Metabolismo de Fenilpropanoides Maduracion

Phenylpropanoid Metabolism in Ripening Fruits
Rupinder Singh, Smita Rastogi, and Upendra N. Dwivedi
Abstract: Ripening of fleshy fruit is a differentiation process involving biochemical and biophysical changes that lead
to the accumulation of sugars and subsequent changes in tissue texture. Also affected are phenolic compounds, which
confer color, flavor/aroma, and resistance to pathogen invasion and adverse environmental conditions. These phenolic
compounds, which are the products of branches of the phenylpropanoid pathway, appear to be closely linked to fruit
ripening processes. Three key enzymes of the phenylpropanoid pathway, namely phenylalanine ammonia lyase, Omethyltransferase, and cinnamyl alcohol dehydrogenase (CAD) have been reported to modulate various end products
including lignin and protect plants against adverse conditions. In addition, peroxidase, the enzyme following CAD
in 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 the
relationships between phenylpropanoid metabolism and fruit ripening processes. Understanding the role of these enzymes
in fruit ripening and their manipulation may possibly be valuable for delineating the regulatory network that controls the
expression of ripening genes in fruit. This review elucidates the functional characterization of these key phenylpropanoid
biosynthetic enzymes/genes during fruit ripening processes.
Introduction
Fruit ripening is a developmentally regulated process resulting
from the coordination of numerous biochemical and physiological
changes within the fruit tissue that culminates in changes in fruit
firmness, color, taste, aroma, and texture of fruit flesh (Figure 1)
(Brummell and Harpster 2001; Vicente and others 2006; Singh
and others 2007). Textural changes that lead to softening of fruits
have drawn attention to the putative involvement of several enzymes able to act and modify its structure in a developmental and
coordinated way (Brady 1987; Rose and others 1998; Sozzi and
others 1998).
Phenolic compounds produced by the phenylpropanoid pathway contribute to fruit pigmentation and the disease resistance
response found in many fleshy fruits during ripening (Figure 2).
In olive fruits, several simple and complex phenolics have been
reported to be effective against pathogenic bacteria (Chowdhury
and others 1997). Caffeic acid has been found to be the most effective agent, although oleuropein, the major phenolic constituent
of olives, also exhibits bactericidal action (Ruiz-Barba and others
1991). Moreover, phenolic compounds protect plants by acting
as feeding deterrents to insects (Nahrstedt 1990). For example,
flavonoids such as luteolin, naringenin, phloretin, quercetin 3rhamnoside, and myricetin-3-rhamnoside, have been shown to
act as feeding deterrents to aphids (Dreyer and Jones 1981).
MS 20100054 Submitted 1/16/2010, Accepted 3/7/2010. Authors Singh and

Dwivedi 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: upendradwivedi@hotmail.com).
In addition, a complex polymer, lignin, is also a product of phenylpropanoid metabolism. Lignin represents a major carbon sink in vascular plants and is derived from 3
lignin precursors termed as monolignols (hydroxycinnamyl
alcohols), namely, p-coumaryl alcohol, coniferyl alcohol, and
sinapyl alcohol. The biosynthesis of monolignols involves 2 specific steps branching off the general phenylpropanoid pathway.
The phenylpropanoid pathway drives the carbon flow from
the aromatic amino acid L-phenylalanine (L-Phe) or, in some
cases, L-tyrosine (L-Tyr) (Rosler and others 1997), for the
production of 4-coumaroyl CoA (or a respective thiol ester
in the presence of other 4-hydroxycinnamates). The synthesis of monolignols (lignin monomers) involves several hydroxylation/methylation and oxidation/reduction reactions (Whetten
and Sederoff 1995) (Figure 3). Numerous enzymes, namely, a
family of oxygen-dependent cytochrome P450 hydroxylases and
S-adenosylmethionine-dependent methyltransferases, act on the
free cinnamic acids and their CoA esters which are converted to
p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. After
synthesis in cytoplasm, monolignols are transported to the cell wall
as glucoside derivatives formed in a reaction catalyzed by uridine
diphosphate glucose coniferyl alcohol glucosyltransferase (UDPGT), where these glucoside derivatives are hydrolyzed by coniferin
-glucosidase (CBG). The last major step in lignin biosynthesis
is monolignol dehydrogenation in a reaction catalyzed by peroxidase (POD), laccase (LAC), polyphenol oxidase (PO), or coniferyl
alcohol oxidase (CAO) followed by polymerization by oxidative
coupling (Boerjan and others 2003). Peroxidases are involved in
the oxidation of phenolic compounds in cell walls, polymerization of lignin and suberin, and have also been shown to degrade
flavonoids in noncell systems (processed food) as well as in vivo. As
lignin polymerizes, it serves as a matrix around the polysaccharide
398 Comprehensive Reviews in Food Science and Food Safety r Vol. 9, 2010

c 2010 Institute of Food Technologists
doi 10.1111/j.1541-4337.2010.00116.x
Phenylpropanoid metabolism in ripening fruits . . .
S
H
I
K
I
M
A
T
E
Phosphoenolpyruvate
Glycolysis
P
A
T
H
W
A
Y
Phenylalanine
P
H
E
N
Y
L
P
R
O
P
A
N
O
I
D
P
A
T
H
W
A
Y
F
L
A
V
O
N
O
I
D
P
A
T
H
W
A
Y
Benzoic acid
derivatives
Cinnamate
Esters of
organic acids
4-coumarate
MONOLIGNOLS
(p-coumaryl alcohol, coniferyl alcohol, sinapyl alcohol)
malonylCoA
Lignin
(Biopolymer of aromatic subunits
forming an integral part of the
secondary cell walls of plants)
Carbohydrate
metabolism
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)]
Isoflavonoids & flavonoids

(Polyphenols having
3-phenylchromen-4-one and
2-phenylchromen-4-one backbones,
respectively)
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)
Proanthocyanidins
(Polymers of flavonoids)
Figure 1Schematic overview of branch pathways of shikimate, phenylpropanoid metabolism, and flavonoid biosynthesis in plants leading to the
synthesis of flavonoids.

Vol. 9, 2010 r Comprehensive Reviews in Food Science and Food Safety 399
Caffeic acid
(Source of O-diphenols
oxidizable, fungitoxic)
Zwitterionic anthocyanin
(Protectant)
OH
C
OH
C
OH
C
OH
OH
OH
OH
C
NH2
PAL
C4H
HCH
OMT
F5H
OH
OH
OCH3 HO
OH
Phenylalanine Cinnamic Acid p-Coumaric Acid Caffeic Acid
OMT
OH
Ferulic Acid
Esterification of host
cell wall
(Chemical barrier)
OCH3 H3CO
OH
OCH3
OH
5 OH-Ferulic Acid
Sinapic Acid
Stress lignin
(Physical Barrier to fungus)
Figure 2The involvement of phenolic compounds in the expression of resistance to pathogen infection. The diagram shows the family of compounds
known as phenylpropanoids and their route of synthesis from phenylalanine through the phenylpropanoid pathway. PAL-phenylalanine ammonia
lyase, 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 rigidity

and compressive strength as well as rendering the walls hydrophobic and water-impermeable (Whetten and Sederoff 1995; Rastogi
and Dwivedi 2003a). Limiting the carbon flow down the monolignol pathway should enhance the availability of coumaroyl CoA
esters for chalcone synthase that catalyzes the 1st step in flavonoid
biosynthesis.
The characteristics of lignin differ among cell walls, tissues,
and plant organs (Rastogi and Dwivedi 2003b; Grabber and others 2004). Lignin biosynthesis is coordinated and regulated during fruit ripening which provides structural strength to cells and
disease resistance (Lagrimini 1991; Chapple and Carpita 1998;
Abdel-Massih and others 2007; Seymour and others 2008). Aromatic rings of lignin are deposited within the cell wall carbohydrate matrix, which is often oriented within the plane of the cell
wall (Atalla and Agarwal 1985). Studies have suggested that the
increase in firmness of loquat fruit (Eriobotrya japonica) involves a
coordinated regulation of lignin biosynthesis and cellulose hydrolysis (Cai and others 2006). Lignin in a soft fruit like strawberry
has been detected in the achenes (combination of seed and ovary
tissue) and in the vascular bundles that connect the achenes to the
central pith (Suutarinen and others 1998).
Caffeoyl CoA OMT (CCoAOMT) is induced in elicited cells
synthesizing phenylpropanoid phytoalexins and, therefore, has
been implicated in the defense response of plant cells (Schmitt
and others 1991). In another study, the analysis of the effect of
high CO2 treatment on phenolic metabolism and ripening-related
changes in cherimoya fruit (Annona cherimola) demonstrated no
change in the total polyphenol levels. Upon exposure to air, however, there was a slight decrease in lignin content in CO2 -treated
fruits but the levels remained significantly higher compared to airtreated controls (Maldonado and others 2002). The data indicated
that high CO2 treatment promoted changes in lignin degradation. It was hypothesized that cell walls maintaining more lignin
deposits could modulate the strength of cellcell adhesion (Assis
and others 2001). Furthermore, in considering the insecticidal effects attributed to enriched CO2 treatment and the importance of
phenylpropanoid compounds in general defense strategies, it has
been suggested that the maintenance of these compounds in CO2 treated fruit may be an advantage in pathogen defense (Assis and

OH
C
NH2
TAL
O
OH
OH
OH
S-CoA
COAMP
OH
NH2
PAL
B
4CL
C4H
C
4CL
CCR
D
OH
OH
CAD
OH
OH
C3H
G
OH
CCoA3H
OH
COAMP
4CL
4CL
OH
OH
S-CoA
OH
OH
OH
OH
CCoAOMT
OMT
OH
COAMP
4CL
4CL
OCH3
OCH3
OH
OH
CCR
CAD
OCH3
OH
CHO
SCoA
OCH3
OH
OH
OCH3
OH
F5H
OH
COAMP
HO
OCH3
HO
OCH3 HO
OH
OH
OCH3
P
OH
CCoAOMT
OH
COAMP
OCH3 H3CO
OH
SCoA
4CL
4CL
H3CO
OCH3 H3CO
OH
OMT
O
CCR
4CL
4CL
Lignin and
Lignans
Synthesis
CHO
SCoA
OCH3 H3CO
OH
CCR
R
OH
OCH3 H3CO
OH
CHO
CAD
S
OH
OCH3 H3CO
OCH3
OH
Figure 3An 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-coumarate
CoA ligase; CCR, cinnamoyl CoA reductase; CCoAOMT, caffeoyl CoA 3-O-methyltransferase; F5H, ferulate 5-hydroxylase; ?, yet unconfirmed reactions.


qualities in addition to structural and defense-related functions
(Chen and others 2006).
PAL is considered to be a key regulatory enzyme for
flavonoid/anthocyanin biosynthesis during fruit ripening (Martinez and others 1996). Flavonoids are synthesized by the phenylpropanoid pathway in which the amino acid phenylalanine
(substrate of PAL) is used to produce 4-coumaroyl CoA. This combines with malonyl CoA to yield chalcones (flavonoid precursors
with 2 phenyl rings). Conjugate ring closure of chalcones results in
a 3-ring structure, the typical form of flavonoids. The metabolic
pathway continues through a series of enzymatic modifications
to yield several flavonoid classes, including the flavonols, dihydroflavonols, and anthocyanins (Mintz-Oron and others 2008).
Indeed, PAL activation is considered as product-specific, such as
for lignin or tannin or anthocyanin biosynthesis.
Interestingly in loquat fruit, chilling injury, such as an increase
in postharvest firmness at low temperatures, has been found to be
attributable to an increase in PAL activity, lignin, and fiber contents as well as adherence of peel with flesh and development of
a leathery (juiceless) pulp. Significantly, no augmentation of lignification and PAL activity was reported at 12 C. Lignin content
of flesh tissue of the fruit was enhanced on ethylene (159%) and
1-MCP (63%) treatment as well as in the control samples (139%).
The loquat fruit firmness increased steadily even after harvest at
20 C, thus indicating that it was not a low-temperaturedependent response (Cai and others 2006). The results indicate
that the presence of ethylene is required not only for inducing
synthesis of the PAL enzyme, but probably also for maintaining
its continuous high activity. Although lignification is a known
response to physical impacts in mangosteen fruit (Garcinia mangostana) (Ketsa and Atantee 1998), some fruits develop increased
levels of lignin during storage. Although the increase noted in
apple (Marlett 2000) and pear (Martin-Cabrejas and others 1994)
fruits were relatively small, the most notable case is that of another rosaceous fruit, the loquat (E. japonica). In this fruit, tissue
lignification may result in greater flesh firmness, toughness of the
texture, or loss of juiciness (Cai and others 2006). Recently, it
was suggested that the increase in pericarp firmness of mangosteen fruit resulted from induction of lignin synthesis, associated
Phenylalanine Ammonia Lyase (PAL)
PAL (EC 4.3.1.5), a cytosolic protein in vascular plants, is the with an increase in pal and pod gene expression and its respective
initial enzyme in the monolignol biosynthetic pathway that cat- enzyme activities (Dangcham and others 2008).
alyzes the deamination of L-phenylalanine to transcinnamate, a
precursor of various phenylpropanoids, such as lignins, coumarins, Diversity and functional conservation of pal gene
pal gene has been cloned, as well as characterized, from many
flavonoids, UV protectants, and furanocoumarin phytoalexins.
The reaction is generally considered to represent a key point at plant tissues (Boudet 2007) (Table 1). pal appears to exist univerwhich carbon flux into this pathway is controlled (Hanson and sally in higher plants as a family of genes and the presence of pal isoHavir 1981; Jones 1984). PAL enzyme is one of the most in- forms is a common observation. The significance of this diversity is
tensively studied in plant secondary metabolism (Hrazdina 1992; unclear, but evidence for a degree of metabolic channeling within
Lewis and others 1999). Phenylpropanoid metabolism is related phenylpropanoid metabolism suggests that partitioning of phototo the plant defense system and early studies in Phaseolus vulgaris synthate into particular branches of phenylpropanoid metabolism
had reported an increase in PAL activity and concentration of to- may involve labile multienzyme complexes that include specific
tal phenols due to the presence of pathogens (Bolwell and others isoforms of PAL (Sreelakshmi and Sharma 2008).
In melon fruit, pal was shown to be transcriptionally induced
1985). Treatment of apple with prohexadionecalcium has been
reported to result in alterations in the phenylpropanoid biosynthe- both in response to fruit ripening and wounding (Given and othsis pathway that also enhanced disease resistance (Roemmelt and ers 1988). Regulation of pal gene expression in this fruit is a
others 2002). Results from the subcellular-localization in grape coordinated process in response to both ethylene and an ethyleneberry revealed that PAL was present in the cell walls, secondarily independent wound signal. pal gene expression followed the exthickened walls, and the parenchyma cells of the berry mesocarp pression kinetics similar to that of the ethylene biosynthetic genes
cells, whereas 4-coumarate:CoA ligase (4CL) involved in the for- during fruit development. In contrast, ethylene biosynthetic genes
mation of CoA thioesters of cinnamic acids was present predomi- showed different induction kinetics compared to pal expression in
nantly in the secondarily thickened walls and the parenchyma cells response to wounding (Diallinas and Kanellis 1994). However, acof mesocarp vascular tissue that were closely associated with fruit tivation of PAL has also been observed in response to several types
others 2001). It may be presumed that there could be a concomitant increase in expression of genes coding for putative enzymes
of secondary metabolism including a gene associated with host
defense response. Interestingly, there is a further unusual instance
of lignification in foods, and that is an increase in lignin contents of some fruits during ripening after harvest. The enzymes of
the phenylpropanoid pathway involved in lignification have not
only been shown in tissues undergoing active lignin synthesis, but
have also been associated with nonlignified tissues or poorly lignified systems such as cell suspension cultures, suggesting that the
enzymes may be involved in other secondary metabolites.
In recent years, results based on a transgenic approach have
led to propositions of newer concepts to the classical phenylpropanoid pathway. There are several genes which are involved in
the phenylpropanoid pathway but only very few genes, namely,
phenylalanine ammonia lyase (pal) (MacLean and others 2007), Omethyltransferase (omt) (Matus and others 2009), cinnamyl alcohol
dehydrogenase (cad) (Aharoni and others 2002), and peroxidases
(pod) (Ketsa and Atantee 1998) were considered to have a potential role in fruit ripening and flavonoid biosynthesis. Flavonoid
metabolism competes directly with pathways leading to lignin and
sinapate ester biosynthesis and is itself composed of a number
of branch pathways leading to isoflavonoids, flavonols, proanthocyanidins (condensed tannins), and anthocyanins (Shirley 1999).
The isolation and cloning of most of the structural flavonoid genes
opens up possibilities to develop plants with tailor-made optimized flavonoid levels and composition. This review provides a
critical insight into the role of key phenylpropanoid biosynthesis genes and enzymes in the structural and subsequently compositional changes during fruit ripening. The numerous effects
that ethylene elicits during ripening are also comprehensively discussed. There are considerable gaps in our knowledge with respect to the role of phenylpropanoid metabolizing enzymes in
fruit ripening processes and it is anticipated that fresh ideas arising
from this article will shed light and facilitate other and future researchers in understanding emerging concepts in the regulation of
ripening.


Table 1Summary of experimental results cited in the literature, which correlate mRNA accumulation and enzymatic activity of phenylalanine ammonia
lyase (PAL) with ripening in selected fruits.
Serial nr
Plant
description
Accession
nr
1.
Vitis vinifera
EF192469
2.
Prunus avium
AF036948
3.
Musa acuminata
EU104680
4.
Eriobotrya japonica
EF685344
5.
Malus X domestica
AF494403
6.
Pyrus communis
DQ230992
7.
Citrus limon
U43338
8.
Fragaria X ananassa
AB360390
9.
Rubus idaeus
AF237955
10.
Ipomoea batatas
M29232
11.
Cucumis melo
X76130
12.
Lycopersicon esculentum
M83314
Maximum
identity
with fruits
Maximum
identity
other plants
Enzyme
activity
Prunus avium
AF036948 (80%)
Pyrus communis
DQ230992 (88%)
Camellia sinensis
D26596 (79%)
Robinia pseudoacacia
EU650628 (80%)
Sparvoli and others

(1994)
Wiersma and Wu
(1998) (P. avium)
Citrus clementina X
Citrus reticulate
AJ238754 (83%)
Pyrus communis
DQ901399 (95%)
Pyrus communis
DQ230992 (97%)
Prunus avium
AF036948 (88%)
Populus trichocarpa
EU603319 (84%)
Wang and others

(2007)
Quercus suber
AY443341 (80%)
Populus euramericana
AJ698920 (79%)
Populus tomentosa
EU760386 (78%)
Shan and others

(2008)
Venisse and others
(2002)
Fischer and others
(2007)
C. clementina X C.
reticulate
AJ238753 (77%)
Rubus idaeus
AF237955 (92%)
Prunus avium
AF036948 (83%)
Lycopersicon
esculentum
M83314 (80%)
Populus trichocarpa
EU603320 (81%)
Lo Piero and others

(2006)
Strissel and others

(2005)
Qian and others
(2008) (Pyrus
pyrifolia)
Lafuente and others
(2003)
Lotus japonicas
AB283033 (79%)
Camellia sinensis
D26596 (78%)
Nicotiana tabacum
(Samsun NN)
X78269 (79%)
Trifolium pretense
DQ073811 (78%)
S. tuberosum X63103
(92%)
Cheng and Breen

(1991)
Kumar and Ellis
(2001)
Tanaka and others
(1989)
Jiang and Joyce

(2003)
Nita-Lazar and others
(2004)
Singh and others
(1998)
Diallinas and Kanellis

(1994)
Lee and others (1992)
Given and others

(1988)
Sreelakshmi and
Sharma (2008)
Ipomoea nil
AF325496 (80%)
of stresses, including CO2 treatment (Ke and Salveit 1989) and

low temperature (Martnez-Tellez and Lafuente 1997). Similarly,
in minimally processed lettuces, the evaluation of initial induction kinetics and the time to reach maximum PAL levels revealed
higher levels of induction by combining different kinds of stresses
(wounding plus ethylene) (Lopez-Galvez and others 1996). These
results signify that ethylene produced in response to biological
stress could be a signal for plants to activate defense mechanisms
against potential pathogens. PAL is also an important source of
ammonia for plant tissues (Lewis and others 1999). In a study on
cherimoya fruit (A. cherimola), PAL activity has been reported to
increase with a high ammonia demand and decreased in fruit with
low rate of ammonia assimilation thereby reflecting the metabolic
status imposed by storage in different conditions (Maldonado and
others 2002). The possible involvement of PAL activity in the supply of important metabolic compounds for early events of ripening
was anticipated (Assis and others 2001).
PAL-regulated flavonoid/anthocyanin accumulation

PAL has been implicated in 2 major problems, rapid pericarp
browning and fruit decay, which both decrease the market value of
fruits. It was believed that PAL activity enhanced the accumulation
of phenol compounds in rambutan fruit (Nephelium lappaceum) by
polyphenol oxidase (PPO) and/or peroxidase led to the appearance of brown products (Ke and Saltveit 1988; Cantos and others
2002; Yingsanga and others 2008). Enhanced PAL activity has also
been suggested to play a role in ethylene-mediated anthocyanin
accumulation and enhanced strawberry fruit color development
(Jiang and Joyce 2003). Parallel changes in anthocyanin accumulation and PAL activity apparently reflect control of anthocyanin
synthesis by PAL, presumably through the supply of component
cinnamic acid molecules. It is noteworthy that 2 peaks of PAL
activity were reported in strawberry fruits, one in green fruit and

mRNA
accumulation
Chen and others

(2006)
Manganaris and
others (2007) (P.
salicina)
Promyou and others
(2007)
Cai and others (24)
the other in nearly ripe fruit (Cheng and Breen 1991). The 1st
peak was suggested to be involved in the synthesis of flavonoids
(condensed tannins) and phenolics that took place during early
fruit development, whereas the 2nd activity peak was associated
with the anthocyanin accumulation that occurred during later
stages of fruit ripening (Macheix and others 1990). In orange
fruit, the expression of a putative anthocyanin transporting glutathione S-transferase (GST) was correlated with the expression of
the pal, chalcone synthase (chs), dihydroflavonol 4-reductase (dfr),
and UDP glucose, flavonol 3-O-glucosyltransferase (ufgt) genes
under cold stress (Lo Piero and others 2005, 2006). As is the case
for many secondary metabolite biosynthetic proteins, an apparent redundancy with the anthocyanin-transporting GSTs in grape
berries (Vitis vinifera) was reported, and this redundancy was seen
with numerous functional copies of biosynthetic enzymes including PAL (Conn and others 2008). It is quite clear that, even for
much-studied old pathways like flavonoid biosynthesis, these
are exciting times.
In an earlier study, expression of 7 genes of the anthocyanin
biosynthetic pathway (pal, chs, chi, f3h, dfr, ldox, and ufct) was
determined in Shiraz grape berries (V. vinifera). In flowers and
grape berry peels, expression of all of the genes, except ufct, was
detected up to 4 wk postflowering, followed by a reduction in this
expression 6 to 8 wk postflowering. Expression of chs, chi, f3h, dfr,
ldox, and ufct then increased 10 wk postflowering, coinciding with
the onset of anthocyanin synthesis. The results obtained in the
study provide additional evidence for the correlation between the
expression of structural flavonoid pathway genes and anthocyanin
production during fruit development. In grape berry flesh, no pal
or ufct expression was detected at any stage of development, but
chs, chi, f3h, dfr, and ldox were expressed up to 4 wk postflowering.
These results indicated that the onset of anthocyanin synthesis in
ripening grape berry peel coincides with a coordinated increase in

expression of a number of genes in the anthocyanin biosynthetic
pathway, suggesting the involvement of regulatory genes. ufct is
regulated independently as compared to other genes, suggesting
that in grapes it could be a major control point in this pathway
(Boss and others 1996). These studies emphasize the complex
nature of flavonoid regulation in fruits, at least at the biosynthetic
gene level, and the potential problems in correlating genotypes
and phenotypes.
Schaffer and others (2007) observed that the genes involved in
the 1st steps of phenylpropanoid pathway were ethylene-responsive
and pal1 being one of the candidate genes exhibited a rapid increase
of expression in apple fruit. Transcript patterns of the 2 pal genes
in loquat fruit (Eriobotrya japonica) differed with a sharp increase
in ejpal1 transcripts late in fruit development. The opposite trend
occurred with ejpal2, where it was strongly expressed in young
fruit and not detectable at maturity (Shan and others 2008). This
suggested that ejpal2 might be more heavily involved in phenylpropanoid synthesis (including lignin synthesis) during early stages
of fruit development, when there was considerable increase in
vascular tissues. At later stages, this declined as the fruit matured,
whereas, in contrast, ejpal1 was thought to be more involved in
induction of flavonoid synthesis and lignification of the mature
fruit during ripening. In addition, manipulation of the flavonoid
pathway by antisense expression modulated PAL activity through
transcriptional and posttranscriptional mechanisms in strawberry
(Griesser and others 2008). It appears that chs and pal genes are
crucial for flavonoid synthesis and the enhanced expression of one
or both of these genes during development could specifically be
associated with higher flavonoid content at maturity.
Promyou and others (2007) reported that Sucrier banana
coated with polyethylene parafilm wax (20%) showed a delay of
peel spotting which was significantly not associated with a change
of total free phenolics in peel or with PPO activity, but was accompanied by reduced in vitro PAL activity. Results suggested that
the delay in peel spotting, after surface coating, was a result, at
least in part, of reduced PAL activity. Similarly in another study
on banana, the results suggested that the induction of PAL during low-temperature storage was regulated at transcriptional and
translational levels, and was related to a reduction in CI symptoms. Northern and Western blot analyses revealed that mRNA
transcripts of mapal1 and mapal2 and PAL protein levels during
storage increased, reaching a peak at about day 8, and finally decreased at chilling temperature. Prior to low-temperature storage,
pretreatment with propylene could alleviate CI and enhance PAL
activity, protein amount, and mRNA transcripts of mapal1 and
mapal2. Moreover, changes in PAL activity, protein content, and
accumulation of mapal1 and mapal2 exhibited almost the same
patterns (Wang and others 2007). Thus, the PAL activation by
propylene or chilling temperature may be attributed to the synthesis of new PAL protein. The results appear to suggest that the
accumulation of pal transcript can serve as a molecular marker for
chilling tolerance in banana fruit.
In raspberry (Rubus idaeus), development of fruit color and flavor was dependent on PAL encoded by a family of 2 genes (ripal1
and ripal2). Although expression of both genes was detected in
all tissues examined, ripal1 was associated with early fruit ripening events, whereas expression of ripal2 correlated more with later
stages of flower and fruit development. Determination of the absolute levels of the 2 transcripts in various tissues showed that
ripal1 transcripts were 3- to 10-fold more abundant than those of
ripal2 in leaves, shoots, roots, young fruits, and ripe fruits. The
2 ripal genes, therefore, appeared to be controlled by different
regulatory mechanisms (Kumar and Ellis 2001). Although fruits

at 2 stages differed in their chemistry, determination of the exact
role played by each ripal isoform in supporting accumulation of
specific phenylpropanoid products in fruits would require detailed
metabolite profiling.
Promising outlook of PAL

Two alleles of the pal gene identified in loquat fruit were divergently regulated during fruit development (Shan and others 2008).
This would have implications well beyond lignification, where the
direct relationship between PAL and lignin is still not very strong.
Cherimoya fruit (A. cherimola) had exhibited an increase in PAL activity without significant increase in lignin synthesis, even though
this enzyme is part of the phenylpropanoid pathway (Assis and
others 2001). The extent to which this gene may regulate the
pathway is different in different tissues; however, the mechanism
is still unclear. To further unravel the role of pal genes during
ripening in various fruit systems, the tissue-specific and developmental expression of each gene family member has to be studied.
Moreover, modulation of PAL activity, which caused the reduced
level of the cinnamic acid derivatives, has already opened new
avenues (Shirsat and Nair 1986). The identification and characterization of pal genes from a fruit cDNA library would certainly
create an opportunity to explore the possible functions of multiple pal genes during fruit development. To resolve their respective
roles, it would be informative to selectively silence pal genes and
monitor the resulting transgenic phenotypes. Therefore, it should
be feasible to scrutinize the functions of the pal gene family, and
the cis-acting elements involved in selective expression during fruit
development. A possible function of PAL during ripening could
be the catalysis of the 1st reaction toward the formation of compounds participating in the aroma of ripe fruit. The emerging
theme from recent studies is that the pal promoter is able to integrate the complex set of developmental and environmental signals
in order to finely adapt pal gene expression to the diverse functions
of 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-methyl
derivatives and S-adenosyl homocysteine (Ibrahim and others
1998). SAM-dependent O-methylation is catalyzed by OMTs
and involves the transfer of the methyl group of S-adenosylL-methionine (AdoMet) to the hydroxyl group of an acceptor
molecule, with the formation of its methyl ether derivative and
S-adenosyl-L-homocysteine as products such as lignin, flavonoids,
phenylpropanoids, and alkaloids (Dwivedi and others 1994; Pichersky and Gang 2000; Rastogi and Dwivedi 2006). OMTs play
a critical role in the biosynthesis of many classes of compounds
required for plant growth, aroma generation, and plant defense.
There are several hundred O-methylated flavonoids which occur in
plants, and these range from mono- to polymethylated compounds
belonging to the chalcones, flavones, isoflavones, and flavonols, as
well as their dihydro derivatives (Wollenweber and Dietz 1981).
Combined biochemical and molecular analyses of volatile components produced by fruit have demonstrated that their biogenesis
forms an integral part of ripening. Several omt cDNA clones have
been reported from different plant species, which share common
structural as well as physicochemical features. The phylogenetic
analysis of plant omt sequences suggests that plant omts may have
diverged from a common ancestral gene, through gene duplication

Figure 4Role of OMTs in plant-specialized

metabolism conversions catalyzed by COMT.
COMT
COMT
OH
OCH3 HO
OH
OH
OCH3
H3CO
OCH3
OH
OH
R-COOH:Caffeic Acid
R-COOH:Ferulic Acid
R-COOH:5-Hydroxyferulic Acid
R-COOH:Sinapic Acid
R-CHO:Caffeoyl Aldehyde R-CHO:Coniferyl Aldehyde R-CHO:5-Hydroxyconiferyl Aldehyde
R-CHO:Sinapyl Aldehyde
R-COH:Caffeoyl Alcohol
R-COH:Coniferyl Alcohol R-COH:5-Hydroxyconiferyl Alcohol R-COH:Sinapyl Alcohol
COOH
OH
OMT
OH
OH
OH
OCH3
Catechol
OCH3
Guaiacol
OH
Ferulic Acid
[A]
OH
OH
OMT
OCH3
OMT
OH
OH
Caffeic Acid
Figure 5O-methylation of caffeic acid, catechol,

and protocatechuic aldehyde to their respective
methylated products, ferulic, guaiacol, and
vanillin, by the action of O-methyltransferase.
COOH
CHO
Protocatechuic
Aldehyde
[B]
CHO
Vanillin
[C]
CCoAOMT (EC 2.1.1.104), which catalyzes the meta-Omethylation of caffeoyl CoA to form feruloyl CoA, appears to
play an important role in the formation of the coniferyl alcohol
moieties that are precursors for lignification (Dwivedi and Campbell 1995; Vander and others 2000). CCoAOMT catalyzes 1 of 2
alternative methylation steps of the phenylpropanoid biosynthesis
pathway which leads to the synthesis of diverse secondary products such as lignin, flavonoids, and isoflavonoids (Ye and Varner
Classification of plant OMT
OMTs are widespread throughout the plant kingdom and found 1995) and this methylation step is highly regulated (Hahlbrock and
in all lignin-producing plants. O-methylation of the phenyl- Scheel 1989).
propanoid and flavonoid groups of compounds is catalyzed by
distinct classes of OMTs.
Proposed methyl acceptor classes and groups
The molecular and biochemical data available so far provide the
Caffeic acid 3-O-methyltransferase (COMT, EC 2.1.1.68) catalyzes the O-methylation of aromatic diols and is involved in basis for a meaningful classification of the plant omt gene superfamlignification (Rastogi and Dwivedi 2008) or may have other ily. There exist some 36 omt cDNA clones, which are subdivided
physiological functions like flavor generation (Collendavelloo and into 5 groups encoding the methylation of the lignin precursors,
others 1981; Pellegrini and others 1993) (Figure 4). Interest- caffeic and 5-hydroxyferulic acids (Group 1), flavonoids (Group 2),
ingly gene evolution studies in Clarkia breweri have suggested that and phenylpropanoids (such as the coumarins Group 3). Group 4 is
isoeugenol OMT (iemt) gene that catalyzes the methylation of primarily comprised of simple phenols and anthocyanins, whereas
eugenol and isoeugenol to form the volatiles methyleugenol and Group 5 encompasses polyketides and other acetate/malonateisomethyleugenol had arisen from comt gene. It was suggested that derived compounds (Table 2). Each group of OMT is further
OMT substrate preference could be regulated by a few amino classified into Class A and Class B. Based on the class of subacid residues, and new OMTs with different substrate specifici- strate methylation, Class A OMTs methylate phenylpropanoid
ties could evolve from an existing OMT by mutation of several compounds, whereas Class B OMTs methylate flavonoid comamino acids (Wang and Pichersky 1999). The evolution of new pounds. Although the chemical mechanisms of methyl transfer
OMTs with new substrate specificities is relatively simple; it is not reactions are identical, OMTs differ in their selectivity with resurprising that OMTs with similar substrate specificities evolved spect to the stereochemistry of the methyl acceptor molecules, as
independently in different plant lineages more than once. These well as the substitution pattern of their phenolic hydroxyl groups
broad-specificity enzymes may be recruited for new metabolic (Ibrahim and others 1998). Some of the known OMTs display
pathways, followed by further evolution toward more specific and strict specificities toward their acceptor substrate as well as to the
efficient catalysts. It follows that gene duplication (or even poly- position of substrate methylation. In contrast, other OMTs, espeploidy) is an important factor in this concept as it provides the cially those catalyzing the methylation of catechol (O-dihydroxy)
raw material for the acquisition of new biosynthetic pathways. It moiety substrates, exhibit surprisingly broad substrate specificities.
is somewhat surprising that developing strawberry fruits display OMTs have been shown to be multifunctional enzymes that could
high OMT activity levels toward caffeic acid, protocatechuic alde- also catalyze transformations in 2 different biosynthetic pathways
hyde, and catechol resulting in ferulic acid, guaiacol, and vanillin such as the alkaloid and phenylpropanoid pathways (Li and others 1997) or aroma biosynthesis (Lavid and others 2002) and,
(Figure 5A to 5C), respectively.
and mutation, to yield the various functional enzyme groups currently recognized in plants (Ibrahim 1997). It would be interesting
to group plant omt cDNA genes according to a functional trait
that reflects the substrate preferences of their encoded proteins,
and could be used as the basis for classification of this supergene
family.


Table 2Classification of omt cDNA clones encoding the methylation of allel with other reactions involving the methylation of caffeic acid,
phenylpropanoid compounds (Class A) and omt cDNA clones encoding catechol, anthocyanidin, or other as yet unidentified O-diphenols
the methylation of flavonoid compounds (Class B).
that increase during fruit ripening.
Groups
Group 1
Group 2
Group 3
Group 4
Group 5
OMT cDNA clones encoding

methylation of
phenylpropanoid
compounds Class A
Caffeic/5-hydroxyferulic acid
CoA esters of
caffeic/5-hydroxyferulic
acid
Phenylpropanoids
(Coumarins,
furanocoumarins)
Phenolic compounds (Simple
phenols, benzoic acids,
phenolic esters)
Polyketides and other
acetate/malonate derived
compounds
OMT cDNA clones encoding

methylation of flavonoid
compounds Class B
Flavonols (flavones)
Chalcones (flavanones)
Pterocarpans and their
isoflavone precursors
Flavans and anthocyanins
(although none has been
reported)
incidentally, both phenylpropanoid and flavonoid compounds

share some structural similarities in which the phenolic B ring
and carbons 2, 3, and 4 of flavonoids are derived from phenylpropanoids (Ibrahim and others 1998). There are now documented cases from different plant species of apparent evolution
from COMTs of new OMTs with new substrate specificities. As
more such cases are described, and an understanding of the effects
of specific amino acids on the active site develops, it may become
possible to use a COMT as the starting point in designing an
OMT that can act on a particular substrate of interest.
Despite a particular specificity for phenolic substrates, plant
OMTs share highly conserved domains (Dwivedi and Campbell
1995; Joshi and Chiang 1998). To date, more than 10 distinct
groups of SAMOMTs that utilize SAM and a variety of substrates have been described in higher plants. A comparison of
the amino acid sequence of 56 SAMOMTs from different plants
showed that plant OMTs fall into 2 distinct groups, Pl-OMT I and
Pl-OMT II. The length of Pl-OMT I enzymes varies from 231
to 248 amino acids, whereas the length of Pl-OMT II enzymes is
344 to 383. Unlike Pl-OMT I members that are known to utilize
only a pair of substrates, the members of the Pl-OMT II group
can accept a variety of substrates and are multifunctional enzymes
(Li and others 1997). Multiple OMTs displaying small but defined
substrate discrimination could be found within the same plant
and even within the same tissue (Gang and others 2001, 2002).
Recently, in anise (Pimpinella anisum) seeds and leaves, phenylpropene t-anethole has been shown to impart the characteristic
sweet aroma. A cDNA encoding t-anol/isoeugenol synthase 1
(ais1), which is an NADPH-dependent enzyme that can biosynthesize t-anol and isoeugenol from coumaryl acetate and coniferyl
acetate, respectively, was cloned. In addition, t-anol/isoeugenol
OMT 1 (aimt1), an enzyme that converts t-anol or isoeugenol to
t-anethole or methylisoeugenol, respectively, via methylation of
the p-OH group was also successfully cloned. The genes encoding
ais1 and aimt1 were expressed throughout the plant and incidentally their transcript levels were highest in developing fruits. The
AIMT1 protein sequence exhibited significant homology to basil
(Ocimum basilicum) and Clarkia breweri phenylpropene OMTs, but
unlike these enzymes, which do not show large discrimination
between substrates with isomeric propenyl side chains, AIMT1
showed a 10-fold preference for t-anol over chavicol and for
isoeugenol over eugenol (Koeduka and others 2009). Therefore, it
could be that furaneol methylation occurs as a result of and in par-
Biological significance of OMTs in volatile generation

Volatile compounds from the phenylpropanoid pathway, for example estragole (and eugenol), are likely to be produced from
phenylalanine. The final step in the estragole biosynthesis is predicted to involve a methyltransferase class of enzymes but still
some of the enzymatic steps are still poorly understood. Apart
from OMT that add a methyl group, several enzymes belonging to
the SABATH family (S-adenosyl-L-methionine carboxyl methyltransferase) have also been reported to exhibit a similar function.
Significantly, both add the methyl group using methionine as a
donor and the main pathway for ethylene biosynthesis departs from
methionine and is converted to S-adenosyl methionine (SAM),
amino-cyclopropane carboxylic acid (ACC), and ethylene in 3
consecutive reactions catalyzed by the enzymes SAM-synthetase,
ACC-synthase (ACCS), and ACC-oxidase (ACCO), respectively.
SAM is a primary metabolite, crucial in polyamine metabolism and
the main methyl group donor in many reactions, such as those of
lignin biosynthesis, nucleic acid, flavonoids, phenylpropenes, alkaloids, and protein methylation. Two (SABATH1 and 4) out of 6
members of the SABATH family showed an increase of expression
upon the treatment of ethylene in apple during fruit ripening. Of
the 7 O-methyl transferases in apple, omt7 was ethylene-induced
and quite similar to comt (Gowri and others 1991) and, therefore, could possibly be instrumental in catalyzing the final step in
estragole 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 cultivar and species, which contribute significantly to flavor (Schieberle
and Hofmann 1997). In apple, a total of 186 candidate genes mined
from expressed sequence tags (EST) databases have been thought
to be involved in the production of ester, polypropanoid, and
terpene volatile compounds during ripening. Based on sequence
similarity and gene expression patterns, 17 candidate genes including omt have been identified as ethylene control points for
aroma production during the fruit ripening process (Schaffer and
others 2007). In papaya, huge numbers of genes have been reported which are involved in the development of volatiles during
fruit ripening. Papaya has 30 candidate genes for the lignin synthesis 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 ccr
gene (Ehlting and others 2005). Moreover, papaya has 2 genes in
the family ccoaomt, which are presumed to convert caffeic acid to
ferulic acid (Ming and others 2008).
OMT-mediated anthocyanin biosynthesis
Ripening-related gene sequences that code for proteins involved
in key metabolic events including anthocyanin biosynthesis, have
been isolated from strawberry (Manning 1998) and were not found
to be active in green fruits. The flavonoid biosynthesis pathway
leads to anthocyanin formation and it is noteworthy that there
is cross-linking and interdependence of the flavonoid and lignin
biosynthesis pathways during the ripening process (Figure 6). A
thorough knowledge of the interconnecting pathways of lignin
biosynthesis is required for the rational designing of metabolic engineering strategies. Higher levels of omt transcripts have been observed during fruit ripening of various cultivars of berries, which
accumulated methoxylated forms of anthocyanins more abundantly than nonmethoxylated forms. It was assumed that cyanidin

OH
C4H
p-Coumaric acid
HOOC
PAL
HOOC
HOOC Cinnamic acid
NH3 Phenylalanine
General phenylpropanoid pathway

4CL
OH
HSAOC
OH
HO
COOH
H2C
OH
STS
HO
Trihydroxychalcone
Tetrahydroxychalcone
HO
Stilbene
F3'H
Nf F3'5'H
HO
HO
OH
UFGT
Delphinidin
O-Glc
OH
IOMT
OH
Flavonol glycosides
R
OCH3
I2'H
OH
HO
OH
Petunidin-3-glucoside
O
O
OH
Guc
OMT
HO
OCH3
HO
OCH3
IFR
Delphinidin-3-glucoside
O
OH
OH
OH
HO
RT
OCH3
OMT
HO
Isoflavone
R'
O-Glc-O-Rha
OH
OH
OH
OH
UFGT
R'
OH
HO
OH
Cyanidin
Flavonols
FSLI
OH
IFS
OH
LAR
Catechins
Catechins LAR Leucocyanidin Flavonols Leucodelphinidin
OH
LDOX
LDOX OH BAN
BAN
OH
OH Rha-O
O
H
R
HO
Phf
Dhk
DFR
OH
Aurones
F3H
F3'5'H Dhm
DFR
FLSI
F3H
Flavanone
OH
F3H
Flavonols FSLI Dhq
OH
O
CH
CHI
CHI
O
OH
HO
OH
OH
OH
OH
Naringenin Chalcone
HO
OH
OH
OH
OH
COSCoA
MalonylCoA
4-Coumaroyl-CoA
CHS
CHS
HO
CHS/CHR
OCH3
Guc
OMT
OH
Cyanidin-3-glucoside
OCH3
OH
HO
OCH3
Malvidin-3-glucoside
VR
DMID
Peonidin-3-glucoside
2'-hydroxy Isoflavanone
HO
OCH3
Isoflavanoids
Figure 6Metabolic pathway and key steps of the flavonoid biosynthesis pathway leading to anthocyanin formation. The figure also depicts the
cross-linking and interdependence of flavonoid and lignin biosynthesis pathways. Acronyms of the compounds reported in the figure stand for the
following: 4CL, 4-coumaroyl CoA ligase; I2 H, isoflavone 2 -hydroxylase; BAN, anthocyanidin reductase; C4H, cinnamate-4-hydroxylase; CHI, chalcone
isomerase; 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, flavonoid
3 -hydroxylase; F3H, flavanone 3-hydroxylase; FLS1, flavonol synthase; IFR, isoflavone reductase; IFS, isoflavone synthase; IOMT, isoflavone
O-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, vestitone
reductase.


methylation occurred as a nondirected side-effect action of caffeic acid methylation, which increased during fruit ripening. The
evolution of the ratio of the transcriptional level omt/ufgt through
ripening and the relative abundance of methoxylated anthocyanin
was compatible with a role of OMT in the methoxylation of
the B-ring of anthocyanin in grapevines (Castellarin and Gaspero
2007). The cumulative expression of the transcription factors may
explain the quantitative variation in anthocyanin content, which
probably conceals the presence of additional factors involved in
the process.
Aroma biosynthesis in strawberry

Strawberries are a rich source of phenolic compounds but
produce low levels of lignin; hence, the OMT enzyme in wild
strawberry could be involved in the synthesis of phenols and
its derivatives. During early stages of fruit ripening, nontannin
flavonoids and mainly condensed tannins accumulated to high
levels and gave strawberry a characteristic astringent flavor (Cheng
and Breen 1991). During later stages of fruit ripening, when fruit
started to ripen, other flavonoids such as anthocyanins (mainly
pelargonidin glucoside), flavonols, and cinnamoyl--d-glucose accumulated to high levels (Latza and others 1996; Manning 1998;
Moyano and others 1998; Aharoni and others 2000; Deng and
Davis 2001). It may be proposed that other regulatory mechanisms
(not related to transcriptional control) are governing flavonoid synthesis at least during the initial stages of ripening. In response to
external ethylene, apple fruits showed a normal climacteric burst
and produced increasing concentrations of ester, polypropanoid,
HO
Figure 7(A) Chemical structures of substrates

and products of enzymatic reactions catalyzed
by Fragaria x ananassa O-methyltransferase
(FaOMT). HDMF, 4-hydroxy-2,5-dimethyl3(2H)-furanone; DMMF, 2,5-dimethyl4-methoxy-3(2H)-furanone. (B) Substrates and
products of OMT enzyme that are acting in a
branch of the phenylpropanoid pathway
depicting the dual function of FaOMT in
strawberry fruits.
OH
and terpene volatile compounds (Dandekar and others 2004). Interestingly, due to the huge number of flavor compounds found in
strawberry, Zabetakis and Holden (1997) suggested that it was not
possible for each substance to have its own enzymes. Accordingly, the side activity of COMT involved in phenylpropanoid
metabolism of strawberry fruits was thought to be responsible
for the formation of 2,5-dimethyl-4-methoxy-3(2H)-furanone
(DMMF). Consistent with this view, it was reported that a single OMT from Chrysosplenium americanum could methylate both
flavonoid and phenylpropanoid compounds (Gauthier and others
1998). Of the 15 volatiles in strawberry, 4-hydroxy-2,5-dimethyl3(2H)-furanone (HDMF) was regarded as vital, but it was methylated further by FaOMT (Fragaria X ananassa OMT) to DMMF
during the ripening process (Wein and others 2001) (Figure 7A).
HDMF was indispensable because of its high concentration (up
to 55 mg kg1 strawberry fruit FW) (Larsen and others 1992)
and low odor threshold (10 ppb in water) (Schieberle and Hofmann 1997). The reduction of faomt gene expression altered the
HDMF/DMMF ratio, resulting in a near-depletion of the DMMF
pool, thus confirming the importance of FaOMT in the DMMF
formation. faomt encoded a sequence of 365 amino acids and accepted a substrate spectrum for compounds containing O-diphenol
structures (Wein and others 2002).
In addition, the dual function of this enzyme in the secondary
metabolism was proved as faomt down-regulation and also affected
the concentration of feruloyl 1-O--d-glucose and caffeoyl 1-O-d-glucose, suggesting that it was also involved in the methylation
of the caffeoyl group (Lunkenbein and others 2006) (Figure 7B).
S-adenosyl-L- S-adenosyl-Lmethionine homocysteine
HO
OH
OCH3
HDMF
O
FaOMT
OH
DMMF
[A]
HO
COOH
H3CO
COOH
FaOMT
HO
Caffeic Acid
S-adenosyl-L- S-adenosyl-Lmethionine homocysteine
Ferulic Acid
O
OH
HO
O
HO
HO
O
HO
Caffeoyl -D-glucose
H3CO
OH OH
OH
O
HO OH
HO
OH
Feruloyl -D-glucose
[B]


Ripening related gene sequences that code for proteins involved
in key metabolic events including anthocyanin biosynthesis were
isolated from strawberry and were not found to be active in green
fruits (Manning 1998). Caffeic acid is usually not an intermediate
in anthocyanin pigmentation biosynthesis and methoxyfuraneol
formation in strawberry. But cyanidin, an anthocyanin precursor,
which possesses an O-dihydroxyphenol structure similar to that of
caffeic acid, might be recognized by the OMT (Wein and others 2002). Similarly, many enzymes of secondary metabolism are
known to recognize more than one substrate, although they often
have different catalytic rates toward them (Wang and Pichersky
1999). It has been postulated that this phenomenon was probably due to the evolution of ancestor genes involved in primary
metabolism, such as the comt involved in lignification (Pichersky
and Gang 2000). Because of the expression pattern of faomt in different stages of fruit ripening, it was assumed that in the beginning
of fruit development FaOMT could be involved in lignification
of the vascular bundles in the expanding fruit. Subsequently, in
the later stages (ripe fruit), FaOMT activity probably provided
the precursors for achene lignification (Lunkenbein and others
2006). On the other hand, FaOMT is also instrumental in the
biosynthesis of strawberry volatiles, because it efficiently catalyzes
protocatechuic aldehyde to vanillin during ripening. Vanillin has
been reported to contribute to strawberry flavor in both wild and
commercial strawberry cultivars (Hirvi and Honkanen 1982). Recently, an enone oxidoreductase (FaQR) involved in the HDMF
formation was isolated from a crude strawberry fruit extract and
the corresponding gene cloned. It represented a very promising
target for biotechnological engineering for flavonoid biosynthesis
(Raab and others 2006).
It is still unknown whether one enzyme with relatively low
substrate specificity is able to catalyze the transfer of a methyl group
to the substrates, or if it is a mixture of more than one enzyme
which methylates different substrates. The availability of cDNAs
coding for the respective enzymes and their functional expression
will be valuable in attempts to answer this question. The genes
involved in aroma biosynthesis are not coordinately regulated by
ethylene, but typically only the 1st and final steps are ethyleneregulated suggesting important transcriptional regulation points
for aroma production in fruits during the ripening process. A
better understanding of the enzymes involved in the formation of
methoxyfuraneol will assist classical breeding and biotechnological
efforts 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 to
the corresponding alcohols, the last step in the biosynthesis of
monolignols (Wyrambik and Grisebach 1975; Boudet and others
1995; Roth and others 1997; Boerjan and others 2003). In both
naturally occurring mutants and transgenic plants with depleted
levels of CAD, increased incorporation of cinnamaldehydes into
lignin was observed. The cross-linking and physical properties of
cinnamaldehyde-rich lignins differed from that of normal lignins
(Salentijn and others 2003). However, CAD was also expressed
in response to stress (Galliano and others 1993), pathogen elicitors (Campbell and Ellis 1992), and wounding (MacLean and
others 2007). However, CAD has been reported to express itself
even in cells that do not make lignin (OMalley and others 1992;
Grima-Pettenati and others 1994). The cytochemistry studies of
defense responses to Xanthomonas campestris in cassava fruit showed
considerable changes in the metabolism of parenchymal cell walls,

involving the accumulation of lignin, flavonoids, and polysaccharides which was thought to be due to cad induction (Kpemoua
and others 1996). CAD is therefore regulated by both developmental and environmental stimuli, much like other well-studied
enzymes of phenylpropanoid metabolism (Whetten and Sederoff
1995).
Molecular characterization of CAD expression

cad cDNA isolated from various angiosperms has been shown to
share extensive nucleotide sequence homology suggesting cad gene
has been conserved during evolution (Halpin and others 1994).
Blanco-Portales and others (2002) classified strawberry fruit CAD
as zinc-containing alcohol dehydrogenase due to occurrence of a
consensus amino acid sequence at positions ranging from 60 to
83. Concurrent gene expression in receptacle and achene tissues
using DNA microarrays in strawberry fruit revealed a total of 1701
cDNA clones (comprising 1100 ESTs and 601 unsequenced cDNAs). Analysis of expression ratios identified 66 out of the 259
(25%) achene-related clones and 80 out of 182 (44%) receptaclerelated clones with more than a 4-fold difference in expression between the 2 tissue types. Different cad and ccr clones isolated were
thought to be involved in the lignification process in the receptacle.
Enzymatic activity assays with a recombinant protein encoded by a
strawberry cad gene homologue, identified as ripening-regulated,
retained CAD activity and was immunolocalized to the vascular
tissue in the receptacle. Interestingly, a strawberry cDNA showed
homology to the tobacco and Arabidopsis ethylene-responsive element binding factor (ERFs), suggesting a role for ethylene in
late achene development (Aharoni and OConnell 2002). Similar
results were earlier reported by quantitative real time PCR (QRTPCR) data in same fruit suggesting a relationship of a strawberry
FxaCAD1 enzyme with a lignification process to both vascular
development and achene maturation. Thus, as fruit ripened, the
achenes underwent strong lignifications of thick pericarp (PerkinsVeazie 1995).
Strawberry fruit traverse through 4 different stages (green,
white, turning, and red) of fruit development. Interestingly, CAD
expression decreased after the green stage (white and turning
stages) before increasing again at the red stage (Sozzi and others 1998). Detailed analysis of cad expression in different strawberry tissues during ripening using RNA gel blots complemented
with microarray data showing elevated levels of cad transcript in
the red stage. Expression of cad was detected in achenes and receptacles (fruits with no achenes), petioles, leaves, and flowers.
Because cad was strongly expressed in the ripening receptacle tissue, it was suspected that some of them might be actively expressed
in the vascular bundles and associated with lignification. Using a
primary antistrawberry CAD polyclonal antiserum, Aharoni and
others (2002) showed that the corresponding protein was localized
specifically to immature xylem cells undergoing active lignification. It has been observed that the lignin in CAD-deficient plants
was more susceptible to chemical extraction and showed improved
pulping characteristics (Russell and others 2000; Ruel and others
2001). Thus, the differences in cad gene expression could be related
to lignin composition, being richer in aldehydes and more susceptible to enzymatic degradation in the soft cultivar. Modifications
of lignin by downregulation or overexpression of cad in strawberry
showed that severe changes in CAD activity had a striking effect
on the composition of lignin too. In strawberry fruit maturation,
due to the higher quantity of CAD in firm cultivars, there was a
strong lignification of thick pericarp of fruit (Salentijn and others
2003).

A cDNA clone, mdcad1, encoding putative cad from apples
(Malus X domestica Borkh. cv Fuji) was characterized and the
clone contained an open reading frame of 325 amino acid residues,
which showed more than 80% identity with Eucalyptus cad1. mdcad1 mRNA was detectable in vegetative tissues and was strongly
expressed in the fruit (Sung-Hyun and others 1999). The transcription level of mdcad1 in apple initially increased with mechanical wounding only when the pathogen attacked the plant
and thereafter endogenous salicylic acid (SA) was produced in the
pathogen-induced necrotic tissue. The expression pattern of mdcad1 mRNA in the fruit peel after light exposure increased until
1 d after light exposure and remained stable thereafter, suggesting
that mdcad1 was light-inducible. The increased level of SA could
perhaps strongly induce the expression of mdcad1 to catalyze the
synthesis of cinnamyl alcohol. The increased lignin content in
the attacked tissue thus served as a barrier against any pathogen
invasion. The induction of mdcad1 expression by wounding and
further induction by SA, yet not by ethylene and jasmonic acid,
suggested that the induction of mdcad1 transcripts could follow
the SA-dependent pathway of plant defense. Southern blot hybridization showed that there were either 1 or a few copies of
cad genes in apples (Sung-Hyun and others 1999). Although the
secondary metabolites present in the fruit peel could act as protectants against changing environmental conditions and in deterring
pathogens, these could also play a role in attracting seed-dispersing
frugivors.
CAD and lignin accumulation

Most transgenic studies on CAD have shown that loss of activity resulted in changes in lignin composition rather than lignin
content (Baucher and others 1999). But the results in the literature on CAD are still vague, with lignin composition sometimes
being affected and at times not, in cad antisense transgenic plants
(Boudet 2000). In accordance with this theory, the increase in
lignin content of postharvest loquat fruit (E. japonica) tissue was
paralleled by a rise in CAD. Incidentally, the direct proportionality relationship between CAD activity and lignin accumulation
might be missing, responding in a way analogous to other enzymes
of the pathway. Modulation of the levels of ejcad1 transcripts by
ethylene treatment or low-temperature conditioning in E. japonica
were particularly associated with changes in lignification during
ripening. Expression of ejcad1 increased markedly 4 d after exposure to 0 C. These results support a view that lignification is
stimulated by low temperature, possibly mediated by a stimulation
in CAD activity (Shan and others 2008). Increase in expression
levels of ejcad1 preceded the major increase in fruit firmness and
lignifications, portentous of some role in loquat fruit ripening,
despite the lack of homology with cad gene of tobacco associated
with lignification (Damiani and others 2005). Because cad gene
was isolated from fruit flesh, the possibility arises that these have a
more fruit-dependent role, or that fruit tissue has variants in these
and other genes associated with tissue structural changes. In grape
berry (V. vinifera), 4 CAD isozymes exhibited preferential mRNA
accumulation in skin and or skin/pulp during ripening (Grimplet
and others 2007). The expression patterns were related to vascular bundle formation, which occurred specifically in these tissues
similar to that observed in strawberry (Aharoni and others 2002).
However, CADs may also be involved in the synthesis of cinnamyl
alcohol derivatives responsible for fruit flavor (taste and aroma)
apart from lignin formation (Mitchell and Jelenkovic 1995).
Various branches of the phenylpropanoid pathway including
those associated with the biosynthesis of lignin, hydroxycinnamates
(ferulic, sinapic, caffeic, and 4-coumaric acids) and flavonoids, have

been reported to be closely linked (Salentijn and others 2003). It
cannot be ruled out that CAD acting in the lignin biosynthesis pathway is associated with other functions like biosynthesis of
flavor compounds during the ripening process (Mitchell and Jelenkovic 1995) apart from the primary role of providing structural
strength to the cells and disease resistance. Variations in the flux
through the pathway may lead to the biosynthesis of a different
pool of hydroxycinnamic acids or aldehydes with a putative effect
on flavor or on cell wall-bound hydroxycinnamates (Kroon and
Williamson 1999). As cad genes have been isolated from fruit flesh,
this raises the possibility that they could have a more fruit-specific
role. Available data show an encouraging relationship between
components of the phenylpropanoid pathway and firmness development in fruits. Hence, this strongly suggests that lignification
being a conventional synthetic process in the fruit ripening and cad
gene could have a central role to play during the process. Therefore, CAD turns out to be a suitable candidate among the battery
of lignin biosynthetic enzymes in modifying fruit ripening rates.
The Ever-Growing Phenylpropanoid Pathway

With the emerging evidence of the possible role of the phenylpropanoid pathway in fruit ripening and the suggestion that the
various branches of the phenylpropanoid pathway appear to be
closely linked. Consequently, the role of the phenylpropanoid
pathway is turning out to be crucial during the ripening process.
Furthermore, the possible biological functions of other putative
genes of the phenylpropanoid pathway is being evaluated which
at present remains elusive.
In the lignin branch of the phenylpropanoid pathway, cinnamoyl
CoA reductase (CCR, EC 1.2.1.44) is the 1st enzyme and is responsible for the conversion of hydroxycinnamic acid CoA esters
to the corresponding hydroxycinnamaldehydes. CCR plays a major role in determining total lignin content and quality of soluble
phenolic content in tomato (Van der Rest and others 2006). Thus,
downregulation of CCR might cause variations in the flux through
the pathway leading to the synthesis of a different pool of hydroxycinnamic acids or aldehydes with a putative effect on flavor or
on cell wall-bound hydroxycinnamates (Kroon and Williamson
1999). In strawberry, expression of the ccr and cad genes differed
between the cultivars, ccr being lower in the firm cultivar and
cad in the soft cultivar (Salentijn and others 2003). Modifications
of lignin by downregulation of CCR decreased the total lignin
content (Chapple and Carpita 1998). Thus, the differences in cad
gene expression could be related with lignin composition, being
richer in aldehydes and more susceptible to enzymatic degradation in the soft cultivar. Consistently, downregulation of CCR
in tomato through RNA interference (RNAi) led to quantitative
and qualitative changes in the soluble phenolic content of extracts
from fruit and vegetative organs and an increase in the antioxidant
capacity of the plant extracts (Van der Rest and others 2006). Of
the 5 isogenes of ccr identified in grape berry (V. vinifera), only 2
genes showed seed specific expression and 1 showed peel-specific
expression, whereas 2 others exhibited mixed expression in the
pulp/skin or skin/seed (Grimplet and others 2007).
Cinnamate-4-hydroxylase (C4H, EC 1.14.13.11), a cytochrome P-450-linked monooxygenase, is a key enzyme in the
phenylpropanoid pathway, responsible for hydroxylation of cinnamic acid to p-coumaric acid. In Korean black raspberry (Rubus
sp.), QRT-PCR studies indicated that the c4h gene had a differential expression pattern during fruit development, that is, gene
expression was first detected in green fruit, markedly reduced in


yellow fruit, and increased in red and black fruit stages, which
was concomitant with flavonoid content. In contrast, the content
of anthocyanins during the progression of ripening dramatically
increased suggesting that the c4h gene in raspberry was instrumental in color development at the later stages of fruit ripening,
whereas the expression of the c4h gene during the early stages
might be related to the accumulation of flavonols (Myung-Hwa
and others 2008). In Valencia sweet orange (Citrus sinensis), 2 c4h
genes were described coding a constitutively expressed C4H2 enzyme that plays a normal role in the phenylpropanoid pathway
in contrast to a wound-induced C4H1 isoform (Betz and others
2001). Chen and others (2006) determined that C4H enzyme
activity was 10-fold the PAL activity, although C4H was located
immediately downstream pal in the biosynthetic pathway during
grape berry development. Ikegami and others (2005) reported
that inhibition of flavonoid biosynthetic gene expression of c4h
coincided with loss of astringency in pollination-constant, nonastringent (PCNA)-type persimmon fruit.
Another enzyme, 4-coumarate coenzyme A ligase (4CL, EC
6.2.1.12), catalyzes the conversion of 3 cinnamic acid derivatives
to their corresponding coenzyme A esters in a 2-step reaction.
The reaction is considered to be a branch point between general phenylpropanoid metabolism and pathways leading to end
products such as lignin. 4CL plays a particularly important role in
plant defense reactions because of its position, joining the phenylpropanoid pathway with lignin and flavonoid branch pathways. In
fact, different isoforms of 4CL direct carbon flow to the diverse
pathways of phenylpropanoid metabolism according to different
substrate preferences. For example, 4CL3 (a class II 4CL) has
a high affinity for 4-coumarate, whereas 4CL1 and 4CL2 have
strong affinities for 4-caffeate (Stuible and others 2000). Thus,
Ehlting and others (1999) hypothesized that the primary function of class II 4CL is to channel 4-coumarate to the flavonoid
pathway. In raspberry (Rubus idaeus), 3 classes of raspberry 4cl
cDNAs (ri4cl1, ri4cl2, and ri4cl3) were isolated. Based on phylogenetic classification, expression patterns, and recombinant protein
activities, the different ri4cl genes were thought to participate in
different biosynthetic pathways leading to the biosynthesis of various phenylpropanoid-derived metabolites that help to create flavor
and color in raspberry fruit (Kumar and Ellis 2003a,b). Phenylpropanoid genes (fapal, fac4h, and fa4cl) in strawberry had a similar
2 phase expression pattern with a decrease of transcript levels at
the W stage (partially ripe) (Almeida and others 2007). Expression
of ej4cl in white-fleshed Baisha (BS) loquat (E. japonica) fruit increased during the 1st 6 d, again slightly preceding a rise in enzyme
activity, whereas expression in ripening red-fleshed Luoyangqing
(LYQ) fruit remained at a relatively low level. The relatively high
4CL activity, as well as the corresponding gene expression in BS
fruit, indicated that further effort is necessary to find lignificationspecific 4cl gene family members in loquat (Shan and others 2008).
Prior to CAD in the lignin biosynthetic pathway, PAL and 4CL
both have a role in a wider range of biosynthetic pathways, including that of flavonoid production. Because of this, we might not
expect relationships of these genes with lignification to be direct
or particularly sensitive.
Chalcone Synthase
Chalcone synthase (CHS, EC 2.3.1.74) is the enzyme responsible for catalyzing the 1st committed step of the flavonoid biosynthesis pathway. CHS is an acyltransferase enzyme that catalyzes
the condensation of 4-coumaroyl CoA to the 1st flavonoid naringenin chalcone, in the presence of 3 molecules of malonyl CoA

(Lucheta and others 2007). The flavonoid skeleton, synthesized

by CHS, is converted to chalcones, flavanones, flavonols, anthocyanins, and proanthocyanidins (Velasco and others 2007). Quality
traits of raspberry fruits (Rubus) such as aroma and color derive
in part from the polyketide derivatives, benzalacetone and dihydrochalcone, respectively. The formation of these metabolites
during fruit ripening is the result of the activity of polyketide synthases (PKS), benzalcetone synthase, and CHS (Kumar and Ellis
2003a,b). Hairpin RNA (ihpRNA) silencing led to reduced levels of chs mRNA and enzymatic CHS activity in strawberry fruit.
The levels of anthocyanins were down-regulated and precursors of
the flavonoid pathway were shunted to the phenylpropanoid pathway leading to a large increase in levels of (hydroxy) cinnamoyl
glucose esters (Hoffmann and others 2006). In citrus fruit (Citrus
unshiu Marc.), mRNA levels of the flavonoid biosynthetic pathway
genes (citchs1 and citchs2) exhibited high transcript levels in young
tissues and low in senescent tissues during fruit development. The
high expression of flavonoid biosynthetic genes and high accumulation of flavonoids in young fruits suggested that flavonoids were
synthesized in the early developmental stage (Moriguchi and others 2001). Recently, a study in grapes during ripening has shown
chs2 expression increased by light treatment and it appeared that
this response was concomitant with the expression of leucoanthocyanidin oxidase (ldox), omt, and ufgt, because of their remarkably
similar expression profiles (Matus and others 2009). Surprisingly,
in tomato it was found that ectopic expression of chs and f3h in
conjunction 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 another study, concomitant ectopic expression of chs, chi, f3h, and fls
in tomato fruit resulted in increased levels of flavonol accumulation in both peel and flesh tissues (Verhoeyen and others 2002).
In apple fruit, the relationship of CHS activity with anthocyanin
synthesis in peel revealed that the flavonoid content was relatively
high and constant from fruitlet to maturation stage (Ju and others 1995) whereas UV-B and low temperature were important
factors for anthocyanin accumulation in apple fruit skin by inducing the expression of chs and anthocyanidin synthase (ans) genes
(Ubi and others 2006). chs has been an attractive target for genetic
engineering and there are numerous instances of co-suppression
or downregulation of this gene in order to modify flower color
toward pure white as a result of a complete absence of flavonoids.
Peroxidases
Peroxidase (POD, EC 1.11.1.7) catalyzes the polymerization of
phenylpropanoid precursors of lignin and are involved in the last
step of lignin formation. However, it has been difficult to differentiate between peroxidase isozymes associated with lignification
and those involved in biochemical activities (Whetten and Sederoff
1995). Ryugo (1964) followed the changes in lignin and phenolic
precursors in the developing peach pit and suggested it was a good
system for lignin synthesis studies. In peach, a 2-fold increase in
total peroxidase activity during lignifications was reported which
was established by an increase in a number of basic isozymes in
lignifying tissues. Although an increase in the amount and diversity of basic peroxidases was observed during lignification, other
enzymes involved in producing phenylpropanoid precursors may
play a more important role in controlling lignin formation (Abeles
and Biles 1991). Dalet and Cornu (1988) reported no differences in
the amount or distribution of peroxidase isozymes in cherry clones
with different degrees of lignifications. Interestingly, the ejpod gene
cloned from E. japonica exhibited only 1 of 6 amino acid residues

associated with lignin synthesis and even lower identity with pod
involved in lignin synthesis in other plants, but significantly had a
close temporal association with the ripening-associated lignification in loquat fruit (Shan and others 2008). Different peroxidase
isoforms typically have different kinetic properties in vitro prompting the question whether, and to what extent, these peroxidases
help define the lignin composition, and thus structure, in the cell
wall. In rambutan (N. lappaceum) fruit, the rapid desiccation of the
spinterns compared to the peel appeared to be the main reason for
the rapid browning of the spinterns. But higher activity of POD
observed, owing to higher rates of oxygen transmission into spinterns as compared to the peel, was also thought to play a central
role (Yingsanga and others 2008).
In tomato, it was suggested that POD isozymes located within
the fruit exocarp may have a dual role in restricting fruit expansion through cross-linking of cell wall components and producing a protective barrier in the epidermis (Andrews and
others 2002). The presence of wall-bound POD activity in
mature fruit confirmed earlier findings and supports the notion
that POD-mediated stiffening of the exocarp cell walls leads to
the cessation of fruit growth (Thompson and others 1998; Andrews and others 2000). POD activity of undamaged pericarp of
mangosteen fruit did not change, while that of damaged pericarp increased slightly during the 1st 2 h after impact, and rapidly
thereafter. The rapid increase in POD occurred concomitantly
with increased lignin content and firmness of damaged pericarp
(Ketsa and Atantee 1998). Impact may increase the activity of POD
and simultaneously damage the tonoplast of vacuoles resulting in
leakage of phenolics and contact with POD. The end result may
be synthesis of lignin, and this may form complexes with other
compounds such as carbohydrates, proteins, and pectins resulting
in strong lignin complexes, and in turn increased firmness (Iiyama
and others 1994).
(U.S.A.) in dairy foods (Schrader 2007). Flavor compounds have

numerous 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 secondary
metabolism is likely to move us much more rapidly toward a new
era of flavonoid biochemistry research.
As with any good model, each new piece of information appears to raise a number of unanticipated and intriguing questions.
Major results indicate that no single gene or enzyme can account
for the major events that underlie fruit softening and that many
of the potentially responsible genes and their corresponding cell
wall modifying proteins are members of large gene families that
exhibit overlapping patterns of expression and possibly redundant biochemical action (Bennett and Labavitch 2008). Moreover,
experiments with antisense RNA will facilitate to explicate the
involvement of phenylpropanoid metabolism during fruit development and ripening. At the same time, new techniques are providing the opportunity to consider flavonoid biosynthesis, not
as an assemblage of independent components, but as part of a
large, complex and tightly orchestrated metabolic network. A
great deal of work of functional genomics, coupled with contemporary postharvest approaches, is still needed in elucidating
the mechanisms involved in ripening, which would facilitate a
more systematic understanding of the process. Utilizing information from tomato genomic resources may allow us to develop a
model to predict approaches to modulate various aspects of ripening in a wide range of fleshy fruit-bearing species. Overall, the
information reported in this review demonstrates the potential
of genetic engineering for the improvement of shelf-life, aroma,
and taste properties of horticultural products. However, most if
not all of these are related so far to basic studies at the laboratory
level, they provide only proof of principle that engineering one
of several of these genes could be of practical interest.
Future Prospective and Conclusion

Phenolic compounds, especially phenylpropanoids and
flavonoids, play an important role in plant growth and development as well as in plant interactions with the environment. Overall,
the data reported in this review demonstrate that many genes participating in monolignol biosynthesis have the potential for the
maintenance of firmness and improvement of aroma/organoleptic
properties of fruits. Relationships between components of the
lignin synthesis pathway, lignifications, and development of firmness in various fruits do exist and these strongly suggest that lignification could be a conventional synthetic process in various fruits.
Lignin forms complexes with other compounds such as carbohydrates, proteins, and pectins resulting in strong lignin complexes
(Iiyama and others 1994). The incorporated lignin imparts rigidity
to cell wall, providing a close connection between the carbohydrate matrix and the cellulose polymers. This supports the role
for lignin in helping maintain cell wall structure (Hu and others
1999). An integrated approach on lignin, monolignol precursors,
associated enzymes, and genes could provide a consistent model
of lignification during fruit ripening.
Identification of the biological and pharmacological activities of
flavor compounds has been gathering momentum in recent years.
Today, the total market for flavors and fragrances is estimated at
US$ 18 billion annually, with market shares between the flavor
and fragrance businesses being almost equal (Guentert 2007). The
percentage of natural flavors with respect to all added flavors has
increased 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%
Acknowledgments
The financial assistance from the Dept. of Biotechnology
(DBT), New Delhi, India (in the form of DBT-SRF to Rupinder
Singh), is gratefully acknowledged. We are also thankful to DSTFIST, CSIR (NMITLI), and UP Government (Under Centre of
Excellence in Biochemistry and Biotechnology) for their financial
support in the form of infrastructural facilities.
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Metabolismo de Fenilpropanoides Maduracion

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Phenylpropanoid Metabolism in Ripening Fruits

Rupinder Singh, Smita Rastogi, and Upendra N. Dwivedi

MS 20100054 Submitted 1/16/2010, Accepted 3/7/2010. Authors Singh and

Phenylpropanoid metabolism in ripening fruits . . .

Isoflavonoids & flavonoids

Phenylpropanoid metabolism in ripening fruits . . .

Phenylalanine Cinnamic Acid p-Coumaric Acid Caffeic Acid

components of some plant cell walls, providing additional rigidity

Phenylpropanoid metabolism in ripening fruits . . .

Phenylpropanoid metabolism in ripening fruits . . .

Phenylpropanoid metabolism in ripening fruits . . .

Sparvoli and others

Wang and others

Shan and others

Lo Piero and others

Strissel and others

Cheng and Breen

Jiang and Joyce

Diallinas and Kanellis

Given and others

of stresses, including CO2 treatment (Ke and Salveit 1989) and

PAL-regulated flavonoid/anthocyanin accumulation

Chen and others

Phenylpropanoid metabolism in ripening fruits . . .

regulatory mechanisms (Kumar and Ellis 2001). Although fruits

Promising outlook of PAL

Phenylpropanoid metabolism in ripening fruits . . .

Figure 4Role of OMTs in plant-specialized

Figure 5O-methylation of caffeic acid, catechol,

Phenylpropanoid metabolism in ripening fruits . . .

that increase during fruit ripening.

OMT cDNA clones encoding

OMT cDNA clones encoding

incidentally, both phenylpropanoid and flavonoid compounds

Biological significance of OMTs in volatile generation

Phenylpropanoid metabolism in ripening fruits . . .

HOOC Cinnamic acid

General phenylpropanoid pathway

Flavonols FSLI Dhq

Phenylpropanoid metabolism in ripening fruits . . .

Aroma biosynthesis in strawberry

Figure 7(A) Chemical structures of substrates

S-adenosyl-L- S-adenosyl-Lmethionine homocysteine

S-adenosyl-L- S-adenosyl-Lmethionine homocysteine

Phenylpropanoid metabolism in ripening fruits . . .

Cinnamyl Alcohol Dehydrogenase (CAD)

Molecular characterization of CAD expression

Phenylpropanoid metabolism in ripening fruits . . .

CAD and lignin accumulation

(ferulic, sinapic, caffeic, and 4-coumaric acids) and flavonoids, have

The Ever-Growing Phenylpropanoid Pathway

Phenylpropanoid metabolism in ripening fruits . . .

(Lucheta and others 2007). The flavonoid skeleton, synthesized

Phenylpropanoid metabolism in ripening fruits . . .

(U.S.A.) in dairy foods (Schrader 2007). Flavor compounds have

Future Prospective and Conclusion

Phenylpropanoid metabolism in ripening fruits . . .

Collendavelloo J, Legrand M, Geoffroy P, Barthelemy J, Fritig B. 1981.

Phenylpropanoid metabolism in ripening fruits . . .

O-methyltransferase specific for a C7-C8 propenyl side chain. Plant Physiol

Phenylpropanoid metabolism in ripening fruits . . .

Raab T, Lopez-Raez JA, Klein D, Caballero JL, Moyano E, Schwab W,

Phenylpropanoid metabolism in ripening fruits . . .

F, Tao Q, Si-Ammour A, Harkins T, Lackey A, Perbost C, Taillon B, Stella