Biochemistry of Apple Aroma : A Review

Apple quality is determined by att ributes such as appearance, fi rmness and fl avour, as well as by the absence of physiological and pathological disorders. However, the concept of quality in fruits has evolved, and increasing attention is currently given to sensory att ributes to achieve higher acceptance by consumers (1). Flavour is one the most important and distinctive features of apples, and it is determined by both taste and aroma (2,3). While taste is mainly determined by sugars and organic acids, aroma is a complex mixture of many volatile compounds whose composition is specifi c to the species and oft en to the variety (3–5). The volatile aroma compounds in apple have been studied for more than 50 years. In this period, more than 300 compounds have been identifi ed (6–8), with only a few that contribute signifi cantly to the fruit aroma. They mostly include esters, alcohols, aldehydes, ketones and ethers (6). Aldehydes predominate in immature apples (8–10), but their content decreases as the fruit matures, a period in which the concentration of alcohols and esters increases, the latt er being the main volatile compounds in ripe apples (11,12). All the volatile compounds are of great importance for the complete characteristic aroma profi le of apples (8). Their composition and concentration diff er among varieties (4,13,14), and their production can also be aff ected by several factors before, during and aft er the harvest.


Introduction
Apple quality is determined by att ributes such as appearance, fi rmness and fl avour, as well as by the absence of physiological and pathological disorders.However, the concept of quality in fruits has evolved, and increasing attention is currently given to sensory att ributes to achieve higher acceptance by consumers (1).Flavour is one the most important and distinctive features of apples, and it is determined by both taste and aroma (2,3).While taste is mainly determined by sugars and organic acids, aroma is a complex mixture of many volatile compounds whose composition is specifi c to the species and oft en to the variety (3)(4)(5).The volatile aroma compounds in apple have been studied for more than 50 years.In this period, more than 300 compounds have been identifi ed (6)(7)(8), with only a few that contribute signifi cantly to the fruit aroma.They mostly include esters, alcohols, aldehydes, ketones and ethers (6).Aldehydes predominate in immature apples (8)(9)(10), but their content decreases as the fruit matures, a period in which the concentration of alcohols and esters increases, the latt er being the main volatile compounds in ripe apples (11,12).All the volatile compounds are of great importance for the complete characteristic aroma profi le of apples (8).Their composition and concentration diff er among varieties (4,13,14), and their production can also be aff ected by several factors before, during and aft er the harvest.

Volatile Aroma Compounds in Apple
In apples, the profi le of volatile compounds changes with maturation; aldehydes predominate at the beginning, then the content of alcohols starts to increase considerably, and fi nally the profi le is dominated by esters (15).Therefore, it is important to discuss exactly how the cultivar and biotic and abiotic factors aff ect the profi le of aldehydes, alcohols and esters in apples.
Fatt y acids with 16 and 18 carbons are the most predominant in apples (C16:0, C18:0, C18:1, C18:2 and C18:3) (71), which are the principal substrates for the production of volatiles (72).The lipid content and concentration of fatt y acids in pre-and postharvest climacteric apples are similar, reaching their maximum concentration in the climacteric period (71)(72)(73)(74)(75).The most important amino acids for the biosynthesis of volatile compounds in apples are OTH=odour threshold; GD=Golden Delicious, GS=Granny Smith, MG=Mondial Gala, GR=Golden Reinders, FJ=Fuji, PL=Pink Lady; tr=traces those with branched chains (leucine, isoleucine and valine), although in other fruits, phenylalanine, tyrosine and tryptophan are important as well (76,77).The concentration of most amino acids decreases with the maturation of apples (78)(79)(80)(81) due to the synthesis and metabolism of proteins (78).During storage, an initial reduction in the content of amino acids occurs, without signifi cant change aft erwards (78).On the other hand, there are two main groups of flavour compounds that come directly from carbohydrate metabolism: terpenoids and furanones (3).
The following section describes the main pathways involved in the biosynthesis of volatile compounds in apples from fatt y acids, amino acids and carbohydrates.

Fatt y acid metabolism
In apples, β-oxidation and the lipoxygenase (LOX) pathway are the two main enzymatic systems in the catabolism of fatt y acids for the formation of aldehydes, alcohols and esters (32,34,44,82), the former being more important in intact fruit (16,83) and the latt er in cut fruit (5,84).Nonetheless, as apples mature, the rates of both lipid synthesis and degradation increase, causing a change in membrane fl uidity, thereby increasing its permeability to diff erent substrates (71,83).This, along with the breakdown of chloroplasts, which release fatt y acids such as linoleic (C18:2) and linolenic (C18:3) acids (71), make the LOX pathway an alternative to β-oxidation of the whole fruit, which is confi rmed by high activity of the LOX pathway enzymes during apple development (34,54,73,85).

β-Oxidation of fatt y acids
β-Oxidation is the main pathway involved in the degradation of fatt y acids, and in plants it is mainly performed in peroxisomes (86)(87)(88), which contain all the necessary enzymes.During β-oxidation, fatt y acids are activated to their corresponding acyl coenzyme A (CoA) by acyl-CoA synthase in a reaction requiring adenosine triphosphate (ATP), Mg 2+ and coenzyme A with sulfh ydryl functional group (CoASH) (89,90).The acyl-CoA is then imported into the peroxisome.
The main cycle of β-oxidation, known as the core β-oxidation cycle, includes four enzymatic reactions (87) performed by three proteins: (i) acyl-CoA oxidase, (ii) a multifunctional protein containing domains responsible for four enzymatic activities (2-trans-enoyl-CoA hydratase, l-3-hydroxyacyl-CoA dehydrogenase, d-3-hydroxyacyl-CoA epimerase and ∆ 3 ,∆ 2 -enoyl-CoA isomerase), and (iii) l-3-ketoacyl-CoA thiolase (89,90).In the fi rst reaction of the main cycle of β-oxidation, acyl-CoA is transformed into trans-2-enoyl-CoA by acyl-CoA oxidase.This reaction requires fl avin adenine dinucleotide (FAD) as a cofactor and O 2 as an electron acceptor.The O 2 is reduced to H 2 O 2 , which is degraded by catalase inside the peroxisome (90).In the second reaction, 2-trans-enoyl-CoA hydratase catalyzes the hydration of trans-2-enoyl-CoA to 3-hydroxyacyl-CoA, which is oxidized to 3-ketoacyl-CoA by l-3-hydroxyacyl-CoA dehydrogenase in the third reaction of the cycle, requiring NAD + as a cofactor.In the fourth reaction of the β-oxidation cycle, 3-ketoacyl-CoA thiolase catalyzes the breakage of the thiol end of 3-ketoacyl-CoA resulting in one molecule of acetyl-CoA and one of acyl-CoA aft er the removal of two carbons, which return to the β-oxidation cycle.This cycle is repeated with the oxidative removal of two carbon atoms in the form of acetyl--CoA from the carboxyl end of a fatt y acid until it is completely oxidized (Fig. 1; 86,87,[89][90][91].The fi nal product of the β-oxidation of fatt y acids with odd number of carbon atoms is an acyl-CoA in which the fatt y acid has fi ve carbon atoms.The products of oxidation and breakdown are acetyl-CoA and propionyl-CoA (91).The enzymes of the main β-oxidation cycle are able to catabolize linear saturated fatt y acids or those with double trans bonds in the ∆ 2 position (86,89,90).Some linear fatt y acids with double cis bonds have odd number of carbon atoms and they form enoyl-CoA molecules that cannot be metabolized by the enzymes of the main β-oxidation cycle.Three auxiliary enzymes have been identifi ed: (i) a ∆ 3 ∆ 2 -enoyl-CoA isomerase that converts 3-cis-or 3-trans--enoyl-CoA to 2-trans-enoyl-CoA, which can be incorporated into the main β-oxidation cycle (90), (ii) a 2,4-dienoyl--CoA reductase that catalyzes the conversion of 2-trans,-4-cis-dienoyl or 2-trans,4-trans-dienoyl-CoA into 3-trans--enoyl-CoA, and (iii) a ∆ 3,5 ∆ 2,4 -dienoyl-CoA isomerase that catalyzes the conversion of 3,5-dienoyl-CoA to 2,4-dienoyl-CoA (86,87).All these enzymes are required for the conversion of fatt y acids in plants (90).Linoleic and linolenic acids have double bonds with a cis confi guration and an even number of carbon atoms.Fig. 1 shows the enzymes, cofactors and products involved in the degradation of stearic, oleic and linoleic acids, all present in apples, through the β-oxidation pathway (86,87,(89)(90)(91).The β-oxidation of long-chain fatt y acids produces shorter acids such as acetic, butanoic and hexanoic acids, which can be reduced to their corresponding alcohols ( 16) before being esterifi ed with acyl-CoA by the alcohol acyltransferase (AAT) enzyme.The combination of acyl-CoA molecules with diff erent alcohols results in an important range of esters.However, given that acetyl-CoA is the main acyl-CoA produced in β-oxidation, most of the esters are acetate esters (23).
The genes MdLOX1a and MdLOX5e were found to be involved in the production of volatiles in Alkmene, Discovery, McIntosh, Royal Gala and Prima apples.More recently, four genes (LOX1a and -c and LOX2a and -b) were related to Golden Delicious apple aroma (106).The genes MdLOX2 and MdLOX5 were expressed in leaves, fl owers and fruits of Golden Delicious and McIntosh, whereas the remaining genes were expressed in a diff erent way or were absent from tissues.LOX genes exclusively expressed in fruits were not found (96).Of the 22 LOXs, 17 were expressed in Jonagold apple peel (107).However, litt le is known about the specifi c function of each LOX isoform (96).
Products of the LOX reaction can be converted to different compounds, through at least six pathways (100).One of these pathways is through the hydroperoxide lyase (HPL) enzyme (108) (Fig. 2; 16,29,32,63,109).HPL is an enzyme belonging to the cytochrome P450 CYP74B/C family and acts on hydroperoxides with no need for cofactors (63) to form short-chained aldehydes (6 or 9 carbon atoms) (16,109).Some HPL enzymes break only 9-HPOs (110), others act only on 13-HPOs, and others have dual specifi city (111), which can infl uence the aroma profi le of the fruit (5).However, the 9-LOX hydroperoxide derivatives are not totally understood (107).In the apple genome, a total of 39 MdALDH genes were identified (112).
Aldehydes (6 or 9 carbon atoms) are subsequently reduced to the corresponding alcohol by the enzyme alcohol dehydrogenase (ADH) (63).ADH is an oxidoreductase that catalyzes the reversible reduction of aldehydes to alcohols (68,113,114), and its direction is infl uenced by the pH.However, at physiological pH, the reaction favours the production of alcohols (29).ADH acts on a wide range of linear, branched and cyclic alcohols (29), showing preference for the former in apples (28,115).It also requires the presence of the reduced coenzymes NADH and NADPH, which possess two diastereotopic hydrogens, pro-R and pro-S; the substrate can be att acked from both ends resulting in (S) or (R) alcohols.Most ADH enzymes form (S) alcohols (116).

Amino acid metabolism
Amino acids are precursors of volatile aromatic compounds such as aldehydes, alcohols, acids and esters, being the second most important source of volatile compounds in the aroma of fruits and vegetables (5,32,68).In apples, the production of branched-chain esters has been reported from the branched amino acids isoleucine (Ile), leucine (Leu) and valine (Val) (17,137).These amino acids are branched compounds of aliphatic nature and are synthesized in chloroplasts (138,139).Free amino acids in cells originate from proteolysis (140) Golden Delicious Ethephon increased the LOX activity in the apple treated with 1-MCP and stored for 14 weeks under regular atmosphere (127)

Hydroperoxide lyase (HPL)
On the tree Pink Lady HPL activity increased up to 1 month before harvest (54) Golden Reinders HPL activity decreased in apple skin with a slight increase at the beginning of climacteric stage, it remained lower and unchanged in the pulp (119)

At harvest
Royal Gala HPL enzyme was insensitive to ethylene (40) Golden Delicious Expression of the MdHPL gene was significantly enhanced by ethylene treatment, while 1-MCP treatment had no significant eff ect (121)

During storage
Pink Lady 7 months under low or ultra-low oxygen: higher activity of HPL under ultra-low oxygen storage ( Fuji 5-7 months under ultra-low oxygen: higher activity in the skin aft er 5 months, and in the pulp aft er 7 months Golden Reinders 5-7 months under ultra-low oxygen: no diff erence in the activity in the pulp (45)

Alcohol dehydrogenase (ADH)
On the tree Fuji ADH activity decreased with ripening on the tree (34) Jonagold ADH activity increased until harvest (85)

At harvest
Greensleeves Maximum activity at harvest; insensitive to ethylene (44)

Fuji
Higher activity of ADH in the pulp than in the skin (123) Royal Gala Of 10 genes, only ADH1 gene expression was decreased by ethylene (40)

Golden Reinders
The activity remained stable and increased in the climacteric stage (119) Golden Delicious Ethylene treatment increased the expression of MdADH3 gene, while the expression of MdADH1 decreased.1-MCP treatment increased the expression of MdADH1, while there was no significant eff ect on MdADH2 and MdADH3 (127)

During storage
Mondial Gala 6 months under regular or controlled atmosphere: higher ADH activity under controlled than under regular atmosphere (129)  in the synthesis of branched amino acids and the fi rst step of their degradation (138,139).Leu is degraded in plant mitochondria, and Val and Ile might also be degraded in this organelle (138,139) or at least converted into their corresponding ketoacids (90,139) because some of the enzymes required for the catabolism of Leu and Ile are also found in peroxisomes (141).The degradation of branched amino acids helps to maintain the balance between NAD + and NADH+H + .Moreover, the produced acyl-CoA serves as an energy source for ATP production (142).
The catabolism of amino acids has been well documented in bacteria and yeasts (18,(142)(143)(144).In fruits, it has been demonstrated that the reactions for the derivation of volatile compounds from branched amino acids follow the pathway found in some of these microorganisms.In apples (17,67,137) and fruits such as melon (31), these pathways have been elucidated by the exogenous addition of labelled amino acids.The addition of labelled Leu, Ile, and Val produces a diff erent ester patt ern in different apple varieties, which indicates that the isozymes diff er in their substrate selectivity (137).
In the second, 2-oxo acids can be converted to fatt y acids by oxidative decarboxylation.First, the α-keto acid is irreversibly converted to a branched-chain acyl-CoA (2-methyl butanoil-CoA, 4-methyl butyl-CoA and 2-methyl propyl-CoA from Ile, Leu and Val, respectively) (141,149) by the action of branched-chain α-keto acid dehydrogenase (BCKDH), which substitutes a CO 2 from the 2-oxo acid for the CoA cofactor with NAD + reduction (140).Then, the acyl-CoA can be converted to a fatt y acid by phosphate acetyltransferase (PAT) (18,140).Branched fatty acids can be transformed into alcohols and esters by the reduction of the acid to its corresponding aldehyde (67).Alcohols formed by the Ehrlich pathway can be es-terifi ed with acyl-CoA by AAT to form branched-chain esters (8,17,137).
In plant tissues (90) and in yeasts (150), it has been reported that 2-methyl-2-butenyl-CoA derived from the catabolism of Ile can be degraded to acetyl-CoA and propyl-CoA through β-oxidation reactions (90).However, these esters derived from Ile by this pathway have not been reported in apples.In melon, the formation of propanoate from 2-methylbutyl by the addition of Ile has been reported (31).
In the third pathway, α-oxo acids can also be reduced to hydroxy acids by a hydroxyacid dehydrogenase (HDH), which is specifi c for each α-oxo acid (18,142,145).Hydroxy acids do not contribute to aroma, but they allow the conversion of NADH to NAD + (140).Fig. 3 shows the formation of volatile compounds from the catabolism of l-isoleucine (17,18,31,67,90,140,142,143,151).The levels of precursor amino acids do not always explain the formation of the corresponding esters, therefore, it has been suggested that the selectivity of enzymes preceding AAT has an important role in the composition of the formed esters (49).

Terpenoids and phenylpropenes
Terpenoids are synthesized via two parallel pathways: (i) mevalonate (MVA) pathway, which operates in the cytosol and starts with the condensation of two acetyl--CoA molecules, and (ii) methylerythritol phosphate (MEP) pathway active in the plastids, which starts with pyruvate and glyceraldehyde 3-phosphate as precursors to form 1-de oxyxylulose 5-phosphate.Both MVA and MEP routes result in the formation of isopentyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMADP), intermediaries from which all terpenes are derived.Sesquiterpenes (C15) and triterpenes are produced via MVA pathway, while monoterpenes (C10), diterpenes (C20) and tetraterpenes (C40) are produced via MEP pathway (3,63,64,68,69,152,153).The catalytic conversion of the terpene precursors is carried out by terpene synthase enzymes.Nieuwenhuizen et al. (154) reported that genome of cultivated apple (Malus domestica) contains 55 putative terpene synthase genes, ten of which appear to be functional.
Monoterpenes and sesquiterpenes are the main fruit aroma components from the isoprenoid family in the apple fruit.The acyclic branched sesquiterpene (E,E)-α--farnesene, synthesized predominantly in epidermal and hypodermal cell layers of the fruit (155), is the most associated with ripe apple fruit (154,156,157).However, α-farnesene has also been associated with superfi cial scald in the skin of several apple cultivars (155).According to Pechous and Whitaker (158) (E,E)-α-farnesene is produced during storage of apple and its production decreases aft er about 2 months of storage.Other terpenes occur in very low amounts in fruit and floral and vegetative tissues.
Phenylpropenes, such as eugenol, estragole and isoestragole, also contribute to apple flavour and aroma.These compounds are derived from the phenylpropanoid pathway (162).Estragole imparts a spicy/aromatic flavour to some apple varieties like Ellison's Orange, D'Arcy Spice and Fenouillet, and an aniseed note to fresh Royal Gala apple (162).

Factors Aff ecting the Production of Volatiles
There are several factors that aff ect the synthesis of volatile aromatic compounds in apple.These factors may be classifi ed as preharvest, harvest or postharvest factors, depending on the period of time during apple growth when they are relevant.The eff ect of these factors on the production of aroma volatiles in apple is described in this section.

Preharvest factors
The aroma of an apple greatly depends on the variety.Diff erences in the concentrations of volatile com-pounds among diff erent apple varieties give them a characteristic aroma patt ern (4,12).Table 2 shows the main aromatic volatile compounds in diff erent apple varieties.It shows that 2-methylbutyl acetate is the main ester in apples such as Bisbee Red Delicious (10), Redchief Delicious (35) and Fuji (48,123), whereas butyl acetate is the main ester in Golden Delicious (163,164), Royal Gala (165) and Mondial Gala apples (53,124).
Environmental factors can also aff ect the composition of volatiles in apples.Likewise, it has been reported that the profi le of volatile compounds can be aff ected by the weather (166), geographic location (167) and cultural practices (168).
Although the aroma of apples is a highly heritable feature only minimally infl uenced by the harvest, Dunemann et al. (52) reported that a variety can develop a different aromatic compound profi le in diff erent production years (14,34,169).
The volatile compounds of the aroma are not produced in signifi cant amounts during apple growth, but they increase during the climacteric period, when the production of ethylene induces a series of physical and chemical changes in the expression of certain genes and the activity of certain enzymes (170,171).While total esters are found in very low concentrations when endogenous ethylene levels are low, ester production increases rapidly as soon as ethylene synthesis starts (75).Given that the production of esters is regulated by ethylene (172), inhibitors of this hormone added in the preharvest stage reduce the production of volatiles in the fruit.18,31,67,90,140,142,143,151.BCAT=branched-chain amino acid transferase, GDH=glutamate dehydrogenase, PDC=piruvate decarboxylase, BCKD=branched--chain α-ketoacid dehydrogenase, HDH=2-hydroxy-3-methyl-pentanoic acid dehydrogenase, BCCoDH=2-methyl branched-chain acyl-CoA dehydrogenase, PAT=phosphate acetyltransferase, AldDH=aldehyde dehydrogenase, ADH=alcohol dehydrogenase, AAT= alcohol acyltransferase 1-Methylcyclopropene (1-MCP) is a powerful ethylene inhibitor.It has been observed that its application reduces the production of volatiles in apples of diff erent varieties such as Golden Delicious (173), Fuji (174), Gala (175), Anna (176) and Greensleeves transgenic apple (44,172).1-MCP reduces the transcription and translation of the gene MdAAT2 in Golden Delicious apples with a subsequent decrease in the production of esters (127).Aminoethoxyvinylglycine, another inhibitor of ethylene production, has also been shown to reduce volatiles in apples (164,(177)(178)(179).

Harvest factors
The concentration of volatile compounds in apple greatly increases as maturation advances (15,180).Maturity at the time of harvest is one of the main factors aff ecting the quality of the apple during and aft er storage.Harvesting of apples before physiological maturity normally implies low volatile levels (181)(182)(183).An apple harvested in a climacteric stage will produce more volatiles during storage compared to preclimacteric fruit or overly mature fruit (164,184,185).

Postharvest factors
Apples can be successfully stored under regular or controlled atmosphere for several months (186).However, storage conditions may reduce volatile compound biosynthesis in apples (184,187) depending on the type of storage atmosphere employed and the length of the storage period (184,188).Modifi ed atmospheres, especially those with ultra-low oxygen (p(O 2 )<1 kPa), cause a reduction in the biosynthesis of linear chain volatiles due to a reduction in the concentration of alcohols and their esters (37,179), except for ethanol and its derivatives, which are produced in apple under anaerobic conditions (10,187).The synthesis and degradation of fatt y acids also decline (15,17,184) due to the reduction in β-oxidation, LOX activity and ethylene biosynthesis (22).However, the availability of substrates, more than the lack of enzymatic activity, is the most important factor in the suppression of volatile compounds during and aft er storage under controlled atmosphere (48,53,123,124).Low oxygen concentration during storage has litt le eff ect on the biosynthesis of branched esters (22,35,179,187).
On the other hand, a storage atmosphere with high CO 2 concentration suppresses the production of linear and branched aromatic compounds, probably by inhibiting tricarboxylic acid, from which certain precursor amino acids derive (184).The reduction in aroma production can also be due to a low respiration rate in apples under controlled atmosphere, which can deplete stored energy metabolites such as ATP and NADPH, required for the biosynthesis and desaturation of fatt y acids (189,190).
During postharvest, concentration of esters may decrease due to their hydrolyzation by carboxylesterases to form their respective acids and alcohols, and by their diffusion in the environment (37)(38)(39)(40)(41).However, a low ester content can be caused rather by the lack of alcohols as precursors than by esterase activity, or diff usion (15,37,191).

Addition of Biosynthetic Precursors of Volatile Compounds to Apples
The exogenous application of precursors is an alternative strategy for aroma regeneration in refrigerated apples.Substrate availability is an important factor in the recovery of aroma in apples stored for long periods (15,36,124).It has been demonstrated that precursors added to whole apples diff use towards the internal tissues of the fruit (46).However, the exogenous application of substrates in apple has been used more to elucidate the metabolic pathways (16,17,192) than to increase the production of volatile compounds (193).
Precursors of volatile compounds have been added to diff erent tissues of apples, and although intact apples and tissue samples metabolize substrates in the same way, most researchers prefer to use the intact fruit (17,137,163).An intact apple and tissues are placed in a closed glass container, and substrate vapours diff use through the tissue.The volatile compounds released by the fruit are carried by a continuous air current and recovered with a trap for subsequent analysis using gas chromatography.The incubation period lasts 24 hours or more and the evaluation period lasts several days (9,16,17,43,46,179).The use of cortical or epidermal tissue disks, usually 9 to 12 mm in diameter and 1 to 3 mm thick, has the advantage of allowing easier access of the precursors, reducing the incubation time to less than 24 hours.However, its disadvantage is the requirement of a buff ering system to maintain the viability of the fruit cells (194).The recovery of the volatile compounds can be performed by dynamic (16,17,67,195) or static headspace (127,196,197), and the volatile compounds are analyzed by gas chromatography.The addition of fatt y acids, amino acids, aldehydes and alcohols stimulates the biosynthesis of volatile compounds in diff erent apple varieties.

Fatt y acid addition
The addition of fatt y acids causes the formation of aldehydes, alcohols and their corresponding esters, in addition to the formation of new shorter fatt y acids by the β-oxidation pathway, which stimulates a wide range of volatile compounds (43,47).The optimal fatt y acid concentration to be added decreases with the length of the chain.The addition of fatt y acids at a concentration above the optimum can cause a decrease in the biosynthesis of aldehydes and alcohols, and at very high concentrations, fatt y acids can cause browning of the tissues and acetaldehyde formation (47).
The addition of propionic acid to Golden Delicious apples that were stored for 8 months under controlled atmosphere led to the formation of propanal and propyl and propionate esters, usually absent from the fruit.However, no change in the total concentration of volatiles occurred.On the other hand, butanoic acid was converted to butanoate esters (46).In this variety, which was stored for 7 months under low oxygen concentration conditions, butanoic acid increased the biosynthesis of ethyl esters and butyl butyrate (184).
The addition of fatt y acids with 2-5 carbons to Golden Delicious apples stored under regular or controlled atmosphere for two months was found to cause an increase in the content of aldehydes corresponding to these acids, except for those of acetic acid and aldehydes derived from the β-oxidation of endogenous fatt y acids.Under regular atmosphere, aldehydes are transformed to esters, but under controlled atmosphere, apparently the increased CO 2 content causes a change in ADH activity, with a reduction in alcohol that limits the production of esters (9).

Amino acid addition
The addition of amino acids increases the production of volatile compounds with a structure similar to that of the side chain of the added amino acid (31).Even though amino acids are a secondary source of substrates for the biosynthesis of volatile compounds in apples, very few studies have reported the addition of amino acids as substrates for the production of volatile compounds in this fruit (17,66,67).Several amino acids such as leucine, valine, phenylalanine (65) and isoleucine (198) have been added to plantain, and methionine has been added to melon (31).

Aldehyde addition
In addition to contributing to apple aroma, aldehydes are intermediate compounds between fatt y acids and alcohols (47).Vapours of aldehydes with 3 to 6 carbon atoms added to recently harvested intact Golden Delicious apples considerably increased the content of the corresponding acetate esters, indicating that they were efficiently converted to alcohols (195).Additionally, in Golden Delicious apples that were recently harvested or stored for 5 months under ultra-low oxygen conditions, butanal vapours induced the formation of butanol, butyl acetate and butyl butyrate (179).In fruit stored for 8 months under ultra-low oxygen conditions, butanal was transformed into butanol or butanoic acid, increasing the production of esters such as pentyl and butyl butanoate (184).In another study on Red Delicious and Granny Smith apples, deuterium-labelled trans-2-hexenal and cis-3-hexenal aldehydes, added as vapour, produced a mixture of hexyl esters (trans-3-hexenyl and cis-2-hexenyl) and 1-hexanol in both varieties.Moreover, only in Red Delicious apples, ethyl, propyl and butyl butanoate esters were formed and in Granny Smith trans-3-hexenol, cis-3-hexenol and trans-2-hexenol alcohols were exclusively formed (16).In these varieties, deuterium-labelled hexanal was converted to 1-hexanol and to hexyl and hexanoate esters (16).

Alcohol addition
Alcohols with 2-8 carbon atoms have been added alone or as a mixture.However, because high-molecular--mass substrates evaporate poorly and diff use only weakly through cellular membranes (37,46,83), the results obtained with 7-and 8-carbon alcohols have not been suc cessful.Ester production is proportional to the concentration of the added alcohol, and the concentration of acyl-CoA is not a limiting factor (43,192), although the diff erence in the aroma composition among varieties can be due to differences in the preference for acyl-CoA molecules (47).The fruit can effi ciently catabolize alcohols with 8 or few-er carbons exogenously added aft er 8 months under ultra-low oxygen, suggesting that ADH remains active (184).However, aft er 7 months under ultra-low oxygen, intact Golden Delicious apples exposed to a mixture of alcohols synthesized only 20 % of volatiles compared to apples stored under regular atmosphere (179).With the addition of an alcohol, mostly acetate esters are produced (43).However, at the same time, a decrease in the formation of other esters, especially those with 4 or more carbon atoms, can be observed in a kind of antagonism between substrates (46,47,192).Given that the addition of a single alcohol at high concentration can inhibit the production of esters from other alcohols (194), these compounds have been added in equimolar solutions.However, the addition of alcohols leads to high alcohol concentration in tissues, which may deteriorate the apple (43).
Alcohols with 2-6 carbon atoms added individually to Golden Delicious (184) and as a mixture to Cox's Orange Pippin apples (83) were converted to their corresponding acetate esters, with butanol and pentanol being the quickest and most esterifi ed in Cox's Orange Pippin apples.In another study with this variety, although hexanol formed hexyl acetate, ethanol did not produce ethyl acetate but accumulated in the tissues (37).However, Dixon (194) reports that ethanol forms ethyl acetate when added individually but not when added in a mixture.Ethanol and methanol added separately to Red Delicious apples form the corresponding ethyl and methyl esters (192).In Golden Delicious apples stored for 5 months under ultra-low oxygen and exposed to 3-to 6-carbon linear alcohols, isobutanol and isopentanol added separately increased the biosynthesis of acetate esters (mostly) and butanoate (to a lesser degree) (179).
Alcohols have also been added to tissues of apples from diff erent varieties.In Golden Delicious skin disks treated with 1-pentanol, 1-hexanol, trans-2-hexenol and cis-2-hexenol 65 days aft er full fl owering, cis-and trans-2--hexenyl acetate, butanoate and hexanoate esters were formed.Pentyl and hexyl esters were only formed 15 days aft er full fl owering of fruit (127).In disks of diff erent tissues from Red Chief Delicious apples, the addition of pentanol formed pentanal and pentyl acetate in higher concentrations, as well as pentyl propionate, butyrate, pentanoate and hexanoate esters at lower concentrations.
None of these compounds was present in untreated fruit.The production of volatiles decreased from the skin towards the centre of the fruit (197).In other studies, the addition of 1-to 6-carbon linear alcohols to pulp disks and skin from recently harvested Red Delicious apples achieved the maximum production of esters with 1-butanol converting to butyl and butanoate esters and 1-pentanol converting almost exclusively to pentyl esters, while methanol and ethanol had very low esterifi cation rate (192).All alcohols formed acetate esters, and ethanol, propanol and butanol also formed their corresponding aldehydes (192).
Primary 2-to 6-carbon alcohols, when added separately or in equimolar solution to apple skin disks, were converted to their acetate ester in nine apple varieties including Fuji, Golden Delicious, Red Delicious, Granny Smith and Cox's Orange Pippin (194).In skin disks of this last variety, in addition to primary 2-to 6-carbon alcohols, 2-methyl-1-propanol and 2-methyl-1-butanol also formed acetate esters and 1-butanol also formed butyl butanoate ester, whereas 2-propanol and 2-butanol did not form any new product (37).Also, the addition of deuterium-labelled hexanol and 2-methyl-1-butanol to diced Red Delicious apples stored for 5 months under controlled atmosphere increased the production of their corresponding acetate esters (67).

Ester addition
Aldehydes and fatt y acids are diffi cult to manipulate (43), so esters of short-chain fatt y acids have been used as precursor sources in apples because they can diff use more easily.In apples, esters are hydrolyzed by carboxylesterase, releasing the fatt y acid and the corresponding alcohol (43).It has been suggested that these enzymes remain active in apples even aft er 8 months of stora ge under ultra-low oxygen conditions (184).In Cox's Orange Pippin apples, methyl heptanoate was mostly converted to pentyl acetate, and at 10-to 20--fold lower concentrations to propyl acetate, whereas the production of heptyl acetate was insignifi cant or absent, confi rming that heptanoic acid completes one or two β-oxida tion cycles.The same occurred when other methyl esters with a fraction of 4-to 8-carbon acids were added.Methyl octanoate was converted to butyl and hexyl acetate and propyl and butyl butanoate (43).On the other hand, the exposure of Red Delicious and Granny Smith apples to ethyl 2-methylbutanoate-d 3 vapours formed 2-methylbutanoate esters, mostly butyl-2-methyl butanoate (18 and 49 % in Red Delicious and Granny Smith, respectively) and hexyl-2-methyl butanoate (17 and 27 %).However, Red Delicious formed 2-methylbutane esters (mostly acetate, 49 %) and 2-methyl-2-butenyl esters (mostly acetate, 2 %), while in Granny Smith only traces of the former where detected and the latt er were not detected at all (17).
The exposure of Calvilla, Golden Delicious and Starking apple skin disks to methyl ether (butanoate to hexanoate) caused the release of fatt y acids, and those with more than 4 carbons completed 1 or 2 β-oxidation cycles, with the loss of 2 carbons in each cycle, forming acetate esters with the corresponding alkyl fraction (acetate esters from propyl to hexyl) (47).

Conclusions
The production of volatile aroma compounds in apples is the result of a combination of complex metabolic pathways with diverse physiological processes and control mechanisms in the fruit metabolism.Their production also varies due to genetic factors, culture practices, crop maturity and storage conditions.Especially important is the eff ect of compounds that suppress ethylene production such as 1-methylcyclopropene, as is the reduction in the respiration rate at low O 2 and/or high CO 2 atmosphere.In these cases, a decrease in the production of adenosine triphosphate molecules reduces the synthesis of fatt y acids, the main precursors of volatile compounds.As demonstrated, although the availability of substrates for the production of volatile compounds is a critical factor, it is not the only one aff ecting the aroma of apples, and it can be controlled in both whole and cut apples.However, up to this point, there is litt le knowledge regardi ng the metabolic pathways, the genes encoding the enzymes involved, the mechanisms controlling genetic expression and enzymatic activity, or even the exact metabolism of exogenously added precursors, especially in freshly cut apples.The exogenous addition of substrates for the production of volatile compounds is an area of potential development, especially in lightly processed apples (sliced, in cubes or cylinders), given the increasing demand for freshly cut fruit on the market.All these are areas of opportunity in which bett er knowledge can help us understand, and therefore exploit, the production of volatile compounds in the apple.

Table 1 .
Proportion of aldehydes in the total volatiles identifi ed in each apple variety GD=Golden Delicious, BD=Bisbee Spur Delicious, FJ=Fuji, GRU=Gold Rush, GS=Granny Smith, PL=Pink Lady, GRE=Golden Reinders; ULO=ultra-low oxygen

Table 3 .
. The branched-chain amino transferase (BCAT) enzyme catalyzes the last step Important enzymes involved in the production of volatile aroma compounds of apple fruit, and their changes at preharvest, harvest and postharvest stages Fig 2. Lipoxygenase pathway in the production of acetate esters from the catabolism of linoleic and linolenic acids.Adaptations based on 16,29,32,63,109.LOX=lipoxygenase, HPL=hydroperoxide lyase, ADH=alcohol dehydrogenase, ALDH=aldehyde dehydrogenase, AOR=alquenal oxidoreductase, 3-2EI=cis-3: trans-2-enal isomerase, AAT=alcohol acyltransferase Jonagold LOX activity increased until 160 days aft er full bloom and then remained constant until harvest (85) Pink Lady LOX activity increased in climacteric stage; subsequent reduction of activity (54) Golden Reinders LOX activity decreased in the skin until harvest; it remained low and unchanged in the pulp (119) Golden Delicious and McIntosh LOX2b, -3b and -5e genes were up-regulated by fruit ripening; LOX1a was detected in the last ripening stage only in McIntosh (96) Golden Reinders 7 months under ultra-low oxygen: partial inhibition of LOX enzyme activity

Table 3 .
-continued Biosynthesis of volatile aroma compounds from isoleucine catabolism.Adaptation based on 17,