8 food biochemistry and food phần 111

In apple fruits, malic acid is converted to pyruvate by the action of NADP-malic enzyme, and pyruvate subsequently converted to ethanol by the action of pyruvate decarboxylase and alcoho

is converted to lactate-by-lactate dehydrogenase using NADH

as the reducing factor, and generating NAD Accumulation

of 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 activadecarboxyla-tion of pyruvate

decarboxylase under low pH conditions in the cytosol It is

also likely that the increase in concentration of pyruvate in the

cytoplasm may stimulate pyruvate decarboxylase directly

Ac-etaldehyde is reduced to ethanol by alcohol dehydrogenase using

NADH as the reducing power Thus, acetaldehyde and ethanol

are common volatile components observed in the headspace of

fruits indicative of the occurrence of anaerobic respiration

Cy-tosolic acidification is a condition that stimulates deteriorative

reactions By removing lactate through efflux and converting

pyruvate 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, respiratory

activity 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 resres-piration was observed

in 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 oxaloacetate

was detectable in Hamlin orange juice An increase in the

activ-ity levels of pyruvate decarboxylase, alcohol dehydrogenase and

malic enzyme was noticed in parallel with the decline in

pyru-vate and accumulation of ethanol In apple fruits, malic acid is

converted to pyruvate by the action of NADP-malic enzyme,

and pyruvate subsequently converted to ethanol by the action of

pyruvate decarboxylase and alcohol dehydrogenase The

alco-hol dehydrogenase in apple can use NADPH as a cofactor, and

NADP is regenerated during ethanol production, thus driving

malate utilisation Ethanol is either released as a volatile or can

be 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 as

carbon precursors for the biosynthesis of amino acids, nucleic

acids, secondary plant products and so on The PPP shares many

of the sugar phosphate intermediates with the glycolytic

path-way (Fig 27.4) The PPP is characterised by the interconversion

of 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 and

6-phosphogluconate dehydrogenase (Fig 27.4) In the first step,

glucose-6-phosphate is converted to 6-phosphogluconate by the

removal 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-carbon sugar phosphate This reaction involves the removal of a car-bon dioxide molecule along with the formation of NADPH Ribulose-5-phosphate undergoes several metabolic conversions

to yield fructose-6-phosphate Fructose-6-phosphate can then be converted back to 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 comcom-plete oxidation of a glucose molecule

Despite the differences in the reaction sequences, the gly-colytic pathway and the PPP intermediates can interact with one another and share common intermediates Intermediates of both the 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 chloroplast and cytoplasm Cytosolic glucose-6-phosphate dehydrogenase activity is strongly inhibited by NADPH Thus, the ratio of NADP to NADPH could be the regulatory control point for the enzyme function The chloroplast-localised enzyme is regu-lated differently through oxidation and reduction, and reregu-lated to the photosynthetic process 6-Phosphogluconate dehydrogenase exists as distinct cytosol- and plastid-localised isozymes The PPP is a key metabolic pathway related to biosyn-thetic reactions, antioxidant enzyme function and general stress tolerance of the fruits Ribose-5-phosphate is used in the biosynthesis of nucleic acids and erythrose-4- phosphate

is channelled into phenyl propanoid pathway leading to the biosynthesis 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 of sugars in fruits during ripening has been related to the function

of PPP In mangoes, an increase in the levels of pentose sugars observed during ripening has been related to increased activity

of PPP Increases in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were observed during ripening of mango

NADPH is a key component required for the proper function-ing of the antioxidant enzyme system (Fig 27.4) Durfunction-ing growth, stress conditions, fruit ripening and senescence, free radicals are generated within the cell Activated forms of oxygen, such as superoxide, 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 of antioxidants and antioxidant enzymes Simple antioxidants such

as ascorbate and vitamin E can scavenge the free radicals and protect the tissue Anthocyanins and other polyphenols may also serve as simple antioxidants In addition, the antioxidant enzyme 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 is immediately acted upon by catalase, generating water Hydro-gen peroxide can also be removed by the action of peroxidases

Transketolase

Xylulose-5-phosphate Ribose-5-phosphate

Ribulose-5-phosphate

6-Phosphogluconate

Glucose-6-phosphate dehydrogenase

Antioxidant (enzyme) system

Mitochondria chloroplast Membrane degradation

SOD

POX CAT APX MDHAR

DHAR GR

6-phosphogluconate dehydrogenase

Glyceraldehyde-3-phosphate

Oxidative pentose phosphate pathway

Pyruvate

Isoprenoids (carotenoids)

Nucleic acid

Glucose-6-phosphate

CO 2

NADPH

NADPH

NADP +

2 H +

H2O

H2O

H2O2

O2 O2

-NADP+ NADPH

NADPH GSSG

NADPH pool

Glycolysis NADP

NADP

Pentose phosphate isomerase Epimerase

Transaldolase

Erythrose-4-phosphate Fructose-6-phosphate

Phenyl propanoid pathway

Chalcone

Anthocyanins

Figure 27.4 Oxidative pentose phosphate pathway in plants NADPH generated from the pentose phosphate pathway is channeled into the

antioxidant enzyme system, where the regeneration of oxidised intermediates requires NADPH GSH, reduced glutathione; GSSG, oxidised glutathione; 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 (usually having 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 upon

by ascorbate peroxidase, which uses ascorbate as the hydro-gen donor for the reaction, resulting in water formation The oxidised ascorbate is regenerated by the action of a series of enzymes (Fig 27.4) These include monodehydroascorbate re-ductase (MDHAR) and dehydroascorbate rere-ductase (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 GSH

by 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 enzymes are also compartmentalised in chloroplast, mitochondria and cy-toplasm The functioning of the antioxidant enzyme system is crucial to the maintenance of fruit quality through preserving cellular structure and function (Meir and Bramlage 1988, Ahn

et al 2002)

Lipid Metabolism

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

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 oil

content 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 fatty

acids are exported into the endoplasmic reticulum where they are

esterified with glycerol-3-phosphate by the action of a number

of enzymes to form the triglyceride The triglyceride-enriched

regions then are believed to bud off from the endoplasmic

retic-ulum as the oil body The oil body membranes are different

from other cellular membranes, since they are made up of only a

single layer of phospholipids The triglycerides are catabolised

by the action of triacylglycerol lipases with the release of fatty

acids The fatty acids are then broken down into acety CoA units

throughβ-oxidation.

Even though phospholipids constitute a small fraction of the

lipids in fruits, the degradation of phospholipids is a key

fac-tor that controls the progression of senescence As in several

senescing systems, there is a decline in phospholipids as the

fruit undergoes senescence With the decline in phospholipid

content, there is a progressive increase in the levels of

neu-tral lipids, primarily diacylglycerols, free fatty acids and fatty

aldehydes 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 the

formation of gel phase or non-bilayer lipid structures (micelles)

These changes can make the membranes leaky, thus resulting

in the loss of compartmentalisation, and ultimately, senescence

(Paliyath and Droillard 1992)

Membrane lipid degradation occurs by the tandem action of

several enzymes, one enzyme acting on the product released by

the previous enzyme in the sequence Phospholipase D (PLD)

is the first enzyme of the pathway which initiates phospholipids

catabolism and is a key enzyme of the pathway (Fig 27.6)

PLD acts on phospholipids liberating phosphatidic acid and the

respective headgroup (choline, ethanolamine, glycerol,

inosi-tol) Phosphatidic acid, in turn, is acted upon by phosphatidate

phosphatase 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 the

enzyme lipolytic acyl hydrolase liberating free fatty acids

Un-saturated fatty acids with a cis-1,4- pentadiene structure (linoleic

acid, linolenic acid) are acted upon by lipoxygenase (LOX)

caus-ing the peroxidation of fatty acids This step may also cause the

production of activated oxygen species such as singlet oxygen,

superoxide and peroxy radicals and so on The peroxidation

products of linolenic acid can be 9-hydroperoxy linoleic acid

or 13-hydroperoxy linoleic acid The hydroperoxylinoleic acids

undergo cleavage by hydroperoxide lyase resulting in several

products including hexanal, hexenal andω-keto fatty acids (keto

group 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 esters that constitute components of flavour volatiles that are charac-teristic to fruits The free fatty acids and their catabolites (fatty aldehydes, fatty alcohols, alkanes, etc.) can accumulate in the membrane causing membrane destabilisation (formation of gel phase, non-bilayer structures, etc.) An interesting regulatory feature of this pathway is the very low substrate specifity of enzymes that act downstream from PLD for the phospholipids Thus, phosphatidate phosphatase, lipolytic acyl hydrolase and LOX do not directly act on phospholipids, though there are ex-ceptions to this rule Therefore, the degree of membrane lipid catabolism will be determined by the extent of activation of PLD (Fig 27.5)

The membrane lipid catabolic pathway is considered as an autocatalytic pathway (Fig 27.5) The destabilisation of the membrane can cause the leakage of calcium and hydrogen ions from the cell wall space, as well as the inhibition of calcium-and proton ATPases, the enzymes responsible for maintaining a physiological calcium and proton concentration within the cy-toplasm (calcium concentration below micromolar range, pH in the 6–6.5 range) Under conditions of normal growth and devel-opment, these enzymes pump the extra calcium- and hydrogen ions that enter the cytoplasm from storage areas such as apoplast and the ER lumen in response to hormonal and environmental stimulation using ATP as the energy source The activities of calcium- and proton ATPases localised on the plasma membrane, the endoplasmic reticulum and the tonoplast are responsible for pumping 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 of calcium and hydrogen ions PLD is stimulated by low pH and calcium concentration over 10µM Thus, if the cytosolic con-centrations of these ions progressively increase during ripening

or senescence, the membranes are damaged as a consequence However, this is an inherent feature of the ripening process

in fruits, and results in the development of ideal organoleptic qualities that makes them edible The uncontrolled membrane deterioration can result in the loss of shelf life and quality in fruits (Paliyath et al 2008)

The properties and regulation of the membrane degradation pathway are increasingly becoming clear Enzymes such as PLD and LOX are very well studied There are several isoforms of PLD designated as PLD alpha, PLD beta, PLD gamma and so

on The expression and activity levels of PLD alpha are much higher than that of the other PLD isoforms Thus, PLD alpha is considered as a housekeeping enzyme; however, it is also de-velopmentally regulated (Pinhero et al 2003) The regulation of PLD activity is an interesting feature PLD is normally a soluble enzyme The secondary structure of PLD shows the presence of a

segment of around 130 amino acids at the N-terminal end,

desig-nated as the C2 domain This domain is characteristic of several enzymes and proteins that are integral components of the hor-mone signal transduction system In response to hormonal and environmental stimulation and the resulting increase in cytosolic calcium concentration, C2 domain binds calcium and transports

Ethylene receptor

C2H4

Gene expression

Phospholipid Outside

Inside

Outside Inside

Ca

Ca

Ca Ca

Ca

Ca Ca

Ca Ca Ca

H H

H

H

H H H

H

Ca

Ca PLD PLD

PLD

PLD

Phospholipase D

Increased cytosolic

Ca2+, H+

Phosphatidic acid

Diacylglycerols

Phosphatidate phosphatase

Free fatty acids

Fatty aldehydes Calmodulin

Lipoxygenase Alkanes

Peroxidized fatty acids Free radicals

Gel phase formation, reduced membrane fluidity

Leakage

Damage to Ca 2+

-H+ATPase

Lipolytic acyl hydrolase

Ca2 Autocatalytic

Figure 27.5 Diagrammatic representation of the autocatalytic pathway of phospholipid degradation that occur during fruit ripening/harvest

stress in horticultural produce.

PLD to the membrane where it can initiate membrane lipid degradation The precise relation between the stimulation of the ethylene receptor and PLD activation is not fully understood, but could involve the release of calcium and migration of PLD

to the membrane, formation of a metabolising enzyme complex (metabolon) with other lipid degrading enzymes of the pathway

as well as calmodulin PLD alpha appear to be the key enzyme responsible for the initiation of membrane lipid degradation in tomato fruits (Pinhero et al 2003) Antisense inhibition of PLD alpha in tomato fruits resulted in the reduction of PLD activity and consequently, an improvement in the shelf life, firmness, soluble solids and lycopene content of the ripe fruits (Whitaker

et al 2001, Pinhero et al 2003, Oke et al 2003, Paliyath et al

2008a) There are other phospholipid degrading enzymes such

as phospholipase C and phospholipase A2 Several roles of these enzymes in signal transduction processes have been extensively reviewed (Wang 2001, Meijer and Munnik 2003)

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

Proteolysis and Structure Breakdown

in 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 the chloroplast With the disorganisation of the thylakoid, globular structures termed as plastoglobuli accumulate within the chloro-plast stroma, which are rich in degraded lipids The degradation

of chloroplasts and chlorophyll result in the unmasking of other

coloured pigments and is a prelude to the state of ripening and

development of organoleptic qualities Mitochondria, which are

also 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 the

phytol 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 The

removal 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 several

other 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 of

several proteases The enzyme ribulose-bis-phosphate

carboxy-lase/oxygenase (Rubisco), the key enzyme in photosynthetic

carbon fixation, is the most abundant protein in chloroplast

Rubisco levels also decline during ripening/senescence due to

proteolysis The amino acids resulting from the catabolism of

proteins may be translocated to regions where they are needed

for biosynthesis In fruits, they may just enrich the soluble

frac-tion with amino acids

SECONDARY PLANT PRODUCTS

AND FLAVOUR COMPONENTS

Secondary plant products are regarded as metabolites that are

derived from primary metabolic intermediates through

well-defined biosynthetic pathways The importance of the secondary

plant products to the plant or organ in question may not readily

be obvious, but these compounds appear to have a role in the

interaction of the plant with the environment The secondary

plant products may include non-protein amino acids, alkaloids,

isoprenoid components (terpenes, carotenoids, etc.), flavonoids

and 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 perspective

of fruit quality, the important secondary plant products include

isoprenoids, anthocyanins and ester volatiles

Isoprenoid Biosynthesis

In general, isoprenoids possess a basic five-carbon skeleton in

the form of 2-methyl-1,3-butadiene (isoprene), which

under-goes condensation to form larger molecules There are two

distinct pathways for the formation of isoprenoids: the

ac-etate/mevalonate pathway (Bach et al 1999) localised in the

cytosol 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 Coenzyme

A Through the condensation of three acetyl CoA molecules,

a key component of the pathway, 3-hydroxy-3-methyl-glutaryl

CoA (HMG CoA) is generated HMG-CoA undergoes

reduc-tion in the presence of NADPH mediated by the key regulatory enzyme of the pathway HMG CoA reductase (HMGR), to form mevalonate Mevalonate undergoes a two-step phosphorylation

in the presence of ATP, mediated by kinases, to form isopen-tenyl pyrophosphate (IPP), the basic five carbon condensational unit 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 C20 pyrophosphates give rise to diterpenes Monoterpenes are ma-jor volatile components of fruits In citrus fruits, these include components such as limonene, myrcene, pinene and so on occur-ring in various proportions Derivatives of monoterpenes such

as geranial, neral (aldehydes), geraniol, linalool, terpineol (al-cohols), geranyl acetate, neryl acetate (esters) and so on are also ingredients of the volatiles of citrus fruits Citrus fruits are es-pecially rich in monoterpenes and derivatives Alpha-farnesene

is a major sesquiterpene (C15) component evolved by apples The catabolism of alpha-farnesene in the presence of oxygen into oxidised forms has been implicated as a causative feature in the 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 encoded

by a multigene family (Lichtenthaler et al 1997) The HMGR

genes (hmg1, hmg2, hmg3, etc.) are nuclear encoded and can

be differentiated from each other by the sequence differences

at the 3-untranslated regions of the cDNAs There are three distinct genes for HMGR in tomato and two in apples The dif-ferent HMGR end products may be localised in difdif-ferent cellular compartments and are synthesised differentially in response to hormones, environmental signals, pathogen infection and so on

In tomato fruits, the level of hmg1 expression is high during

early stage of fruit development when cell division and expan-sion processes are rapid, when it requires high levels of sterols for incorporation into the expanding membrane compartments

The expression of hmg2 which is not detectable in young fruits

increases 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 is

a membrane-localised enzyme, and the activity is detectable

in the endoplasmic reticulum, plastid and mitochondrial mem-branes It is likely that HMGR may have undergone proteolytic cleavage 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 cytosolic enzyme Similarly, farnesene synthase, the enzyme which con-verts farnesyl pyrophosphate to alpha-farnesene in apples, is a cytosolic enzyme Thus, several enzymes may act in concert

at the cytoplasm/endoplasmic reticulum boundary to synthesise isoprenoids