fruit ripening phenomena—an

Critical Reviews in Food Science and Nutrition, 47:1–19 (2007)Copyright C©© Taylor and Francis Group, LLCISSN: 1040-8398DOI: 10.1080/10408390600976841

Fruit Ripening Phenomena—AnOverview

V. PRASANNA, T. N. PRABHA, and R. N. THARANATHANDepartment 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 normalhealth. Fruits are widely distributed in nature. One of the limiting factors that influence their economic value is the relativelyshort ripening period and reduced post-harvest life. Fruit ripening is a highly coordinated, genetically programmed, and anirreversible phenomenon involving a series of physiological, biochemical, and organoleptic changes, that finally leads to thedevelopment of a soft edible ripe fruit with desirable quality attributes. Excessive textural softening during ripening leads toadverse effects/spoilage upon storage. Carbohydrates play a major role in the ripening process, by way of depolymerizationleading to decreased molecular size with concomitant increase in the levels of ripening inducing specific enzymes, whosetarget differ from fruit to fruit. The major classes of cell wall polysaccharides that undergo modifications during ripeningare starch, pectins, cellulose, and hemicelluloses. Pectins are the common and major components of primary cell wall andmiddle lamella, contributing to the texture and quality of fruits. Their degradation during ripening seems to be responsible fortissue softening of a number of fruits. Structurally pectins are a diverse group of heteropolysaccharides containing partiallymethylated D-galacturonic acid residues with side chain appendages of several neutral polysaccharides. The degree ofpolymerization/esterification and the proportion of neutral sugar residues/side chains are the principal factors contributingto their (micro-) heterogeneity. Pectin degrading enzymes such as polygalacturonase, pectin methyl esterase, lyase, andrhamnogalacturonase are the most implicated in fruit-tissue softening. Recent advances in molecular biology have provideda better understanding of the biochemistry of fruit ripening as well as providing a hand for genetic manipulation of the entireripening process. It is desirable that significant breakthroughs in such related areas will come forth in the near future, leadingto considerable societal benefits.

Keywords fruit ripening, cell wall polysaccharides, pectin, pectic enzymes, polygalacturonase

INTRODUCTION

Fruits constitute a commercially important and nutritionallyindispensable food commodity. They are edible seed vessels orreceptacles developed from a mature, fertilized ovary. They arehighly specialized organs in higher plants offering a great varietyof aesthetic qualities with their complex/delicate aroma, pleas-ant taste, exotic colors, succulence, flavor, and texture. Theyplay a vital role in human nutrition, by supplying the necessarygrowth factors essential for maintaining normal health. Nutri-tionally, they are known for their high energy, roughage value,minerals, vitamins (B-complex, C and K in some instances), β-carotene (pro-vitamin A), and phenolics (antioxidants). Fruitsare widely distributed in nature and depending upon their dis-

Address correspondence to R. N. Tharanathan, Department of Biochem-istry and Nutrition, Central Food Technological Research Institute, Mysore,Karnataka 570020, India. E-mail: [email protected]

tribution, they are classified into tropical, subtropical, and tem-perate fruits [Table 1].

Fruits are harvested at complete maturity. They are self-sufficient with their own catalytic machinery to maintain an inde-pendent life, even when detached from the parent plant. Based ontheir respiratory pattern and ethylene biosynthesis during ripen-ing, harvested fruits can be further classified as climacteric andnon-climacteric type [Table 2]. Climacteric fruits, harvested atfull maturity, can be ripened off the parent plant. The respirationrate and ethylene formation though minimal at maturity, raisedramatically to a climacteric peak, at the onset of ripening, afterwhich it declines (Gamage and Rehman, 1999). Non-climactericfruits are not capable of continuing their ripening process, oncethey are detached from the parent plant. Also, these fruits pro-duce a very small quantity of endogenous ethylene, and do notrespond to external ethylene treatment. Non-climacteric fruitsshow comparatively low profile and a gradual decline in their res-piration pattern and ethylene production, throughout the ripen-ing process (Gamage and Rehman, 1999).

1

2 V. PRASANNA ET AL.

Table 1 Classification of fruits based on distribution

Temperate Sub-tropical Tropical

Apple Avocado AnnonaApricot Lime BananaCherry Litchi GuavaGrapes Mandarin JackfruitKiwi fruit Olive MangoPeach Orange PapayaPear Passion fruit PineapplePlums Persimmon SapotaStrawberry PomegranateMelon

FRUIT RIPENING

Fruit ripening is a highly co-ordinated, genetically pro-grammed, and an irreversible phenomenon involving a series ofphysiological, biochemical, and organoleptic changes that leadto the development of a soft and edible ripe fruit with desirablequality attributes. A wide spectrum of biochemical changes suchas increased respiration, chlorophyll degradation, biosynthesisof carotenoids, anthocyanins, essential oils, and flavor and aromacomponents, increased activity of cell wall-degrading enzymes,and a transient increase in ethylene production are some of themajor changes involved during fruit ripening (Brady, 1987)

The color change during fruit ripening is due to the unmask-ing of previously present pigments by degradation of chlorophylland dismantling of the photosynthetic apparatus and synthesisof different types of anthocyanins and their accumulation invacuoles, and accumulation of carotenoids such as β-carotene,xanthophyll esters, xanthophylls, and lycopene (Tucker andGrierson, 1987; Lizada, 1993). The increase in flavor and aromaduring fruit ripening is attributed to the production of a complexmixture of volatile compounds such as ocimene and myrcene(Lizada, 1993), and degradation of bitter principles, flavanoids,tannins, and related compounds (Tucker and Grierson, 1987).The taste development is due to a general increase in sweet-ness, which is the result of increased gluconeogenesis, hy-drolysis of polysaccharides, especially starch, decreased acid-

Table 2 Classification of fruits

Climacteric fruits Non-Climacteric fruits

Apple CherryApricot CucumberBanana GrapeGuava GrapefruitKiwifruit LemonMango LimePapaya LitchiPassion fruit MandarinPeach MelonPear OrangePersimmon PineapplePlum PomegranateSapodilla RaspberryTomato Strawberry

ity, and accumulation of sugars and organic acids resulting inan excellent sugar/acid blend (Lizada, 1993; Grierson, Tucker,and Robertson, 1981; Selvaraj, Kumar, and Pal, 1989). Themetabolic changes during fruit ripening include increase inbiosynthesis and evolution of the ripening hormone, ethylene(Yang and Hoffman, 1984), increase in respiration mediated bymitochondrial enzymes, especially oxidases and de novo synthe-sis of enzymes catalyzing ripening specific changes (Tucker andGrierson, 1987). Alteration of cell structure involves changes incell wall thickness, permeability of plasma membrane, hydra-tion of cell wall, decrease in the structural integrity, and increasein intracellular spaces (Tucker and Grierson, 1987; Redgwell,MacRae, Hallet, Fischer, Perry, and Harker, 1997).

The major textural changes resulting in the softening of fruitare due to enzyme-mediated alterations in the structure andcomposition of cell wall, partial or complete solubilization ofcell wall polysaccharides such as pectins and cellulose (Tuckerand Grierson, 1987), and hydrolysis of starch and other storagepolysaccharides (Selvaraj et al., 1989; Fuchs, Pesis and Zauber-man, 1980). The changes in gene expression during ripeninginvolves the appearance of new “ripening- specific” mRNAs,tRNA, rRNA, poly A+RNA, and proteins, and the disappear-ance of some mRNAs (Tucker and Grierson, 1987; Grierson,Slater, Spiers, and Tucker, 1985; Wong, 1995). However, somemRNAs are found to remain constant throughout the ripeningprocess (Gomez-Lim, 1997). These changes during fruit ripen-ing are activated by plant hormones.

ROLE OF FRUIT RIPENING HORMONE

Ethylene, a fruit ripening phytohormone, in minute amountscan trigger many events of cell metabolism including initiationof ripening and senescence, particularly in a climacteric fruit.Ethylene, which is synthesized autocatalytically at levels as lowas 0.01 µl L–1and 0.05 µl L–1 triggers the ripening processin mango and banana, respectively (Johnson, Sharp, Milne andOosthuyse, 1997). A number of reviews have been published onthe role of ethylene in fruit ripening, particularly in mangoes aswell as its biogenesis (Adams and Yang, 1979; Kende, 1993).Fruits treated with exo-polygalacturonase or other cell wall hy-drolases or their products have been shown to elicit ethylene pro-duction (Baldwin and Pressey, 1990; Kim, Gross and Solomos,1987). This response is not fruit specific (Kim et al., 1987). Incultured pear cells it was shown that the pectic oligomers mightalso induce and regulate ethylene biosynthesis (Campbell andLabavitch, 1991).

The pathway for ethylene biosynthesis has been eluci-dated in apple, and other fruits such as avocado, banana, andtomato (Kende, 1993; Yang and Hoffman, 1984). The firststep is the conversion of S-adenosylmethionine (SAM) to 1-aminocyclopropane carboxylic acid (ACC) by the enzyme ACCsynthase (Fig. 1). At the onset of fruit ripening, expressionof multiple ACC synthase genes are activated, resulting in in-creased production of ACC. In most cases, it is the ACC synthase

FRUIT RIPENING PHENOMENON 3

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. Inhibi-tion of ethylene biosynthesis by antisense RNA for ACC syn-thase (Oeller, Min-Wong, Taylor, Pike, and Theologis, 1991)and ACC oxidase (Hamilton, Lycett and Grierson, 1990) wasdemonstrated first in the tomato fruit. Deamination of ACC toα-ketobutyrate by over-expressing ACC deaminase enzyme alsoinhibited ethylene formation and fruit ripening (Klee, Hayford,Kretzmer, Barry, and Kishore, 1991). The resultant transgenicfruit did not overripe as normal controls, though some colorchange occurred and a mere ethylene boost triggered back allthe ripening related biochemical changes in a similar way as innormal fruit (Hamilton et al., 1990; Oeller et al., 1991). Recentlythe cDNA encoding for ACC oxidase has been isolated andcharacterized from mango (Zainal, Tucker, and Lycett, 1999).Down regulation of ACC synthase and ACC oxidase genes inmango are now being used for extending the shelf life of thisfruit.

TEXTURAL SOFTENING DURING RIPENING

Fruit ripening is associated with textural alterations, whichare dramatic in climacteric fruits. Textural change is the ma-jor event in fruit softening, and is the integral part of ripening,which is the result of enzymatic degradation of structural aswell as storage polysaccharides (Tucker and Grierson, 1987,Grierson et al., 1981, Bartley and Knee, 1982; Hulme, 1971).Depending upon their inherent composition and nature, dif-ferent 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 tex-tural softening from “stone hard” stage to a “soft pulpy” stage,whereas apple and citrus fruits do not exhibit such a drasticsoftening, though they undergo textural modifications duringripening. An overview of fruit ripening with special referenceto textural softening has been diagrammatically represented inFig. 2.

Fruit texture is influenced by various factors like struc-tural integrity of the primary cell wall and the middlelamella, accumulation of storage polysaccharides, and the tur-gor pressure generated within cells by osmosis (Jackman andStanley, 1995). Change in turgor pressure, and degradationof cell wall polysaccharides and starch determine the extent

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

of fruit softening (Brady, 1987; Tucker and Grierson, 1987;Grierson et al., 1981). In citrus fruit, softening is mainlyassociated with change in turgor pressure, a process asso-ciated with the post harvest dehydration and/or loss of drymatter. Starch is the bulk polysaccharide present in somefruits (mango and banana), and its enzymatic hydrolysis re-sults in pronounced loosening of cell structure and sweetnessdevelopment.

The major classes of cell wall polysaccharides that undergomodifications during ripening are pectins, cellulose, and hemi-celluloses. In fruits, which are known for excessive softening,the cell walls are thoroughly modified by solubilization, de-esterification, and depolymerization, accompanied by an exten-sive loss of neutral sugars and galacturonic acid, followed bysolubilization of the remaining sugar residues and oligosaccha-rides (Voragen, Pilnik, Thibault, Axelos and Renard, 1995).

The process of textural softening is of commercial importanceas it directly dictates fruit shelf life and quality (Tucker, 1993).This should be considered to avoid mechanical damage duringharvesting and transportation. The textural properties of fruits ingeneral play a very significant role in the consumer acceptabil-ity. The increased interest in controlling the textural qualitiesof fruit stimulated further research on the biochemistry of thecell wall, with particular reference to cell wall polysaccharidesand their degradation (Jackman and Stanley, 1995; Van Buren,1979). The textural qualities of fruits are attributed to its inher-ent composition, particularly the cell wall composition. Figure 3shows the schematic representation of the levels of structure thatcontribute to fruit texture. The “textural” characteristics are at-tributed to the mechanical properties of the final organ, which inturn depends on contributions and interactions of different levelsof structure (Waldron, Smith, Parr, Ng, and Parker, 1997). At-tempt to understand the molecular mechanism of fruit softeninghas led to the investigation of cell wall polymers, their com-positional changes and the related cell-wall degrading enzymesduring ripening (Knee, 1978).


Figure 3 Schematic representation of the levels of structure that contributeto 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 galac-tomannans; gel formers such as gums and mucilages; and phys-iological information carriers like antigens, in general, are anextremely diverse set of biopolymers, which play a very im-portant role as structural elements. Fruit polysaccharides, upontheir degradation, play a crucial role in textural softening duringripening.

Polysaccharides from different sources vary in their chem-ical-biological, physico-chemical, and structural–functionalcharacteristics (Tharanathan, Muralikrishna, Salimath, andRaghavendra Rao, 1987). Plant polysaccharides play a majorrole in storage, mobilization of energy and in maintaining celland tissue integrity due to their structural and water bindingcapacity. Cell wall polysaccharides differ widely in their phys-ical / nutritional properties and have the greatest potential forstructural diversity (Aman and Westerlund, 1996). They reg-ulate the utilization of other dietary components in the food.Recently plant polysaccharides have emerged as important,bioactive, natural products exhibiting a number of biologicalproperties. They are capable of regulating gene expression andhost-defense mechanism by the generation of elicitor-active oli-gogalacturonide fragments from the cell wall (Ridley, O’Neill,and Mohnen, 2001).

Cell wall is an active organelle, vital to cell growth,metabolism, transport, attachment, shape, cell resistance, andstrength. The old notion of the cell wall being static, inert, anda mere load-bearing structure has changed to the newer conceptof the dynamic nature of the cell wall (Jackman and Stanley,1995).

Fruit pulp or the mesocarp is the edible part of the fruit, andis composed of thin-walled storage parenchymatous cells (50–500 µm). These cells are characterized by a prominent cell wallconsisting of complex network of polysaccharides and proteins,which gives mechanical strength to the tissues. The primary cellwall contains 35% pectin, 25% cellulose, 20% hemicelluloseand 10% structural, hydroxyproline-rich protein (Brownleader

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

Pectic Polysaccharides and Fruit Softening

Pectins are the common components of the primary cell walland middle lamella contributing to the fruit texture. Pectin con-tent varies from fruit to fruit and pectins from fruits are usedfor commercial purposes, eg. apple, guava and citrus [Table 3]Whitaker, 1984; El-Zoghbi, 1994; Nwanekezi, Alawube, andMkpolulu, 1994; Thakur, Singh and Handa, 1997).

The word “Pectin” originated from the Greek word “Pec-tos” meaning, “gelled.” Native pectin plays an important rolein the consistency of fruit and also in textural changes duringripening, storage, cooking, or irradiation and other processingoperations. Tissue softening is attributed to enzymatic degra-dation and solubilization of the protopectin (Sakai, Sakomoto,Hallaert, and Vandamme, 1993). Pectins are likely to be thekey substances involved in the mechanical strength of the pri-mary cell wall and are important to the physical structure of theplant (Sirisomboon, Tanaka, Fujitha, and Kojima, 2000). Theirdegradation during ripening seems to be responsible for tissuesoftening, as reported for a number of fruits including tomato(Poovaiah and Nukuya, 1979; Seymour, Harding, Taylor, Hob-son, and Tucker, 1987), kiwi (Redgwell, Melton, and Brasch,1992), apple (De Vries, Voragen, Rombouts, and Pilnik, 1984),

Table 3 Pectin content of some fruit tissues

% Pectin contentFruits Tissue (fresh weight basis )

African Mango Pulp 0.72Apple Pulp 0.5–1.6Apple Pomace 1.5–2.5Avocado Pulp 0.73Banana Pulp 0.7–1.2Cashew Pomace 1.28Cherries Pulp 0.24–0.54Guava Pulp 0.26–1.2Lemon Pulp 2.5–4.0Lemon Peel 5.0Litchi Pulp 0.42Mango Pulp 0.66–1.5Orange Pulp 1.35Orange Peel 3.5–5.5Papaya Pericarp 0.66–1.0Passion fruit Pulp 0.5Passion fruit Rind 2.1– 3.0Peach Pulp 0.1–0.9Pineapple Pulp 0.04–0.13Strawberry Pulp 0.14– 0.44Tomato Pulp 0.2–0.6


and bush butter (Missang, Renard, Baron, and Drilleau, 2001a).The major changes in the cell wall structure are the dissolutionof middle lamella and primary cell wall during ripening. Thus,elucidation of the chemical structure of pectin is essential inunderstanding its role in plant growth/development and duringripening of fruits (Thakur et al., 1997).

Parenchymatous tissues are thought to consist principallyof calcium salts of pectic substances, which are deposited inearly stages of the cell growth, specifically when the area ofthe cell wall is increasing (Voragen et al., 1995). Pectic sub-stances are prominent structural constituents of primary cellwall and middle lamella and are the sole polysaccharides inmiddle lamella, along with some cellulose microfibrils, whilethey may be virtually absent in secondary walls (Van Buren,1979). Middle lamella are heat labile and their dissolution re-sults in the separation of plant cells. Ultrastructural studies inripening fruits have also shown that cell wall breakdown was ac-companied by dissolution of middle lamella and gradual dissolu-tion of fibrillar network of primary cell wall (Ben-Arie, Sonego,and Frankel, 1979; Crookes and Grierson, 1983; Jackman andStanley, 1995). Deesterified pectins in the middle lamella areassociated with calcium ions, and its removal also usually leadsto cell separation (Aman and Westerlund, 1996). The associa-tion involves binding of two or more polymeric chains, in theform of a corrugated egg-box (Fig. 4), with interstices in whichcalcium ions are packed and coordinated, creating an “egg-box”system (Grant, Morris, Rees, Smith, and Tom, 1973). Specificbinding of the divalent cations to pectins in an “Egg box model”leads to a firm cohesion between the chains (Grant et al., 1973).Calcium treatment inhibited softening of fruits due to an in-crease in cohesion of pectin network (Krall and McFeeters,1998). Generally, pectins in the cell wall are cross-linked throughionic interaction (Mac Dougall, Brett, Morris, Rigby, Ridoutand Ring, 2001). Due to this ability to form co-ordinationcomplexes with Ca2+, chelator soluble pectins are of specialinterest as they increase fruit firmness (Jimenez, Rodriguez,Fernandez-Caro, Guillen, Fernandez-Bolanos, and Heredia,2001).

Pectins are structural, acidic, homo-/heteropolysaccharidesobtained commercially from fruits but present universally inplant cell wall matrices. They are structurally diverse het-eropolysaccharides containing partially methylated galacturonicacid residues, methyl esterified pectins, deesterified pectic acidsand their salts, pectates [Table 4] and the neutral polysaccha-rides, which lack galacturonan backbone, i.e., arabinogalactans,arabinans, and galactans (Fig. 5). Several neutral plant polysac-charides are also grouped under pectins mainly because of theirassociation with acidic pectins as side chains to the main galac-

Figure 4 Egg-Box model depicting association of pectins with Ca++ ions.

Table 4 Chemical nature of pectic substances present in plant cell walls

Pecticsubstances Chemical nature

Pectic substances Group of colloidal, complex polysaccharides ofGalA

Protopectin Water-insoluble parent pectic substancesPectic acids Pectic substances free from methyl ester groupsPectates Normal or acid salts of pectic acidsPectinic acids Pectic substances partially esterified with methyl

groupsHigh methoxyl pectins Highly esterified (>50% esterified) pectinic

acidsLow methoxyl pectins Less esterified (<50% esterified) pectinic acidsPectinates Normal or acid salts of pectinic acids

turonan backbone. They may also be present as free polymers(Brownleader et al., 1999).

The pectin chain, α-D-galacturonans, i.e., α-D-galac-turanoglycans or poly(α-D-galactopyranosyluronic acid), con-sists largely of D-galacturonic acid linked by α (1 → 4) linkages(BeMiller, 1986). The carboxyl groups of pectin are partiallyesterified with methanol and the hydroxyl groups are partiallyacetylated with acetic acid (Pilnik and Voragen, 1970). Theyoccur mainly in chair L-form and as both C-1and C-4 hydroxylgroups are on the axial position, the polymer formed is a trans1,4-polygalacturonan (Sakai et al., 1993).

During ripening, softening of fruit is caused by the con-version of protopectin, the insoluble, high molecular weightparent 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 phe-nomenon is attributed to textural softening during ripening(Doreyappa Gowda, and Huddar, 2001). Protopectin increasesbefore physiological maturity, but decreases during mango fruitripening (Tandon and Kalra, 1984). The inter-relation betweendifferent pectic substances and their degradation is shown inFig. 6.

The degree of polymerization, degree of esterification, andthe proportion of neutral sugar side chains are the principalfactors contributing to the heterogeneity of pectic polysac-charides (Rexova-Benkova and Markovic, 1976). Pectins, likeother polysaccharides, are both polydisperse and polymolecu-lar, mainly due to their heterogeneous nature in both molec-ular weight and chemical structure (Bartley and Knee, 1982;BeMiller, 1986).

Three types of pectic polysaccharides have been structurallycharacterized. Homogalacturonans (HG) consist solely of linearchain of 1 → 4 linked α-D-galacturonans (see Fig. 5), in whichsome of the carboxylic groups are methyl esterified. They arefound to be ∼100 nm in length. It is a polysaccharide isolatedfrom only a few plant sources such as sunflower heads and seeds,sisal, the bark of amabilis fir, jack fruit and apple fruit (Pilnik andVoragen, 1970). It has also been isolated from the cell wall ofrice endosperm, primary cell wall of Rosa, sycamore (McNeil,Darvill, Fry, and Albersheim, 1984; Voragen et al., 1995), and


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 releasedfrom the heterogeneous pectic substances by the conditions em-ployed during extraction (Voragen et al., 1995).

Rhamnogalacturonan-I (RG-I) is primarily responsible forthe chemical and structural diversity of the pectins. It is themajor component of the primary cell wall and middle lamella ofdicotyledonous plants (McNeil, Darvill, and Albersheim, 1980).They consist mainly of the backbone of the repeating disaccha-ride units → 4)-α-D-GalA-(1 → 2)-α-L-Rha-(1→ (BeMiller,1986). Insertion of rhamnose in the main chain forms a ‘T’shaped “kink” in the polygalacturonan chain (Fig. 7), whichminimizes the frequency of interaction with adjacent polymericchains (Grant et al., 1973). Galacturonic acid residues typically


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 ofgalacturonic acid was reported in sugar beet pectins (Renard,Crepeau, and Thibault, 1999). Rhamnose residues are found asbranch 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-4with side chains consisting of D-galactose and/or L-arabinoseresidues. Small amounts of fucose, glucuronic acid, and 4-O-methyl β-D-glucuronic acid units are also found linked to rham-nose units (McNeil et al., 1980, O’Neill, Albersheim and Darvill,1990). RG-I was reported from a number of fruits includingtomato (Seymour, Colquhoun, Dupont, Parsley, and Selvendran,1990), grape (Nunan, Sims, Bacic, Robinson and Fincher, 1998;Saulnier, Brillouet, and Joseleau, 1988), apple (Schols, Posthu-mus and Voragen, 1990), pear (Schols and Voragen, 1994), kiwi(Redgwell et al., 1992), and raspberry (Stewart, Iannetta, andDavies, 2001), although the nature and length of the neutralsugar side chain varied.

Rhamnogalacturonan-II (RG-II) is invariably present as a mi-nor component of the cell wall, and has extremely complexstructures. It is not structurally related to RG-I, since it con-tains a high proportion of rhamnosyl residues, which occuras terminal (1 → 3) as well as branched (1 → 2, 3, 4,) units(Voragen et al., 1995). RG-II is a polysaccharide containing ahomogalacturonan backbone composed of at least eight 1 → 4linked α-D-galacturonic acid residues having side chains mainlycomposed of twelve glycosyl residues including several rare“diagnostic” monosaccharides such as apiose, 2-O-methyl-α-L-fucose, 2-O-methyl-α-D-xylose, aceric acid, KDO (2-keto-3-deoxy-D-manno-octulosonic acid), and Dha (3-deoxy-D-lyxo-heptulosaric acid) (Vidal, Williams, O’Neill and Pellerin, 2001).Recently, it was shown that RG-II is present predominately as adimer (O’Neill et al., 1996). These dimers are found cross-linkedby borate-diol esters, through apiosyl residues and play an im-

portant role in the structure and function of pectins (O’Neill et al.,1996). RG-II has been isolated from primary cell walls of tomato(O’Neill et al., 1990), apple (O’Neill et al., 1990) and kiwi fruit( Redgwell, Melton and Brasch, 1990). High amounts of RG-IIare present in fruit juices (Doco, Williams, Vidal and Pellerin,1997). RG-II binds heavy metals and has immuno-modulatingactivities, which has stimulated further research on structure ofRG-II and its role in human nutrition and health (Ridley et al.,2001).

Substituted galacturonans are a diverse group of pecticpolysaccharides that contain a backbone of linear 1,4-linkedα-D-galacturonic acid residues, substituted with other sugarresidues. Xylogalacturonans, in which β-D-xylose residues areattached to C-3 of the galacturonan backbone, are found in applepectin (Schols, Bakx, Schipper, and Voragen, 1995).

Regarding the neutral sugar side chains, considerable varia-tions were found in the nature, type, length, and structure of theside chains attached to the rhamnosyl residues of rhamnogalac-turonans (see Fig. 5). Usually, the ratio of rhamnose to galac-turonic acid is 1 : 40, as reported for citrus pectin (Zhan et al.,1998). Side chains composed of D-galactose and L-arabinoseoccur 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 dis-tributed 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 throughC-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 rham-nose. The presence of galacturonans rich in xylose has also beenreported in apple (Schols et al., 1990; De Vries et al., 1983). Theproportion of branched rhamnose residues varies with fruits,20–40% in grapes, tomato, and kiwi fruit, while it varies from


25–100% in apple (Voragen et al., 1995). RGs branched withseveral neutral polymers such as arabinans, galactans, and ara-binogalactans 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; Strasserand Amado, 2001).

Arabinans are branched polysaccharide chains composed ofα-(1 → 5) linked L-arabinose residues that contain single (ter-minal) L-arabinose side chains, linked to O-3 or O-2 positionof the main chain (see Fig. 5) (Whitaker, 1984; Voragen et al.,1995). They resemble a “comb-like” structure. Arabinan associ-ated pectins have been isolated from apple and has been recentlycharacterized from sugar beet pulp (Oesterveld et al., 2000).

Galactans are linear chains of β-(1 → 4) linked D-galactoseresidues (see Fig. 5). They occur as oligosaccharide chains at-tached to the rhamnose residues of the RG backbone (McNeilet al., 1980).

Arabinogalactans (AG) are heteropolymers of D-galactoseand L-arabinose residues (see Fig. 5). Two structurally differ-ent forms of arabinogalactans are found in plants. AG-I is asimple polysaccharide composed of chains of β-(1 → 4) D-galactose with single L-arabinose residues linked to O-3 of thegalactose (Whitaker, 1984; Smith, 1999). They have been iso-lated from different fruits including apple (Ovodov et al., 1971),kiwi (Redgwell et al., 1990), tomato (Seymour et al., 1990) andpineapple (Smith and Harris, 1995). AG-II is a complex andbranched polysaccharide, consisting of β-(1 → 3) D-galactoselinked to β-(1 → 6) D-galactose at O-6. The O-3 and O-6 posi-tions of the side chains are in turn linked to terminal L-arabinoseresidues (Whitaker, 1984; Strasser and Amado, 2001). Theypossess freeze-inhibition, water holding, and adhesive proper-ties. Plant arabinogalactans are known for their multifacetedphysiological and functional characteristics.

The pectic polymers of the primary cell wall have a rela-tively higher proportion of neutral oligosaccharide chains ontheir backbone (i.e., highly substituted pectins) and these sidechains are much longer than those of the pectins of middlelamella (Sakai et al., 1993; Selvendran, 1985). The side chainsare not distributed regularly but are concentrated in some regionscalled “hairy regions.” Highly esterified and slightly branchedrhamnogalacturonan, the “smooth regions,” are present in mid-dle 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 moleculeslink to protein, hemicellulose, and cellulose.

The acidic and neutral pectins carry non-sugar substituents,essentially methanol, acetic acid, phenolic acids, and amidegroups, and contribute further to the structural diversity ofpectins (Mac Dougall et al., 2001). The esterification of galactur-onic acid carboxyl with methanol or acetic acid is a very impor-tant structural characteristic of pectins. The degree of methyla-tion (DM) is defined as the percentage of carboxylic acid groupsesterified with methanol. The degree of acetylation (DAc) is de-fined as the percentage of galacturonic acid residues esterifiedwith one acetyl group (Voragen et al., 1995). Chelator-soluble

pectins have high DM and DAc than those extracted with al-kali, which is mainly due to the liberation by saponification ofmethyl ester and acetyl groups by alkali (Thomas and Thibault,2002). Phenolic acids, especially ferulic and p-coumaric acidsare found esterified to the non-reducing ends of the neutral ara-binose / galactose residues. These non-sugar substituents, espe-cially the ferulic acid facilitate oxidative cross-linking betweenpectins or with other polysaccharides in the cell walls, by theformation of diferuloyl bridges, which would limit wall exten-sibility (Brownleader et al., 1999) and play a significant role ingrowth regulation and defense mechanism.

Pectins are extracted from plant material using a wide varietyof extracting media. Some pectins are soluble in water indicatinglittle 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 suchas oxalates, hexametaphosphate, EDTA, CDTA, EGTA, etc. forextracting pectins. Chelating treatment is often combined withheating, and this treatment does not give a real proof for thepresence of Ca-bridges, as heating cleaves pectic backbone ir-respective of pH (Fry, 1986). At cold condition and neutral pH,CDTA removes all the Ca-bridges from the pectins, renderingits solubilization. Pectins are abundantly found in fruits, mod-erately in leafy vegetables and in low levels in cultured tissues(Fry, 1986) and originate from the middle lamella (Thomas andThibault, 2002). These pectins are found complexed with cal-cium ions (Thomas and Thibault, 2002). Pectins extracted withHCl (pH 1.5), had a wider molecular weight range with a peakmolecular weight slightly lower than that extracted with 0.5%EDTA or 0.25% ammonium oxalate. This suggests that acidmight hydrolyse pectins during extraction and EDTA or ammo-nium oxalate may be preferred for pectin extraction (Pathak,Chang, and Brown, 1988). Cold sodium carbonate (containingsodium borohydride) treatment would cause hydrolysis of interpolymeric ester bonds with negligible β-elimination degradation(Thomas and Thibault, 2002). It solubilizes the CDTA-insolublepectins and suggests that inter polymeric ester bonds help to holdpectins in the cell wall (Fry, 1986).

A simple model consisting of five different types of pectinwas proposed based on their molecular interactions with othercell wall constituents and extraction behavior (Chang, Tsai, andChang, 1993). The five different types of pectins present in planttissues are S-, A-, B-, C-, P-types [Table 5].

Table 5 Different types of pectins based on molecular interaction andextractability

BondingTypes (molecular interaction) Extractions

S-type pectins weak bonds/Van der waalsforce

with cold water

A-type pectins ionic interactions with cold chelating agentsB-type pectins intensive hydrogen bonding with hot waterC-type pectins hydrogen bonds and ionic

interactionswith hot chelating agents

P-type pectins covalent bonds with dilute acid /alkali


Pectins form gels under certain conditions and this propertyhas made them as useful additive in jams, jellies, and mar-malades, as well as in confectionery industries as stabilizersfor acid milk products (Voragen, 1995). They are used in a num-ber of foods as thickeners, texturizers, emulsifiers, etc. (Thakuret al., 1997). In recent years, pectin has been used as a fat orsugar replacer in low-calorie foods (Thakur et al., 1997). Theyhave a wide application in pharmaceutical industries, mainlybecause of their activities like antidiarrhea, antibacterial, an-tiviral, wound-healing, detoxicant, regulation, and protection ofgastrointestinal tract, delay in gastric emptying, lowering bloodcholesterol level, and glucose metabolism (Voragen et al., 1995;Thakur et al., 1997; Baker, 1997). Also, it is the major con-stituent in the fruit cell wall that undergoes drastic degradationby the carbohydrate hydrolases, during ripening, leading to fruitsoftening.

Changes in Pectic Polysaccharides during Ripening

During ripening fruits loose firmness, and unless the fruitis dehydrated, the osmotic properties of the cell and the turgorpressure usually remain constant. While in plant tissues, it isassumed that turgor pressure alone is not contributing to theloss of firmness, instead it is the result of changes in the cellwall polysaccharides (Van Buren, 1979). Much work done torelate chemical changes in cell walls to fruit softening has beenfocused towards the characterization of changes in pectic sub-stances (Krall and McFeeters, 1998). Pectins are the lone cellwall polysaccharides that are easily soluble in water and due tothis property they can be deesterified and depolymerized mostlyby enzymatic reactions. Also, retardation of textural softeningby the addition of Ca++ ions to fruit is related to the abilityof divalent cations to form calcium bridges between the pecticpolysaccharide chains (Krall and McFeeters, 1998). A limiteddegradation of the pectic polymers might be due to the methy-lation of galacturonic acid groups or their accessibility for de-polymerization (Voragen et al., 1995).

Loss of firmness during heat treatment of acid fruit has beenattributed to acid hydrolysis of glycosidic bonds in cell wallpolysaccharides. It was suggested earlier that hydrolysis of neu-tral sugar glycosidic bonds was involved in the softening process.Arabinofuranosyl linkages are most labile while glycosidic link-ages between galacturonans are most stable in pectins (Voragenet al., 1995). However in acidic pH (pH 2.5-4.5) hydrolysis ofgalacturonans occurs faster than neutral sugars, as inferred bythe loss of uronic acid residues from the cell wall, while the neu-tral sugars are still found associated with the pectic substances.Thus, the possible mechanism of softening during ripening atacidic condition is the hydrolysis of pectin (Krall and McFeeters,1998).

Changes in the proportion and characteristics of pectic sub-stances are reported in many fruits (Kertesz, 1951). Duringripening, the progressive loss of firmness is the result of a grad-ual solubilization of protopectin in the cell walls to form pectin

and other products (Grierson et al., 1981; John and Dey, 1986;Sakai et al., 1993). Solubilization followed by depolymerizationand deesterification of pectic polysaccharides is the most appar-ent 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 andCantliffe, 1989; Ranwala, Suematsu and Masuda, 1992), bellpepper (Gross, Watada, Kang, Kim, Kim and Lee, 1986), straw-berry (Huber, 1984; Nogata, Ohta and Voragen, 1993), kiwifruit(Redgwell et al., 1992), bush butter (Missang, Renard, Baron andDrilleau, 2001b), apricot (Femenia, Sanchez, Simal and Rosello,1998), melon (Rose, Hadfield, 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 esterifi-cation, a lower average molecular weight, and decreased neutralsugar content compared to pectins from unripe fruits (Huber andLee, 1986).

Cell Wall Hydrolases in Relation to Fruit Softening

The changes in the cell wall composition, which accompanyfruit softening during ripening, are due to the action of car-bohydrate hydrolases. They act on cell wall polymers, result-ing in their degradation. Most of these enzymes are present inlow levels and are constitutive throughout fruit development andripening (Tucker, 1993), but during ripening, generally all thehydrolases increase in activity, particularly cell wall hydrolases,showing a peak activity at climacteric stage.

Among cell wall hydrolases, pectin-degrading enzymes aremostly implicated in fruit softening. Increased solubilization ofthe pectic substances, progressive loss of tissue firmness, anda rapid rise in the PG activity accompany normal ripening inmany fruits. (Brady, 1987; Tucker, 1993; Fischer and Bennett,1991; Pressey, 1986a). Since the pectic polymers begin to ac-quire solubility only after PG has become active, it is believedthat this enzyme is involved in the breakdown of the insolublecomplex polysaccharides by reducing the length of the chainscross-linked by calcium (Wong, 1995). A positive correlation be-tween the appearance of PG and initiation of softening is shownin a number of fruits like guava (El-Zoghbi, 1994), papaya (Paulland Chan, 1983), and mango (Roe and Bruemmer, 1988). PGand PME activity increased remarkably in peach, tomato, andpear (Tucker and Grierson, 1987). In apple and strawberry fruitsthe mechanism of solubilization of polyuronide is thought to bedifferent 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 depoly-merization of acidic pectins by polygalacturonase. However, ex-periments with transgenic tomatoes have shown that even thoughPG is important for the degradation of pectins, it is not the soledeterminant of tissue softening during ripening (Gray, Picton,Shabbeer, Schuch, and Grierson, 1992). PG antisense constructsfor various tomato lines have little effect on the fruit characteris-tics, viz, reduced susceptibility to cracking, and decay and other


damages at the later stages of ripening (Gray et al., 1992). Nowthe focus is on the hydrolysis of neutral sugar side chains, whichmay weaken the complex network of cell wall polymers, thuscontributing to textural softening (Smith and Harris, 1995). Thevariation in pectins from different sources is mainly attributedto the arrangement of these neutral sugar side chains resultingin configurational rearrangements.

The loss of neutral sugar side chains from pectin is one of themost important features occurring during ripening. Substantialvariation in the cell wall composition among fruits and fruittissues exists. Further, their metabolism in relation to softeningalso varies from fruit to fruit (Gross and Sams, 1984). Out of 17types of economically important fruits, 14 types showed a netloss of neutral sugars, galactose, and arabinose, from the cell wallduring ripening. No such loss of neutral sugars occurs in ripeningplum and cucumber fruits (Gross and Sams, 1984). The mutanttomato fruit (‘rin’) containing little or no PG activity showed asubstantial loss of galactose from the cell wall suggesting thatthis loss is not due to the action of PG. This evidence suggeststhat other cell wall hydrolases, especially glycosidases, play animportant role in the textural softening during ripening (Grayet al., 1992).

One novel approach to elucidate the role of enzymes in cellwall degradation and softening is to employ antisense RNAtechnology. This technology was one of the first molecular ap-proaches used for delaying fruit ripening (Bansal, 2000). It hasbeen possible to obtain firmer tomatoes with longer shelf-lifeby specific suppression of PG gene expression with antisenseRNA (Smith et al., 1988). Pectin methyl esterase (PME) sup-pression resulted in increased solid content in tomato (Tieman,Harriman, Ramamohan and Handa, 1992). The genes coding forPG, PME, and other enzymes have been cloned in tomato (Grayet al., 1992) and other fruits (Bansal, 2000).

A wide range of cell wall hydrolases are identified in fruit tis-sues (Fischer and Bennett, 1991; Ahmed and Labavitch, 1980;Fry, 1995; Wallner and Walker, 1975). The major hydrolasesinvolved in polysaccharide dissolution in vivo can be broadlyclassified into 2 types of hydrolases; viz, glycanases and glycosi-dases [Table 6]. Glycanases (glycanohydrolases) by definitionare a class of enzymes cleaving high molecular weight poly-mers (polysaccharides) into shorter chains, while glycosidases(glycohydrolases) generally act on shorter chain oligosaccha-rides, which may be homo- or heterooligomers or glycoproteinsor glycolipids. Recently, it has been reported that temperatureplays a crucial role in the activities of these cell wall hydrolases(Reddy and Srivastava, 1999).

Enzymes Related to Pectin Dissolution In Vivo

Pectolytic enzymes are widespread in plants, fungi, and bac-teria. They constitute a unique group of enzymes that are re-sponsible for the degradation of pectin and pectic substances inplant cell walls [Table 7]. They act on plant tissues, especiallyon the main polyuronide chains of pectins and eventually cause

Table 6 Different types of carbohydrate hydrolases in fruits

Glycanases Glycosidases

Polygalacturonase α-MannosidasePectin methyl esterase α-GalactosidaseCellulase β-GalactosidaseHemicellulase α-GlucosidaseAmylase β-GlucosidaseMannanase α-HexosaminidaseGalactanase β-HexosaminidaseGlucanase α-XylosidaseArabinanase β-XylosidaseXylanase α-ArabinosidaseRhamnogalacturonase β-Arabinosidase

cell lysis. The other enzymes such as arabinanase, galactanase,and β-galactosidase act on the side chains of the galacturonidebackbone, eventually degrading the entire pectic substance.

Pectic enzymes have been used for the clarification of winessince the beginning of the 19th century. They are industriallyuseful enzymes for extraction, clarification, and liquefactionof fruit juices and wines (Chauhan, Tyagi, and Singh, 2001).They are also used in the fabric industry to soak plant fibers andin the paper making industry to solve the retention problemsby de-clogging the pulps (Sakai et al., 1993). They hydrolysethe pectic substances and aid in the flocculation of suspendedparticles and clarification of wines and juices (Chauhan etal., 2001). Recently, immobilized pectic enzymes are gainingimportance in this area (Alkorta, Garbisu, Llama, and Serra,1998). PG from a fungal source is commercially utilized inthe fruit juice industries. One of the technically importantdifferences between PG from tomato and fungal source is theinhibition of the latter by some vegetable extracts, which mayrender them useless in the preparation of vegetable maceratesfor baby foods. Thus, fruit PGs are gaining importance.

Pectin-degrading enzymes are classified, based on their modeof action on pectin and pectic substances into PG, PME, pectatelyase, 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 enzymeplaying a significant role in pectin dissolution in vivo (Poovaiahand Nukuya, 1979). PG is a hydrolytic enzyme, which acts onpectic acid (polygalacturonic acid, PGA). It hydrolyses the α-1,4-glycosidic bonds between the galacturonic acid residues ingalacturonans.

Based on their mode of action, PGs are classified into exo-PG(exo-poly (1,4 α-D-galacturonide) galacturonohydrolase, EC3.2.1.67) and endo-PG (endo-poly (1,4 α-D-galacturonide) gly-canohydrolase, EC 3.2.1.15). The former catalyses the hydroly-sis of the glycosidic bonds between the de-esterified galacturo-nans from the non-reducing end, which results in the release ofgalacturonic acid as the major reaction product. The rate of hy-drolysis depends on the degree of polymerization and it increaseswith increase in the molecular size of the substrate (Pressey and


Table 7 Classification of pectin-degrading enzymes

Enzymes Substrate Products Mechanism

Pectin Methyl Esterase Pectin Pectic acid + Methanol HydrolysisPolygalacturonasesProtopectinase Protopectin Pectin HydrolysisEndo-PG Pectic acid Oligogalacturonides HydrolysisExo-PG Pectic acid Monogalacturonides HydrolysisOligogalacturonide hydrolase Trigalacturonic acid Monogalacturonides Hydrolysis� 4:5 unsaturated

Oligogalacturonide hydrolase� 4:5 (galacturonide)n Unsaturated monogalacturonide +

galacturonides (n-1)Hydrolysis

Endopolymethyl galacturonase Pectin Methyl-oligogalacturonides HydrolysisRhamnogalacturonase Pectin α-(1,2)linked L-Rha, α-(1,4) linked

D-GalHydrolysis

Rhamnogalacturonanacetylesterase

Pectin (Hairy region) Pectin + Acetic acid Hydrolysis

Pectin acetyl esterase Lyases Pectins (Smooth region) Unsaturated oligogalacturonides HydrolysisEndopectate lyases Pectic acid Unsaturated oligogalacturonides Trans eliminationExopectate lyases Pectic acid Unsaturated digalacturonides Trans eliminationOligogalacturonide lyases Unsaturated digalacturonate Unsaturated monogalacturonides Trans eliminationEndopectin lyases Pectin Unsaturated oligogalacturonides Trans elimination

Arabinanaseα-L-Arabino-furanosidase Arabinans α-L-Arabinose HydrolysisEndoarabinanase (1,5)-α-Arabinans Arabinose and higher

oligosaccharidesHydrolysis

Galactanaseβ-D-Galactanase Galactans β-D-Galactose Hydrolysis

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

Figure 8 Mode of action of pectin degrading enzymes.

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


the chain. Some fruits like apple, Freestone peach, and persim-mon possess only exo-PG, while other fruits such as apple, avo-cado, Clingstone peach, lemon, mango, musk melon, raspberry,and kiwi contain only endo-PG (Lang and Dornenburg, 2000).Cucumber, papaya, passion fruit, peach, pear, strawberry, andtomato contain both endo- and exo-PGs (Lang and Dornenburg,2000). The marked difference in the textural characteristics ofthe two types of peaches (Clingstone and Freestone) is attributedto the differences in PG (Pressey, 1986b; Pressey and Avants,1978). The extent and rate of textural softening during ripeningis directly related to PG composition, i.e., extensive softeningoccurs if endo- or both endo- and exo-PG are present and lim-ited softening occurs if only exo-PG is present (Bartley, 1978;Huber, 1984).

It is generally accepted that PG is primarily responsible fordissolution of the middle lamella during fruit ripening (Jackmanand Stanley, 1995; Voragen et al., 1995). There is a clear correla-tion between the appearance of PG and the onset of dissolutionof middle lamella and the primary cell wall during ripening(Crookes and Grierson, 1983). PG alone is sufficient to dissolvemiddle lamella in apple, but both PG and cellulase are requiredin pear for dissolution.

One of the most characteristic changes during fruit ripen-ing is decrease in firmness. This has been shown to be associ-ated with an increased activity of pectic enzymes, particularlyPG (Crookes and Grierson, 1983; Watkins, Haki, and Frenkel,1988). An increase in the total PG activity prior to the respira-tory climacteric stage of the tomato suggested that this enzymemight play a role in initiating the ripening process (Poovaiahand Nukuya, 1979). However, no detectable endo-PG, an en-zyme thought to play a role in tomato softening, was found inpre-climacteric tomato and appearance of endo-PG in tomatoesafter the onset of climacteric ethylene was reported (Griersonet al., 1985; Baldwin and Pressey, 1990). The absence of PG inunripe fruits and appearance near the onset of ripening with in-creased activity during ripening, along with concomitant pectindegradation suggest that this enzyme is implicated in pectin sol-ubilization. The appearance of soluble pectin was the result of anincreased activity of PG during ripening (Tucker and Grierson,1987). This suggests that fruit softening is regulated by the ac-cumulation of PG and the rate of splitting of pectin. PG acts onthe de-esterified portion of the galacturonan chains, particularlyon those glycosidic bonds, which have the carboxylic groupsadjacent to the glycosidic linkage, and free from esterification.

PG was first found in ripe tomato fruit, and it still remainsthe richest plant source of the enzyme (Pressey, 1986c; Wong,1995). Recently, an increase in PG activity with a peak at the cli-macteric stage in mango (Prabha, Yashoda, Prasanna, Jagadeesh,and Bimba Jain, 2000), capsicum (Priyasethu, Prabha, and Tha-ranathan, 1996), and banana (Prabha and Bhagyalakshmi, 1998)was reported from our lab. An increase in PG activity in seven In-dian mango cultivars during ripening was also reported (Selvarajand Kumar, 1989). In climacteric fruits, whose texture altersconsiderably during ripening, a maximum loss of firmness wasdirectly 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 ex-plants, and seedlings are also reported to contain PG, althoughtheir biochemical/physiological aspects may differ considerablyfrom those of fruit (Pressey, 1986b).

Recently, isozymes of PG were reported from banana (Pathakand 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 endo-acting (Pressey and Avants, 1973b), splitting glycosidic bondsrandomly and releasing oligogalacturonides (Ali and Brady,1982). Both isozymes have pH optima in the acidic range andSDS-PAGE suggests that PG1 is a dimer of PG2 (Tucker, Robert-son, and Grierson, 1980). Later studies suggest that PG1 is pro-duced by the combination of both PG2 and a β-subunit (con-verter) (Pressey, 1986a; 1986b). Both PGs are glycosylated. TwoPG2 isoenzymes (PG2A and PG2B) have been characterized andare the product of post-translational modification or glycosyla-tion. It was shown that these two PG2 isozymes have similarpolypeptides, but have differences in the degree of glycosyla-tion (DellaPenna and Bennett, 1988). DellaPenna and cowork-ers (DellaPenna, Lashbrook, Toenjes, Giovannoni, Fischer, andBennett, 1990) demonstrated that all the PG isozymes arise bydifferential processing of a single gene product. The physio-logically active form of PG in tomato is PG1, which is activeenough to carryout both solubilization and depolymerization(DellaPenna et al., 1990).

Multiple forms appear due to genetic variants (allelic), ge-netically independent proteins, or heteropolypeptide chains thatare bound non-covalently. However conformational differences,covalent alteration, or conjugation may also cause multiplicityof enzymes (Dey and Del Campillo, 1984). The significance ofthese multiple forms may be related to the complex nature of thepectic substrates and their modification during ripening (Presseyand Avants, 1972). The PG gene was the first to be cloned fromtomato for studying textural regulation in ripening fruit and thetransformed tomato with PG antisense gene resulted in improvedfruit with firmer texture and an extended shelf life (Smith et al.,1988). This gave remarkable clues regarding the role of PG infruit cell wall metabolism. However, despite similar catalyticproperties, PGs differ from fruit to fruit, thus reducing the per-cent homology of the PG genes. Thus it is necessary to studythis enzyme individually in the fruit of choice.

Methods for quantification assay of PG have been well doc-umented (Pressey, 1986b). PG activity is generally measuredby an increase in reducing equivalents. The more recent spec-trophotometric method for quantification of reducing equivalentis by using 2-cyanoacetamide (Pressey, 1986c). Measurement ofviscosity changes using an Oswald viscometer is less convenientfor routine measurement but still it is useful in distinguishingbetween endo- and exo-splitting PGs. This is by comparing therate of decrease in viscosity with rate of hydrolysis, as measuredby increase in reducing equivalents. An endo-splitting enzymecauses around 50% reduction in viscosity when only 3–5% ofthe glycosidic bonds are cleaved, while an exo-splitting enzyme


causes similar reduction in viscosity with as much as 10–15%of the glycosidic bond cleavage. Other difference between theseenzymes is in the nature of product formed, at the beginning ofthe reaction. The endo-splitting enzyme does not produce lowmolecular weight products, whereas an exo-splitting enzymeresults in low molecular weight products.

Due to the presence of rhamnose in almost all fruit pectins, PGalone is not sufficient for pectin degradation. It seems that otherglycanases, such as rhamnogalacturonase, are also responsiblefor the degradation of rhamnogalacturonan backbone.

Rhamnogalacturonase (RGase)

Rhamnogalacturonase is an enzyme that catalyses the hy-drolysis of glycosidic bonds between galacturonic acid andrhamnose units in RG backbone, the “hairy regions” of manyfruit pectins (Schols and Voragen, 1994; Colquhoun, de Ruiter,Schols, and Voragen, 1990). The products are oligomers with al-ternating galacturonic acid and rhamnose units, rhamnose form-ing the non-reducing end (Schols and Voragen, 1994). RGase ac-tivity enhances strongly when the ester groups are de-esterifiedand the side chains are removed (Schols and Voragen, 1994).RGase activity is hindered by O-acetyl group. Thus, they actalong with rhamnogalacturonan acetylesterase, which splits offacetyl groups from the “hairy regions” of pectin (Voragen et al.,1995). Recently, the probable presence of RGase was also re-ported for bush butter fruit (Missang et al., 2001b). These cellwall glycanases (PG and RGase) appear to be more active on de-esterified pectins than esterified pectins (Seymour et al., 1987).Therefore, de-esterification is the most important reaction andis catalyzed by a unique group of enzymes, the pectin methylesterase.

Pectin Methylesterase (PME)

PME (Pectin pectylhydrolase, EC 3.1.1.11) catalyses the hy-drolysis of pectin methyl ester groups, resulting in deesterifica-tion. PME is specific for galacturonide esters and its action isto remove methoxyl groups from methylated pectin by nucle-ophilic attack. This results in the formation of an acyl enzyme in-termediate with the release of methanol, followed by deacylation(hydrolysis) to generate the enzyme and a carboxylic acid. PMEsof plant origin exhibit an action pattern that results in the forma-tion of carboxylate groups along the pectin chain (Wong, 1995).De-esterification appears to proceed linearly along the chainresulting in blocks of free carboxyl groups (Rexova-Benkovaand Markovic, 1976). It appears that PME preferentially attacksmethyl ester bonds of a galacturonate unit next to non-esterifiedgalacturonate unit (Pilnik and Voragen, 1970). Thus, they de-esterify the esterified pectic substances, making them vulnerablefor PG action. Its action may be a prerequisite for the action ofPG during ripening.

PME activity was detected in fruits like apple, banana, cherry,citrus, grape, papaya, peach, pear, tomato, and strawberry (Pilnikand Voragen, 1970). The activity of PME increases as mature

green tomatoes pass through different color stages to becomefull red. Unripe fruits are rich in PME, while ripe fruits are richin 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 (Selvarajand Kumar, 1989; Aina and Oladunjoye, 1993) or remain con-stant (Ahmed and Labavitch, 1980; Ashraf, Khan, Ahmed, andElahi, 1981) during fruit ripening. PME has been purified andcharacterized in few ripening fruits (Pressey and Avants, 1972;Tucker, Robertson and Grierson, 1982). Several PME isozymeshave been identified in tomato (Tucker, Robertson, and Grierson,1982). The slow ripening of “Abu-Samaka” mango in spite ofhigh PG activity, suggests a key role to PME in controlling fruitsoftening (Abu-Sarra and Abu-Goukh, 1992).

PME also acts on commercial methylated pectin (citrus) toliberate the carboxyl group and methanol. The activity may beassayed by estimating the released methanol chromatographi-cally. A continuous spectrophotometric assay has been devel-oped based on the reaction of PME on pectins in the presence ofa pH indicator bromothymol blue. The carboxylic groups pro-duced by hydrolysis of ester groups lower the pH, causing theindicator dye to change the color (Doner, 1986).

By genetic engineering, it has been shown that PME maynot be the sole determinant of softening, and other enzymesmay be involved in textural softening. But an increase in thetotal soluble solid was a very important and significant findingin ripening tomato as demonstrated from PME suppression byantisense construct (Gray et al., 1992; Tieman et al., 1992).

Lyases

The lyases or trans eliminases cleave the glycosidic bond bytrans β-elimination mechanism, i.e., elimination of hydrogenfrom the C-4 and C-5 position of the aglycone portion of thesubstrate (Whitaker, 1984). It is known that in alkaline medium,pectin undergoes deesterification, accompanied by degradationby β-elimination reaction. Similar splitting of glycosidic bondsalso occurs in neutral pH at elevated temperature. These enzymesare absent in fruit but are present only in microorganisms.

Pectate lyases (PL) catalyses the cleavage of de-esterified oresterified galacturonate units by a trans β-elimination of hydro-gen from the C-4and C-5 positions of galacturonic acid. Exo-PL (exo-poly 1,4 α-galacturonide) lyase, EC 4.2.2.9) acts fromthe non-reducing end, whereas endo-PL (endo-poly 1,4 α-Dgalacturonide) lyase, EC 4.2.2.2) acts randomly on de-esterifiedgalacturonans. Pectin lyase (PNL) (EC 4.2.2.10) catalyzes thecleavage of esterified galacturonate units by trans β-elimination.All PNLs studied so far are endo-enzymes, acting randomly(Wong, 1995).

Arabinanase

Arabinanase are of two types; arabinofuranosidase (EC3.2.1.55) and endo-arabinanase (EC 3.2.1.99). They reduce thedegree of branching and increase the polymer–polymer asso-ciation (Whitaker, 1984). Endo-arabinanase hydrolyses linear


arabinan in a random fashion producing oligomers of shorterlengths. Arabinofuranosidase degrades branched arabinan to alinear chain by splitting of terminal α-1,3-linked arabinofura-nosyl side chains and sequentially breaks the α-1,5 links at thenon-reducing end of linear arabinan. This enzyme hydrolysesthe terminal non-reducing arabinofuranosyl groups from a rangeof arabinose-containing polysaccharides such as, arabinogalac-tans, arabinans, and arabinoxylans. The substrates most widelyused for the assay of arabinofuranosidase are p-nitrophenyl-α-L-arabinofuranoside, phenyl-α-L-arabinofuranoside and β-L-arabinan. The release of L-arabinose is quantitated either byreducing group estimation or by HPLC.

Galactanase

Galactanases are of two types; endo-galactanase (EC3.2.1.89), which catalyses the random cleavage of the β-1,4linkages of galactan chains and other galactanases (EC 3.2.1.90),which also randomly hydrolyses the β-1,3 and β-1,6 linkagesof galactans, present as side chains in pectins. An increase in theactivity of arabinanase and galactanase in mango, banana, andcapsicum was reported (Prabha et al., 2000; Priyasethu et al.,1996; Bhagyalakshmi, Prabha, Yashoda, Prasanna, Jagadeeshand Tharanathan, 2002). Recently, exo-(1-4)-β-galactanase waspurified and characterized from tomato (Carey et al., 1995).

β-Galactosidase. It is very well understood by molecularevidence that PG activity alone is not responsible for the degra-dation of pectins to the extent that occurs during fruit ripen-ing. Initial softening was not correlated with the increase of PGactivity in ripening apples. Further, in ripening inhibitor mu-tant ‘rin’ tomato, little or no PG activity was detected, but asubstantial amount of galactose was lost indicating the involve-ment of other enzymes. The apparent absence of PG in somefruits that soften normally has implied other alternative mech-anisms of cell wall dissolution (Ranwala et al., 1992; Grosset al., 1986). This evidence stimulated further research on thisglycosidase. This enzyme is also implicated in pectin dissolu-tion by way of deglycosylating the galactan, which is gener-ally present in pectin–type of polymers. Thus, loss of neutralsugars has become a general feature of fruit ripening (Grossand Sams, 1984). This loss of neutral sugar residues is separateand independent of polyuronide solubilization during ripening,and independent of PG activity, suggesting the involvement ofβ-galactosidase/galactanase, which have been associated withmany ripening fruits (Carey et al., 1995; Rose et al., 1998)

β−Galactosidase (EC 3.2.1.23), a glycosidase, acts on shortchain oligomers of galactose units present either as glycoprotein,glycolipid, or hetero-/homopolysaccharides. This enzyme par-tially degrades the pectic and hemicellulosic components of thecell wall and is possibly related to the breakdown of polysac-charides at over-ripening. β-Galactosidase was detected in awide variety of fruit systems (Dey and Del Campillo, 1984). In-crease in β-galactosidase activity during ripening was reportedin many fruits (Bartley, 1974; John and Dey, 1986). It was re-ported that this enzyme also increases during the developmental

stages of fruits like mango (Rahman, Akhter and Absar, 2000).This enzyme has been purified from a number of fruits includ-ing tomato (Pressey, 1983), apple (Ross, Wegrzyn, MacRae andRedgwell, 1994), orange (Burns, 1990), muskmelon (Ranwalaet al., 1992), coffee berry (Golden, John and Kean, 1993), sweetcherry (Andrews and Li, 1994), sapota (Dore Raju and KarunaKumar, 1996), and “Harumanis” mango (Ali, Armugam andLazan, 1995). This enzyme is incapable of degrading nativegalactans in citrus fruit (Burns, 1990). However, in some fruitslike tomato (Pressey, 1983), muskmelon (Ranwala et al., 1992),and apple (Ross et al., 1994), they attack native galactan poly-mers. In most studies of fruit β-galactosidase, the syntheticsubstrate, para-nitrophenyl-β-D-galactopyranoside was widelyused. The other substrates used for assaying the activity werephenyl-β-D-galactopyranoside, arabinogalactans, galactoman-nan and lactose.

Other Hydrolases Implicated in Fruit Softening

Hemicelluloses are (neutral) polysaccharides extracted by al-kaline solutions from the cell wall residues after the extraction ofpectic polysaccharides. The inert, insoluble, crystalline cell wallmaterial remaining after the hemicellulose extraction, which ismainly composed of β-glucose, is cellulose (Van Buren, 1979).

An apparent dissolution of the middle lamella and cell wallfibrillar network due to cellulolytic activity in ripening of av-ocado, pear, and apple has been demonstrated (Knee, 1973).Ripening associated changes involving dramatic decrease inthe molecular size of hemicellulose are reported in tomato(Huber, 1983), strawberry (Nogata et.al., 1993), pepper (Grosset al., 1986), muskmelon (McCollum et al., 1989), melon (Roseet al., 1998), mango (Mitcham and McDonald, 1992), andpeach (Hegde and Maness, 1998). The amount of hemicellu-lose decreased steadily during ripening of many fruits includ-ing mango (Mitcham and McDonald, 1992). Decline or lossof substantial levels of characteristic monomers of hemicellu-loses viz. glucose, xylose, and mannose occur during ripening offruits.

Little is known about the enhancement of cellulase or hemi-cellulase activity in relation to fruit softening. Cellulase is amultienzyme system composed of several enzymes, viz. endo-glucanase (EC 3.2.1.4), exo-glucanase (EC 3.2.1.91) and glu-cosidase (EC 3.2.1.21) (Sobotka and Stelzig, 1974). Endo-glucanase hydrolyses the β-1,4-link between adjacent glucoseresidues at random positions. Exo-glucanase breaks the bond atnon-reducing ends of the chain, producing glucose or cellobiose(dimers of β-1,4-linked glucose), whereas β-glucosidase splitscellobiose into glucose molecules.

Cellulase activity increased during ripening of avocado,peach, strawberry, tomato, and papaya (Hobson, 1981). Cel-lulase levels in unripe fruit are generally low and increase dra-matically during ripening. The loss of firmness, climacteric riseof respiration and ethylene evolution in ripening fruit were di-rectly correlated with marked increase in cellulase activity (Roe


and Bruemmer, 1981; Abu-Sarra and Abu-Goukh, 1992). Cel-lulase activity in normal and non-ripening mutants of tomatosuggests that this enzyme has no primary role in fruit soften-ing (Poovaiah and Nukuya, 1979). However, cellulase has beenimplicated in the softening process in tomato (Hobson, 1981).Cellulase activity was reported in several Indian mango cul-tivars, which increased during ripening (Selvaraj and Kumar,1989). No cellulase activity was detected in pears (Ahmed andLabavitch, 1980).

Xylanases (EC 3.2.1.8) catalyse the hydrolysis of β-1,4-xylan. β-1,4-D-endo-xylanase and β-1,4-D-exo-xylanase arereported as cell wall degrading enzymes from fruits includ-ing banana (Prabha and Bhagyalakshmi, 1998) and capsicum(Priyasethu et al., 1996). In papaya during ripening, a clearcorrelation between polygalacturonase and xylanase activities,climacteric rise in respiration and ethylene evolution and fruitsoftening were demonstrated (Paull and Chan, 1983). Man-nanase catalyses the hydrolysis of mannan polymer in capsicum(Priyasethu et al., 1996) and mango (Prabha et al., 2000). Xy-lanase, arabinanase and mannanase are localized both in solubleand bound form, which will increase during ripening. It was in-teresting to note that arabinanase, galactanase, and mannanasewere very prominent enzymes in mango fruit, having activitypeaks at climacteric stage of ripening (Bhagyalakshmi et al.,2002; Prabha et al., 2000). Among glycosidases, the promi-nent enzymes found in ripening fruit were β-hexosaminidase,α-mannosidase and α- and β-galactosidases (Priyasethu et al.,1996).

α-Amylase (EC 3.2.1.1) and β-amylase (EC 3.2.1.2) are thetwo amylases in plant tissues capable of metabolizing starch, α-amylases hydrolyse the α-1,4-linkages of amylose at random toproduce a mixture of glucose and maltose, whereas β-amylaseattacks only the penultimate linkage from the non-reducing endand thus releases only maltose. These enzymes are unable todegrade the α-(1 → 6) branch points of amylopectin, which arecatalyzed by debranching enzymes. Amylase activity increasesto some extent during ripening of many fruits (Fuchs et al., 1980;Tucker and Grierson, 1987). Mango and banana are the majorstarch 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 texturalsoftening during ripening (Bhagyalakshmi et al., 2002).

BIOTECHNOLOGICAL IMPLICATIONS ANDFUTURE PROSPECTS

The world population is expanding at a faster rate than thatof food production. Thus increasing the availability of foodhas become a vexing problem. This problem of population–food-imbalance can be solved either by limiting populationgrowth or by increasing food supplies. But both require con-siderable amount of capital and time to achieve. Consider-ing the nutritional and pharmacological significance, fruitswill play an important role in the nutrition of the world

population. A major step contributing towards this is toprevent the fruit loss between the time of harvesting andconsumption.

Fruits are an important part of a healthy and balanced diet.They provide us with essential vitamins, proteins, minerals, andfibers. They are also aesthetically pleasing to our eye and olfac-tory sense organs. However, unlike most other food commodi-ties, fruits are also living organisms, even after harvesting. Asa result of their biological nature, they are subjected to phys-ical, chemical, and microbiological spoilages. Current storagefacilities of fruits usually employ refrigeration alone, or cou-pled with controlled or modified atmospheres, in which O2 ismaintained at low level, and CO2 level at high. These tech-niques are expensive and can result in damage to the storedfruits. In addition, the fruits have to be treated at each harvestand also individually. Nevertheless, the economic viability ofsuch technology depends on local, regional and national socioe-conomic conditions, as well as national and international tradepolicies.

Recent advent in molecular biology has provided a betterunderstanding of the biochemistry of fruit ripening as well asproviding a hand for genetic manipulation of the entire ripen-ing process. These techniques are used to investigate the roleof hydrolases in cell wall degradation, ethylene synthesis, pig-ment synthesis, starch degradation, and hence fruit softeningduring ripening. These techniques could be used to manipu-late the ripening process genetically, and have significant com-mercial advantages. Currently, such manipulations are restrictedonly to some fruits such as tomato fruit. The resultant fruit hasa longer shelf life and are more resistant to disease and crack-ing during transportation. The major obstacle to achieving thisaim in other fruits is the lack of fundamental biochemistry oftheir ripening process. In future, more aspects of ripening maybe cracked-down and can be used to manipulate the fruits to ouradvantage.

One more aspect is that the plant enzymes are gaining greaterimportance in modern food biotechnology. The important dif-ference between enzymes of fruit and fungal source is the inhi-bition of the latter by some vegetable extracts, which may renderthem useless in the preparation of vegetable macerates for babyfoods. Not only this, usage of microbial products such as en-zymes is decreasing day-by-day because of awareness by theconsumers. Thus prospects for plant enzymes in the future arevery promising. A constant research and development effort inorder to optimize plant enzymes will lead to products that areeven better than those of earlier. It is desirable that significantbreakthroughs will naturally be made in the near future, muchto the benefit of the society at large.

REFERENCES

Abu-Sarra, A.F., and Abu-Goukh, A.A. 1992. Changes in pectinesterase, poly-galacturonase and cellulase activity during mango fruit ripening. Journal ofHorticultural Science, 67:561–568.


Adams, D.O., and Yang, S.F. 1979. Ethylene biosynthesis: Identification of1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversionof methionine to ethylene. Proceedings of National Academy of Science, USA,76:170–174.

Ahmed, A.E.R., and Labavitch, J.M. 1980. Cell wall metabolism in ripeningfruit. II. Changes in carbohydrate-degrading enzymes in ripening ‘Bartlett’pears. Plant Physiology, 65:1014–1016.

Aina, J.O., and Oladunjoye, O.O. 1993. Respiration, pectolytic activity and tex-tural changes in ripening African mango (Irvingia gabonensis) fruits. Journalof the Science of Food and Agriculture, 63:451–454.

Ali, Z.M., and Brady, C.J. 1982. Purification and characterization of the poly-galacturonases of tomato fruits. Australian Journal of Plant Physiology,9:155–169.

Ali, Z.M., Armugam, S., and Lazan, H. 1995. β-Galactosidase and its signifi-cance in ripening mango fruit. Phytochemistry, 38:1109–1114.

Alkorta, I., Garbisu, C., Llama, M.J., and Serra, J.L. 1998. Industrial applicationsof pectic enzymes: A review. Process Biochemistry, 33:21–28.

Aman, P., and Westerlund, E. 1996. Cell wall polysaccharides: Structural, chem-ical, and analytical aspects. In: Eliasson, A.C. (Ed.). Carbohydrates in Food.pp. 191–226. Marcel Dekker Inc., New York.

Andrews, P.K., and Li, S., 1994. Partial purification and characterization of β-D-galactosidase from sweet cherry, a non-climacteric fruit. Journal of Agri-cultural and Food Chemistry, 42:2177–2182.

Ashraf, M., Khan, N., Ahmed, M., and Elahi, M. 1981. Studies on thepectinesterase activity and some chemical constituents of some Pakistanimango varieties during storage-ripening. Journal of Agricultural and FoodChemistry, 29:526–528.

Baker, R.A. 1997. Reassessment of some fruit and vegetable pectin levels. Jour-nal of Food Science, 62:225–229.

Baldwin, E.A., and Pressey, R. 1990. Exopolygalacturonase elicits ethyleneproduction in tomato. HortScience, 25:779–780.

Bansal, K.C. 2000. Biotechnology for improving product quality in horticulturalcrops. In: Chadha, K. L, Ravindran, P.N and Leela Sahijram (Eds.) Biotechnol-ogy in Horticultural and Plantation Crops. pp. 201–218. Malhotra PublishingHouse, New Delhi, India.

Bartley, I.M. 1974. β-Galactosidase activity in ripening apples. Phytochemistry,13:2107–2111.

Bartley, I.M. 1978. Exo-polygalacturonase of apple. Phytochemistry, 17:213–216.

Bartley, I.M., and Knee, M. 1982. The chemistry of textural changes in fruitduring storage. Food Chemistry, 9:47–58.

BeMiller, J.N. 1986. An introduction to pectins: Structure and properties. In:Fishman, M.L., and Jen, J.J. (Eds.) Chemistry and Function of Pectins. pp. 2–12. Amer. Chem. Soc., Washington D.C.

Ben-Arie, R., Sonego, L., and Frankel, C. 1979. Changes in pectic substances inripening pears. Journal of American Society of Horticulture Science, 104:500–505.

Bhagylakshmi, N., Prabha, T.N., Yashoda, H.M., Prasanna, V., Jagadeesh, B.H.,and Tharanathan, R.N. 2002. Biochemical studies related to textural regulationduring ripening of banana and mango fruit. Acta Horticulturae, 575:717–724.

Boothby, D. 1983. Pectic substances in developing and ripening plum fruits.Journal of the Science of Food and Agriculture, 34:1117–1122.

Brady, C.J. 1987. Fruit ripening. Annual Review of Plant Physiology, 38:155–178.

Brownleader, M.D., Jackson, P., Mobasheri, A., Pantelides, A.T., Sumar, S.,Trevan, M., and Dey, P.M. 1999. Molecular aspects of cell wall modificationsduring fruit ripening. Critical Reviews in Food science and Nutrition, 39:149–164.

Burns, J. K. 1990. α- and β-galactosidase activities in juice vesicles of storedValencia oranges. Phytochemistry, 29:2425–2429.

Campbell, A.D., and Labavitch, J.M. 1991. Induction and regulation of ethylenebiosynthesis by pectic oligomers in cultured pear cells. Plant Physiology,97:699–705.

Carey, A.T., Holt, K., Picard, S., Wilde, R., Tucker, G.A., Bird, C.R., Schuch, W.,and Seymour, G.B. 1995. Tomato exo-(1–4)-β-D-galactanase. Plant Physiol-ogy, 108:1099–1107.

Chang, C.Y., Tsai, Y.R., and Chang, W.H. 1993. Models for the interactionsbetween pectin molecules and other cell-wall constituents in vegetable tissues.Food Chemistry, 48:145–157.

Chauhan, S.K., Tyagi, S.M., and Singh, D. 2001. Pectinolytic liquefaction ofapricot, plum and mango pulps for juice extraction. International Journal ofFood Properties, 4:103–109.

Colquhoun, I.J., de Ruiter, G.A., Schols, H.A., and Voragen, A.G.J. 1990. Iden-tification by n. m. r. spectroscopy of oligosaccharides obtained by treatmentof the hairy regions of apple pectin with rhamnogalacturonase. CarbohydrateResearch, 206:131–144.

Crookes, P.R., and Grierson, D. 1983. Ultrastructure of tomato fruit ripeningand the role of polygalacturonase isoenzymes in cell wall degradation. PlantPhysiology, 72:1088–1093.

Darvill, A.G., McNeil, M., and Albersheim, P. 1978. Structure of plant cell wallsVIII. A new pectic polysaccharide. Plant Physiology, 62:418–422.

De Vries J.A., den Uijl, C.H., Voragen, A.G.J., Rombouts, F.M., and Pilnik, W.1983. Structural features of the neutral side chains of apple pectic substances.Carbohydrate Polymers, 3:193–205.

De Vries, J.A., Voragen, A.G.J., Rombouts, F.M., and Pilnik, W. 1984. Changesin the structure of apple pectic substances during ripening and storage. Car-bohydrate Polymers, 4:3–14.

DellaPenna, D., Lashbrook, C.C., Toenjes, K., Giovannoni, J.J., Fischer, R.L.,and Bennett, A.B. 1990. Polygalacturonase isoenzymes and pectin depoly-merization in transgenic rin tomato fruit. Plant Physiology, 94:1882–1886.

DellaPenna, D., and Bennett, A.B. 1988. In vitro synthesis and processing oftomato fruit polygalacturonase. Plant Physiology, 86:1057–1063.

Dey, P.M., and Del Campillo, E. 1984. Biochemistry of the multiple forms ofglycosidases in plants. Advances in Enzymology, 56:141–249.

Doco, T., Williams, P., Vidal, S., and Pellerin, P. 1997. RhamnogalacturonanII, a dominant polysaccharide in juices produced by enzymic liquefaction offruits and vegetables. Carbohydrate Research, 297:181–186.

Doner, L.W. 1986. Analytical methods for determining pectin composition. In:Fishman, M.L., and Jen, J.J. (Eds.) Chemistry and Function of Pectins. pp.13–21. Amer. Chem. Soc., Washington D.C.

Dore Raju, M.R., and Karuna Kumar, M. 1996. Purification and characterizationof β-D-galactosidase from the pulp of sapota (Achrus sapota) fruit. Journalof Food Science and Technology, 33:36–40.

Doreyappa Gowda, I.N., and Huddar, A.G. 2001. Studies on ripening changes inmango (Mangifera indica L.) fruits. Journal of Food Science and Technology,38:135–137.

El-Zoghbi, M. 1994. Biochemical changes in some tropical fruits during ripen-ing. Food Chemistry, 49:33–37.

Femenia, A., Sanchez, E.S., Simal, S., and Rosello, C. 1998. Developmentaland ripening-related effects on the cell wall of apricots (Prunus armeniaca)fruit. Journal of the Science of Food and Agriculture, 77:487–493.

Fisher, R.L., and Bennett, A.B. 1991. Role of cell wall hydrolases in fruitripening. Annual Review of Plant Physiology and Plant Molecular Biology,42:675–703.

Fry, S.C. 1986. Cross-linking of matrix polymers in the growing cell walls ofangiosperms. Annual Review of Plant Physiology, 37:165–186.

Fry, S.C. 1995. Polysaccharide-modifying enzymes in the plant cell wall. AnnualReview of Plant Physiology and Plant Molecular Biology, 46:497–520.

Fuchs, Y., Pesis, E., and Zauberman, G. 1980. Changes in amylase activity,starch and sugar contents in mango fruit pulp. Horticultural Science, 13:155–160.

Gamage, T.V., and Rehman, M.S. 1999. Post harvest handling of foods of plantorigin. In: Rehman, M.S. (Ed.) Handbook of Food Preservation. pp. 11–46.Marcel Dekker Inc., New York.

Golden, K.D., John, M.A., and Kean, E.A. 1993. β-Galactosidase from Coffeaarabica and its role in fruit ripening. Phytochemistry, 34:355–360.

Gomez-Lim, M.A. 1997. Postharvest physiology. In: Litz, R. E. (Ed.) TheMango; Botany, Production and Uses. pp. 425–445. CAB International,Florida.

Grant, G.T., Morris, E.R., Rees, D.A., Smith, P.J.C., and Tom, D. 1973.Biological interactions between polysaccharides and divalent cations: TheEgg-Box model. FEBS Letters, 32:195–198.


Gray, J., Picton, S., Shabbeer, J., Schuch, W., and Grierson, D. 1992. Molecularbiology of fruit ripening and its manipulation with antisense genes. PlantMolecualr Biology, 19:69–87.

Grierson, D., Slater, A., Spiers, J., and Tucker, G.A. 1985. The appearance ofpolygalacturonase mRNA in tomatoes; one of a series of changes in geneexpression during development and ripening. Planta, 163:263–271.

Grierson, D., Tucker, G.A., and Robertson, N.G. 1981. The molecular biologyof ripening. In: Friend, J., and Rhodes, M.J.C., (Eds.). Recent Advances inthe Biochemistry of Fruits and Vegetables. pp. 149–160. Academic Press,London.

Gross, K.C., and Sams, C.E. 1984. Changes in cell wall neutral sugar composi-tion during fruit ripening: A Species survey. Phytochemistry, 23:2457–2461.

Gross, K.C., Watada, A.E., Kang, M.S., Kim, S.D., Kim, K.S., and Lee, S.W.1986. Biochemical changes associated with the ripening of hot pepper fruit.Physiology Plantarum, 66:31–36.

Hamilton, A.J., Lycett, G.W., and Grierson, D. 1990. Antisense gene that inhibitssynthesis of the hormone ethylene in transgenic plants. Nature, 346:284–287.

Hegde, S., and Maness, N.O. 1996. Sugar composition of pectin and hemicellu-lose extracts of peach fruit during softening over two harvest seasons. Journalof American Society of Horticulture Science, 121:1162–1167.

Hegde, S., and Maness, N.O. 1998. Changes in apparent molecular mass ofpectin and hemicellulose extracts during peach softening. Journal of AmericanSociety of Horticulture Science, 123:445–456.

Hobson, G.E. 1981. Enzymes and texture changes during ripening. In: Friend,J., and Rhodes, M.J.C., (Eds.). Recent Advances in the Biochemistry of Fruitsand Vegetables. pp. 123–132. Academic Press, London.

Huber, D.J. 1983. Polyuronide degradation and hemicellulose modifications inripening tomato fruit. Journal of American Society of Horticulture Science,108:405–409.

Huber, D.J. 1984. Strawberry fruit softening: The potential roles of polyuronidesand hemicelluloses. Journal of Food Science, 49:1310–1315.

Huber, D.J., and Lee, J.H. 1986. Comparative analysis of pectins from peri-carp and locular gel in developing tomato fruit. In: Fishman, M., and Jen, J.(Eds.). Chemistry and Functions of Pectins. pp. 141–156. Amer. Chem. Soc.,Washington D.C., USA.

Hulme, A.C. 1971. The mango. In: Hulme, A.C. (Ed.). The Biochemistry ofFruits and their Products. Vol. 2. pp. 233–254. Academic Press, London &New York.

Jackman, R.L., and Stanley, D.W. 1995. Perspectives in the textural evaluationof plant foods. Trends in Food Science and Technology, 6:187–194.

Jimenez, A., Rodriguez, R., Fernandez-caro, I., Guillen, R., Fernandez-Bolanos,J., and Heredia, A. 2001. Olive fruit cells wall: Degradation of pecticpolysaccharides during ripening. Journal of Agricultural and Food Chem-istry, 49:409–415.

John, M.A., and Dey, P.M. 1986. Postharvest changes in fruit cell wall. Advancesin Food Research, 30:139–193.

Johnson, G.I., Sharp, J.L., Milne, D.L., and Oosthuyse, S.A. 1997. Post-harvesttechnology and quarantine treatments. In: Litz, R. E. (Ed.) The Mango:Botany, Production and Uses. pp. 447–507. CAB International, Florida.

Kende, H. 1993. Ethylene biosynthesis. Annual Review of Plant Physiology andPlant Molecular Biology, 44:283–307.

Kertesz, Z.I. 1951. The Pectic Substances. Interscience Publishers Inc., NewYork.

Kim, J., Gross, K.C., and Solomos, T. 1987. Characterization of the stimula-tion of ethylene production by galactose in tomato (Lycopersicon esculentumMill.) fruit. Plant Physiology, 85:804–807.

Klee, H.J., Hayford, M.B., Kretzmer, K.A., Barry, G.F., and Kishore, G.M.1991. Control of ethylene synthesis by expression of a bacterial enzyme intransgenic tomato plants. The Plant Cell, 3:1187–1193.

Knee, M. 1973. Polysaccharide changes in cell walls of ripening apples. Phyto-chemistry, 12:1543–1549.

Knee, M. 1978. Properties of polygalacturonate and cell cohesion in apple fruitcortical tissue. Phytochemistry, 17:1257–1260.

Krall, S.M., and McFeeters, R.F. 1998. Pectin hydrolysis: effect of tempera-ture, degree of methylation, pH and calcium on hydrolysis rates. Journal ofAgricultural and Food Chemistry, 46:1311–1315.

Lang, C., and Dornenburg, H. 2000. Perspectives in the biological function andthe technological application of polygalacturonases. Applied Microbiologyand Biotechnology, 53:366–375.

Lizada, C. 1993. Mango. In: Seymour, G.B., Taylor, J.E., and Tucker, G.A.(Eds.) Biochemistry of Fruit Ripening. pp. 255–271. Chapman and Hall,London.

Mac Dougall, A.J., Brett, G.M., Morris, V.J., Rigby, N.M., Ridout, M.J., andRing, S.G. 2001. The effect of peptide-pectin interactions on the gelationbehaviour of a plant cell wall pectin. Carbohydrate Research, 335:115–126.

McCollum, T.G., Huber, D.J., and Cantliffe, D.J. 1989. Modification ofpolyuronides and hemicellulose during muskmelon fruit softening. Physi-ology Plantarum, 76:303–308.

McNeil, M., Darvill, A.G., and Albersheim, P. 1980. The structure of plant cellwalls. X. Rhamnogalacturonan I, a structurally complex pectic polysaccha-ride in the walls of suspension-cultured sycamore cells. Plant Physiology,66:1128–1134.

McNeil, M., Darvill, A.G., Fry, S., and Albersheim, P. 1984. Structure andfunction of the primary cell walls of plants. Annual Review of Biochemistry,53:625–663.

Missang, C.E., Renard, C.M.G.C., Baron, A., and Drilleau, J-F. 2001a. Cellwall polysaccharides of bush butter (Dacryodes edulis (G Don) HJ Lam) fruitpulp and their evolution during ripening. Journal of the Science of Food andAgriculture, 81:773–780.

Missang, C.E., Renard, C.M.G.C., Baron, A., and Drilleau, J-F. 2001b. Changesin the pectic fraction of bush butter (Dacryodes edulis (G Don) H J Lam)fruit pulp during ripening. Journal of the Science of Food and Agriculture,81:781–789.

Mitcham, E.J., and McDonald, R.E. 1992. Cell wall modification during ripeningof ‘Keitt’ and ‘Tommy Atkins’ mango fruit. Journal of American Society ofHorticulture Science, 117:919–924.

Nogata, Y., Ohta, H., and Voragen, A.G.J. 1993. Polygalacturonase in strawberryfruit. Phytochemistry, 34:617–620.

Nunan, K.J., Sims, I.M., Bacic, A., Robinson, S.P., and Fincher, G.B. 1998.Changes in cell wall composition during ripening of grape berries. PlantPhysiology, 118:783–792.

Nwanekezi, E.C., Alawube, O.C.G., and Mkpolulu, C.C.M. 1994. Characteriza-tion of pectic substances from selected tropical fruits. Journal of Food Scienceand Technology, 31:159–161.

O’Neill, M.A., Albersheim, P., and Darvill, A.G. 1990. The pectic polysaccha-rides of primary cell walls. In: Dey, P.M. (Ed.) Methods in Plant Biochemistry,Vol. 2. pp. 415–441. Academic Press, London.

O’Neill, M.A., Warrenfeltz, D., Kates, K., Pellerin, P., Doco, T., Darvill, A.G.,and Albersheim, P. 1996. Rhamnogalacturonan-II, a pectic polysaccharide inthe walls of growing plant cell, forms a dimer that is covalently cross-linkedby a borate ester. In vitro conditions for the formation and hydrolysis of thedimer. Journal of Biological Chemistry, 271:22923–22930.

Oeller, P.W., Min-Wong, L., Taylor, L.P., Pike, D.A., and Theologis, A. 1991.Reversible inhibition of tomato fruit senescence by antisense RNA. Science,254:437–439.

Oesterveld, A., Beldman, G., Schols, H.A., and Voragen, A.G.J. 2000. Char-acterization of arabinose and ferulic acid rich pectic polysaccharides andhemicelluloses from sugar beet pulp. Carbohydrate Research, 328:185–197.

Ovodov, Y.S., Ovodova, R.G., Bondarenko, O.D., and Krasikova, I.N. 1971. Thepectic substances of zosteraceae. Carbohydrate Research, 18:311–318.

Pathak, L., Chang, K.C., and Brown, G. 1988. Isolation and characterization ofpectin in sugar beet pulp. Journal of Food Science, 53:830–833.

Pathak, N., and Sanwal, G.G. 1998. Multiple forms of polygalacturonase frombanana fruits. Phytochemistry, 48:249–255.

Paull, R.E., and Chan, N.J. 1983. Postharvest variation in cell wall degrading en-zymes of papaya (Carica papaya L.) during fruit ripening. Plant Physiology,72:382–385.

Pilnik, W., and Voragen, A.G.J. 1970. Pectic substances and other uronides. In:Hulme, A.C. (Ed.) The Biochemistry of Fruits and their Products. Vol. I. pp.53–87. Academic Press, London & New York.


Poovaiah, B.W., and Nukuya, A. 1979. Polygalacturonase and cellulase enzymesin the normal Rutgers and mutant rin tomato fruits and their relationship tothe respiratory climacteric. Plant Physiology, 64:534–537.

Prabha, T.N., and Bhagyalakshmi, N. 1998. Carbohydrate metabolism in ripen-ing banana fruit. Phytochemistry, 48:915–919.

Prabha, T.N., Yashoda, H.M., Prasanna, V., Jagadeesh, B.H., and Bimba Jain,M.V., 2000. Carbohydrate metabolism in relation to textural softening duringfruit ripening. Trends in Carbohydrate Chemistry, 6:89–95.

Pressey, R. 1983. β-Galactosidase in ripening tomatoes. Plant Physiology,71:132–135.

Pressey, R. 1986a. Changes in polygalacturonase isoenzymes and converter intomatoes during ripening. HortScience, 21:1183–1185.

Pressey, R. 1986b. Extraction and assay of tomato polygalactur-onases.HortScience, 21:490–492.

Pressey, R. 1986c. Polygalacturonases in higher plants. In: Fishman, M.L., andJen, J.J. (Eds.) Chemistry and Function of Pectins. pp. 157–174. Amer. Chem.Soc., Washington D.C.

Pressey, R., and Avants, J.K. 1972. Multiple forms of pectinesterase in tomatoes.Phytochemistry, 11:3139–3142.

Pressey, R., and Avants, J.K. 1973a. Separation and characterization of en-dopolygalacturonase and exopolygalacturonase from peaches. Plant Physiol-ogy, 52:252–256.

Pressey, R., and Avants, J.K. 1973b. Two forms of polygalacturonase in tomatoes.Biochimica et Biophysica Acta, 309:363–369.

Pressey, R., and Avants, J.K. 1975. Cucumber polygalacturonase. Journal ofFood Science, 40:937–939.

Pressey, R., and Avants, J.K. 1976. Pear polygalacturonases. Phytochemistry,15:1349–1351.

Pressey, R., and Avants, J.K. 1978. Difference in polygalacturonase compositionof Clingstone and Freestone peaches. Journal of Food Science, 43:1415–1417,1423.

Priyasethu, K.M., Prabha, T.N., and Tharanathan, R.N. 1996. Post-harvest bio-chemical changes associated with the softening phenomenon in Capsicumannuum fruits. Phytochemistry, 42:961–966.

Rahman, M.M., Akhter, T., and Absar, N. 2000. Activities of glucosidase andgalactosidase enzymes during fruit development of ten mango varieties. In-dian Journal of Plant Physiology, 5:374–376.

Ranwala, A.P., Suematsu, C., and Masuda, H. 1992. The role of β-galactosidasesin the modification of cell wall components during muskmelon fruit ripening.Plant Physiology, 100:1318–1325.

Reddy, Y.V., and Srivastava, G.C. 1999. Regulation of cell wall softening en-zymes during ripening of mango fruits. Indian Journal of Plant Physiology,4:194–196.

Redgwell, R.J., MacRae, E.A., Hallet, I., Fischer, M., Perry, J., and Harker, R.1997. In vivo and in vitro swelling of cell walls during fruit ripening. Planta,203:162–173.

Redgwell, R.J., Melton, L.D., and Brasch, D.J. 1990. Cell-wall changes in ki-wifruit following postharvest ethylene treatment. Phytochemistry, 29:399–407.

Redgwell, R.J., Melton, L.D., and Brasch, D.J. 1992. Cell wall dissolutionin kiwifruit (Actinidia deliciosa). Solubilization of pectic polymers. PlantPhysiology, 98:71–81.

Renard, C.M.C.G., Crepeau, M.J., and Thibault, J.F. 1999. Glucuronic aciddirectly linked to galacturonic acid in the rhamnogalacturonan backbone ofbeet pectins. European Journal of Biochemistry, 266:566–574.

Rexova-Benkova, L., and Markovic, O. 1976. Pectic enzymes. Advances inCarbohydrate Chemistry and Biochemistry, 33:323–385.

Ridley, B.L., O’Neill, M.A., and Mohnen, D. 2001. Pectins: structure, biosyn-thesis, and oligogalacturonide-related signaling. Phytochemistry, 57:929–967.

Roe, B., and Bruemmer, J.H. 1981. Changes in pectic substances and enzymesduring ripening and storage of ‘Keitt’ mangos. Journal of Food Science,46:186–189.

Rose, J.K.C., Hadfield, K.A., Labavitch, J.M., and Bennett, A.B. 1998. Tempo-ral sequence of cell wall disassembly in rapidly ripening melon fruit. PlantPhysiology, 117:345–361.

Ross, G.S., Wegrzyn, T., MacRae, E.A., and Redgwell, R.J. 1994. Apple β-galactosidase. Activity against cell wall polysaccharides and characterizationof a related cDNA clone. Plant Physiology, 106:521–528.

Sakai, T., Sakamoto, T., Hallaert, J., and Vandamme, E.J. 1993. Pectin, pecti-nase and protopectinase: Production, properties and application. Advances inApplied Microbiology, 39:213–294.

Saulnier, L., Brillouet, J.M., and Joseleau, J.P. 1988. Structural studies of pecticsubstances from the pulp of grape berries. Carbohydrate Research, 182:63–78.

Schols, H.A., and Voragen, A.G.J. 1994. Occurrence of pectic hairy regions invarious plant cell wall materials and their degradability by rhamnogalactur-onase. Carbohydrate Research, 256:83–95.

Schols, H.A., Bakx, E.J., Schipper, D., and Voragen, A.G.J. 1995. A xylogalac-turonan subunit present in the modified hairy regions of apple pectin. Carbo-hydrate Research, 279:265–279.

Schols, H.A., Posthumus, M.A., and Voragen, A.G.J. 1990. Structural features ofhairy regions of pectins isolated from apple juice produced by the liquefactionprocess. Carbohydrate Research, 206:117–129.

Selvaraj, Y., and Kumar, R. 1989. Studies on fruit softening enzymes andpolyphenol oxidase activity in ripening mango (Mangifera indica L.) fruit.Journal of Food Science and Technology, 26:218–222.

Selvaraj, Y., Kumar, R., and Pal, D.K. 1989. Changes in sugars, organic acids,amino acids, lipid constituents and aroma characteristics of ripening mango(Mangifera indica L.) fruit. Journal of Food Science and Technology, 26:308–313.

Selvendran, R.R. 1985. Developments in the chemistry and biochemistry ofpectic and hemicellulosic polymers. Journal of Cell Science, 2(Suppl.):51–88.

Seymour, G.B., Colquhoun, I.J., Dupont, M.S., Parsley, K.R., and Selvendran,R.R. 1990. Composition and structural features of cell wall polysaccharidesfrom tomato fruits. Phytochemistry, 29:725–731.

Seymour, G.B., Harding, S.E., Taylor, A.J., Hobson, G.E., and Tucker, G.A.1987. Polyuronide solubilization during ripening of normal and mutant tomatofruit. Phytochemistry, 26:1871–1875.

Sirisomboon, P., Tanaka, M., Fujitha, S., and Kojima, T. 2000. Relationship be-tween the texture and pectin constituents of Japanese pear. Journal of TextureStudies, 31:679–690.

Smith, B.G., and Harris, P.J. 1995. Polysaccharide composition of unlignifiedcell walls of pineapple (Ananas comosus L. Merr.) fruit. Plant Physiology,107:1399–1409.

Smith, C.J. 1999. Carbohydrate biochemistry. In: Lea, P. J., and Leegood, R.C. (Eds.) Plant Biochemistry and Molecular Biology. pp. 81–118. II edition.John Willey & Sons, New York.

Smith, C.J.S., Watson, C.F., Ray, J., Bird, C.R., Morris, P.C., Schuch, W., andGrierson, D. 1988. Antisense RNA inhibition of polygalacturonase gene ex-pression in transgenic tomatoes. Nature, 334:724–726.

Sobotka, F.E., and Stelzig, D.A. 1974. An apparent cellulase complex in tomato(Lycopersicon esculentum) fruit. Plant Physiology, 53:759–763.

Stevens, B.J.H., and Selvendran, R.R. 1984. Structural features of cell- wallpolymers of the apple. Carbohydrate Research, 135:155–166.

Stewart, D., Iannetta, P.P.M., and Davies, H.V. 2001. Ripening-related changes inraspberry cell wall composition and structure. Phytochemistry, 56:423–428.

Strasser, G.R., and Amado, R. 2001. Pectic substances from red beet(Beta vulgaris L. var. conditiva) Part-I. Structural analysis of rhamnogalac-turonan I using enzymic degradation and methylation analysis. CarbohydratePolymers, 44:63–70.

Tandon, D.K., and Kalra, S.K. 1984. Pectin changes during the development ofmango fruit cv Dashehari. Journal of Horticultural Science, 59:283–286.

Thakur, B.R., Singh, R.K., and Handa, A.K. 1997. Chemistry and uses of pectin-A review. Critical Reviews of Food Science and Nutrition, 37:47–73.

Tharanathan, R.N., Muralikrishna, G., Salimath, P.V., and Raghavendra Rao,M.R. 1987. Plant carbohydrates- an overview. Proceedings of Indian Academyof Sciences (Plant Sci.), 97:81–155.

Thomas, M., and Thibault, J.F. 2002. Cell wall polysaccharides in the fruits ofJapanese quince (Chaenomeles japonica): extraction and preliminary charac-terization. Carbohydrate Polymers, 49:345–355.


Tieman, D.M., Harriman, R.W., Ramamohan, G., and Handa, A.K. 1992. Anantisense pectin methylesterase gene alters pectin chemistry and soluble solidsin tomato fruit. The Plant Cell, 4:667–679.

Tucker, G.A. 1993. Introduction. In: Seymour, G.B., Taylor, J.E., and Tucker,G.A. (Eds.). Biochemistry of Fruit Ripening. pp. 1–51. Chapman and Hall,London.

Tucker, G.A., and Grierson, D. 1987. Fruit ripening. In: Davies, D. (Ed.). TheBiochemistry of Plants. Vol. 12. pp. 265–319. Academic Press Inc., New York.

Tucker, G.A., Robertson, N.G., and Grierson, D. 1980. Changes in polygalactur-onase isoenzymes during the “ripening” of normal and mutant tomato fruit.European Journal of Biochemistry, 112:119–124.

Tucker, G.A., Robertson, N.G., and Grierson, D. 1982. Purification and changesin activities of tomato pectinesterase isoenzymes. Journal of the Science ofFood and Agriculture, 3:396–400.

Van Buren, J.P. 1979. The chemistry of texture in fruits and vegetables. Journalof Texture Studies, 10:1–23.

Vidal, S., Williams, P., O’Neill, M.A., and Pellerin, P. 2001. Polysaccharidesfrom grape berry cell walls. Part I: tissue distribution and structural character-ization of the pectic polysaccharides. Carbohydrate Polymers, 45:315–323.

Voragen, A.G.J., Pilnik, W., Thibault, J-F, Axelos, M.A.V., and Renard,C.M.C.G. 1995. Pectins. In: Stephen, A.M. (Ed.) Food Polysaccharides andtheir Applications. pp. 287–339. Marcel Dekker Inc., New York.

Waldron, K.W., Smith, A.C., Parr, A.J., Ng, A., and Parker, M.L. 1997. Newapproaches to understanding and controlling cell separation in relation tofruit and vegetable texture. Trends in Food Science and Technology, 8:213–221.

Wallner, S.J., and Walker, J.E. 1975. Glycosidases in cell wall-degrading extractsof ripening tomato fruits. Plant Physiology, 55:94–98.

Watkins, C.B., Haki, J.M., and Frenkel, C. 1988. Activities of polygalactur-onase, α-D-mannosidase, and α-D- and β-D-galactosidase in ripening tomato.HortScience, 23:192–194.

Whitaker, J.R. 1984. Pectic substances, pectic enzymes and haze formation infruit juices. Enzyme and Microbial Technology, 6:341–349.

Wong, D.W.S. 1995. Pectic enzymes. In: Food Enzymes. Structure and Mecha-nisms. pp. 212–236. Chapman and Hall, New York,

Yang, S.F., and Hoffman, N.E. 1984. Ethylene biosynthesis and its regulation inhigher plants. Annual Review of Plant Physiology, 35:155–189.

Zainal, Z., Tucker, G.A., and Lycett, G.W. 1999. Isolation and characteriza-tion of cDNA encoding 1-ACC oxidase from mango (Mangifera indica.L). Asia Pacific Journal of Molecular Biology and Biotechnology, 7:53–59.

Zhan, D., Janssen, P., and Mort, A.J. 1998. Scarcity or complete lack of singlerhamnose residues interspersed within the homogalacturonan regions of citruspectin. Carbohydrate Research, 308:373–380.

fruit ripening phenomena—an

Documents