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Volatile compounds play key roles in fruit, as they are involved in defense against pests and diseases, act as a cue to attract or repel seed dispersers, and are major components determining fruit flavor. Fruit volatile production is highly dynamic and tightly coordinated along the process of ripening, with most compounds showing dramatic changes in their biosynthesis in a short lapse of time. Ripe fruits typically produce a blend consisting of a few hundreds of volatile compounds of different chemical nature which originate from several independent metabolic pathways (Granell and Rambla 2013). In most cases these pathways are not new to the plant, and the same or similar volatiles are also produced by other plant organs. Some pathways are preferentially activated in the fruit, and some of them are expressed only in certain species. Volatiles produced by fruit appear to have played an important role in defining eating habits (i.e., frugivore vs. herbivore) and other plant-insect interactions (Dweck et al. 2015).
THE MAIN PATHWAYS RESPONSIBLE FOR THE BIOSYNTHESIS OF FRUIT VOLATILES
Unlike flower scent, which is produced and emitted by the intact flower or flower organs, many of the volatile compounds emitted by the fruit are produced after physical tissue disruption, like that produced during overripening or in the mouth when chewing the fruit. In fact, the volatile profile produced by the intact fruit is dramatically different from that of the same fruit after being cut into pieces, and even more after complete homogenization. This fact, together with the techniques used for volatile extraction and determination, is in some extent responsible for the divergence observed in the scientific literature regarding fruit volatile profiles (Rambla et al. 2015). Therefore, different approaches should be considered when analyzing fruit volatiles depending on the aim of the study. For instance, when studying frugivore attraction, focus should be set on the compounds emitted by the whole intact fruit, while when studying fruit flavor, the tissues should be homogenized to some extent, somewhat mimicking the chewing process taking place in the mouth. The same consideration applies for the identification of the enzymes and genes involved in these processes.
Fruit volatile compounds are most often produced by means of several independent metabolic pathways from different substrates previously accumulated in the fruit during its development. These substrates include several amino acids, essential fatty acids and some other healthy/beneficial compounds such as carotenoids. In fact, most of the volatile precursors have positive effects on the frugivores feeding on them. Therefore it has been proposed that fruit volatile compounds could act as sensory cues for health and nutritional value for their predators (Klee and Giovannoni 2011).
In general terms, the volatile metabolic pathways are essentially the same in most plant organs. Nevertheless, according to the specific organ function they are activated differently, each organ producing a characteristic and often dramatically different volatile profile. As in all plant organs, fruit volatile biosynthesis is a developmentally and environmentally regulated process. The volatile profile in the fruit shows ample modifications along development and is also modulated by the environmental conditions. In most species, the process of ripening implies a dramatic shift in volatile production. Below, we are going to focus on the ripe fruit, summarizing the most universal metabolic pathways leading to fruit volatile production.
10.2.1 Fatty Acid Derivatives
Volatiles derived from fatty acid metabolism are among the most ubiquitous compounds. Although also emitted by the intact fruit, a burst of these compounds is produced upon tissue disruption. They are typically oxygenated linear molecules, most often aldehydes, ketones or alcohols, originated from the cleavage of fatty acids, preferentially linoleic and linolenic acids. These include the so called green leaf volatiles (Chapter 9), which are C6 molecules providing green aromatic notes to the fruit flavor. Longer chain molecules tend to provide fatty notes and, in the case of some C9 compounds, cucumber-like aroma.
Although other metabolic pathways also lead to the production of fatty acid derivatives in fruit, such as fatty acid biosynthesis (as in the case of methyl ketone synthases/acylsulfurylases) or p-oxidation and a-oxidation of preformed fatty acids, the most important and well-studied metabolic pathway involved in their biosynthesis is the lipoxygenase pathway. The initial step in this pathway is lipid hydrolysis by a lipase to release the free fatty acids (Garbowicz et al. 2018). Lipoxygenases (LOX) are fatty acid dioxygenases that catalyze the dioxygenation of polyunsaturated fatty acids with a (lZ,4Z)-pentadiene moiety, such as the Cl8 linoleic and linolenic acids, producing the corresponding hydroperoxides, which are further metabolized by hydroperoxide lyases (HPL) to produce a volatile aldehyde and an oxoacid. Depending on the position where the fatty acid is oxygenated, lipoxygenases are classified as 13-LOX or 9-LOX. The resulting hydroperoxides are cleaved by means of 13-HPL or 9-HPL respectively. Depending on the species, different activities are predominant. For instance, the most abundant compounds produced in the tomato fruit are hexanal and (Z)-3-hexenal, which are produced by the 13-LOX gene TomLoxC, while 9-LOX activity is responsible for the production of several C9 volatiles in cucumber, providing this fruit its characteristic aroma. Most plants have several isoforms of lipoxygenases, but usually only one or a few of them are involved in volatile biosynthesis in the fruit. The aldehydes produced can be reversibly converted into alcohols by alcohol dehydrogenases (ADH). These enzymes also participate in the reduction to alcohols of the aldehydes synthesized by means of other metabolic pathways. Eventually, the alcohols produced can be further metabolized by means of alcohol acyl transferases to produce esters (Klee and Tieman 2018).
10.2.2 Phenylpropanoids AND Benzenoids
This group comprises a large class of volatile compounds with an aromatic ring in their chemical structure that derives from the amino acid phenylalanine. Each compound produces a different perception. For instance, eugenol and cinnamaldehyde provide spicy notes, phenylacetaldehyde is pungent, guaiacol is smoky or pharmaceutical, while 2-phenylethanol is sweet and floral. This group also includes vanillin, the world’s most used flavor additive.
In general terms, most volatile compounds in the fruit are either only synthesized during ripening or show a dramatic increase when the fruit ripens. Nevertheless, this is not always the general rule in benzenoids. In some species like tomato, although some phenylalanine-derived volatiles such as 2-phenylethanol increase during ripening, several volatile phenylpropanoids such as guaiacol or methyl salicylate often decrease dramatically, thus suggesting a role of the latter compounds in mechanisms of defense against pests or pathogens, or in discouraging frugivores from feeding on the unripe fruit (Tikunov et al. 2013).
Most compounds in this group are synthesized by means of the phenylpropanoid pathway, also leading to the synthesis of lignin and many other important fruit plant metabolites such as antho- cyanins. The first step in this pathway is the conversion of phenylalanine into (£)-cinnamic acid by means of an 1 -phenylalanine ammonia-lyase (PAL), from which metabolism is driven to the biosynthesis of a variety of both volatile and nonvolatile metabolites. Nevertheless, a few other volatiles are synthesized by alternate metabolic pathways, such as phenylacetaldehyde or 2-phenyl- ethanol, which are directly produced from phenylalanine (Granell and Rambla 2013).
10.2.3 Branched-Chain Volatiles
These are originally small molecules originated in the metabolic pathway of branched-chain amino acids leucine, isoleucine and valine. Most of these compounds are aldehydes, alcohols or acids, and impact fruit flavor in a number of species, including tomato, banana, apple and strawberry. Most of them tend to provide the fruit roast/burned or cheese-like aromatic notes. Branched-chain alcohols can also be used as precursors for the biosynthesis of esters. In the case of banana, branched-chain esters are the compounds providing the dominant aroma notes to the fruit (Pino et al. 2017).
Their biosynthesis has not yet been completely unraveled. Branched-chain amino acid transaminases SIBCAT1, SIBCAT2 and CmBCATl catalyzing the conversion of the amino acids into the corresponding a-ketoacids have been identified in tomato and melon respectively. Such a-ketoacids, either obtained from amino acid catabolism or by de novo synthesis, have been proposed to be the most relevant precursors of the volatile compounds, which would be obtained by decarboxylation to produce aldehydes and eventually, after further modification, the corresponding alcohols, acids and esters (Gonda et al. 2010).
Biosynthesis of volatile esters is mediated by alcohol acyl transferases (AAT), which catalyze the acyl esterification of alcohols by means of an acyl-CoA thioester donor. AATs are mid-size families of enzymes from the BAHD superfamily with different tissue-specific pattern producing both volatile and nonvolatile esters. Several AATs have been identified as responsible of the production of fruit volatile esters, such as SAAT in strawberry (Aharoni et al. 2000), CmAATl, СтААТЗ and CmAAT4 in melon (El-Sharkawy et al. 2005), VpAATl in mountain papaya (Balbontin et al. 2010) or MpAATl in apple (Dunemann et al. 2012). Methyl esters are a specific type of esters synthesized by S-adenosyl-1 -methionine dependent O-methyltransferases, which make up the SABATH family of enzymes. Several genes from this family have been described such as SAMT in tomato, which is involved in the biosynthesis of methyl salicylate (Tieman et al. 2010), or FaOMT in strawberry catalyzing the methylation of furaneol into mesifurane (Zorrilla- Fontanesi et al. 2012).
The most common substrates for ester biosynthesis are fatty acid derivatives and branched- chain volatiles. Most esters provide fruity notes and sometimes even the characteristic flavor to some fruit species. Esters are predominant in the volatile profile of many fruit such as strawberry, peach, banana, or the climacteric varieties of melon. Interestingly, this ability to synthesize a large amount of diverse esters does not rely on large gene families. For instance, in wild strawberry Fragaria vesca only six AATs were identified after sequencing the whole genome (Shulaev et al. 2011). Nevertheless, other species lack almost completely ester compounds in their volatile profile, as in the case of tomato fruit. Interestingly, both cultivated tomato and all the red-fruited related wild species show a high esterase activity which degrade a set of (for humans) unpleasant esters which are synthesized in both the red- and green- fruited species, but which only accumulate in the green-fruited species showing a lower esterase activity (Goulet et al. 2012).
Terpenoids constitute the largest class among the plant volatile compounds. Although some terpenoids are linear, most of them are cyclic molecules. Fruit terpenes can be found either as hydro- carbonated molecules or further modified, often as aldehydes, alcohols, ketones or even esters (Gonzalez-Mas et al. 2019).
These metabolites are often abundant in the vegetative tissues of many plant species, and also have an important role in the volatile profile of some fruit such as mango, different Citrus species, where monoterpenes are dominant or, in a lesser extent, strawberry and some white grape varieties. Nevertheless, in general terms their relevance in fruit is less important than in vegetative tissues. For instance, mono- and sesquiterpenoids are dominant in the volatile profile of tomato leaves, as they accumulate at high levels in glandular trichomes where they have a key role in plant defense against pests and pathogens (Lopez-Gresa et al. 2017). However, terpenoids represent only a tiny part of the tomato fruit volatile blend (Rambla et al. 2017).
Biosynthesis of terpenoid volatiles is produced by a mid-size family of terpene synthases by means of two alternate metabolic pathways localized in the plastids and the cytosol respectively, and they originate from the five carbon precursors isopentenyl diphosphate (IPP) and dimethylal- lyl diphosphate (DMAPP). These serve as substrates to prenyltransferases to produce C10 geranyl diphosphate (GPP) and C15 farnesyl diphosphate (FPP), which are the precursors of monoterpenes and sesquiterpenes respectively (Chapter 8). Terpene synthases are responsible for the production of volatile mono- and sesquiterpenes; TPSs specifically involved in the synthesis of either mono- or sesquiterpenes are the rule, but TPS enzymes with both activities have also been described (Aharoni et al. 2004). Correlation between expression levels of biosynthetic genes and volatile production suggests that terpenoid synthesis would be regulated transcriptionally, as observed for the CsTpsl gene in orange (Sharon-Asa et al. 2003), VvVal, VvGerD and VvTer in grape (Lucker et al. 2004) and FaNESl in strawberry (Aharoni et al. 2004). It is interesting again that species producing a range of terpenes in fruit can do it w'ith a small number of terpene synthases, i.e., just three NES and 4 PINS in wild strawberry (Shulaev et al. 2011). Similarly, in the octoploid strawberry, 89% of the biosynthesis of the terpene geranyl acetate is due to the expression of the homeologs from F. vesca (Edger et al. 2019).
Volatile apocarotenoids are a small family of linear or cyclic norisoprenoid molecules, which can therefore be considered as terpenoid compounds. Their distinctive characteristic is that they are originated from the cleavage of tetraterpenes. Apocarotenoids are not usually among the most abundant compounds in the volatile profile of fruits, but in many fruit species they have an impact on their flavor and aroma, as the human olfactory receptors are extremely sensitive to these compounds, particularly to the cyclic ones. Apocarotenoids tend to provide fruity, floral or sweet notes to the fruit, and are therefore considered as desirable compounds for consumer flavor perception (Goff and Klee 2006).
Apocarotenoids are biosynthesized by a small family of carotenoid cleavage dioxygenases (CCD), and produce either linear or cyclic compounds depending on the chemical structure of the substrate. These are promiscuous enzymes which can recognize carotenes after ^-carotene in the carotene biosynthesis pathway, and have the ability to cleave double bonds in cyclic carotenoids in the 9-Ю (9',Ю') or 7-8 (7',8') positions, and linear carotenoids also in the 5,6 (5',6') positions, thus liberating and oxygenated Cl3, C10 or C8 volatile moiety respectively (Ilg et al. 2009).
Regulation of their biosynthesis seems to be predominantly due to substrate availability, as CCD expression has been detected in the fruit in early developmental stages when no volatile apocarotenoids are produced at significant levels. In fact, their biosynthesis mainly takes place after chloro- plast conversion into chromoplasts, where carotenoids accumulate, and after membrane degradation or tissue disruption, which favors their contact with the CCD enzymes (Vogel et al. 2010). It is interesting to note that despite their hydrolytic origin, some volatile apocarotenoids in fruit are later converted in nonvolatile forms by conjugation to sugars (Wirth et al. 2001).