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Floral Scents and Fruit Aromas: Functions, Compositions, Biosynthesis, and Regulation

生物合成途径

Biosynthetic Pathways

PART 01

萜类生物合成途径

Terpenoids are the largest group of volatile compounds in plants (Muhlemann et al., 2014; Ramya et al., 2018). Based on C5 isoprenoid units, terpenoids are classified as C5 (hemiterpenes), C10 (monoterpenes), C15 (sesquiterpenes), C20 (diterpenes), C25 (sesterpenes), C30 (triterpenes), C40 (tetraterpenes), and > C40 (polyterpenes) (Martin et al., 2003; Ashour et al., 2010). Terpenoids are derived from isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP). Terpenoids are classified based on the molar ratio of IPP to DMAPP: monoterpenes (1:1); sesquiterpenes and sterols (2:1); and diterpenes, carotenoids, and polyterpenes (3:1) (Gershenzon and Kreis, 1999). In higher plants, the C5 unit is generated by the mevalonic acid (MVA) and 2-c-methylerythritol 4-phosphate (MEP) pathways. In the cytosol, IPP is generated from the mevalonic acid (MVA) pathway, beginning with the condensation of acetyl-CoA (Newman and Chappell, 1999). In plastids, IPP is formed by the MEP pathway, beginning with pyruvate and glyceraldehyde-3-phosphate. In cytosol and plastid, isopentenyl diphosphate isomerase is responsible for the reversible conversion of IPP to DMAPP (Phillips et al., 2008; Jin et al., 2020).

Monoterpenes constitute an important class of aromatic compounds; they are the principal constituents of the scents of flowers and fruits (Croteau et al., 2000). Monoterpenes include limonene, (E)-β-ocimene, myrcene, linalool, and α- and β-pinene (Piechulla and Effmert, 2010; Dudareva et al., 2013; Oyama-Okubo et al., 2013). In plants, monoterpenes are synthesized in plastids (Dudareva and Pichersky, 2000; Chen and Tholl, 2011). IPP is the precursor of geranyl diphosphate (GPP) and geranylgeranyl pyrophosphate (GGPP). Monoterpenes are derived from geranyl diphosphate (GPP, C10); GPP synthase catalyzes the conversion of one IPP and one DMAPP molecule to GPP through a head-to-tail condensation reaction (Poulter and Rilling, 1981; Ogura and Koyama, 1998). Sesquiterpenes are synthesized in the cytosol by the MVA pathway. In this pathway, two IPP molecules and one DMAPP molecule are condensed to produce farnesyl diphosphate by farnesyl pyrophosphate synthase. Finally, cytosolic sesquiterpene synthases catalyze the conversion of farnesyl diphosphate to sesquiterpenes (Chen and Tholl, 2011; Monson, 2013). Notably, in snapdragon flowers, sesquiterpenes are synthesized via the MEP pathway (Vranová et al., 2013).

与其他类异戊二烯类似，类胡萝卜素与单萜和二萜共享生物合成途径；它们来源于IPP。在质体中(Lichtenthaler et al.， 1997)，在香叶二磷酸合成酶的催化下，将三个IPP分子依次加到一个DMAPP分子上，合成20碳分子GGPP。类胡萝卜素途径的第一步是两个GGPP分子在植物烯合成酶的催化下缩合产生初级类胡萝卜素——植物烯。植物烯经过几个酶促反应生成类胡萝卜素化合物(图1;Zhou and Pichersky，2020)。

FIGURE 1. Terpenoid biosynthetic pathway. Terpenoid precursors (acetyl-CoA and pyruvate) enter the MVA pathway in the cytosol to produce sesquiterpenes or the MEP pathway in plastids to generate monoterpenes, diterpenes, and carotenoids. The enzymes and intermediates of both pathways are shown. DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; FPPS, farnesyl pyrophosphate synthase; GGPP, geranylgeranyl pyrophosphate; GGPS, geranylgeranyl pyrophosphate synthase; GPP, geranyl diphosphate; GPPS, geranyl diphosphate synthase; IDI, isopentenyl diphosphate isomerase; IPP, isopentenyl diphosphate; PSY, phytoene synthase; TPS, terpenoid synthase.

Terpenoid concentrations in flowers and fruits vary according to developmental stage. For example, in berries, terpenes accumulate during the early stages of development. Terpene compounds synthesized during this stage might be precursors for the final products (Kalua and Boss, 2009). In lavender (L. angustifolia), terpenoid quantities are closely related to flower maturity (Li et al., 2019).

Carotenoids are important components of fruit aroma and important precursors of volatile norisoprenoids, which influence the aroma profile of fruits despite their presence at low levels (Watanabe et al., 2009). In some tomato and watermelon varieties, the degradation of carotenoids into lycopene pigment (red) produces geranial, a lemon-scented monoterpene aldehyde (Azulay et al., 2005; Crops et al., 2005). Furthermore, the degradation of β-carotene and lycopene in “Sui hong” papaya fruit results in a pleasant aromatic odor (Jing et al., 2015). In cashew apple (Anacardium occidentale L.) juice, the thermal degradation of carotenoids produces an aroma profile of 33 active odor volatiles, such as 1,2,3,5-tetramethylbenzene, naphthalene, and p-xylene (Queiroz et al., 2014). Carotenoids are also involved in floral scent in Osmanthus fragrans (Baldermann et al., 2010; Xi et al., 2021). β-ionone, a carotenoid derivative, is produced by the flowering plants Rosa moschata, Thymus vulgaris, Viola tricolor, Medicago marina, and Myrtus communis (Paparella et al., 2021).

PART 02

苯丙/苯环型途径

Phenylpropanoids and benzenoids are the second largest class of plant VOCs (Knudsen and Gershenzon, 2006). Only phenylpropanoids containing a reduced carboxyl group at C9 (i.e., aldehydes, alcohols, or alkenes) and/or alkyl addition to hydroxyl groups of the benzyl ring or to the carboxyl group (i.e., ethers and esters) are considered volatiles (Dudareva and Pichersky, 2000). According to the side-chain length, aromatic compounds are classified as phenylpropanoids (C6–C3), phenylpropanoid-related (C6–C2), and benzenoids (C6–C1) (Muhlemann et al., 2014; Lackus et al., 2021; Figure 2B).

苯丙素和苯类化合物是植物挥发性有机化合物的第二大类别(Knudsen和Gershenzon, 2006)。只有在C9上含有还原羧基(即醛、醇或烯烃)和/或在苯基环的羟基或羧基(即醚和酯)上添加烷基的苯丙素才被认为是挥发物(Dudareva和Pichersky, 2000)。根据侧链长度，芳香族化合物可分为苯丙类化合物(C6-C3)、苯丙类化合物(C6-C2)和苯类化合物(C6-C1) (Muhlemann et al.， 2014;Lackus et al.， 2021;图2 b)。

Phenylpropanoids and benzenoids are produced from the aromatic amino acid phenylalanine (Phe) via the shikimate pathway (Maeda and Dudareva, 2012; Yoo et al., 2013). The first step in phenylpropanoid and benzenoid biosynthesis is catalyzed by phenylalanine ammonia-lyase (PAL), the entry-point enzyme of the general phenylpropanoid biosynthetic pathway, which converts Phe to trans-cinnamate (CA) (Bate et al., 1994; Boatright et al., 2004; Orlova et al., 2006).

FIGURE 2. (A) Phenylpropanoid and benzenoid biosynthesis. All shikimate pathway enzymes and intermediates in the cytosol and peroxisome are shown. BALDH, benzaldehyde dehydrogenase; CA, trans-cinnamate; CHD, cinnamoyl-CoA hydratase/dehydrogenase; CFAT, coniferyl alcohol acyltransferase; C4H, cinnamate 4-hydroxylase; CNL, cinnamate-CoA ligase; 4CL, 4-hydroxycinnamoyl CoA ligase; EGS, eugenol synthase; IEMT, isoeugenol O-methyltransferase; IGS, isoeugenol synthase; PAL, phenylalanine ammonia lyase; KAT, 3-ketoacyl thiolase 1.(B) Chemical structures of benzenoid (C6–C1) compounds. A, methylbenzene; B, benzaldehyde; C, methyl 2-hydroxybenzoate (methyl salicylate); D, benzyl (E)-2-methyl-2-butenoate (benzyl tiglate); E, 4-hydroxy-3-methoxybenzaldehyde (vanillin). Phenylpropanoid-related (C6–C2) compounds: F, ethylbenzene; G, phenylacetaldehyde; H, acetophenone; I, 2-phenylethanol; J, 2-phenylacetonitrile. Phenylpropanoid (C6–C3) compounds: K (E)-cinnamic aldehyde; L, phenylpropanol.

To synthesize phenylpropanoids, CA is hydroxylated by cinnamate 4-hydroxylase (C4H), producing p-coumarate. Consequently, 4-coumaroyl CoA ligase catalyzes the conversion of p-coumarate into p-coumaroyl-CoA (Deng and Lu, 2017). A series of enzymatic reactions is required for the formation of coniferyl alcohol, which is acetylated by coniferyl alcohol acyltransferase and converted to coniferyl acetate. The NADPH-dependent reductases eugenol synthase and isoeugenol synthase (IGS) catalyze the conversion of coniferyl acetate to eugenol and isoeugenol, respectively (Ferrer et al., 2008; Huang et al., 2020). Eugenol and isoeugenol are methylated by isoeugenol O-methyltransferases to produce the volatiles methyleugenol and methyl isoeugenol (Gang et al., 2002; Dudareva et al., 2013; Figure 2A).

To synthesize benzenoids, the propyl side chain of CA is shortened by two carbons. Multiple chain-shortening pathways have been proposed: two nonoxidative pathways in the cytosol (CoA-dependent or CoA-independent), and a β-oxidative pathway in peroxisomes (Widhalm and Dudareva, 2015). The CoA-dependent non-oxidative pathway involves the conversion of CA to cinnamoyl-CoA by 4-hydroxycinnamoyl CoA ligase. Thereafter, hydratase catalyzes cinnamoyl-CoA to 3-hydroxy-3-phenyl-propanoyl-CoA, from which benzaldehyde is produced by lyase. In the CoA-independent non-oxidative pathway, CA is converted by hydratase to 3-hydroxyphenyl-propionic acid, which is reduced by lyase to benzaldehyde (Widhalm and Dudareva, 2015). The last step is the production of benzoic acid by benzaldehyde dehydrogenase, as characterized in Snapdragon flowers (Anthirrhinum majus) (Long et al., 2009) and proposed in petunia flowers (Kim et al., 2019).

The β-oxidative pathway has been described in petunia (Petunia hybrida) flowers and Arabidopsis (A. thaliana) (Moerkercke et al., 2009; Qualley et al., 2012). The β-oxidative pathway includes shortening of the propyl side chain in three enzymatic steps. First, CA is converted by cinnamate-CoA ligase to cinnamoyl-CoA. The bifunctional cinnamoyl-CoA hydratase/dehydrogenase, which is responsible for two intermediate steps (hydration and oxidation), converts cinnamoyl-CoA to 3-oxo-3-phenyl-propanoyl-CoA (Qualley et al., 2012). Finally, 3-oxo-3-phenyl-propanoyl-CoA is converted by 3-keto-acyl CoA-thiolase to benzoyl-CoA (Moerkercke et al., 2009). Benzoyl-CoA is a precursor of volatile and nonvolatile benzenoids (Lackus et al., 2021). Unfortunately, the steps and several enzymes of the benzenoid pathway are unknown.

在矮牵牛(petunia hybrida)花和拟南芥(A. thaliana)中已经描述了β-氧化途径(Moerkercke et al.， 2009;Qualley et al.， 2012)。β-氧化途径包括在三个酶促步骤中缩短丙基侧链。首先，CA通过肉桂酸辅酶a连接酶转化为肉桂酰辅酶a。双功能肉桂酰辅酶a水合酶/脱氢酶负责两个中间步骤(水合和氧化)，将肉桂酰辅酶a转化为3-氧-3-苯基丙酰辅酶a (Qualley等，2012)。最后，3-氧-3-苯基-丙酰辅酶a被3-酮酰基辅酶a硫基水解酶转化为苯甲酰辅酶a (Moerkercke et al.， 2009)。苯甲酰辅酶a是挥发性和非挥发性苯类化合物的前体(Lackus等人，2021)。不幸的是，苯途径的步骤和几种酶是未知的。

The phenylpropanoid and benzenoid biosynthetic pathways compete for Phe (opposite relationship). For example, in Phlox subulata cultivars, the emission of larger quantities of benzenoids will produce little or no phenylpropanoid volatiles and vice versa (Majetic and Sinka, 2013). Because phenylpropanoids and their derivatives are widely distributed in flowers (Ramya et al., 2017) and fruits (Neelam et al., 2020), further investigations of their synthesis mechanisms are warranted.

PART 03

脂肪酸衍生物途径

Fatty acids are the major precursors of volatile compounds in floral scents and fruit aromas; they are catabolized via the lipoxygenase (LOX) pathway (Sanz et al., 1997). The LOX pathway is important for the production of aromatic compounds. Polyunsaturated fatty acids—linoleic (C18:2) and linolenic (C18:3) acids—are the main substrates of LOX in plant tissues (Espino-Díaz et al., 2016). Linoleic and linolenic acids are produced from phospholipids, triacylglycerols, and glycolipids by acyl hydrolases. Soluble cytosolic LOXs are divided into two major subfamilies: 9- and 13-LOXs (Baysal and Demirdöven, 2007; Viswanath et al., 2020). LOXs catalyze the addition of oxygen to polyunsaturated fatty acids at the 9 or 12 position, yielding unsaturated 9- or 13-hydroperoxides. Thereafter, 9-hydroperoxide lyase and 13-hydroperoxide lyase convert 9- and 13-hydroperoxides to C9 and C6 aldehydes, respectively. These C9 and C6 aldehydes are reduced by alcohol dehydrogenases (ADH) to C9 and C6 alcohols, whereas C6 alcohols are converted into esters by alcohol acyltransferase (Jerković et al., 2019). The divergence of esters depends on substrate availability, enzyme specificity, and ATT gene variation (Sanz et al., 1997; Espino-Díaz et al., 2016). Jasmonic acid can be generated from 13-hydroperoxy through a separated branch via production of an unstable epoxide by allene oxide synthase, followed by a series of cyclization reduction reactions (Muhlemann et al., 2014; Ramya et al., 2018; Figure 3).

脂肪酸是花香和水果香气中挥发性化合物的主要前体;它们通过脂氧合酶(LOX)途径分解代谢(Sanz et al.， 1997)。LOX途径对芳香族化合物的生成具有重要意义。多不饱和脂肪酸-亚油酸(C18:2)和亚麻酸(C18:3) -是植物组织中LOX的主要底物(Espino-Díaz et al.， 2016)。亚油酸和亚麻酸是由磷脂、三酰基甘油和糖脂通过酰基水解酶产生的。可溶性细胞质LOXs分为两个主要亚家族:9-和13-LOXs (Baysal and Demirdöven, 2007;Viswanath et al.， 2020)。LOXs催化多不饱和脂肪酸在9或12位的氧加成，生成不饱和的9-或13-氢过氧化物。然后，9-氢过氧化物裂解酶和13-氢过氧化物裂解酶分别将9-和13-氢过氧化物转化为C9和C6醛。这些C9和C6醛通过醇脱氢酶(ADH)还原为C9和C6醇，而C6醇通过醇酰基转移酶转化为酯(jerkovic et al.， 2019)。酯类的分化取决于底物利用率、酶特异性和ATT基因变异(Sanz等，1997;Espino-Díaz et al.， 2016)。茉莉酸可以由13-超氧化氢通过一个独立的分支，由丙二烯氧化物合成酶(AOS)生成不稳定的环氧化物，然后进行一系列环化还原反应(Muhlemann et al.， 2014;Ramya et al.， 2018;图3)。

FIGURE 3. Fatty acid derivative biosynthetic pathway. Fatty acid precursors (linoleic and linolenic acids) enter the LOX pathway and are converted to 9- and 13-hydroperoxide, which are oxidized and converted to volatiles by hydroperoxide lyase (HPL) and alcohol dehydrogenase (ADH). AAT, alcohol acyltransferase; ADH, alcohol dehydrogenase; AOS, allene oxide synthase; HPL, hydroperoxide lyase; LOX, lipoxygenase; PUFAs, polyunsaturated fatty acids.

图3    脂肪酸衍生物生物合成途径。脂肪酸前体(亚油酸和亚麻酸)进入LOX途径，转化为9-和13-氢过氧化物，经氢过氧化物裂解酶(HPL)和醇脱氢酶(ADH)氧化转化为挥发物。AAT，醇酰基转移酶；ADH，醇脱氢酶；AOS，丙二烯氧化物合成酶;HPL，过氧化氢裂解酶；LOX,脂氧合酶；PUFAs，多不饱和脂肪酸。

PART 04

氨基酸衍生物途径

Amino acids are precursors for various plant aromatic secondary metabolites (Vogt, 2010), such as aldehydes, alcohols, acids, and esters. There are three main aromatic amino acids in plants, which are the precursors for various secondary metabolites: Phe, tyrosine (Tyr), and tryptophan (Trp). The amino acid Phe is a precursor for multiple functional secondary metabolites: phenylpropanoids, flavonoids, cell wall lignin, anthocyanins, and various other compounds (Tzin and Galili, 2010b; Qian et al., 2019). Chorismate is converted by chorismate mutase (CM) to prephenate as the first step in Phe biosynthesis (Mobley et al., 1999). The conversion of chorismate to Phe (via prephenate and arogenate) is catalyzed by prephenate aminotransferase and arogenate dehydratase, respectively (Cho et al., 2007; Yamada et al., 2008; Maeda et al., 2010). Furthermore, plants can produce Phe through a microbe-like phenylpyruvate pathway involving prephenate dehydratase; flux is increased when the entry point to the arogenate pathway is limiting (Yoo et al., 2013). The volatile Tyr has the same biosynthetic pathway as Phe, involving derivation from chorismate and arogenate. The last step is conversion by arogenate dehydrogenase (TyrA) to Tyr (Schenck et al., 2020; Yokoyama et al., 2021). This pathway has been explained in plants such as tobacco (Gaines et al., 1982), sorghum (Connelly and Conn, 1986), and Arabidopsis (Rippert and Matringe, 2002; Rippert et al., 2009). Alternatively, Tyr biosynthesis can involve prephenate conversion to 4-hydroxyphenylpyruvate by prephenate dehydrogenase and possibly TyrA2 (Rippert and Matringe, 2002). Thereafter, 4-hydroxyphenylpyruvate is converted to Tyr by aminotransferases (e.g., 4-hydroxyphenylpyruvate amino transferase) (Tzin and Galili, 2010a).

氨基酸是各种植物芳香次生代谢物的前体(Vogt, 2010)，如醛、醇、酸和酯。植物中主要有三种芳香氨基酸，它们是各种次生代谢产物的前体:苯丙氨酸(Phe)、酪氨酸(Tyr)和色氨酸(Trp)。氨基酸苯丙氨酸是多种功能性次级代谢物的前体：苯丙素、类黄酮、细胞壁木质素、花青素和各种其他化合物(Tzin和Galili, 2010b;Qian et al.， 2019)。作为Phe生物合成的第一步，分支酸被分支酸变位酶 (CM)转化为预苯酸盐(Mobley et al.， 1999)。分支酸转化为苯丙氨酸(通过预苯酸酯和前酪氨酸)分别由预苯酸氨基转移酶和砷酸脱水酶催化(Cho et al.， 2007;Yamada et al.， 2008;Maeda et al.， 2010)。此外，植物可以通过类似微生物的苯丙酮酸途径产生苯丙氨酸，其中主要通过预苯酸脱水酶;当前酪氨酸路径的入口点受到限制时，通量增加(Yoo et al.， 2013)。挥发性酪氨酸具有与苯丙氨酸相同的生物合成途径，包括从分支酸和前酪氨酸衍生。最后一步是由芳酸脱氢酶(TyrA)转化为Tyr (Schenck et al.， 2020;Yokoyama等人，2021)。这一途径在烟草(Gaines et al.， 1982)、高粱(Connelly and Conn, 1986)和拟南芥(Rippert and Matringe, 2002;Rippert et al.， 2009)。或者，Tyr的生物合成可以涉及预苯酸脱氢酶和可能的TyrA2将预苯酸转化为4-羟基苯基丙酮酸(Rippert和matriinge, 2002)。之后，4-羟基苯基丙酮酸通过转氨酶(如4-羟基苯基丙酮酸氨基转移酶)转化为Tyr (Tzin和Galili, 2010a)。

The first committed step of volatile Trp biosynthesis is the transfer of an amino group of glutamine to chorismate to produce anthranilate and pyruvate; this is performed by anthranilate synthase (ASα and ASβ) (Tzin and Galili, 2010a). Thereafter, anthranilate phosphoribosylanthranilate transferase (AnPRT) converts anthranilate and phosphoribosyl pyrophosphate into phosphoribosylanthranilate and inorganic pyrophosphate. The next step is conversion of phosphoribosylanthranilate into L-(O-carboxyphenylamino)-L-deoxyribulose-5-phosphate (CdRP); this is performed by phosphoribosylanthranilate isomerase. Furthermore, indole-3-glycerol phosphate synthase catalyzes the conversion of CdRP to indole-3-glycerol phosphate (Li et al., 1995). The last two steps in the Trp biosynthetic pathway are catalyzed by Trp synthase (TS), which has alpha (TSα) and beta (TSβ) subunits. Indole-3-glycerol phosphate is converted by TSα into indole and glyceraldehyde-3-phosphate (α-reaction). Finally, indole is transported to TSβ, which catalyzes its condensation with serine (β-reaction) to generate Trp (Miles, 2001; Weber-Ban et al., 2001; Parthasarathy et al., 2018; Figure 4).

FIGURE 4. Phe, Tyr, and Trp biosynthetic pathways. Chorismate is the precursor of Phe, Tyr and Trp. Two distinctive pathways starting from the same precursor, Chorismate, where Phe and Tyr share some common intermediates (i.e., prephenate and arogenate) and Trp is produced from a separated branched-chain. ADT, arogenate dehydratase; AnPRT, anthranilate phosphoribosylanthranilate transferase; ASα; ASβ, anthranilate synthase; CdRP, L-(O-carboxyphenylamino)-l-deoxyribulose-5-phosphate; CM, chorismate mutase; HPP-AT, 4-hydroxyphenylpyruvate aminotransferase; IGP, indole-3-glycerol phosphate; IGPS, indole-3-glycerol phosphate synthase; PAI, phosphoribosylanthranilate isomerase; PAT, prephenate aminotransferase; PDH, prephenate dehydrogenase; PDT, prephenate dehydratase; Phe, phenylalanine; PPi, inorganic pyrophosphate; PPY-AT, phenylpyruvate aminotransferase; PRA, phosphoribosylanthranilate; PRPP, phosphoribosyl pyrophosphate; Trp, tryptophan; TSα, tryptophan synthase alpha subunit; TSβ, tryptophan synthase beta subunit; Tyr, tyrosine; TyrA, arogenate dehydrogenase.

Amino acids undergo deamination or transamination, forming the corresponding α-keto acid, a key intermediate in the conversion of amino acids to volatiles (Gonda et al., 2010; Pott et al., 2019); this reversible step is catalyzed by branched-chain aminotransferases (Klee, 2010). For example, in tomato, six of these enzymes are present in chloroplasts, mitochondria, and the cytoplasm (Kochevenko et al., 2012). Decarboxylation is the most likely route for conversion of α-keto acids to aldehyde volatiles, followed by SDR-mediated synthesis of alcohols (Klee, 2010).

Various synthesis mechanisms of Phe-derived volatiles, such as phenylacetaldehyde and 2-phenylethanol, which contribute to fruit flavors and floral scents have been studied. For instance, in tomato plants, aromatic L-amino acid decarboxylases convert Phe to phenethylamine, the first step in phenylacetaldehyde and 2-phenylethanol synthesis (Tieman et al., 2006). Conversion of phenylacetaldehyde to 2-phenylethanol by phenylacetaldehyde reductases is the last step in the pathway (Tieman et al., 2007). Notably, P. hybrida contains a bi-functional decarboxylase/amine oxidase enzyme that directly converts Phe to phenylacetaldehyde (Kaminaga et al., 2006). Additionally, the synthesis of 2-phenylethanol (which has a pleasant fragrance) has been studied in rose (Sakai et al., 2007). Branched-chain alcohols, carbonyls, and esters are produced by metabolism of the amino acids leucine, isoleucine, valine, alanine, and aspartic acid (Heath and Reineccius, 1986; Sanz et al., 1997). Volatile esters are the largest group of volatile compounds produced by fruits. Fruit esters are formed by the esterification of alcohols and acyl CoA, which are derived from fatty acid and amino acid metabolism. This reaction is catalyzed by alcohol acyltransferase (Sanz et al., 1997). In post-climacteric banana slices, amino acids are converted to branched-chain alcohols and esters by aminotransferases, decarboxylases, and ADH (Sanz et al., 1997). The biosynthesis of higher alcohols is linked to amino acid deamination (Herrero et al., 2006).

花和果实挥发性化合物的调控

01

影响挥发性有机化合物释放的因素

PART 01

花香挥发物

The emission of floral scents is regulated by time of release, environmental variables (light and temperature), tissue type, and plant status (age, physiological state, and sex). These factors stimulate olfactory communication (Raguso, 2008).

The circadian rhythm controls the emission patterns of floral scents in response to the day/night cycle (Picazo-Aragonés et al., 2020). Diurnal and nocturnal emission patterns have been reported in plant species. Some VOCs are emitted independently of environmental conditions; they are influenced by the endogenous biological clock (Zeng et al., 2017). The floral scents emitted by Myrtaceae species at night attract nocturnal bees (Cordeiro et al., 2019). In Hoya carnosa, the circadian rhythm is also expressed during exposure to continuous dark (Fenske and Imaizumi, 2016). The impact of the circadian clock on the release patterns of volatile terpenoids and phenylpropanoids/benzenoids has been reported; however, few studies have focused on volatile fatty acid derivatives (Zeng et al., 2017). For example, the circadian clock and light promote the diurnal emission of monoterpenes from orchid (Phalaenopsis violacea) flowers (Chuang et al., 2017). The endogenous circadian clock in petunia flower is proposed to regulate the rhythmic emission of volatile phenylpropanoids and benzenoids (Cheng et al., 2016). Moreover, an important fatty acid derivative, jasmonic acid, is controlled by the circadian clock (Zhang Y. et al., 2019).

Volatile compounds are synthesized and emitted from petunia petal limbs during the evening and shortly after anthesis; such emission is drastically decreased when limbs are not supplied with exogenous Phe, and post-pollination-derived ethylene causes the same effect (Dexter et al., 2007). In a single snapdragon flower, the upper and lower petal lobes release high levels of myrcene, ocimene, linalool, and nerolidol (Dudareva et al., 2003; Nagegowda et al., 2008). The cuticle of petunia flowers prevents the release of almost 50% of internal VOCs; a decrease in cuticle thickness influences the synthesis, cellular distribution, and mass transfer resistance of VOCs (Liao et al., 2021).

Floral VOCs respond to changes in environmental conditions, such as temperature, light, water availability, and soil nutrients. As demonstrated by Dötterl et al. (2005), volatile emissions by Silene latifolia decrease during hot and dry summer months, whereas low temperatures decrease scent emission in Pyrola grandiflora (Knudsen, 1994). Moreover, abiotic stresses influence plant-pollinator and plant-herbivore interactions through different floral VOCs (Campbell et al., 2019).

The developmental stage of flowers affects VOC emission patterns. The changes that occur in petal cells from budding to full blooming, along with the intercellular spaces, modulate VOC evaporation in jasmine (J. sambac) flowers (Chen et al., 2020).

PART 01

水果香气挥发物

Many factors regulate aroma emission by fruits. Fruit genotype influences flavor (Kongor et al., 2016). The final flavor profile is affected by environmental conditions such as climate, sunlight, soil, fruit ripening, harvesting time, and post-harvesting processes (Kongor et al., 2016). Environmental stresses (e.g., temperature and drought) influence fruit metabolism and aromatic compound content (Romero et al., 2021).

许多因素调节着水果的香气释放。水果基因型影响风味(Kongor et al.， 2016)。最终的风味特征受气候、阳光、土壤、水果成熟、收获时间和收获后过程等环境条件的影响(Kongor et al.， 2016)。环境胁迫(如温度和干旱)影响果实代谢和芳香化合物含量(Romero et al.， 2021)。

The VOC profiles of fruit varieties change according to maturation stage. Terpenoids dominate the aroma profile in some fruits during ripening, such as apple (Liu et al., 2021), apricot (Karabulut et al., 2018), and peach (Wei et al., 2021). In grape, some phenylpropanoids increase with maturation (Kambiranda et al., 2016). Furthermore, fatty acid and amino acid-related compounds increase during the maturation of apple (Liu et al., 2021) and apricot (Karabulut et al., 2018). Therefore, maturation is vital for VOC emission in fruits and affects commercial production.

Pre-harvest factors (water, sunlight, fertilization, and chemical application) influence fruit quality characteristics, including flavor (Ahmed et al., 2013). For example, the use of biofertilizers influences the fruit aroma profile and can improve flavors. Biofertilizer treatment promotes the production of volatile compounds in strawberry (Duan et al., 2021). Treatment of pomegranate and peach with salicylic acid, a natural plant phenolic, improves their quality, including flavor (Erogul and Özsoydan, 2020). Pesticides (e.g., triazole pesticides) can negatively affect fruit flavor compounds (Xiao et al., 2020).

Post-harvest techniques (e.g., chemical applications, radiation, cold, heat, and controlled storage atmospheres) are used to suppress disease and enhance fruit quality during storage (Ahmed et al., 2013). Nitric oxide, salicylic acid, 1-methylcyclopropene, and oxalic acid are used for fruit crops, altering their VOC contents (Bambalele et al., 2021). The application of chemical treatments, individually or in combination, can also alter VOC contents. Controlled atmosphere (CA) treatment of fruit aims to prevent the development of bitter pit disease by altering the aromatic contents (Ahmed et al., 2013). The application of CA treatment in combination with 1-methylcyclopropene to “Fuji” apples stored at room temperature promotes the emission of volatiles (Lu et al., 2018). Ethephon increases and 1-methylcyclopropene decreases the production of volatile compounds in apple (Kondo et al., 2005). Intriguingly, the combination of ethephon with methyl jasmonate decreases or increases VOCs in apple cultivars (Kondo et al., 2005). A combination of methyl jasmonate and ethanol significantly decreases the levels of volatiles in raspberry and enhances the aroma of strawberry (Blanch et al., 2011).

The flavor properties of fruits and their products are affected by ultraviolet radiation in grape (Del-Castillo-Alonso et al., 2021), mango (Wang et al., 2020), strawberry (Warner et al., 2021) and pepper fruit (Ma et al., 2021). Cold storage is used for fruit preservation worldwide. However, chilling injury and aroma loss are problems for commercial fruit production. During cold storage, VOC levels are significantly reduced because of precursor metabolism suppression; this occurs in banana (Zhu et al., 2018b), grape (Matsumoto and Ikoma, 2015), mango (Singh et al., 2004), tomato (Bai et al., 2022), and avocado (García-Rojas et al., 2016). CA storage of fruits is a common preservation technology involving the control of O2 and CO2 levels. This method increases the levels of some volatile compounds, while decreasing the levels of other volatile compounds. The storage of mango in a high level of CO2 reduces the monoterpene and sesquiterpene levels, while increasing the levels of esters and norisoprenoids (Lalel et al., 2003b). High-pressure and decompression technology, ethylene scavengers, ozone (O3), active packaging, plasma treatment, high-voltage electrostatic field, and pulse-CA with modified atmosphere preservation have been evaluated for fruit preservation (Fang and Wakisaka, 2021).

葡萄(Del-Castillo-Alonso et al.， 2021)、芒果(Wang et al.， 2020)、草莓(Warner et al.， 2021)和胡椒果实(Ma et al.， 2021)中的紫外线辐射会影响水果及其制品的风味特性。冷藏在世界范围内被用于水果保鲜。然而，冷害和香气损失是商品水果生产中存在的问题。在冷藏过程中，由于前体代谢受到抑制，VOC水平显著降低;这种情况发生在香蕉(Zhu等人，2018b)、葡萄(Matsumoto和Ikoma, 2015)、芒果(Singh等人，2004)、西红柿(Bai等人，2022)和鳄梨(García-Rojas等人，2016)中。水果CA贮藏是一种常见的保鲜技术，涉及对O2和CO2水平的控制。这种方法增加了一些挥发性化合物的水平，同时降低了其他挥发性化合物的水平。芒果在高二氧化碳水平下的储存降低了单萜烯和倍半萜烯的水平，同时增加了酯类和降异戊二烯类的水平(Lalel等，2003b)。已经对高压减压技术、乙烯清除剂、臭氧(O3)、活性包装、等离子体处理、高压静电场和脉冲CA与改性气氛保存的水果保存进行了评估(Fang和Wakisaka, 2021)。

Heat treatment is performed after harvesting to control insects, inhibit ripening, and induce resistance to chilling injury (Lu et al., 2007). Heat processing influences fruit aromatic compound contents (e.g., it significant increases off-flavors in watermelon juice) (Aboshi et al., 2020). Drying is used for fruit conservation; it also influences their VOC contents. In banana, ester and aldehyde levels decrease rapidly in response to drying, whereas alcohol levels initially increase then decrease (Saha et al., 2018). The volatile profiles of strawberry subjected to different drying methods have been reported (Abouelenein et al., 2021). Most commercial fruits undergo post-harvest treatments or preservation; however, the effects on fruit aromas are unclear.

02

影响挥发性有机化合物释放的糖苷、乙烯和酶

Glycosides control volatile emission by flowers and fruits (Schwab et al., 2015). Glycoside volatiles are odorless and release free aroma volatiles upon hydrolysis by β-glucosidase (Yauk et al., 2014; Schwab et al., 2015). Glycosides enhance the flavor of grape wine and tea; they also modify fruit aroma during maturation, storage, and processing (Birtic et al., 2009; Garcia et al., 2013; Yauk et al., 2014; Ohgami et al., 2015).

Ethylene production accelerates the release of VOCs during fruit ripening. For example, the overexpression of a ripening inhibitor (rin) gene led to decreased ethylene production in tomato fruit, thereby reducing volatile levels. Fruits with antisense aminocyclopropanecarboxylic acid synthase have low levels of several volatiles. Fruits with downregulated phytoene synthase have lower levels of carotenoid-derived volatiles, whereas fruits with antisense pectin methylesterase have a lowered level of methanol (Baldwin et al., 2000).

乙烯的产生加速了水果成熟过程中挥发性有机化合物的释放。例如，成熟抑制剂(rin)基因的过度表达导致番茄果实中乙烯产量减少，从而降低挥发性物质水平。具有反义氨基环丙烷羧酸合酶的果实具有低水平的几种挥发物。含有下调的植物烯合成酶的水果含有较低水平的类胡萝卜素衍生挥发物，而含有反义果胶甲基酯酶的水果含有较低水平的甲醇(Baldwin等人，2000)。

03

挥发性有机化合物生物合成的遗传和表观遗传调控

PART 01

Genes

基因

Discovery of important genes in volatile biosynthetic pathways has revealed the molecular mechanisms underlying regulation of floral scent and fruit aroma. Multiple studies have focused on isolating terpenoid-related genes in various plant species. For example, in Lilium “Siberia,” three terpenoid-related genes (LoTPS2, LoTPS4 and LoTPS5) have been isolated and characterized. Among them, LoTPS2 catalyzes (E, E)-α-farnesene, LoTPS4 generates D-limonene and β-myrcene, and LoTPS5 produces squalene (Abbas et al., 2020a,b). Regarding phenylpropanoids and benzenoids, only the genes responsible for the biosynthesis of phenylacetaldehyde and 2-phenylethanol have been discovered (Picazo-Aragonés et al., 2020) (e.g., RhPAAS in rose) (Roccia et al., 2019). Little is known regarding the genes responsible for the synthesis of fatty acid and amino acid derivatives.

挥发性生物合成途径中重要基因的发现揭示了花香和果实香气调控的分子机制。从多种植物中分离萜类相关基因已成为研究热点。例如，在百合“西伯利亚”中，三个萜类相关基因(LoTPS2, LoTPS4和LoTPS5)已被分离并鉴定。其中，LoTPS2催化(E, E)-α-法尼烯，LoTPS4生成d -柠檬烯和β-月桂烯，LoTPS5生成角鲨烯(Abbas et al.， 2020a,b)。关于苯丙素和苯类，目前只发现了负责苯乙醛和2-苯乙醇生物合成的基因(picazo-Aragonés 等人，2020)(例如玫瑰中的RhPAAS) (Roccia等人，2019)。关于负责脂肪酸和氨基酸衍生物合成的基因所知甚少。

Transcriptome analysis is used to identify genes involved in the metabolism of floral scents and fruit aromas. There have been reports of transcriptome and genes underlying the regulation of floral scents and fruit aromas of rose (R. chinensis “Old Blush” and R. odorata var. gigantean) (Guo et al., 2017), orchid (C. sinense) (Yang F. X. et al., 2021), orchid (Vanda Mimi Palmer) (Toh et al., 2017), peony (Paeonia suffruticosa Andr.) (Zhang et al., 2021), Lilium “Siberia” (Hu et al., 2017), water lily (Zhang et al., 2020), carnation (D. caryophyllus) (Tanase et al., 2012), lavender (L. angustifolia) (Li et al., 2019), jasmine (J. sambac) (Chen et al., 2020), Polianthes tuberosa (Fan et al., 2018) and the Chinese narcissus (He et al., 2020), banana (Kaur et al., 2021), apple “Ruixue” (Liu et al., 2021), grape (V. vinifera) (Sun et al., 2019), mango (Xin et al., 2021), and apricot (Zhang Q. et al., 2019). The identification of sets of genes involved in VOC synthesis will provide insight into the corresponding metabolic pathways.

PART 02

Epigenetics

表观遗传学

The formation of VOCs is modulated by DNA methylation, histone protein modifications, and chromatin structure remodeling (Picazo-Aragonés et al., 2020). Cold storage of tomato plant reduces its flavor content; it also causes DNA methylation changes (Zhang et al., 2016). Chromatin-based regulatory mechanisms in petunia flower affect genes involved in phenylpropanoid metabolism, implicating histone acetylation in VOC synthesis (Patrick et al., 2021).

PART 03

Transcription Factors

转录因子

Numerous transcription factors (TFs) regulate the production of volatile compounds. In petunia (P. hybrida) flowers, TFs regulate the phenylpropanoid/benzenoid network. For example, ODORANT1 (ODO1), a member of the R2R3-type MYB family, regulates the synthesis of precursors in the shikimate pathway; it also controls the transcription of entry points to the Phe and phenylpropanoid pathways. Suppression of ODO1 expression reduces the transcript levels of multiple genes in this pathway: DAHPS, EPSPS, PAL, CM, and SAMS (Verdonk et al., 2005). ODO1 activates the ABC transporter promoter (ATP-binding cassette transporter), which is localized to the plasma membrane (Van Moerkercke et al., 2012); it also regulates the emission of volatile compounds (Adebesin et al., 2017). The TF Lilium hybrid ODO1 (LhODO1) was isolated from Oriental and Asiatic hybrid lilies; it regulates the production of phenylpropanoids/benzenoids (Yoshida et al., 2018).

许多转录因子(TFs)调节挥发性化合物的产生。在矮牵牛(P. hybrida)花中，TFs调节苯丙/苯类网络。例如，作为R2R3型MYB家族成员的ODORANT1 (ODO1)调节莽草酸途径中前体的合成；它还控制苯丙氨酸和苯丙类化合物途径入口点的转录。抑制ODO1表达可降低该通路中多个基因的转录水平：DAHPS、EPSPS、PAL、CM和SAMS (Verdonk et al.， 2005)。ODO1激活定位于质膜的ABC转运子启动子(ATP结合盒转运子)(Van Moerkercke et al.， 2012)；它还调节挥发性化合物的释放(Adebesin et al.， 2017)。从东方和亚洲百合杂交中分离到TF百合杂种ODO1 (LhODO1);它调节苯丙素/苯类化合物的产生(吉田等人，2018)。

EMISSION OF BENZENOIDS II (EOBII), a member of the R2R3-type MYB family, regulates ODO1 and activates the promoter of the biosynthetic gene isoeugenol synthase (Spitzer-Rimon et al., 2010; Colquhoun et al., 2011a; Van Moerkercke et al., 2011). Petunia EOBI (R2R3-MYB–like) interacts with regulatory genes; it acts both downstream of EOBII and upstream of ODO1 (Spitzer-Rimon et al., 2012). Numerous genes in the shikimate and phenylpropanoid pathways [5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHP synthase), chorismate synthase, CM, arogenate dehydratase, and PAT), and scent-related genes (PAL, isoeugenol synthase, and benzoic acid/salicylic acid carboxyl methyltransferase] are downregulated by the silencing of EOBI expression (Spitzer-Rimon et al., 2012). The interaction between PhERF6 and EOBI negatively regulates benzenoid biosynthesis in petunia (Liu et al., 2017). Moreover, in petunia flower and tobacco, the overexpression of PAP1 (R2R3 MYB-type TF) increases the levels of anthocyanins and volatile compounds (phenylpropanoids/benzenoids) (Xie et al., 2006; Zvi et al., 2008). In rose, overexpression of PAP1 increases the levels of volatile phenylpropanoids/benzenoids and volatile terpenoids (Zvi et al., 2012). Furthermore, MYB4TF represses cinnamate-4-hydroxylase in the phenylpropanoid pathway, influencing the production of phenylpropanoid compounds in petunia (Colquhoun et al., 2011b). In the Cymbidium cultivar “Sael Bit,” CsMYB1 positively regulates genes responsible for the synthesis of phenylpropanoids/benzenoids and esters. CsMYB1 is highly expressed in petals and columns, particularly at the fully open flower stage, when compared with sepals and labella (Ramya et al., 2019).

Six TF families are implicated in the regulation of terpenoid biosynthesis: AP2/ERF, ARF, bHLH, bZIP, MYB, and WRKY (Picazo-Aragonés et al., 2020). MYC2 (bHLH family) induces the expression of two sesquiterpene synthase genes (TPS11 and TPS21) in Arabidopsis (Hong et al., 2012). In rose flowers, RhMYB1 is involved in floral scent biosynthesis (Yan et al., 2011). In Syringa oblata flowers, two R2R3-MYB TFs are upregulated at the bud and flowering developmental stages; they are involved in the biosynthesis of terpenoids and monoterpenoids (Zheng et al., 2015). In Hedychium coronarium, HcMYB3-6 (scent related R2R3-MYB TFs) is highly expressed at full flowering stages, but its expression levels are low in other plant organs (Yue et al., 2015). MYBs from H. coronarium and S. oblata control the biosynthesis of terpenoid volatile compounds (Ramya et al., 2017). In H. coronarium, several HcMYB genes regulate terpenoid and benzenoid biosynthesis (Abbas et al., 2021b); HcARF5 regulates β-ocimene production (Abbas et al., 2021a). Some monoterpenes in sweet osmanthus (O. fragrans) are regulated by OfWRKY genes (Ding et al., 2020), and OfERF61 regulates β-ionone biosynthesis (Han et al., 2019). In Phalaenopsis orchids, multiple TFs (e.g., PbbHLH4, PbbHLH6, PbbZIP4, PbERF1, PbERF9, and PbNAC1) regulate the expression of PbGDPS and its downstream putative monoterpene synthases genes, PbTPS5 and PbTPS10 (Chuang et al., 2017). CrWRKY1 positively regulates the biosynthesis of terpenoid indole alkaloid in Catharanthus roseus (Suttipanta et al., 2011). Artemisinin, a sesquiterpene, is regulated by AabZIP1 in Artemisia annua (Zhang et al., 2015). Linalool synthesis in Cinnamomum osmophloeum is controlled by CoWRKY (Lin et al., 2014). The overexpression of CpMYC2 increases the linalool level, while overexpression of CpbHLH13 increases the β-caryophyllene level, in wintersweet (Chimonanthus praecox L.) flowers (Aslam et al., 2020).

Several TFs linked to fruit VOCs have been characterized. For example, in wine grape, VviWRKY40 regulates monoterpenoid glycosylation (Li et al., 2020). E-geraniol synthesis is positively regulated by CitERF71 in sweet orange (Li et al., 2017). In ripe kiwifruit (Actinidia arguta), NAC and ETHYLENE-INSENSITIVE3-like TF binding sites in the terpene synthase-1 protein (AaTPS1) promoter activate its expression; the absence of NAC (AaNAC2, AaNAC3 and AaNAC4) reduces the AcTPS1 transcript and protein levels, as well as the monoterpene volatile content (Nieuwenhuizen et al., 2015). Further studies of TFs will provide insights into the mechanisms underlying regulation of fruit aroma production. The major TFs that regulate VOCs are listed in Table 2.

TABLE 2. Major transcription factors that regulate VOC production.

展    望

Studies of floral and fruit aromas have generated extensive information concerning their volatile compounds, functions, biosynthesis, and regulation. Moreover, new techniques and methods have accelerated the discovery of VOCs and their synthetic enzymes, genes, and TFs, thereby enhancing the broader understanding of VOC biosynthesis in plants. However, several unresolved issues warrant further investigation.

对花和水果香气的研究已经产生了大量关于其挥发性化合物、功能、生物合成和调控的信息。此外，新的技术和方法加速了VOCs及其合成酶、基因和TF的发现，从而增强了对植物中VOC生物合成的更广泛的认识。然而，有几个尚未解决的问题需要进一步调查。

The aromas of flowers and fruits serve as signals to pollinators or fruit eaters; however, most horticultural varieties and cultivars are selected by human preference. The identification of VOCs relevant to human sensory preference is important to ensure that consumer demand is met. Additionally, biotechnological modification of the aromatic characteristics of plants or engineering of synthesis pathways in microbial cell factories could increase aromatic metabolite production for commercial exploitation.

Studies of the composition, synthesis, and regulation of floral scents and fruit aromas have focused on major plants, such as petunia (floral scents), tomato, apple (fruit aromas), and model plants. Little information is available concerning other aromatic plant species, possibly because of scarce genome information and difficulties with genetic transformation. The rapid development of genomic techniques, particularly gene editing, enables research regarding genome functions and regulatory mechanisms in a range of plant species. Flowers differ from edible fruits and may be less restricted by concerns involving public opinion and governmental regulation. Thus, genome editing can be applied to produce flowering plants with novel aromas and phenotypes, as well as improved sensory properties.

Most studies concerning the functions of fruit VOCs have focused on the biological activities of VOCs against microorganisms and insects, or on the attraction of animal dispersers; investigations of flower VOCs have focused on pollinator attraction. However, the effects of flower and fruit VOCs on human health are unknown. While VOCs improve fruit/flower edible/aromatic qualities, they also have potential for improving human physical and mental health. The utility of flower fragrances and fruit aromas as natural adjuvant therapies for chronic diseases or other health problems warrants further research.

补 充 材 料

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2022.860157/full#supplementary-material

脚注

1. https://www.statista.com/statistics/264001/worldwide-production-of-fruit-by-variety

论文引用

Citation: Mostafa S, Wang Y, Zeng W and Jin B (2022) Floral Scents and Fruit Aromas: Functions, Compositions, Biosynthesis, and Regulation. Front. Plant Sci. 13:860157. doi: 10.3389/fpls.2022.860157

Received: 22 January 2022; Accepted: 09 February 2022;
Published: 10 March 2022.