2.

REVIEW OF LITERATURE

2.1 Studies on Oil Palm

In the plant kingdom fatty acids are the main source of

triglycerides, which are stored in the endosperm or cotyledons. However the fruit

tissues of oil palm, olive and avocado are exceptions to this. The oil reserves of

fatty seeds fuel the germination of seeds by the way of glyoxylate metabolism for

their energy mobilization. But in the case of oil palm, avocado and olive

mesocarp oil reserve does not participate in the process of seed germination and

the actual physiological role of these reserves is yet a debatable issue. The

possible answer is to interpret this reserve as a part of its dispersal mechanism

through biotic means. Man draws upon these natural resources to meet his

dietary requirements. Of the several hundred varieties of plants known to have

oil bearing seeds, only a few are significant commercially. They are Soyabean,

Groundnut, Cotton seed, Sunflower, Coconut, Oil palm, Olive, Sesame, Linseed,

Rapeseed, Castor and Tung. Of these the last three are used in industrial

purpose (Gurr, 1980). Animals also contribute significantly to the fat and oil pool.

9
Butter, lard, tallow and fish oil are the major animal fats that are commercially

important. In order to satisfy the growing needs for fats and oils as food and oleo

chemicals it is imperative to understand the mechanism of biogenesis of oil,

composition, structure and utilization of oil-bearing tissues. The plant

biochemistry pertaining to oil -bearing tissues has not received sufficient

attention in spite of their commercial importance. The available literature

confines largely to the composition analysis of lipids of the oleaginous tissue.

Cytochemical analysis of mature oleaginous tissues in the active

phase of oil deposition reveals the cytoplasm packed with spherical organelles

that react strongly with lipid stains and are undoubtedly sites of oil storage within

the cell. It may therefore be mentioned that the oil is stored in discrete

intracellular spherical organelles and are not in free form. There has been

confusion about the nomenclature of these oil - bearing organelles. The sub

cellular microscopic bodies containing neutral lipids have been variously referred

as spherosomes (Frey-Wyssling et al 1963; Jacks et aI., 1967; Rest and

Vaughan 1972; Wanner and Theimer 1978; Mohankumar et aI., 1990; Murphy,

2000). The controversy on the nomenclature of oil bodies has been resolved to a

greater extend as a result of electron microscopic studies of the structure of

oleosomes and their origin and formation (Mohankumar et al., 1990). In a

detailed electron microscopic examination of E.guineenis fruit, Mohankumar

th
(1992) reported that oleosomes were apparent from 8 week after pollination (8

10
WAP). Filamentous granular ribosome aggregations were found as electron

dense structures distributed throughout the cytosole at this stage. They appear

as irregular contour of nascent lipid droplets devoid of a limiting membrane. A

close proximity of these nascent droplets and rough endoplasmic reticulum was

also noticed. Average size of the oleosome of the oil palm fruit was found to fall

in the range of 0.3 to 0.6 ~l diameter and the thickness of the limiting membrane

of the globule varied from 2-4 nm. These observations are in line with those of

earlier workers (Boatman and Crambie, 1958; Wanner and Theimer 1978;

Mohankumar et aI., 1990; Murphy 2000). The available studies on the

morphology of the oleosomes of oil palm fruit indicate that they are delimited by

distinct half unit membranes as opposed to the ubiquitous tripartite membranes

of the other sub cellular organelles of plant and animal origin.

Mesocarp of oil palm fruit has been studied mostly from the

commercial angle, as it is a source of palm oil. Total lipids and their composition

and accumulations during fruit development therefore have been the main focus

of studies by various authors (Crombie and Hardman, 1958; Thomas et aI.,

.
1971, 00 et aI., 1985, 1986; Bafor and Osagie, 1986, 1988a; 1988b, 1989;

Mohankumar et aI., 1990; George and Arumughan, 1991; Ekpa et aI., 1994). The

first comprehensive report on lipid accumulation on oil palm fruit mesocarp was

from Crombie and Hardman (1958) for the dura variety of West African origin.

According to them the lipid accumulation in the mesocarp was delayed until the

11
full development of the seed (at about 19 weeks after pollination) and was then

extremely rapid with the major part of the lipid formed within a week. They also

studied the formation of carbohydrate in developing fruit and showed that starch,

sucrose and reducing sugars remain almost constant. More elaborate studies on

the lipid classes and their component fatty acids were reported in a series of

papers by Bafor and Osagie (1986, 1988a, 1988b, 1989) for Nigerian oil palm

fruits of the dura variety. The total lipid was found to accumulate between 18 -

22 weeks after pllination, which is almost in line with the report of Crombie and

Hardman (Bafor and Osagie, 1986). The subsequent reports on the details of the

nonpolar and polar lipids indicated that while polar lipids constituted the major

part of the lipids in the early stages of fruit development, the nonpolar lipids

accounted for 97% of the total lipids of the mature mesocarp (Bafor and Osagie,

1988a, 1988b). 00 et al. (1985) and 00 et al. (1986) examined the composition

of lipid classes in developing oil palm fruit mesocarp grown in Malaysia.

According to them the oil accumulation commenced from 12-13 weeks after

flowering and continued until the fruit ripened (20 weeks). They also observed

that the lipid continued to be deposited till the detachment of fruits from the

bunch. George and Arumughan (1991) examined the distribution of lipid classes

in the exocarp and mesocarp of three varieties of oil palm fruits. They noticed an

extremely high content of phospholipids and glycolipids in the exocarp. The lipids

of different process streams from a typical palm oil mill were also characterized

recently (George and Arumughan, 1992). It is clear that palm oil constituents

12
about 50% of the mature fresh mesocarp of the oil palm fruit are the most

important commercial product on which the oil palm industry depends. The

constituent fatty acids of palm oil have invariably been the subject of study when

the mesocarp of oil palm fruit is examined. Now it is well established that palm oil

contains major fatty acids such as myristic (1.3%), palmitic (47%), stearic (46%),

oleic (38.7%), linoleic (11 %) and linolenic (0.5%) (00 et ai., 1986; Bafor and

Osagie, 1986). From the fatty acid composition it may be noted that palm oil

contains saturated and unsaturated fatty acids in the ratio 1: 1 and therefore it

can be called a medium unsaturated fat. However, nutritional significance of

palm oil lies in its minor constituents. Though crude palm oil contains only 1% of

minor components (carotenoids, tocopherols, sterols, phospholipids, glycolipids,

hydrocarbons) they are significant from the nutritional and quality points of view

(Goh et ai., 1985; Arumughan et ai., 1989). For instance, raw palm oil is the

richest natural source of a and ~ carotene (provitamin A) and also is a rich

source of tocopherols (Vitamin E). Minor constituents of palm oil were reviewed

by Goh et ai., (1985). The edible red palm oil became more nutritional than other

vegetable oils because of its high vitamin content, provitamin A and vitamin E. In

order to get wide market for edible palm oil the intensity of the deep red colour is

reduced to light yellow by bleaching. The excess ~ - carotene is used as a

vitamin supplement in world market. Apart from its edible quality palm oil is used

for the manufacture of soaps and candles. Industrially palm oil is used

extensively in tin plate industry protecting cleaned iron surfaces before tin is

13
applied. Oil is used as a lubricant in textile and rubber industry. The palm kernel

oil is extracted from the kernel or endosperm contained a high amount of

saturated fatty acid namely lauric acid similar to coconut oil. Kernel oil is used as

an edible fat and in making ice cream, baked goods and confectionaries and for

the manufacture of soaps and detergents. Oil cake is used as a good livestock

feed. Quantitative changes of the non-lipid constituents such as cellulose; lignin,

hemiecellulose and pectic substances of mesocarp of the developing tenera

fruits were examined by Mohankumar et ai, (1994). It is clear from the study that

in the mesocarp, structural constituents increased slowly during the early stages

of fruit development followed by a steep increase in the mature stages. In mature

mesocarp, lignin and cellulose constituted 18-20%. Physicochemical and

structural characterization of alkali soluble lignin from oil palm trunk and empty

fruit bunch fibres were studies by Sun et al. (1999). The role of lignifying

enzymes Phenylalanine ammonia lyase (PAL), Cinnamyl alcohol - NADPH -

dehydrogenase (CAD) and Peroxidase (POD) in the shell synthesis of dura fruit

has also been extensively studied (Salini and Mohankumar 2001).

2.1.1 Oil Palm fruit lipase:

Though oil palm is a highly profitable source of palm oil, there are

inherent problems associated with fruit ripening, harvesting, handling, and

processing. The most important of them is the excessive production of free fatty

acids in the fruit and subsequently in the oil under improper handling and

14
processing conditions. The free fatty acid level in the oil is the main criterion that

determines the quality and therefore price of this commodity. From the economic

angle the main objective of the planter is to harvest properly ripened fruit

bunches and the processor to produce palm oil with minimum free fatty acid. It

has been a common knowledge since the beginning of the oil palm fruit

processing that free fatty acid formation is associated with several factors.

However, the exact cause has not been traced till recently. Handling and bruising

of oil palm fruit bunches led to the increase in free fatty acid content of the oil

(00, 1981; Arumughan et aI., 1989). A major factor for this was attributed to

enzymatic hydrolysis without much experimental evidence (Degraaf, 1966;

Hartley, 1979). The empirical nature of this observation could be summed up in

the words of Hartley. "It is believed that the fat is protected from the lipase in the

fruit by the membranes of the vacuoles and these may be ruptured either

mechanically or by low temperature" (Hartley, 1979). Earlier attempt to find

evidence for the presence of endogenous lipase in the oil palm fruit did not yield

results (00, 1981; Tombs and Stubbs, 1982). 00 (1981) tried to isolate the

lipase enzyme from the homogenate of ripe palm fruit mesocarp. He could not

detect the activity in the aqueous extract. As recently as 1982, Tombs and

Stubbs failed to detect the enzyme lipase in oil palm fruit and therefore they

attributed to the invading microorganisms for the hydrolysis of endogenous

triacylglycerol of the fruit mesocarp. For the first time, Abigor et al (1985)

demonstrated the activity of endogenous lipase in the mature oil palm fruit

15
mesocarp. The success of their experiment was mainly because they focused on

the fat pad of the mesocarp homogenate instead of the aqueous extract. The

authors could partially purify the enzyme and carried out experiments related to

substrate specificity for the enzyme. They noticed that this enzyme hydrolysed

palm oil triacylglycerols faster than triacylglycerols of other oils. Henderson and

Osbourne (1991) in their attempt to characterize the oil palm fruit lipase and to

study the factors that influence the enzyme activity presented further evidence

for the existence of an active lipase in the oil palm fruit mesocarp. The couple of

invitro studies of the enzyme so far reported could not provide evidence as to the

localization of this enzyme in the intact fruit and the factors that trigger its invivo

activity. Mohankumar et al (1990) conducted a detailed study on in vivo lipase

activity in oil palm fruit and confirmed its association with oleosome membranes.

2.1.2 Oil palm kernel

Oil palm fruit is unique in having two distinct energy reserves, one

in the fruit mesocarp (palm oil) and the other in the endosperm (palm kernel oil).

It is much more striking that both the reserves are in the form of storage lipids

with distinct chemical compositions. Though both of these tissues are

commercially significant sources of edible oils, the former is more important due

to greater volume of production. The endosperm in the oil palm fruit has an

added role as reserve energy for the embryo during the process of germination.

In spite of its commercial significance, the lipids of fruit mesocarp or any other

16
constituent of the mesocarp do not contribute to the seed germination, its role in

the dissipation of fruit notwithstanding.

Unlike other oil seeds the process of germination of oil palm seed

is more complex involving the co-operative interaction of tissues such as the

endosperm and haustorium and the shoot of growing seedling. Haustorium has

been reported to be a simple absorbing tissue developed during germination

during germination of seeds of Arecaceae family (Davis, 1966). In the case of oil

palm seed, apart from the biotransformation of fat to sugars, translocation of the

constituents and sites of transformation therefore are significant to understand

the mechanism of lipid utilization as a whole. Boatman and Crambie (1958) first

examined the fatty acid metabolism in the developing seedling of the oil palm.

They brought out distinct difference in the translocation of the endosperm fat as

compared to other fatty seeds. The oil palm haustorium considered as a fat

categorizing organ was also shown to be a fat absorbing one. They also showed

that shorter chain fatty acids were utilized faster by the haustorium. 00 and

Stumpf (1983a; 1983b) in their attempt to study the mechanism of lipid utilization

in ther germinating oil palm seed reported the presence of enzymes of the

glyxoylate cycle. Mohankumar (1992) studied the interaction of endosperm,

haustoria and shoot in fat metabolism of germinating seed biochemically.

Activities of the major enzymes of glyoxylate cycle viz isocitrate lyase and malate

synthase were found in haustorial tissues during earlier stages of germination.

17
These observations were further supported by localization of glyoxysomes in the

haustorial tissues by TEM. The foregoing review, pertaining to oil palm fruit,

reveals that oil palm fruit has been examined mostly from the commercial angle

and therefore most of the literature cited here are related to formation of oil in the

mesocarp, its fatty acid composition and lipase action with respect to fruit

development and variety. It may also be noted that the studies on formation and

synthesis of non lipid constituents and structural constituents of fruit during

development, which also contribute to physiology of the fruit, have not been

conducted systematically so far.

2.2 Oil palm fruit processing

The versatile nature of lipid composition and chemical constituents

of palm oil has given it more food value than other edible oils. The nutritive value

of red palm oil has got greater significance as the richest natural source of p-
carotene and a rich source of tocopherols. The local inhabitants of Africa

traditionally extracted crude palm oil from the fleshy fibrous mesocarp by boiling

and squeezing the fruits, which was technically called "soft oil production"

(Hartley, 1979). This crude methodology gained wider acceptance in other oil

palm growing countries with their own indigenous mechanization and

modernization. Considering the fast growing nature of the palm as well as its

high productivity a working group of RRL (T) has designed and demonstrated a

processing mill for the extraction of edible grade palm oil from fresh fruit bunches

18
for meeting the requirements of small scale farmers. Even though the principle of

the processing unit showed similarity with the traditional soft oil production, its

mechanization at each step of processing i.e., sterilization and striping of fruits;

hydraulic and screw pressing; clarification and centrifugation makes it more

sophisticated than the traditional methods (Fig. 4). Today this wet extraction

method becomes popular among the farmers of oil palm growing states of India.

As far as Kerala is concerned the high density of population and the non

availability of vast cultivable land at a minimum of 200 hectares makes the

current methodology impractical to local farmers. Thereby the small-scale

farmers would not have accepted oil palm as an alternative crop.

2.3 Lignification in plant tissues

Lignin is one of the major organic materials in the biosphere, being

second only to cellulose in abundance (Grisebach, 1981). It is synthesized from

phenylpropanoid units by a complex sequence of reactions, some catalyzed by

enzymes involved in general phenylpropanoid metabolism and other by branch

path enzymes specific for lignan and lignin biosynthesis. The term 'lignin' derived

from the Latin word 'lignum' meaning wood is indicative of its physical texture.

Lignin forms an essential component of the woody stems of arborescent plants

in which their amount ranges from 15-36%. However they are not restricted

exclusively to arborescent plants but are found as integral cell wall constituents

of all vascular plants including the herbaceous varieties. Their presence has

19
 Harvest

Sterilization

Stripping

Digestion

Hydraulic pressing

Clarification

Crude palm oil

Purification

Raw palm oil

(Edible grade)

Fig. 4 Steps involved in the production of edible

grade red palm oil by wet method
been demonstrated in tissues associated with stem, foliage, roots, seed coats

and fruits (Sarkanen and Ludwig, 1971). Because of its wide array of functional

diversity in plant system this constituent remains unique compared to other

structural components. Initially lignin plays an important role in intricate internal

transport of water, nutrients and metabolites. Secondly, lignin impart rigidity to

the cell wall and act as a permanent bounding agent between cells generating a

composite structure outstandingly resistant towards impact compression and

bending. Finally lignified tissues effectively resist attacks by microbes by

impeding penetration of destructive enzymes into the cell wall. The exact

definition and differentiation of lignin from other polyphenolic plant constituents

has remained a matter of debate until recently. Brown (1964) has defined lignin

as a polymeric natural product arising from an enzyme initiated dehydrogenative

polymerization of three primary precursors: trans - coniferyl, trans, sinapyl, and

trans - coumaryl alcohols. The rapid concurrent progress in the clarification of

biogenetic pathways leading to the formation of primary lignin precursors further

helped to consolidate the picture of lignin polymerization process for getting

meaningful definition of lignin from other polyphenolic plant constituents has

remained a matter of debate until recently, Brown (1964( has defined lignin as a

polymeric natural product arising, from an enzyme initiated dehydrogenative

polymerization of three primary precursors: trans - coniferyl, trans, sinapyl, and

trans coumaryl alcohols. The rapid concurrent progress in the clarification of

biogenetic pathways leading to the formation of primary lignin precursors further

20
helped to consolidate the picture of lignin polymerization process for getting

meaningful definition of lignin as phenolic polymer. The chemical structure

proposed by Freudenberg (1965) and his coworkers for spruce wood lignin has

substantial merits in providing a reasonable picture of the molecular architecture

of lignin and in forming a basis of mapping the cause of various chemical

reactions.

2.3.1 Detection of lignin in plant tissues:

Due to the presence of certain insoluble polyphenols often related

to tannins of flavanols in plant tissues, identification of lignin becomes a difficult

task. These constituents have certain characteristics common with lignin and as

a consequence they have been frequently confused with the latter. In

microscopic examination of tissue sections, lignin can be recognized

histochemically by Weisner and Maule's colour reactions as purple red and

brown colour deposits on cell walls respectively under visible light. The

controversy of identification of lignin in plant tissues based on its chemical group

has been resolved to a great extent as a result of histochemical studies under

light and electron microscopy. Weisner reaction has universal applicability to

guaiacyl Iignins, which reacts with the coniferaldehyde groups in lignin forming a

purple red colour. But the reaction may be weak or even absent in lignin

containing high amount of syringyl units. Maule's reaction is positive only for

lignin containing significant amount of syringyl units (Sarkanen and Ludwig,

1971). Microscopic methods based on the use of transmitted monochromatic UV

21
light are also applicable for the most accurate measurement of lignin distribution

in tissue sections. These histochemical methods have been used to identify the

types of lignins: guaiacyl lignin (G) and syringyl (8) lignin in plant tissues by

subsequent workers. (Faulkner and Kimmins, 1975; Pearse, 1980; Gahan,

1984). In angiosperms, the lignin consists of both guaiacyl and syringyl units in

approximately equal amounts. Analytical investigations of structure of lignin have

shown that the composition of lignin from gymnosperms is different from that of

angiosperms. Whereas gymnosperms are mainly made up of guaiacyl units

originated form coniferul alcohol, lignin from angiosperm dicotyledons contains

guaiacyl - syringyl units originated from both coniferyl and sinapyl alcohols.

Erythrina cristagtalli is an angiosperm that produces only guaiacyl lignin

(gymnosperm type) but has typical angiosperm lignin biosynthetic enzymes

(Campbell and 8ederoff, 1996). The difference in lignin composition can be

considered as a criterion for classifying them histochemically using specific

techniques. The variation in lignin composition is also reflected biochemically in

the substrate specificity involved in the biosynthesis of lignin precursors. Recent

investigations have shown that mosses do not contain true lignin but do contain

other cell wall constituents such as sphagnum acid (Grisebach, 1981).

2.3.2 Lignin biosynthesis

Lignin heterogeneity could be directly related to enzyme diversity

and specificity. Lignin heterogeneity within the plant could result from temporal

and spatial distribution of isoforms for given steps in the biosynthetic pathway.

22
The concept that lignin is derived from polymerization of coniferyl alcohol dates
th
from the later 19th and early 20 centruries (Freudenberg, 1965). Doubts still

remain about the degree to which the pathway follows the same sequence in

different plant species or different cell types within a species. Three monolignols

differing only in the substitution pattern on the aromatic ring can be polymerized

into lignin (Fig. 5). The relative abundance of the different monolignol residues in

lignin varies between species and within species, as does the total lignin content.

the mechanisms that control this variation are not well understood. The

mechanism and enzymology of monolignols into lignin have not yet been clearly

defined (0 'Malley et aI., 1993; Dean and Eriksson, 1994; Savidge et aI., 1994).

Confieryl alcohol and other monolignols are derived from

phenylalanine in a multistep process (Fig. 6). Several other major classes of

plant products in addition to lignin are derived from phenylalanine including

flavonoids, coumarines, stilbenses and benzoic acid derivatives (Dixon and

Paiva, 1995; Holton and Cornish, 1995). The initial step in the biosynthesis of all

these compounds are shared through the general phenylpropanoid pathway. The

phenylpropanoid compound are so named because of the basic structure of a 3-

carbon side chain on an aromatic ring, which is derived from L-phenylalanine

(Fig. 6). The monolignols themselves are relatively toxic, unstable compounds

that do not accumulate in high levels within the living plants. Glyoxylation of the

phenolic hydroxyl groups to produce monolignol glucosides stabilizes the

23
 (7) I"'"
0 H
(7) I
0
CCR

H
(7) 1
0
CCR

H
"C " "c" <>C "

¢ oH
p-coumaraldehyde
~ OH
con~eraldehyde
~h~ OH
slnapaldehyde

00 I <AD

p-coumaryl alcohol
00

con~eryl
lOAO
alcohol
00 lOAO
slnapyl alcohol

POD
POD

Lignin

Fig. 5 Pathway of lignin synthesis

 OCH 3 H3CO CH 3

OH OH OH
P-coumaryl alcohol coniferyl alcohol sinapyl alcohol

WVVV'
1
polyme~ WVVV'
1
polyme~ WVVV'
1
polyme~

¢O-R
~OCH' H'CO~OCH'
O-R O-R
p-hydroxyphenyl guaiacyl residue syringyl residue
residue in lignin in lignin in lignin

Fig. 6 Chemical structure of monolignols

compound and makes them non-toxic. The glucosides probably serves as both

storage and transport forms of monolignols (Whetten and Sederoff, 1995).

Enzymes capable of cleaving the glycosidic bonds exist in lignifying cells of many

species of plants. The free monolignols are believed to be polymerized to lignin

by a free radical mechanism that is initiated through oxidation of monolignols by

cell wall bound oxidases. Enzymic reactions of lignin biosynthesis and the

mechanism of its control and implications for the genetic improvement of plants

have been extensively reviewed by several authors (Sederoff et aI., 1994;

Whetten and Sederoff, 1995; Campbell and Sederoff, 1996). At different steps in

the biosynthetic pathway, several enzymes have been identified that could carry

out similar reactions during development. Multiple enzymes may also indicate

diversity of function or alternate pathways as biosynthesis.

Determination of phenylalanine to cinnamate: PAL is one of the most

intensively studied enzymes in plant secondary metabolism because of the key

role it plays in phenylpropanoid biosynthesis. Deamination of phenylalanine to

cinnamate is catalyzed by the enzyme PAL (EC 4.3.1.5). An analogous enzyme

activity (tyrosine ammonia lyase-TAL) that deaminates tyrosine to form p-

coumarate has been detected mainly in grasses. This activity frequently

copurifies with PAL activity and the question remains whether they are two

different proteins or simply two activities of a single polypeptide. As PAL may

show TAL activity, it is pertinent to ask whether it is closely related to any other

24
plant enzyme. Even though there are close structural and mechanistic

resemblances between PAL and histidine ammonia lyase (HAL) from bacteria

and mammals, a distinct enzyme tryptophan ammonia lyase has not been

described from any source (Hanson and Havier, 1981). Functional PAL enzyme

has been expressed from a parsley c-DNA in Escherichia coli, the same

experiment could be performed with a maize c-DNA to test for PAL and TAL

activities in the same polypeptide (Schulz et aI., 1989). Extensive purification of

PAL from cell suspension cultures of bean and alfalfa showed that these species

expressed multiple forms of PAL with different kinetic properties. The individual

PAL isozymes display Michaelis - Menten kinetics, but the mixture of isozymes

show the negative co-operativity previously considered characteristics of PAL

(Bolwell et aI., 1985; Jorrin and Dixon, 1990). PAL catalyzes the committed step

in phenylpropanoid metabolism and as such is well suited to playa regulatory

role in controlling biosynthesis of all phenylpropanoid compounds including lignin

(Northcote, 1985). Transgenic plants with modified levels of PAL activity have

provided opportunities to test hypotheses about the role of PAL in plant

metabolism and development (Elkind et aI., 1990). Bate et ai, (1994), analyzed

phenylpropanoid metabolites in transgenic tobacco plants with decreasing

amounts of PAL activity and found that lignin content is not greatly affected until

PAL activity is reduced to 20-25% of wild type levels. Levels of chlorogenic acid

(a caffeic acid esters) and rutine (a flavonoid glycoside) in contrast are affected

by small changes in PAL activity. A correlation of PAL activity with lignin

25
concentration in maIze internode during development has been reported by

Morrison et aL (1994). As the first enzyme of phenylpropanoid metabolism,

catalyzing the transamination of ammoia from L-phenylalanine to from trans -

cinnamic acid, a precursor of lignin biosynthesis, PAL isolated from different

sources have been discussed in detail by various authors (Hanson and Havier,

1981; Cunha, 1987). More detailed information on the physiological role of this

enzyme in lignin biosynthesis and defense mechanism of plant tissues were

reported in subsequent papers (Cunha, 1987; Goliber, 1989; Morrison and

Buxton, 1993; Morrison et aL, 1994; Sewalt et aL, 1997; Jung et aL, 1999).

Hydroxylation of cinnamic acids: Cinnamic acid formed by deamination is

then converted by a sequence of hydroxylation and methylation reactions to

several substituted acids that can be activated as their corresponding esters of

CoA. These activated acids can then enter different biosynthetic pathways

leading to lignin, flavonoids, stillbenes, benzoic acids and other compounds

(Grisebach, 1981). Hydroxylation of cinnamic acid to p-counmaric acid is

catalyzed by cinnamate 4-hydroxylase (C 4 H; EC 1.14.13.11), cytochrome pA50

lined mono-oxygenase. Molecular oxygen is cleaved during this reaction with one

oxygen atom added to the aromatic ring and the other reduced to water. C4 H has

been purified and characterized to different degrees from several plant species.

Feeding experiments using phenylalanine and cinnamic acids produced in situ

by PAL more readily than exogenously supplied cinnamic acids (Czichi and

26
Kindl, 1997; Hrazdina and Wagner, 1985). These findings suggest that

cinnamate is preferentially transferred from PAL to C 4 H, rather than coming to

equilibrium with cytosol and diffusing to C4H. This preferential transfer of

intermediates between the enzymes of a pathway is an example of metabolic

channeling (Srere, 1987; Hrazdina and Jensen, 1992). The degree to which

channeling occurs in the remainder to the monolignol biosynthetic pathway is as

yet unknown, but there have long been hypothesis that channeling might be

important in regulation of monolignol biosynthesis. Little is known about

coumarate 3-hydroxylase (C 3 H), the enzyme that catalyzes the dyroxylation of p-

coumarate to from coffeate (Whetten and Sederoff, 1995).

Methylation of phenolic acids: Caffeic acid is methylated to from ferulic acid

by caffeic 3-o-methyltransferase (C-OMT; EC 2.1.1.68) using S-adenosyl

methionine as the methyl group donor. This methylation reaction limits the

reactivity of the 3-hydroxy groups, thus reducing the number the sites on the

aromatic ring that can from bonds to other monolignol molecules during

polymerization. The same enzyme is also believed to catalyze the methylation of

5-hydroxyferulate to sinapate. C-OMT is clearly implicated in the synthesis of

monolignols based on genetic evidence from both monocots and dicots. The C-

OMTs from different species show preference for different substrates.

Comparison of C-OMT activity assayed in crude extracts from gymnosperms and

angiosperms lead to the conclusion that gymnosperm C-OMT typically shows

27
preferential activity with caffeate over 5-hydroxyferulate, whereas angiosperm C-

GMT shows greater activity with 5-hydroxyferulate as substrate (Kuroda et aL,

1981). Conifers generally contain primarily guaiacyl lignin, whereas angiosperms

can produce guaiacyl - syringyl lignin; the difference in substrate specificity of C-

GMT has been proposed to playa role in determining the monomer composition

of lignin (Gross, 1985; Higuchi, 1985). Some gymnosperms produce guaiacyl-

syringyl lignin and some angiosperms species produce only guaiacyl lignin

challenging the generalization about the distribution of guaiacyl-syringyl lignin

across taxa (Lewis and Yamamoto, 1990). An enzyme distinct from C-GMT,

caffeoyl -CoA 3-0-methyltransferase (CCoA-GMT; Ec 2.1.1.104) has been

identified in connection with the defense response in several dicot plant species

(Kuhnl el aL, 1989; Pakusch et aL, 1989). Based on the kinetics of the induction

of enzyme activity in cell cultures and the correlation of CCoA - GMT activity with

lignification, Ye et al. (1994) proposed that CCoA - GMT plays a role in

methylation of both caffeoyl CoA and 5-hydroxyferuloyl CoA during monolignol

biosynthesis, Hydroxylation of ferulate to 5-hydroxyferulate is catalyzed (FsH).

This enzyme has proven extremely difficult to work with; little has been published

since the first reported assay of F5 H activity in polar microsomes (Grand, 1984).

FsH has been implicated in the differences in lignin composition between

angiosperms and gymnosperms. F5 H activity is necessary for the hydroxylation

of ferulate, an essential step for the formation of sinapyl alcohol.

28
 Formation of GoA thioesters: 4-Coumarate: CoA ligase (4CL, EC 6.2.1.12)

catalyzes the formation of CoA thioesters of cinnamic acids in the biosynthesis of

a wide variety of phenolic derivatives including benzoic acid, condensed tannins,

flavonoids and the cinnamyl alcohols (Gross, 1985). 4CL depends strictly on

ATP, and the reaction resembles the activation of fatty acids, proceeding through

an intermediate acyladenylate, which reacts with CoA to from the thioester. 4CL

. from most plants shows low activity with sinapic acid (Kutsuki el aI., 1982b). Two

alternative pathways have been proposed to account for the production of

sinapyl alcohol. Higuchi (1985) suggested a route to sinapaldehyde through

hydroxylation of ferulate, activation of 5-hydroxyferulate to the CoA thioester,

reduction to 5-hydroxyconiferaldehyde, and methylation to sinapaldehyde 4CL

preparations from several angiosperm and gymnosperm species have shown

activity with 5-hydroxyferulate (Knobloch and Hahlbrock, 1977; Kutsuki et aI.,

1982b; Luderitz et aI., 1982; Grand et aI., 1983) Ye et aI., (1994) suggested a

pathway from caffeic acid through caffeoyl - CoA, feruloyl - CoA 5-

hydroxyferuloyl-CoA and sinapyl - CoA, based on the ability of CCoA-GMT to

methylate the CoA thioesters. This alternative pathway to sinapyl alcohol

requires hydroxylation of feruloyl - CoA to 5-hydroxyferuloyl - CoA before

methylation by CCoA-GMT. Reduction of hydroxycinnamoyl-CoA thioesters to

the corresponding aldehydes is catalyzed by cinnamoyl - CoA reductase (CCR;

EC 1.2.1.44). CCR does not generally exhibit much specificity for one

hydroxycinnamoyl-CoA substrate over another, although feruloyl - CoA is

29
reported to be best substrate for CCR (Gross and Kreiten, 1975; Wengenmayer

et aI., 1976; Sarni et aL, 1984; Goffner et aI., 1992). Goffner et al. (1992)

hypothesizsed that CCR plays a key regulatory role in lignin biosynthesis as the

first committed step in the production of monolignols from phenyl propanoid

metabolites.

Reduction of cinnamyl GoA estes to cinnamyl alcohols: The reduction of

hydroxycinnamaldehydes to hydroxycinnamyl alcohols is catalyzed by cinnamyi

alcohol - NADPH- dehydrogenase (CAD; EC 1.1.1.195). CAD has been

considered to be an indicator of lignin biosynthesis because of its specific role at

the end of monolignol biosynthetic pathway (Walter et aI., 1988). However, CAD

is expressed in cells that do not make lignin (O'Malley et aI., 1992; Grima-

Pettenati et aI., 1994). CAD is also expressed in response to stress, pathogen

elicitors and wounding (Campbell and Ellis, 1992a; Galliano et aI., 1993). CAD is

therefore regulated by both developmental and environmental pathways, much

like other well studied enzymes of phenylpropanoid metabolism. The catalystic

property of CAD in reducing the three cinnamaldehydes (sinapaldehyde, p-

coumaraldehyde and confieraldehyde) to the corresponding cinnamyl alcohols,

the direct monomeric precursors of lignin polymer has been well established in

tobacco, maize, populus and eucalyptus by biochemical studies (Grand et aI.,

1985; Halpin et aI., 1992; Morrison and Buxton, 1993; Grima - Pettenati et aI.,

1993; Hawkins and Boudet 1994; Morrison et al 1994). Due to its critical role in

30
lignin biosynthesis, CAD is also a potential target enzyme for biotechnology

directed towards modulating the quantity and quality of lignin in plants (Grima-

Pettenati et aI., 1993). Differences in substrate affinities of CAD enzymes from

angiosperms and gymnosperms may playa role in controlling the formation of

different types of lignin. (Kutsuki et aI., 1982a). Isoforms of CAD with markedly

different sustrate affinities are detected in such species as soybean, wheat,

eucalyptus and salix (Wyrambik and Grisebach, 1975; Mansell et aI., 1976;

Pillonel et aI., 1992; Goffner et aI., 1992). Many species however, are believed to

contain a single form of the enzyme. MacKay et al. (1995) identified a single

CAD enzyme encoded by a single gene in loblolly pine. CAD preparations from

gymnosperms are generally much more active on coniferaldehyde, whereas

angiosperm CAD preparations show more equal activities with coniferaldehyde

and sinapaldehyde (Gross, 1985). The monolignol p-hydroxycinnamyl alcohol,

conoferyl alcohol and sinapyl alcohol are relatively toxic and unstable

compounds. Glycosylation on the phenolic hydroxyl group, a reaction performed

by UDP-glucose: coniferyl alcohol glucosyltransferase (EC 2.4.1.111) forms the

monolignol glucosides p-hydroxycinnamyl alcohol glucoside, coniferin, and

syringin respectively. These glucosides accumulate in some species of plants

most notably in conifers. Experiments with labeled coniferin have shown that this

compound can act as a lignin precursor in a variety of species. However the role

of coniferin and other cinnamyl alcohol glucosides in lignification remains

uncertain.

31
Polymerization of cinnamyl alcohols to lignin : The polymerization of

monolignols (4-coumaryl, coniferyl and sinapyl alcohols) is initiated by oxidation

of the phenolic hydroxyl groups of these monomers yielding mesomeric phenoxy

radicals. Coupling of these radicals leads to dilignols, which after re-oxidation

and radical coupling are converted to oligomeric intermediates, which finally

combine to form the lignin macromolecule. Two different classes of enzymes

such as peroxidase (EC 1.11.1.7) and laccases (EC 1.10.3.2) have been

proposed to perform the polymerization of monolignols to lignin (Higuchi,1985;

O'Malley et aI., 1993; Dean and Eriksson, 1994). The evidence in support to the

involvement of both type of enzyme is extensive butcircumstantial. Laccase, an

oxygen-dependent oxidase containing four copper atoms, and peroxidase, an

H2 0 2 -dependent hemoprotein, are both capable of oxidizing monolignols to free

radicals in vitro (Whetten and Sederoff, 1975). The evidence for and against

involvement of peroxidase and laccase in lignification has recently been

reviewed (O'Malley et aI., 1993; Dean and Eriksson, 1994; Mc Dougall et aI.,

1994). A convincing answer to this question will require demonstration of

changes in lignin content or composition upon experimental manipulation of

enzyme activity, either in transgenic plants or in mutants defective in specific

enzymes. Such experiments are under way in several laboratories. The role of

these enzymes in lignification process has been suggested based on correlation

with lignification process. New approaches, such as Nuclear Magnetic

Reasonance (NMR), Raman microprobe studies for the analysis of lignin

32
structure in situ, have provided support for the concept that lignin polymerization

is not random but organized (Atalla and Agarwal, 1985; Agarwal and Atalla,

1986; Lewis et al., 1987). The participation of laccase and peroxidase in lignin

formation was estimated by the oxidation of syringaldazine to the purple

tetramethoxy azo-p-methylene quinone by laccase and oxygen or by peroxidase

and H2 0 2 (Grisebach, 1981).

Peroxidase : Peroxidases have been implicated in a variety of physiological

process such as polymerization of cinnamyl alcohol precursors to lignin, repairing

response to wounding, biosynthesis and polymerization of extensin and auxin

metabolism (Whetten and Sederoff, 1995, Espelie et aI., 1986; Fry, 1987;

Riquelme and Cardemil, 1995). They are widely distributed in plant tissues and

are particularly abundant in cell walls where they are probably involved in cell

wall lignification. (Fry, 1980). However it has been difficult to tell which

peroxidase isozymes are associated with lignification and which with other

biochemical activities. A number of peroxidase isozymes have been found as

soluble, ionically bound and covalently bound forms (Haard, 1973; Gkinis and

Fennema, 1978; Mc Dougall, 1993; Ingham et aI., 1998). The soluble forms are

cytoplasmic, whereas bound forms are generally thought to be associated with

particulate components such as plant cell walls and some organelles eg:

mitochondria (Haard, 1973, Moulding et aI., 1987; Van Huystee and Zheng,

1995). Due to their suggested involvement in normal balance, ethylene

33
biosynthesis, membrane integrity and respiration control, soluble (cytoplasmic)

peroxidases have been implicated in the metabolic control of ripening and

senescence of fruits (Grambow, 1986; Ingham, 1998). There have been number

of studies investigating changes in peroxidase activity during fruit ripening

(Haard, 1973; Kumar and Goswami, 1985; Miesley et aI., 1991; Marin and Cano,

1992). Cell wall located peroxidase activity has a role in cell wall formation. It is

thought to play to key role in controlling the deposition of lignin in vascular tissue

and this has been supported by the cytochemical localization of peroxidase

activity in the secondary wall and adjacent primary wall (Helper et aI., 1972). Cell

wall associated peroxidases were extracted selectively from fibre bearing tissue

dissected from the stem of Linum usitatissimum and their activities correlated

with extent of fibre lignification (Mc dougall, 1992). The onset of fibre lignification

was accompanied by increases in the levels of peroxidases both ionically and

covalently bound to the wall. Abeles and Biles (1991) characterized the role of

total peroxidases (soluble and bound) in lignifcation. However it is not clear

whether there is any relationship between soluble and bound forms of

peroxidases in encdocarp lignification. A correlation of soluble peroxidase activity

with lignification in needles of Picea abies was established by Polle et al. (1994).

Accumulation of lignin and seasonal variations of soluble peroxidase activities

were studied during needle development. Sub-cellular localization of peroxidase

with guaiacol indicated that high activities were present in lignifying cell walls.

Their results suggest that soluble peroxidases participate in lignin formation like

34
that of bound forms of peroxidase. In non lignified primary cell walls, peroxidase

in more likely to be involved in the cross linking of cinnamic acids such as ferulic

acids which are attached to cell wall polymers (Brett and Waldron, 1996; Negrel

et aI., 1996).

2.4 Oil Palm Fruit Pectinase (OPFP)

The post harvest storage of a fleshy fruit expressed a plethora of

biochemical changes during its storage. The major visual expression during

storage is excessive softening. Pectinase (P) otherwise called Polygalacturonase

(PG) and Pectinmethylesterase (PME) were considered as the primary

hydrolases involved in the softening process (Knegt et aI., 1988; King and

O'Donoghue 1995; Sethu et ai, 1996; Andreasen et ai, 1999; Ait Barka et ai,

2000). In addition a variety of other enzymes have been assigned roles in fruit

cell wall metabolism, such as xylanase and glycosidase. It has been suggested

that the complex series of modifications in ripening fruit cell walls may be the

result of an orchestrated action of several classes of enzymes during fruit

ripening (Fisher and Bennet, 1991). A multidirectional variation in tissue

softening has been observed in peach during ripening (Maness et ai, 1992). The

biochemical bases of differential softening of mango fruits were reported as the

differential distribution of PG in the mesocarp tissue (Lazan and Ali, 1993). On

the view of enzymatic action pattern towards the substrates, PG is classified into

endo-type (endo PG) and exo-type (exoPG). Endo PG is known as the key

35
enzyme involved in the breakage of fruit cell walls during ripening. Subsequent to

the purification and characterization of PG during ripening the PG gene encoding

the polypeptide have been extensively studied in recent years (Giovannoni et al

1990). Although there is only a single gene for the catalytic PG polypeptide the

total PG activity isolated from the ripe tomato fruits is attributable to a mixture of

several closely related, post translationally derived isozymes PG 1 , PG2 A, PG2 B,

PG2A and PG2 B isozymes accumulate late in the ripening process and are each

composed of a single catalytic PG polypeptide differing only in the degree of

glycosylation (Zheng et al, 1992). PG 1 isozyme accumulates during ripening and

it is thought to be composed of one or two catalytic polypeptides tightly

associated with an ancillary glycoprotein (Knegt et al, 1988). Since this enzyme

has been extensively studied in the ripening phase of majority of fruits and

vegetables as a marker, the potentiality of this enzyme in biotechnological

application has gained wide attention.

36