Review Of

Literature

Germination is the resumption of metabolic activity in ths seed. The seed tissues

undergo a lot of changes involving rehydration. utilization of natrient reserves and the

gradual development of synthetic systems which enable the yclng plant to assume an

autotrophic existence. Seed germination is also frequently zccompanied by marked

increase in the level of activity of certain enzymes and disintegrzion of reserve material, . ..

an increase in the quantity of nucleic acid and bee amino 5--

there must be sufficient oxygen and suitable temperature for the unhindered progress of

gemination, Thus, the changes in the various components fn the seeds with the

commencement of germination became the subject matter of r e w c h and investigation.

But the role of light on the metabolic activity in the seed during germination was not

ardently attempted by scientists, though the effects of light on germination as a stimulant

was experimented since 1930's by Kinzel (192q; ~ inca id ( l93 j ) and Flint and Mc

Allister (1935).

Seed germination was one of the first developmental responses to be recognised

as being capable of photoregulation. Effects of light on germinarion had been the subject

matter of serious study and research since the last quarter of the 19th century and the first

' a l r e c o r d e d experimental demonstration was published in

that the seeds of Bull~arda aquatica would not germinate in darhess. By the turn of the

centuq. photoblastism became an established phenomenon and this sort of photo I

regulation of seed germination was canied out in a wide r a n 5 of seeds e.g., Kinzel

-J

(1926) recorded over 600 species which were influenced by light. The role of

phytochrome - the main photoreceptor pigment for photomorphogenic reactions was

i/ discovered by Borth wick, Hendricks and their colleagues (1952).

! >

The main photoreceptor pigment, Phytochrome, exists in two photoreversible

/

forms. One form, Pr, absorbs red light at 660nm, and is thereby converted to the second

form. Pfr. actlve in promoting germination in the lettuce seeds. Pfr shows peak

absorpt~on in the far red. and under far red i!iumination it is converted to Pr again. White

light can affect germination in one of two ways - either it will stimulate germination or

~nhibit germination. Somerimes no changes occur. These phenomenons are

characterised as positively photoblastic and negatively photoblastic. This stimulato~y and

inhibitory activities of white light are caused by different spectral regions.

Photophysiology of seed germination and ed wavelengths of light had been

reached in 1940's. Three effective spectral regions were recognised (a) blue (420nm) (b)

red (65Onm) and (c) far red (75Onm). Red light was found to terminate dormancy in

seeds and promote germination where as blue light and far red light were found to inhibit

./ germination (Borthwick et a1 1952). Although positively photoblastic seeds can be

caused to germinate by white light, this can be reversed by subsequent treatment with far -

red light. Like,wise,inhibition of germination of a negatively photoblastic seed by white

light can be reversed by application with redlight. These spectral regions were similiarly

effective for both 11ght promotion and light inhibitor seed? (Evsna" /

\--_-- - 3

In a series of papers published between 1930 and 1937 Flint and Mc Allister

C /

(19131937) demonstrated that shorter wavelengths of light promote germination in

V

sensitive seeds and that longer wavelengths are suppressive. Kincaid (1935) studied the

effect of light on the germination of Tobacco seeds and found that as little as 0.Olsec of

sunlight was effective in stimulating germination and even moonlight could stimulate. ..

the gemination of Amarathus For the usually light - -

\

Inhibited seeds of Phacelia, Resuehr observed light stimulation of germination at 640nm,

and 5 inhibitory zones, at 350 m, 450nm, 475 - 490nm and above 1000nm. Orange light

R 2 h a s been reported to stimulate germination of

d a n environmental factor influencing germination

relationship between llght and seed dormancy was described by

The inhibitory character of blue light, like far red has led to a plethora of studies

and research among scientists, both modem and pre twentieth century, eg., in lettuce

seeds blue light inhibitgermination when given for a 2 hour period starting six hours

after promotion by red light and in Nemophila in- maximum inhibition by blue light

occurs when seeds are Irradiated from the twentieth to the fourtieth hour after the effort %d -- - - - x . BWg! of imbibition (Malcoste et a1, 1 9 9 Action spectrum show major inhibitory activity at

( . . . _ A 472 - 480nm. Ultraviolet (365nm) is also inhibitory. There is no coincidence between

the action spectrum and the absorption spectrum for phytochrome, strongly suggesting

that this pigment is not responsible for the inhibition by the blue light.

However, plant response to irradiation on other aspects are also studied, e.g. blue

light - induced phospho&ation o f a plasma membrane - associated protein in Lea - mays 1 J

L. was detected by Palmer et al(1993). They showed that blue light induces a variety of

photomorphogenic responses in higher plants, among them phototropic curvature, the

bending of seedlings toward a uni directional light source etc. Regulation of blue - light - ~JWS .

induced proton pumpins in V'a f& L. Guard - cell protoplasts -studled by

d

Mawson in 1993. He has also investigated the energetic contribution from guard cell

chloroplasts and mitocondria to blue light induced proton pumping by Vicia --v faba guard \/

cell - protoplasts(Mawson, 1993).

_~.---_ ! ,,

I"

T k sensitivity of seeds to light increases with time 'f i m b i b i t i o n ! ( l i a r i a

If seeds are given a light stimulus while imbibed and then dried, the

r/

ct~mulatory effect is retained(Kincaid. 1935). In Tobacco seeds Kicaid (1935) found that

high intensities were effective after short periods of imbibition, while low light intensities

were most effective afier 4 days. .leer 10 days of imbibition, the seeds no longer

responded to illumination. Temperature can also play a role in t h w q i t i v i t y . - .*

@ Effect of Temperature on of oats was described by ~dwards( 193gand -7 - i - .~ ... '

~ y ' t t e n b e r s , ~ 2 8 ~ $ n n k l a n d that in Sinapis --. awensis sensitivity to far red --.

light changed during imbibition.

31 In Sinapis ensis, dark germination of between 0 - 7% in various batches

-. /Pi/.- resulted in germination of behveen 1 and 70% following the same standard red

' \

'I irradiation(Frankland_ 1976).) An unusual response to light has been found in Bromus @% --

\ derilis, in which g~rmination is inhibited by red light. and this inhibition is re\.ersed by

The effect of the mother plant has been investigated b d ~ o l l e r 2 germination of lettuce seed could be pre conditioned by the light

regime under which the parent plants were grown. Shropshire (1973) studied more

precisely in Arabidopsis thaliana seeds, which had varying dark germination depending

on whether the floral primordia had been irradiated by fluorescent or incandescent light.

The effect of light on germination was studied in a number of plant species like

lettuce (Lactuca sativa), Iepidium virginicum, Nicotiana tobacum and various amaranthus .- -. . -- - ~ . ~ -

Q? specieq,(Mayer and Poijakoff. .. Mayber 19*$ For lettuce and lepidivn the P R C ~ S ~

.. spectral pea<s for germination stimulation and inhibition have been established by short

illumination, showing stimulation at 670nm and inhibition at 730nm. An important

finding was the reversibility of both stimulation and germination inhibition

J by alternating illumination shown by Borthwick et. a1 (1952).

The different photomorphogenic responses in plants like photoblastism,

flowering, etiolation and unfolding of the plumular hook of bean seedlings and pigment

formation in certain fruit and leaves etc. show a very similiar action

of seeds" Mayer A M and Poljakoff Wayber A1982, 3rd edition. Pergamon .

The action spectrum is that associated with the plant pigment

L L

phytochrome (p), \\hose existence was deduced from the biological response of various

4 tissues to illumination . Butler et. al (1959) showed that there are chanses in the

absorption spectrum of an intact tissue, coleoptiles, as a result of illumination

with red or far red light

%,WY. Numerous areas of the effect of - phytochrome on germination of different seeds - $,>

f .. were done . . by a number of modem scientists e.g. Amaral and Takaki (1995 =died@ . the role of phytochrome in seed germination of B i d 9 pilosa and their results indicate

/-

- 1 ' that phytochrome is responsible for the control of seed germination in Biden - pilosa and -

the level of pre,existing active form of phytochrome (Pfr) above the threshold switch the *

, seed to germinate in darkness. The control of 1ight.environrnent and phytochrome in the cG4":: r-

-. .~ *jp ( 8

'ALL 4.G d, '~

,.,*% germination of Piper .~. auritum . was doneiby Orozcosegovia, iSanchez Coronado and, < .~. - - - - - > - ,',.,'. ,. -. - _I- , ..,,

1.

Vazquezyanes (1993). Phytochrome control o f the development of ascorbate oxidase

<"L.!c. . &c;ll I

1; et. activity in mustard (Sinap~s *a L) cotyledoq were done by Hayashi,,Reiko: bforohashi, ,<~,<' ... - s . (.< . - j , ' v ,- &d Yukib (1993). The activity of ascorbate oxidase (AOX) in mustard c o ~ l e d o n s tvas

'- ' markedly increased by irradiation with continuous far red light. The involvement of

'1 phytochrome in this light mediated response was demonstrated by redifar red reversibility

The molecular structure of ?hytochrome is described by Pratt, (1982) and Quail

et.al (1983). The t ~ o forms of phytochrome PR and PFR differ in their molecular

configuration and PFR form of phytochrome is associated with cellular membranes /

(Song. 1983; Kendrick. 1983). The transition of phytochrome from one form to another

has also been proved. These transfomations tak lace via a number of + >q ' intermediates(Kendrick and Spreuit. 1974):; These intermediaries of phytochmme

~. ,-.l

transformation are involved in the photo - inhibition of germination when dry seeds are

&k . illuminated by red light e.g. ~ i n a ~ i s @ i r t l e ~ - and Frankland, 1 9 8 9 Different populations - of Poa trivialis from different habitats required different PFR /TR total ratios, in order that

'54'; gemination could occur jHilton et.al: 1984) The response of seeds to light is modified " -1 J

by various internal and external factors. Osmotic stress, the presence of growth

promoters or growth inhibitors, the oxygen tension and other factors, all can change the

duration and intensities of light required to evoke a certain response. Even the spectrum

of radiation to which the parent plant has been exposed during seed formation. affects the

- response of the seeds to light,

1 . > The catabol~sm or rather the breakdown process of prote~ns, carbohydrates, lipids \ &4."*' i 0

1 : - t L L and other reserve materials with its own distinct chemical composition during seed \ ( 2 ' . .,...,&

<&.',C "

-. - n..v gemination enabling the plant to have an autotrophic existence, has been i&vesiiga&& , , .*u r,,<s

..& L 7

The major part of the protein of the seedlings is in the

containing only a small proportion of the total. In niung bean seeds geminating in the

! . dark, only about 5% of proteins was left in the cotyledons ai the end o f the

1 b?, 6daysyam1kawa, - - 1979). The disappearence of the storage proteins is associated with -- ~ncreased proteolytic act~vity not only in cereals but also in legumes(Ashton, 1976;) ' - ,

, ,Matlie, 1982)) In mung bean the major endopeptidase is completely absent in -- ungerminated seeds and appears on the third da!- of germmation by de novo synthesis. In

* f&# pea ((Guadiola - and Sutcliffc, Vamcr, . .-c 1913)and also mung bean

7 M~namlkawa, 1979)increase in the proteolytic activity in cotyledons during germinat~on @+) 4& -

did not occur in the absence of attached axis orsans. On the other hand, the development -- . . . w? of pmteolytic activity in French bean G o m o and Srinivasan, Q?A and ~ u ~ i d ( ~ e e c h a a f t _--. .. I ,.~ /' &! " ~ > r s o n , as not dependent on the presence of attached axis. /

The protein content at any stage represented a net value, being the resultant of

degradative and biosynthetic reactions, inclusive of turnover. Protein bioqpthetic I/

/ reactions are characteristic of the developing axis in geminating seeds (Bewley, 1982).

P Studies with the embryonic axis of Phaseolus vulgaris have shown that germinating seeds

M?, can utilise ribosome formed during the maturation phas In Peas, also

there is evidence for the preservation of functionally active ribosomes during seed

~l desiccation @'aulson and Beevers, A limited extent of synthesis occurs also in the

cotyledons of germinating seeds, in particular during the reorganisation, or de - - novo -\ e. formation of elaboration of hydrolytic enzymes

. , ....

7 ( ( ~ a ~ e r and although the emphasis w!. .+ --. ,,

is on the degradation of protei

.. Amino acids formed by degradation of storage proteins could be reutilized for the

synthesis of the metabolically active proteins. Free amino acids present in dry seed might

i. also have been utilized in the formation of proteins. It was also possible that

cotyledonary tissues brought about the synthesis of amino acids from nitrate nitrogen, for __ .

R? which evidence exists in developing legume seeds and Muntz, 1 9 3 Nitrate --

accumulation and nitrate reductase activity in vegetative tissues of winged bean were -. . ~ ~. . a- al . . . . .

? studied by Gildebrand. Harper and Hyrnouitz, ( 1 9 8 9 Nitrate. nitrite and ammonium --s-.

-'\

can be assimilated by plant tissues not only in the light, hut also in the dark ( ~ u f f a k a g

P&! 1982)! Factors influencing nitrate reductase activity in winged bean were studied by ' , - -

-c& ac- Munjal, ,&adam and SalunkheK1983k Allantoin and Allantoic acids, not asparagine and R? - -

C " glutamine are the principal forms of the nitrogen transported from nodulated roots to the , - . - , , A&,

&? shoots of the soyabean an important role during pod

1 formation and seed - ~ a i n ~ d --- /

that in the winged bean, under nitrogen utilizing conditions (nodu1ation)about 80% of the

organic nitrogen of the xylem sap was as rcide this proportion dropped to less than 10% 9%' under nitrate utilising conditions.

Not only was the major part of the seedling protein held back in the cotyledon, a

concentration level at which protein occ '/ in the axis of the developing seedling was '?P kept low, contrasting with the increased level at which phosphorus was maintained in the

plumule and radicle. The resenre tissue of legumes is distinctive in containing unique

proteins which do not occur in other parts of the plant and which function as storage

oroteins. The metabolic form of protein in the cotvledons of the oulses mav constitute

only a small part of the total protein. According

upto 80% of the total proteins in legume seeds is constituted of reserve proteins, the axis

proteins are constituted essentially of metabolic and structural proteins;

-0 ?',

the presence of storage proteins

of dicotyledonous plants have been

most seeds the seed coat is inert and

of the system during germination

In certazl instances, the seed coat does involve living components which

may affect the exchangs wlth the environment profoundly. ~evepthecless, the seed coat V Y

may have a profound effect on germination througfi its permeability properties to water,

various solutes and gas?. The family Leguminosae is one of the most commonly known ~ .- .

%?, to possess seeds with inpermeable \

chemical inhibitors of germination was recognised h m early times _ " ..~ ,.,

/----- Yw >@orttrrndc and has the capacity to suppress the development of 6be%( . . . .. . enzyme activity involwd in the degradation of storage reserves was demonstrated by

... -. . .- .~.. . .. ,

. Dav~es and Chapman (1979) and by Slack

7 bean hull conmbuted about 50% of tannln P-4. - these tannins inhibited ryptic activity. The seed coat of soyabean is characterised by a

higher percentage of d o h y d r a t e s (21%) than cotyledons (14.6%) but this is made up -'-z

largely of crude fibre analysed pea seed c'x * , .. .. . -. I *---

cotyledons and testa for Ca, Mg, K iind P coqcluded that the developing seedling does not

1 d e h e any mmeralr from the seed coat. (Hocking L L I ! the seed coats of - --- Lupinus albus and Lupinus angustifoliLs for several micro and macro elements. The L- -

I amount in the testa ransed from 0.2% . to .. 18% of the , total in the mature seed, Ca being an

r U ,.' -. - / --

r\C. \ exception with over one half (56 - 61%) in the testa. Analysis o f the shed testa led .--

i

b\.~ocking (1980).to - conclude that the loss of minerals from the testa during germination / -

was negligible. p ' \ I , 4- c;ge, : > r-.4"' ' i

G r e a k down of storage proteins in the cotyledons is accompanied by the

appearence of new proteins in other parts of the seedling. Seeds contain a variety of

proteolytic enzymes some of which are present in the dry seeds while others appear

during germination. The proteolytic enzymes can be divided into proteinases and

/ peptidises depending on the size of the molecule which is at tackeg The development of

proteolytic activity appears to be under hormonal control, the hormons originating in the.

"L f+:ie or embryo(Davis and Chapman, 1979). The proteolytic enzymes and peptidases of

LQ germinating seeds show great diversity with regard to their specificity for peptide .

linkages, their pH optima and their response to inhibitors. @ayer and M a r b a c . h , s . ~ . . b$-? 1 :, ! -/ m u n t z et al, 19851, The enzymes responsible for proteolytic break down initially present ' . ' /-.:'"-.

/~ - in the protein bodies may be s>nthesised, or activated. in the protein bodies themselves or J may be synthesised elsewhere in the cell and transported into the protein bodies

vulgaris two alkaline peptidases are present in L '11 Q&AZ

the cotyledons of the dry seed. \\hose activity falls during gemination. However, the

activity of three peptidases and proteinases which are also present in dry seeds rises kc;‘% 1 2 ,

1 . during germinat~on((Mikkonen. 1986 In the endosperm of castor bean initial hydrolysis 2 3 of storage proteln is due to the activity of a preformed protease which becomes active by

/

MI. hydration during germination c (G~fford et aI 1986) ~reakPdown 4 of storage proteins

involves, in its initial stages, limited proteolysis of some, but not all of these proteins as . t

seen in castor bean, where the 2 storage proteins glycinin and beta - conglycinin, which

are broken down at quiet different rates, beta - conglycinin being broken down first

e . S O et al, 1986j .- Endogenous inhibitors of trypsin, fhymotrypsin a n d g a p y a r e

'... ~ . ~ . .

7: present (Vogel et I . 1968:,,Ryas73 in many seeds.

A lot of e~~erimentafion had been carried out with regard to the detailed

mechanism of protein metabolism during germination. The early work on nitrogen " !. r/ b7 metabol~sm both by and Prianishnlkov(l951) and more recent work + L L - - - ,-'--1

summansed by ,Oaks and BldweIl ( 1 9 7 9 and @k_s&a showed that it is possible to '.. induce amide formation in germinating seeds in the dark by feeding them with

amtnonium salts. If sugars were also fed to the seedlings this led to a marked sparing

-1

@%' actlon on proteln break down (oaks- Feeding of both glucose and ammonla .. - nitrogen led to zreatly increased amide formation. The chief arnides formed are

J J glutamine and asparagme depending on the plants (Chibnall, 1939: Lea and Joy, 1983).

Although appreciable amounts of amides and ureides are stored in the shoot

as estimated that 75 - 90%of rhe imported amide or ureide is rapidly metabolised.

The products of catabolism are reassimilated and used in the synthesis of the insoluble J

organic fractions. Egami er a1 (1957) showed that the small amount of nitrate present in J

Vigna seeds disappear as germination proceeded. Yamamoto (195j)shoned. in the same u

seeds, that asparagine present in the cotyledons disappears and instead appears in the

d y p o c o t y l and plumule. Glutamine -~ . ~ ~ . is formed -. from gutamic acid c - and ammonia, in t b 5L.A . r &

presence of the enzyme glutamine synthetase and ATP, the reaction being energy - .~~~ - - - -

J 4

requiring. Asparagine formation is less clear (Lea and Joy, 1983). Yamamoto (1955) ~ ..

showed that in Vigna hypocotyl, asparagine is formed if the hypocotyls are fed with . .

aspartic acid, ammonia and ATP. Although rapidly synthesized under conditions of

, nitrogen excess, the catabolism of asparagine is apparently very 7'

concluded on the basis of ''c :racer experiments with wheat seedlings that "asparagine is

a dead - end metabolite whose only major metabolic role is as a constituent of protein".

Other work with cotton seedlfng roots demonstrated the metabolic stability of the carbon

q ) ~ f ~ ~ a r r g i n e ( er and Fonde,. 1 9 7 d Hcdever in these studies the important atom of e; /-~

asparaghe (i.e., amide-S) ~vas not labelled and thus they have little relevance to the real

role of asparagine.

Three poss~ble routs o i asparagine breakdown have been suggested Lea and L -1 .- -

&f,, Forden, 1975i The only enz>me definitely characterised is asparaginase, initially _ - ._._- 2 . . ~

detected in the roots and iarer investigated in the maturing seeds of luplnskea 9 -

&7 $?wden , 19762 The enzyme has a high Km for asparagme of appro~~mately lOmM, . --====- although the phloem contains approximately 30mM, is substrate specific, and has no

action on glutamine or its anaiosue. Despite the fact that cenain species of lupins have

very high levels of asparagine in their developing cotyledons. initial enzymological and

immunological studies have sugsested that the enzyme is not universally present in all

p& * species of lupin nor in all legumes (Lea, Miflin &

W A second enzyme Aspxagine transaminase, leads to the removal of 2 - amino N

J of asparagine leaving 2 - ososuccinamic acid (Streeter, 197-11. Asparagine could act as

an am~de donor in any of the rs3ctions shown for glutamine. particularly in the synthesis

of carbamoyl phosphate for the ionnation o f arginine.

Q&?- Asparagine is rapidly metabolised in shoots and reproductive organs.

and associates found that 75 and 15% o f the ''c asparagine supplied to pea leaves was

metabolised within 3.5hour in rhe light and dark, respectively. Although aspartic acid

Li and other amino acids were labelled, the major product that accumulated was 2 - hydroxy

succinamate. The latter was metabolised slowly in the light and more rapidly in the dark.

During seed development over 80% of the asparagine supplied to lupin fruits was broken

down with "N appearing in the amide N of glutamine and asparagine and in 13 other

J amino acids (Atkins, Pate, Sharkey, 1975).

j '"~ -

There is evidence to support several pathways of,'~sn)cat~bolism. Asparaginase \ . ~

which catalyses the removal of the amide - N - has been and nodules &

J developing seeds (Sodek, Lea and Miflin, 1980) &ler et a1 suggested that asparagine

3 - . 1 . 07 b m ; . ' r r 0 ~ .

could act directly as an . amide .. , group . - donor ..--.-- similiar in role to glutamine. @hflin and L g ~ /' 9

' I t r . (19809 reported asparagine aminotransferase activity. Germinating seeds contain

, , ~ ~ e n z y m e s , causing hydrolysis of amide bond, glutaminase and asparaginase(Sodek et al, &?.J &a. ,- boa. 1980). Another type of transfer enzymes concerned with amino acid metabolis_m is '.'.'y 'J,!"

,L*~: ' constituted by the transaminases, which transfer amino groups from amino acids to keto : :;. " -..

4 ; , i . . acidpowden, 1965) The presence of transaminases in a variety of seeds has been sho\m \b\:? . - \\ ' = ~~ d

PL9- b @ z h and Evidence for the presence of enzymes cataliging the - ~ -

reverse reactions from glutamine to pyruvic or oxaloacetic acid in wheat germination was

p*?. brought byCCruickshank and .

transamination reaction glutamic - oxaloacetic acid and its reverse reaction in oat

embryos. The former action took place at three times the rate of the latter. Glutamic

oxoloacetic acid transaminase of the embtyo expressed per unit of protein

increased steadily during germination.

- # I- Dry seeds contain very little ffee @no acids., The growth of the embryo in the

germinating seed is dependent on a supply of amino acids for its protein synthesis. The /-

- 1 $b -amino acid pool increases during germination (Klein, 1955). The main source for these -.

-. - amino acids is the storage protein, but its amino acid ratio need not be the same as that of

the newely synthesised seedling protein and apparently inter conversion of rhe amino i acids occurs. The main pathways for such kterconversion are the transasmination and I

4 I deamination reactions. Virtanen et al, (1953) gave shown that in germinating pea seeds / homoserine, which is absent in the dry seeds both in its free form and in protein, is

synthesised in the first 24hour. of g e ~ i n a t i o n . Arginine is also synthesized de novo in -1-

\ 'Y w geminating peas(qhargoa1 and The synthesis and interconversion of

.. - I I

amino acids are apparently the same as in other plant tissues and proceed via the same . pathways and amino acid families (Forest ?nd Wightman, 1971, 1972; Miflin and Lea /

P,, v+&h& 5 r ~ pp 1 a 0 - 4 ~ 3 . and Joy, 1983).? J

1

Asparagine metabolism and nitrogen distribution during protein de-adation in &oa.

sugar starved maize root t ~ p s were done by Brouquisse, James, Prader and ~ a ~ m o n ; i ) -- -- -- --

(1992). Asparagine, which is a good marker of protein and amino acid degradation under

stress condition, accumulated considerably until 45 hour of starvation and accounted for

~ 7 - 50% of the n i t r ~ e n released by protein degradation.i(~eidelb,1991i) Tonin and Sodek / - - - -

V

(1990) cultured immature soyabean (Glycine max) in vitro with glutamine, asparagine

and allantoin as source of N, and the activities of asparaginase, allantoinase and 7 A 4 '

glutamine synthetase measured over eight days. @min and ~odek,(l9* found that -- .-

allantoinase and glutamine synthetase activity increased independent of the N source but

asparaginase activity increased markedly in cotyledons grown on asparagine.

,' Chapleo and Hall (1989), in the biology department of Southampton University

has worked m nodulated w~nged bean (Psophocarpus .. tetragonolobus) a& found that

levels of amino acids in xylem sap were greater for plants supplied with a complete

nutrient solution, than those grown without applied nitrate, particularly for asparagine,

glutamine and proline. Changes in the activities of N metabolism in developing seeds of v'

Luplnus luteus by Ratajczak(l986) showed that during the seeds development upto the

dehydration stage, the highest activities of asparaginase , glutamate dehydrogenase and k--\

aspartate ami transfer were noted in the seed coat. In the embryo maximum g,.. /

asparaginase activity was detected within the period of the most intense syntnesis of - storage proteins in the cotyledons.

Allantoinase and asparaginase activities in developing beans seeds haseolus 9- r/ /

vulgans) have been studied by Hostalio, Sodek and Valio (1985). The high activities of

these enzymes found in the cotyledons during seed development suggested that they may

play an important role in the N metobolism of these seeds. Some activity of both enzymes

was also found in the seed coat and embryo axis, and asparaginase activity was

particularly high in the seed coat towards the end of seed development. Changes in

activlt~es of enzymes of "nitrogen metabolism " in seed coats and cotyledons during - - - - CJm bLtC.-(sL %

embryo development m pea seeds (Pisum sanvurn) 9 don by Murray and Kennedy j (1980r In the seed coats of developing pea seeds, the maximal activities of

asparaginase and aspartate are attained early in development, before the embryo has

expanded to fill the embryo sac. These two em-me activities could account for the --C

early absence of asparagine and aspartate from the fluid secreated by the seed coats into

the embryo sac.

There have been extensive chemical studies on starches from the seeds of cereals .~ ~.~

< and legumes and there are recent reviews on their chemistxy(Banks and Green

.~~. ~ . .~ ~

~- ~. ,. ! / ' 1

and WJ-p, 1980)jbio-chemistry (French ,1974,(~uffus, 1979;'Qfeiss and / . - .. .

-\

19-i)' and f accumulation. Some

', - tissues are capable of starch synthesis because they possess chloroplasts. Developing

cotyledons of dicotyledonous species commonly contain chloroplasts that may be

C converted to strach grains. In some dicotyledons there is no starch any where in the seed

i..

at maturity, but on germination, starch can be detected microscopically(Mlodzianowski __--- .~

~~1 a n d x 5 ) . .~. or chemically (saini and Matheson. 1981)) . - ~.

.- ~ ..~.

. Seeds consis$ of various organs and tissues in which the starch may have $4 ' :,. P

" +!'I.'' .different characteristics. e g in the embryo and endosperm of waxy cereal grains. Starch o_ l)x . . . . ~ . ~ A - .4------z ' - .-_-.___- . . ~.

! ah- in both chloroplasts and myloplasts disappears from the pericarp early in the

~7 .development of barley and wheat grain In some dicotyledons

there is no starch anywhere in the seed at maturity, but on gemination, starch c& be

p+,' - detected microscopically ( M l o d i i a n o w s ~ w e s l o w s X a , 1 9 9 0 i chemically . . . ~.

- r7 Matheson, 1981b Other carbohbdrate reserves, for e.g. galacto mannan in fennugreek ,-------h '.-'

or sucrose and galacrosyl sucrose in yellow lupin are then utilised for starch \

N 7 synthesis I~a in i and Matheson, 19819 This synthesis occurs in the dark and is also found

in species that already contain reserve starch in their cotyledons: (14C) glucose was ,,.

incorporated into starch in pea cotyledons following imbibition and gemintio<:iuliano

(. 4 and Vmu, 1969). --

pr , Stach which occurs as granules is composed of an essentially unbranched , > '

' component of ( 1 -> 4) alpha glucan (amylose) and a branched component

11,

(mylopedin), in which the branches are ( 1->6) alpha linked. The mount of the -. -

> , *

. ^ unbranched can be estimated from the isopotential absorption of iodine by potentiometric

'1 . ' i- . t , '<itration of the solubilised starch granules. The long (r-*-alpha glucan-chain of

-- :'@ \.';C Ipmylose bind iodine more strongly than the shorter changes of the amylopectin.

&j4 >.! L ,-

$4 Solubilised starches from most mature seed sources have been separated into an insoluble b,

fraction equated w~th amylose and soluble fraction equated with amylopectin by c L,,!

complexing w~th compounds such as n - butanol and thymol. Improved separation has

resulted from the application of alternative procedures such as ultracentrifugation and - ,-'

' 1 , . gel- chromatography [(Greenwood and Thomson, and the affinity - -.< . . ~~. , \ , - ~ ~ - ? of concanavalin A for amylopectiq'(Chan1979 q, \ ",

\ \ ,

Starch is normally broken down by alpha and beta amylases. The changes in the

6 enzymes involved in the starch break down during the germination of peas was done by - Shain and Mayer (1968$! Low activity of beta amylase is present in the dry seed, but no - alpha amylase activity could be detected. The activity of

I

increase only hvo days after imbibition. Break down of - - - 2. ~

A

\rib:: 7 , $?sin and Dekker. (19661, The starch is deposited in plastids during maturation ,and the .q C ' ,'

,v2' t b q t lamellar structure of the plastids as the starch granules grow to full

, "TG" 2 K. i

size(Opik, 1968). During seed germmat~on, starch gains in direct contact with P cytoplasm and appear rugged on the surface, indicating in situ hydrolysis(Bain and

I/

Mercer, 1966). The major reduction of starch content in germinating peas occurs after 8

days of germination as determined by chemical analysis and ultra structural examination J s p -. z.. w7. (Bain and Mercer, 1966, Juliano and Verner. 1969) The hydmlysis,stm@provides . - - .- -

h-

" energy and synthetic requirements of the seedling. Phosphorylase activity is high in the \w 1 . I _ - - .y' ,, , ., .

%\\- early stages of gemination increased upto 8 days and then declines. The alpha - amylase

C activity is negligible at early stages of germination, increases rapidly to 80 fold of the

I, J '- phosphorylase activity in starch break down at 10 - 12 days and then d e ~ r e a s e ~ e r a t a et\,

'-

d

Maltase activity coincides with alpha - amylase activity indicating a co-

J operative role of both enzymes in the tissue (Nomura et al, 1969).

In Phaseolus v u l e s - seeds alpha galactosidase activity is high in the embryo and

low in the cotyledons of the dry seeds. During germination its activity falls in the - - .

@$?emblyonic axis and nses in The enzyme

endobetamannanase acts upon galactomannans and mannans which IS further broken

down by a beta mannosidase which is produced in the cell walls of the cotyledon - -- -"\

@&!Q~ewle~ and Halmer. he enzyme mannosidase a c d e x t r a cellularly to form , -_ .. - &': mannose and which are absorbed -~ ---

the storage material in the endosperm is mannan. In coconut and date palm, the distal

end of the single cotyledon develops into a haustorium during germination. The

epithelial cells of this haustorium are rich in organelles and the endosperm cells adjacent

to the haustorlum are the first ones to be digested en as on, 1983, 1985, De Mason et) - -

The breakaown of the mannan in the endospenn appears to be regulated by .. . - J

the haustorium. The presence of invertase has been demonstrated in a number of

? germinating seeds, e.g. barley (Prentice, . germination and is responsible for sucrose break1 wn A art of sucrose is metabolised 4. by glycosyl

w;*i iU.J . formed during of carbohydrate kc - I p a t G * .

mobilisation is the relationship between axis and cotyledons in contT01ling the rate of 3 ' -- - - - , - - - - - I"

7 - ( r ' J

breakdown. Some of the arguments about this can be found in articles by Murray (1984) - - - - - - - - j--'--- C

and ((almer (1985). - ---sl

! \?,

The lipids which are present in lipid bodies or spherosomes, contain all of the

enzymes required for the breakdown of lipids to fatty acids and glycerol - (lipases). In

Arachis cotyledons enzyme systems have been shown which convert glycerol to glycerol - phosphate, which is then converted to trios hos hate and then to pymvic acid or to ~A p w, sugars@t&pf The bulk of the fatty acids formed following

. .

lipase action are broken down by the process of beta - oxidationfiesu~tin~ in the

cleavage of two carbon units in the form of acetyl - CoA, which can enter the

tricarboxylic acid cycle, which requires both CoA and ATP. Beta oxidation was shown

&?. to occur in extracts of various see he beta oxidation activity was J

shown to be associated with In many seeds

disappearance of lipids is accompanied by the appearance of carbohydrates. But lipids ~- - -

are not always converted to carbohydrates during germinationc(~&&an and' 9 .-.~ ~~ ~. ~.

$bi -3, a n d p m b i e and Comber, followed lipid breakdown in two different

C - . .~ ~ ~~ C

seeds. ltrullus and the oz~palm Elaeis guineensis. In f i r n u s lipids are rapidly f..~ - . f~

broken down in the cotyledons and the breakdown products utilised in respiration. There

a does not appear to be conversion to carbohydrates. In Elaeis seeds, the lipids which are 1 - located in the endosperm are utilised during germination and no conversion to

5 carbohydrate occurs. In E&is lipas2are present only in the shoot of the seedling ( 0 0 --__

b6!g ' and Stumpf, 1983). &l W L " ~ - -. -

Some lipase activity may be assumed to be associated with the membrane of lipid

bodies or spherosomes. But in the lipid bodies of dry seeds this association has not ... ~ . .~ --~

" w: .- - -. 2 always been possible ( ~ u a n ~ and Moreau, 1978). There are several lipases present in - ,

&-I seeds, which differ in pH optimum for their activity. (&<*,19$howed -.. that in ~- ~.

homogenates of soyabean cotyledons three different activities of lipase can

demonstrated an acid lipase with pH optimum of 4.9, a neutral lipase with optimum

between pH 6.0 and pH 7.5 and an alkaline lipase with an optimum at pH 9.0. 4 Lipase activity changes during germination and the course of the change differs in

J 7 / different parts of the seed. Yamada, 1957 showed the changes in activity in the neutral 1

lipase in the endosperm and the embryo of germinating castor beans (Ricinus

Communis). Lipase activity in the embryo reaches a peak after 24 hour germination, ! while in the endospenn it only begins to increase after about 50hour. In Douglas fir

.

- . 1968) lipase appears to be associated with the lipid bodies. Sub cellular location .- <.- -

I -- ~

of lipase varies in different seeds. In rape and mustard cotyledons it is associated with

c 4 w" lipid bodies (Lin and Huang, 1973f,Lin et al, 1983). In soyabean cotyledons the alkaline

.--- - / 11pare a assoc~ated wlth glyoxysomes and the neutral one w l the lip~d bod~es

+.L \ 1982)

The fatty acids formed by hydrolysis from the glycerides are further metabolised

by the glyoxylate cycle. The main function of glyoxylate cycle is to convert lipids to

carbohydrates. The glyoxylate cycle has

w? example Arachis and Ricinus - J --

b/$. Velasco, 1 9 6 0 ; ~ m m o t o -- ~

of the enzymes involved in this cycle has been shown to increase during germination of

? A , seedsangle . 0th malate synthetase and '..

during germination which are completely absent in dry seeds.

. ~ . . .kdJI< &,-&J plrh J I ! i , u ;-i i' r~ .

!. '? ~. " ,, ,, l a (!4w 'up,< k q ' JL", Xtl \JC L.

i

Id

p4 7 The g ly~~ysomes are first described by V --.

C M glyoxysomes are a special class of micro bodies found in cells, they have a density , '

L - greater than mitochondria and can be separated from the latter by differential or density

gradlent centnfugatlon. Glyoxysomes have a single boundary membrane and

re charactensed by the occufnce of catalase, uricase and glycolate oxidase. Glyoxisomes

also contain the enzymes required for the glycolate cycle as well as the enzymes required

for beta oxidation. transamination and often a malate dehydrogenase. Glyoxisomes are

formed during germination. Their formation is clearly linked to the onset of lipid

utilisation. The origin and nature of microbodies in general and glyoxisomes have been

\?,&reviewed by et.al (1983).,Jt has been domenstrated that ..

glyoxysomal malate dehydrogenase in c'otyledons of germinating pumpkin seeds is

synthesised in the cytosol, and 1s incorporated into the organelle after processing /----- --

( t ~ a m a ~ u c h l e t . ~ 1 9 8 7 ) ? The presence and characteristics of glyoxysomes has been - - .

.; '. reported in many s e e d s q ~ a ~ e r and Shain, 1979, XIayer and Marbach , 1 9 8 y although 3 Q, d-' u. -

the most detailed studies are those on the glyoxysomes of the castor bean endosperm. An

unusual feature of the glyoxysomes is their disapperance after the lipid reserve of the - seed has been broken down. There is some evidence that glyoxysomes are converted to

T&( peroxysomes (Beevers, 1979; Huang et.al , 1983).-, I. --

,; Chlorophyll synthesis is also controlled by light. The plastid is the characteristic , I" . ..

&\- . . plant organelle. The term plastid covers a wide range of structurally different, but ,,

~'

1 , .!, i . basically inter convertable bodies of varying function. The most important, both i .'. % \

C 1 numerically and metabolically, is the chloroplasf which contains the photo id synthetic 1 . \

! i machinery of the cell. Other plastids include the starch storing amyloplasts ,the flower , ,

I

' 1 co:;ur producing chromoplasts and the etioplasts of the dark grown plant. Plastids are

.-- nornally about 5 micrometer in diameter, although their ovecall shape appears to be /

I rciclvely flexible. Plashds contain their own discrete types of DNA. nbosomes and : I

wLii-jfer RNA. Photosynthetic enzymes are synthesised within the plastids.

I . .

Meristematic tissues of higher plants contain no mature plasids, but they contain 1 f j. .

prc plastids. Proplastids are relatively v e q small unstructured bodies which developing i' . > .. 1 -

to sature plastids as the cell in which they reside develop. If such development takes j . ,. .. i "

p l z e in the light, the characteristic chloroplast with its grana and thylacoid membranes ; : f i ,

.:;

is 5rmed, whergas, if the development occurs in darkness a very different structure is i 2 C-- , - 2

prduced, the etioplast. Etioplasts are usually smaller than mature chloroplasts, I

'- riel-srtheless, have a characteristic internal structure which is normally d o m . b y .~ a , -_ j

.p ', ; ; ~4'. 1ar:i central paracrystalline formation known as prolamell ody (Gunning e t . y / .: .- .-

7

)>, . , . -

The major photosynthetic pigments chlorophyll a and chlorophyll b are h' 7 L

sp 1.: i n ~ ~ n a t e l y associated with the thylacold membranes of the mature chloroplastid, as are - - ( . ', - the enzymes and other electron - carrying proteins responsible for photosynthetic b-

II -, phnsphorelation. z The enzymfresponsiblr for carbo&ioxide fixation are present m the

& ,' I /--

suorna of the chloroplast. The etioplasts of dark grown angiosperms contain no '3

I

chlorophyll and it is only upon illumination that chlorophyll systhesis occurs. The ' T

conversion of an etioplasts to a mature chloroplasts is a very important aspect of : photomorphogenesis. Light is known to regulate the synthesis of very many components

I of ihe chloroplast, and also to have an important role in the structural changes that occur. 1

/