenic manipulation of monoterpene biosynthesis in plants. Trends Plant Sci, 7, 366–373. MANRIQUEZ D., EL-SHARKAWY I., FLORES F.B., EL-YAHYAOUI F., REGAD F., BOUZAYEN M., LATCHÉ A. AND PECH J.C. (2006) Two highly divergent alcohol dehydrogenases of melon exhibit fruit ripening-specific expression and distinct biochemical characteristics. Plant Mol Biol, 61, 675–685. MATHIEU S., TERRIER N., PROCUREUR J., BIGEY F. AND GUNATA Z. (2005) A carotenoid cleavage dioxygenase from Vitis vinifera L.: functional characterization and expression during grape berry development in relation to C13-norisoprenoid accumulation. J Exp Bot, 56, 2721–2731. MATSUI K., UJITA C., FUJIMOTO S., WILKINSON J., HIATT B., KNAUF V., KAJIWARA T. AND FEUSSNER I. (2000) Fatty acid 9- and 13-hydroperoxide lyases from cucumber. FEBS Lett, 481, 183–188. MATSUI K., FUKUTOMI S., WILKINSON J., HIATT B., KNAUF V. AND KAJWARA T. (2001) Effect of overexpression of fatty acid 9-hydroperoxide lyase in tomatoes (Lycopersicon esculentum Mill.). J Agric Food Chem, 49, 5418–5424. MCCASKILL D. AND CROTEAU R. (1998) Some caveats for bioengineering terpenoid metabolism in plants. Trends Biotech, 16, 349–355. MCGARVEY D.J. AND CROTEAU R. (1995) Terpenoid metabolism. Plant Cell, 7, 1015–1026. MITA G., QUARTA A., FASANO P., DE PAOLIS A., DI SANSEBASTIANO G.P., PERROTTA C., IANNACONE R., BELFIELD E., HUGHES R., TSESMETZIS N., CASEY R. AND SANTINO A. (2005)

Genes involved in the biosynthesis of aroma volatiles

269

Molecular cloning and characterization of an almond 9-hydroperoxide lyase, a new CYP74 targeted to lipid bodies. J Exp Bot, 56, 2321–2333. NUÑEZ-PALENIUS H.G., CANTLIFFE D.J., HUBER D.J., CIARDI J. AND KLEE H.J. (2006) Transformation of a muskmelon ‘Galia’ hybrid parental line (Cucumis melo L. var. reticulatus Ser.) with an antisense ACC oxidase gene. Plant Cell Rep, 25, 198–205. OHARA K., UJIHARA T., ENDO T., SATO F. AND YAZAKI K. (2003) Limonene production in tobacco with Perilla limonene synthase cDNA. J Exp Bot. 54, 2635–2642. OKE M., PINHERO R.G. AND PALIYATH G. (2003) The effects of genetic transformation of tomato fruit with antisense phospholipase D cDNA on the quality characteristics of fruits and their processed products. Food Biotech, 17, 163–182. PALIYATH G. AND DROILLARD M.J. (1992) The mechanisms of membrane deterioration and disassembly during senescence. Plant Physiol Biochem, 30, 789–812. PEREZ A.G., RIOS J.J., SANZ C. AND OLIAS J.M. (1992) Aroma components and free amino acids in strawberry variety Chandler during ripening. J Agric Food Chem, 40, 2232–2235. PICHERSKY E. AND GANG D.R. (2000) Genetics and biochemistry of secondary metabolites in plants: an evolutionary perspective. Trends Plant Sci, 5, 439–445. PICHERSKY, E., DUDAREVA, N. (2007) Scent engineering: toward the goal of controlling how flowers smell. Trends Biotech, 25, 105–110. PICTON S., GRAY J., BARTON S., ABUBAKAR U., LOWE A. AND GRIERSON D. (1993) cDNA cloning and characterisation of novel ripening-related mRNAs with altered patterns of accumulation in the ripening inhibitor (rin) tomato ripening mutant. Plant Mol Biol, 23, 193–207. PINHERO R.G., ALMQUIST K.C., NOVOTNA Z. AND PALIYATH G. (2003) Developmental regulation of phospholipase D in tomato fruits. Plant Physiol Biochem, 41, 223–240. RAAB T., LOPEZ-RAEZ J.A., KLEIN D., CABALLERO J.L., MOYANO E., SCHWAB W. AND MUNOZBLANCO J. (2006) FaQR, required for the biosynthesis of the strawberry flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone, encodes an enone oxidoreductase. Plant Cell, 18, 1023–1037. RÖMER S., FRASER P.D., KIANO J.W., MISAWA N., SCHUCH W. AND BRAMLEY P.M. (2000) Elevation of provitamin A content of transgenic tomato plants. Nat Biotech, 18, 1167–1171. ROSATI C., AQUILANI R., DHARMAPURI S., PALLARA P., MARUSIC C., TAVAZZA R., BOUVIER F., CAMARA B. AND GIULIANO G. (2000) Metabolic engineering of β-carotene and lycopene content in tomato fruit. Plant J, 24, 413–419. ROSCHER R., BRINGMANN G., SCHREIER P. AND SCHWAB W. (1998) Radiotracer studies on the formation of 2,5-dimethyl-4-hydroxy-3(2H)-furanone in detached ripening strawberry fruits. J Agric Food Chem, 46, 1488–1493. SALAS J.J., SANCHEZ C., GARCIA-GONZALEZ D.L. AND APARICIO R. (2005) Impact of the suppression of lipoxygenase and hydroperoxide lyase on the quality of the green odor in green leaves. J Agric Food Chem, 53, 1648–1655. SANZ C., OLIAS J.M. AND PEREZ A.G. (1997) Aroma biochemistry of fruits and vegetables. In: Phytochemistry of Fruit and Vegetables (Ed F.A. Tomas-Barberan), pp. 125–155. Oxford Science Publications, Oxford. SARRY J.-E. AND GUNATA Z. (2004) Plant and microbial glycoside hydrolases: volatile release from glycoside aroma precursors. Food Chem, 87, 509–521. SCHIJLEN E.G., RIC DE VOS C.H., VAN TUNEN A.J. AND BOVY A.G. (2004) Modification of flavonoid biosynthesis in crop plants. Phytochemistry, 65, 2631–2648. SCHWAB W. AND ROSCHER R. (1997) 4-Hydroxy-3(2H)-furanones: Natural and Maillard products. Recent Res. Dev. Phytochem, 1, 643–673. SCHWARTZ S.H., TAN B.C., GAGE D.A., ZEEVAART J.A. AND MCCARTY D.R. (1997) Specific oxidative cleavage of carotenoids by VP14 of maize. Science, 276, 1872–1874. SHALIT M., GUTERMAN I., VOLPIN H., BAR E., TAMARI T., MENDA N., ADAM Z., ZAMIR D., VAINSTEIN A., WEISS D., PICHERSKY E. AND LEWINSOHN E. (2003) Volatile ester formation in roses. Identification of an acetyl-coenzyme A. Geraniol/Citronellol acetyltransferase in developing rose petals. Plant Physiol, 131, 1868–1876.

270

Fruit and vegetable flavour

SHARON-ASA L., SHALIT M., FRYDMAN A., BAR E., HOLLAND D., OR E., LAVI U., LEWINSOHN E. AND EYAL Y. (2003) Citrus fruit flavor and aroma biosynthesis: isolation, functional

characterization, and developmental regulation of Cstps1, a key gene in the production of the sesquiterpene aroma compound valencene. Plant J, 36, 664–674. SIMKIN A.J., SCHWARTZ S.H., AULDRIDGE M., TAYLOR M.G. AND KLEE H.J. (2004a) The tomato carotenoid cleavage dioxygenase 1 genes contribute to the formation of the flavor volatiles beta-ionone, pseudoionone, and geranylacetone. Plant J, 40, 882–892. SIMKIN A.J., UNDERWOOD B.A., AULDRIDGE M., LOUCAS H.M., SHIBUYA K., SCHMELZ E., CLARK D.G. AND KLEE H.J. (2004b) Circadian regulation of the PhCCD1 carotenoid cleavage dioxygenase controls emission of beta-ionone, a fragrance volatile of petunia flowers. Plant Physiol, 136, 3504–3514. SOULEYRE E.J., GREENWOOD D.R., FRIEL E.N., KARUNAIRETNAM S. AND NEWCOMB R.D. (2005) An alcohol acyl transferase from apple (cv. Royal Gala), MpAAT1, produces esters involved in apple fruit flavor. FEBS J, 272, 3132–3144. SPEIRS J., LEE E., HOLT K., YONG-DUK K., STEELE SCOTT N., LOVEYS B. AND SCHUCH W. (1998) Genetic manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the balance of some flavor aldehydes and alcohols. Plant Physiol, 117, 1047–1058. SPEIRS J., CORRELL R. AND CAIN P. (2002) Relationship between ADH activity, ripeness and softness in six tomato cultivars. Scientia Horticulturae, 93, 137–142. TADMOR Y., FRIDMAN E., GUR A., LARKOV O., LASTOCHKIN E., RAVID U., ZAMIR D. AND LEWINSOHN E. (2002) Identification of malodorous, a wild species allele affecting tomato aroma that was selected against during domestication. J Agric Food Chem, 50, 2005– 2009. TAN B.C., SCHWARTZ S.H., ZEEVAART J.A. AND MCCARTY D.R. (1997) Genetic control of abscisic acid biosynthesis in maize. Proc Natl Acad Sci USA, 94, 12235–12240. TESNIÈRE C. AND VERRIÈS C. (2000) Molecular cloning and expression of cDNAs encoding alcohol dehydrogenases from Vitis vinifera L. during berry development. Plant Science, 157, 77–88. THOLL D. (2006) Terpene synthases and the regulation, diversity and biological roles of terpene metabolism. Curr Opin Plant Biol, 9, 297–304. TIEMAN D., TAYLOR M., SCHAUER N., FERNIE A.R., HANSON A.D. AND KLEE H.J. (2006) Tomato aromatic amino acid decarboxylases participate in synthesis of the flavor volatiles 2phenylethanol and 2-phenylacetaldehyde. Proc Natl Acad Sci USA, 103, 8287–8292. TIJET N., SCHNEIDER C., MULLER B.L. AND BRASH A.R. (2001) Biogenesis of volatile aldehydes from fatty acid hydroperoxides: molecular cloning of a hydroperoxide lyase (CYP74C) with specificity for both the 9- and 13-hydroperoxides of linoleic and linolenic acids. Arch Biochem Biophys, 386, 281–289. VAN DER STRAETEN D., RODRIGUES POUSADA R.A., GIELEN J. AND VAN MONTAGU M. (1991) Tomato alcohol dehydrogenase. Expression during fruit ripening and under hypoxic conditions. FEBS Lett, 295, 39–42. WANG C., CHIN C.K., HO C.T., HWANG C., POLASHOCK J. AND MARTIN C.E. (1996) Changes of fatty acids and fatty acid-derived flavor compounds by expressing the yeast ∆-9 desaturase gene in tomato. J Agric Food Chem, 44, 3399–3402. WANG C., XING J., CHIN C.K., HO C.T. AND MARTIN C.E. (2001) Modification of fatty acids changes the flavor volatiles in tomato leaves. Phytochemistry, 58, 227–232. WANG J. AND DE LUCA V. (2005) The biosynthesis and regulation of biosynthesis of Concord grape fruit esters, including ‘foxy’ methylanthranilate. Plant J, 44, 606–619. WEI S., MARTON I., DEKEL M., SHALITIN D., LEWINSOHN E., BRAVDO B.-A. AND SHOSEYOV O. (2004) Manipulating volatile emission in tobacco leaves by expressing Aspergillus niger beta-glucosidase in different subcellular compartments. Plant Biotech J, 2, 341–350. WEIN M., LAVID N., LUNKENBEIN S., LEWINSOHN E., SCHWAB W. AND KALDENHOFF R. (2002) Isolation, cloning and expression of a multifunctional O-methyltransferase capable of forming 2,5-dimethyl-4-methoxy-3(2H)-furanone, one of the key aroma compounds in strawberry fruits. Plant J, 31, 755–765.

Genes involved in the biosynthesis of aroma volatiles

271

WYLLIE S.G. AND LEACH D.N. (1990) Aroma volatiles of Cucumis melo cv ‘Golden Crispy’.

J Agric Food Chem, 38, 2042–2044. YAHYAOUI F.E.L., WONGS-AREE C., LATCHÉ A., HACKETT R., GRIERSON D. AND PECH J.-C.

(2002) Molecular and biochemical characteristics of a gene encoding an alcohol acyltransferase involved in the generation of aroma volatile esters during melon ripening. Eur J Biochem, 269, 2359–2366.