Phenylpropanoid Metabolism in Ripening Fruits

Phenylpropanoid Metabolism in Ripening FruitsRupinder Singh, Smita Rastogi, and Upendra N. Dwivedi

Abstract: Ripening of fleshy fruit is a differentiation process involving biochemical and biophysical changes that leadto the accumulation of sugars and subsequent changes in tissue texture. Also affected are phenolic compounds, whichconfer color, flavor/aroma, and resistance to pathogen invasion and adverse environmental conditions. These phenoliccompounds, which are the products of branches of the phenylpropanoid pathway, appear to be closely linked to fruitripening processes. Three key enzymes of the phenylpropanoid pathway, namely phenylalanine ammonia lyase, O-methyltransferase, and cinnamyl alcohol dehydrogenase (CAD) have been reported to modulate various end productsincluding lignin and protect plants against adverse conditions. In addition, peroxidase, the enzyme following CADin the phenylpropanoid pathway, has also been associated with injury, wound repair, and disease resistance. However,the role of these enzymes in fruit ripening is a matter of only recent investigation and information is lacking on therelationships between phenylpropanoid metabolism and fruit ripening processes. Understanding the role of these enzymesin fruit ripening and their manipulation may possibly be valuable for delineating the regulatory network that controls theexpression of ripening genes in fruit. This review elucidates the functional characterization of these key phenylpropanoidbiosynthetic enzymes/genes during fruit ripening processes.

IntroductionFruit ripening is a developmentally regulated process resulting

from the coordination of numerous biochemical and physiologicalchanges within the fruit tissue that culminates in changes in fruitfirmness, color, taste, aroma, and texture of fruit flesh (Figure 1)(Brummell and Harpster 2001; Vicente and others 2006; Singhand others 2007). Textural changes that lead to softening of fruitshave drawn attention to the putative involvement of several en-zymes able to act and modify its structure in a developmental andcoordinated way (Brady 1987; Rose and others 1998; Sozzi andothers 1998).

Phenolic compounds produced by the phenylpropanoid path-way contribute to fruit pigmentation and the disease resistanceresponse found in many fleshy fruits during ripening (Figure 2).In olive fruits, several simple and complex phenolics have beenreported to be effective against pathogenic bacteria (Chowdhuryand others 1997). Caffeic acid has been found to be the most ef-fective agent, although oleuropein, the major phenolic constituentof olives, also exhibits bactericidal action (Ruiz-Barba and others1991). Moreover, phenolic compounds protect plants by actingas feeding deterrents to insects (Nahrstedt 1990). For example,flavonoids such as luteolin, naringenin, phloretin, quercetin 3-rhamnoside, and myricetin-3-rhamnoside, have been shown toact as feeding deterrents to aphids (Dreyer and Jones 1981).

MS 20100054 Submitted 1/16/2010, Accepted 3/7/2010. Authors Singh andDwivedi are with Dept. of Biochemistry, Lucknow Univ., Lucknow 226007, India.Author Rastogi is with Dept. of Biotechnology, Integral Univ., Lucknow 226026,India. Direct inquiries to author Dwivedi (E-mail: [email protected]).

In addition, a complex polymer, lignin, is also a prod-uct of phenylpropanoid metabolism. Lignin represents a ma-jor carbon sink in vascular plants and is derived from 3lignin precursors termed as “monolignols” (hydroxycinnamylalcohols), namely, p-coumaryl alcohol, coniferyl alcohol, andsinapyl alcohol. The biosynthesis of monolignols involves 2 spe-cific steps branching off the general phenylpropanoid pathway.The phenylpropanoid pathway drives the carbon flow fromthe aromatic amino acid L-phenylalanine (L-Phe) or, in somecases, L-tyrosine (L-Tyr) (Rosler and others 1997), for theproduction of 4-coumaroyl CoA (or a respective thiol esterin the presence of other 4-hydroxycinnamates). The synthe-sis of monolignols (lignin monomers) involves several hydroxy-lation/methylation and oxidation/reduction reactions (Whettenand Sederoff 1995) (Figure 3). Numerous enzymes, namely, afamily of oxygen-dependent cytochrome P450 hydroxylases andS-adenosylmethionine-dependent methyltransferases, act on thefree cinnamic acids and their CoA esters which are converted top-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. Aftersynthesis in cytoplasm, monolignols are transported to the cell wallas glucoside derivatives formed in a reaction catalyzed by uridinediphosphate glucose coniferyl alcohol glucosyltransferase (UDP-GT), where these glucoside derivatives are hydrolyzed by coniferinβ-glucosidase (CBG). The last major step in lignin biosynthesisis monolignol dehydrogenation in a reaction catalyzed by peroxi-dase (POD), laccase (LAC), polyphenol oxidase (PO), or coniferylalcohol oxidase (CAO) followed by polymerization by oxidativecoupling (Boerjan and others 2003). Peroxidases are involved inthe oxidation of phenolic compounds in cell walls, polymeriza-tion of lignin and suberin, and have also been shown to degradeflavonoids in noncell systems (processed food) as well as in vivo. Aslignin polymerizes, it serves as a matrix around the polysaccharide

398 Comprehensive Reviews in Food Science and Food Safety � Vol. 9, 2010c© 2010 Institute of Food Technologists®

doi 10.1111/j.1541-4337.2010.00116.x

Phenylpropanoid metabolism in ripening fruits . . .

Phosphoenolpyruvate

Phenylalanine

Cinnamate

4-coumarate

MONOLIGNOLS(p-coumaryl alcohol, coniferyl alcohol, sinapyl alcohol)

Chalcone(Aromatic ketone that forms the central core for a variety of important

biological compounds)

Flavones[Flavonoids based on the backbone of 2-phenylchromen-4-one

(2-phenyl-1-benzopyran-4-one)]

Leucoanthocyanidins(Chemically flavan-3,4-diols which are colorless compounds

related to anthocyanidins and anthocyanins)

Anthocyanins(Water-soluble vacuolar pigments which are glucosides of

anthocyanidins and may appear red, purple or blue according to pH)

SHIKI

MATE

PATHWAY

PHENYLPROPANOID

PATHWAYFLAVONOID

PATHWAY

Glycolysis

CarbohydratemetabolismmalonylCoA

Esters of organic acids

Benzoic acid derivatives

Isoflavonoids & flavonoids (Polyphenols having

3-phenylchromen-4-one and 2-phenylchromen-4-one backbones,

respectively)

Proantho-cyanidins

(Polymers of flavonoids)

Lignin(Biopolymer of aromatic subunits

forming an integral part of the secondary cell walls of plants)

Figure 1–Schematic overview of branch pathways of shikimate, phenylpropanoid metabolism, and flavonoid biosynthesis in plants leading to thesynthesis of flavonoids.

c© 2010 Institute of Food Technologists® Vol. 9, 2010 � Comprehensive Reviews in Food Science and Food Safety 399


C

NH2

O OH

CO OH

CO OH

OH

CO OH

OH

OH

CO OH

OH

OCH3

CO OH

OH

OCH3HO

CO OH

OH

OCH3H3CO

PAL C4H HCH OMT F5H OMT

Sinapic Acid5 OH-Ferulic AcidFerulic AcidCaffeic Acidp-Coumaric AcidCinnamic AcidPhenylalanine

Stress lignin(Physical Barrier to fungus)

Zwitterionic anthocyanin (Protectant)

Caffeic acid(Source of O-diphenols oxidizable, fungitoxic)

Esterification of host cell wall

(Chemical barrier)

Figure 2–The involvement of phenolic compounds in the expression of resistance to pathogen infection. The diagram shows the family of compoundsknown as phenylpropanoids and their route of synthesis from phenylalanine through the phenylpropanoid pathway. PAL-phenylalanine ammonialyase, C4H-cinnamic acid 4-hydroxylase, HCH-hydroxycinnamate 3-hydroxylase, OMT-O-methyl transferase, F5H-ferulic acid-5-hydroxylase.

components of some plant cell walls, providing additional rigidityand compressive strength as well as rendering the walls hydropho-bic and water-impermeable (Whetten and Sederoff 1995; Rastogiand Dwivedi 2003a). Limiting the carbon flow down the mono-lignol pathway should enhance the availability of coumaroyl CoAesters for chalcone synthase that catalyzes the 1st step in flavonoidbiosynthesis.

The characteristics of lignin differ among cell walls, tissues,and plant organs (Rastogi and Dwivedi 2003b; Grabber and oth-ers 2004). Lignin biosynthesis is coordinated and regulated dur-ing fruit ripening which provides structural strength to cells anddisease resistance (Lagrimini 1991; Chapple and Carpita 1998;Abdel-Massih and others 2007; Seymour and others 2008). Aro-matic rings of lignin are deposited within the cell wall carbohy-drate matrix, which is often oriented within the plane of the cellwall (Atalla and Agarwal 1985). Studies have suggested that theincrease in firmness of loquat fruit (Eriobotrya japonica) involves acoordinated regulation of lignin biosynthesis and cellulose hydrol-ysis (Cai and others 2006). Lignin in a soft fruit like strawberryhas been detected in the achenes (combination of seed and ovary

tissue) and in the vascular bundles that connect the achenes to thecentral pith (Suutarinen and others 1998).

Caffeoyl CoA OMT (CCoAOMT) is induced in elicited cellssynthesizing phenylpropanoid phytoalexins and, therefore, hasbeen implicated in the defense response of plant cells (Schmittand others 1991). In another study, the analysis of the effect ofhigh CO2 treatment on phenolic metabolism and ripening-relatedchanges in cherimoya fruit (Annona cherimola) demonstrated nochange in the total polyphenol levels. Upon exposure to air, how-ever, there was a slight decrease in lignin content in CO2-treatedfruits but the levels remained significantly higher compared to air-treated controls (Maldonado and others 2002). The data indicatedthat high CO2 treatment promoted changes in lignin degrada-tion. It was hypothesized that cell walls maintaining more lignindeposits could modulate the strength of cell–cell adhesion (Assisand others 2001). Furthermore, in considering the insecticidal ef-fects attributed to enriched CO2 treatment and the importance ofphenylpropanoid compounds in general defense strategies, it hasbeen suggested that the maintenance of these compounds in CO2-treated fruit may be an advantage in pathogen defense (Assis and

400 Comprehensive Reviews in Food Science and Food Safety � Vol. 9, 2010 c© 2010 Institute of Food Technologists®


C

NH2

O OH

C

NH2

O OH

CO OH

CO OH

OH

COAMP

OH

CO S-CoA

OH

CO H

OH

OH

OH

CO OH

OH

OH

COAMP

OH

OH

S-CoA

OH

OH

CO OH

OH

OCH3

COAMP

OH

OCH3

SCoA

OH

OCH3

CO OH

OH

OCH3HO

COAMP

OH

OCH3HO

SCoA

OH

OCH3HO

CO OH

OH

OCH3H3CO

COAMP

OH

OCH3H3CO

SCoA

OH

OCH3H3CO

CHO

OH

OCH3H3CO

OH

OCH3H3CO

OH

OH

OCH3

OH

CHO

OH

OCH3H3CO

CHO

OH

OCH3

Lignin and Lignans

Synthesis

A

B C D GE

H I

K L MJ

N O P

Q R S T

?

?

F

PAL

TAL

C4H

4CL

4CL CCR CAD

4CL CCR CAD

4CL

4CL CCR CAD

CCoA3H

CCoAOMT

CCoAOMT

C3H

OMT

F5H

OMT

CCR

4CL

4CL

4CL

4CL

4CL

Figure 3–An overview of the monolignol biosynthetic pathway (A) tyrosine, (B) L-phenylalanine, (C) cinnamic acid, (D) p-coumaric acid, (E)p-coumaroyl CoA, (F) p-coumaraldehyde, (G) p-coumaryl alcohol, (H) caffeic acid, (I) caffeoyl CoA, (J) ferulic acid, (K) feruloyl CoA, (L) coniferaldehyde,(M) coniferyl alcohol, (N) 5-hydroxyferulic acid, (O) 5-hydroxyferuloyl CoA, (P) 5-hydroxyconiferaldehyde, (Q) sinapic acid, (R) sinapoyl CoA, (S)sinapaldehyde, (T) sinapyl alcohol. TAL, tyrosine ammonia lyase; PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarateCoA ligase; CCR, cinnamoyl CoA reductase; CCoAOMT, caffeoyl CoA 3-O-methyltransferase; F5H, ferulate 5-hydroxylase; ?, yet unconfirmed reactions.



others 2001). It may be presumed that there could be a concomi-tant increase in expression of genes coding for putative enzymesof secondary metabolism including a gene associated with hostdefense response. Interestingly, there is a further unusual instanceof lignification in foods, and that is an increase in lignin con-tents of some fruits during ripening after harvest. The enzymes ofthe phenylpropanoid pathway involved in lignification have notonly been shown in tissues undergoing active lignin synthesis, buthave also been associated with nonlignified tissues or poorly lig-nified systems such as cell suspension cultures, suggesting that theenzymes may be involved in other secondary metabolites.

In recent years, results based on a transgenic approach haveled to propositions of newer concepts to the classical phenyl-propanoid pathway. There are several genes which are involved inthe phenylpropanoid pathway but only very few genes, namely,phenylalanine ammonia lyase (pal) (MacLean and others 2007), O-methyltransferase (omt) (Matus and others 2009), cinnamyl alcoholdehydrogenase (cad) (Aharoni and others 2002), and peroxidases(pod) (Ketsa and Atantee 1998) were considered to have a poten-tial role in fruit ripening and flavonoid biosynthesis. Flavonoidmetabolism competes directly with pathways leading to lignin andsinapate ester biosynthesis and is itself composed of a numberof branch pathways leading to isoflavonoids, flavonols, proantho-cyanidins (condensed tannins), and anthocyanins (Shirley 1999).The isolation and cloning of most of the structural flavonoid genesopens up possibilities to develop plants with tailor-made opti-mized flavonoid levels and composition. This review provides acritical insight into the role of key phenylpropanoid biosynthe-sis genes and enzymes in the structural and subsequently com-positional changes during fruit ripening. The numerous effectsthat ethylene elicits during ripening are also comprehensively dis-cussed. There are considerable gaps in our knowledge with re-spect to the role of phenylpropanoid metabolizing enzymes infruit ripening processes and it is anticipated that fresh ideas arisingfrom this article will shed light and facilitate other and future re-searchers in understanding emerging concepts in the regulation ofripening.

Phenylalanine Ammonia Lyase (PAL)PAL (EC 4.3.1.5), a cytosolic protein in vascular plants, is the

initial enzyme in the monolignol biosynthetic pathway that cat-alyzes the deamination of L-phenylalanine to transcinnamate, aprecursor of various phenylpropanoids, such as lignins, coumarins,flavonoids, UV protectants, and furanocoumarin phytoalexins.The reaction is generally considered to represent a key point atwhich carbon flux into this pathway is controlled (Hanson andHavir 1981; Jones 1984). PAL enzyme is one of the most in-tensively studied in plant secondary metabolism (Hrazdina 1992;Lewis and others 1999). Phenylpropanoid metabolism is relatedto the plant defense system and early studies in Phaseolus vulgarishad reported an increase in PAL activity and concentration of to-tal phenols due to the presence of pathogens (Bolwell and others1985). Treatment of apple with prohexadione–calcium has beenreported to result in alterations in the phenylpropanoid biosynthe-sis pathway that also enhanced disease resistance (Roemmelt andothers 2002). Results from the subcellular-localization in grapeberry revealed that PAL was present in the cell walls, secondarilythickened walls, and the parenchyma cells of the berry mesocarpcells, whereas 4-coumarate:CoA ligase (4CL) involved in the for-mation of CoA thioesters of cinnamic acids was present predomi-nantly in the secondarily thickened walls and the parenchyma cellsof mesocarp vascular tissue that were closely associated with fruit

qualities in addition to structural and defense-related functions(Chen and others 2006).

PAL is considered to be a key regulatory enzyme forflavonoid/anthocyanin biosynthesis during fruit ripening (Mar-tinez and others 1996). Flavonoids are synthesized by the phenyl-propanoid pathway in which the amino acid phenylalanine(substrate of PAL) is used to produce 4-coumaroyl CoA. This com-bines with malonyl CoA to yield chalcones (flavonoid precursorswith 2 phenyl rings). Conjugate ring closure of chalcones results ina 3-ring structure, the typical form of flavonoids. The metabolicpathway continues through a series of enzymatic modificationsto yield several flavonoid classes, including the flavonols, dihy-droflavonols, and anthocyanins (Mintz-Oron and others 2008).Indeed, PAL activation is considered as product-specific, such asfor lignin or tannin or anthocyanin biosynthesis.

Interestingly in loquat fruit, chilling injury, such as an increasein postharvest firmness at low temperatures, has been found to beattributable to an increase in PAL activity, lignin, and fiber con-tents as well as adherence of peel with flesh and development ofa leathery (juiceless) pulp. Significantly, no augmentation of ligni-fication and PAL activity was reported at 12 ◦C. Lignin contentof flesh tissue of the fruit was enhanced on ethylene (159%) and1-MCP (63%) treatment as well as in the control samples (139%).The loquat fruit firmness increased steadily even after harvest at20 ◦C, thus indicating that it was not a low-temperature-dependent response (Cai and others 2006). The results indicatethat the presence of ethylene is required not only for inducingsynthesis of the PAL enzyme, but probably also for maintainingits continuous high activity. Although lignification is a knownresponse to physical impacts in mangosteen fruit (Garcinia man-gostana) (Ketsa and Atantee 1998), some fruits develop increasedlevels of lignin during storage. Although the increase noted inapple (Marlett 2000) and pear (Martin-Cabrejas and others 1994)fruits were relatively small, the most notable case is that of an-other rosaceous fruit, the loquat (E. japonica). In this fruit, tissuelignification may result in greater flesh firmness, toughness of thetexture, or loss of juiciness (Cai and others 2006). Recently, itwas suggested that the increase in pericarp firmness of mangos-teen fruit resulted from induction of lignin synthesis, associatedwith an increase in pal and pod gene expression and its respectiveenzyme activities (Dangcham and others 2008).

Diversity and functional conservation of pal genepal gene has been cloned, as well as characterized, from many

plant tissues (Boudet 2007) (Table 1). pal appears to exist univer-sally in higher plants as a family of genes and the presence of pal iso-forms is a common observation. The significance of this diversity isunclear, but evidence for a degree of metabolic channeling withinphenylpropanoid metabolism suggests that partitioning of photo-synthate into particular branches of phenylpropanoid metabolismmay involve labile multienzyme complexes that include specificisoforms of PAL (Sreelakshmi and Sharma 2008).

In melon fruit, pal was shown to be transcriptionally inducedboth in response to fruit ripening and wounding (Given and oth-ers 1988). Regulation of pal gene expression in this fruit is acoordinated process in response to both ethylene and an ethylene-independent wound signal. pal gene expression followed the ex-pression kinetics similar to that of the ethylene biosynthetic genesduring fruit development. In contrast, ethylene biosynthetic genesshowed different induction kinetics compared to pal expression inresponse to wounding (Diallinas and Kanellis 1994). However, ac-tivation of PAL has also been observed in response to several types



Table 1–Summary of experimental results cited in the literature, which correlate mRNA accumulation and enzymatic activity of phenylalanine ammonialyase (PAL) with ripening in selected fruits.

Maximum MaximumPlant Accession identity identity mRNA Enzyme

Serial nr description nr with fruits other plants accumulation activity

1. Vitis vinifera EF192469 Prunus aviumAF036948 (80%)

Camellia sinensisD26596 (79%)

Sparvoli and others(1994)

Chen and others(2006)

2. Prunus avium AF036948 Pyrus communisDQ230992 (88%)

Robinia pseudoacaciaEU650628 (80%)

Wiersma and Wu(1998) (P. avium)

Manganaris andothers (2007) (P.salicina)

3. Musa acuminata EU104680 Citrus clementina XCitrus reticulateAJ238754 (83%)

Populus trichocarpaEU603319 (84%)

Wang and others(2007)

Promyou and others(2007)

4. Eriobotrya japonica EF685344 Pyrus communisDQ901399 (95%)

Quercus suberAY443341 (80%)

Shan and others(2008)

Cai and others (24)

5. Malus X domestica AF494403 Pyrus communisDQ230992 (97%)

Populus euramericanaAJ698920 (79%)

Venisse and others(2002)

Strissel and others(2005)

6. Pyrus communis DQ230992 Prunus aviumAF036948 (88%)

Populus tomentosaEU760386 (78%)

Fischer and others(2007)

Qian and others(2008) (Pyruspyrifolia)

7. Citrus limon U43338 C. clementina X C.reticulateAJ238753 (77%)

Populus trichocarpaEU603320 (81%)

Lo Piero and others(2006)

Lafuente and others(2003)

8. Fragaria X ananassa AB360390 Rubus idaeusAF237955 (92%)

Lotus japonicasAB283033 (79%)

Cheng and Breen(1991)

Jiang and Joyce(2003)

9. Rubus idaeus AF237955 Prunus aviumAF036948 (83%)

Camellia sinensisD26596 (78%)

Kumar and Ellis(2001)

Nita-Lazar and others(2004)

10. Ipomoea batatas M29232 LycopersiconesculentumM83314 (80%)

Nicotiana tabacum(Samsun NN)X78269 (79%)

Tanaka and others(1989)

Singh and others(1998)

11. Cucumis melo X76130 – Trifolium pretenseDQ073811 (78%)

Diallinas and Kanellis(1994)

Given and others(1988)

12. Lycopersicon esculentum M83314 Ipomoea nilAF325496 (80%)

S. tuberosum X63103(92%)

Lee and others (1992) Sreelakshmi andSharma (2008)

of stresses, including CO2 treatment (Ke and Salveit 1989) andlow temperature (Martınez-Tellez and Lafuente 1997). Similarly,in minimally processed lettuces, the evaluation of initial induc-tion kinetics and the time to reach maximum PAL levels revealedhigher levels of induction by combining different kinds of stresses(wounding plus ethylene) (Lopez-Galvez and others 1996). Theseresults signify that ethylene produced in response to biologicalstress could be a signal for plants to activate defense mechanismsagainst potential pathogens. PAL is also an important source ofammonia for plant tissues (Lewis and others 1999). In a study oncherimoya fruit (A. cherimola), PAL activity has been reported toincrease with a high ammonia demand and decreased in fruit withlow rate of ammonia assimilation thereby reflecting the metabolicstatus imposed by storage in different conditions (Maldonado andothers 2002). The possible involvement of PAL activity in the sup-ply of important metabolic compounds for early events of ripeningwas anticipated (Assis and others 2001).

PAL-regulated flavonoid/anthocyanin accumulationPAL has been implicated in 2 major problems, rapid pericarp

browning and fruit decay, which both decrease the market value offruits. It was believed that PAL activity enhanced the accumulationof phenol compounds in rambutan fruit (Nephelium lappaceum) bypolyphenol oxidase (PPO) and/or peroxidase led to the appear-ance of brown products (Ke and Saltveit 1988; Cantos and others2002; Yingsanga and others 2008). Enhanced PAL activity has alsobeen suggested to play a role in ethylene-mediated anthocyaninaccumulation and enhanced strawberry fruit color development(Jiang and Joyce 2003). Parallel changes in anthocyanin accumu-lation and PAL activity apparently reflect control of anthocyaninsynthesis by PAL, presumably through the supply of componentcinnamic acid molecules. It is noteworthy that 2 peaks of PALactivity were reported in strawberry fruits, one in green fruit and

the other in nearly ripe fruit (Cheng and Breen 1991). The 1stpeak was suggested to be involved in the synthesis of flavonoids(condensed tannins) and phenolics that took place during earlyfruit development, whereas the 2nd activity peak was associatedwith the anthocyanin accumulation that occurred during laterstages of fruit ripening (Macheix and others 1990). In orangefruit, the expression of a putative anthocyanin transporting glu-tathione S-transferase (GST) was correlated with the expression ofthe pal, chalcone synthase (chs), dihydroflavonol 4-reductase (dfr),and UDP glucose, flavonol 3-O-glucosyltransferase (ufgt) genesunder cold stress (Lo Piero and others 2005, 2006). As is the casefor many secondary metabolite biosynthetic proteins, an appar-ent redundancy with the anthocyanin-transporting GSTs in grapeberries (Vitis vinifera) was reported, and this redundancy was seenwith numerous functional copies of biosynthetic enzymes includ-ing PAL (Conn and others 2008). It is quite clear that, even formuch-studied “old” pathways like flavonoid biosynthesis, theseare exciting times.

In an earlier study, expression of 7 genes of the anthocyaninbiosynthetic pathway (pal, chs, chi, f3h, dfr, ldox, and ufct) wasdetermined in Shiraz grape berries (V. vinifera). In flowers andgrape berry peels, expression of all of the genes, except ufct, wasdetected up to 4 wk postflowering, followed by a reduction in thisexpression 6 to 8 wk postflowering. Expression of chs, chi, f3h, dfr,ldox, and ufct then increased 10 wk postflowering, coinciding withthe onset of anthocyanin synthesis. The results obtained in thestudy provide additional evidence for the correlation between theexpression of structural flavonoid pathway genes and anthocyaninproduction during fruit development. In grape berry flesh, no palor ufct expression was detected at any stage of development, butchs, chi, f3h, dfr, and ldox were expressed up to 4 wk postflowering.These results indicated that the onset of anthocyanin synthesis inripening grape berry peel coincides with a coordinated increase in



expression of a number of genes in the anthocyanin biosyntheticpathway, suggesting the involvement of regulatory genes. ufct isregulated independently as compared to other genes, suggestingthat in grapes it could be a major control point in this pathway(Boss and others 1996). These studies emphasize the complexnature of flavonoid regulation in fruits, at least at the biosyntheticgene level, and the potential problems in correlating genotypesand phenotypes.

Schaffer and others (2007) observed that the genes involved inthe 1st steps of phenylpropanoid pathway were ethylene-responsiveand pal1 being one of the candidate genes exhibited a rapid increaseof expression in apple fruit. Transcript patterns of the 2 pal genesin loquat fruit (Eriobotrya japonica) differed with a sharp increasein ejpal1 transcripts late in fruit development. The opposite trendoccurred with ejpal2, where it was strongly expressed in youngfruit and not detectable at maturity (Shan and others 2008). Thissuggested that ejpal2 might be more heavily involved in phenyl-propanoid synthesis (including lignin synthesis) during early stagesof fruit development, when there was considerable increase invascular tissues. At later stages, this declined as the fruit matured,whereas, in contrast, ejpal1 was thought to be more involved ininduction of flavonoid synthesis and lignification of the maturefruit during ripening. In addition, manipulation of the flavonoidpathway by antisense expression modulated PAL activity throughtranscriptional and posttranscriptional mechanisms in strawberry(Griesser and others 2008). It appears that chs and pal genes arecrucial for flavonoid synthesis and the enhanced expression of oneor both of these genes during development could specifically beassociated with higher flavonoid content at maturity.

Promyou and others (2007) reported that “Sucrier” bananacoated with polyethylene parafilm wax (20%) showed a delay ofpeel spotting which was significantly not associated with a changeof total free phenolics in peel or with PPO activity, but was ac-companied by reduced in vitro PAL activity. Results suggested thatthe delay in peel spotting, after surface coating, was a result, atleast in part, of reduced PAL activity. Similarly in another studyon banana, the results suggested that the induction of PAL dur-ing low-temperature storage was regulated at transcriptional andtranslational levels, and was related to a reduction in CI symp-toms. Northern and Western blot analyses revealed that mRNAtranscripts of mapal1 and mapal2 and PAL protein levels duringstorage increased, reaching a peak at about day 8, and finally de-creased at chilling temperature. Prior to low-temperature storage,pretreatment with propylene could alleviate CI and enhance PALactivity, protein amount, and mRNA transcripts of mapal1 andmapal2. Moreover, changes in PAL activity, protein content, andaccumulation of mapal1 and mapal2 exhibited almost the samepatterns (Wang and others 2007). Thus, the PAL activation bypropylene or chilling temperature may be attributed to the syn-thesis of new PAL protein. The results appear to suggest that theaccumulation of pal transcript can serve as a molecular marker forchilling tolerance in banana fruit.

In raspberry (Rubus idaeus), development of fruit color and fla-vor was dependent on PAL encoded by a family of 2 genes (ripal1and ripal2). Although expression of both genes was detected inall tissues examined, ripal1 was associated with early fruit ripen-ing events, whereas expression of ripal2 correlated more with laterstages of flower and fruit development. Determination of the ab-solute levels of the 2 transcripts in various tissues showed thatripal1 transcripts were 3- to 10-fold more abundant than those ofripal2 in leaves, shoots, roots, young fruits, and ripe fruits. The2 ripal genes, therefore, appeared to be controlled by different

regulatory mechanisms (Kumar and Ellis 2001). Although fruitsat 2 stages differed in their chemistry, determination of the exactrole played by each ripal isoform in supporting accumulation ofspecific phenylpropanoid products in fruits would require detailedmetabolite profiling.

Promising outlook of PALTwo alleles of the pal gene identified in loquat fruit were diver-

gently regulated during fruit development (Shan and others 2008).This would have implications well beyond lignification, where thedirect relationship between PAL and lignin is still not very strong.Cherimoya fruit (A. cherimola) had exhibited an increase in PAL ac-tivity without significant increase in lignin synthesis, even thoughthis enzyme is part of the phenylpropanoid pathway (Assis andothers 2001). The extent to which this gene may regulate thepathway is different in different tissues; however, the mechanismis still unclear. To further unravel the role of pal genes duringripening in various fruit systems, the tissue-specific and develop-mental expression of each gene family member has to be studied.Moreover, modulation of PAL activity, which caused the reducedlevel of the cinnamic acid derivatives, has already opened newavenues (Shirsat and Nair 1986). The identification and charac-terization of pal genes from a fruit cDNA library would certainlycreate an opportunity to explore the possible functions of multi-ple pal genes during fruit development. To resolve their respectiveroles, it would be informative to selectively silence pal genes andmonitor the resulting transgenic phenotypes. Therefore, it shouldbe feasible to scrutinize the functions of the pal gene family, andthe cis-acting elements involved in selective expression during fruitdevelopment. A possible function of PAL during ripening couldbe the catalysis of the 1st reaction toward the formation of com-pounds participating in the aroma of ripe fruit. The emergingtheme from recent studies is that the pal promoter is able to inte-grate the complex set of developmental and environmental signalsin order to finely adapt pal gene expression to the diverse functionsof phenylpropanoid biosynthetic products.

O-Methyltransferases (OMTs)Methyltransferases are ubiquitous enzymes that catalyze the

transfer of a methyl group from S-adenosyl-L-methionine (SAM)to an acceptor substrate, generating O-, N-, S-, and C-methylderivatives and S-adenosyl homocysteine (Ibrahim and others1998). SAM-dependent O-methylation is catalyzed by OMTsand involves the transfer of the methyl group of S-adenosyl-L-methionine (AdoMet) to the hydroxyl group of an acceptormolecule, with the formation of its methyl ether derivative andS-adenosyl-L-homocysteine as products such as lignin, flavonoids,phenylpropanoids, and alkaloids (Dwivedi and others 1994; Pich-ersky and Gang 2000; Rastogi and Dwivedi 2006). OMTs playa critical role in the biosynthesis of many classes of compoundsrequired for plant growth, aroma generation, and plant defense.There are several hundred O-methylated flavonoids which occur inplants, and these range from mono- to polymethylated compoundsbelonging to the chalcones, flavones, isoflavones, and flavonols, aswell as their dihydro derivatives (Wollenweber and Dietz 1981).Combined biochemical and molecular analyses of volatile com-ponents produced by fruit have demonstrated that their biogenesisforms an integral part of ripening. Several omt cDNA clones havebeen reported from different plant species, which share commonstructural as well as physicochemical features. The phylogeneticanalysis of plant omt sequences suggests that plant omts may havediverged from a common ancestral gene, through gene duplication



R

OH

OH

R

OCH3

OHR-COOH:Caffeic AcidR-CHO:Caffeoyl AldehydeR-COH:Caffeoyl Alcohol

R-COOH:Ferulic AcidR-CHO:Coniferyl AldehydeR-COH:Coniferyl Alcohol

R

OCH3

OH

HO

R

OCH3

OH

H3CO

R-COOH:5-Hydroxyferulic AcidR-CHO:5-Hydroxyconiferyl AldehydeR-COH:5-Hydroxyconiferyl Alcohol

R-COOH:Sinapic AcidR-CHO:Sinapyl AldehydeR-COH:Sinapyl Alcohol

COMTCOMT

Figure 4–Role of OMTs in plant-specializedmetabolism conversions catalyzed by COMT.

OH

OH

OH

OCH3

Catechol Guaiacol

OMT

OH

OH

OH

OCH3OMT

CHO CHOVanillinProtocatechuic

Aldehyde

COOH

OH

OHCaffeic Acid

COOH

OCH3

OHFerulic Acid

OMT

[A] [B] [C]

Figure 5–O-methylation of caffeic acid, catechol,and protocatechuic aldehyde to their respectivemethylated products, ferulic, guaiacol, andvanillin, by the action of O-methyltransferase.

and mutation, to yield the various functional enzyme groups cur-rently recognized in plants (Ibrahim 1997). It would be interestingto group plant omt cDNA genes according to a functional traitthat reflects the substrate preferences of their encoded proteins,and could be used as the basis for classification of this supergenefamily.

Classification of plant OMTOMTs are widespread throughout the plant kingdom and found

in all lignin-producing plants. O-methylation of the phenyl-propanoid and flavonoid groups of compounds is catalyzed bydistinct classes of OMTs.

Caffeic acid 3-O-methyltransferase (COMT, EC 2.1.1.68) cat-alyzes the O-methylation of aromatic diols and is involved inlignification (Rastogi and Dwivedi 2008) or may have otherphysiological functions like flavor generation (Collendavelloo andothers 1981; Pellegrini and others 1993) (Figure 4). Interest-ingly gene evolution studies in Clarkia breweri have suggested thatisoeugenol OMT (iemt) gene that catalyzes the methylation ofeugenol and isoeugenol to form the volatiles methyleugenol andisomethyleugenol had arisen from comt gene. It was suggested thatOMT substrate preference could be regulated by a few aminoacid residues, and new OMTs with different substrate specifici-ties could evolve from an existing OMT by mutation of severalamino acids (Wang and Pichersky 1999). The evolution of newOMTs with new substrate specificities is relatively simple; it is notsurprising that OMTs with similar substrate specificities evolvedindependently in different plant lineages more than once. Thesebroad-specificity enzymes may be recruited for new metabolicpathways, followed by further evolution toward more specific andefficient catalysts. It follows that gene duplication (or even poly-ploidy) is an important factor in this concept as it provides theraw material for the acquisition of new biosynthetic pathways. Itis somewhat surprising that developing strawberry fruits displayhigh OMT activity levels toward caffeic acid, protocatechuic alde-hyde, and catechol resulting in ferulic acid, guaiacol, and vanillin(Figure 5A to 5C), respectively.

CCoAOMT (EC 2.1.1.104), which catalyzes the meta-O-methylation of caffeoyl CoA to form feruloyl CoA, appears toplay an important role in the formation of the coniferyl alcoholmoieties that are precursors for lignification (Dwivedi and Camp-bell 1995; Vander and others 2000). CCoAOMT catalyzes 1 of 2alternative methylation steps of the phenylpropanoid biosynthesispathway which leads to the synthesis of diverse secondary prod-ucts such as lignin, flavonoids, and isoflavonoids (Ye and Varner1995) and this methylation step is highly regulated (Hahlbrock andScheel 1989).

Proposed methyl acceptor classes and groupsThe molecular and biochemical data available so far provide the

basis for a meaningful classification of the plant omt gene superfam-ily. There exist some 36 omt cDNA clones, which are subdividedinto 5 groups encoding the methylation of the lignin precursors,caffeic and 5-hydroxyferulic acids (Group 1), flavonoids (Group 2),and phenylpropanoids (such as the coumarins Group 3). Group 4 isprimarily comprised of simple phenols and anthocyanins, whereasGroup 5 encompasses polyketides and other acetate/malonate-derived compounds (Table 2). Each group of OMT is furtherclassified into Class “A” and Class “B.” Based on the class of sub-strate methylation, Class “A” OMTs methylate phenylpropanoidcompounds, whereas Class “B” OMTs methylate flavonoid com-pounds. Although the chemical mechanisms of methyl transferreactions are identical, OMTs differ in their selectivity with re-spect to the stereochemistry of the methyl acceptor molecules, aswell as the substitution pattern of their phenolic hydroxyl groups(Ibrahim and others 1998). Some of the known OMTs displaystrict specificities toward their acceptor substrate as well as to theposition of substrate methylation. In contrast, other OMTs, espe-cially those catalyzing the methylation of catechol (O-dihydroxy)moiety substrates, exhibit surprisingly broad substrate specificities.OMTs have been shown to be multifunctional enzymes that couldalso catalyze transformations in 2 different biosynthetic pathwayssuch as the alkaloid and phenylpropanoid pathways (Li and oth-ers 1997) or aroma biosynthesis (Lavid and others 2002) and,



Table 2–Classification of omt cDNA clones encoding the methylation ofphenylpropanoid compounds (Class “A”) and omt cDNA clones encodingthe methylation of flavonoid compounds (Class “B”).

Groups

OMT cDNA clones encodingmethylation of

phenylpropanoidcompounds Class “A”

OMT cDNA clones encodingmethylation of flavonoid

compounds Class “B”

Group 1 Caffeic/5-hydroxyferulic acid Flavonols (flavones)Group 2 CoA esters of

caffeic/5-hydroxyferulicacid

Chalcones (flavanones)

Group 3 Phenylpropanoids(Coumarins,furanocoumarins)

Pterocarpans and theirisoflavone precursors

Group 4 Phenolic compounds (Simplephenols, benzoic acids,phenolic esters)

Flavans and anthocyanins(although none has beenreported)

Group 5 Polyketides and otheracetate/malonate derivedcompounds

–

incidentally, both phenylpropanoid and flavonoid compoundsshare some structural similarities in which the phenolic B ringand carbons 2, 3, and 4 of flavonoids are derived from phenyl-propanoids (Ibrahim and others 1998). There are now docu-mented cases from different plant species of apparent evolutionfrom COMTs of new OMTs with new substrate specificities. Asmore such cases are described, and an understanding of the effectsof specific amino acids on the active site develops, it may becomepossible to use a COMT as the starting point in designing anOMT that can act on a particular substrate of interest.

Despite a particular specificity for phenolic substrates, plantOMTs share highly conserved domains (Dwivedi and Campbell1995; Joshi and Chiang 1998). To date, more than 10 distinctgroups of SAM–OMTs that utilize SAM and a variety of sub-strates have been described in higher plants. A comparison ofthe amino acid sequence of 56 SAM–OMTs from different plantsshowed that plant OMTs fall into 2 distinct groups, Pl-OMT I andPl-OMT II. The length of Pl-OMT I enzymes varies from 231to 248 amino acids, whereas the length of Pl-OMT II enzymes is344 to 383. Unlike Pl-OMT I members that are known to utilizeonly a pair of substrates, the members of the Pl-OMT II groupcan accept a variety of substrates and are multifunctional enzymes(Li and others 1997). Multiple OMTs displaying small but definedsubstrate discrimination could be found within the same plantand even within the same tissue (Gang and others 2001, 2002).Recently, in anise (Pimpinella anisum) seeds and leaves, phenyl-propene t-anethole has been shown to impart the characteristicsweet aroma. A cDNA encoding t-anol/isoeugenol synthase 1(ais1), which is an NADPH-dependent enzyme that can biosyn-thesize t-anol and isoeugenol from coumaryl acetate and coniferylacetate, respectively, was cloned. In addition, t-anol/isoeugenolOMT 1 (aimt1), an enzyme that converts t-anol or isoeugenol tot-anethole or methylisoeugenol, respectively, via methylation ofthe p-OH group was also successfully cloned. The genes encodingais1 and aimt1 were expressed throughout the plant and inciden-tally their transcript levels were highest in developing fruits. TheAIMT1 protein sequence exhibited significant homology to basil(Ocimum basilicum) and Clarkia breweri phenylpropene OMTs, butunlike these enzymes, which do not show large discriminationbetween substrates with isomeric propenyl side chains, AIMT1showed a 10-fold preference for t-anol over chavicol and forisoeugenol over eugenol (Koeduka and others 2009). Therefore, itcould be that furaneol methylation occurs as a result of and in par-

allel with other reactions involving the methylation of caffeic acid,catechol, anthocyanidin, or other as yet unidentified O-diphenolsthat increase during fruit ripening.

Biological significance of OMTs in volatile generationVolatile compounds from the phenylpropanoid pathway, for ex-

ample estragole (and eugenol), are likely to be produced fromphenylalanine. The final step in the estragole biosynthesis is pre-dicted to involve a methyltransferase class of enzymes but stillsome of the enzymatic steps are still poorly understood. Apartfrom OMT that add a methyl group, several enzymes belonging tothe SABATH family (S-adenosyl-L-methionine carboxyl methyl-transferase) have also been reported to exhibit a similar function.Significantly, both add the methyl group using methionine as adonor and the main pathway for ethylene biosynthesis departs frommethionine and is converted to S-adenosyl methionine (SAM),amino-cyclopropane carboxylic acid (ACC), and ethylene in 3consecutive reactions catalyzed by the enzymes SAM-synthetase,ACC-synthase (ACCS), and ACC-oxidase (ACCO), respectively.SAM is a primary metabolite, crucial in polyamine metabolism andthe main methyl group donor in many reactions, such as those oflignin biosynthesis, nucleic acid, flavonoids, phenylpropenes, al-kaloids, and protein methylation. Two (SABATH1 and 4) out of 6members of the SABATH family showed an increase of expressionupon the treatment of ethylene in apple during fruit ripening. Ofthe 7 O-methyl transferases in apple, omt7 was ethylene-inducedand quite similar to comt (Gowri and others 1991) and, there-fore, could possibly be instrumental in catalyzing the final step inestragole biosynthesis (Schaffer and others 2007).

Because of the relative abundance of volatiles present in a fruit,each individual volatile acts as a “fingerprint” of a particular culti-var and species, which contribute significantly to flavor (Schieberleand Hofmann 1997). In apple, a total of 186 candidate genes minedfrom expressed sequence tags (EST) databases have been thoughtto be involved in the production of ester, polypropanoid, andterpene volatile compounds during ripening. Based on sequencesimilarity and gene expression patterns, 17 candidate genes in-cluding omt have been identified as ethylene control points foraroma production during the fruit ripening process (Schaffer andothers 2007). In papaya, huge numbers of genes have been re-ported which are involved in the development of volatiles duringfruit ripening. Papaya has 30 candidate genes for the lignin syn-thesis pathway, with an intermediate number of genes for cad [18],pal [9], f5h [4], c4h [2], and c3h [2], but only 1 comt and 1 ccrgene (Ehlting and others 2005). Moreover, papaya has 2 genes inthe family ccoaomt, which are presumed to convert caffeic acid toferulic acid (Ming and others 2008).

OMT-mediated anthocyanin biosynthesisRipening-related gene sequences that code for proteins involved

in key metabolic events including anthocyanin biosynthesis, havebeen isolated from strawberry (Manning 1998) and were not foundto be active in green fruits. The flavonoid biosynthesis pathwayleads to anthocyanin formation and it is noteworthy that thereis cross-linking and interdependence of the flavonoid and ligninbiosynthesis pathways during the ripening process (Figure 6). Athorough knowledge of the interconnecting pathways of ligninbiosynthesis is required for the rational designing of metabolic en-gineering strategies. Higher levels of omt transcripts have been ob-served during fruit ripening of various cultivars of berries, whichaccumulated methoxylated forms of anthocyanins more abun-dantly than nonmethoxylated forms. It was assumed that cyanidin



HSAOC

O

OH

OH

OHHO

OH O

NfE

Dhq

Leucocyanidin

OH

OHO

OH

OH

OH

Phf

Dhk

Flavonols

Dhm

Leucodelphinidin

OH

OHO

OH

OH

OH

OH

OH

OHO

OH

O

OH

OH

Guc

OCH3

OH

OCH3

OCH3

OH

HOH

OHO

OH

O

OH

Guc

OCH3

OH

H

Catechins

Flavonols

Catechins

OMT

OMT

OMT

F3H

F3'H

F3HF3H

F3'5'H

F3'5'HFSLI

LAR LAR

DFR DFRFLSI

LDOX LDOX

UFGTUFGT

BAN

Peonidin-3-glucoside

Cyanidin-3-glucoside

Delphinidin-3-glucoside

Delphinidin

Petunidin-3-glucoside

Malvidin-3-glucoside

Cyanidin

Naringenin Chalcone

4-Coumaroyl-CoA

CHS

BAN

O

OH

HO

R'

R

O

OH

OHFSLI

RT

OH OH

H O

HO

O OH

H O

O

R O

HO

OH

O

R O

HO

OCH3

O

R O

HO

OCH3HO

O

OCH3

HO

O

O

R O

HO

OCH3HO

HO OH

OH

O

CH

R

OH

HO

O

OH

Stilbene

H2CCOOH

COSCoA+

MalonylCoA Trihydroxychalcone

Flavanone

Isoflavone

Isoflavanoids

VRDMID

IFR

I2'H

CHI

IFS

IOMT

Tetrahydroxychalcone

Aurones

CHS/CHR

STSCHS

Flavonol glycosides

Flavonols

O

O-Glc-O-Rha

OH

R'

R

O-Glc

Rha-O

O

NH3HOOCHOOCHOOC

OH PAL

PhenylalanineCinnamic acidp-Coumaric acid

C4H

4CLGeneral phenylpropanoid pathway

OH

OH

HO

O

OH

2'-hydroxy Isoflavanone

CHI

Figure 6–Metabolic pathway and key steps of the flavonoid biosynthesis pathway leading to anthocyanin formation. The figure also depicts thecross-linking and interdependence of flavonoid and lignin biosynthesis pathways. Acronyms of the compounds reported in the figure stand for thefollowing: 4CL, 4-coumaroyl CoA ligase; I2′H, isoflavone 2′-hydroxylase; BAN, anthocyanidin reductase; C4H, cinnamate-4-hydroxylase; CHI, chalconeisomerase; CHR, chalcone reductase; CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase; Dhk, dihydrokaempferol; Dhm, dihydromyricetin; Dhq,dihydroquercetin; DMID, 7,2′-dihydroxy, 4′-methoxyisoflavonol dehydratase; E, eriodictyol; F3′5′H, flavonoid 3′ ,5′-hydroxylase; F3′H, flavonoid3′-hydroxylase; F3H, flavanone 3-hydroxylase; FLS1, flavonol synthase; IFR, isoflavone reductase; IFS, isoflavone synthase; IOMT, isoflavoneO-methyltransferase; LAR, leucoanthocyanidin; LDOX, leucoanthocyanidin dioxygenase; Nf, naringenin flavanone; PAL, phenylalanine ammonia lyase;Phf, pentahydroxyflavanone; RT, rhamnosyl transferase; STS, stilbene synthase; UFGT UDP, glucose, flavonol 3-O-glucosyltransferase; VR, vestitonereductase.



methylation occurred as a nondirected side-effect action of caf-feic acid methylation, which increased during fruit ripening. Theevolution of the ratio of the transcriptional level omt/ufgt throughripening and the relative abundance of methoxylated anthocyaninwas compatible with a role of OMT in the methoxylation ofthe B-ring of anthocyanin in grapevines (Castellarin and Gaspero2007). The cumulative expression of the transcription factors mayexplain the quantitative variation in anthocyanin content, whichprobably conceals the presence of additional factors involved inthe process.

Aroma biosynthesis in strawberryStrawberries are a rich source of phenolic compounds but

produce low levels of lignin; hence, the OMT enzyme in wildstrawberry could be involved in the synthesis of phenols andits derivatives. During early stages of fruit ripening, nontanninflavonoids and mainly condensed tannins accumulated to highlevels and gave strawberry a characteristic astringent flavor (Chengand Breen 1991). During later stages of fruit ripening, when fruitstarted to ripen, other flavonoids such as anthocyanins (mainlypelargonidin glucoside), flavonols, and cinnamoyl-β-d-glucose ac-cumulated to high levels (Latza and others 1996; Manning 1998;Moyano and others 1998; Aharoni and others 2000; Deng andDavis 2001). It may be proposed that other regulatory mechanisms(not related to transcriptional control) are governing flavonoid syn-thesis at least during the initial stages of ripening. In response toexternal ethylene, apple fruits showed a normal climacteric burstand produced increasing concentrations of ester, polypropanoid,

and terpene volatile compounds (Dandekar and others 2004). In-terestingly, due to the huge number of flavor compounds found instrawberry, Zabetakis and Holden (1997) suggested that it was notpossible for each substance to have its “own” enzymes. Accord-ingly, the side activity of COMT involved in phenylpropanoidmetabolism of strawberry fruits was thought to be responsiblefor the formation of 2,5-dimethyl-4-methoxy-3(2H)-furanone(DMMF). Consistent with this view, it was reported that a sin-gle OMT from Chrysosplenium americanum could methylate bothflavonoid and phenylpropanoid compounds (Gauthier and others1998). Of the 15 volatiles in strawberry, 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) was regarded as vital, but it was methy-lated further by FaOMT (Fragaria X ananassa OMT ) to DMMFduring the ripening process (Wein and others 2001) (Figure 7A).HDMF was indispensable because of its high concentration (upto 55 mg kg−1 strawberry fruit FW) (Larsen and others 1992)and low odor threshold (10 ppb in water) (Schieberle and Hof-mann 1997). The reduction of faomt gene expression altered theHDMF/DMMF ratio, resulting in a near-depletion of the DMMFpool, thus confirming the importance of FaOMT in the DMMFformation. faomt encoded a sequence of 365 amino acids and ac-cepted a substrate spectrum for compounds containing O-diphenolstructures (Wein and others 2002).

In addition, the dual function of this enzyme in the secondarymetabolism was proved as faomt down-regulation and also affectedthe concentration of feruloyl 1-O-β-d-glucose and caffeoyl 1-O-β-d-glucose, suggesting that it was also involved in the methylationof the caffeoyl group (Lunkenbein and others 2006) (Figure 7B).

O

OO

O

OO

O OH

HO OH

OHHO

O

O OCH3

S-adenosyl-L-methionine

S-adenosyl-L-homocysteine

FaOMT

HDMF

DMMF

[A]

COOHHO

HO

COOHH3CO

HO

OH

HO

HO

OO

OH

HO OH

H3CO

HO

OO

HO

O O

Caffeic Acid Ferulic AcidS-adenosyl-L-

methionineS-adenosyl-L-homocysteine

FaOMT

OHOH

OH

Caffeoyl -D-glucoseβ Feruloyl -D-glucoseβ

[B]

Figure 7–(A) Chemical structures of substratesand products of enzymatic reactions catalyzedby Fragaria x ananassa O-methyltransferase(FaOMT). HDMF, 4-hydroxy-2,5-dimethyl-3(2H)-furanone; DMMF, 2,5-dimethyl-4-methoxy-3(2H)-furanone. (B) Substrates andproducts of OMT enzyme that are acting in abranch of the phenylpropanoid pathwaydepicting the dual function of FaOMT instrawberry fruits.



Ripening related gene sequences that code for proteins involvedin key metabolic events including anthocyanin biosynthesis wereisolated from strawberry and were not found to be active in greenfruits (Manning 1998). Caffeic acid is usually not an intermediatein anthocyanin pigmentation biosynthesis and methoxyfuraneolformation in strawberry. But cyanidin, an anthocyanin precursor,which possesses an O-dihydroxyphenol structure similar to that ofcaffeic acid, might be recognized by the OMT (Wein and oth-ers 2002). Similarly, many enzymes of secondary metabolism areknown to recognize more than one substrate, although they oftenhave different catalytic rates toward them (Wang and Pichersky1999). It has been postulated that this phenomenon was proba-bly due to the evolution of ancestor genes involved in primarymetabolism, such as the comt involved in lignification (Picherskyand Gang 2000). Because of the expression pattern of faomt in dif-ferent stages of fruit ripening, it was assumed that in the beginningof fruit development FaOMT could be involved in lignificationof the vascular bundles in the expanding fruit. Subsequently, inthe later stages (ripe fruit), FaOMT activity probably providedthe precursors for achene lignification (Lunkenbein and others2006). On the other hand, FaOMT is also instrumental in thebiosynthesis of strawberry volatiles, because it efficiently catalyzesprotocatechuic aldehyde to vanillin during ripening. Vanillin hasbeen reported to contribute to strawberry flavor in both wild andcommercial strawberry cultivars (Hirvi and Honkanen 1982). Re-cently, an enone oxidoreductase (FaQR) involved in the HDMFformation was isolated from a crude strawberry fruit extract andthe corresponding gene cloned. It represented a very promisingtarget for biotechnological engineering for flavonoid biosynthesis(Raab and others 2006).

It is still unknown whether one enzyme with relatively lowsubstrate specificity is able to catalyze the transfer of a methyl groupto the substrates, or if it is a mixture of more than one enzymewhich methylates different substrates. The availability of cDNAscoding for the respective enzymes and their functional expressionwill be valuable in attempts to answer this question. The genesinvolved in aroma biosynthesis are not coordinately regulated byethylene, but typically only the 1st and final steps are ethylene-regulated suggesting important transcriptional regulation pointsfor aroma production in fruits during the ripening process. Abetter understanding of the enzymes involved in the formation ofmethoxyfuraneol will assist classical breeding and biotechnologicalefforts to improve the aroma of fruits.

Cinnamyl Alcohol Dehydrogenase (CAD)CAD (EC 1.1.1.195) is an NADP(H)-specific oxidoreductase

catalyzing the reversible conversion of cinnamyl aldehydes tothe corresponding alcohols, the last step in the biosynthesis ofmonolignols (Wyrambik and Grisebach 1975; Boudet and others1995; Roth and others 1997; Boerjan and others 2003). In bothnaturally occurring mutants and transgenic plants with depletedlevels of CAD, increased incorporation of cinnamaldehydes intolignin was observed. The cross-linking and physical properties ofcinnamaldehyde-rich lignins differed from that of normal lignins(Salentijn and others 2003). However, CAD was also expressedin response to stress (Galliano and others 1993), pathogen elic-itors (Campbell and Ellis 1992), and wounding (MacLean andothers 2007). However, CAD has been reported to express itselfeven in cells that do not make lignin (O’Malley and others 1992;Grima-Pettenati and others 1994). The cytochemistry studies ofdefense responses to Xanthomonas campestris in cassava fruit showedconsiderable changes in the metabolism of parenchymal cell walls,

involving the accumulation of lignin, flavonoids, and polysaccha-rides which was thought to be due to cad induction (Kpemouaand others 1996). CAD is therefore regulated by both develop-mental and environmental stimuli, much like other well-studiedenzymes of phenylpropanoid metabolism (Whetten and Sederoff1995).

Molecular characterization of CAD expressioncad cDNA isolated from various angiosperms has been shown to

share extensive nucleotide sequence homology suggesting cad genehas been conserved during evolution (Halpin and others 1994).Blanco-Portales and others (2002) classified strawberry fruit CADas zinc-containing alcohol dehydrogenase due to occurrence of aconsensus amino acid sequence at positions ranging from 60 to83. Concurrent gene expression in receptacle and achene tissuesusing DNA microarrays in strawberry fruit revealed a total of 1701cDNA clones (comprising 1100 ESTs and 601 unsequenced cD-NAs). Analysis of expression ratios identified 66 out of the 259(25%) achene-related clones and 80 out of 182 (44%) receptacle-related clones with more than a 4-fold difference in expression be-tween the 2 tissue types. Different cad and ccr clones isolated werethought to be involved in the lignification process in the receptacle.Enzymatic activity assays with a recombinant protein encoded by astrawberry cad gene homologue, identified as ripening-regulated,retained CAD activity and was immunolocalized to the vasculartissue in the receptacle. Interestingly, a strawberry cDNA showedhomology to the tobacco and Arabidopsis ethylene-responsive el-ement binding factor (ERFs), suggesting a role for ethylene inlate achene development (Aharoni and O’Connell 2002). Similarresults were earlier reported by quantitative real time PCR (QRT-PCR) data in same fruit suggesting a relationship of a strawberryFxaCAD1 enzyme with a lignification process to both vasculardevelopment and achene maturation. Thus, as fruit ripened, theachenes underwent strong lignifications of thick pericarp (Perkins-Veazie 1995).

Strawberry fruit traverse through 4 different stages (green,white, turning, and red) of fruit development. Interestingly, CADexpression decreased after the green stage (white and turningstages) before increasing again at the red stage (Sozzi and oth-ers 1998). Detailed analysis of cad expression in different straw-berry tissues during ripening using RNA gel blots complementedwith microarray data showing elevated levels of cad transcript inthe red stage. Expression of cad was detected in achenes and re-ceptacles (fruits with no achenes), petioles, leaves, and flowers.Because cad was strongly expressed in the ripening receptacle tis-sue, it was suspected that some of them might be actively expressedin the vascular bundles and associated with lignification. Using aprimary antistrawberry CAD polyclonal antiserum, Aharoni andothers (2002) showed that the corresponding protein was localizedspecifically to immature xylem cells undergoing active lignifica-tion. It has been observed that the lignin in CAD-deficient plantswas more susceptible to chemical extraction and showed improvedpulping characteristics (Russell and others 2000; Ruel and others2001). Thus, the differences in cad gene expression could be relatedto lignin composition, being richer in aldehydes and more suscep-tible to enzymatic degradation in the soft cultivar. Modificationsof lignin by downregulation or overexpression of cad in strawberryshowed that severe changes in CAD activity had a striking effecton the composition of lignin too. In strawberry fruit maturation,due to the higher quantity of CAD in firm cultivars, there was astrong lignification of thick pericarp of fruit (Salentijn and others2003).



A cDNA clone, mdcad1, encoding putative cad from apples(Malus X domestica Borkh. cv Fuji) was characterized and theclone contained an open reading frame of 325 amino acid residues,which showed more than 80% identity with Eucalyptus cad1. md-cad1 mRNA was detectable in vegetative tissues and was stronglyexpressed in the fruit (Sung-Hyun and others 1999). The tran-scription level of mdcad1 in apple initially increased with me-chanical wounding only when the pathogen attacked the plantand thereafter endogenous salicylic acid (SA) was produced in thepathogen-induced necrotic tissue. The expression pattern of md-cad1 mRNA in the fruit peel after light exposure increased until1 d after light exposure and remained stable thereafter, suggestingthat mdcad1 was light-inducible. The increased level of SA couldperhaps strongly induce the expression of mdcad1 to catalyze thesynthesis of cinnamyl alcohol. The increased lignin content inthe attacked tissue thus served as a barrier against any pathogeninvasion. The induction of mdcad1 expression by wounding andfurther induction by SA, yet not by ethylene and jasmonic acid,suggested that the induction of mdcad1 transcripts could followthe SA-dependent pathway of plant defense. Southern blot hy-bridization showed that there were either 1 or a few copies ofcad genes in apples (Sung-Hyun and others 1999). Although thesecondary metabolites present in the fruit peel could act as protec-tants against changing environmental conditions and in deterringpathogens, these could also play a role in attracting seed-dispersingfrugivors.

CAD and lignin accumulationMost transgenic studies on CAD have shown that loss of ac-

tivity resulted in changes in lignin composition rather than lignincontent (Baucher and others 1999). But the results in the litera-ture on CAD are still vague, with lignin composition sometimesbeing affected and at times not, in cad antisense transgenic plants(Boudet 2000). In accordance with this theory, the increase inlignin content of postharvest loquat fruit (E. japonica) tissue wasparalleled by a rise in CAD. Incidentally, the direct proportion-ality relationship between CAD activity and lignin accumulationmight be missing, responding in a way analogous to other enzymesof the pathway. Modulation of the levels of ejcad1 transcripts byethylene treatment or low-temperature conditioning in E. japonicawere particularly associated with changes in lignification duringripening. Expression of ejcad1 increased markedly 4 d after ex-posure to 0 ◦C. These results support a view that lignification isstimulated by low temperature, possibly mediated by a stimulationin CAD activity (Shan and others 2008). Increase in expressionlevels of ejcad1 preceded the major increase in fruit firmness andlignifications, portentous of some role in loquat fruit ripening,despite the lack of homology with cad gene of tobacco associatedwith lignification (Damiani and others 2005). Because cad genewas isolated from fruit flesh, the possibility arises that these have amore fruit-dependent role, or that fruit tissue has variants in theseand other genes associated with tissue structural changes. In grapeberry (V. vinifera), 4 CAD isozymes exhibited preferential mRNAaccumulation in skin and or skin/pulp during ripening (Grimpletand others 2007). The expression patterns were related to vascu-lar bundle formation, which occurred specifically in these tissuessimilar to that observed in strawberry (Aharoni and others 2002).However, CADs may also be involved in the synthesis of cinnamylalcohol derivatives responsible for fruit flavor (taste and aroma)apart from lignin formation (Mitchell and Jelenkovic 1995).

Various branches of the phenylpropanoid pathway includingthose associated with the biosynthesis of lignin, hydroxycinnamates

(ferulic, sinapic, caffeic, and 4-coumaric acids) and flavonoids, havebeen reported to be closely linked (Salentijn and others 2003). Itcannot be ruled out that CAD acting in the lignin biosynthe-sis pathway is associated with other functions like biosynthesis offlavor compounds during the ripening process (Mitchell and Je-lenkovic 1995) apart from the primary role of providing structuralstrength to the cells and disease resistance. Variations in the fluxthrough the pathway may lead to the biosynthesis of a differentpool of hydroxycinnamic acids or aldehydes with a putative effecton flavor or on cell wall-bound hydroxycinnamates (Kroon andWilliamson 1999). As cad genes have been isolated from fruit flesh,this raises the possibility that they could have a more fruit-specificrole. Available data show an encouraging relationship betweencomponents of the phenylpropanoid pathway and firmness de-velopment in fruits. Hence, this strongly suggests that lignificationbeing a conventional synthetic process in the fruit ripening and cadgene could have a central role to play during the process. There-fore, CAD turns out to be a suitable candidate among the batteryof lignin biosynthetic enzymes in modifying fruit ripening rates.

The Ever-Growing Phenylpropanoid PathwayWith the emerging evidence of the possible role of the phenyl-

propanoid pathway in fruit ripening and the suggestion that thevarious branches of the phenylpropanoid pathway appear to beclosely linked. Consequently, the role of the phenylpropanoidpathway is turning out to be crucial during the ripening process.Furthermore, the possible biological functions of other putativegenes of the phenylpropanoid pathway is being evaluated whichat present remains elusive.

In the lignin branch of the phenylpropanoid pathway, cinnamoylCoA reductase (CCR, EC 1.2.1.44) is the 1st enzyme and is re-sponsible for the conversion of hydroxycinnamic acid CoA estersto the corresponding hydroxycinnamaldehydes. CCR plays a ma-jor role in determining total lignin content and quality of solublephenolic content in tomato (Van der Rest and others 2006). Thus,downregulation of CCR might cause variations in the flux throughthe pathway leading to the synthesis of a different pool of hydrox-ycinnamic acids or aldehydes with a putative effect on flavor oron cell wall-bound hydroxycinnamates (Kroon and Williamson1999). In strawberry, expression of the ccr and cad genes differedbetween the cultivars, ccr being lower in the firm cultivar andcad in the soft cultivar (Salentijn and others 2003). Modificationsof lignin by downregulation of CCR decreased the total lignincontent (Chapple and Carpita 1998). Thus, the differences in cadgene expression could be related with lignin composition, beingricher in aldehydes and more susceptible to enzymatic degrada-tion in the soft cultivar. Consistently, downregulation of CCRin tomato through RNA interference (RNAi) led to quantitativeand qualitative changes in the soluble phenolic content of extractsfrom fruit and vegetative organs and an increase in the antioxidantcapacity of the plant extracts (Van der Rest and others 2006). Ofthe 5 isogenes of ccr identified in grape berry (V. vinifera), only 2genes showed seed specific expression and 1 showed peel-specificexpression, whereas 2 others exhibited mixed expression in thepulp/skin or skin/seed (Grimplet and others 2007).

Cinnamate-4-hydroxylase (C4H, EC 1.14.13.11), a cy-tochrome P-450-linked monooxygenase, is a key enzyme in thephenylpropanoid pathway, responsible for hydroxylation of cin-namic acid to p-coumaric acid. In Korean black raspberry (Rubussp.), QRT-PCR studies indicated that the c4h gene had a differ-ential expression pattern during fruit development, that is, geneexpression was first detected in green fruit, markedly reduced in



yellow fruit, and increased in red and black fruit stages, whichwas concomitant with flavonoid content. In contrast, the contentof anthocyanins during the progression of ripening dramaticallyincreased suggesting that the c4h gene in raspberry was instru-mental in color development at the later stages of fruit ripening,whereas the expression of the c4h gene during the early stagesmight be related to the accumulation of flavonols (Myung-Hwaand others 2008). In Valencia sweet orange (Citrus sinensis), 2 c4hgenes were described coding a constitutively expressed C4H2 en-zyme that plays a normal role in the phenylpropanoid pathwayin contrast to a wound-induced C4H1 isoform (Betz and others2001). Chen and others (2006) determined that C4H enzymeactivity was 10-fold the PAL activity, although C4H was locatedimmediately downstream pal in the biosynthetic pathway duringgrape berry development. Ikegami and others (2005) reportedthat inhibition of flavonoid biosynthetic gene expression of c4hcoincided with loss of astringency in pollination-constant, nonas-tringent (PCNA)-type persimmon fruit.

Another enzyme, 4-coumarate coenzyme A ligase (4CL, EC6.2.1.12), catalyzes the conversion of 3 cinnamic acid derivativesto their corresponding coenzyme A esters in a 2-step reaction.The reaction is considered to be a branch point between gen-eral phenylpropanoid metabolism and pathways leading to endproducts such as lignin. 4CL plays a particularly important role inplant defense reactions because of its position, joining the phenyl-propanoid pathway with lignin and flavonoid branch pathways. Infact, different isoforms of 4CL direct carbon flow to the diversepathways of phenylpropanoid metabolism according to differentsubstrate preferences. For example, 4CL3 (a class II 4CL) hasa high affinity for 4-coumarate, whereas 4CL1 and 4CL2 havestrong affinities for 4-caffeate (Stuible and others 2000). Thus,Ehlting and others (1999) hypothesized that the primary func-tion of class II 4CL is to channel 4-coumarate to the flavonoidpathway. In raspberry (Rubus idaeus), 3 classes of raspberry 4clcDNAs (ri4cl1, ri4cl2, and ri4cl3) were isolated. Based on phyloge-netic classification, expression patterns, and recombinant proteinactivities, the different ri4cl genes were thought to participate indifferent biosynthetic pathways leading to the biosynthesis of vari-ous phenylpropanoid-derived metabolites that help to create flavorand color in raspberry fruit (Kumar and Ellis 2003a,b). Phenyl-propanoid genes (fapal, fac4h, and fa4cl) in strawberry had a similar2 phase expression pattern with a decrease of transcript levels atthe W stage (partially ripe) (Almeida and others 2007). Expressionof ej4cl in white-fleshed Baisha (BS) loquat (E. japonica) fruit in-creased during the 1st 6 d, again slightly preceding a rise in enzymeactivity, whereas expression in ripening red-fleshed Luoyangqing(LYQ) fruit remained at a relatively low level. The relatively high4CL activity, as well as the corresponding gene expression in BSfruit, indicated that further effort is necessary to find lignification-specific 4cl gene family members in loquat (Shan and others 2008).Prior to CAD in the lignin biosynthetic pathway, PAL and 4CLboth have a role in a wider range of biosynthetic pathways, includ-ing that of flavonoid production. Because of this, we might notexpect relationships of these genes with lignification to be director particularly sensitive.

Chalcone SynthaseChalcone synthase (CHS, EC 2.3.1.74) is the enzyme responsi-

ble for catalyzing the 1st committed step of the flavonoid biosyn-thesis pathway. CHS is an acyltransferase enzyme that catalyzesthe condensation of 4-coumaroyl CoA to the 1st flavonoid narin-genin chalcone, in the presence of 3 molecules of malonyl CoA

(Lucheta and others 2007). The flavonoid skeleton, synthesizedby CHS, is converted to chalcones, flavanones, flavonols, antho-cyanins, and proanthocyanidins (Velasco and others 2007). Qualitytraits of raspberry fruits (Rubus) such as aroma and color derivein part from the polyketide derivatives, benzalacetone and di-hydrochalcone, respectively. The formation of these metabolitesduring fruit ripening is the result of the activity of polyketide syn-thases (PKS), benzalcetone synthase, and CHS (Kumar and Ellis2003a,b). “Hairpin” RNA (ihpRNA) silencing led to reduced lev-els of chs mRNA and enzymatic CHS activity in strawberry fruit.The levels of anthocyanins were down-regulated and precursors ofthe flavonoid pathway were shunted to the phenylpropanoid path-way leading to a large increase in levels of (hydroxy) cinnamoylglucose esters (Hoffmann and others 2006). In citrus fruit (Citrusunshiu Marc.), mRNA levels of the flavonoid biosynthetic pathwaygenes (citchs1 and citchs2) exhibited high transcript levels in youngtissues and low in senescent tissues during fruit development. Thehigh expression of flavonoid biosynthetic genes and high accumu-lation of flavonoids in young fruits suggested that flavonoids weresynthesized in the early developmental stage (Moriguchi and oth-ers 2001). Recently, a study in grapes during ripening has shownchs2 expression increased by light treatment and it appeared thatthis response was concomitant with the expression of leucoantho-cyanidin oxidase (ldox), omt, and ufgt, because of their remarkablysimilar expression profiles (Matus and others 2009). Surprisingly,in tomato it was found that ectopic expression of chs and f3h inconjunction with chi led to the expected increase in peel flavonols,but was not sufficient to upregulate flavonol accumulation in flesh(pericarp and columella) tissues (Colliver and others 2002). In an-other study, concomitant ectopic expression of chs, chi, f3h, and flsin tomato fruit resulted in increased levels of flavonol accumula-tion in both peel and flesh tissues (Verhoeyen and others 2002).In apple fruit, the relationship of CHS activity with anthocyaninsynthesis in peel revealed that the flavonoid content was relativelyhigh and constant from fruitlet to maturation stage (Ju and oth-ers 1995) whereas UV-B and low temperature were importantfactors for anthocyanin accumulation in apple fruit skin by induc-ing the expression of chs and anthocyanidin synthase (ans) genes(Ubi and others 2006). chs has been an attractive target for geneticengineering and there are numerous instances of co-suppressionor downregulation of this gene in order to modify flower colortoward pure white as a result of a complete absence of flavonoids.

PeroxidasesPeroxidase (POD, EC 1.11.1.7) catalyzes the polymerization of

phenylpropanoid precursors of lignin and are involved in the laststep of lignin formation. However, it has been difficult to differ-entiate between peroxidase isozymes associated with lignificationand those involved in biochemical activities (Whetten and Sederoff1995). Ryugo (1964) followed the changes in lignin and phenolicprecursors in the developing peach pit and suggested it was a goodsystem for lignin synthesis studies. In peach, a 2-fold increase intotal peroxidase activity during lignifications was reported whichwas established by an increase in a number of basic isozymes inlignifying tissues. Although an increase in the amount and diver-sity of basic peroxidases was observed during lignification, otherenzymes involved in producing phenylpropanoid precursors mayplay a more important role in controlling lignin formation (Abelesand Biles 1991). Dalet and Cornu (1988) reported no differences inthe amount or distribution of peroxidase isozymes in cherry cloneswith different degrees of lignifications. Interestingly, the ejpod genecloned from E. japonica exhibited only 1 of 6 amino acid residues



associated with lignin synthesis and even lower identity with podinvolved in lignin synthesis in other plants, but significantly had aclose temporal association with the ripening-associated lignifica-tion in loquat fruit (Shan and others 2008). Different peroxidaseisoforms typically have different kinetic properties in vitro prompt-ing the question whether, and to what extent, these peroxidaseshelp define the lignin composition, and thus structure, in the cellwall. In rambutan (N. lappaceum) fruit, the rapid desiccation of thespinterns compared to the peel appeared to be the main reason forthe rapid browning of the spinterns. But higher activity of PODobserved, owing to higher rates of oxygen transmission into spin-terns as compared to the peel, was also thought to play a centralrole (Yingsanga and others 2008).

In tomato, it was suggested that POD isozymes located withinthe fruit exocarp may have a dual role in restricting fruit ex-pansion through cross-linking of cell wall components and pro-ducing a protective barrier in the epidermis (Andrews andothers 2002). The presence of “wall-bound” POD activity inmature fruit confirmed earlier findings and supports the notionthat POD-mediated “stiffening” of the exocarp cell walls leads tothe cessation of fruit growth (Thompson and others 1998; An-drews and others 2000). POD activity of undamaged pericarp ofmangosteen fruit did not change, while that of damaged peri-carp increased slightly during the 1st 2 h after impact, and rapidlythereafter. The rapid increase in POD occurred concomitantlywith increased lignin content and firmness of damaged pericarp(Ketsa and Atantee 1998). Impact may increase the activity of PODand simultaneously damage the tonoplast of vacuoles resulting inleakage of phenolics and contact with POD. The end result maybe synthesis of lignin, and this may form complexes with othercompounds such as carbohydrates, proteins, and pectins resultingin strong lignin complexes, and in turn increased firmness (Iiyamaand others 1994).

Future Prospective and ConclusionPhenolic compounds, especially phenylpropanoids and

flavonoids, play an important role in plant growth and develop-ment as well as in plant interactions with the environment. Overall,the data reported in this review demonstrate that many genes par-ticipating in monolignol biosynthesis have the potential for themaintenance of firmness and improvement of aroma/organolepticproperties of fruits. Relationships between components of thelignin synthesis pathway, lignifications, and development of firm-ness in various fruits do exist and these strongly suggest that ligni-fication could be a conventional synthetic process in various fruits.Lignin forms complexes with other compounds such as carbohy-drates, proteins, and pectins resulting in strong lignin complexes(Iiyama and others 1994). The incorporated lignin imparts rigidityto cell wall, providing a close connection between the carbohy-drate matrix and the cellulose polymers. This supports the rolefor lignin in helping maintain cell wall structure (Hu and others1999). An integrated approach on lignin, monolignol precursors,associated enzymes, and genes could provide a consistent modelof lignification during fruit ripening.

Identification of the biological and pharmacological activities offlavor compounds has been gathering momentum in recent years.Today, the total market for flavors and fragrances is estimated atUS$ 18 billion annually, with market shares between the flavorand fragrance businesses being almost equal (Guentert 2007). Thepercentage of natural flavors with respect to all added flavors hasincreased to 90% (EU) and 80% (U.S.A.) in beverages, to 80%(EU and U.S.A.) in savory foods, and to 50% (EU) and 75%

(U.S.A.) in dairy foods (Schrader 2007). Flavor compounds havenumerous functional properties (including antioxidative, analgesic,digestive) and hence will continue to be vital natural ingredients.The ability to now consider flavonoid enzymes, in 3 dimensions,and to examine the interdependence of the pathways of secondarymetabolism is likely to move us much more rapidly toward a newera of flavonoid biochemistry research.

As with any good model, each new piece of information ap-pears to raise a number of unanticipated and intriguing questions.Major results indicate that no single gene or enzyme can accountfor the major events that underlie fruit softening and that manyof the potentially responsible genes and their corresponding cellwall modifying proteins are members of large gene families thatexhibit overlapping patterns of expression and possibly redun-dant biochemical action (Bennett and Labavitch 2008). Moreover,experiments with antisense RNA will facilitate to explicate theinvolvement of phenylpropanoid metabolism during fruit devel-opment and ripening. At the same time, new techniques are pro-viding the opportunity to consider flavonoid biosynthesis, notas an assemblage of independent components, but as part of alarge, complex and tightly orchestrated metabolic network. Agreat deal of work of functional genomics, coupled with con-temporary postharvest approaches, is still needed in elucidatingthe mechanisms involved in ripening, which would facilitate amore systematic understanding of the process. Utilizing informa-tion from tomato genomic resources may allow us to develop amodel to predict approaches to modulate various aspects of ripen-ing in a wide range of fleshy fruit-bearing species. Overall, theinformation reported in this review demonstrates the potentialof genetic engineering for the improvement of shelf-life, aroma,and taste properties of horticultural products. However, most ifnot all of these are related so far to basic studies at the laboratorylevel, they provide only proof of principle that engineering oneof several of these genes could be of practical interest.

AcknowledgmentsThe financial assistance from the Dept. of Biotechnology

(DBT), New Delhi, India (in the form of DBT-SRF to RupinderSingh), is gratefully acknowledged. We are also thankful to DST-FIST, CSIR (NMITLI), and UP Government (Under Centre ofExcellence in Biochemistry and Biotechnology) for their financialsupport in the form of infrastructural facilities.

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Phenylpropanoid Metabolism in Ripening Fruits