Critical Reviews in Food Science and Nutrition, 47:119 (2007) Copyright C Taylor and Francis Group, LLC ISSN:

1040-8398 DOI: 10.1080/10408390600976841

Fruit Ripening Phenomena An Overview

V. PRASANNA, T. N. PRABHA, and R. N. THARANATHAN
Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore 570020, Karnataka, India

Fruits constitute a commercially important and nutritionally indispensable food commodity. Being a part of a balanced diet, fruits play a vital role in human nutrition by supplying the necessary growth regulating factors essential for maintaining normal health. Fruits are widely distributed in nature. One of the limiting factors that inuence their economic value is the relatively short ripening period and reduced post-harvest life. Fruit ripening is a highly coordinated, genetically programmed, and an irreversible phenomenon involving a series of physiological, biochemical, and organoleptic changes, that nally leads to the development of a soft edible ripe fruit with desirable quality attributes. Excessive textural softening during ripening leads to adverse effects/spoilage upon storage. Carbohydrates play a major role in the ripening process, by way of depolymerization leading to decreased molecular size with concomitant increase in the levels of ripening inducing specic enzymes, whose target differ from fruit to fruit. The major classes of cell wall polysaccharides that undergo modications during ripening are starch, pectins, cellulose, and hemicelluloses. Pectins are the common and major components of primary cell wall and middle lamella, contributing to the texture and quality of fruits. Their degradation during ripening seems to be responsible for tissue softening of a number of fruits. Structurally pectins are a diverse group of heteropolysaccharides containing partially methylated D-galacturonic acid residues with side chain appendages of several neutral polysaccharides. The degree of polymerization/esterication and the proportion of neutral sugar residues/side chains are the principal factors contributing to their (micro-) heterogeneity. Pectin degrading enzymes such as polygalacturonase, pectin methyl esterase, lyase, and rhamnogalacturonase are the most implicated in fruit-tissue softening. Recent advances in molecular biology have provided a better understanding of the biochemistry of fruit ripening as well as providing a hand for genetic manipulation of the entire ripening process. It is desirable that signicant breakthroughs in such related areas will come forth in the near future, leading to considerable societal benets. Keywords fruit ripening, cell wall polysaccharides, pectin, pectic enzymes, polygalacturonase

INTRODUCTION Fruits constitute a commercially important and nutritionally indispensable food commodity. They are edible seed vessels or receptacles developed from a mature, fertilized ovary. They are highly specialized organs in higher plants offering a great variety of aesthetic qualities with their complex/delicate aroma, pleasant taste, exotic colors, succulence, avor, and texture. They play a vital role in human nutrition, by supplying the necessary growth factors essential for maintaining normal health. Nutritionally, they are known for their high energy, roughage value, minerals, vitamins (B-complex, C and K in some instances), carotene (pro-vitamin A), and phenolics (antioxidants). Fruits are widely distributed in nature and depending upon their dis-

Address correspondence to R. N. Tharanathan, Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore, Karnataka 570020, India. E-mail: tharanathan@yahoo.co.uk

tribution, they are classied into tropical, subtropical, and temperate fruits [Table 1]. Fruits are harvested at complete maturity. They are selfsufcient with their own catalytic machinery to maintain an independent life, even when detached from the parent plant. Based on their respiratory pattern and ethylene biosynthesis during ripening, harvested fruits can be further classied as climacteric and non-climacteric type [Table 2]. Climacteric fruits, harvested at full maturity, can be ripened off the parent plant. The respiration rate and ethylene formation though minimal at maturity, raise dramatically to a climacteric peak, at the onset of ripening, after which it declines (Gamage and Rehman, 1999). Non-climacteric fruits are not capable of continuing their ripening process, once they are detached from the parent plant. Also, these fruits produce a very small quantity of endogenous ethylene, and do not respond to external ethylene treatment. Non-climacteric fruits show comparatively low prole and a gradual decline in their respiration pattern and ethylene production, throughout the ripening process (Gamage and Rehman, 1999). 1

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Table 1 Temperate Apple Apricot Cherry Grapes Kiwi fruit Peach Pear Plums Strawberry Melon Classication of fruits based on distribution Sub-tropical Avocado Lime Litchi Mandarin Olive Orange Passion fruit Persimmon Pomegranate Tropical Annona Banana Guava Jackfruit Mango Papaya Pineapple Sapota

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FRUIT RIPENING Fruit ripening is a highly co-ordinated, genetically programmed, and an irreversible phenomenon involving a series of physiological, biochemical, and organoleptic changes that lead to the development of a soft and edible ripe fruit with desirable quality attributes. A wide spectrum of biochemical changes such as increased respiration, chlorophyll degradation, biosynthesis of carotenoids, anthocyanins, essential oils, and avor and aroma components, increased activity of cell wall-degrading enzymes, and a transient increase in ethylene production are some of the major changes involved during fruit ripening (Brady, 1987) The color change during fruit ripening is due to the unmasking of previously present pigments by degradation of chlorophyll and dismantling of the photosynthetic apparatus and synthesis of different types of anthocyanins and their accumulation in vacuoles, and accumulation of carotenoids such as -carotene, xanthophyll esters, xanthophylls, and lycopene (Tucker and Grierson, 1987; Lizada, 1993). The increase in avor and aroma during fruit ripening is attributed to the production of a complex mixture of volatile compounds such as ocimene and myrcene (Lizada, 1993), and degradation of bitter principles, avanoids, tannins, and related compounds (Tucker and Grierson, 1987). The taste development is due to a general increase in sweetness, which is the result of increased gluconeogenesis, hydrolysis of polysaccharides, especially starch, decreased acidTable 2 Classication of fruits Non-Climacteric fruits Cherry Cucumber Grape Grapefruit Lemon Lime Litchi Mandarin Melon Orange Pineapple Pomegranate Raspberry Strawberry

ity, and accumulation of sugars and organic acids resulting in an excellent sugar/acid blend (Lizada, 1993; Grierson, Tucker, and Robertson, 1981; Selvaraj, Kumar, and Pal, 1989). The metabolic changes during fruit ripening include increase in biosynthesis and evolution of the ripening hormone, ethylene (Yang and Hoffman, 1984), increase in respiration mediated by mitochondrial enzymes, especially oxidases and de novo synthesis of enzymes catalyzing ripening specic changes (Tucker and Grierson, 1987). Alteration of cell structure involves changes in cell wall thickness, permeability of plasma membrane, hydration of cell wall, decrease in the structural integrity, and increase in intracellular spaces (Tucker and Grierson, 1987; Redgwell, MacRae, Hallet, Fischer, Perry, and Harker, 1997). The major textural changes resulting in the softening of fruit are due to enzyme-mediated alterations in the structure and composition of cell wall, partial or complete solubilization of cell wall polysaccharides such as pectins and cellulose (Tucker and Grierson, 1987), and hydrolysis of starch and other storage polysaccharides (Selvaraj et al., 1989; Fuchs, Pesis and Zauberman, 1980). The changes in gene expression during ripening involves the appearance of new ripening- specic mRNAs, tRNA, rRNA, poly A+ RNA, and proteins, and the disappearance of some mRNAs (Tucker and Grierson, 1987; Grierson, Slater, Spiers, and Tucker, 1985; Wong, 1995). However, some mRNAs are found to remain constant throughout the ripening process (Gomez-Lim, 1997). These changes during fruit ripening are activated by plant hormones.

ROLE OF FRUIT RIPENING HORMONE Ethylene, a fruit ripening phytohormone, in minute amounts can trigger many events of cell metabolism including initiation of ripening and senescence, particularly in a climacteric fruit. Ethylene, which is synthesized autocatalytically at levels as low as 0.01 l L1 and 0.05 l L1 triggers the ripening process in mango and banana, respectively (Johnson, Sharp, Milne and Oosthuyse, 1997). A number of reviews have been published on the role of ethylene in fruit ripening, particularly in mangoes as well as its biogenesis (Adams and Yang, 1979; Kende, 1993). Fruits treated with exo-polygalacturonase or other cell wall hydrolases or their products have been shown to elicit ethylene production (Baldwin and Pressey, 1990; Kim, Gross and Solomos, 1987). This response is not fruit specic (Kim et al., 1987). In cultured pear cells it was shown that the pectic oligomers might also induce and regulate ethylene biosynthesis (Campbell and Labavitch, 1991). The pathway for ethylene biosynthesis has been elucidated in apple, and other fruits such as avocado, banana, and tomato (Kende, 1993; Yang and Hoffman, 1984). The rst step is the conversion of S-adenosylmethionine (SAM) to 1aminocyclopropane carboxylic acid (ACC) by the enzyme ACC synthase (Fig. 1). At the onset of fruit ripening, expression of multiple ACC synthase genes are activated, resulting in increased production of ACC. In most cases, it is the ACC synthase

Climacteric fruits Apple Apricot Banana Guava Kiwifruit Mango Papaya Passion fruit Peach Pear Persimmon Plum Sapodilla Tomato

FRUIT RIPENING PHENOMENON

Figure 1

Pathway for ethylene biosynthesis and metabolism.

activity, which determines the rate of ethylene biosynthesis. ACC is then oxidized to ethylene by ACC oxidase. Inhibition of ethylene biosynthesis by antisense RNA for ACC synthase (Oeller, Min-Wong, Taylor, Pike, and Theologis, 1991) and ACC oxidase (Hamilton, Lycett and Grierson, 1990) was demonstrated rst in the tomato fruit. Deamination of ACC to -ketobutyrate by over-expressing ACC deaminase enzyme also inhibited ethylene formation and fruit ripening (Klee, Hayford, Kretzmer, Barry, and Kishore, 1991). The resultant transgenic fruit did not overripe as normal controls, though some color change occurred and a mere ethylene boost triggered back all the ripening related biochemical changes in a similar way as in normal fruit (Hamilton et al., 1990; Oeller et al., 1991). Recently the cDNA encoding for ACC oxidase has been isolated and characterized from mango (Zainal, Tucker, and Lycett, 1999). Down regulation of ACC synthase and ACC oxidase genes in mango are now being used for extending the shelf life of this fruit.

Figure 2 An overview of fruit ripening with particular emphasis on textural softening. Control points at ethylene (1) and post-ethylene (2) levels.

TEXTURAL SOFTENING DURING RIPENING Fruit ripening is associated with textural alterations, which are dramatic in climacteric fruits. Textural change is the major event in fruit softening, and is the integral part of ripening, which is the result of enzymatic degradation of structural as well as storage polysaccharides (Tucker and Grierson, 1987, Grierson et al., 1981, Bartley and Knee, 1982; Hulme, 1971). Depending upon their inherent composition and nature, different fruits soften at different rates and to varying degrees (Tucker and Grierson, 1987). Fruits such as mango, papaya, avocado, sapota, and banana undergo drastic and extensive textural softening from stone hard stage to a soft pulpy stage, whereas apple and citrus fruits do not exhibit such a drastic softening, though they undergo textural modications during ripening. An overview of fruit ripening with special reference to textural softening has been diagrammatically represented in Fig. 2. Fruit texture is inuenced by various factors like structural integrity of the primary cell wall and the middle lamella, accumulation of storage polysaccharides, and the turgor pressure generated within cells by osmosis (Jackman and Stanley, 1995). Change in turgor pressure, and degradation of cell wall polysaccharides and starch determine the extent

of fruit softening (Brady, 1987; Tucker and Grierson, 1987; Grierson et al., 1981). In citrus fruit, softening is mainly associated with change in turgor pressure, a process associated with the post harvest dehydration and/or loss of dry matter. Starch is the bulk polysaccharide present in some fruits (mango and banana), and its enzymatic hydrolysis results in pronounced loosening of cell structure and sweetness development. The major classes of cell wall polysaccharides that undergo modications during ripening are pectins, cellulose, and hemicelluloses. In fruits, which are known for excessive softening, the cell walls are thoroughly modied by solubilization, deesterication, and depolymerization, accompanied by an extensive loss of neutral sugars and galacturonic acid, followed by solubilization of the remaining sugar residues and oligosaccharides (Voragen, Pilnik, Thibault, Axelos and Renard, 1995). The process of textural softening is of commercial importance as it directly dictates fruit shelf life and quality (Tucker, 1993). This should be considered to avoid mechanical damage during harvesting and transportation. The textural properties of fruits in general play a very signicant role in the consumer acceptability. The increased interest in controlling the textural qualities of fruit stimulated further research on the biochemistry of the cell wall, with particular reference to cell wall polysaccharides and their degradation (Jackman and Stanley, 1995; Van Buren, 1979). The textural qualities of fruits are attributed to its inherent composition, particularly the cell wall composition. Figure 3 shows the schematic representation of the levels of structure that contribute to fruit texture. The textural characteristics are attributed to the mechanical properties of the nal organ, which in turn depends on contributions and interactions of different levels of structure (Waldron, Smith, Parr, Ng, and Parker, 1997). Attempt to understand the molecular mechanism of fruit softening has led to the investigation of cell wall polymers, their compositional changes and the related cell-wall degrading enzymes during ripening (Knee, 1978).

V. PRASANNA ET AL.

et al., 1999). Whitaker (1984) reported the cell wall composition and percent pectin present in some ripe fruits, such as pear, tomato, apple, and date. Neutral sugar composition of fruit cell wall varies from fruit to fruit, and marked changes in their composition occur during ripening (Gross and Sams, 1984). Most of these changes are attributed to the action of cell wall (carbohydrate) hydrolases.

Pectic Polysaccharides and Fruit Softening

Figure 3 Schematic representation of the levels of structure that contribute to the fruit texture.

PLANT CELL WALL AND ITS COMPONENTS Plant Cell Wall Polysaccharides Plant cell wall polysaccharides, such as pectins, celluloses, hemicelluloses; reserve polysaccharides like starch and galactomannans; gel formers such as gums and mucilages; and physiological information carriers like antigens, in general, are an extremely diverse set of biopolymers, which play a very important role as structural elements. Fruit polysaccharides, upon their degradation, play a crucial role in textural softening during ripening. Polysaccharides from different sources vary in their chemical-biological, physico-chemical, and structuralfunctional characteristics (Tharanathan, Muralikrishna, Salimath, and Raghavendra Rao, 1987). Plant polysaccharides play a major role in storage, mobilization of energy and in maintaining cell and tissue integrity due to their structural and water binding capacity. Cell wall polysaccharides differ widely in their physical / nutritional properties and have the greatest potential for structural diversity (Aman and Westerlund, 1996). They regulate the utilization of other dietary components in the food. Recently plant polysaccharides have emerged as important, bioactive, natural products exhibiting a number of biological properties. They are capable of regulating gene expression and host-defense mechanism by the generation of elicitor-active oligogalacturonide fragments from the cell wall (Ridley, ONeill, and Mohnen, 2001). Cell wall is an active organelle, vital to cell growth, metabolism, transport, attachment, shape, cell resistance, and strength. The old notion of the cell wall being static, inert, and a mere load-bearing structure has changed to the newer concept of the dynamic nature of the cell wall (Jackman and Stanley, 1995). Fruit pulp or the mesocarp is the edible part of the fruit, and is composed of thin-walled storage parenchymatous cells (50 500 m). These cells are characterized by a prominent cell wall consisting of complex network of polysaccharides and proteins, which gives mechanical strength to the tissues. The primary cell wall contains 35% pectin, 25% cellulose, 20% hemicellulose and 10% structural, hydroxyproline-rich protein (Brownleader

Pectins are the common components of the primary cell wall and middle lamella contributing to the fruit texture. Pectin content varies from fruit to fruit and pectins from fruits are used for commercial purposes, eg. apple, guava and citrus [Table 3] Whitaker, 1984; El-Zoghbi, 1994; Nwanekezi, Alawube, and Mkpolulu, 1994; Thakur, Singh and Handa, 1997). The word Pectin originated from the Greek word Pectos meaning, gelled. Native pectin plays an important role in the consistency of fruit and also in textural changes during ripening, storage, cooking, or irradiation and other processing operations. Tissue softening is attributed to enzymatic degradation and solubilization of the protopectin (Sakai, Sakomoto, Hallaert, and Vandamme, 1993). Pectins are likely to be the key substances involved in the mechanical strength of the primary cell wall and are important to the physical structure of the plant (Sirisomboon, Tanaka, Fujitha, and Kojima, 2000). Their degradation during ripening seems to be responsible for tissue softening, as reported for a number of fruits including tomato (Poovaiah and Nukuya, 1979; Seymour, Harding, Taylor, Hobson, and Tucker, 1987), kiwi (Redgwell, Melton, and Brasch, 1992), apple (De Vries, Voragen, Rombouts, and Pilnik, 1984),
Table 3 Fruits African Mango Apple Apple Avocado Banana Cashew Cherries Guava Lemon Lemon Litchi Mango Orange Orange Papaya Passion fruit Passion fruit Peach Pineapple Strawberry Tomato Pectin content of some fruit tissues Tissue Pulp Pulp Pomace Pulp Pulp Pomace Pulp Pulp Pulp Peel Pulp Pulp Pulp Peel Pericarp Pulp Rind Pulp Pulp Pulp Pulp % Pectin content (fresh weight basis ) 0.72 0.51.6 1.52.5 0.73 0.71.2 1.28 0.240.54 0.261.2 2.54.0 5.0 0.42 0.661.5 1.35 3.55.5 0.661.0 0.5 2.1 3.0 0.10.9 0.040.13 0.14 0.44 0.20.6

FRUIT RIPENING PHENOMENON

and bush butter (Missang, Renard, Baron, and Drilleau, 2001a). The major changes in the cell wall structure are the dissolution of middle lamella and primary cell wall during ripening. Thus, elucidation of the chemical structure of pectin is essential in understanding its role in plant growth/development and during ripening of fruits (Thakur et al., 1997). Parenchymatous tissues are thought to consist principally of calcium salts of pectic substances, which are deposited in early stages of the cell growth, specically when the area of the cell wall is increasing (Voragen et al., 1995). Pectic substances are prominent structural constituents of primary cell wall and middle lamella and are the sole polysaccharides in middle lamella, along with some cellulose microbrils, while they may be virtually absent in secondary walls (Van Buren, 1979). Middle lamella are heat labile and their dissolution results in the separation of plant cells. Ultrastructural studies in ripening fruits have also shown that cell wall breakdown was accompanied by dissolution of middle lamella and gradual dissolution of brillar network of primary cell wall (Ben-Arie, Sonego, and Frankel, 1979; Crookes and Grierson, 1983; Jackman and Stanley, 1995). Deesteried pectins in the middle lamella are associated with calcium ions, and its removal also usually leads to cell separation (Aman and Westerlund, 1996). The association involves binding of two or more polymeric chains, in the form of a corrugated egg-box (Fig. 4), with interstices in which calcium ions are packed and coordinated, creating an egg-box system (Grant, Morris, Rees, Smith, and Tom, 1973). Specic binding of the divalent cations to pectins in an Egg box model leads to a rm cohesion between the chains (Grant et al., 1973). Calcium treatment inhibited softening of fruits due to an increase in cohesion of pectin network (Krall and McFeeters, 1998). Generally, pectins in the cell wall are cross-linked through ionic interaction (Mac Dougall, Brett, Morris, Rigby, Ridout and Ring, 2001). Due to this ability to form co-ordination complexes with Ca2+ , chelator soluble pectins are of special interest as they increase fruit rmness (Jimenez, Rodriguez, Fernandez-Caro, Guillen, Fernandez-Bolanos, and Heredia, 2001). Pectins are structural, acidic, homo-/heteropolysaccharides obtained commercially from fruits but present universally in plant cell wall matrices. They are structurally diverse heteropolysaccharides containing partially methylated galacturonic acid residues, methyl esteried pectins, deesteried pectic acids and their salts, pectates [Table 4] and the neutral polysaccharides, which lack galacturonan backbone, i.e., arabinogalactans, arabinans, and galactans (Fig. 5). Several neutral plant polysaccharides are also grouped under pectins mainly because of their association with acidic pectins as side chains to the main galac-

Table 4

Chemical nature of pectic substances present in plant cell walls Chemical nature Group of colloidal, complex polysaccharides of GalA Water-insoluble parent pectic substances Pectic substances free from methyl ester groups Normal or acid salts of pectic acids Pectic substances partially esteried with methyl groups Highly esteried (>50% esteried) pectinic acids Less esteried (<50% esteried) pectinic acids Normal or acid salts of pectinic acids

Pectic substances Pectic substances Protopectin Pectic acids Pectates Pectinic acids High methoxyl pectins Low methoxyl pectins Pectinates

Figure 4

Egg-Box model depicting association of pectins with Ca++ ions.

turonan backbone. They may also be present as free polymers (Brownleader et al., 1999). The pectin chain, -D-galacturonans, i.e., -D-galacturanoglycans or poly(-D-galactopyranosyluronic acid), consists largely of D-galacturonic acid linked by (1 4) linkages (BeMiller, 1986). The carboxyl groups of pectin are partially esteried with methanol and the hydroxyl groups are partially acetylated with acetic acid (Pilnik and Voragen, 1970). They occur mainly in chair L-form and as both C-1and C-4 hydroxyl groups are on the axial position, the polymer formed is a trans 1,4-polygalacturonan (Sakai et al., 1993). During ripening, softening of fruit is caused by the conversion of protopectin, the insoluble, high molecular weight parent pectin into soluble polyuronides (John and Dey, 1986). This tightly bound protopectin is degraded into soluble pectins, which are found loosely bound to the cell walls. This phenomenon is attributed to textural softening during ripening (Doreyappa Gowda, and Huddar, 2001). Protopectin increases before physiological maturity, but decreases during mango fruit ripening (Tandon and Kalra, 1984). The inter-relation between different pectic substances and their degradation is shown in Fig. 6. The degree of polymerization, degree of esterication, and the proportion of neutral sugar side chains are the principal factors contributing to the heterogeneity of pectic polysaccharides (Rexova-Benkova and Markovic, 1976). Pectins, like other polysaccharides, are both polydisperse and polymolecular, mainly due to their heterogeneous nature in both molecular weight and chemical structure (Bartley and Knee, 1982; BeMiller, 1986). Three types of pectic polysaccharides have been structurally characterized. Homogalacturonans (HG) consist solely of linear chain of 1 4 linked -D-galacturonans (see Fig. 5), in which some of the carboxylic groups are methyl esteried. They are found to be 100 nm in length. It is a polysaccharide isolated from only a few plant sources such as sunower heads and seeds, sisal, the bark of amabilis r, jack fruit and apple fruit (Pilnik and Voragen, 1970). It has also been isolated from the cell wall of rice endosperm, primary cell wall of Rosa, sycamore (McNeil, Darvill, Fry, and Albersheim, 1984; Voragen et al., 1995), and

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Figure 5

Structure of pectic substances.

Figure 6

Inter-relationship of pectic substances.

recently from citrus (Zhan, Janssen, and Mort, 1998). However, it has been viewed that the homogalacturonan might be released from the heterogeneous pectic substances by the conditions employed during extraction (Voragen et al., 1995). Rhamnogalacturonan-I (RG-I) is primarily responsible for the chemical and structural diversity of the pectins. It is the major component of the primary cell wall and middle lamella of dicotyledonous plants (McNeil, Darvill, and Albersheim, 1980). They consist mainly of the backbone of the repeating disaccharide units 4)--D-GalA-(1 2)--L-Rha-(1 (BeMiller, 1986). Insertion of rhamnose in the main chain forms a T shaped kink in the polygalacturonan chain (Fig. 7), which minimizes the frequency of interaction with adjacent polymeric chains (Grant et al., 1973). Galacturonic acid residues typically

FRUIT RIPENING PHENOMENON

Figure 7

T-shaped kinking of the pectin molecule.

are not substituted with mono- or oligosaccharides side chains, but a single glucuronic acid substitution on C-3 position of galacturonic acid was reported in sugar beet pectins (Renard, Crepeau, and Thibault, 1999). Rhamnose residues are found as branch points for the attachment of neutral sugar side chains (Mac Dougall et al., 2001; McNeil et al., 1980). Almost 50% of the 1 2 linked rhamnose residues are branched at O-4 with side chains consisting of D-galactose and/or L-arabinose residues. Small amounts of fucose, glucuronic acid, and 4-Omethyl -D-glucuronic acid units are also found linked to rhamnose units (McNeil et al., 1980, ONeill, Albersheim and Darvill, 1990). RG-I was reported from a number of fruits including tomato (Seymour, Colquhoun, Dupont, Parsley, and Selvendran, 1990), grape (Nunan, Sims, Bacic, Robinson and Fincher, 1998; Saulnier, Brillouet, and Joseleau, 1988), apple (Schols, Posthumus and Voragen, 1990), pear (Schols and Voragen, 1994), kiwi (Redgwell et al., 1992), and raspberry (Stewart, Iannetta, and Davies, 2001), although the nature and length of the neutral sugar side chain varied. Rhamnogalacturonan-II (RG-II) is invariably present as a minor component of the cell wall, and has extremely complex structures. It is not structurally related to RG-I, since it contains a high proportion of rhamnosyl residues, which occur as terminal (1 3) as well as branched (1 2, 3, 4,) units (Voragen et al., 1995). RG-II is a polysaccharide containing a homogalacturonan backbone composed of at least eight 1 4 linked -D-galacturonic acid residues having side chains mainly composed of twelve glycosyl residues including several rare diagnostic monosaccharides such as apiose, 2-O-methyl-L-fucose, 2-O-methyl--D-xylose, aceric acid, KDO (2-keto-3deoxy-D-manno-octulosonic acid), and Dha (3-deoxy-D-lyxoheptulosaric acid) (Vidal, Williams, ONeill and Pellerin, 2001). Recently, it was shown that RG-II is present predominately as a dimer (ONeill et al., 1996). These dimers are found cross-linked by borate-diol esters, through apiosyl residues and play an im-

portant role in the structure and function of pectins (ONeill et al., 1996). RG-II has been isolated from primary cell walls of tomato (ONeill et al., 1990), apple (ONeill et al., 1990) and kiwi fruit ( Redgwell, Melton and Brasch, 1990). High amounts of RG-II are present in fruit juices (Doco, Williams, Vidal and Pellerin, 1997). RG-II binds heavy metals and has immuno-modulating activities, which has stimulated further research on structure of RG-II and its role in human nutrition and health (Ridley et al., 2001). Substituted galacturonans are a diverse group of pectic polysaccharides that contain a backbone of linear 1,4-linked -D-galacturonic acid residues, substituted with other sugar residues. Xylogalacturonans, in which -D-xylose residues are attached to C-3 of the galacturonan backbone, are found in apple pectin (Schols, Bakx, Schipper, and Voragen, 1995). Regarding the neutral sugar side chains, considerable variations were found in the nature, type, length, and structure of the side chains attached to the rhamnosyl residues of rhamnogalacturonans (see Fig. 5). Usually, the ratio of rhamnose to galacturonic acid is 1 : 40, as reported for citrus pectin (Zhan et al., 1998). Side chains composed of D-galactose and L-arabinose occur most frequently, while D-xylose, D-glucose, D-mannose, D-apiose, and L-fucose occur rarely in plant pectins (Darvill, McNeil, and Albersheim, 1978). These side chains are distributed discontinuously rather than continuously in pectins. The branching occurs at C-2 (Ovodov, Ovodova, Bondarenko, and Krasikova, 1971) or C-3 (De-Vries, den Ujil, Voragen, Rombouts, and Pilnik, 1983) of galacturonic acid or through C-4 (Stevens and Selvendran, 1984) or C-3 (Darvill et al., 1978) of rhamnose. Arabinose and galactose constitute oligo/polysaccharide units substituting the hydroxyl groups of rhamnose. The presence of galacturonans rich in xylose has also been reported in apple (Schols et al., 1990; De Vries et al., 1983). The proportion of branched rhamnose residues varies with fruits, 2040% in grapes, tomato, and kiwi fruit, while it varies from

V. PRASANNA ET AL.

25100% in apple (Voragen et al., 1995). RGs branched with several neutral polymers such as arabinans, galactans, and arabinogalactans were reported for some pectins (Nunan et al., 1998; Saulnier et al., 1988; Schols et al., 1990; De Vries et al., 1983; Oesterveld, Beldman, Schols, and Voragen, 2000; Strasser and Amado, 2001). Arabinans are branched polysaccharide chains composed of -(1 5) linked L-arabinose residues that contain single (terminal) L-arabinose side chains, linked to O-3 or O-2 position of the main chain (see Fig. 5) (Whitaker, 1984; Voragen et al., 1995). They resemble a comb-like structure. Arabinan associated pectins have been isolated from apple and has been recently characterized from sugar beet pulp (Oesterveld et al., 2000). Galactans are linear chains of -(1 4) linked D-galactose residues (see Fig. 5). They occur as oligosaccharide chains attached to the rhamnose residues of the RG backbone (McNeil et al., 1980). Arabinogalactans (AG) are heteropolymers of D-galactose and L-arabinose residues (see Fig. 5). Two structurally different forms of arabinogalactans are found in plants. AG-I is a simple polysaccharide composed of chains of -(1 4) Dgalactose with single L-arabinose residues linked to O-3 of the galactose (Whitaker, 1984; Smith, 1999). They have been isolated from different fruits including apple (Ovodov et al., 1971), kiwi (Redgwell et al., 1990), tomato (Seymour et al., 1990) and pineapple (Smith and Harris, 1995). AG-II is a complex and branched polysaccharide, consisting of -(1 3) D-galactose linked to -(1 6) D-galactose at O-6. The O-3 and O-6 positions of the side chains are in turn linked to terminal L-arabinose residues (Whitaker, 1984; Strasser and Amado, 2001). They possess freeze-inhibition, water holding, and adhesive properties. Plant arabinogalactans are known for their multifaceted physiological and functional characteristics. The pectic polymers of the primary cell wall have a relatively higher proportion of neutral oligosaccharide chains on their backbone (i.e., highly substituted pectins) and these side chains are much longer than those of the pectins of middle lamella (Sakai et al., 1993; Selvendran, 1985). The side chains are not distributed regularly but are concentrated in some regions called hairy regions. Highly esteried and slightly branched rhamnogalacturonan, the smooth regions, are present in middle lamella, whereas highly branched rhamnogalacturonan, the hairy regions are present in primary cell wall (Selvendran, 1985). In a plant cell wall, the side chains of the pectin molecules link to protein, hemicellulose, and cellulose. The acidic and neutral pectins carry non-sugar substituents, essentially methanol, acetic acid, phenolic acids, and amide groups, and contribute further to the structural diversity of pectins (Mac Dougall et al., 2001). The esterication of galacturonic acid carboxyl with methanol or acetic acid is a very important structural characteristic of pectins. The degree of methylation (DM) is dened as the percentage of carboxylic acid groups esteried with methanol. The degree of acetylation (DAc) is dened as the percentage of galacturonic acid residues esteried with one acetyl group (Voragen et al., 1995). Chelator-soluble

pectins have high DM and DAc than those extracted with alkali, which is mainly due to the liberation by saponication of methyl ester and acetyl groups by alkali (Thomas and Thibault, 2002). Phenolic acids, especially ferulic and p-coumaric acids are found esteried to the non-reducing ends of the neutral arabinose / galactose residues. These non-sugar substituents, especially the ferulic acid facilitate oxidative cross-linking between pectins or with other polysaccharides in the cell walls, by the formation of diferuloyl bridges, which would limit wall extensibility (Brownleader et al., 1999) and play a signicant role in growth regulation and defense mechanism. Pectins are extracted from plant material using a wide variety of extracting media. Some pectins are soluble in water indicating little or no binding to the other cell wall components (Fry, 1986). It is assumed that pectins are held together by calcium bridges. This forms the basis for the wide use of chelating agents such as oxalates, hexametaphosphate, EDTA, CDTA, EGTA, etc. for extracting pectins. Chelating treatment is often combined with heating, and this treatment does not give a real proof for the presence of Ca-bridges, as heating cleaves pectic backbone irrespective of pH (Fry, 1986). At cold condition and neutral pH, CDTA removes all the Ca-bridges from the pectins, rendering its solubilization. Pectins are abundantly found in fruits, moderately in leafy vegetables and in low levels in cultured tissues (Fry, 1986) and originate from the middle lamella (Thomas and Thibault, 2002). These pectins are found complexed with calcium ions (Thomas and Thibault, 2002). Pectins extracted with HCl (pH 1.5), had a wider molecular weight range with a peak molecular weight slightly lower than that extracted with 0.5% EDTA or 0.25% ammonium oxalate. This suggests that acid might hydrolyse pectins during extraction and EDTA or ammonium oxalate may be preferred for pectin extraction (Pathak, Chang, and Brown, 1988). Cold sodium carbonate (containing sodium borohydride) treatment would cause hydrolysis of inter polymeric ester bonds with negligible -elimination degradation (Thomas and Thibault, 2002). It solubilizes the CDTA-insoluble pectins and suggests that inter polymeric ester bonds help to hold pectins in the cell wall (Fry, 1986). A simple model consisting of ve different types of pectin was proposed based on their molecular interactions with other cell wall constituents and extraction behavior (Chang, Tsai, and Chang, 1993). The ve different types of pectins present in plant tissues are S-, A-, B-, C-, P-types [Table 5].
Table 5 Different types of pectins based on molecular interaction and extractability Types S-type pectins A-type pectins B-type pectins C-type pectins P-type pectins Bonding (molecular interaction) weak bonds/Van der waals force ionic interactions intensive hydrogen bonding hydrogen bonds and ionic interactions covalent bonds Extractions with cold water with cold chelating agents with hot water with hot chelating agents with dilute acid /alkali

FRUIT RIPENING PHENOMENON

Pectins form gels under certain conditions and this property has made them as useful additive in jams, jellies, and marmalades, as well as in confectionery industries as stabilizers for acid milk products (Voragen, 1995). They are used in a number of foods as thickeners, texturizers, emulsiers, etc. (Thakur et al., 1997). In recent years, pectin has been used as a fat or sugar replacer in low-calorie foods (Thakur et al., 1997). They have a wide application in pharmaceutical industries, mainly because of their activities like antidiarrhea, antibacterial, antiviral, wound-healing, detoxicant, regulation, and protection of gastrointestinal tract, delay in gastric emptying, lowering blood cholesterol level, and glucose metabolism (Voragen et al., 1995; Thakur et al., 1997; Baker, 1997). Also, it is the major constituent in the fruit cell wall that undergoes drastic degradation by the carbohydrate hydrolases, during ripening, leading to fruit softening.

and other products (Grierson et al., 1981; John and Dey, 1986; Sakai et al., 1993). Solubilization followed by depolymerization and deesterication of pectic polysaccharides is the most apparent change occurring during ripening of many fruits like pear (Ben-Arie et al., 1979), apple (De Vries et al., 1984), tomato (Seymour et al., 1987), muskmelon (McCollum, Huber and Cantliffe, 1989; Ranwala, Suematsu and Masuda, 1992), bell pepper (Gross, Watada, Kang, Kim, Kim and Lee, 1986), strawberry (Huber, 1984; Nogata, Ohta and Voragen, 1993), kiwifruit (Redgwell et al., 1992), bush butter (Missang, Renard, Baron and Drilleau, 2001b), apricot (Femenia, Sanchez, Simal and Rosello, 1998), melon (Rose, Hadeld, Labavitch and Bennett, 1998), peach (Hegde and Maness, 1996), and olive fruit (Jimenez et al., 2001). Pectins from ripe fruit exhibited a lower degree of esterication, a lower average molecular weight, and decreased neutral sugar content compared to pectins from unripe fruits (Huber and Lee, 1986).

Changes in Pectic Polysaccharides during Ripening Cell Wall Hydrolases in Relation to Fruit Softening During ripening fruits loose rmness, and unless the fruit is dehydrated, the osmotic properties of the cell and the turgor pressure usually remain constant. While in plant tissues, it is assumed that turgor pressure alone is not contributing to the loss of rmness, instead it is the result of changes in the cell wall polysaccharides (Van Buren, 1979). Much work done to relate chemical changes in cell walls to fruit softening has been focused towards the characterization of changes in pectic substances (Krall and McFeeters, 1998). Pectins are the lone cell wall polysaccharides that are easily soluble in water and due to this property they can be deesteried and depolymerized mostly by enzymatic reactions. Also, retardation of textural softening by the addition of Ca++ ions to fruit is related to the ability of divalent cations to form calcium bridges between the pectic polysaccharide chains (Krall and McFeeters, 1998). A limited degradation of the pectic polymers might be due to the methylation of galacturonic acid groups or their accessibility for depolymerization (Voragen et al., 1995). Loss of rmness during heat treatment of acid fruit has been attributed to acid hydrolysis of glycosidic bonds in cell wall polysaccharides. It was suggested earlier that hydrolysis of neutral sugar glycosidic bonds was involved in the softening process. Arabinofuranosyl linkages are most labile while glycosidic linkages between galacturonans are most stable in pectins (Voragen et al., 1995). However in acidic pH (pH 2.5-4.5) hydrolysis of galacturonans occurs faster than neutral sugars, as inferred by the loss of uronic acid residues from the cell wall, while the neutral sugars are still found associated with the pectic substances. Thus, the possible mechanism of softening during ripening at acidic condition is the hydrolysis of pectin (Krall and McFeeters, 1998). Changes in the proportion and characteristics of pectic substances are reported in many fruits (Kertesz, 1951). During ripening, the progressive loss of rmness is the result of a gradual solubilization of protopectin in the cell walls to form pectin The changes in the cell wall composition, which accompany fruit softening during ripening, are due to the action of carbohydrate hydrolases. They act on cell wall polymers, resulting in their degradation. Most of these enzymes are present in low levels and are constitutive throughout fruit development and ripening (Tucker, 1993), but during ripening, generally all the hydrolases increase in activity, particularly cell wall hydrolases, showing a peak activity at climacteric stage. Among cell wall hydrolases, pectin-degrading enzymes are mostly implicated in fruit softening. Increased solubilization of the pectic substances, progressive loss of tissue rmness, and a rapid rise in the PG activity accompany normal ripening in many fruits. (Brady, 1987; Tucker, 1993; Fischer and Bennett, 1991; Pressey, 1986a). Since the pectic polymers begin to acquire solubility only after PG has become active, it is believed that this enzyme is involved in the breakdown of the insoluble complex polysaccharides by reducing the length of the chains cross-linked by calcium (Wong, 1995). A positive correlation between the appearance of PG and initiation of softening is shown in a number of fruits like guava (El-Zoghbi, 1994), papaya (Paull and Chan, 1983), and mango (Roe and Bruemmer, 1988). PG and PME activity increased remarkably in peach, tomato, and pear (Tucker and Grierson, 1987). In apple and strawberry fruits the mechanism of solubilization of polyuronide is thought to be different due to the absence of endo-PG (Voragen et al., 1995). PG activity was not detected in plum fruit (Boothby, 1983). In ripening fruits, much attention was focused on the depolymerization of acidic pectins by polygalacturonase. However, experiments with transgenic tomatoes have shown that even though PG is important for the degradation of pectins, it is not the sole determinant of tissue softening during ripening (Gray, Picton, Shabbeer, Schuch, and Grierson, 1992). PG antisense constructs for various tomato lines have little effect on the fruit characteristics, viz, reduced susceptibility to cracking, and decay and other

10

V. PRASANNA ET AL.
Table 6 Different types of carbohydrate hydrolases in fruits Glycosidases -Mannosidase -Galactosidase -Galactosidase -Glucosidase -Glucosidase -Hexosaminidase -Hexosaminidase -Xylosidase -Xylosidase -Arabinosidase -Arabinosidase

damages at the later stages of ripening (Gray et al., 1992). Now the focus is on the hydrolysis of neutral sugar side chains, which may weaken the complex network of cell wall polymers, thus contributing to textural softening (Smith and Harris, 1995). The variation in pectins from different sources is mainly attributed to the arrangement of these neutral sugar side chains resulting in congurational rearrangements. The loss of neutral sugar side chains from pectin is one of the most important features occurring during ripening. Substantial variation in the cell wall composition among fruits and fruit tissues exists. Further, their metabolism in relation to softening also varies from fruit to fruit (Gross and Sams, 1984). Out of 17 types of economically important fruits, 14 types showed a net loss of neutral sugars, galactose, and arabinose, from the cell wall during ripening. No such loss of neutral sugars occurs in ripening plum and cucumber fruits (Gross and Sams, 1984). The mutant tomato fruit (rin) containing little or no PG activity showed a substantial loss of galactose from the cell wall suggesting that this loss is not due to the action of PG. This evidence suggests that other cell wall hydrolases, especially glycosidases, play an important role in the textural softening during ripening (Gray et al., 1992). One novel approach to elucidate the role of enzymes in cell wall degradation and softening is to employ antisense RNA technology. This technology was one of the rst molecular approaches used for delaying fruit ripening (Bansal, 2000). It has been possible to obtain rmer tomatoes with longer shelf-life by specic suppression of PG gene expression with antisense RNA (Smith et al., 1988). Pectin methyl esterase (PME) suppression resulted in increased solid content in tomato (Tieman, Harriman, Ramamohan and Handa, 1992). The genes coding for PG, PME, and other enzymes have been cloned in tomato (Gray et al., 1992) and other fruits (Bansal, 2000). A wide range of cell wall hydrolases are identied in fruit tissues (Fischer and Bennett, 1991; Ahmed and Labavitch, 1980; Fry, 1995; Wallner and Walker, 1975). The major hydrolases involved in polysaccharide dissolution in vivo can be broadly classied into 2 types of hydrolases; viz, glycanases and glycosidases [Table 6]. Glycanases (glycanohydrolases) by denition are a class of enzymes cleaving high molecular weight polymers (polysaccharides) into shorter chains, while glycosidases (glycohydrolases) generally act on shorter chain oligosaccharides, which may be homo- or heterooligomers or glycoproteins or glycolipids. Recently, it has been reported that temperature plays a crucial role in the activities of these cell wall hydrolases (Reddy and Srivastava, 1999).

Glycanases Polygalacturonase Pectin methyl esterase Cellulase Hemicellulase Amylase Mannanase Galactanase Glucanase Arabinanase Xylanase Rhamnogalacturonase

cell lysis. The other enzymes such as arabinanase, galactanase, and -galactosidase act on the side chains of the galacturonide backbone, eventually degrading the entire pectic substance. Pectic enzymes have been used for the clarication of wines since the beginning of the 19th century. They are industrially useful enzymes for extraction, clarication, and liquefaction of fruit juices and wines (Chauhan, Tyagi, and Singh, 2001). They are also used in the fabric industry to soak plant bers and in the paper making industry to solve the retention problems by de-clogging the pulps (Sakai et al., 1993). They hydrolyse the pectic substances and aid in the occulation of suspended particles and clarication of wines and juices (Chauhan et al., 2001). Recently, immobilized pectic enzymes are gaining importance in this area (Alkorta, Garbisu, Llama, and Serra, 1998). PG from a fungal source is commercially utilized in the fruit juice industries. One of the technically important differences between PG from tomato and fungal source is the inhibition of the latter by some vegetable extracts, which may render them useless in the preparation of vegetable macerates for baby foods. Thus, fruit PGs are gaining importance. Pectin-degrading enzymes are classied, based on their mode of action on pectin and pectic substances into PG, PME, pectate lyase, and pectin lyase (Fig. 8) (Wong, 1995; Sakai et al., 1993; Chauhan et al., 2001). Polygalacturonase (PG) PG, an important pectolytic glycanase, is the primary enzyme playing a signicant role in pectin dissolution in vivo (Poovaiah and Nukuya, 1979). PG is a hydrolytic enzyme, which acts on pectic acid (polygalacturonic acid, PGA). It hydrolyses the 1,4-glycosidic bonds between the galacturonic acid residues in galacturonans. Based on their mode of action, PGs are classied into exo-PG (exo-poly (1,4 -D-galacturonide) galacturonohydrolase, EC 3.2.1.67) and endo-PG (endo-poly (1,4 -D-galacturonide) glycanohydrolase, EC 3.2.1.15). The former catalyses the hydrolysis of the glycosidic bonds between the de-esteried galacturonans from the non-reducing end, which results in the release of galacturonic acid as the major reaction product. The rate of hydrolysis depends on the degree of polymerization and it increases with increase in the molecular size of the substrate (Pressey and

Enzymes Related to Pectin Dissolution In Vivo Pectolytic enzymes are widespread in plants, fungi, and bacteria. They constitute a unique group of enzymes that are responsible for the degradation of pectin and pectic substances in plant cell walls [Table 7]. They act on plant tissues, especially on the main polyuronide chains of pectins and eventually cause

FRUIT RIPENING PHENOMENON

Table 7 Enzymes Pectin Methyl Esterase Polygalacturonases Protopectinase Endo-PG Exo-PG Oligogalacturonide hydrolase 4:5 unsaturated Oligogalacturonide hydrolase Endopolymethyl galacturonase Rhamnogalacturonase Rhamnogalacturonan acetylesterase Pectin acetyl esterase Lyases Endopectate lyases Exopectate lyases Oligogalacturonide lyases Endopectin lyases Arabinanase -L-Arabino-furanosidase Endoarabinanase Galactanase -D-Galactanase Pectin Protopectin Pectic acid Pectic acid Trigalacturonic acid 4:5 (galacturonide)n Pectin Pectin Pectin (Hairy region) Pectins (Smooth region) Pectic acid Pectic acid Unsaturated digalacturonate Pectin Arabinans (1,5)--Arabinans Classication of pectin-degrading enzymes Substrate Products Pectic acid + Methanol Pectin Oligogalacturonides Monogalacturonides Monogalacturonides Unsaturated monogalacturonide + galacturonides (n-1) Methyl-oligogalacturonides -(1,2)linked L-Rha, -(1,4) linked D-Gal Pectin + Acetic acid Unsaturated oligogalacturonides Unsaturated oligogalacturonides Unsaturated digalacturonides Unsaturated monogalacturonides Unsaturated oligogalacturonides -L-Arabinose Arabinose and higher oligosaccharides -D-Galactose Mechanism Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis Hydrolysis Trans elimination Trans elimination Trans elimination Trans elimination Hydrolysis Hydrolysis

11

Galactans

Hydrolysis

Avants, 1975), and it interrupts at the branching point. Exo-PG action causes a large increase in the formation of reducing groups and a slow decrease in viscosity. From the long polygalacturonan chain mere removal of terminal galacturonic acid residue does not show much effect on pectin solubility (Pressey and Avants, 1978). Thus, this enzyme is not involved in ripening, as pectate degradation does not occur. However, some evidence suggests

a possible implication of this enzyme in fruit ripening (John and Dey, 1986). Recently, exo-PG in tomato was found to elicit ethylene production, which in turn triggers the ripening process (Baldwin and Pressey, 1990). On the other hand, endo-PG depolymerizes pectic acid randomly, resulting in a rapid decrease in viscosity and therefore an involvement in the ripening process. The rate of hydrolysis decreases with decrease in the length of

Figure 8

Mode of action of pectin degrading enzymes.

12

V. PRASANNA ET AL.

the chain. Some fruits like apple, Freestone peach, and persimmon possess only exo-PG, while other fruits such as apple, avocado, Clingstone peach, lemon, mango, musk melon, raspberry, and kiwi contain only endo-PG (Lang and Dornenburg, 2000). Cucumber, papaya, passion fruit, peach, pear, strawberry, and tomato contain both endo- and exo-PGs (Lang and Dornenburg, 2000). The marked difference in the textural characteristics of the two types of peaches (Clingstone and Freestone) is attributed to the differences in PG (Pressey, 1986b; Pressey and Avants, 1978). The extent and rate of textural softening during ripening is directly related to PG composition, i.e., extensive softening occurs if endo- or both endo- and exo-PG are present and limited softening occurs if only exo-PG is present (Bartley, 1978; Huber, 1984). It is generally accepted that PG is primarily responsible for dissolution of the middle lamella during fruit ripening (Jackman and Stanley, 1995; Voragen et al., 1995). There is a clear correlation between the appearance of PG and the onset of dissolution of middle lamella and the primary cell wall during ripening (Crookes and Grierson, 1983). PG alone is sufcient to dissolve middle lamella in apple, but both PG and cellulase are required in pear for dissolution. One of the most characteristic changes during fruit ripening is decrease in rmness. This has been shown to be associated with an increased activity of pectic enzymes, particularly PG (Crookes and Grierson, 1983; Watkins, Haki, and Frenkel, 1988). An increase in the total PG activity prior to the respiratory climacteric stage of the tomato suggested that this enzyme might play a role in initiating the ripening process (Poovaiah and Nukuya, 1979). However, no detectable endo-PG, an enzyme thought to play a role in tomato softening, was found in pre-climacteric tomato and appearance of endo-PG in tomatoes after the onset of climacteric ethylene was reported (Grierson et al., 1985; Baldwin and Pressey, 1990). The absence of PG in unripe fruits and appearance near the onset of ripening with increased activity during ripening, along with concomitant pectin degradation suggest that this enzyme is implicated in pectin solubilization. The appearance of soluble pectin was the result of an increased activity of PG during ripening (Tucker and Grierson, 1987). This suggests that fruit softening is regulated by the accumulation of PG and the rate of splitting of pectin. PG acts on the de-esteried portion of the galacturonan chains, particularly on those glycosidic bonds, which have the carboxylic groups adjacent to the glycosidic linkage, and free from esterication. PG was rst found in ripe tomato fruit, and it still remains the richest plant source of the enzyme (Pressey, 1986c; Wong, 1995). Recently, an increase in PG activity with a peak at the climacteric stage in mango (Prabha, Yashoda, Prasanna, Jagadeesh, and Bimba Jain, 2000), capsicum (Priyasethu, Prabha, and Tharanathan, 1996), and banana (Prabha and Bhagyalakshmi, 1998) was reported from our lab. An increase in PG activity in seven Indian mango cultivars during ripening was also reported (Selvaraj and Kumar, 1989). In climacteric fruits, whose texture alters considerably during ripening, a maximum loss of rmness was directly correlated with a rapid increase in PG (Roe and Bruem-

mer, 1981; Abu-Sarra and Abu-Goukh, 1992; Pressey, 1986a). Apart from fruits, other plant parts like roots, stem, leaf explants, and seedlings are also reported to contain PG, although their biochemical/physiological aspects may differ considerably from those of fruit (Pressey, 1986b). Recently, isozymes of PG were reported from banana (Pathak and Sanwal, 1998), strawberry (Nogata et al., 1993), pear (Pressey and Avants, 1976), and peach (Pressey and Avants, 1973a). In tomato, PG exists in two forms and both are endoacting (Pressey and Avants, 1973b), splitting glycosidic bonds randomly and releasing oligogalacturonides (Ali and Brady, 1982). Both isozymes have pH optima in the acidic range and SDS-PAGE suggests that PG1 is a dimer of PG2 (Tucker, Robertson, and Grierson, 1980). Later studies suggest that PG1 is produced by the combination of both PG2 and a -subunit (converter) (Pressey, 1986a; 1986b). Both PGs are glycosylated. Two PG2 isoenzymes (PG2A and PG2B) have been characterized and are the product of post-translational modication or glycosylation. It was shown that these two PG2 isozymes have similar polypeptides, but have differences in the degree of glycosylation (DellaPenna and Bennett, 1988). DellaPenna and coworkers (DellaPenna, Lashbrook, Toenjes, Giovannoni, Fischer, and Bennett, 1990) demonstrated that all the PG isozymes arise by differential processing of a single gene product. The physiologically active form of PG in tomato is PG1, which is active enough to carryout both solubilization and depolymerization (DellaPenna et al., 1990). Multiple forms appear due to genetic variants (allelic), genetically independent proteins, or heteropolypeptide chains that are bound non-covalently. However conformational differences, covalent alteration, or conjugation may also cause multiplicity of enzymes (Dey and Del Campillo, 1984). The signicance of these multiple forms may be related to the complex nature of the pectic substrates and their modication during ripening (Pressey and Avants, 1972). The PG gene was the rst to be cloned from tomato for studying textural regulation in ripening fruit and the transformed tomato with PG antisense gene resulted in improved fruit with rmer texture and an extended shelf life (Smith et al., 1988). This gave remarkable clues regarding the role of PG in fruit cell wall metabolism. However, despite similar catalytic properties, PGs differ from fruit to fruit, thus reducing the percent homology of the PG genes. Thus it is necessary to study this enzyme individually in the fruit of choice. Methods for quantication assay of PG have been well documented (Pressey, 1986b). PG activity is generally measured by an increase in reducing equivalents. The more recent spectrophotometric method for quantication of reducing equivalent is by using 2-cyanoacetamide (Pressey, 1986c). Measurement of viscosity changes using an Oswald viscometer is less convenient for routine measurement but still it is useful in distinguishing between endo- and exo-splitting PGs. This is by comparing the rate of decrease in viscosity with rate of hydrolysis, as measured by increase in reducing equivalents. An endo-splitting enzyme causes around 50% reduction in viscosity when only 35% of the glycosidic bonds are cleaved, while an exo-splitting enzyme

FRUIT RIPENING PHENOMENON

13

causes similar reduction in viscosity with as much as 1015% of the glycosidic bond cleavage. Other difference between these enzymes is in the nature of product formed, at the beginning of the reaction. The endo-splitting enzyme does not produce low molecular weight products, whereas an exo-splitting enzyme results in low molecular weight products. Due to the presence of rhamnose in almost all fruit pectins, PG alone is not sufcient for pectin degradation. It seems that other glycanases, such as rhamnogalacturonase, are also responsible for the degradation of rhamnogalacturonan backbone. Rhamnogalacturonase (RGase) Rhamnogalacturonase is an enzyme that catalyses the hydrolysis of glycosidic bonds between galacturonic acid and rhamnose units in RG backbone, the hairy regions of many fruit pectins (Schols and Voragen, 1994; Colquhoun, de Ruiter, Schols, and Voragen, 1990). The products are oligomers with alternating galacturonic acid and rhamnose units, rhamnose forming the non-reducing end (Schols and Voragen, 1994). RGase activity enhances strongly when the ester groups are de-esteried and the side chains are removed (Schols and Voragen, 1994). RGase activity is hindered by O-acetyl group. Thus, they act along with rhamnogalacturonan acetylesterase, which splits off acetyl groups from the hairy regions of pectin (Voragen et al., 1995). Recently, the probable presence of RGase was also reported for bush butter fruit (Missang et al., 2001b). These cell wall glycanases (PG and RGase) appear to be more active on deesteried pectins than esteried pectins (Seymour et al., 1987). Therefore, de-esterication is the most important reaction and is catalyzed by a unique group of enzymes, the pectin methyl esterase.

green tomatoes pass through different color stages to become full red. Unripe fruits are rich in PME, while ripe fruits are rich in hydrolase enzymes. Activity of PME was shown to decrease (El-Zoghbi, 1994; Roe and Bruemmer, 1981; Prabha et al., 2000; Abu-Sarra and Abu-Goukh, 1992), or increase (Selvaraj and Kumar, 1989; Aina and Oladunjoye, 1993) or remain constant (Ahmed and Labavitch, 1980; Ashraf, Khan, Ahmed, and Elahi, 1981) during fruit ripening. PME has been puried and characterized in few ripening fruits (Pressey and Avants, 1972; Tucker, Robertson and Grierson, 1982). Several PME isozymes have been identied in tomato (Tucker, Robertson, and Grierson, 1982). The slow ripening of Abu-Samaka mango in spite of high PG activity, suggests a key role to PME in controlling fruit softening (Abu-Sarra and Abu-Goukh, 1992). PME also acts on commercial methylated pectin (citrus) to liberate the carboxyl group and methanol. The activity may be assayed by estimating the released methanol chromatographically. A continuous spectrophotometric assay has been developed based on the reaction of PME on pectins in the presence of a pH indicator bromothymol blue. The carboxylic groups produced by hydrolysis of ester groups lower the pH, causing the indicator dye to change the color (Doner, 1986). By genetic engineering, it has been shown that PME may not be the sole determinant of softening, and other enzymes may be involved in textural softening. But an increase in the total soluble solid was a very important and signicant nding in ripening tomato as demonstrated from PME suppression by antisense construct (Gray et al., 1992; Tieman et al., 1992). Lyases The lyases or trans eliminases cleave the glycosidic bond by trans -elimination mechanism, i.e., elimination of hydrogen from the C-4 and C-5 position of the aglycone portion of the substrate (Whitaker, 1984). It is known that in alkaline medium, pectin undergoes deesterication, accompanied by degradation by -elimination reaction. Similar splitting of glycosidic bonds also occurs in neutral pH at elevated temperature. These enzymes are absent in fruit but are present only in microorganisms. Pectate lyases (PL) catalyses the cleavage of de-esteried or esteried galacturonate units by a trans -elimination of hydrogen from the C-4and C-5 positions of galacturonic acid. ExoPL (exo-poly 1,4 -galacturonide) lyase, EC 4.2.2.9) acts from the non-reducing end, whereas endo-PL (endo-poly 1,4 -D galacturonide) lyase, EC 4.2.2.2) acts randomly on de-esteried galacturonans. Pectin lyase (PNL) (EC 4.2.2.10) catalyzes the cleavage of esteried galacturonate units by trans -elimination. All PNLs studied so far are endo-enzymes, acting randomly (Wong, 1995). Arabinanase Arabinanase are of two types; arabinofuranosidase (EC 3.2.1.55) and endo-arabinanase (EC 3.2.1.99). They reduce the degree of branching and increase the polymerpolymer association (Whitaker, 1984). Endo-arabinanase hydrolyses linear

Pectin Methylesterase (PME) PME (Pectin pectylhydrolase, EC 3.1.1.11) catalyses the hydrolysis of pectin methyl ester groups, resulting in deesterication. PME is specic for galacturonide esters and its action is to remove methoxyl groups from methylated pectin by nucleophilic attack. This results in the formation of an acyl enzyme intermediate with the release of methanol, followed by deacylation (hydrolysis) to generate the enzyme and a carboxylic acid. PMEs of plant origin exhibit an action pattern that results in the formation of carboxylate groups along the pectin chain (Wong, 1995). De-esterication appears to proceed linearly along the chain resulting in blocks of free carboxyl groups (Rexova-Benkova and Markovic, 1976). It appears that PME preferentially attacks methyl ester bonds of a galacturonate unit next to non-esteried galacturonate unit (Pilnik and Voragen, 1970). Thus, they deesterify the esteried pectic substances, making them vulnerable for PG action. Its action may be a prerequisite for the action of PG during ripening. PME activity was detected in fruits like apple, banana, cherry, citrus, grape, papaya, peach, pear, tomato, and strawberry (Pilnik and Voragen, 1970). The activity of PME increases as mature

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V. PRASANNA ET AL.

arabinan in a random fashion producing oligomers of shorter lengths. Arabinofuranosidase degrades branched arabinan to a linear chain by splitting of terminal -1,3-linked arabinofuranosyl side chains and sequentially breaks the -1,5 links at the non-reducing end of linear arabinan. This enzyme hydrolyses the terminal non-reducing arabinofuranosyl groups from a range of arabinose-containing polysaccharides such as, arabinogalactans, arabinans, and arabinoxylans. The substrates most widely used for the assay of arabinofuranosidase are p-nitrophenyl-L-arabinofuranoside, phenyl--L-arabinofuranoside and -Larabinan. The release of L-arabinose is quantitated either by reducing group estimation or by HPLC.

Galactanase Galactanases are of two types; endo-galactanase (EC 3.2.1.89), which catalyses the random cleavage of the -1,4 linkages of galactan chains and other galactanases (EC 3.2.1.90), which also randomly hydrolyses the -1,3 and -1,6 linkages of galactans, present as side chains in pectins. An increase in the activity of arabinanase and galactanase in mango, banana, and capsicum was reported (Prabha et al., 2000; Priyasethu et al., 1996; Bhagyalakshmi, Prabha, Yashoda, Prasanna, Jagadeesh and Tharanathan, 2002). Recently, exo-(1-4)--galactanase was puried and characterized from tomato (Carey et al., 1995). -Galactosidase. It is very well understood by molecular evidence that PG activity alone is not responsible for the degradation of pectins to the extent that occurs during fruit ripening. Initial softening was not correlated with the increase of PG activity in ripening apples. Further, in ripening inhibitor mutant rin tomato, little or no PG activity was detected, but a substantial amount of galactose was lost indicating the involvement of other enzymes. The apparent absence of PG in some fruits that soften normally has implied other alternative mechanisms of cell wall dissolution (Ranwala et al., 1992; Gross et al., 1986). This evidence stimulated further research on this glycosidase. This enzyme is also implicated in pectin dissolution by way of deglycosylating the galactan, which is generally present in pectintype of polymers. Thus, loss of neutral sugars has become a general feature of fruit ripening (Gross and Sams, 1984). This loss of neutral sugar residues is separate and independent of polyuronide solubilization during ripening, and independent of PG activity, suggesting the involvement of -galactosidase/galactanase, which have been associated with many ripening fruits (Carey et al., 1995; Rose et al., 1998) Galactosidase (EC 3.2.1.23), a glycosidase, acts on short chain oligomers of galactose units present either as glycoprotein, glycolipid, or hetero-/homopolysaccharides. This enzyme partially degrades the pectic and hemicellulosic components of the cell wall and is possibly related to the breakdown of polysaccharides at over-ripening. -Galactosidase was detected in a wide variety of fruit systems (Dey and Del Campillo, 1984). Increase in -galactosidase activity during ripening was reported in many fruits (Bartley, 1974; John and Dey, 1986). It was reported that this enzyme also increases during the developmental

stages of fruits like mango (Rahman, Akhter and Absar, 2000). This enzyme has been puried from a number of fruits including tomato (Pressey, 1983), apple (Ross, Wegrzyn, MacRae and Redgwell, 1994), orange (Burns, 1990), muskmelon (Ranwala et al., 1992), coffee berry (Golden, John and Kean, 1993), sweet cherry (Andrews and Li, 1994), sapota (Dore Raju and Karuna Kumar, 1996), and Harumanis mango (Ali, Armugam and Lazan, 1995). This enzyme is incapable of degrading native galactans in citrus fruit (Burns, 1990). However, in some fruits like tomato (Pressey, 1983), muskmelon (Ranwala et al., 1992), and apple (Ross et al., 1994), they attack native galactan polymers. In most studies of fruit -galactosidase, the synthetic substrate, para-nitrophenyl--D-galactopyranoside was widely used. The other substrates used for assaying the activity were phenyl--D-galactopyranoside, arabinogalactans, galactomannan and lactose.

Other Hydrolases Implicated in Fruit Softening Hemicelluloses are (neutral) polysaccharides extracted by alkaline solutions from the cell wall residues after the extraction of pectic polysaccharides. The inert, insoluble, crystalline cell wall material remaining after the hemicellulose extraction, which is mainly composed of -glucose, is cellulose (Van Buren, 1979). An apparent dissolution of the middle lamella and cell wall brillar network due to cellulolytic activity in ripening of avocado, pear, and apple has been demonstrated (Knee, 1973). Ripening associated changes involving dramatic decrease in the molecular size of hemicellulose are reported in tomato (Huber, 1983), strawberry (Nogata et.al., 1993), pepper (Gross et al., 1986), muskmelon (McCollum et al., 1989), melon (Rose et al., 1998), mango (Mitcham and McDonald, 1992), and peach (Hegde and Maness, 1998). The amount of hemicellulose decreased steadily during ripening of many fruits including mango (Mitcham and McDonald, 1992). Decline or loss of substantial levels of characteristic monomers of hemicelluloses viz. glucose, xylose, and mannose occur during ripening of fruits. Little is known about the enhancement of cellulase or hemicellulase activity in relation to fruit softening. Cellulase is a multienzyme system composed of several enzymes, viz. endoglucanase (EC 3.2.1.4), exo-glucanase (EC 3.2.1.91) and glucosidase (EC 3.2.1.21) (Sobotka and Stelzig, 1974). Endoglucanase hydrolyses the -1,4-link between adjacent glucose residues at random positions. Exo-glucanase breaks the bond at non-reducing ends of the chain, producing glucose or cellobiose (dimers of -1,4-linked glucose), whereas -glucosidase splits cellobiose into glucose molecules. Cellulase activity increased during ripening of avocado, peach, strawberry, tomato, and papaya (Hobson, 1981). Cellulase levels in unripe fruit are generally low and increase dramatically during ripening. The loss of rmness, climacteric rise of respiration and ethylene evolution in ripening fruit were directly correlated with marked increase in cellulase activity (Roe

FRUIT RIPENING PHENOMENON

15

and Bruemmer, 1981; Abu-Sarra and Abu-Goukh, 1992). Cellulase activity in normal and non-ripening mutants of tomato suggests that this enzyme has no primary role in fruit softening (Poovaiah and Nukuya, 1979). However, cellulase has been implicated in the softening process in tomato (Hobson, 1981). Cellulase activity was reported in several Indian mango cultivars, which increased during ripening (Selvaraj and Kumar, 1989). No cellulase activity was detected in pears (Ahmed and Labavitch, 1980). Xylanases (EC 3.2.1.8) catalyse the hydrolysis of -1,4xylan. -1,4-D-endo-xylanase and -1,4-D-exo-xylanase are reported as cell wall degrading enzymes from fruits including banana (Prabha and Bhagyalakshmi, 1998) and capsicum (Priyasethu et al., 1996). In papaya during ripening, a clear correlation between polygalacturonase and xylanase activities, climacteric rise in respiration and ethylene evolution and fruit softening were demonstrated (Paull and Chan, 1983). Mannanase catalyses the hydrolysis of mannan polymer in capsicum (Priyasethu et al., 1996) and mango (Prabha et al., 2000). Xylanase, arabinanase and mannanase are localized both in soluble and bound form, which will increase during ripening. It was interesting to note that arabinanase, galactanase, and mannanase were very prominent enzymes in mango fruit, having activity peaks at climacteric stage of ripening (Bhagyalakshmi et al., 2002; Prabha et al., 2000). Among glycosidases, the prominent enzymes found in ripening fruit were -hexosaminidase, -mannosidase and - and -galactosidases (Priyasethu et al., 1996). -Amylase (EC 3.2.1.1) and -amylase (EC 3.2.1.2) are the two amylases in plant tissues capable of metabolizing starch, amylases hydrolyse the -1,4-linkages of amylose at random to produce a mixture of glucose and maltose, whereas -amylase attacks only the penultimate linkage from the non-reducing end and thus releases only maltose. These enzymes are unable to degrade the -(1 6) branch points of amylopectin, which are catalyzed by debranching enzymes. Amylase activity increases to some extent during ripening of many fruits (Fuchs et al., 1980; Tucker and Grierson, 1987). Mango and banana are the major starch containing fruits (15 to 20%, on fresh weight basis), where starch is almost completely hydrolyzed to free sugars, thus contributing to loosening of the cell structure and textural softening during ripening (Bhagyalakshmi et al., 2002).

BIOTECHNOLOGICAL IMPLICATIONS AND FUTURE PROSPECTS The world population is expanding at a faster rate than that of food production. Thus increasing the availability of food has become a vexing problem. This problem of population food-imbalance can be solved either by limiting population growth or by increasing food supplies. But both require considerable amount of capital and time to achieve. Considering the nutritional and pharmacological signicance, fruits will play an important role in the nutrition of the world

population. A major step contributing towards this is to prevent the fruit loss between the time of harvesting and consumption. Fruits are an important part of a healthy and balanced diet. They provide us with essential vitamins, proteins, minerals, and bers. They are also aesthetically pleasing to our eye and olfactory sense organs. However, unlike most other food commodities, fruits are also living organisms, even after harvesting. As a result of their biological nature, they are subjected to physical, chemical, and microbiological spoilages. Current storage facilities of fruits usually employ refrigeration alone, or coupled with controlled or modied atmospheres, in which O2 is maintained at low level, and CO2 level at high. These techniques are expensive and can result in damage to the stored fruits. In addition, the fruits have to be treated at each harvest and also individually. Nevertheless, the economic viability of such technology depends on local, regional and national socioeconomic conditions, as well as national and international trade policies. Recent advent in molecular biology has provided a better understanding of the biochemistry of fruit ripening as well as providing a hand for genetic manipulation of the entire ripening process. These techniques are used to investigate the role of hydrolases in cell wall degradation, ethylene synthesis, pigment synthesis, starch degradation, and hence fruit softening during ripening. These techniques could be used to manipulate the ripening process genetically, and have signicant commercial advantages. Currently, such manipulations are restricted only to some fruits such as tomato fruit. The resultant fruit has a longer shelf life and are more resistant to disease and cracking during transportation. The major obstacle to achieving this aim in other fruits is the lack of fundamental biochemistry of their ripening process. In future, more aspects of ripening may be cracked-down and can be used to manipulate the fruits to our advantage. One more aspect is that the plant enzymes are gaining greater importance in modern food biotechnology. The important difference between enzymes of fruit and fungal source is the inhibition of the latter by some vegetable extracts, which may render them useless in the preparation of vegetable macerates for baby foods. Not only this, usage of microbial products such as enzymes is decreasing day-by-day because of awareness by the consumers. Thus prospects for plant enzymes in the future are very promising. A constant research and development effort in order to optimize plant enzymes will lead to products that are even better than those of earlier. It is desirable that signicant breakthroughs will naturally be made in the near future, much to the benet of the society at large.

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