Food Biochemistry and Food Processing (Simpson/Food Biochemistry and Food Processing) || Biochemistry of Fruits

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Part 5Fruits, Vegetables, and Cereals

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27Biochemistry of Fruits

Gopinadhan Paliyath, Krishnaraj Tiwari, Carole Sitbon, and Bruce D. Whitaker

IntroductionBiochemical Composition of Fruits

Carbohydrates, Storage and Structural ComponentsLipids and BiomembranesProteinsOrganic Acids

Fruit Ripening and SofteningCarbohydrate Metabolism

Cell Wall DegradationStarch DegradationGlycolysisCitric Acid CycleGluconeogenesisAnaerobic RespirationPentose Phosphate Pathway

Lipid MetabolismProteolysis and Structure Breakdown in Chloroplasts

Secondary Plant Products and Flavour ComponentsIsoprenoid BiosynthesisAnthocyanin BiosynthesisEster Volatile Biosynthesis

General ReadingReferences

Abstract: Fruits are major ingredients of human diet and pro-vide several nutritional ingredients including carbohydrates, vita-mins and functional food ingredients such as soluble and insolublefibers, polyphenols and carotenoids. Biochemical changes duringfruit ripening make the fruit edible by making them soft, changingthe texture through the breakdown of cell wall, converting acids orstored starch into sugars and causing the biosynthesis of pigmentsand flavour components. Fruits are processed into several productsto preserve these qualities.

INTRODUCTIONBecause of various health benefits associated with the consump-tion of fruits and various products derived from fruits, these areat the centre stage of human dietary choices in recent days. Theselection of trees that produce fruits with ideal edible qualityhas been a common process throughout human history. Fruitsare developmental manifestations of the seed-bearing structuresin plants, the ovary. After fertilisation, the hormonal changes in-duced in the ovary result in the development of the characteristicfruit that may vary in ontogeny, form, structure and quality. Pomefruits such as apple and pear are developed from the develop-ment of the thalamus in the flower. Drupe fruits, such as cherry,peach, plum, apricot and so on, are developed from the ovarywall (mesocarp) enclosing a single seed. Berry fruits such astomato possess the seeds embedded in a jelly-like pectinaceousmatrix, with the ovary wall developing into the flesh of the fruit.Cucumbers and melons develop from an inferior ovary. Citrusfruits belong to the class hesperidium, where the ovary walldevelops as a protective structure surrounding the juice-filledlocules that become the edible part of the fruit. In strawberry,the seeds are located outside the fruit, and it is the receptacle ofthe ovary (central portion) that develops into the edible part. Thebiological purpose of the fruit is to attract vectors that help in thedispersal of the seeds. For this, the fruits have developed variousorganoleptic (stimulatory to organs) characteristics that includeattractive colour, flavour and taste. The biochemical characteris-tics and pathways in the fruits are developmentally structured toachieve these goals. The nutritional and food qualities of fruitsarise as a result of the accumulation of components derived fromthese intricate biochemical pathways. In terms of production and

Food Biochemistry and Food Processing, Second Edition. Edited by Benjamin K. Simpson, Leo M.L. Nollet, Fidel Toldra, Soottawat Benjakul, Gopinadhan Paliyath and Y.H. Hui.C© 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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534 Part 5: Fruits, Vegetables, and Cereals

volume, tomato, orange, banana and grape are the major fruitcrops used for consumption and processing around the world(Kays 1997).

BIOCHEMICAL COMPOSITIONOF FRUITSFruits contain a large percentage of water, which can often ex-ceed 95% by fresh weight. During ripening, activation of sev-eral metabolic pathways often leads to drastic changes in thebiochemical composition of fruits. Fruits such as banana storestarch during development, and hydrolyse the starch to sugarsduring ripening, that also results in fruit softening. Most fruitsare capable of photosynthesis, store starch and convert them tosugars during ripening. Fruits such as apple, tomato, grape andso on have a high percentage of organic acids, which decreasesduring ripening. Fruits also contain large amounts of fibrousmaterials such as cellulose and pectin. The degradation of thesepolymers into smaller water-soluble units during ripening leadsto fruit softening as exemplified by the breakdown of pectinin tomato and cellulose in avocado. Secondary plant productsare major compositional ingredients in fruits. Anthocyanins arethe major colour components in grape, blueberry, apple andplum; carotenoids, specifically lycopene and carotene, are themajor components that impart colour in tomato and watermelon.Aroma is derived from several types of compounds that includemonoterpenes (as in lime, orange), ester volatiles (ethyl, methylbutyrate in apple, isoamyl acetate in banana), simple organicacids such as citric and malic acids (citrus fruits, apple) andsmall chain aldehydes such as hexenal and hexanal (cucumber).Fruits are also rich in vitamin C. Lipid content is quite low infruits, the exceptions being avocado and olives, in which tri-acylglycerols (oils) form the major storage components. Theamounts of proteins are usually low in most fruits.

Carbohydrates, Storage and StructuralComponents

As the name implies, carbohydrates are organic compounds con-taining carbon, hydrogen and oxygen. Basically, all carbohy-drates are derived by the photosynthetic reduction of CO2 tothe pentoses (ribose, ribulose) and hexoses (glucose, fructose),which are also intermediates in the metabolic pathways. Poly-merisation of several sugar derivatives leads to various storage(starch, inulin) and structural components (cellulose, pectin).

During photosynthesis, the glucose formed is converted tostarch and stored as starch granules. Glucose and its isomer fruc-tose, along with phosphorylated forms (glucose-6-phosphate,glucose-1,6- diphosphate, fructose-6-phosphate and fructose-1,6-diphosphate), can be considered to be the major metabolichexose pool components that provide carbon skeleton for thesynthesis of carbohydrate polymers. Starch is the major storagecarbohydrate in fruits. There are two molecular forms of starch,amylose and amylopectin and both components are present in thestarch grain. Starch is synthesised from glucose phosphate by theactivities of a number of enzymes designated as ADP-glucosepyrophosphorylase, starch synthase and a starch-branching en-

zyme. ADP-glucose pyrophosphorylase catalyses the reactionbetween glucose-1-phosphate and ATP that generates ADP-glucose and pyrophosphate. ADP-glucose is used by starch syn-thase to add glucose molecules to amylose or amylopectin chain,thus increasing their degree of polymerisation. By contrast tocellulose that is made up of glucose units in β-1,4-glycosidiclinkages, the starch molecule contains glucose linked by α-1,4-glycosidic linkages. The starch branching enzyme introducesglucose molecules through α-1,6- linkages which further gets ex-tended into linear amylose units with α-1,4- glycosidic linkages.Thus, the added glucose branch points (α-1,6-linkages) serve assites for further elongation by starch synthase, thus resulting ina branched starch molecule, also known as amylopectin.

Cell wall is a complex structure composed of cellulose andpectin, derived from hexoses such as glucose, galactose, rham-nose and mannose, and pentoses such as xylose and arabinose,as well as some of their derivatives such as glucuronic andgalacturonic acids (Negi and Handa 2008). A model proposedby Keegstra et al. (1973) describes the cell wall as a polymericstructure constituted by cellulose microfibrils and hemicelluloseembedded in the apoplastic matrix in association with pecticcomponents and proteins. In combination, these componentsprovide the structural rigidity that is characteristic to the plantcell. Most of the pectin is localised in the middle lamella. Cel-lulose is biosynthesised by the action of β-1,4-glucan synthaseenzyme complexes that are localised on the plasma membrane.The enzyme uses uridine diphosphate glucose (UDPG) as asubstrate, and by adding UDPG units to small cellulose units,extends the length and polymerisation of the cellulose chain. Inaddition to cellulose, there are polymers made of different hex-oses and pentoses known as hemicelluloses, and based on theircomposition, they are categorised as xyloglucans, glucoman-nans and galactoglucomannans. The cellulose chains assembleinto microfibrils through hydrogen bonds to form crystallinestructures. In a similar manner, pectin is biosynthesised fromUDP-galacturonic acid (galacturonic acid is derived from galac-tose, a six carbon sugar), as well as other sugars and derivativesand includes galacturonans and rhamnogalacturonans that formthe acidic fraction of pectin. As the name implies, rhamnogalac-turonans are synthesised primarily from galacturonic acid andrhamnose. The acidic carboxylic groups complex with calciumthat provide the rigidity to the cell wall and the fruit. The neu-tral fraction of the pectin comprises polymers such as arabinans(polymers of arabinose), galactans (polymers of galactose) orarabinogalactans (containing both arabinose and galactose). Allthese polymeric components form a complex three-dimensionalnetwork stabilised by hydrogen bonds, ionic interactions involv-ing calcium, phenolic components such as diferulic acid andhydroxyproline-rich glycoproteins (Fry 1986, Negi and Handa2008). It is also important to visualise that these structures arenot static and the components of cell wall are constantly beingturned over in response to growth conditions.

Lipids and Biomembranes

By structure, lipids can form both structural and storagecomponents. The major forms of lipids include fatty acids,

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27 Biochemistry of Fruits 535

diacyl- and triacylglycerols, phospholipids, sterols, and waxesthat provide an external barrier to the fruits. Fruits in generalare not rich in lipids with the exception of avocado and olivesthat store large amounts of triacylglycerols or oil. As gener-ally observed in plants, the major fatty acids in fruits includepalmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2) andlinolenic (18:3) acids. Among these, oleic, linoleic and linolenicacids possess an increasing degree of unsaturation. Olive oil isrich in triacylglycerols containing the monounsaturated oleicacid and is considered as a healthy ingredient for humanconsumption.

Compartmentalisation of cellular ingredients and ions is anessential characteristic of all life forms. The compartmentalisa-tion is achieved by biomembranes, formed by the assembly ofphospholipids and several neutral lipids that include diacylglyc-erols and sterols, the major constituents of the biomembranes.Virtually, all cellular structures include or are enclosed bybiomembranes. The cytoplasm is surrounded by the plasmamembrane, the biosynthetic and transport compartments suchas the endoplasmic reticulum and Golgi bodies form an in-tegral network of membranes within the cell. Photosynthesis,which converts light energy into chemical energy, occurs on thethylakoid membrane matrix in the chloroplast, and respiration,which further converts chemical energy into more usable forms,occurs on the mitochondrial cristae. All these membranes havetheir characteristic composition and enzyme complexes to per-form their designated function.

The major phospholipids that constitute the biomembranesinclude phosphatidylcholine, phosphatidylethanolamine, phos-phatidylglycerol and phosphatidylinositol. Their relative pro-portion may vary from tissue to tissue. In addition, metabolicintermediates of phospholipids such as phosphatidic acid, dia-cylglycerols, free fatty acids and so on are also present in themembrane in lower amounts. Phospholipids are integral func-tional components of hormonal and environmental signal trans-duction processes in the cell. Phosphorylated forms of phos-phatidylinositol such as phosphatidylinositol-4- phosphate andphosphatidylinositol-4,5-bisphosphate are formed during signaltransduction events, though their amounts can be very low. Themembrane also contains sterols such as sitosterol, campesteroland stigmasterol, as well as their glucosides, and they are ex-tremely important for the regulation of membrane fluidity andfunction (Whitaker 1988, 1991, 1993, 1994).

Biomembranes are bilamellar layers of phospholipids. Theamphipathic nature of phospholipids having hydrophilic headgroups (choline, ethanolamine, etc.) and hydrophobic fatty acylchains, thermodynamically favour their assembly into bilamellaror micellar structures when exposed to an aqueous environment.In a biomembrane, the hydrophilic headgroups are exposed tothe external aqueous environment. The phospholipid composi-tion between various fruits may differ, and within the same fruit,the inner and outer lamella of the membrane may have a dif-ferent phospholipids composition. Such differences may causechanges in polarity between the outer and inner lamellae of themembrane, and lead to the generation of a voltage across themembrane. These differences usually become operational dur-ing signal transduction events.

An essential characteristic of the membrane is its fluidity.The fluid-mosaic model of the membrane (Singer and Nicholson1972) depicts the membrane as a planar matrix comprising phos-pholipids and proteins. The proteins are embedded in the mem-brane bilayer (integral proteins) or are bound to the periphery(peripheral proteins). The nature of this interaction results fromthe structure of the proteins. If the proteins have a much largerproportion of hydrophobic amino acids, they would tend to be-come embedded in the membrane bilayer. If the protein containsmore hydrophilic amino acids it may tend to prefer a more aque-ous environment, and thus remain as a peripheral protein. Inaddition, proteins may be covalently attached to phospholipidssuch as phosphatidylinositol. Proteins that remain in the cytosolmay also become attached to the membrane in response to anincrease in cytosolic calcium levels. The membrane is a highlydynamic entity. The semi-fluid nature of the membrane allowsfor the movement of phospholipids in the plane of the mem-brane, and between the bilayers of the membrane. The proteinsare also mobile within the plane of the membrane. However, thisprocess is not always random and is regulated by the functionalassembly of proteins into metabolons (functional assembly ofenzymes and proteins, e.g., photosynthetic units in thylakoidmembrane, respiratory complexes in the mitochondria, cellu-lose synthase on plasma membrane, etc.), their interactions withthe underlying cytoskeletal system (network of proteins such asactin and tubulin), and the fluidity of the membrane.

The maintenance of homeostasis (life processes) requires themaintenance of the integrity and function of discrete membranecompartments. This is essential for the compartmentalisation ofions and metabolites, which may otherwise destroy the cell. Forinstance, calcium ions are highly compartmentalised within thecell. The concentration of calcium is maintained at the millimo-lar levels within the cell wall compartment (apoplast), endoplas-mic reticulum and the tonoplast (vacuole). This is achieved byenergy-dependent transport of calcium from the cytoplasm intothese compartments by ATPases. As a result, the cytosolic cal-cium levels are maintained at low micromolar (<1 µM) levels.Maintenance of this concentration gradient across the membraneis a key requirement for the signal transduction events, as reg-ulated entry of calcium into the cytosol can be achieved simplyby opening calcium channels. Calcium can then activate severalcellular biochemical reactions that mediate the response to thesignal. Calcium is pumped back into the storage compartmentswhen the signal diminishes in intensity. In a similar manner,cytosolic pH is highly regulated by the activity of proton AT-Pases. The pH of the apoplast and the vacuole is maintained nearfour, whereas the pH of the cytosol is maintained in the rangeof 6–6.5. The pH gradient across the membrane is a key fea-ture that regulates the absorption, or extrusion of other ions andmetabolites such as sugars. The cell could undergo senescenceif this compartmentalisation is lost.

There are several factors that affect the fluidity of the mem-brane. The major factor that affects the fluidity is the type andproportion of acyl chain fatty acids of the phospholipids. At agiven temperature, a higher proportion of unsaturated fatty acylchains (oleic, linoleic, linolenic) in the phospholipids can in-crease the fluidity of the membrane. An increase in saturated

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fatty acids such as palmitic and stearic acids can decrease thefluidity. Other membrane components such as sterols, and degra-dation products of fatty acids such as fatty aldehydes, alkanesand so on can also decrease the fluidity. Based on the physiolog-ical status of the tissue, the membrane can exist in either a liquidcrystalline state (where the phospholipids and their acyl chainsare mobile) or a gel state where they are packed as rigid orderedstructures and their movements are much restricted. The mem-brane usually has co-existing domains of liquid crystalline andgel phase lipids depending on growth conditions, temperature,ion concentration near the membrane surface and so on. Thetissue has the ability to adjust the fluidity of the membrane byaltering the acyl lipid composition of the phospholipids. For in-stance, an increase in the gel phase lipid domains resulting fromexposure to cold temperature could be counteracted by increas-ing the proportion of fatty acyl chains having a higher degreeof unsaturation, and, therefore, a lower melting point. Thus, themembrane will tend to remain fluid even at a lower temper-ature (Whitaker 1991, 1992, 1993, 1994). An increase in gelphase lipid domains can result in the loss of compartmentalisa-tion. The differences in the mobility properties of phospholipidacyl chains can cause packing imperfections at the interface be-tween gel and liquid crystalline phases, and these regions canbecome leaky to calcium ions and protons that are highly com-partmentalised. The membrane proteins are also excluded fromthe gel phase into the liquid crystalline phase. Thus, during ex-aminations of membrane structure by freeze fracture electronmicroscopy, the gel phase domains can appear as regions devoidof proteins (Paliyath and Thompson 1990).

Proteins

Fruits, in general, are not very rich sources of proteins. Duringthe early growth phase of fruits, the chloroplasts and mitochon-dria are the major organelles that contain structural proteins.The structural proteins include the light-harvesting complexes inchloroplast or the respiratory enzyme/protein complexes in mito-chondria. Ribulose-bis-phosphate carboxylase/oxygenase (Ru-bisco) is the most abundant enzyme in photosynthetic tissues.Fruits do not store proteins as an energy source. The green fruitssuch as bell peppers and tomato have a higher level of chloroplastproteins.

Organic Acids

Organic acids are major components of some fruits. The acid-ity of fruits arises from the organic acids that are stored in thevacuole, and their composition can vary depending on the typeof fruit. In general, young fruits contain more organic acids,which may decline during maturation and ripening due to theirconversion to sugars (gluconeogenesis; eg. conversion of malicacid into glucose during ripening of apple). Some fruit familiesare characterised by the presence of certain organic acids. Forexample, fruits of oxalidaceae members (e.g., starfruit, Averrhoacarambola) contain oxalic acid, and fruits of the citrus family,rutaceae, are rich in citric acid. Apples contain malic acid andgrapes are characterised by the presence of tartaric acid. In gen-

eral, citric and malic acids are the major organic acids of fruits.Grapes contain tartaric acid as the major organic acid. Duringripening, these acids can enter the citric acid cycle and undergofurther metabolic conversions.

l-(+)tartaric acid is the optically active form of tartaric acidin grape berries. A peak in acid content is observed beforethe initiation of ripening, and the acid content declines on afresh weight basis during ripening. Tartaric acid can be biosyn-thesised from carbohydrates and other organic acids. Radiola-belled glucose, glycolate and ascorbate were all converted totartarate in grape berries. Malate can be derived from the citricacid cycle or through carbon dioxide fixation of pyruvate bythe malic enzyme (NADPH-dependent malate dehydrogenase).Malic acid, as the name implies, is also the major organic acidin apples.

FRUIT RIPENING AND SOFTENING

Fruit ripening is a physiological event that results from a verycomplex and interrelated biochemical changes that occur in thefruits. Ripening is the ultimate stage of the development ofthe fruit, which entails the development of ideal organolepticcharacters such as taste, colour and aroma that are importantfeatures of attraction for the vectors (animals, birds, etc.) re-sponsible for the dispersal of the fruit, and thus the seeds, in theecosystem. Human beings have developed an agronomic sys-tem of cultivation, harvest and storage of fruits with ideal foodqualities. In most cases, the ripening process is very fast, andthe fruits undergo senescence resulting in the loss of desirablequalities. An understanding of the biochemistry and molecu-lar biology of the fruit ripening process has resulted in devel-oping biotechnological strategies for the preservation of post-harvest shelf life and quality of fruits (Negi and Handa 2008,Paliyath et al. 2008a).

A key initiator of the ripening process is the gaseous planthormone ethylene. In general, all plant tissues produce a low,basal, level of ethylene. Based on the pattern of ethylene produc-tion and responsiveness to externally added ethylene, fruits aregenerally categorised into climacteric and non-climacteric fruits.During ripening, the climacteric fruits show a burst in ethyleneproduction and respiration (CO2 production). Non-climactericfruits show a considerably low level of ethylene production. Inclimacteric fruits (apple, pear, banana, tomato, avocado, etc.),ethylene production can reach levels of 30–500 ppm (parts permillion, microlitre/L), whereas in non-climacteric fruits (orange,lemon, strawberry, pineapple, etc.) ethylene levels usually arein the range of 0.1–0.5 ppm. Ethylene can stimulate its ownbiosynthesis in climacteric fruits, known as autocatalytic ethy-lene production. As well, the respiratory carbon dioxide evolu-tion increases in response to ethylene treatment, termed as therespiratory climacteric. Climacteric fruits respond to externalethylene treatment by accelerating the respiratory climactericand time required for ripening, in a concentration-dependentmanner. Non-climacteric fruits show increased respiration in re-sponse to increasing ethylene concentration without acceleratingthe time required for ripening.

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Methyl thioribose

1-Aminocyclopropane-1-carboxylic acid

ACC synthase

S-Adenosyl methionine (SAM)

Methionine adenosyl transferase

Methionine

Ethylene biosynthetic pathway

ACC oxidase

Ethylene

Ethylene receptor

Fruit ripening/senescence

Figure 27.1. Summary of ethylene biosynthesis and action duringfruit ripening. ACC, 1-Aminocyclopropane 1- Carboxylic Acid.

Ethylene is biosynthesised through a common pathwaythat uses the amino acid methionine as the precursor (Yang1981, Fluhr and Mattoo 1996) (Fig. 27.1). The first reac-tion of the pathway involves the conversion of methionine toS-adenosyl methionine (SAM) mediated by the enzyme me-thionine adenosyl transferase. SAM is further converted into1-aminocyclopropane-1-carboxylic acid (ACC) by the enzymeACC synthase. The sulphur moiety of methylthioribose gener-ated during this reaction is recycled back to methionine by theaction of a number of enzymes. ACC is the immediate precur-sor of ethylene and is acted upon by ACC oxidase to generateethylene. ACC synthase and ACC oxidase are the key controlpoints in the biosynthesis of ethylene. ACC synthase is a solubleenzyme located in the cytoplasm, with a relative molecular massof 50 kDa (kiloDalton). ACC oxidase is found to be associatedwith the vacuolar or mitochondrial membrane. Using molecularbiology tools, a cDNA (complementary DNA representing thecoding sequences of a gene) for ACC oxidase was isolated fromtomato (Hamilton et al. 1991) and is found to encode a proteinwith a relative molecular mass of 35 kDa. There are severalisoforms of ACC-synthase. These are differentially expressed inresponse to wounding, other stress factors and at the initiationof ripening. ACC oxidase reaction requires Fe2+, ascorbate andoxygen.

Regulation of the activities of ACC synthase and ACC oxidaseis extremely important for the preservation of shelf life and qual-

ity in fruits. Inhibition of the ACC synthase and ACC oxidasegene expression by the introduction of their respective antisensecDNAs resulted in delayed ripening and better preservation ofthe quality of tomato (Hamilton et al. 1990, Oeller et al. 1991)and apple (Hrazdina et al. 2000) fruits. ACC synthase, whichis the rate-limiting enzyme of the pathway, requires pyridoxal-5-phosphate as a cofactor, and is inhibited by pyridoxal phos-phate inhibitors such as aminoethoxyvinylglycine (AVG) andaminooxy acetic acid (AOA). Field application of AVG as agrowth regulator (RetainTM, Valent Biosciences, Chicago) hasbeen used to delay ripening in fruits such as apples, peaches andpears. Also, commercial storage operations employ controlledatmosphere with very low oxygen levels (1–3%) for long-termstorage of fruits such as apples to reduce the production ofethylene, as oxygen is required for the conversion of ACC toethylene.

In response to the initiation of ripening, several biochemi-cal changes are induced in the fruit, which ultimately resultsin the development of ideal texture, taste, colour and flavour.Several biochemical pathways are involved in these processesas described in the subsequent text.

Carbohydrate Metabolism

Cell Wall Degradation

Cell wall degradation is the major factor that causes soften-ing of several fruits. This involves the degradation of cellulosecomponents, pectin components or both. Cellulose is degradedby the enzyme cellulase or β-1,4-glucanase. Pectin degradationinvolves the enzymes pectin methylesterase, polygalacuronase(pectinase) and β-galactosidase (Negi and Handa 2008). Thedegradation of cell wall can be reduced by the application ofcalcium as a spray or drench in apple fruits. Calcium bindsand cross-links the free carboxylic groups of polygalacturonicacid components in pectin. Calcium treatment, therefore, alsoenhances the firmness of the fruits.

The activities of both cellulase and pectinase have been ob-served to increase during ripening of avocado fruits and result intheir softening. Cellulase is an enzyme with a relative molecularmass of 54.2 kDa and formed by extensive post-translational pro-cessing of a native 54 kDa protein involving proteolytic cleavageof the signal peptide and glycosylation (Bennet and Christo-pherson 1986). Further studies have shown three isoforms ofcellulose ranging in molecular masses between 50 and 55 kDa.These forms are associated with the endoplasmic reticulum,the plasma membrane and the cell wall (Dallman et al. 1989).The cellulase isoforms are initially synthesised at the style endof the fruit at the initiation of ripening, and the biosynthesisprogressively increases towards the stalk end of the fruit withthe advancement of ripening. Degradation of hemicelluloses(xyloglucans, glucomannans and galactoglucomannans) is alsoconsidered as an important feature that leads to fruit softening.Degradation of these polymers could be achieved by cellulasesand galactosidases.

Loss of pectic polymers through the activity of polygalactur-onases (PG) is a major factor involved in the softening of fruits

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such as tomato. There are three major isoforms of PG responsiblefor pectin degradation in tomato, designated as PG1, PG2a andPG2b (Fischer and Bennet 1991). PG1 has a relative molecularmass of 100 kDa, and is the predominant form at the initia-tion of ripening. With the advancement of ripening, PG2a andPG2b isoforms increase, becoming the predominant isoformsin ripe fruit. The different molecular masses of the isozymesresult from the post-translational processing and glycosylationof the polypeptides. PG2a (43 kDa) and PG2b (45 kDa) appearto be the same polypeptide with different degrees of glycosyla-tion. PG1 is a complex of three polypeptides, PG2a, PG2b anda 38kDa subunit known as the β-subunit. The 38 kDa subunit isbelieved to exist in the cell wall space where it combines withPG2a and PG2b forming the PG1 isoform of PG. The increasein activity of PG1 is related to the rate of pectin solubilisationand tomato fruit softening during the ripening process.

Research into the understanding of the regulation of biosyn-thesis and activity of PG using molecular biology tools hasresulted in the development of strategies for enhancing the shelflife and quality of tomatoes. PG mRNA was one of the firstripening-related mRNAs isolated from tomato fruits. All thedifferent isoforms of PGs are encoded by a single gene. ThePG cDNA which has an open reading frame of 1371 bases en-codes a polypeptide having 457 amino acids, which includesa 24 amino acid signal sequence (for targeting to the cell wallspace) and a 47 amino acid pro-sequence at the N-terminal end,which are proteolytically removed during the formation of theactive PG isoforms. A 13 amino acid long C-terminal peptideis also removed resulting in a 373 amino acid long polypeptide,which undergoes different degrees of glycosylation resulting inthe PG2a and PG2b isozymes. Complex formation among PG2a,PG2b and the 38-kDa subunit in the apoplast results in the PG1isozyme (Grierson et al. 1986, Bird et al. 1988). In response toethylene treatment of mature green tomato fruits which stim-ulates ripening, the levels of PG mRNA and PG are found toincrease. These changes can be inhibited by treating tomatoeswith silver ions, which interfere with the binding of ethylene toits receptor and initiation of ethylene action (Davies et al. 1988).Thus, there is a link between ethylene, PG synthesis and fruitsoftening.

Genetic engineering of tomato with the objective of regulat-ing PG activity has yielded complex results. In the rin mutantof tomato which lacks PG and does not soften, introduction ofa PG gene resulted in the synthesis of an active enzyme; how-ever, this did not cause fruit softening (Giovannoni et al. 1989).As a corollary to this, introduction of the PG gene in the anti-sense orientation resulted in near total inhibition of PG activity(Smith et al. 1988). In both these cases, there was very little ef-fect on fruit softening, suggesting that factors other than pectinde-polymerisation may play an integral role in fruit softening.Further studies using a tomato cultivar such as UC82B (Krameret al. 1992) showed that antisense inhibition of ethylene biosyn-thesis or PG did indeed result in lowered PG activity, improvedintegrity of cell wall and increased fruit firmness during fruitripening. As well, increased activity of pectin methylesterase,which removes the methyl groups from esterified galacturonicacid moieties, may contribute to the fruit softening process.

The activities of pectin degrading enzymes have been relatedto the incidence of physiological disorders such as “mealiness”or “wooliness” in mature unripened peaches that are stored at alow temperature. The fruits with such a disorder show a lack ofjuice and a dry texture. De-esterification of pectin by the activityof pectin methyl esterase is thought to be responsible for thedevelopment of this disorder. Pectin methyl esterase isozymeswith relative molecular masses in the range of 32 kDa have beenobserved in peaches, and their activity increases after 2 weeksof low temperature storage. Polygalacuronase activity increasesas the fruit ripens. The ripening fruits which possess both poly-galacturonase and pectin methyl esterase do not develop mealysymptoms when stored at low temperature implicating the poten-tial role of pectin degradation in the development of mealinessin peaches.

There are two forms of PG in peaches, the exo- and endo-PG.The endo-PG are the predominant forms in the freestone typeof peaches, whereas the exo-PG are observed in the mesocarpof both freestone and clingstone varieties of peaches. As thename implies, exo-PG remove galacturonic acid moieties ofpectin from the terminal reducing end of the chain, whereas theendo-PG can cleave the pectin chain at random within the chain.The activities of these enzymes increase during the ripeningand softening of the fruit. Two exo-PG isozymes have beenidentified in peach, having a relative molecular mass of near66 kDa. The exo-acting enzymes are activated by calcium. Peachendo-PG is observed to be similar to the tomato endo-PG. Thepeach endo-PG is inhibited by calcium. The freestone peachespossess enhanced activities of both exo-PG and endo-PG leadingto a high degree of fruit softening. However, the clingstonevarieties with low levels of endo-PG activity do not soften as thefreestone varieties. In general, fruits such as peaches, tomatoes,strawberries, pears and so on, which soften extensively, possesshigh levels of endo-PG activity. Apple fruits which remain firmlack endo-PG activity.

Starch Degradation

Starch is the major storage form of carbohydrates. During ripen-ing, starch is catabolised into glucose and fructose, which entersthe metabolic pool where they are used as respiratory substratesor further converted to other metabolites (Fig. 27.2). In fruitssuch as banana, the breakdown of starch into simple sugars isassociated with fruit softening. There are several enzymes in-volved in the catabolism of starch. α-amylase hydrolyses amy-lose molecules by cleaving the α-1,4-linkages between sug-ars providing smaller chains of amylose termed as dextrins.β-amylase is another enzyme that acts upon the glucan chainreleasing maltose, which is a diglucoside. The dextrins as wellas maltose can be further catabolised to simple glucose unitsby the action of glucosidases. Starch phosphorylase is anotherenzyme, which mediates the phosphorylytic cleavage of termi-nal glucose units at the non-reducing end of the starch moleculeusing inorganic phosphate, thus releasing glucose-1-phosphate.The amylopectin molecule is not only degraded in a similarmanner to amylose but also involves the action of de-branching

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Glucose-1-phosphate

ADP-glucosepyrophosphorylase

UDP-glucosepyrophosphorylase

ADP-glucose

Starch synthase

Amylopectin

Branchingenzyme

α-1-4-Glucanprimer

α-Glucosidase

α-GlucosidaseGlucose

Glucose

Glucose-1-phosphate

Metabolic pool

Photosynthesis

Carbohydrate metabolism in fruits

Maltose

Maltose

(Amylose)

Hexokinase

Dextrins

α-Glucosidase

β-AmylaseH2O

α-Amylase

β-A

myl

ase

ATP

ATP

ADP

UDP-glucose

Fructose-6-phosphate

Sucrosephosphatesynthase

Sucrose-6-phosphate

Phosphatase Pi

Sucrose

Starch

UDP-glucose

UDP-glucosepyrophosphorylase

Starchphosphorylase

InvertaseSucrosesynthase

Fructose

Glucose+ fructose

PPi

PPi

PPi

UTP

UTP

UDP

Figure 27.2. Carbohydrate metabolism in fruits. UDP, Uridine diphosphate; UTP, Uridine triphosphate.

enzymes which cleaves the α-1,6-linkages in amylopectin andreleases linear units of the glucan chain.

In general, starch is confined to the plastid compartments offruit cells, where it exists as granules made up of both amyloseand amylopectin molecules. The enzymes that catabolise starchare also found in this compartment and their activities increaseduring ripening. The glucose-1-phosphate generated by starchdegradation (Fig. 27.2) is mobilised into the cytoplasm whereit can enter into various metabolic pools such as that of gly-colysis (respiration), pentose phosphate pathway (PPP) or forturnover reactions that replenish lost or damaged cellular struc-tures (cell wall components). It is important to visualise thatthe cell always tries to extend its life under regular develop-mental conditions (the exceptions being programmed cell deathwhich occurs during hypersensitive response to kill invadingpathogens, thus killing both the pathogen and the cell/tissue;formation of xylem vessels, secondary xylem tissues, etc.), and

the turnover reactions are a part of maintaining the homeostasis.The cell ultimately succumbs to the catabolic reactions duringsenescence. The compartmentalisation and storage of chemicalenergy in the form of metabolisable macromolecules are all theinherent properties of life, which is defined as a struggle againstincreasing entropy.

The biosynthesis and catabolism of sucrose is an importantpart of carbohydrate metabolism. Sucrose is the major form oftransport sugar and is translocated through the phloem tissuesto other parts of the plant. It is conceivable that carbon dioxidefixed during photosynthesis in leaf tissues may be transportedto the fruits as sucrose during fruit development. Sucrose isbiosynthesised from glucose-1-phosphate by three major steps(Fig. 27.2). The first reaction involves the conversion of glucose-1-phosphate to UDP-glucose by UDP-glucose pyrophospho-rylase in the presence of UTP (Uridine triphosphate). UDP-glucose is also an important substrate for the biosynthesis of

P1: SFK/UKS P2: SFK



cell wall components such as cellulose. UDP-glucose is con-verted to sucrose-6-phosphate by the enzyme sucrose phosphatesynthase (SPS), which utilises fructose-6-phosphate during thisreaction. Finally, sucrose is formed from sucrose-6-phosphateby the action of phosphatase with the liberation of the inorganicphosphate.

Even though sucrose biosynthesis is an integral part of starchmetabolism, sucrose often is not the predominant sugar that ac-cumulates in fruits. Sucrose is further converted into glucoseand fructose by the action of invertase, which are character-istic to many ripe fruits. By the actions of sucrose synthaseand UDP-glucose pyrophosphorylase, glucose-1-phosphate canbe regenerated from sucrose. As well, sugar alcohols suchas sorbitol and mannitol formed during sugar metabolism aremajor transport and storage components in apple and olive,respectively.

Biosynthesis and catabolism of starch has been extensivelystudied in banana, where prior to ripening, it can account for20–25% by fresh weight of the pulp tissue. All the starch degrad-ing enzymes, α-amylase, β-amylase, α-glucosidase and starchphosphorylase, have been isolated from banana pulp. The ac-tivities of these enzymes increase during ripening. Concomitantwith the catabolism of starch, there is an accumulation of thesugars, primarily, sucrose, glucose and fructose. At the initiationof ripening, sucrose appears to be the major sugar component,which declines during the advancement of ripening with a si-multaneous increase in glucose and fructose through the actionof invertase (Beaudry et al. 1989). Mango is another fruit whichstores large amounts of starch. The starch is degraded by theactivities of amylases during the ripening process. In mango,glucose, fructose and sucrose are the major forms of simple sug-ars (Selvaraj et al. 1989). The sugar content is generally veryhigh in ripe mangoes and can reach levels in excess of 90%of the total soluble solids content. By contrast to the bananas,the sucrose levels increase with the advancement of ripening inmangoes, potentially due to gluconeogenesis from organic acids(Kumar and Selvaraj 1990). As well, the levels of pentose sugarsincrease during ripening, and could be related to an increase inthe activity of the PPP.

Glycolysis

The conversion of starch to sugars and their subsequentmetabolism occur in different compartments. During the de-velopment of fruits, photosynthetically fixed carbon is utilisedfor both respiration and biosynthesis. During this phase, thebiosynthetic processes dominate. As the fruit matures and be-gin to ripen, the pattern of sugar utilisation changes. Ripeningis a highly energy-intensive process. And this is reflected inthe burst in respiratory carbon dioxide evolution during ripen-ing. As mentioned earlier, the respiratory burst is characteristicof some fruits which are designated as climacteric fruits. Thepost-harvest shelf life of fruits can depend on their intensity ofrespiration. Fruits such as mango and banana possess high levelof respiratory activity and are highly perishable. The applicationof controlled atmosphere conditions having low oxygen levels

and low temperature have thus become a routine technology forthe long-term preservation of fruits.

The sugars and sugar phosphates generated during thecatabolism of starch are metabolised through the glycolysis andcitric acid cycle (Fig. 27.3). Sugar phosphates can also be chan-nelled through the PPP, which is a major metabolic cycle thatprovides reducing power for biosynthetic reactions in the form ofNADPH, as well as supplying carbon skeletons for the biosyn-thesis of several secondary plant products. The organic acidsstored in the vacuole are metabolised through the functional re-versal of respiratory pathway and is termed as gluconeogenesis.Altogether, sugar metabolism is a key biochemical characteristicof the fruits.

In the glycolytic steps of reactions (Fig. 27.3), glucose-6-phosphate is isomerised to fructose-6-phosphate by the enzymehexose-phosphate isomerase. Glucose 6-phosphate is derivedfrom glucose-1-phosphate by the action of glucose phosphatemutase. Fructose-6-phosphate is phosphorylated at the C1 posi-tion, yielding fructose-1,6- bisphosphate. This reaction is catal-ysed by the enzyme phosphofructokinase (PFK) in the pres-ence of ATP. Fructose-1,6-bisphosphate is further cleaved intotwo three carbon intermediates, dihydroxyacetone phosphateand glyceraldehyde-3-phosphate, catalysed by the enzyme al-dolase. These two compounds are interconvertible through anisomerisation reaction mediated by triose phosphate isomerase.Glyceraldehyde-3-phosphate is subsequently phosphorylated atthe C1 position using orthophosphate, as well as oxidised usingNAD, to generate 1,3-diphosphoglycerate and NADH. In thenext reaction, 1,3-diphosphoglycerate is dephosphorylated byglycerate-3-phosphate kinase in the presence of ADP, along withthe formation of ATP. Glycerate-3-phosphate formed during thisreaction is further isomerised to 2-phosphoglycerate in the pres-ence of phosphoglycerate mutase. In the presence of the enzymeenolase, 2-phosphoglycerate is converted to phosphoenol pyru-vate (PEP). Dephosphorylation of phosphoenolpyruvate in thepresence of ADP by pyruvate kinase yields pyruvate and ATP.Metabolic fate of pyruvate is highly regulated. Under normalconditions, it is converted to acetyl CoA, which then enters thecitric acid cycle. Under anaerobic conditions, pyruvate can bemetabolised to ethanol, which is a by-product in several ripeningfruits.

There are two key regulatory steps in glycolysis, one mediatedby PFK and the other by pyruvate kinase. In addition, thereare other types of modulation involving cofactors and enzymestructural changes reported to be involved in glycolytic control.ATP levels increase during ripening. However, in fruits, thisdoes not cause a feed back inhibition of PFK as observed inanimal systems. There are two isozymes of PFK in plants, onelocalised in plastids and the other localised in the cytoplasm.These isozymes regulate the flow of carbon from the hexosephosphate pool to the pentose phosphate pool. PFK isozymesare strongly inhibited by PEP. Thus, any conditions that maycause the accumulation of PEP will tend to reduce the carbonflow through glycolysis. By contrast, inorganic phosphate is astrong activator of PFK. Thus, the ratio of PEP to inorganicphosphate would appear to be the major factor that regulates theactivity of PFK and carbon flux through glycolysis. Structural

P1: SFK/UKS P2: SFK



Glucose Glucose-6-phosphate

Dihydroxyacetone phosphateFructose-6-phosphate

Phosphofructokinase

Fructose-1,6-bisphosphate

1,3-Diphosphoglycerate

Glycerate-3-phosphate kinase

3-Phosphoglycerate 2-Phosphoglycerate

Phosphoenol pyruvate

Pyruvate dehydrogenase

Pyruvate

Acetyl CoA

Anaerobic metabolism

Breakdown of sugars: Glycolysis/citric acid cycle

Citric acid cycle

CO2

CO2

CO2

Phosphoglycerate mutase

EthanolAlcohol

dehydrogenasePyruvate

decarboxylase

Citrate synthaseCitrate

Aconitase

Isocitrate

Isocitratedehydrogenase

Malatedehydrogenase

Oxaloacetate

Malate

Fumarase

Fumarate

Succinatedehydrogenase Succinate

Coash Succinate thiokinase

Succinyl CoA

COA-SH

α-Ketoglutarate

Acetaldehyde

Enolase

AldolaseTriose phosphate

isomerase

Glyceraldehyde-3-phosphate

Glyceraldehyde-3-phosphatedehydrogenase

Hexokinase

Hexosephosphate isomeraseATP

ATP

ATP

ATP

ADP

ADP

NAD

NAD

NAD

NAD

NAD

NAD

FAD

FADH2

NADH

NADH

NADH

NADH

NADH

ATP ADP

NADH

ADP

ADP

Figure 27.3. Catabolism of sugars through glycolytic pathway and citric acid cycle.

alteration of PFK, which increases the efficiency of utilisationof fructose-6-phosphate, is another means of regulation that canactivate the carbon flow through the glycolytic pathway.

Other enzymes of the glycolytic pathway are involved inthe regulation of starch/sucrose biosynthesis (Figs. 27.2 and27.3). Fructose-1,6-bisphosphate is converted back to fructose-6-phosphate by the enzyme fructose-1,6-bisphosphatase, alsoreleasing inorganic phosphate. This enzyme is localised inthe cytosol and chloroplast. Fructose-6-phosphate is converted

to fructose-2,6-bisphosphate by fructose-6-phosphate 2-kinasewhich can be dephosphorylated at the 2 position by fructose-2,6-bisphosphatase. Fructose-6-phosphate is an intermediary insucrose biosynthesis (Fig. 27.2). SPS is regulated by reversiblephosphorylation (a form of post-translational modification thatinvolves addition of a phosphate moiety from ATP to an OH-amino acid residue in the protein, such as serine or threonine, me-diated by a kinase, and dephosphorylation mediated by a phos-phatase) by SPS kinase and SPS phosphatase. Phosphorylation

P1: SFK/UKS P2: SFK



of the enzyme makes it less active. Glucose-6-phosphate is anallosteric activator (a molecule that can bind to an enzyme andincrease its activity through enzyme subunit association) of theactive form of SPS (dephosphorylated). Glucose-6-phosphate isan inhibitor of SPS kinase and inorganic phosphate is an inhibitorof SPS phosphatase. Thus, under conditions when glucose-6-phosphate/inorganic phosphate ratio is high, the active form ofSPS will dominate favouring sucrose phosphate biosynthesis.These regulations are highly complex and may be regulated bythe flux of other sugars in several pathways.

The conversion of PEP to pyruvate mediated by pyruvate ki-nase is another key metabolic step in the glycolytic pathway andis irreversible. Pyruvate is used in several metabolic reactions.During respiration, pyruvate is further converted to acetyl coen-zyme A (acetyl CoA), which enters the citric acid cycle throughwhich it is completely oxidised to carbon dioxide (Fig. 27.3).The conversion of pyruvate to acetyl CoA is mediated by the en-zyme complex pyruvate dehydrogenase, and is an oxidative stepthat involves the formation of NADH from NAD. Acetyl CoAis a key metabolite and starting point for several biosyntheticreactions (fatty acids, isoprenoids, phenylpropanoids, etc.).

Citric Acid Cycle

The citric acid cycle involves the biosynthesis of several organicacids, many of which serve as precursors for the biosynthesisof several groups of amino acids. In the first reaction, oxaloac-etate combines with acetyl CoA to form citrate, and is mediatedby citrate synthase (Fig. 27.3). In the next step, citrate is con-verted to isocitrate by the action of aconitase. The next two stepsin the cycle involve oxidative decarboxylation. The conversionof isocitrate to α-ketoglutarate involves the removal of a car-bon dioxide molecule and reduction of NAD to NADH. Thisstep is catalysed by isocitrate dehydrogenase. α-ketoglutarate isconverted to succinyl-CoA by α-ketoglutarate dehydrogenase,along with the removal of another molecule of carbon dioxideand the conversion of NAD to NADH. Succinate, the next prod-uct, is formed from succinyl CoA by the action of succinyl CoAsynthetase that involves the removal of the CoA moiety and theconversion of ADP to ATP. Through these steps, the completeoxidation of the acetyl CoA moiety has been achieved with theremoval of two molecules of carbon dioxide. Thus, succinateis a four-carbon organic acid. Succinate is further converted tofumarate and malate in the presence of succinate dehydrogenaseand fumarase, respectively. Malate is oxidised to oxaloacetateby the enzyme malate dehydrogenase along with the conversionof NAD to NADH. Oxaloacetate then can combine with anothermolecule of acetyl CoA to repeat the cycle. The reducing powergenerated in the form of NADH and FADH (succinate dehydro-genation step) is used for the biosynthesis of ATP through theelectron transport chain in the mitochondria.

Gluconeogenesis

Several fruits store large amounts of organic acids in their vac-uole and these acids are converted back to sugars during ripen-ing, a process termed as gluconeogenesis. Several irreversible

steps in the glycolysis and citric acid cycle are bypassed dur-ing gluconeogenesis. Malate and citrate are the major organicacids present in fruits. In fruits such as grapes, where there is atransition from a sour to a sweet stage during ripening, organicacids content declines. Grape contains predominantly tartaricacid along with malate, citrate, succinate, fumarate and severalorganic acid intermediates of metabolism. The content of or-ganic acids in berries can affect their suitability for processing.High acid content coupled with low sugar content can result inpoor-quality wines. External warm growth conditions enhancethe metabolism of malic acid in grapes during ripening and couldresult in a high tartarate/malate ratio, which is considered idealfor vinification.

The metabolism of malate during ripening is mediated by themalic enzyme, NADP-dependent malate dehydrogenase. Alongwith a decline in malate content, there is a concomitant increasein the sugars, suggesting a possible metabolic precursor prod-uct relationship between these two events. Indeed, when grapeberries were fed with radiolabelled malate, the radiolabel couldbe recovered in glucose. The metabolism of malate involves itsconversion to oxaloacetate mediated by malate dehydrogenase,the decarboxylation of oxaloacetate to PEP catalysed by PEP-carboxykinase, and a reversal of glycolytic pathway leading tosugar formation (Ruffner et al. 1983). The gluconeogenic path-way from malate may contribute only a small percentage (5%)of the sugars, and a decrease in malate content could primarilyresult from reduced synthesis and increased catabolism throughthe citric acid cycle. The inhibition of malate synthesis by the in-hibition of the glycolytic pathway could result in increased sugaraccumulation. Metabolism of malate in apple fruits is catalysedby NADP-malic enzyme, which converts malate to pyruvate. Inapples, malate appears to be primarily oxidised through the cit-ric acid cycle. Organic acids are important components of citrusfruits. Citric acid is the major form of the acid followed by malicacid and several less abundant acids such as acetate, pyruvate,oxalate, glutarate, fumarate and so on. In oranges, the acidityincreases during maturation of the fruit and declines during theripening phase. Lemon fruits, by contrast, increase their acidcontent through the accumulation of citrate. The citrate levels invarious citrus fruits range from 75% to 88%, and malate levelsrange from 2% to 20%. Ascorbate is another major component ofcitrus fruits. Ascorbate levels can range from 20 to 60 mg/100 gjuice in various citrus fruits. The orange skin may possess150–340 mg/100 g fresh weight of ascorbate, which may notbe extracted into the juice.

Anaerobic Respiration

Anaerobic respiration is a common event in the respirationof ripe fruits and especially becomes significant when fruitsare exposed to low temperature. Often, this may result fromoxygen-depriving conditions induced inside the fruit. Underanoxia, ATP production through the citric acid cycle and mito-chondrial electron transport chain is inhibited. Anaerobic res-piration is a means of regenerating NAD, which can drivethe glycolyic pathway and produce minimal amounts of ATP(Fig. 27.3). Under anoxia, pyruvate formed through glycolysis

P1: SFK/UKS P2: SFK



is converted to lactate-by-lactate dehydrogenase using NADHas the reducing factor, and generating NAD. Accumulationof lactate in the cytosol could cause acidification, and un-der these low pH conditions, lactate dehydrogenase is in-hibited. The formation of acetaldehyde by the decarboxyla-tion of pyruvate is stimulated by the activation of pyruvatedecarboxylase under low pH conditions in the cytosol. It isalso likely that the increase in concentration of pyruvate in thecytoplasm may stimulate pyruvate decarboxylase directly. Ac-etaldehyde is reduced to ethanol by alcohol dehydrogenase usingNADH as the reducing power. Thus, acetaldehyde and ethanolare common volatile components observed in the headspace offruits indicative of the occurrence of anaerobic respiration. Cy-tosolic acidification is a condition that stimulates deteriorativereactions. By removing lactate through efflux and convertingpyruvate to ethanol, cytosolic acidification can be avoided.

Anaerobic respiration plays a significant role in the respira-tion of citrus fruits. During early stages of growth, respiratoryactivity predominantly occurs in the skin tissue. Oxygen up-take by the skin tissue was much higher than the juice vesicles(Purvis 1985). With advancing maturity, a decline in aerobic res-piration and an increase in anaerobic respiration was observedin Hamlin orange skin (Bruemmer 1989). In parallel with this,the levels of ethanol and acetaldehyde increased. As well, a de-crease in the organic acid substrates pyruvate and oxaloacetatewas detectable in Hamlin orange juice. An increase in the activ-ity levels of pyruvate decarboxylase, alcohol dehydrogenase andmalic enzyme was noticed in parallel with the decline in pyru-vate and accumulation of ethanol. In apple fruits, malic acid isconverted to pyruvate by the action of NADP-malic enzyme,and pyruvate subsequently converted to ethanol by the action ofpyruvate decarboxylase and alcohol dehydrogenase. The alco-hol dehydrogenase in apple can use NADPH as a cofactor, andNADP is regenerated during ethanol production, thus drivingmalate utilisation. Ethanol is either released as a volatile or canbe used for the biosynthesis of ethyl esters of volatiles.

Pentose Phosphate Pathway

Oxidative PPP is a key metabolic pathway that provides re-ducing power (NADPH) for biosynthetic reactions as well ascarbon precursors for the biosynthesis of amino acids, nucleicacids, secondary plant products and so on. The PPP shares manyof the sugar phosphate intermediates with the glycolytic path-way (Fig. 27.4). The PPP is characterised by the interconversionof sugar phosphates with three (glyceraldehyde-3-phosphate),four (erythrose-4-phosphate), five (ribulose-, ribose-, xylulose-phosphates), six (glucose-6-phosphate, fructose-6-phosphate)and seven (sedoheptulose-7-phosphate) carbon long chains.

The PPP involves the oxidation of glucose-6-phosphate,and the sugar phosphate intermediates formed are recycled.The first two reactions of PPP are oxidative reactions medi-ated by the enzymes glucose-6-phosphate dehydrogenase and6-phosphogluconate dehydrogenase (Fig. 27.4). In the first step,glucose-6-phosphate is converted to 6-phosphogluconate by theremoval of two hydrogen atoms by NADP to form NADPH.In the next step, 6-phosphogluconate, a six-carbon sugar acid

phosphate, is converted to ribulose-5-phosphate, a five-carbonsugar phosphate. This reaction involves the removal of a car-bon dioxide molecule along with the formation of NADPH.Ribulose-5-phosphate undergoes several metabolic conversionsto yield fructose-6-phosphate. Fructose-6-phosphate can then beconverted back to glucose-6-phosphate by the enzyme glucose-6-phosphate isomerase and the cycle repeated. Thus, six com-plete turns of the cycle can result in the complete oxidation of aglucose molecule.

Despite the differences in the reaction sequences, the gly-colytic pathway and the PPP intermediates can interact with oneanother and share common intermediates. Intermediates of boththe pathways are localised in plastids, as well as the cytoplasm,and intermediates can be transferred across the plastid mem-brane into the cytoplasm and back into the chloroplast. Glucose-6-phosphate dehydrogenase is localised both in the chloroplastand cytoplasm. Cytosolic glucose-6-phosphate dehydrogenaseactivity is strongly inhibited by NADPH. Thus, the ratio ofNADP to NADPH could be the regulatory control point forthe enzyme function. The chloroplast-localised enzyme is regu-lated differently through oxidation and reduction, and related tothe photosynthetic process. 6-Phosphogluconate dehydrogenaseexists as distinct cytosol- and plastid-localised isozymes.

The PPP is a key metabolic pathway related to biosyn-thetic reactions, antioxidant enzyme function and generalstress tolerance of the fruits. Ribose-5-phosphate is used inthe biosynthesis of nucleic acids and erythrose-4- phosphateis channelled into phenyl propanoid pathway leading to thebiosynthesis of the amino acids phenylalanine and tryptophan.Phenylalanine is the metabolic starting point for the biosynthe-sis of flavonoids and anthocyanins in fruits. Glyceraldehyde-3-phosphate and pyruvate serve as the precursors for the iso-prenoid pathway localised in the chloroplast. Accumulation ofsugars in fruits during ripening has been related to the functionof PPP. In mangoes, an increase in the levels of pentose sugarsobserved during ripening has been related to increased activityof PPP. Increases in glucose-6-phosphate dehydrogenase and6-phosphogluconate dehydrogenase activities were observedduring ripening of mango.

NADPH is a key component required for the proper function-ing of the antioxidant enzyme system (Fig. 27.4). During growth,stress conditions, fruit ripening and senescence, free radicals aregenerated within the cell. Activated forms of oxygen, such assuperoxide, hydroxyl and peroxy radicals, can attack enzymes,proteins, nucleic acids and lipids, causing structural and func-tional alterations of these molecules. Under most conditions,these are deleterious changes, which are nullified by the action ofantioxidants and antioxidant enzymes. Simple antioxidants suchas ascorbate and vitamin E can scavenge the free radicals andprotect the tissue. Anthocyanins and other polyphenols mayalso serve as simple antioxidants. In addition, the antioxidantenzyme system involves the integrated function of several en-zymes. The key antioxidant enzymes are superoxide dismutase(SOD), catalase, ascorbate peroxidase and peroxidase. SOD con-verts superoxide into hydrogen peroxide. Hydrogen peroxide isimmediately acted upon by catalase, generating water. Hydro-gen peroxide can also be removed by the action of peroxidases.

P1: SFK/UKS P2: SFK



Sedoheptulose-7-phosphare

Transketolase

Xylulose-5-phosphate Ribose-5-phosphate

Ribulose-5-phosphate

6-Phosphogluconate

Glucose-6-phosphate dehydrogenase

Antioxidant (enzyme) system

Mitochondriachloroplast

Membranedegradation

SOD

POXCAT

APX

MDHAR

DHARGR

6-phosphogluconatedehydrogenase

Glyceraldehyde-3-phosphate

Oxidative pentose phosphate pathway

Pyruvate

Isoprenoids(carotenoids)

Nucleicacid

Glucose-6-phosphate

CO2

NADPH

NADPH

NADP+

2 H+H2O

H2OH2O2O2 O2-

NADP+ NADPH

NADPH GSSG

GSH DHA

ASA MDHA

NADPHpool

Glycolysis

NADP

NADP

Pentose phosphateisomerase

Epimerase

Transaldolase

Erythrose-4-phosphate

Fructose-6-phosphate

Phenyl propanoidpathway

Chalcone

Anthocyanins

Figure 27.4. Oxidative pentose phosphate pathway in plants. NADPH generated from the pentose phosphate pathway is channeled into theantioxidant enzyme system, where the regeneration of oxidised intermediates requires NADPH. GSH, reduced glutathione; GSSG, oxidisedglutathione; ASA, reduced ascorbate; MDHA, monodehydroascorbate; DHA, dehydroascorbate; GR, glutathione reductase; DHAR,dehydroascorbate reductase; MDHAR, monodehydroascorbate reductase; SOD, superoxide dismutase; CAT, catalase; POX, peroxidase;APX, ascorbate peroxidase.

A peroxidase uses the oxidation of a substrate molecule (usuallyhaving a phenol structure, C–OH, which becomes a quinone,C = O, after the reaction) to react with hydrogen peroxide, con-verting it to water. Hydrogen peroxide can also be acted uponby ascorbate peroxidase, which uses ascorbate as the hydro-gen donor for the reaction, resulting in water formation. Theoxidised ascorbate is regenerated by the action of a series ofenzymes (Fig. 27.4). These include monodehydroascorbate re-ductase (MDHAR) and dehydroascorbate reductase (DHAR).Dehydroascorbate is reduced to ascorbate using reduced glu-tathione (GSH) as a substrate, which itself gets oxidised (GSSG)during this reaction. The oxidised GSH is reduced back to GSHby the activity of GSH reductase using NADPH. Antioxidant

enzymes exist as several functional isozymes with differing ac-tivities and kinetic properties in the same tissue. These enzymesare also compartmentalised in chloroplast, mitochondria and cy-toplasm. The functioning of the antioxidant enzyme system iscrucial to the maintenance of fruit quality through preservingcellular structure and function (Meir and Bramlage 1988, Ahnet al. 2002).

Lipid Metabolism

Among fruits, avocado and olive are the only fruits that sig-nificantly store reserves in the form of lipid triglycerides. Inavocado, triglycerides form the major part of the neutral lipid

P1: SFK/UKS P2: SFK



fraction, which can account for nearly 95% of the total lipids.Palmitic (16:0), palmitoleic (16:1), oleic (18:1) and linoleic(18:2) acids are the major fatty acids of triglycerides. The oilcontent progressively increases during maturation of the fruit,and the oils are compartmentalised in oil bodies or oleosomes.The biosynthesis of fatty acids occurs in the plastids, and the fattyacids are exported into the endoplasmic reticulum where they areesterified with glycerol-3-phosphate by the action of a numberof enzymes to form the triglyceride. The triglyceride-enrichedregions then are believed to bud off from the endoplasmic retic-ulum as the oil body. The oil body membranes are differentfrom other cellular membranes, since they are made up of only asingle layer of phospholipids. The triglycerides are catabolisedby the action of triacylglycerol lipases with the release of fattyacids. The fatty acids are then broken down into acety CoA unitsthrough β-oxidation.

Even though phospholipids constitute a small fraction of thelipids in fruits, the degradation of phospholipids is a key fac-tor that controls the progression of senescence. As in severalsenescing systems, there is a decline in phospholipids as thefruit undergoes senescence. With the decline in phospholipidcontent, there is a progressive increase in the levels of neu-tral lipids, primarily diacylglycerols, free fatty acids and fattyaldehydes. In addition, the levels of sterols may also increase.Thus, there is an increase in the ratio of sterol:phospholipids.Such changes in the composition of membrane can cause theformation of gel phase or non-bilayer lipid structures (micelles).These changes can make the membranes leaky, thus resultingin the loss of compartmentalisation, and ultimately, senescence(Paliyath and Droillard 1992).

Membrane lipid degradation occurs by the tandem action ofseveral enzymes, one enzyme acting on the product released bythe previous enzyme in the sequence. Phospholipase D (PLD)is the first enzyme of the pathway which initiates phospholipidscatabolism and is a key enzyme of the pathway (Fig. 27.6).PLD acts on phospholipids liberating phosphatidic acid and therespective headgroup (choline, ethanolamine, glycerol, inosi-tol). Phosphatidic acid, in turn, is acted upon by phosphatidatephosphatase which removes the phosphate group from phospha-tidic acid with the liberation of diacylglycerols (diglycerides).The acyl chains of diacylglycerols are then de-esterified by theenzyme lipolytic acyl hydrolase liberating free fatty acids. Un-saturated fatty acids with a cis-1,4- pentadiene structure (linoleicacid, linolenic acid) are acted upon by lipoxygenase (LOX) caus-ing the peroxidation of fatty acids. This step may also cause theproduction of activated oxygen species such as singlet oxygen,superoxide and peroxy radicals and so on. The peroxidationproducts of linolenic acid can be 9-hydroperoxy linoleic acidor 13-hydroperoxy linoleic acid. The hydroperoxylinoleic acidsundergo cleavage by hydroperoxide lyase resulting in severalproducts including hexanal, hexenal and ω-keto fatty acids (ketogroup towards the methyl end of the molecule). For example,hydroperoxide lyase action on 13-hydroperoxylinolenic acid re-sults in the formation of cis-3-hexenal and 12-keto-cis-9- dode-cenoic acid. Hexanal and hexenal are important fruit volatiles.The short-chain fatty acids may feed into catabolic pathway(β-oxidation) that results in the formation of short-chain acyl

CoAs, ranging from acetyl CoA to dodecanoyl CoA. The short-chain acyl CoAs and alcohols (ethanol, propanol, butanol, pen-tanol, hexanol, etc.) are esterified to form a variety of estersthat constitute components of flavour volatiles that are charac-teristic to fruits. The free fatty acids and their catabolites (fattyaldehydes, fatty alcohols, alkanes, etc.) can accumulate in themembrane causing membrane destabilisation (formation of gelphase, non-bilayer structures, etc.). An interesting regulatoryfeature of this pathway is the very low substrate specifity ofenzymes that act downstream from PLD for the phospholipids.Thus, phosphatidate phosphatase, lipolytic acyl hydrolase andLOX do not directly act on phospholipids, though there are ex-ceptions to this rule. Therefore, the degree of membrane lipidcatabolism will be determined by the extent of activation of PLD(Fig. 27.5).

The membrane lipid catabolic pathway is considered as anautocatalytic pathway (Fig. 27.5). The destabilisation of themembrane can cause the leakage of calcium and hydrogen ionsfrom the cell wall space, as well as the inhibition of calcium-and proton ATPases, the enzymes responsible for maintaining aphysiological calcium and proton concentration within the cy-toplasm (calcium concentration below micromolar range, pH inthe 6–6.5 range). Under conditions of normal growth and devel-opment, these enzymes pump the extra calcium- and hydrogenions that enter the cytoplasm from storage areas such as apoplastand the ER lumen in response to hormonal and environmentalstimulation using ATP as the energy source. The activities ofcalcium- and proton ATPases localised on the plasma membrane,the endoplasmic reticulum and the tonoplast are responsible forpumping the ions back into the storage compartments. In fruits(and other senescing systems), with the advancement in ripen-ing and senescence, there is a progressive increase in leakage ofcalcium and hydrogen ions. PLD is stimulated by low pH andcalcium concentration over 10 µM. Thus, if the cytosolic con-centrations of these ions progressively increase during ripeningor senescence, the membranes are damaged as a consequence.However, this is an inherent feature of the ripening processin fruits, and results in the development of ideal organolepticqualities that makes them edible. The uncontrolled membranedeterioration can result in the loss of shelf life and quality infruits (Paliyath et al. 2008).

The properties and regulation of the membrane degradationpathway are increasingly becoming clear. Enzymes such as PLDand LOX are very well studied. There are several isoforms ofPLD designated as PLD alpha, PLD beta, PLD gamma and soon. The expression and activity levels of PLD alpha are muchhigher than that of the other PLD isoforms. Thus, PLD alpha isconsidered as a housekeeping enzyme; however, it is also de-velopmentally regulated (Pinhero et al. 2003). The regulation ofPLD activity is an interesting feature. PLD is normally a solubleenzyme. The secondary structure of PLD shows the presence of asegment of around 130 amino acids at the N-terminal end, desig-nated as the C2 domain. This domain is characteristic of severalenzymes and proteins that are integral components of the hor-mone signal transduction system. In response to hormonal andenvironmental stimulation and the resulting increase in cytosoliccalcium concentration, C2 domain binds calcium and transports

P1: SFK/UKS P2: SFK



Ethylenereceptor

C2H4

Geneexpression

Phospholipid

OutsideInside

OutsideInside

Ca

Ca

Ca

Ca

CaCa

Ca

Ca

CaCa

HH

H

H

H

HH

H

Ca

Ca

PLD

PLD

PLD

PLD

Phospholipase D

Increased cytosolicCa2+, H+

Phosphatidic acid

Diacylglycerols

Phosphatidatephosphatase

Free fatty acids

Fatty aldehydes

Calmodulin

Lipoxygenase

Alkanes

Peroxidized fattyacidsFree radicals

Gel phaseformation, reducedmembrane fluidity

Leakage

Damage to Ca2+

-H+ATPase

Lipolytic acylhydrolase

Ca2

Autocatalytic

Figure 27.5. Diagrammatic representation of the autocatalytic pathway of phospholipid degradation that occur during fruit ripening/harveststress in horticultural produce.

PLD to the membrane where it can initiate membrane lipiddegradation. The precise relation between the stimulation of theethylene receptor and PLD activation is not fully understood,but could involve the release of calcium and migration of PLDto the membrane, formation of a metabolising enzyme complex(metabolon) with other lipid degrading enzymes of the pathwayas well as calmodulin. PLD alpha appear to be the key enzymeresponsible for the initiation of membrane lipid degradation intomato fruits (Pinhero et al. 2003). Antisense inhibition of PLDalpha in tomato fruits resulted in the reduction of PLD activityand consequently, an improvement in the shelf life, firmness,soluble solids and lycopene content of the ripe fruits (Whitakeret al. 2001, Pinhero et al. 2003, Oke et al. 2003, Paliyath et al.2008a). There are other phospholipid degrading enzymes suchas phospholipase C and phospholipase A2. Several roles of theseenzymes in signal transduction processes have been extensivelyreviewed (Wang 2001, Meijer and Munnik 2003).

LOX exists as both soluble and membranous forms in tomatofruits (Todd et al. 1990). Very little information is available onphosphatidate phosphatase and lipolytic acyl hydrolase in fruits.

Proteolysis and Structure Breakdownin Chloroplasts

The major proteinaceous compartment in fruits is the chloro-plast which is distributed in the epidermal and hypodermal lay-ers of fruits. The chloroplasts are not very abundant in fruits.During senescence, the chloroplast structure is gradually disas-sembled with a decline in chlorophyll levels due to the degra-dation and disorganisation of the grana lamellar stacks of thechloroplast. With the disorganisation of the thylakoid, globularstructures termed as plastoglobuli accumulate within the chloro-plast stroma, which are rich in degraded lipids. The degradationof chloroplasts and chlorophyll result in the unmasking of other

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coloured pigments and is a prelude to the state of ripening anddevelopment of organoleptic qualities. Mitochondria, which arealso rich in protein, are relatively stable and undergo disassem-bly during the latter part of ripening and senescence.

Chlorophyll degradation is initiated by the enzyme chloro-phyllase which splits chlorophyll into chlorophyllide and thephytol chain. Phytol chain is made up of isoprenoid units(methyl-1,3-butadiene), and its degradation products accumu-late in the plastoglobuli. Flavour components such as 6-methyl-5-heptene-2-one, a characteristic component of tomato flavour,are also produced by the catabolism of phytol chain. Theremoval of magnesium from chlorophyllide results in the forma-tion of pheophorbide. Pheophorbide, which possesses a tetrapy-role structure, is converted to a straight chain colourless tetrapyr-role by the action of pheophorbide oxidase. Action of severalother enzymes is necessary for the full catabolism of chloro-phyll. The protein complexes that organise the chlorophyll,the light-harvesting complexes, are degraded by the action ofseveral proteases. The enzyme ribulose-bis-phosphate carboxy-lase/oxygenase (Rubisco), the key enzyme in photosyntheticcarbon fixation, is the most abundant protein in chloroplast.Rubisco levels also decline during ripening/senescence due toproteolysis. The amino acids resulting from the catabolism ofproteins may be translocated to regions where they are neededfor biosynthesis. In fruits, they may just enrich the soluble frac-tion with amino acids.

SECONDARY PLANT PRODUCTSAND FLAVOUR COMPONENTSSecondary plant products are regarded as metabolites that arederived from primary metabolic intermediates through well-defined biosynthetic pathways. The importance of the secondaryplant products to the plant or organ in question may not readilybe obvious, but these compounds appear to have a role in theinteraction of the plant with the environment. The secondaryplant products may include non-protein amino acids, alkaloids,isoprenoid components (terpenes, carotenoids, etc.), flavonoidsand anthocyanins, ester volatiles and several other organic com-pounds with diverse structure. The number and types of sec-ondary plant products are enormous, but, with the perspectiveof fruit quality, the important secondary plant products includeisoprenoids, anthocyanins and ester volatiles.

Isoprenoid Biosynthesis

In general, isoprenoids possess a basic five-carbon skeleton inthe form of 2-methyl-1,3-butadiene (isoprene), which under-goes condensation to form larger molecules. There are twodistinct pathways for the formation of isoprenoids: the ac-etate/mevalonate pathway (Bach et al. 1999) localised in thecytosol and the DOXP pathway (Rohmer pathway, Rohmer et al.1993) localised in the chloroplast (Fig. 27.6). The metabolic pre-cursor for the acetate/mevalonate pathway is acetyl CoenzymeA. Through the condensation of three acetyl CoA molecules,a key component of the pathway, 3-hydroxy-3-methyl-glutarylCoA (HMG CoA) is generated. HMG-CoA undergoes reduc-

tion in the presence of NADPH mediated by the key regulatoryenzyme of the pathway HMG CoA reductase (HMGR), to formmevalonate. Mevalonate undergoes a two-step phosphorylationin the presence of ATP, mediated by kinases, to form isopen-tenyl pyrophosphate (IPP), the basic five carbon condensationalunit of several terpenes. IPP is isomerised to dimethylallylpy-rophosphate (DMAPP) mediated by the enzyme IPP isomerase.Condensation of these two components results in the synthe-sis of C10 (geranyl), C15 (farnesyl) and C20 (geranylgeranyl)pyrophosphates. The C10 pyrophosphates give rise to monoter-penes, C15 pyrophosphates give rise to sesquiterpenes and C20pyrophosphates give rise to diterpenes. Monoterpenes are ma-jor volatile components of fruits. In citrus fruits, these includecomponents such as limonene, myrcene, pinene and so on occur-ring in various proportions. Derivatives of monoterpenes suchas geranial, neral (aldehydes), geraniol, linalool, terpineol (al-cohols), geranyl acetate, neryl acetate (esters) and so on are alsoingredients of the volatiles of citrus fruits. Citrus fruits are es-pecially rich in monoterpenes and derivatives. Alpha-farneseneis a major sesquiterpene (C15) component evolved by apples.The catabolism of alpha-farnesene in the presence of oxygeninto oxidised forms has been implicated as a causative feature inthe development of the physiological disorder superficial scald(a type of superficial browning) in certain varieties of ap-ples such as red Delicious, McIntosh, Cortland and so on(Rupasinghe et al. 2000, 2003).

HMGR is a highly conserved enzyme in plants and is encodedby a multigene family (Lichtenthaler et al. 1997). The HMGRgenes (hmg1, hmg2, hmg3, etc.) are nuclear encoded and canbe differentiated from each other by the sequence differencesat the 3′-untranslated regions of the cDNAs. There are threedistinct genes for HMGR in tomato and two in apples. The dif-ferent HMGR end products may be localised in different cellularcompartments and are synthesised differentially in response tohormones, environmental signals, pathogen infection and so on.In tomato fruits, the level of hmg1 expression is high duringearly stage of fruit development when cell division and expan-sion processes are rapid, when it requires high levels of sterolsfor incorporation into the expanding membrane compartments.The expression of hmg2 which is not detectable in young fruitsincreases during the latter part of fruit maturation and ripening.

HMGR activity can be detected in both membranous and cy-tosolic fractions of apple fruit skin tissue extract. HMGR isa membrane-localised enzyme, and the activity is detectablein the endoplasmic reticulum, plastid and mitochondrial mem-branes. It is likely that HMGR may have undergone proteolyticcleavage releasing a fragment into the cytosol, which also pos-sesses enzyme activity. There is a considerable degree of interac-tion between the different enzymes responsible for the biosyn-thesis of isoprenoids, which may exist as multienzyme com-plexes. The enzyme Farnesyl pyrophosphate synthase, respon-sible for the synthesis of farnesyl pyrophosphate is a cytosolicenzyme. Similarly, farnesene synthase, the enzyme which con-verts farnesyl pyrophosphate to alpha-farnesene in apples, is acytosolic enzyme. Thus, several enzymes may act in concertat the cytoplasm/endoplasmic reticulum boundary to synthesiseisoprenoids.

P1: SFK/UKS P2: SFK



Figure 27.6. Isoprenoid biosynthetic pathway in plants.

HMG CoA reductase expression and activities in apple fruitsare hormonally regulated (Rupasinghe et al. 2001, 2003). Thereare two genes for HMGR in apples designated as hmg1 andhmg2, which are differentially expressed during storage. The ex-pression of hmg1 was constitutive, and the transcripts (mRNA)were present throughout the storage period. In contrast, the ex-pression of hmg2 increased during storage in parallel with theaccumulation of alpha-farnesene. Ethylene production also in-creased during storage. Ethylene stimulates the biosynthesisof alpha-farnesene as evident from the inhibition of alpha-farnesene biosynthesis and the expression of hmg2 by theethylene action inhibitor 1-methylcyclopropene (MCP). Thus,biosynthesis of isoprenoids is a highly controlled process.

Carotenoids, which are major isoprenoid components ofchloroplasts, are biosynthesised through the Rohmer pathway.

The precursors of this pathway are pyruvate and glyceraldehyde-3-phosphate, and through a number of enzymatic steps, 1-deoxy-D-xylulose-5-phosphate (DOXP), a key metabolite of thepathway, is formed. NADPH-mediated reduction of DOXP leadsultimately to the formation of IPP. Subsequent condensation ofIPP and DMAPP are similar as in the classical mevalonate path-way. Carotenoids have a stabilising role in the photosynthetic re-actions. By virtue of their structure, they can accept and stabiliseexcess energy absorbed by the light-harvesting complex. Duringthe early stages of fruit development, the carotenoids have pri-marily photosynthetic function. During fruit ripening, the com-position of carotenoids changes to reveal the coloured xantho-phylls pigments. In tomato, lycopene is the major carotenoidpigment that accumulates during ripening. Lycopene is an inter-mediate of the carotene biosynthetic pathway. In young fruits,

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lycopene formed by the condensation of two geranylgeranyl py-rophosphate (C20) moieties mediated by the enzyme phytoenesynthase is converted to beta-carotene by the action of the en-zyme sesquiterpene cyclase. However, as ripening proceeds, thelevels and activity of sesquiterpene cyclase are reduced lead-ing to the accumulation of lycopene in the stroma. This leadsto the development of red colour in ripe tomato fruits. In yel-low tomatoes, the carotene biosynthesis is not inhibited, and asthe fruit ripens, the chlorophyll pigments are degraded expos-ing the yellow carotenoids. Carotenoids are also major compo-nents that contribute to the colour of melons. Beta-carotene isthe major pigment in melons with an orange flesh. In addition,the contribution to colour is also provided by alpha-carotene,delta-carotene, phytofluene, phytoene, lutein and violaxanthin.In red-fleshed melons, lycopene is the major ingredient, whereasin yellow-fleshed melons, xanthophylls and beta-carotene pre-dominate. Carotenoids provide not only a variety of colour to thefruits but also important nutritional ingredients in human diet.Beta-carotene is converted to vitamin A in the human body andthus serves as a precursor to vitamin A. Carotenoids are strongantioxidants. Lycopene is observed to provide protection fromcardiovascular diseases and cancer (Giovanucci 1999). Lutein,a xanthophyll, has been proposed to play a protective role inthe retina maintaining the vision and prevention of age-relatedmacular degeneration.

Anthocyanin Biosynthesis

The development of colour is a characteristic feature of theripening process, and in several fruits, the colour componentsare anthocyanins biosynthesised from metabolic precursors. Theanthocyanins accumulate in the vacuole of the cell, and are oftenabundant in the cells closer to the surface of the fruit. Antho-cyanin biosynthesis starts by the condensation of three moleculesof malonyl CoA with p-coumaroyl CoA to form tetrahydroxy-chalcone, mediated by the enzyme chalcone synthase (Fig. 27.7).Tetrahydroxychalcone has the basic flavonoid structure C6-C3-C6, with two phenyl groups separated by a three-carbon link.Chalcone isomerase enables the ring closure of chalcone lead-ing to the formation of the flavanone, naringenin that possessesa flavonoid structure having two phenyl groups linked togetherby a heterocyclic ring. The phenyl groups are designated as Aand B and the heterocyclic ring is designated as ring C. Subse-quent conversions of naringenin by flavonol hydroxylases resultin the formation of dihydrokaempferol, dihydromyricetin anddihydroquercetin, which differ in their number of hydroxyl moi-eties. Dihydroflavonol reductase converts the dihydroflavonolsinto the colourless anthocyanidin compounds leucocyanidin,leucopelargonidin and leucodelphinidin. Removal of hydrogensand the induction of unsaturation of the C-ring at C2 and C3,mediated by anthocyanin synthase results in the formation ofcyanidin, pelargonidin and delphinidin, the coloured compounds(Figs. 27.7 and 27.8). Glycosylation, methylation, coumaroyla-tion and a variety of other additions of the anthocyanidins resultin colour stabilisation of the diverse types of anthocyanins seenin fruits. Pelargonidins give orange, pink and red colour, cyani-dins provide magenta and crimson colouration, and delphinidins

provide the purple, mauve and blue colour characteristic to sev-eral fruits. The colour characteristics of fruits may result from acombination of several forms of anthocyanins existing together,as well as the conditions of pH and ions present in the vacuole.

Anthocyanin pigments cause the diverse colouration of grapecultivars resulting in skin colours varying from translucent, redand black. All the forms of anthocyanins along with those withmodifications of the hydroxyl groups are routinely present in thered and dark varieties of grapes. A glucose moiety is attachedat the 3 and 5 positions or at both in most grape anthocyanins.The glycosylation pattern can vary between the European (Vitisvinifera) and North American (Vitis labrusca) grape varieties.Anthocyanin accumulation occurs towards the end of ripening,and is highly influenced by sugar levels, light, temperature, ethy-lene and increased metabolite translocation from leaves to fruits.All these factors positively influence the anthocyanin levels.Most of the anthocyanin accumulation may be limited to epider-mal cell layers and a few of the sub-epidermal cells. In certainhigh anthocyanin containing varieties, even the interior cellsof the fruit may possess high levels of anthocyanins. In the redwine varieties such as merlot, pinot noir and cabernet sauvignon,anthocyanin content may vary between 1500 and 3000 mg/kgfresh weight. In some high-anthocyanin-containing varietiessuch as Vincent, Lomanto and Colobel, the anthocyanin lev-els can exceed 9000 mg/kg fresh weight. Anthocyanins are verystrong antioxidants and are known to provide protection fromthe development of cardiovascular diseases and cancer.

Many fruits have a tart taste during early stage of develop-ment, which is termed as astringency, and is characteristic tofruits such as banana, kiwi, grape and so on. The astringency isdue to the presence of tannins and several other phenolic com-ponents in fruits. Tannins are polymers of flavonoids such ascatechin and epicatechin, phenolic acids (caffeoyl tartaric acid,coumaroyl tartaric acid, etc.). The contents of tannins decreaseduring ripening, making the fruit palatable.

Ester Volatile Biosynthesis

The sweet aroma characteristic to several ripe fruits are due tothe evolution of several types of volatile components that in-clude monoterpenes, esters, organic acids, aldehydes, ketones,alkanes and so on. Some of these ingredients specifically providethe aroma characteristic to fruits and are referred to as characterimpact compounds. For instance, the banana flavour is predom-inantly from isoamyl acetate, apple flavour from ethyl-2 methylbutyrate, and the flavour of lime is primarily due to the monoter-pene limonene. As the name implies, ester volatiles are formedfrom an alcohol and an organic acid through the formation of anester linkage. The alcohols and acids are, in general, productsof lipid catabolism. Several volatiles are esterified with ethanolgiving rise to ethyl derivatives of aliphatic acids (ethyl acetate,ethyl butyrate, etc.).

The ester volatiles are formed by the activity of the enzymeAcyl CoA: alcohol acyltransferase or generally called as alco-holacyltransferase (AAT). In apple fruits, the major aroma com-ponents are ester volatiles (Paliyath et al. 1997). The alcoholcan vary from ethanol, propanol, butanol, pentanol, hexanol and

P1: SFK/UKS P2: SFK



Phenylalanine

p-Coumaryl-CoA p-Coumaric acidCoumaryl-CoA ligase

Chalcone synthase

ChalconeMalonyl-CoA

(3)

Acetyl-CoA

Glycolysis

Glucose

Chalcone isomerase

Naringenin

Flavonol hydroxylase

Dihydrokaempferol DihydromyricetinDihydroquercetin

Leucodelphinidin

Leucopelargonidin

Anthocyanin

Leucocyanidin

DelphinidinPelargondinCyanidin

Trans-cinnamic acid

Cinnmate-4-hydroxylase

Phenylalanine ammonia lyase

Anthocyanin biosynthetic pathway

Dihydroflavonol-4-reductase

Anthocyanin synthase

3-Glucosyl transferase

Methyl transferase

Figure 27.7. Anthocyanin biosynthetic pathway in plants.

so on. The organic acid moiety containing the CoA group canvary in chain length from C2 (acetyl) to C12 (dodecanoyl). AATactivity has been identified in several fruits that include banana,strawberry, melon, apple and so on. In banana, esters are thepredominant volatiles enriched with esters such as acetates andbutyrates. The flavour may result from the combined perceptionof amyl esters and butyl esters. Volatile production increasesduring ripening. The components for volatile biosynthesis mayarise from amino acids and fatty acids. In melons, the volatilecomponents comprise esters, aldehydes, alcohols, terpenes andlactones. Hexyl acetate, isoamyl acetate and octyl acetate are themajor aliphatic esters. Benzyl acetate, phenyl propyl acetate and

phenyl ethyl acetate are also observed. The aldehydes, alcohols,terpenes and lactones are minor components in melons. In mangofruits, the characteristic aroma of each variety is based on thecomposition of volatiles. The variety “Baladi” is characterisedby the presence of high levels of limonene, other monoterpenesand sesquiterpenes, and ethyl esters of even numbered fattyacids. By contrast, the variety “Alphonso” is characterised byhigh levels of C6 aldehydes and alcohols (hexanal, hexanol) thatmay indicate a high level of fatty acid peroxidation in ripe fruits.C6 aldehydes are major flavour components of tomato fruitsas well. In genetically transformed tomatoes (antisense PLD),the evolution of pentanal and hexenal/hexanal was much higher

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8

9

1+

2

1’

2’

3’

4’

5’

6’

B

3

4

C

7

6

OH

OH

OH

OH OH

OCHa

OH OH

OH OH

HO HOO⊕

OH

HO O

5

A

10

Pelargonidin

Cyanidin Peonidin

+

OH

OH

OH

OH

OH

HO O⊕

Delphinidin

Anthocyanidins

Anthocyanins

Antho cyanidins

GlycosylationGalactosylation

+

O⊕+

OCH3

OH

OH

OH

OH

H

Petunidin

O⊕+

OCH3

OCH3

OH

OH

OH

HO

Malvidin

O⊕+

Figure 27.8. Some common anthocyanidins found in fruits and flowers.

after blending, suggesting the preservation of fatty acids in ripefruits. Preserving the integrity of the membrane during ripeningcould help preserve the fatty acids that contribute to the flavourprofile of the fruits and this feature may provide a better flavourprofile for fruits.

GENERAL READING

Buchanan BB et al. (eds.) 2000. Biochemistry and MolecularBiology of Plants. American Society of Plant Physiologists,Bethesda, MD.

Kays SJ. 1997. Postharvest Physiology of Perishable Plant Prod-ucts. Exon Press, Athens.

Paliyath G et al. (eds.) 2008a. Postharvest Biology and Technologyof Fruits, Vegetables and Flowers. Wiley-Blackwell, Iowa.

Seymour GB et al. (eds.) 1991. Biochemistry of Fruit Ripening.Chapman and Hall, London.

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Food Biochemistry and Food Processing (Simpson/Food Biochemistry and Food Processing) || Biochemistry of Fruits