Phenylpropenes: Occurrence, Distribution, and Biosynthesis in Fruit

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Phenylpropenes: Occurrence, distribution and biosynthesis in fruit Ross G Atkinson J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04696 • Publication Date (Web): 23 Dec 2016 Downloaded from http://pubs.acs.org on December 25, 2016

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Journal of Agricultural and Food Chemistry

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Phenylpropenes: Occurrence, distribution and biosynthesis in

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fruit

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Ross G. Atkinson*

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The New Zealand Institute for Plant & Food Research Limited (PFR), Private Bag 92169,

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Auckland 1142, New Zealand

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AUTHOR INFORMATION

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*Corresponding author: Ross Atkinson

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The New Zealand Institute for Plant & Food Research Limited (PFR), Private Bag 92169,

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Auckland 1142, New Zealand

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Telephone: + 64-9-925-7182

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Fax: +64-9-925 7001

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E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT: Phenylpropenes such as eugenol, chavicol, estragole and anethole contribute

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to the flavor and aroma of a number of important herbs and spices. They have been shown to

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function as floral attractants for pollinators, and to have antifungal, and antimicrobial activity.

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Phenylpropenes are also detected as free volatiles and sequestered glycosides in a range of

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economically important fresh fruit species including apple, strawberry, tomato and grape.

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Although they contribute a relatively small percentage of total volatiles compared with esters,

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aldehydes and alcohols, phenylpropenes have been shown to contribute spicy, anise- and

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clove-like notes to fruit. Phenylpropenes are typically found in fruit throughout development

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and to reach maximum concentrations in ripe fruit. Genes involved in the biosynthesis of

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phenylpropenes have been characterized and manipulated in strawberry and apple which has

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validated the importance of these compounds to fruit aroma and may help elucidate other

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functions for phenylpropenes in fruit.

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KEYWORDS: aroma, flavor, fruit, phenylpropene, volatile


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INTRODUCTION

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Phenylpropenes are phenylpropanoid volatiles derived from the amino acid phenylalanine.

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The structural diversity of phenylpropenes is derived from variation to the substituents on the

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benzene ring and in the position of the double bond in the propenyl sidechain. The IUPAC

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names of common phenylpropenes are given in Table 1, along with a number of synonyms

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that have been used in the literature when reporting these compounds. The allylphenols,

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chavicol and eugenol, and their respective double-bond positional isomers, isochavicol and

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isoeugenol, are not particularly volatile. Fortunately, their low solubility in water enables

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facile extraction, from plant tissues, with organic solvents such as diethyl ether.1 In contrast,

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due to methylation of all their hydroxyl groups estragole, anethole, methyleugenol and

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methylisoeugenol are more volatile and can readily be detected by SPME or dynamic

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headspace analysis.2 Glycosylated phenylpropenes are typically purified on Amberlite XAD-

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2 resin and the volatile aglycones released by enzymatic digestion.

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Phenylpropenes have attracted considerable scientific interest as they are key flavor

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constituents of a number of important spices e.g. cloves (Eugenia caryophyllata) and star

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anise (Illicium verum) and aromatic herbs such as sweet basil (Ocimum basilicum) and fennel

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(Foeniculum vulgare). Eugenol is the main constituent (70–90%) of the clove essential oil

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and gives it its distinctive, pungent aroma.3 Star anise contains high proportions of (E)-

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anethole (50–80%) and has a strong anise flavor, with a licorice-like aroma.4 In sweet basil,

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there are chemotypes that accumulate only estragole, only eugenol, estragole and

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methyleugenol in similar amounts, and some that accumulate almost no phenylpropenes at

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all.1,

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flavor.6 The accumulation of high levels of chavicol is relatively rare. Some bay (Pimenta

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racemosa) oils contain high levels of chavicol and eugenol and have notes of cinnamon,

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Fennel contains primarily estragole and (E)-anethole and has a mild, sweet, anise



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clove and nutmeg.7 Phenylpropenes are also key flavor components of processed foodstuffs

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including licorice, teas, sausages and cola flavoured beverages.8-10

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Eugenol has been implicated in contributing to a negative sensory impact in wine with

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‘smoke taint’. In Australia and the USA, exposure of grapes to the smoke of bush fires can

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lead to the production of smoke aromas in wine. GC-MS analysis has shown that levels of

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guaiacol, 4-methylguaiacol, 4-ethylguaiacol and 4-ethylphenol and eugenol are elevated in

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wines produced from fruit from smoke-affected grapevines. Sensory studies identified 'burnt',

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‘smoked, 'leather' and 'earthy’ notes in wines derived from smoke-exposed grapevines but not

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in control wines.11-13 Exogenous application of eugenol and guaiacol in tomato also showed

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these compounds could be absorbed into the fruit. Direct foliar application increased eugenol

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content 200-fold, and 10-fold when plants were in contact with eugenol in the surrounding

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atmosphere.14

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Estragole (methylchavicol) and other phenylpropene volatiles are attractive not only to

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humans, but have also been shown to act as important insect attractants, reviewed in Tan &

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Nishida.15 For example, the males of many tephritid fruit flies respond strongly to

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methyleugenol and the compound is regularly used for monitoring and estimating

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populations.16 Flowers of some orchids produce phenylpropenes that lure male fruit flies and

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also act as floral rewards, which the flies convert to pheromone components.17 In Clarkia

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breweri and Petunia hybrida phenylpropenes are produced in the flowers mainly at night

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which likely relates to the activity of the moth that pollinate these species.18 However,

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phenylpropenes can also act as insect repellents and as defence compounds against

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herbivores.19-21

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Another likely role for phenylpropenes is in plant pathogen defence, with eugenol,

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estragole and anethole, in particular, being reported to have antimicrobial and antifungal

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effects.22-25 However, in most cases the evidence for involvement in pathogen defence is


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indirect as essential oils are extracted from the host plant and their effect on a range of

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pathogens is measured.26 In many cases the pathogens do not infect the host plant.

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Nevertheless, it is reasonable to assume that phenylpropenes have a role in plant defence

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given they show activity against such a wide range of pathogens. Recently, eugenol has been

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shown to enhance resistance of tomato plants to Tomato yellow leaf curl virus (TYLCV).27

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Eugenol application stimulated the production of endogenous nitric oxide (NO) and salicylic

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acid (SA) and enhanced transcription of a host R-gene specific to TYLCV.

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Finally, phenylpropenes have attracted attention through their effects on human health,

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reviewed in Charan Raja et al.28 Eugenol and isoeugenol possess both free radical and nitric

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oxide scavenging activities.29,

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human pathogenic gram-positive and gram-negative bacteria28 and yeast strains.31 Eugenol

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has the ability to inhibit viral replication and reduce viral infection against herpes simplex-1

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and -2 (HSV-2) and in vitro studies suggest it also has potential as an anti-giardial,

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trypanocidal, anti-malarial and anti-leishmanial agent.28

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Eugenol shows antibacterial activity against a range of

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The multiple effects and uses of phenylpropenes described above make them an

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attractive target for study and manipulation in fruit. In contrast to the broad literature on

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phenylpropenes available in herbs, spices and flowers, studies in fruit are not as extensive and

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genetic characterization is still in its infancy. This review summarizes our current knowledge

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of the occurrence of phenylpropenes in fruit, their role in fruit flavor, distribution within the

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fruit as well as how levels change during ripening, and recent genetic studies that have

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increased our understanding of how phenylpropenes are produced in fruit.

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OCCURRENCE OF PHENYLPROPENES IN FRUIT

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The flavor and aroma of ripe fruit arises from a complex mix of sugars, acids and volatile

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compounds with the major proportion of volatiles consisting of esters, aldehydes and



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alcohols. As fruit ripen, flesh firmness decreases, sugar levels increase, acidity drops and

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volatile production rises with all these changes occurring as a result of selection in the wild to

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attract insect dispersers.32-34 More recently, crop domestication and classical breeding have

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also influenced the ‘evolution’ of fruit flavor. However in many cases, modern breeding

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strategies have favored production traits such as yield, uniform ripening and disease

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resistance to the detriment of flavor and flavor diversity.35, 36 Phenylpropenes, sesquiterpenes,

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monoterpenes and sulfur-containing compounds contribute a small percentage of total

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volatiles in fruit, but their high odor activities make them attractive targets for breeding novel

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flavored varieties with minimal yield drag.

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Phenylpropenes are detected as free volatiles and sequestered glycosides in a range of

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fruit (Table 2) including many economically important fresh fruit species such as apple,

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tomato, strawberry and grape. The highest concentrations for individual phenylpropenes were

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found in sour cherry juice (2875 µg/L of eugenol glycoside), Queen Anne’s pocket melon

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(2303 µg/kg eugenol) and in purple passionfruit (1700 µg/kg). The mericarps of many plants

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such as fennel and anise (Pimpinella anisum) are commonly referred as seed spices, although

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anatomically they are fruits. For completeness, and as a point of comparison, Table 2 gives

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some examples of phenylpropene compositions and concentrations in a number of essential

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oils from such ‘dried fruits’. The concentration in these samples (expressed as a percentage of

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compounds found in the essential oil) was typically higher than in fresh fruit, ranging from as

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high as 94% in star anise and 68% in fennel, to ~5% in myrtle and myrobalan.

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In apple, studies have reported the presence of phenylpropenes in ripe fruit with estragole

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being the most commonly detected (Table 2). The first report from Williams & Tucknott37

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described the concentration of estragole in 14 different cultivars including ‘Cox’s Orange

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Pippin’, ‘Golden Delicious’ and ‘Red Delicious’. Concentrations were highest in ‘Spartan’

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and ‘Ellison’s Orange’, which was described as having a recognizable aniseed-like character


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to the aroma. In a study by Fuhrmann & Grosch,38 ‘Cox’s Orange Pippin’ was reported to

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contain only eugenol, and no phenylpropenes were detected in the variety ‘Elstar’. Estragole

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was reported in ‘Gala’ apples in an investigation of volatile changes during storage under

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regular and controlled atmosphere conditions.39 Estragole concentrations were highest after 4

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weeks storage and decreased under both regular and controlled atmosphere at 10 and 20

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weeks. An investigation of three apple varieties from the Madeira Islands revealed an unusual

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apple cultivar ‘Porto Santo’ with high levels of estragole and isoeugenol (15.43% and 0.28%

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of volatiles, respectively) from whole fruit.40 The most recent study of ‘Royal Gala’ apples

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identified estragole as the only phenylpropene detected in the headspace. Six additional

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phenylpropenes were observed after extraction with diethyl ether with (E)-isochavicol (anol)

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and isoeugenol being the most abundant.2

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Two quantitative trait loci (QTLs) controlling the production of estragole in apple were

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identified in a segregating population from a cross between ‘Royal Gala’ (an estragole

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producer) and ‘Granny Smith’ (a non-producer).2 Estragole production showed a normal

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distribution in the population and positive alleles of both QTLs were inherited from the

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‘Royal Gala’ parent. One QTL explaining 9.2% of the variation was located on linkage group

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1 and co-located with the MdoOMT1 gene involved in the biosynthesis of phenylpropenes

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(pathway shown in Figure 1).2 The second QTL was on linkage group 2 and explained a

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larger portion of the variation (24.8%). This QTL was located with 99% confidence in the

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upper 7.5 cM of linkage group 2.2

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Only eugenol and eugenol glycosides have been detected in tomato (Table 2). A study of

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two tomato lines was described by Birtic et al.41 using cultivars ‘Levovil’ (Solanum

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lycopersicum), characterized by large fruits and pharmaceutical sensory attributes, and

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‘Cervil’ (S. lycopersicum var. cerasiforme), a cherry tomato line with high intensity of overall

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aroma. Eugenol was found at much higher levels in ripe fruit of ‘Levovil’ compared with



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‘Cervil’ (121±21 vs 1±1 µg/kg) and eugenol glycosides showed the same pattern (58±4 vs 1±0

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µg/kg). Similar research by Ortiz-Serrano et al.42 compared the volatile and glycosidic

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fractions of two commercial tomato cultivars ‘Moneymaker’ and ‘Raf’. In this case

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‘Moneymaker’ had the highest level of eugenol (305.4 vs 25.5 µg/L) but ‘Raf’ accumulated

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the higher level of glycosides (492.8 vs 212.2 µg/L).

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Two QTLs for eugenol production were observed in a population of 144 recombinant

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inbred lines derived from a cross between ‘Levovil’ vs ‘Cervil’ by Causse et al.43 Eugenol

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was observed at higher concentration in ‘Levovil’ vs ‘Cervil’. QTLs for ‘pharmaceutical’

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aroma and eugenol content co-located on chromosome 2 and on chromosome 9 (in this case

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with guaiacol). Eugenol content was dependent on two loci with epistatic interaction.43 QTLs

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for eugenol production were also identified on chromosomes 2 and 9 by Zanor et al.,44 and

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two further QTLs on chromosome 1 and 4. The QTL on chromosome 1 was later shown to

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co-locate with a glycosyltransferase with activity towards eugenol.45

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Only trace levels of eugenol have been detected in cultivated octoploid varieties of

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strawberry, Fragaria x ananassa46 (Table 2). Cultivated strawberries have large fruits, red

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color, and a pleasant flavor that is usually less appreciated when compared to the more

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fragrant ‘wild’ strawberries. In the diploid woodland strawberry, F. vesca, eugenol and

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safrole were detected by Pet'ka et al.,47 whilst only eugenol was reported by Pyysalo et al.46

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and Ulrich et al.48 In contrast, the hexaploid musk strawberry (F. moschata) contains a much

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wider diversity of phenylpropenes including estragole, chavicol, eugenol, methylisoeugenol

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and high levels of methyleugenol.47

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Two QTLs for eugenol production in cultivated strawberry were reported by Zorrilla-

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Fontanesi et al.49 using an F1 population between lines 1392 (selected for its superior flavor)

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and line 232. Eugenol content was not normally distributed and although eugenol content was

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similar in both parents, significant variation in eugenol content was observed in the progeny


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over multiple years. Hierarchical cluster analysis grouped eugenol with multiple esters, 1-

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decanol, 1-octanol and the terpene alcohols, myrtenol and nerol. One QTL for eugenol

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mapped with other QTLs controlling esters, eugenol and terpenes. The second eugenol QTL

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mapped with QTLs controlling butyl hexanoate, 2-heptanol and 1-hexanol. Both QTLs were

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tentatively associated with putative transcription factors.

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In grape, eugenol and eugenol glycosides have been reported in a range of V. vinifera

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cultivars and wines and Vitis species.50-53 In the two most comprehensive studies on V.

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vinifera, eugenol was measured in 57 Spanish red wines51 and 52 red wines from four

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varieties (Grenache, Tempranillo, Cabernet Sauvignon and Merlot).54 The average

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concentration in wine was 29 µg/L, with a minimum of 4.2 and maximum of 73 µg/L in

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Lopez et al.51 and 3 µg/L, with a minimum of 0.88 and maximum of 15.6 µg/L in Ferreira et

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al.54 A comparison of wines produced from V. vinifera grapes with those from two native

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North American species, V. riparia and V. cinerea indicated a situation similar to cultivated

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strawberry vs wild strawberry. Eugenol was present in much higher concentrations in V.

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riparia (18 µg/L) and V. cinerea (328 µg/L) than the cultivated European wine grape V.

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vinifera (4 µg/L).50 Eugenol can be detected as a bound, glycosylated precursor in grapes, but

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high concentrations in wines are usually associated with contact with oak.50

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In banana and citrus, despite their economic importance, there have been only single

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studies reporting the presence of phenylpropenes in the fruit.55, 56 Eugenol was detected in

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fresh banana fruit (2.65±0.29 ppm), but not in an aqueous essence produced from whole fruit

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puree. In citrus, eugenol-glycoside was reported in lemon peel.56 In most other fresh fruit,

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except kiwifruit, phenylpropenes have been reported in single studies. In kiwifruit,

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phenylpropene glycosides at concentration ranging from 1.02–60.7 µg/kg were reported from

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four species in three studies.57-59

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THE IMPORTANCE OF PHENYLPROPENES TO FRUIT FLAVOR

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Gas chromatography-olfactometry (GC-O), aroma extraction dilution analysis (AEDA),

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Charm analysis and Osme methodology60 have all been used to analyze fruit samples for

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odor-active components. Although they contribute a relatively small percentage of total

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volatiles compared with esters, aldehydes and alcohols, phenylpropenes have been shown by

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these techniques to contribute spicy, anise- and clove-like notes to a range of fruit. Eugenol is

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the most commonly reported odor-active phenylpropene in fruit, followed by isoeugenol,

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chavicol and estragole (Table 3). Dunkel et al.61 reported that eugenol, estragole, isoeugenol,

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methyleugenol and anethole were among 230 compounds termed key food odorants (KFO’s)

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that formed a small group out of circa 10 000 food volatiles. In this meta-analysis, eugenol

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was found in 8.4% of the 227 food samples included, whilst estragole, isoeugenol,

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methyleugenol and anethole were reported in isoeugenol > chavicol in solvent extracted samples and reversed in

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headspace samples (Table 3). Changes in the odor-active volatile compounds in ‘Gala’

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apples were characterized by Osme analysis after cold storage.39 Estragole was identified as

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contributing an anise/licorice note that was perceived more in regular atmosphere stored fruit

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than in controlled atmosphere stored apples. Two other ‘unknown’ compounds were


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perceived as contributing sweet, anise, perfume notes, which may correspond to chavicol or

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eugenol identified in other studies. The highest aroma intensity for all three anise-note

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compounds was in fruit stored for four weeks in regular atmosphere.

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The importance of estragole to apple aroma has been directly validated using transgenic

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‘Royal Gala’ lines down-regulated for expression of the MdoOMT1 gene involved in the

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biosynthesis of phenylpropenes (Figure 1).2 Ripe fruit from these transgenic lines

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accumulated significantly less estragole than ‘Royal Gala’ controls, but otherwise

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accumulated similar levels of volatile esters and aldehydes. Sensory analysis indicated that

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fruit were perceived as having a different odor to control ‘Royal Gala’ fruit. The fruit were

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described as more aromatic/spicy and floral than control fruit. This result was surprising

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given that estragole alone is described as having an anise/spicy character. It suggests that

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estragole interacts with other compounds to influence the aroma of ‘Royal Gala’ fruit.

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In tomato recombinant inbred lines derived from a cross between ‘Levovil’ vs ‘Cervil’, a

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strong correlation between the content of guaiacol (reported as orthomethoxyphenol) and

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eugenol and a ‘pharmaceutical’ aroma was reported by Causse et al.43 Sensory analysis

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associated these compounds with the presence of odors of clove and camphor (Table 3).

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Eugenol was also shown to contribute to the aroma of ‘Levovil’ by Birtic et al.,41 where it

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was described as having a spicy, clove note. In ‘Moneymaker’ and ‘Raf’, hexanal, 3-

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methylbutanol, (E)-2-hexenal, octanal, (Z)-3-hexenol, guaiacol, and eugenol gave positive log

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odor values during ripening,42 which suggested they contributed to the aroma of these

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varieties.

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Odor-active phenylpropenes are not recorded in cultivated strawberry, but are present in

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some wild Fragaria species (Table 3). Freshly picked wild, musk strawberries (F. moschata)

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were described as having green, caramel, seedy and clove-like retronasal notes and overall a

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very intense mango-like, tropical smell.47 GC-O measurements of musk strawberry extract



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revealed three odor-active phenylpropenes, eugenol, chavicol and methyleugenol. Eugenol

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was the second most odor-active compound after mesifuran. The authors concluded that the

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cinnamon smell associated with musk strawberry (German name Zimterdbeere) is likely

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attributable to the high content and olfactory impact of eugenol, methyleugenol and

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methylisoeugenol. In contrast, Ulrich et al.48 did not identify any odor active phenylpropenes

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in F. moschata, but eugenol (with a nutmeg/clove aroma) was detected by GC-O in two F.

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vesca accessions. Eugenol was also detected by GC-O at low intensity in F. ananassa

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‘Elsanta’ but not by GC-MS analysis of the same variety.

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Eugenol is detected at concentrations greater than its sensory threshold in many V.

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vinifera red wine varieties51, 54 and in V. riparia and V. cinerea.50 Eugenol aromas are not

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generally considered a defect in wine, but their presence in unoaked red wines may be

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undesirable.50 The negative sensory impact of eugenol in wine with ‘smoke taint’ has been

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discussed in the introduction to this review.

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The odor activity of phenylpropenes in banana, kiwifruit, snake fruit and mulberry have

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also been reported (Table 3). Eugenol had a high odor activity value in mulberry fruit and

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was considered one of the six key aromas contributing to the mulberry flavor.63 Isoeugenol

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was detected at high nasal impact frequency in three snake fruit cultivars.64 In kiwifruit,

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phenylpropenes were only detected once released from glycosides59 and may not contribute

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to fresh fruit aroma. Although eugenol has been detected by GC-O in one banana study,55

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more work is needed to determine its importance to banana fruit flavor.

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DISTRIBUTION OF PHENYLPROPENES WITHIN FRUIT

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Many plant flavor compounds including phenylpropenes are biosynthesized and accumulated

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in specialized anatomical structures. In sweet basil, biosynthesis of essential oil components

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takes place in glandular trichomes (specifically peltate glands) located mainly on the surface


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of leaves.65 In flowers, volatile biosynthesis is generally believed to occur primarily in the

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epidermal cells of petals, but other cell layers are also likely to be involved.66 In fennel fruit,

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the essential oil is stored in special cavities named oil ducts which are located in the outer

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parts of the mericarp. Raman mapping of transverse and longitudinal sections of fennel fruits,

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showed that anethole was present in most essential oil cells in the mericarp, but also in the

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endosperm, and that the highest concentration of anethole was found at the top of the fruit.67

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Only a small number of studies have investigated the distribution of phenylpropenes in

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fresh fruit, and this does not extend to the cellular level. In Queen Anne’s pocket melon

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concentrations of eugenol, chavicol and isoeugenol were 7–10 fold higher in the skin than

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pulp.68 In three apple varieties from the Madeira Islands, phenylpropenes were found almost

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exclusively in the peel and not the flesh.40 Chassagne et al.69 also detected higher levels of

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glycosidically bound eugenol in the fruit skin of four Passiflora spp. (passionfruit) compared

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to juice. Together these results suggest that the fruit skin (epidermis and hypodermis) is the

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likely site of phenylpropene biosynthesis in fruit.

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Studies on the distribution of phenylpropene production in strawberry fruit have focussed

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on production in achenes versus the receptacle. Strawberries produce a false fruit that

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originates from the expansion of the receptacle of the flower base as a pseudocarp, with the

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one-seeded achenes located on the epidermal layer of the receptacle. The achene is the true

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fruit, and the receptacle, which results from enlargement of the flower receptacle, constitutes

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the fleshy part.70 In F. ananassa ‘Camarosa’ fruit the highest production of eugenol was

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found in achenes compared with receptacles throughout development and ripening when

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compared on a fresh weight basis.70

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CHANGES IN PHENYLPROPENES DURING DEVELOPMENT AND RIPENING



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Phenylpropene production, as well as being shown to be produced in specific tissues and cell

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types, has also been shown to be under strict temporal and developmental control. Emission

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of volatile compounds from flowers often display a rhythmic pattern controlled by a circadian

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clock or regulated by light. Maximum emission of isoeugenol from petunia flowers has been

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shown to reach its maximum at dusk, along with other benzenoid compounds such as

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benzaldehyde, 2-phenylacetaldehyde and benzyl benzoate.18 In sweet basil, there are

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compositional differences in essential oil accumulation between young and mature leaves.

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Methyleugenol was found at higher levels in older leaves (~68%) whilst its precursor,

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eugenol, was found at higher levels in younger leaves (~53%).71 The estragole content in

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fennel fruit also increases early in fruit development reaching a maximum at the waxy fruit

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stage,72 and remaining high after maturation even as the fruit dry and shrivel.72-74

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Table 4 shows examples of phenylpropenes changing during ripening in different fresh

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fruit. In ‘Royal Gala’ apples treated with exogenous ethylene to induce and co-ordinate

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ripening, concentrations of estragole and methyleugenol increased significantly at 7 days

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post-treatment. Eugenol concentrations also increased significantly, however, concentrations

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of chavicol, isochavicol and isoeugenol were similar. The increase in estragole and

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methyleugenol production correlated with an increase in endogenous ethylene production,

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and expression of MdoOMT1 required for their production.2 In a fruit development series,

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very low levels of estragole production were observed in immature fruit (30–120 days after

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anthesis). Estragole production increased significantly only late in apple fruit development,

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when fruit produced endogenous ethylene.

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Two studies have investigated the effects of cold storage on phenylpropene production in

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apple.2, 39 In the study by Plotto et al.39 fruit were stored for 4, 10, and 20 weeks at 1°C in

326

regular atmosphere (RA); controlled atmosphere (CA) for 10 and 20 weeks, or for 16 weeks

327

in CA followed by 4 weeks in RA. After cold-treatment fruit were ripened at 22°C for 5 days.


Page 15 of 39


15 328

Concentrations of estragole were highest in fruit stored for 4 weeks under RA (216 ng/kg/L)

329

compared with longer storage at 10 and 20 weeks (36 and 48 ng/kg/L). Fruit held for 10

330

weeks in CA also had higher concentrations of estragole (44 ng/kg/L) compared with fruit

331

stored for longer periods (25 and 9 ng/kg/L). Yauk et al.2 studied fruit stored for 4 weeks in

332

RA and ripened at room temperature for 1 and 7 days. Under these conditions, production of

333

estragole and methyleugenol increased after 7 days at room temperature which correlated

334

with increased ethylene production in these fruit.

335

In tomato, production of free volatile eugenol increases with ripening in ‘Levovil’ fruit.

336

In green fruit concentrations are low (11 µg/kg), and there is a rapid increase as fruit enter the

337

breaker stage (77 µg/kg). As fruit proceed through pink and into the red stage, concentration

338

continue to increase, but less dramatically.41 Concentrations of eugenol glycoside show a

339

similar pattern over ripening. Free volatile and glycosidically bound eugenol concentrations

340

also increased with ripening in ‘Moneymaker’ and ‘Raf’ fruit.42 However, in these cultivars

341

the most significant increases occurred in bound eugenol between fruit at the green and red

342

stages (Table 4).

343

In strawberry, the concentration of eugenol in the achenes was more than 6-fold higher in

344

green fruit (153.5 ng/g dry weight) than in the red fruit (Table 4). In contrast in receptacles,

345

the production of eugenol increased from the green (6.1 ng/g dry weight) to the red ripe stage

346

(25.9 ng g dry weight).70 The accumulation of eugenol in the receptacle correlated with the

347

increased expression of the eugenol synthase FaEGS2 in the fruit receptacle with ripening,

348

whilst the decrease in concentration in the achenes correlated with reduced expression of two

349

additional synthases — FaEGS1a and FaEGS1b.70

350

In grape, postharvest dehydration of ‘Pinot Noir’ grapes resulted in an increase in aroma

351

compounds such as guaiacol and eugenol (Table 4). Wines made from dehydrated grapes

352

tended to resemble the composition and flavor profile of wines made from grapes left on the



Page 16 of 39

16 353

vine (i.e. with extended ripening).53 Eugenol concentration increased 5-fold in pickling melon

354

as fruit ripened;75 whilst there was little change in phenylpropene glycoside concentrations in

355

two kiwifruit cultivars, as fruit moved from unripe, through ripe to over-ripe59 (Table 4).

356 357

BIOSYNTHESIS AND SEQUESTRATION OF PHENYLPROPENES IN FRUIT

358

The biochemical steps in the biosynthesis of phenylpropenes are shown in Figure 1, and

359

were initially elucidated in basil, petunia, and C. breweri that produce high levels of these

360

compounds; reviewed by Koeduka.76 The initial biosynthetic steps in phenylpropene

361

production are shared with the lignin biosynthetic pathway up to the production of coumaryl

362

alcohol and coniferyl alcohol. The first committed step in phenylpropene production involves

363

the conversion of coniferyl and p-coumaryl alcohols to p-hydroxycinnamyl acetates (Figure

364

1). One enzyme, P. hybrida coniferyl alcohol acyltransferase (PhCFAT), has been shown to

365

catalyze the formation of coniferyl acetate from coniferyl alcohol and acetyl CoA.77 PhCFAT

366

is a member of the BAHD (benzyl alcohol-acetyl-, anthocyanin-O-hydroxy-cinnamoyl-,

367

anthranilate-N-hydroxycinnamoyl/benzoyl-, deacetyl-vindoline) acyltransferase superfamily

368

that catalyze the addition of an acyl moiety from an acyl-coenzyme A (acyl-CoA) donor onto

369

an alcohol acceptor.78 Suppression of PhCFAT in petunia resulted in inhibition of isoeugenol

370

biosynthesis.77 Heterologous over-expression of PhCFAT in transgenic aspen significantly

371

increased the production of eugenol and eugenol glycosides.79

372

The p-hydroxycinnamyl acetates are reduced by NADPH-dependent phenylpropene

373

reductases of the PIP family [pinoresinol–lariciresinol reductase, isoflavone reductase,

374

phenylcoumaran benzylic ether reductase] (Figure 1). Eugenol synthases (EGS), isoeugenol

375

synthases (IGS) and bifunctional synthases (e.g. isochavicol/IGS, chavicol/EGS and

376

EGS/IGS) have been characterized from a number of species including sweet basil, C.

377

breweri and petunia,80,

81

anise,82 and Larrea tridentate.83 The importance of PhIGS1 to


Page 17 of 39


17 378

isoeugenol production in petunia flowers was validated using an RNAi suppression approach.

379

Analysis of the floral volatiles revealed significant reductions in isoeugenol emission (45–

380

90%) compared to wild-type flowers and a corresponding increase in eugenol emission.84

381

Further validation was obtained by examining P. axillaris subsp. parodii flowers that emit

382

neither eugenol nor isoeugenol. Molecular analysis revealed that P. axillaris subsp. parodii

383

flowers contained a functional EGS gene, but the IGS gene was inactive due to a frame-shift

384

mutation. In this case, despite the presence of active EGS, the flowers did not accumulate

385

eugenol but instead accumulated dihydroconiferyl acetate.84

386

Methoxylated phenylpropenes such as estragole and anethole are formed by O-

387

methyltransferases (OMT) using S-adenosylmethionine (SAM) as the methyl donor (Figure

388

1). OMTs, with specificity for phenylpropenes, have been isolated and biochemically

389

characterized from sweet basil;65 C. breweri,85, 86 Rosa chinensis87 and anise.82 OMT genes

390

have been classified into two groups depending on the substrates they use. Class I genes show

391

a preference for caffeic acid, whilst Class 2 genes use a much wider variety of substrates.65

392

As OMT genes utilizing phenylpropenes can be found in both classes, and small changes in

393

sequence can alter enzyme specificity,65 it is difficult to predict OMT substrate preference

394

based on sequence alone.

395

Two recent papers have greatly advanced our understanding of the enzymes responsible

396

for the biosynthesis of phenylpropenes in fresh fruit. In the first paper, three reductases

397

(FaEGS1a, b and FaEGS2) catalyzing the formation of phenylpropenes in strawberry were

398

characterized.70 Recombinant FaEGS1a, b catalyzed the formation of eugenol alone from

399

coniferyl acetate, while FaEGS2 catalyzed the formation of eugenol and isoeugenol. All three

400

enzymes showed different kinetic properties, which the authors suggested might be related to

401

the different endogenous concentrations of coniferyl acetate found in achenes (where

402

FaEGS1a, b are expressed) and the receptacle (where FaEGS2 is expressed). Transient over-



Page 18 of 39

18 403

expression of FaEGS1a in strawberry fruit increased the production of eugenol and 5-

404

methoxyeugenol, as did simultaneous overexpression of FaEGS1b and down-regulation of

405

FaCHS (to increase substrate availability). Co-expression of FaEGS2 with the p19 suppressor

406

of gene silencing (to overcome co-suppression of FaEGS2 in the receptacle) confirmed that

407

FaEGS2 was also a genuine eugenol synthase in vivo.

408

In the second paper, an OMT responsible for the production of estragole in apple was

409

isolated and characterized. The MdoOMT1 gene was shown to co-locate with a major QTL

410

for estragole production and its expression correlated with increased estragole accumulation

411

in ‘Royal Gala’ fruit.2 Biochemical characterization demonstrated that the gene showed

412

activity towards eugenol, chavicol and isoeugenol as well as three structural analogs

413

(dihydrochavicol, dihydroeugenol, and 2-propylphenol). Two alleles isolated from ‘Royal

414

Gala’ showed similar affinities for eugenol, but one allele (MdoOMT1a) showed 2–3 fold

415

higher affinity and catalytic efficiency for chavicol. As described earlier in this review, ripe

416

fruit from transgenic ‘Royal Gala’ lines down-regulated for expression of MdoOMT1

417

accumulated significantly less estragole than ‘Royal Gala’ controls, and also showed altered

418

sensory properties.

419

Sequestration of phenylpropenes as glycosides occurs commonly in fruit (Table 2) and

420

this bound pool represents an important potential source of flavor compounds that can be

421

released during maturation, storage and processing as well as by enzymes, heat or acids.88

422

The production of glycosides is catalyzed by UDP-glycosyltransferases (UGTs) that mediate

423

the transfer of an activated nucleotide sugar to acceptor aglycones (Figure 1). Plants contain

424

large families of UGTs with over 100 genes being described in the genomes of multiple

425

species.89 UGTs, with the ability to glycosylate phenylpropenes, have been isolated from tea

426

(Camellia sinensis),90 Eucalyptus perriniana cultured cells,91 and from the fruit of tomato45

427

and grape.92, 93 CsGT1 from tea and VvGT14a from grape belong to UGT Family 85, EPGT


Page 19 of 39


19 428

from eucalyptus to Family 75, VvGT7a-i from grape to Family 88 and SlUGT5 from tomato

429

to Family 72, suggesting the ability to glycosylate phenylpropenes has evolved independently

430

multiple times. SlUGT5 expression increased in developing and senescing fruit and was also

431

induced after plant infection with Phytophthora infestans. Recombinant SlUGT5 enzyme

432

showed strong affinity for eugenol as a substrate, but also showed activity towards guaiacol,

433

methyl salicylate and benzyl alcohol, indicating it is not a phenylpropene-specific GT.45

434

VvGT7a-h alleles preferentially glycosylated nerol and citronellol,92 whilst VvGT14a

435

glucosylated geraniol, R,S-citronellol and nerol.93 VvGT7 alleles and VvGT14a showed

436

significant catalytic activity towards eugenol.

437

Two papers by Tikunov et al.94,

95

have directly demonstrated the importance of

438

phenylpropanoid/phenylpropene volatile glycosylation to fruit flavor. In tomato, guaiacol,

439

methyl salicylate and eugenol volatiles impart a ‘smoky’ aroma character to the fruit. In

440

mature green fruit, these volatiles are conjugated as hexose-pentose diglycosides that are

441

rapidly cleaved upon tissue damage. As fruit ripen, the diglycosides are converted to

442

triglycosides that are resistant to cleavage.94 The enzyme that converts the cleavable

443

diglycosides into non-cleavable triglycosides was recently identified as NON-SMOKY

444

GLYCOSYLTRANSFERASE 1 (NSGT1) by Tikunov et al.95 The NSGT1 gene was

445

expressed in ripe fruit and co-located with QTLs previously linked with ‘pharmaceutical’

446

aroma and eugenol content on Chromosome IX.43, 44 Structural mutations in the NSGT1 gene

447

were associated with tomato genotypes showing the ‘smoky’ character. The importance of

448

the NSGT1 gene was validated in planta, by constitutive expression of the coding sequence of

449

a functional allele in transgenic ‘Moneymaker’ lines — a cultivar with a ‘smoky’ tomato

450

background. The release of eugenol, guaiacol and methyl salicylate from damaged fruits of

451

the transgenic lines was strongly decreased compared with control fruits, and the transgenic

452

fruit showed a significant reduction in ‘smoky’ aroma.



Page 20 of 39

20 453 454

TRANSCRIPTIONAL CONTROL AND PATHWAY MANIPULATION

455

Until recently, little was known about transcriptional regulation of volatile phenylpropene

456

production at the molecular level. However, several papers, focused on understanding floral

457

scent production in petunia, have started to reveal some of the regulators involved. These

458

include four R2R3-MYB-like transcription factors ODORANT1 (ODO1),96 EMISSION OF

459

BENZENOIDS II (EOBII),97 EMISSION OF BENZENOIDS I (EOBI)98 and PH4.99 Down-

460

regulation of PhODO1, PhEOBI and PhEOBII in transgenic petunia plants strongly reduced

461

levels of isoeugenol and other benzenoids. In contrast, suppression of PH4 reduced volatile

462

emission, but increased the internal pool of volatile compounds including eugenol and

463

isoeugenol. Thus PH4 was required for volatile emission, but not production. Down-

464

regulation of ODO1, EOBI and EOBII each affected the expression of a specific range of

465

structural scent-related genes from both the shikimate and phenylpropanoid pathways. One

466

target of ODO1, designated PhABCG1, was identified by Van Moerkercke et al.66 as

467

encoding an ABC transporter localized on the plasma membrane that might be involved in

468

trafficking volatile compounds between cell layers.

469

Much less in known about the transcriptional regulation of phenylpropene production in

470

fruit. Medina-Puche et al.100 has shown that orthologs of PhODO1 and PhEOBII, but not

471

PhEOBI, are present in the strawberry genome. FaEOBII was expressed more highly in the

472

fruit receptacle than in achenes, and expression increased as fruit ripened from red to over-

473

ripe and eugenol content increased. Transient down-regulation of FaEOBII in fruit

474

receptacles significantly reduced eugenol content and expression of FaEGS2 and FaCAD1

475

(cinnamyl alcohol dehydrogenase 1), the latter encoding an enzyme belonging to the

476

phenylpropanoid pathway. Transient down-regulation of FaMYB10 (an important an

477

important MYB transcription factor that regulates biosynthetic genes from the


Page 21 of 39


21 478

flavonoid/phenylpropanoid pathway) down-regulated FaEOBII expression as well as

479

anthocyanin and eugenol contents in the fruit receptacle.

480

Four further genes in the phenylpropanoid pathway have been shown to have roles in

481

distributing carbon flux to the phenylpropanoid pathway towards phenylpropene

482

biosynthesis: 4-coumarate:CoA ligase (4CL), cinnamoyl-CoA reductase (CCR1), caffeoyl-

483

coenzyme A O-methyltransferase (CCoAOMT1) and chalcone synthase (CHS). 4CL is the

484

third step in the phenylpropanoid pathway and converts hydroxycinnamic acids to their

485

corresponding CoA esters. Transient suppression of OS4CL in O. sanctum leaves caused a

486

reduction in leaf eugenol content, with a concomitant increase in hydroxycinnamic acids.101

487

CCR1 is the first committed step in the biosynthesis of lignin monomers which reduces

488

cinnamyl-CoA thioesters to their respective cinnamaldehydes. Down-regulation of PhCCR1

489

in petunia increased flux through the phenylpropanoid pathway, but internal and emitted

490

pools of phenylpropenes were unaffected.102 CCoAOMT1 methylates caffeoyl-CoA to

491

feruloyl-CoA and 5-hydroxy-feruloyl-CoA to sinapoyl-CoA. Silencing of PhCCoAOMT1 in

492

petunia resulted in a reduction of eugenol production but not of isoeugenol.103

493

In cultivated strawberry fruit, which typically produce only trace levels of

494

phenylpropenes (Table 2), carbon flux from the anthocyanin pathway was re-directed

495

towards production of hydroxycinnamyl alcohols using either transient down-regulation of

496

the FaCHS gene or using a stably transformed line containing an antisense copy of the

497

FaCHS gene.104 In both cases, an increase in volatile phenylpropenes was detected, indicating

498

the presence of functional phenylpropene biosynthetic pathway in cultivated strawberry.

499

Heterologous over-expression of sweet basil ObEGS or petunia PhIGS led to significant

500

increases in levels of chavicol, eugenol, isoeugenol and isochavicol. These results suggest

501

that cultivated strawberry fruit possesses a functional acyl transferase to produce p-

502

hydroxycinnamyl acetates but lack efficient EGS and IGS enzymes. The levels of



Page 22 of 39

22 503

phenylpropenes produced in the transgenic fruit greatly exceeded the threshold for human

504

detection. Although not directly tested, the expectation would be that the sensory properties

505

of the fruit would have been altered.

506 507

FUTURE PROSPECTS

508

Although phenylpropenes have been detected in a wide range of fresh fruit, the majority of

509

research has been carried out in only four: apple, strawberry, tomato, and grape. This may

510

simply reflect their economic importance and consequent research investment, however it is

511

notable that there are few reports of phenylpropenes being found in two other well studied,

512

and commercially important, fresh fruit namely banana and citrus. Further research is

513

required in more species and more cultivars to better understand which phenylpropenes

514

accumulate in fruit, where they are produced, and what changes occur with ripening.

515

Genes involved in the biosynthesis of phenylpropenes have recently been characterized

516

in strawberry, apple and tomato, leveraging off research in model crops such as petunia.

517

Manipulating levels of phenylpropenes in strawberry and apple validated the importance of

518

eugenol and estragole to fruit aroma in these species and may also help elucidate the in planta

519

function(s) for phenylpropenes in fruit. However, for targeted breeding of spicy, anise- and

520

clove-like notes into new fruit cultivars, a better understanding of allelic diversity of key

521

genes involved in the biosynthesis of phenylpropenes will be needed.

522 523

ACKNOWLEDGMENTS

524

I would like to thank the organising committee for the invitation and funding to attend the

525

11th Wartburg Symposium on Flavor Chemistry and Biology and Adam Matich and Andrew

526

Dare for their useful edits in the preparation of this manuscript.

527


Page 23 of 39


23 528

FUNDING SOURCE

529

Research funding was through the New Zealand Ministry of Business, Innovation and

530

Employment and internal PFR investment.

531



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868


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31 869

FIGURE LEGENDS

870

Figure 1. Biosynthetic pathway to phenylpropene production, and genes from fruit that have

871

been linked to their production. AT = acyl transferase, PhR = phenylpropene reductase,

872

OMT = O-methyltransferase and UGT = UDP-glycosyltransferase. Fruit genes were isolated

873

from Fragaria x ananassa (FaMYB10, FaEOBII, FaCHS, FaEGS1a, b, FaEGS2), Pimpinella

874

anisum (AIS1, AIMT1), Malus x domestica (MdoOMT1) and Solanum lycopersicum

875

(SlUGT5, NSGT1).



Page 32 of 39

32

Common name eugenol isoeugenol* chavicol isochavicol* methyleugenol methylisoeugenol*

IUPAC name 2-methoxy-4-(prop-2-en-1-yl) phenol 2-methoxy-4-(prop-1-en-1-yl) phenol 4-(2-prop-1-en-1-yl)phenol 4-(1-prop-1-en-1-yl)phenol 1,2-dimethoxy-4-(prop-2-en-1-yl) benzene 1,2-dimethoxy-4-(prop-1-en-1-yl) benzene

estragole

1-methoxy-4-(prop-2-en-1-yl) benzene

anethole*

1-methoxy-4-(prop-1-en-1-yl) benzene

CAS registry number 97-53-0 97-54-1 501-92-8 539-12-8 93-15-2 93-16-3 140-67-0

104-46-1

Synonyms 4-allylguaiacol, 2-hydroxy-5-allylanisole 4-(1-propenyl) guaiacol, 2-methoxy-4-propenylphenol 4-allylphenol, p-hydroxyallylbenzene anol, 4-hydroxy-1-propenylbenzene eugenol methyl ether, 4-allylveratrole isoeugenol methyl ether, 4-propenylveratrole methylchavicol, isoanethole, 4-allylanisole, chavicol methyl ether, 4-methoxyallyl benzene p-propenylanisole, 4-methoxy-1-propenylbenzene

Table 1. Nomenclature and synonyms of common phenylpropene volatile compounds. * Exist as both cis and trans isomers.


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33

Fresh fruit Common name Apple 14 cultivars ‘Gala’ ‘Elstar’, ‘Cox Orange’ ‘Ponta do Pargo’, ‘Porto Santo’, ‘Santo da Serra’ ‘Royal Gala’

Tomato ‘Cervil, ‘Levovil’ ‘Cervil’, ‘Levovil’

Binomial

Compounds

Concentration

Malus x domestica M. domestica M. domestica M. domestica

estragole estragole eugenol estragole anethole

0.002-0.7 (% of headspace) 9-216 ng/kg/L 0-3.3 µg/kg 0-15.43 (relative % peak area) 0.12-0.28

37

M. domestica

(E)-isochavicol isoeugenol chavicol eugenol estragole methyleugenol (Z)-isochavicol

1057 ng/g 237 140 139 87 38 31

2

Solanum lycopersicum S. lycopersicum

eugenol eugenol eugenol-glc eugenol eugenol-glc

1-5 µg/kg FW 1-121 µg/kg 1-58 25.5-305.4 µg/L 212.1-492.8

43

eugenol eugenol eugenol

46

eugenol safrole eugenol methyleugenol eugenol (E)-methylisoeugenol chavicol chavicol-like estragole anethole safrole eugenol

0.25 mg/kg trace 0.78-1.46 (relative conc.) 0 0 1.09 0.11 mg/kg 0.07 no absolute value given 3.84 mg/kg 1.39 0.4 0.35-0.87 0.06 0.03 not quantified not quantified 25.5 ng/g DW 0.88-15.6 µg/L 4.2-73 µg/L 2.3-5.2 µg/L 28.2 µg/L 29.9 4 µg/L

54

S. lycopersicum

Ref. 39 38 40

41

42

‘Moneymaker’, ‘Raf’ (juice) Strawberry Wild ‘Senga sengana’ Wild ‘Elsanta’ Wild Wild musk Wild

Fragaria vesca F. x ananassa F. vesca F. x ananassa F. virginiana F. moschata F. vesca

Mapping population Wild musk

F. x ananassa F. moschata

‘Camarosa’ Grape 52 red wines 57 red wines ‘Pinot noir’ ‘Falanghina’

F. x ananassa

Cabernet franc /Lemberger (wine) 9 accessions (wine) 10 accessions (wine) ‘Marechal Foch’ (wine) Banana ‘Cavendish’ Citrus Lemon Kiwifruit ‘Hortgem Tahi’

V. vinifera

eugenol eugenol eugenol eugenol eugenol-glc eugenol

V. riparia V. cinerea Interspecific hybrid

eugenol eugenol eugenol

16 328 0.001-0.08 mg/L

Musa sapientum

eugenol

2.65 ppm

55

Citrus limon

eugenol-glc

3 mg from 7.9 kg of peel

56

Actinidia arguta

(E)-isoeugenol-glc eugenol-glc

9.84 µg/kg 1.02

57

Vitis vinifera V. vinifera V. vinifera V. vinifera


48

47

49 47

70

51 53 52

50

105


Page 34 of 39

34 Banana kiwi

A. eriantha

‘Hort16A’

A. chinensis

‘Hayward” Sour cherry ‘Schaarbeekse kriek’ (juice)

A. deliciosa

60.7 µg/kg 4.38 4.05 30.9 µg/kg 12.7 8.6 µg/kg

58

2875 µg/L 108.4 36.4 2302.4 µg/kg 589.8 104.3 25.4 0.57 µg/100 g

106

0.01-0.032 (g/hL anhydrous alcohol) 0.004 0.002-0.006 0.186 0.003 160 µg/L 178-780 µg/L 0.2 % 0.1

Phenylpropenes: Occurrence, Distribution, and Biosynthesis in Fruit

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