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: firstname.lastname@example.org).
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: email@example.com).
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: firstname.lastname@example.org).
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, O-methyltransferase, 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.
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 3-rhamnoside, and myricetin-3-rhamnoside, have been shown to act as feeding deterrents to aphids (Dreyer and Jones 1981).
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) (Rösler 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 (UDP-GT), where these glucoside derivatives are hydrolyzed by coniferin β-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 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.
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 air-treated 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 cell–cell 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 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), O-methyltransferase (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.
Phenylalanine Ammonia Lyase (PAL)
PAL (EC 18.104.22.168), a cytosolic protein in vascular plants, is the initial enzyme in the monolignol biosynthetic pathway that catalyzes the deamination of L-phenylalanine to transcinnamate, a precursor of various phenylpropanoids, such as lignins, coumarins, flavonoids, UV protectants, and furanocoumarin phytoalexins. The reaction is generally considered to represent a key point at which carbon flux into this pathway is controlled (Hanson and Havir 1981; Jones 1984). PAL enzyme is one of the most intensively studied in plant secondary metabolism (Hrazdina 1992; Lewis and others 1999). Phenylpropanoid metabolism is related to the plant defense system and early studies in Phaseolus vulgaris had reported an increase in PAL activity and concentration of total phenols due to the presence of pathogens (Bolwell and others 1985). Treatment of apple with prohexadione–calcium has been reported to result in alterations in the phenylpropanoid biosynthesis pathway that also enhanced disease resistance (Roemmelt and others 2002). Results from the subcellular-localization in grape berry revealed that PAL was present in the cell walls, secondarily thickened walls, and the parenchyma cells of the berry mesocarp cells, whereas 4-coumarate:CoA ligase (4CL) involved in the formation of CoA thioesters of cinnamic acids was present predominantly in the secondarily thickened walls and the parenchyma cells of 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 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-temperature-dependent 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 with an increase in pal and pod gene expression and its respective enzyme activities (Dangcham and others 2008).
Diversity and functional conservation of pal gene
pal gene has been cloned, as well as characterized, from many plant tissues (Boudet 2007) (Table 1). pal appears to exist universally in higher plants as a family of genes and the presence of pal isoforms is a common observation. The significance of this diversity is unclear, but evidence for a degree of metabolic channeling within phenylpropanoid metabolism suggests that partitioning of photosynthate into particular branches of phenylpropanoid metabolism may involve labile multienzyme complexes that include specific isoforms of PAL (Sreelakshmi and Sharma 2008).
Table 1–. Summary of experimental results cited in the literature, which correlate mRNA accumulation and enzymatic activity of phenylalanine ammonia lyase (PAL) with ripening in selected fruits.
In melon fruit, pal was shown to be transcriptionally induced both in response to fruit ripening and wounding (Given and others 1988). Regulation of pal gene expression in this fruit is a coordinated process in response to both ethylene and an ethylene-independent wound signal. pal gene expression followed the expression kinetics similar to that of the ethylene biosynthetic genes during fruit development. In contrast, ethylene biosynthetic genes showed different induction kinetics compared to pal expression in response to wounding (Diallinas and Kanellis 1994). However, activation of PAL has also been observed in response to several types of stresses, including CO2 treatment (Ke and Salveit 1989) and low temperature (Martínez-Téllez 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) (López-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 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.
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-adenosyl-L-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 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.
Classification of plant OMT
OMTs are widespread throughout the plant kingdom and found in all lignin-producing plants. O-methylation of the phenylpropanoid and flavonoid groups of compounds is catalyzed by distinct classes of OMTs.
Caffeic acid 3-O-methyltransferase (COMT, EC 22.214.171.124) catalyzes the O-methylation of aromatic diols and is involved in lignification (Rastogi and Dwivedi 2008) or may have other physiological functions like flavor generation (Collendavelloo and others 1981; Pellegrini and others 1993) (Figure 4). Interestingly gene evolution studies in Clarkia breweri have suggested that isoeugenol OMT (iemt) gene that catalyzes the methylation of eugenol and isoeugenol to form the volatiles methyleugenol and isomethyleugenol had arisen from comt gene. It was suggested that OMT substrate preference could be regulated by a few amino acid residues, and new OMTs with different substrate specificities could evolve from an existing OMT by mutation of several amino acids (Wang and Pichersky 1999). The evolution of new OMTs with new substrate specificities is relatively simple; it is not surprising that OMTs with similar substrate specificities evolved independently in different plant lineages more than once. These broad-specificity enzymes may be recruited for new metabolic pathways, followed by further evolution toward more specific and efficient catalysts. It follows that gene duplication (or even polyploidy) is an important factor in this concept as it provides the raw material for the acquisition of new biosynthetic pathways. It is somewhat surprising that developing strawberry fruits display high OMT activity levels toward caffeic acid, protocatechuic aldehyde, and catechol resulting in ferulic acid, guaiacol, and vanillin (Figure 5A to 5C), respectively.
CCoAOMT (EC 126.96.36.199), which catalyzes the meta-O-methylation 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 1995) and this methylation step is highly regulated (Hahlbrock and Scheel 1989).
Proposed methyl acceptor classes and groups
The molecular and biochemical data available so far provide the basis for a meaningful classification of the plant omt gene superfamily. There exist some 36 omt cDNA clones, which are subdivided into 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 is primarily comprised of simple phenols and anthocyanins, whereas Group 5 encompasses polyketides and other acetate/malonate-derived compounds (Table 2). Each group of OMT is further classified into Class “A” and Class “B.” Based on the class of substrate methylation, Class “A” OMTs methylate phenylpropanoid compounds, whereas Class “B” OMTs methylate flavonoid compounds. Although the chemical mechanisms of methyl transfer reactions are identical, OMTs differ in their selectivity with respect to the stereochemistry of the methyl acceptor molecules, as well as the substitution pattern of their phenolic hydroxyl groups (Ibrahim and others 1998). Some of the known OMTs display strict specificities toward their acceptor substrate as well as to the position of substrate methylation. In contrast, other OMTs, especially those catalyzing the methylation of catechol (O-dihydroxy) moiety substrates, exhibit surprisingly broad substrate specificities. OMTs have been shown to be multifunctional enzymes that could also catalyze transformations in 2 different biosynthetic pathways such as the alkaloid and phenylpropanoid pathways (Li and others 1997) or aroma biosynthesis (Lavid and others 2002) and, 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.
Table 2–. Classification of omt cDNA clones encoding the methylation of phenylpropanoid compounds (Class “A”) and omt cDNA clones encoding the methylation of flavonoid compounds (Class “B”).
OMT cDNA clones encoding methylation of phenylpropanoid compounds Class “A”
OMT cDNA clones encoding methylation of flavonoid compounds Class “B”
Flavans and anthocyanins (although none has been reported)
Polyketides and other acetate/malonate derived compounds
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 SAM–OMTs that utilize SAM and a variety of substrates have been described in higher plants. A comparison of the amino acid sequence of 56 SAM–OMTs 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 parallel with other reactions involving the methylation of caffeic acid, catechol, anthocyanidin, or other as yet unidentified O-diphenols that increase during fruit ripening.
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 , pal , f5h , c4h , and c3h , 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 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-β-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, 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-dimethyl-3(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 kg−1 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-β-d-glucose and caffeoyl 1-O-β-d-glucose, suggesting that it was also involved in the methylation of the caffeoyl group (Lunkenbein and others 2006) (Figure 7B). 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 ethylene-regulated 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.
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%) receptacle-related 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 O’Connell 2002). Similar results were earlier reported by quantitative real time PCR (QRT-PCR) 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 (Perkins-Veazie 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 188.8.131.52) 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 184.108.40.206), 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 220.127.116.11), 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 lignification-specific 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 (CHS, EC 18.104.22.168) 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.
Peroxidase (POD, EC 22.214.171.124) 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).
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% (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.
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 DST-FIST, CSIR (NMITLI), and UP Government (Under Centre of Excellence in Biochemistry and Biotechnology) for their financial support in the form of infrastructural facilities.