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Thèse de doctorat/ PhD Thesis Citation APA:

Glory, M. (1979). The circadian rhythm of polysaccharide synthesis in acetabularia (Unpublished doctoral dissertation). Université libre de Bruxelles, Faculté des sciences, Bruxelles.

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DEPARTEMENT DE BIOLOGIE MOLECULAIRE

LABORATOIRE DE CYTOLOGIE ET EMBRYOLOGIE MOLECULAIRES (Directeur ; Prof. J. BRACHET)

UNIVERSITE LIBRE DE BRUXELLES FACULTE DES SCIENCES

THE CIRCADIAN RHYTHM OF POLYSACCHARIDE SYNTHESIS IN ACETABULARIA.

Mathews GLORY M Sc (Agra)

THESE PRESENTEE EN VUE DE L DBTENTION DU GRADE DE DDCTEUR EN SCIENCES

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1979

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DEPARTEMENT DE BIOLOGIE MOLECULAIRE

LABORATOIRE DE CYTOLOGIE ET EMBRYOLOGIE MOLECULAIRES ( Directeur : Prof. J. BRACHET)

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THE CIRCADIAN RHYTHM OF POLYSACCHARIDE SYNTHESIS IN ACETABULARIA.

Mathews GLORY M Sc (Agra)

THESE PRESENTEE EN VUE DE L'DBTENTIDN DU GRADE DE DDCTEUR EN SCIENCES

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Ackncwiegement Abstract

Chapter 1 General Introdcution ta ths circadian rhythm. 1

2ACETABULARIA 12-

3 Photasynthesis and Polysaccharide Synthesis- 21

4 Nethods _4 2

5 Results I

The circadian Rhythm of Polysaccharide Synthesis 60 6 Results II

The changing Responsiveness to the Norphactins in

Relationship with the Circadian Time 89 7 Results III

The possibility of a Phase Shiftability of the

Circadian Rhythm of Polyasaccharide Synthesis by lAA 100 8 Results IV

DCNU-induced Arrest of Photosynthetic Electron Flow and the Inhibition of Polysaccharide Synthesis 121 9 Results V

The Differential Effect of NADP on Photasynthesis 148 10 General Discussion and Conclusion 163

Summary 176

Bibliography 180

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ACK.NOWLEDGEMENT

I wlsh to express my deep indebtedness to Prof. Or J. Brachet M 0 for having given me the permission to perform this pisce of work in his laboratory under his direction. I a«n particularly thankful to him for providing ail facilitles for modem research in cellular, and molecular biology at my disposai and making the laboratory convenient, confortable and congenlal for this work.

I owe my debt of gratitude to Prof. Dr T. Vanden Driessche 0 Sc for her élégant guidance to this research work and for her unfalllng help and encouragement.

It Is wlth great pleasure that I record here my slncere thanks to Prof. Dr. A Ficq D Sc for her valuable guidance to autoradiography and also for stlmulatlng discussions at varlous occasions.

The author is extremely thankful to Prof., Or. P. Vangansen 0 Sc for the kind permission to électron microscopie work in her laboratory and to Or. n. Geuskens 0 Sc for his valuable collaboration in the experlments wlth électron mlcroscopy.

I am also thankful to ail members of the staff of the Department of Plant Physlology, Université Libre de Bruxelles, for thelr assistance and collaboration in the experlments wlth polarography techniques.

I would llke to aknowledge my hearty thanks to Hr. L. Leteur for the supply of Acetabularia for the experlments.

I an slncerely thankful to Prof Or M. Jacobs 0 Sc. Vrije Universitelt Brussel, who has gone through the manuscrlpt thoroughly and given valuable advlce and crltlcism, My thanks are also due ta Dr. H. Alexander 0 Sc for his helpful suggestions.

I am Indebted beyond ail the posslbllltles of adéquate acknowledgement. ta each and every member of the staff of our laboratory for the cordial help whlch has been rendered during the course of this work.

I would llke to thank Or. E. Perreha Ph 0 and Mrs Meena S Nalr M A for the improvement of the Engllsh text.

I am grateful to Miss M Oppltz for her skllful assistance in dactylography.

The author 1s grateful tO' the Caisse Générale d'Epargne for the award of a scholarship to the reallzation of this thesls and to Miss M Alexandre (directeur du service social. Université Libre de Bruxelles) for having medlated this flnanclal support.

My thanks are also due to the International Cell Research Organization (KTRO) UNESCO, and to the recherche concertée sur programme : "Les mechanismes de l'Irritabilité at du fonctionnement de rythme des

végétaux” (CNRS, France) for their compllmentary awards at different periods.

I should not fail to express my thanks to the St Berchmans' College, Changanacherry, kerala and to the Unlverslty of Kérala for grantlng me an 'extra-ordlnary leave' for the fulfllment of this Investigation.

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aplcobasal gradient of C fructose Incorporation in agreement with others’

flndlng related to Brachet hypothesls.

The clrcadlan rhythm of polysaccharide synthesis in continuous light CLU shows a sllght stimulation compared wlth normal llght-dark cycle CLO}.

rioroever, It la found totally resynchronized when light regimen Is reversad for 10 days. The anucleate calls also malntaln a rhythm.

□ur électron microscopie studles show that NAOP and lAA exert visible affecta on the thylaKolds. We hâve estlmated the surface area occupled by

the polysaccharide grains In the chloroplast surface area and observed a markad déviation of the rhythm of polysaccharide content In the treatad samples.

The photosynthetic inhibitor, GCnu Is found to hâve an inhlbitory affect on polysaccharide synthesis. Exogenoua supply of energy CATP] and reduclng power are found to be ineffective to compensata the Inhlbitory affect of DCnii.

The morphactlna Cantl-auxlns) show a responslveness In relatlonshlp wlth the clrcadlan tlme, the acrophasa Is ahifted frem et 3 to et 0 (advancel. The morphactlna are found rapldly stabillzed In the call.

The affects of lAA alone and together wlth the morphactlna are axamlned. When used alone, lAA shows an Inhlbitory trend on polysaccharide synthesis wlth a change of acrophasa from et 9 to et 12 (delay). When used together wlth the morphactlna in aqulmolar concentration, a compétitive action Is revealed.

lAA's affect on photosynthesls la two-fold. In the mornlng It exerts an Inhibition and In the aftérnoon It has a stlmulatory affect.

NAOP has an Inhlbitory effact on polysaccharide synthesis whlle It shows dlffarentlal affect on photosynthesls.

We conclude that In Acetabularla polysaccharide synthesis Is in accordance wlth a clrcadlan rhythm. The polysaccharide synthesis is dépendant on photosynthesls for the supply of the raw materlal (trlose phosphates) and energy (ATP) and the reduclng power. The aplcobasal gradient also affects the synthesis of polysaccharide.

However, the photosynthesls and polysaccharide synthesis clrcadlan rhythms, are two Indépendant procasses slnce thelr acrophases dlffer and thelr curves are dlsslmllar. Our observation Is a support to the recent évidences on the rôle of cell membranes In the mechanlsm of clrcadlan rhythm.

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CHAPTER I

GENERAL INTRODUCTION TO CIRCADIAN RHYTHN

the clocK strikes the hour and tells the time to none ”

(A S Hoseman)

Historical background

It is well Known that various bio 1 ogica1 ' proces s es are timed with respect to the daily, tidal, monthly or annual cycles of the environment. The importance of time was known long before the emergence of man-made mechanical docks and it was emphasized by Frisius (_+ 1 530 , cited by 100) that it was used in solving the basic problems of navigation.

However, it is only very recently that biologists became aware of what Frisius had revealed so long ago and accepted that there is a built-in mechanism.

There are many migratory birds which hâve indeed good internai docks (101-105) for their successful navigation.

.

Ail living things undergo some vitally important and orderly sequences of changes such as growth, synthesis and dégradation of various materials and reproduction. As they show temporal organization they acquire the ability to measure time or at

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least to tell the day from night. Dbviously, the capacity of an organism to measure time or the length of the day or night or both involves its ability to measure the passage of time.

Rhythmic phenomena, particularly those associated with changes from day to night, hâve stimulated the curiosity of man long before the rise of natural sciences. The sleep movements of plant leaves and petals, sKin colour changes of

crabs, wakefulness in man, emergence of flies from their

pupal cases, rat-running, etc are examples of this fact (Fig1).

NOON MDON NOON

Fig.1 Examples of rhythmic phenomena experimenta 1ly demons- trated to persistunder constant conditions in the laboratory, illustrating diagrammatica1ly the natural phase relationship with respect to the external physical cycles. L, in the

diagram at bottom indicates time of low tide in the crab's habitat. (106)

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The first scientific record is the observation of Linné, the famous swedish naturalist, regarding the opening and closing of flov'jers at different times of the day and this constitutes a set of time marKers for the ’ClocK Flora’ ( 1 0 7 3 .

The organisms usually possess different types of détection and measuring Systems, traditiona 1ly known as senses such as senses of sight, sound, touch, taste, smell and in addition a sense of time too. "A device that measures the elapsed interval between events or that provides for the conventional senses a sériés of events which may be counted may be called a ’clock'." (Einstein 1955 cited by 100).

The sleep movements of flowers and green leaves of plants are so obvious phenomena that they could not escape the attention of early botaniste. In the legumes the alternate folding and expansion of leaves, one cycle being completed in 24 hours, were reported by Sachs (108) and Pfeffer (109).

The earliest intensive studies on solar rhythm during the first decade of this century are those of Pfeffer (110) who has found that legumes possess a persisting daily sleep

rhythm; the time of the day when leaves are expanded is set by the time when the experimental light period commenced.

It may logically be assumed that plants might hâve had some light sensitive reaction which correctly directs the daily rhythm; or the daily rhythm mechanism posseæ the capacity of synchrOniZatiOn with the outside light-dark cycle while the light change is made to occur at any desired time of the day.

Pfeffer also found that daily recurring changes under constant conditions could occur earlier or later day by day, resulting from regular periods deviating a little from the solar days, the so called 'naturel period' (19 hours to 29 hours.)

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K.leinhoohte (111, 1121 who confirmée! Pfeffer’s findings, reported that daily sleep rnovements coula artiflcially be induced to follow alternate light and darkness ranging from about 18 hour to 30 hour days. Further déviation of these limite could not be endured by plants and hence'they broke away revealing their natural periodicity. Therefore, it may be assumed that the organism's daily rhythm is a deep-seated character.

In the light of his experimental results, Pfeffer has proposed an ’autonomous dock hypothesis’, i.e, the period of plant rhythm must dépend on indépendant internai mechanisms.

Bünning's (113] critical experiments, which established the relationship of daily rhythm to température, could constantly support Pfeffer's hypothesis and terminate the then existing controversy held by Stoppel and others (114].

Garner and Allard's early experiments (115] on photoperiodism revealed that plants could produce flovjers at certain seasons, since they are able to measure the length of the day with

sufficient accuracy and thus detect the season of the year.

Time-keeping animais

Among the most striking examples of the dock processes in living organisms is the ability of keeping time found in some insects. The mergence of fruit-fly (éclosion, eedysis] Droso- p hi1 a P s e u d o ob s c u ra, from its puparium (116-119] is an excel­

lent exemple. Although the fly -can complété its development at any time of the day, it emerges only at a certain phase and this éclosion is thus ’gated' (119]. Several earlier workers (120-123] after studying this problem, hâve found

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thiat the emergence Is timed by an internai mechanism (122-1243.

A characteristic time of éclosion of the silk moth, dictated by a b rain-centred photosensitive dock, has been reported

(125). An endogenous biological chronometer has been developped from the studies on the rhythms of celestial navigation of

bees, birds and amphipods (126-129). The initial discoveries of Kalmus (122-123).. Von Frisch (129) that birds and honey-bees measure time using the sun*'s azimuth as compass hâve signifi- cant impact on the study of circadian oscillations. Bees possess

’time sense' and 'time memory* (130) and this true sense of time enables them to return at précisé time of the day to the feeding sation discovered at that time of the previous day

(131-132). In birds and arthopods the celestial navigation

is achieved by continuous compensation for changing the position of the sun in the sky and this indlcates that the animal

possesses a clock-like mechanism (126, 127, 128 & 133). Many invertabrates can find compass directioa with the help of the sun adjusting the angle of the sun according to the time oftheday(134).

Rhythms of naturel frequencies may persist in living things even after they are sealed under constant conditions with respect to every factor for several days. The presence of such rhythms clearly indicates that organisms possess some means of timing these periods. These means are called 'living docks'. A wide variety of organisms ranging from algae to vertebrates show rhythmicity, establishing the fact tha^; most living organisms, beyond ail doubts, exhibit a metabolic

rhythm which is therefore, a universal and ubiquitous phenomenon.

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Most of the biological proces sesinvo1vin g almost ail possible structures and functions are found to exhibit regular

rhythmic variations corresponding to the geophysical cycles.

Thus the rotation of the earth on its polar axis causes the dominant cycle of the day and night; the révolution of the earth around the sun gives rise to the unfailing procession of seasonsj and the movement of the moon in relation to the earth and the sun results in the lunar month as well as tidal cycles.

In recent years some cases hâve been shown to be timed by the lunar influences which particularly affects the marine orga- nisms and those living in the intertidal zones. The tidal

rhythm in the movements of Strombidium, Chromulina & Hautzschia continues even when they are kept in the absence of tides

(136-138].

Familiar to ail are the varions annuel rhythms produced by the orbiting around the sun of the earth with its tilted axis. These phenomena in living things are simple responses to the physical factors in conséquence to their good adaptation to their geOPhysica 1 environment which has rhythmic fluctu­

ations of light, température and océan tides.

X Biological rhythms

Rhythm is a phenomenon (behavioral or physio1ogica1 event]

which repeats itself at regular intervals of time. This broad définition includes ail oscillations which persist, under

constant conditions, for tens of cycles. According to the lehgth

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of the period, the rhythms are classified as diurnal or circadian (24 hours), tidal (about 12.5 hours], semilunar

(about 14.6 days] and lunar (about 29 days].

By définition rhythms hâve the following features : they persist for some time in the absence of diurnal fluctuations in température and illumination; their phase can be altered by a brief disturbance ofthe constant regimen; and their period is relatively independent of the constant external température, at least within the ’ physio1ogica 1 range' (134).

Sweeney (139) defines the circadian rhythm as : "an oscilla­

tion in a biochemical, physio1ogica1 and behavioral fonction which under constant conditions in nature has a period of

exactly 24 hours in phase with the environmenta 1 light and darKness but which continue to oscillate in either continuous light or continuous darKness and constant température with a period of approximately but not exactly 24 hours regardless of an ambiant température”.

Apart from circadian rhythms, living things exhibit many rhythm periods and the frequency of their oscillations may range

from less than a second (brain waves), second and minutes (respiratory and cardiac cycles), to hours (tidal rhythms), to days (circadian and infradian rhythms, to months (menstrual cycles) and to years. Malberg (140) and Reinberg (141) classi­

fied the rhythms as follows : 1. High frequency rhythms 2. Médian frequency rhythms

a. ultradian rhythms b. circadian rhythms c. infradian rhythms 3. Low frequency rhythms

period ranges from a fraction of a, second^ to half an hour.

period changes from half an hour to 2.5 days

period from 1 hour to 20 hours 24 hours approximate1y

1 day to 2.5 days

period is more than 6 days

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Exogenous rhythms occur ae a direct conséquence of the

envirOnmenta 1 changes while endogenous ones are independeht of these changes. The latter involves innate [i.e, endogenous]

PhysiO 1ogica 1 processes.

Most organisms display a temporal modulation within the time span of a day and this is reflected by number of circadian rhythms. In addition, they hâve the ability to utilize changes in the environment to phase their life processes. The term 'circadian' has been coined by Halberg et al [142] (circa = about, dies = a day. Latin].

Studies on biological rhythms hâve brought to light a large number of cases where the highly integrated physiological processes in both lower and higher plants are regulated periodically by an endogenous time-Keeping mechanism. The tremendous increase in scientific research during this

century, particularly the intensive investigation of the past 25 years, has been accompanied by a continuons piling up

of publications on rhythmic phenomena in plants, animais and man himself. There are several booKs, reviews and monographs on this subject [170-187].

A wide variety of high frequericy oscillations exist in

Chemical Systems and the oscillatûry reactions hâve reached the status of a branch of laboratory chemistry only in the , BO's. There are a number of reviews on this topic [143-150].

t

Exemples

Evidences for the existence of a biological dock hâve been accumulating ever since présentation of the first [according' to. Aschoff, 151] doctoral thesis on biological docks by

Julien Joseph Virey[152] pointing out that given drug is not equally indicated at ail hours of the day :

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"... les hypnotiques, les narcotiques, l'opium hors les conjonctures extrêmes, ne seraient pas bien placés dans la matinée lorsque toutes les facultées tendent au réveil;

mais ces remèdes ont une action plus intense et plus salutaires dans la soirée, parce que les forces de la nature aspirent au sommeil et au repos. C'est ainsi que Sydenham prescrivait toujours un parégorique opiatique le soir du jour où il avait donné une purgation ou un émétique, et cet usage est assez imité maintenant pour calmer l'irritation."

The persistent endogenous diurnal rhythms contribute much of the evidence for the biological dock. As circadian

rhythms are found in unicellular and mu 11ice 11u1ar organisms, it is reasonable to assume that the latter inherited the

basic mechanism from their unicellular ancestors.

The universality of daily rhythms has intensively been investigated in recent years and well documented by a number of plant and animal fonctions. The literature is too vast and comprehensive to enumerate it. A selected set of examples are cited to support the ubiquitous

existence of biological rhythms in general and circadian rhythms in particular (Table I, 1533.

Rhythmiclty in Acetabularia

The Acetabularia cell, which is the matériel used in

the présent work on the circadian rhythm of polysaccharide synthesis, displays rhythmic' activi'ties of both short

(2-360 minutes] and long (about 24 hours] duration.

The activities of lactic dehydrogenase and malic dehydrogenase osclllate with a frequency of about 50 minutes. The concentration of 'substance E' (see 154)

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has a frequency betwaen 150 S 360 minutes; the concentration of ATP varies with a much shorter frequency (2-4 minutes) in the presence as well as in the absence of the nucleous

(155).

The daily variations concerning several structural,

physiological and biochemical activities of the Acetabu- 1aria cell, most of them associated with ch 1orop1 asts, hâve been summarized by Bonotto et al (154).

The rhythm of photosynthesis has been extensively

studied and has been shown to display a time-dependent sensitivity to varions antibiotics (actinomycin

ch 1 Oramp heniCO 1, cyc1oheximide, puromycin, rifampcin) to RNAse and to morphactins (anti-auxin) (156-162).

The existence of circadian rhythms in Acetabularia shows that this unicellular organism has a précisé temporal organization (163-169).

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Table 1 (After 153]

RHYTHMIC PHENOMENA IN PLANTS (SELECTED EXAMPLES]

Phenomenon

Bioclectricity Action potentials

Séparation interval Refractory period Change in propagation

direction

Current, transcellular Electric potentials Leaf movements

Rotational Up and down

Stomatal cycling Transpiration Water potential Water uptake Respiration Photosyntlrcsis

Carbon dioxide compensation Carbon dioxide émission Chloroplast shape Enzyme activities

Energy charge Redox potential Nuclear size Mitosis Growth

Flowering stimulus Flower induction Seed germination

Period length

o.i-io s 12 min Circadian Circadian S min, 12 min,

circadian

53 min

Circadian 36 min, 176 min,

circadian min to circadian min to circadian min to circadian 30 min

Circadian Circadian Circadian Circadian Circadian 4-6 h

Semi-circadian Circadian Circadian Circadian Circadian Semi-circadian 20 h

Circadian Circadian Circadian ' Annual Circadian

Reference

Scott, 1962 Pickard, 1973 Pickard, 1972

Paszewski & Zawadzki, 1976 Stoeckel & Pfirsch, 1975 Novak & Sironval, 1976 Aimi & Shibasaki, 1975 Bünning, 1973

Alford & Tibbits, 1971 Alford & Tibbits, 1970 Miller, 1975

Barrs, 1971 Barrs, 1971 Barrs, 1971 Johnsson, 1973

Pallas, Samish & Willmer, 1974 Chia-L ooi & Cumming, 1972 Pallas et al., 1974

Jones, 1973 Wilkins, i960 Busch, 1953

Deitzer, Hopkins & Wagner, 1976

Frosch, Wagner & Cumming, 1973

Frosch et al., 1973 Deitzer et al., 1974

Wagner, Stroebele & Frosch, 1974

Wagner & Frosch, 1974 Biebl, 1974

Bishop & Klein, 1971, 1973 King, 1975

Bail & Dyke, 1954 King, 1975

Cumming, 1972; King, 1975 Zohar, Waisel & Karschon, 1975 Cumming, 1967

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CHAPTER II

ACETABULARIA

Introduction

The giant unicellular alga, Acetabularia which has rightly been designated as "one of the most fascinating living objects in the world" (2Ü0A], is perhaps one of the best Known of the marine algae.

The classification of Acetabularia (Class : ch 1orophyceae, Order : Siphonales, Family : Dasycladacae and Subfamily : AcetabU 1ariacae) has recently been reviewed (202).

The genus is subdivided into two groups based on the presence or absence of an inferior corona on its cap.

The importance of Acetabularia as a tool for modem research has been recognized during the présent century and its réputation as one of the favourite subjects in modem biology exists since the worK of Hammerling (2033.

The early research progress can be dealt under three heads ; ■

13 Biological principles of the nuc1eocytop1asmic relation- ship,

23 Biochemical research introduced in Bruxelles (201F3 andin Wilhemshaven (2043.

33 Molecular biology with spécial reference to Chemical nature of the morphogenetic substances, messenger RNA, site of coding of proteins, etc. üther studies include the turn over of several ribonucleic acids and ribosomes

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13

concerned with those associated with membranes. Chloreplast membranes play an important part in this respect. This

field of research bas been described as "supermo1eeu 1ar biology” (1653.

General morphology

The fully mature Acetabularia cell consists of a rhizoid (base)^ a stalK (middle).and a cap (apex). In addition there are a few (6-10) whorls of fine polychotomous

filaments developed periodically during the early morpho- genesis of the cell, and arranged acropetally at regular intervals.

The rhizoid forms a bail of entanged branches in one of which rests the giant nucléus during the végétative phase of the cell. The nuc1eus - bearing part of the stalk is capable of régénération (206-210).

The stalk is generally 6cm long but it may reach up to 10 cm (A. major). The greatest part of the cell volume is occupied by a large central vacuole which composes the cytoplasm into a narrow pariétal tube. The cytoplasm contains the usual subcellular organelles (ch 1orop1 asts, mitOchondri a, Golgi apparatus, etc.) which are subject to cytoplasmic streaming.

The whorls also contain ch 1 orop1 asts,. mitochondri a and other common constituants of, the cell (211-215) meta- bolically active.

The cap is about 1 cm diameter and is subdivided into

a number of ray-like compartments, each of which accommodâtes

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a secondary nucléus formed during the reproductive phase of the cell and subsequently cysts. As the cap morpho- logy is specles-specific, it provides a direct means to distinguish the different species of Acetabuiaria. Cap formation which is the most conspicuous event in the morphogenesis of the cell, has been studied thoroughly by a number of authors (see 216); in most species each cell produces a single cap. A normal orderly synthesis of RNA and proteins is required for cap, as well as for whorl formation [200 Y).

The comparatively large size of Acetabuiaria qualifies it to be considered as the largest uninucleate and unicellular organism.

Ultra-structure

1. Cell Wall

In natural conditions the cell wall of Acetabuiaria is calcified; but it does not occur in the culture conditions except for some unfavourable conditions. Its u11rastru eture has been studied in detail by many authors (209-214).

2. Primary nucléus

The u1trastructure of the primary nucléus has been studied by several authors (217-223). In the zygote stage (diploid) the nucléus is about 5 jjm in'diameter, but it attains a diameter of 100-300 jjm in an adult plant. It has a net-work of chromatin which is faintly Feulgen positive. A detailed picture of the complex ultrastructural organization of the nucléus and its surroundings is dealt in a recent review

(224).

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Lampbrush chromosomes in the primary nucléus of A. Mediter- ranea hâve been recently reported (225]. The primary nucléus can be isolated and transplanted (226) and stored at room température for 24 hours in artificiel medium (227).

3. Secondary nucléus

When the reproductive cap is fully grown the primary nucléus divides into a sériés of secondary nuclei, as has been first observed by Schulze (207) and confirmed by others (228-233).

The newly formed secondary nuclei are small (+_ 5 ^m in

diameter) and hâve no cytoplasmic border and buds (228/232).

They further divide repeatedly in the stalk and are transported towards the reproductive cap by the upward cytoplasmic streaming (216,232).

According to recent reports (see 234) the secondary nuclei are haploid (233, 235, 236) and control the cyst formation.

They become anchored at fixed positions by numerous microtu- bules (237) which contribute a cytosKeleton with several

dense péri nuclear bodies (224,232,237 & 238) of varying sizes.

4. Chloroplasts

The giant cell of Acetabularia contains several millions (10^) chloroplasts (239,240). The plastid population is heterogeneO us (242) and distributed along a morpho1ogica 1 gradient in the cylindrical cell (220 , 243) .

In the apical, middle and basal parts, the plastid morphology statistically differs with respect to size, shape storage content, number length and stalking of the thylakoids and stroma density (243). From the apex to the base, they

(23)

become progressively larger and charged with stored poly­

saccharides which are almost absent in the apical chlorc- plasts (243.-245 ) .

The apical chloroplasts are active in multiplication (239, 246, 247) in contrast to the basal ones. They are small

and poor in carbohydrate granules. The basal ones are bigger and hâve reduced lamellae and large carbohydrate granules.

The ultra structure of the Acetabularia chloroplast has been described by many authors (212, 220, 228, 243, 248 4- 2 51).

Woodcock and Bogorad (253) report that a very high percentage of the total bulK of the chloroplasts are devoid of DNA and that the highest DNA content is found in the apical ones.

Consequently, it is logical to assume that the majority of the empty chloroplasts are located at the basal région

(234) .

A rejuvenation of the basal chloroplasts has been observed during the formation of the secondary nuclei, by Boloukhère

(228, 243) who demonstrated that the u1trastructure of the chloroplasts dépends not only on the particular région of the cell. where they are located (spatial différentiation), but also on the particular stage of the biological cycle of the alga (temporal différentiation) and on e‘x p e r i me n t a 1 conditions (243) .

1

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I

1 7

5. riitochondria

Work on the mitochondria of Acetabularia has been dons by different authors (214, 228, 243, 252, 254). •

6. Dther organelles

Microtubules bave been found in Acetabularia (221, 228, 237, 238, 255).

Cytoplasts, which represent an anucleate System, bave been studied u11rastructura 11y by Vanden Driessche et al (252).

Specles specificlty : Norphogenet1c substances

Though in ail species of Acetabularia, the cell structure is extremely constant, there is a remarkable species speci- ficity with respect to the morphology of the cap.

Hâmmerling's (see 257) experiments on nucleate and anucleate parts demonstrate that both fragments are capable of

régénération and that the oap produced by the anucleate fragment is always perfectly normal and shows ail the charaeteristic pecu1iarities of the species to which it belonged.

The Work of Hammerling and his co11aborators, mainly based on merotomy experiments, interspecific grafts and trans­

plantation of nuclei^ has lead them to the conclusion that the nucléus produces species spécifie morphogen etic

substances (MS), which are transferred to the cytoplasmj they are stored in the apex and capable of retaining their morphogenetic capacity for several weeks after énucléation Experiments in which the alga was dissected into several

(25)

parts (200 B) confirmée! that the MS mlght be species spécifie gene products.

Brachet's Hypothesis

About 20 years after the first observations of Hammerling (2033 Brachet and his collaborators hâve undertaken a sériés of investigations on the biochernistry of Acetabu 1 ari a

(200 0^ 200 R, 258_- 266) .

A then revolutionary theory had been proposed by Brachet

(200 S) regarding the existence of a relationship between the nucleic acids in the nucléus and the cellular synthesis of proteins. As anucleate Acetabularia can synthesize proteins, including functional enzymes and since the synthesis of

spécifie proteins is under genetic control, Brachet has proposed that the information presented in the nuclear genes must be transported to the cytoplasm in the form of stable molécules of RNA (200 J, K, 200 T—200 Z, 201 A).

According to this concept, Hammerling's species spécifie MS would be identical with stable RNAs of nuclear origin. The

latter hâve been defined as messenger nucleic acids (m-RNAs).

The significance of this work has become évident when the genetic code has been established in E. co1i . It is a logical explanation for the relationship between DNA-RNA- Proteins.

Brachet's hypothesis which is an explanation for the mechanism

i

of cap- formation in Acetabularia, in its présent form, states that : ' the information présent in the genes (nuclear DNA) must be carried to the cytoplasm in an apicobasal gradient, collected at the apex, apparently attach themselves to

receptors located in the cell surface and give the spécifie message for spécifie proteins at the right time to initiâte

(26)

cap formation' (2G0 S, 200 G, 200 H, 200 V, 200 W, 200 Y, 201 B - 201 E] .

This concept has received ample circumstan ci a 1 evidence and adéquate support from recent investigations on the effect of various Chemical and physical factors on Aceta- bu1 aria morphogenesis. Brachet (200 A) emphasized the necessity of isolating highly purified RNA préparations and this has partially been achieved (267, 268). The injection into non-growing Acetabularia fragments, of 40S RNP purified by ultracentrifugation, induces the initiation of morphogenesis, but to a limited extent.

h. ronuTioM or secokuiy mucuui a>id

Fig. 2 Bell cycle of Acetabularia mediterranea

(27)

detail by Puiseux-Dao (216), and Bonotto et al [1543. The fine structural changes are discussed in a recent review

[2693 and will not be dealt with here. since our work is concerned with the circadian rhythm of polysaccharide synthesis in the growing alga only.

Progress in Acetabularia research is summarized in recent books [234, 2703 and review [1543.

A schematic représentation of the cell cycle is given Fig. 2 which is based on recent information [2363j the stage which is used in the présent investigation is indicated as 4.

O 0 o

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21

CHAPTER III

PHOTOSYNTHESIS AND POLYSACCHARIDESYNTHESIS

Chloroplasts are cell arganelles embedded in the cyte- plasm and their primary function is photqsynth esis. They hâve a complex morphology that is of great functional importance.

The chloroplast consists in a system of double membranes : the outer and inner membranes together constitute the

chloroplast "envelope" and enclose an intermembrane space between them. The interior of the chloroplast consists of a matrix called stroma which contains many enzymes and proteins. Besides, there is an organized and covoluted

lamellar system of membranes, the thylaKoid, the arrangement of which differs in different plants. Fig 3 représenta the typical structure of a chloroplast.

Fig. 3. Schematic diagram of the chloroplast structure (300]

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The peeu 1 iarities of the chlaroplast: of Acetabularia hâve been described earlier (Chapter 23. They show versatility of morphology in response te the time of the day, their loeation in the eell, their developmental stage and

wether or not they are under the direet influenee of the nuoleus (3013.

The structure of the Acetabularia chloroplasts has been dealt with by many authors (302“3083,, and their u1trastructure

by others (212, 217, 218, 2203 The u1trastructure has been further studied in the course of the présent work. (see chapter 73

.

Photosynthetic apparatus

The photosynthetic apparatus consists in the two photochemical pigments (PS I & PS II3 which operate at the reducing and

oxidizing ends respectively of the photochemical électron transport chain. Each photosystem is organized into units : the architecture of the photosynthetic unit consists of a set of several hundred antenna chlorophyll molécules that co-operate in harvesting light quanta and channeling them to a single reaction centre. A chlorophyll protein complex containing equal amounts of chlorophyll a & chlorophyll b has been designated as the light harvesting chlorophyll complex (3123 which can feed the excitation energy to either of the two photosystems (Fig 43; it is -embedded in the inner surface of the membrane and bound to proteins.

In each photosystem, a spécial reaction centre chlorophyll (P 680 for PS I & P 700 for PS II3 traps the excitation

energy for the antenna chlorophyll and convert it to Chemical energy .

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23

Chl aj= Chl of PS I Chl ajj= Chl of PS II

Pj. and Pjj = reaction cneter chlorophyll of PSI/PSII Aj and Ajj = primary eloctron

complex.

Fig. 4 A schematlc diagram of the photosy nthetic apparatus (3j 3)r

A unitof 300 chlorophyll [Chl] molécules serves as the reaction center where a single électron transfer act occurs with a quantum requirement of unity. The reaction Products Cformed in a single light flash) of 8 of these units provide the wherewithal for the réduction of one CO^ molécule. Thus one CO^ molécule is reduced for every 2400 chl molécules, but the basic unit contains 300 Chl molécules. If the unit reaction requires two quanta, the unit contains 600 chl molécules and so on.

The smallest photosynthetic structures that can be resolved in the électron microscope are of such a size as to contain one or a small number of photosynthetic units, i.e,

spherical clusters on the lamellar surface of the chloro- plasts.

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a continuous chain of eleotron transport via électron carrying molécules.

A unidirectional or non-cyclic électron flow, which is the normal prooess in photosynthesis, and involves both photosystems is usually aocompanied by the net formation of ATP and NADPH, as well as by the évolution of oxygen.

The non-cyclic type of électron transport can conveniently be subdivided into 3 heads (see Fig 5 A) :

(1} Electron_transport_from_P700_to_NADP

The energy rich excitons of the PS I pigments are

trapped by P70D whose électrons are consequently transferred to aprimary accepter X (P430). The loss of an électron

leaves a hole in P700 (now oxidizedj. The électron is then accepted by a faotor called ferredoxin reducing substance

(FRS) and subsequently transferred to NADP . Thus NADPH is fO rmed.

i2] eleotron transport_from_PS_11_to_P 700

The électrons are available for reducing P700 only when PS II is in turn illuminated and excited. On trapping the

excitation energy, P680 loses an électron as it is transferred to a primary acceptor Q CC550). Thus P680 becomes oxidized.

The eleotron flow from PS II to P700 continues to cyto­

chrome followed by PO- Therefrom cytochrome f C553]

and PC carry the électron further and the latter donatesit to P700.

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25

Fig. 5A. P h Otosynthetic électron transport leading to photophosphorylation and réduction of NADP"^

l=cytochrome b, 2=cytochrome f, 3=X, 4=plastocyanine

[3) _îb2e5_fr°^_ii

The électron required for restoring P680 of PS II to

the reduced State cornes from a water molécule via another Chain of électron carriers. The removal of ail électrons from water résulta in the évolution of molecular oxygen.

Four électrons must be removed from two water molécules to bring about the formation of one molécule of oxygen.

The dehydrogenation of water requires Mg^"^ and chloride ions. The first électron acceptor in this sériés is Z

(Fig 5'A)

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are furnished by one molécule of water. The sequence of the overall reaction is as follows :

2ADP + 2P + 2NADP + 2H^0 + light energy --- >2ATP + 2NADPH2 + 0^

This System of électron light induced movement of électrons from water to NADP is termed non-cyclic électron transport +

and the conséquent formation of ATP is called non-cyclic photophosphorylation.

Cyclic électron transport

Another type of light induced électron flow is the cyclic one : (Fig. 5B) the flow of électrons from P700 of the

excited PS I around a circular chain of électron carries so

+

that the électron ultimately return to reduce P700 . (The cyclic flow of électron is greately stimulated by various oxidative - réduction carriers].

P430

I

Fig.BB : Cyclic flow of électrons (314]

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27

Cyclic électron flow occurs by a shunt or bypass which cornes to play when N A DP is not available to act as the électron acceptor. The e1ectronsejected out of P700 instead

+

of passing to NADP , are shunt into the central électron transport chain probably via cytochrome and passes back to the électron hole of PS I.

Coupled to the cyclic flow there is a phosphorylation of ADP to ATP. It is indépendant of the non-cyclic System and it

requires only the operation of PS I. There is neither évolution of oxygen, nor génération of NADPH.

Proton translocation and gradient

Simu1taneously with the électron transport, hydrogen atoms are extracted from water or near the inside of the membrane and hydrogen ions are extruded into the inner space of the thylakoid : i.e, PS II oxidation takes place inside the thylakoid membrane.

The D^ and protons are released in the intrathy1akoida1 space solution and électrons are driven by the energy of light

through the sequence of carrier molécules.

Having been pumped by the light-driven movement of électrons, the translocated protons are accumulated in the inside, which as a resuit becomes acidic. The inward proton transport across the thylakoid membrane establishes a stable proton concentration

(or pH'} gradient which actually represents a store of free energy mainly in two forms :

(1] the différence in the concentration or Chemical activity of protons (proton concentration is measured in terms of pH).

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Each local gradiant is utilized by a local coupling factor.

The number of molécules of the coupling factor in the chlo- roplast is of the same order of magnitude as the number of électron transport ’chains' (see 315).

Protons diffuse from a région of high concentration to a région of low concentration when there is a pathway through tiie membrane. The net movement of charges across the membrane créâtes a différence in the electrical potentiel. Ail charged particules are affected by the resulting e1eetrostatic field.

The total energy of the proton gradient is the sum of the concentration (or osmotic) component and the electric component.

In chloroplasts, the enzyme coupled for the synthesis of ATP are called the CF -F complex (similar situations exist in

1 °

mitochondrie and bacteria) and protons flow outward this complex 1 leading to the formation of ATP in tne space between the inner and outer membranes.

The chemiosmotic hypothesis (316) explains the couple

of the area of PS II, water splitting reaction and uneven P/e^

values nitchell (316) suggested that the flow of électrons

through a System of carrier molécules drives positively charged hydrogen ions/protons across the membrane of the chloroplast.

Conséquently, an e1eetrochemica 1 gradiant is created across the membrane, consisting of :

(36)

29

Ca) a différence in the hydrogen ion concentration ( ^ pH] and Cb) a différence in the electrical potential C A y]

Fig. 6 Photosynthetic phosphorylation in chloroplasts C3173

Izawa et al C315] who found a corrélation between proton release and ATP formation, demonstrated that an internai proton gradient is an essential intermediary step for ATP formation.

Activation of Enzymes

Light régulâtes the carbon metabolism in green plants ; algae C318-321] and higher plants C322).

Light activâtes two enzymes involved in glucose utilization.

Light activation of the reduced pentose pathway enzyme was first reported in several species of algae (323-325], fot NADP

linked g 1 ycera 1dehyde - 3-hydrogenase. the following enzymes of the photosynthetic carbon metabolism are activated by light (3263

(37)

Ribulose-1-5-diP carboxylase Ribu1Ose-5-diP Kinase

Fructose-&-J diP phosphatase

Sedoheptulose-1-7-diP phosphatase NADP-linKed glyceraIdehyde-3-P dehydrogenase,

when two enzymes are inactivated by light C326] : Glucose-6-P dehydrogenase and P-fructoKinase.'

Bassham (319] concluded that light affects the activity of

several of the reductive pentose pathway enzymes through changes in the levels of ATP and NADH.

Anderson & Avron C327] report that there is some différence in the light modulation of g lyceraldehyde-3-P dehydrogenase and other enzymes because the activation of g lyceraldehyde-3-dehydrogenase occurs much more slowly than the light activation or inactivation of the other enzymes. P hotosynthetic électron transport is re- quired for the light activation of the pea chloroplast enzymes

(327 ] .

Light modulation of chloroplast enzymes has been suggested to

be a reductive process involving the photochemical apparatus(326].

Therefore, a light generated membrane bound réduction might be involved in the regulatory process. The photoreceptor of light modulation of the activity of these enzymes must be the photosynthetic pigment as was demonstrated by action spectra experiments for the activation of g lyceraIdehyde - 3-dehydrogenase

(328 ] .

At least two different System I components are involved in the light modulation :

(1] a Kinase and a dehydrogenase located before the ferredoxin and (2] phosphatase at or beyond ferredoxin.

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31.

Ferredoxin has been implicated in the activation of fructose- 1-6-diphosphatase and sedulose-1-7-diphosphatase [329,330).

Polysaccharide synthesis ; a dark reaction of photosynthesis

\

Photosynthesis, when is the fondamental ele etromagnetic energy conservation process upon which ail life on the earth dépends, results largely in the fixation of CO^-

Carbon .dioXide is converted into carbohydrates, a reaction which, in ail organisme possessing chlorophyl, follows ATP & NADPH

génération by the photosynthetic light reaction.

...-... —...

This is a multistep process which can be explained by means of Chemical and biochemical principles.

Calvin cycle

The reactions involved in the dark phase of photosynthesis, where the reduced pyridine nucletide and ATP produced in the light

phase are used to convert CQ^ into carbohydrate, hâve been worked out by Calvin and his co1laborators [Calvin cycle) Fig.7

CO^ first combines with a five carbon atoms sugar, ribulose

phosphate and an unstable six carbon compound is formed. It soon breaks down into three carbon atoms molécules called phosphogly- ceraldehyde [PGA). Each PGA molécule is then phosphory1ated by ATP and reduced by NADPH^- The resulting energy ri ch - 3-carbon compound' is g 1 yceraIdehyde-3-phosphate [G-3-P).

The G-3-P is utilized in two fundamental ways :

(39)

^6 ADP phosphoribulo , ktnase

\6 ATP

@

(12 molécules) 12 ATP phosphogiveerokinase

12 ADP

12 NADPH + 12H • 12 NADP4-12 Pj

3-phosphoglycéraldehyde (12 molécules)

1,3-diphosphoglyceric acid (12 molécules) 3-phosphogl >ceraldehyde

dehydrogenasc

©

Ribulosc - 5‘phosphule , ,

I I nnulosc (h molécules) phosphate

3 epimerase

phosphoribosc isomerase

triose phosphate isomerase

■ dihydroxyacetonc phosphate (5 molécules)

I aldolasc Fructose I, 6-diphosphate

(3 molécules)

® [phosphatase

r^3HjP04

Fructose 6-phosphate (3 molécules)

@ transkclolasc

I

y>-4

[

Xylulosc 5 -phosphate (2 molécules)

Erythrose 4- phosphate (2 molécules) 4*

(2 molécules)

llhXOSl: PRODUCT Fructose 6- phosphate

(I molécule)

transkctolasc (lü)

. Ribose 5-phosphau:

(2 molécules)

^ |aldolase Sedohcptulosc 1.7 diphosphatc

(2 molécules)

l^2Hp

(§) phosphatase

'^2HjP04

Sedohcptulosc 7-phosphate

Fig. 7 Photosynthetic Carbon Réduction Cycle or Calvin Cycle (331)

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33

1. Most of the molécules are used for the formation of new - ribulose by a sériés of reactions and rearrangements to form a five carbon sugar.

2. Some of the G3P is used as substrate for the reactions leading to the synthesis of glucose.

Respiration

Fig. 8 Synthesis of carbohydrate

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yield energy for doing work or may be used as a raw material for the synthesis of other classes of compounds such as fats, or they may be bound together to form more complex carbohydrates such as sucrose, starch or cellulose. Starch is the most common storage form of carbohydrate in plants (Fig.8].

In order to produce one molécule of hexose sugar (6-carbon sugar], six molécules of CO^ are required.

The carbon réduction cycle can be summarized into Fig 9 ;

Fig. 9 Carbon réduction cycle.

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35

To generate ribulose 1-5-diphosphate from PGA, both NADPH and ATP produced in the light phase are required.

The generally accepted scheme for the light and dark reactions of photosynthesis can be represented as follows (332] :

Electron tninsport

K)., Ilu ADP ATP Iw

l W

1

H..O H ... PS [

elfctnm H..«

t‘(i-(Trmt tinvv

\ADP

Carhoii Fixation ATP ADP CO., + ATP + N'ADPH--- ► C[HOH]

Polysaccharide synthesis

Polysaccharides are polymers of simple sugars resu11ing ’ from the condensation of monosaccharides g lycolytica 1ly linked

together with the élimination of water to form a linear branched Chain. A brief account of the biosynthesis of carbohydrate is given in Goodwin and Mercer C331].

A scheme for the synthesis of polysaccharides in plants is given in Fig. 6, which is a synthetic picture base on Manners (333]

and Goodwin and Mercer (331].

In Acetabularia ch 1 orop1 asts, the polysaccharide (storage type]' are starch and inulin. (349,350].

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C A.RBOHY DRAT S BIOSTNTHISIS IN PIANTS

Fig. 10 A sheme of polysaccharide synthesis in plants. C333 S 3313.

A. Starch synthesis

Starch is a high molecular weight plymer of D-glucose and is the principal reserve carbohydrate in plants. Most starches consist of a mixture of two types of polymères, (amylose & amyiopeetin3, the proportion of which varies in different starches; it is

generally in the range of one part of amylose and 3 parts of amylopectin.

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37

Amylose is a linear polymar af glucose units joined by -D- C1-4]

linkages to yield a linear Chain of several hundred glucose units. The branched polymer of starch is the amylopectin, the molecular size of which ranges from several hundred to millions representing many thousands of D-glucose units per molécule.

The types of linkages are -D-Cl-60 and -D~C1~4]; the former are branch points in the molécule.

In nature, starch occurs in the form of granules in which the amylose and amylopectin molécules are intricately arranged, held together by hydrogen bonding.

The primary molécule of starch synthesis is a D-glucosel oligo­

saccharide. The synthesis of a biological polymer such as starch Cor glycogen] is a fondamental process occuring in ail living Systems and the process can be divided into three headlines ;

1] Activation of the monomeric units

The activated forms of D-glucose units for starch synthesis are D-glucose-1-P. Dridine-diP-D-g 1U CO se, adenosi ne-diP-g luoose and

-D-C1-4] glucan. In plants, D-g lucose-1-P is formed from the Products of Photosynthesis and can be used directly for polymer zation purposes if proper enzymes and acceptors are présent; it can also be converted into uridine-diP-g 1ucose or adenosine-diP .

2 ] Transfer of monomeric units to appropriate acceptor compounds.

The actual monomeric units and the acceptor compound are brought together by a process of diffusion and collision of the enzyme, donor and acceptor molécules. The acceptor substances for starch synthesis contain D-glucosyl units. It is likely that the activated forms of D-glucose and the acceptor molécules are complexed at

(45)

however, close enough together to permit ionic interactions between the donor and accepter. These interactions resuit in a displacement reaction in which the acceptor unit replaces the activating group and a D-glusyl unit is added to the acceptor molécule. Répétition of this process many times leads to the formation of polymer.

3] Patternization of the product during the po lymerization proce ss.

In the patternization process, factors such as steric hindrance in the product which is synthesized, energy of the binding of the activated D-glucose units and of the acceptor to the enzyme, and- the energy of the binding of the product détermine the fianl shape size and structure of the polymer.

Starch granules show a layered or Shell structure (334,335J and they grow by the déposition of material on the outside (apposition) for which much evidence is available (336-338);

however, insertion of molécules inside the granule (intus- suception - 339, cited by 335) has been argued.

Starch synthesis can be promoted in the dark by exogenous sugars such as glucose and a scheme proposed by Walker (340)

(Fig. 11) has found recent support (341)

Glucose enters the normal glycolytic sequence in the cytoplasm and probably enters chloroplasts as triose phosphates.

Inside the chloroplasts, some triose phosphate will be oxidized to PGA yielding ATP in the substrate level phosphorylation. (340).

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39

Exoqenous qlucose

Fig. 11. Starch synthesls in the dark from exogenous glucose.

B. Starch - s Ucrose Interconversion.

The existence of an interrelationship between sucrose meta- bolism and starch synthesis in plants has been recognized for many years (335). Porter (342) has reviewed the relation- ship between sucrose and starch through a sériés of phosphory- lated sugars. An anzymatic linK between the two processes has been established by De Fekete and Cardini (343), who proposed the following scheem : Fig. 12.

Sucrose + UDl*-^

ATP-*-^

UTP*-^

Sti'.rch 4 ADP -<

L'DP u-gluco3c + Fructose

Fig. 12. Reactions involved in the transfer of sucrose to starch (343) .

(47)

Inulin differs from other polysaccharides in having a low molecular weight ( 10000) and in being formed of fructose

as the repeating unit. Several généra of Comositae (Dahlia, Taraxacum, etc) and Gramineae contain inulin as their reserve carbohydrate. However, its presence has also been claimed in Acetabularia chloroplasts (344). Evidence obtained from microorganisms (345) suggests that inulin is formed from

sucrose (see also 331, 333) by a mechanism involving the utilization of the fructose portion of the disaccharide for polysaccharide formation and the libération of the glucose portion(Fig.13).

O-ci

Fig. 13. A schematic représentation of the formation of inulin

It is formed from the fructose portion of the sucrose unit, with the élimination of the glucose portion. (After Donner

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41

The fructose residues are linked together by oxygen bridges between the 1 and 2 carbon atoms of the adjacent hexoses.

The synthesis of inulin is due to transfruetosy1ation reactions (333) The chains formed are shorter than those of starch and contain approximately 28-35 hexose residues.

The structure and metabolism of inulin hâve been reviewed by Hirst (346) and the rôle of various transferases- and carboxy-

lases in the metabo1isms of fructans has been reviewed by tdleman and Jefford (347).

Starch which is a reserve substance of plant origin, is stored in the form of granules, it forms an insoluble source of

energy which can be made available through the action of enzymes. The Chemical composition, crustelline pattern and shape of starch are subject to genetic and physiologica1 factors. The structure of starch granules is immediately connected with the development of the living cell; and the type of starch formed dépends on the availability of water, as well as the concentration of various salts and organic

compounds présent in the plastid or in the cytoplasm, (335,348)

Since starch is formed in plastids ( extrap1 astida 1 starch

formation also exista - see Badenhuizen (335) these structures must contain the enzymes and substrates necessary for its

formation. It is generally accepted that phosphory 1 ase is an important starch synthesizing enzyme and its presence in plastids has been identified by cytochemical studies (335).

Since Acetabularia chloroplasts produce starch and inulin, it can be argued that both are formed from a common source -

sucrose - (see Fig. 10); the glucose portion might be utilized for starch synthesis and the fructan portion for that of inulin However, this will awaits confirmation from biochemical studies

(49)

CHAPTER IV

M E T H 0 D S

The aim of my research work in this laboratory has been to

préparé a D Sc thesis. ,

The initial days Cabout one month and a half] were devoted for making a clear idea of the various techniques and methods used in our laboratory. A primary survey of the current research activities in our laboratory was made in order to choose a

suitable research project. It was décidée), with the help of expert advice of the professera and research workers in our laboratory. to join the "Acetabularia Club".

The élégant guidance of Prof. Vanden Driessche, led me to plan a research project : Circadian rythm of polysacchride synthesis in Acetabularia.

The purpose of my work was to study the circadian variation of polysacchride synthesis by ways of autoradiography.

The isotope used was C fructose. Inhibitors of photosyn- 1 4 thesis were used in order to investigate the possibility that a circadian rythm of polysacchride synthesis exists, besides that of photosynthesis. The conclusions are relevant to the problem of the mechanism of circadian rythmicity, which becomes more and more important in agriculture and in medecine (the

time of drug administration for example). The research was also carried out in anucleate cells, in order to estimate the

influence of the nucléus on polysacchride synthesis.

Our experiments were designed to investigate the circadian rythm of polysacchride synthesis. Some of the several me­

thods used in our laboratory hâve been employed to reach this goal.

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1] Culture of Acetabularia

The technique for the culture of Acetabularia was first proposée! by Hammerling (203) and Beth (400 ). Later, the culture medium and the techniques were improved by several workers (401-407).

In our laboratory the sea water in which Acetabularia

is normally grown cornes from the North Sea; it is filtered through K Seitz filter and stocked in large containers of

«J

300 X 1. The filtered sea water is then sterilised and

transferred to 5 liter storing bottles, which are autoclaved for 10 minutes, (1 Kg/Cm^) and Kept in the dark at 10-12° C for prolonged storage extending upto several weeks. These bottles are provided with basal drainage tubes which facili-

tate easy distribution of this stored sea water into several 250 ml culture flasks.

The "Erdschreiberlosung" (ESL) (203) is the classical medium for the laboratory culture of Acetabularia and other marine algae (408-411).

The following is the composition of the natural culture medium used by Lateur and Bonotto (406).

Lateur and Bonotto's Natural Culture Medium

Na NOg 0.1 gr

Na^HPD^ 0.02 gr

Earth Extract 0.5 gr

Stérile Sea Water 1000.0 m 1

./.

(51)

The earth extract which is used in several laboratories, contains ammonia_andother organic compounds which promote growth {see4123.

The Beckman aminoacid analyser shows that it contains ail aminoacids, ammonia and several unidentified minor com­

pounds (406). The composition of earth sample differs from place to place, as examplified by the samples from Bruxelles and Naples (406). The latter contains a higher level of phenylalanine than the former. Growth of Acetabularia is more rapid with Naples’earth extract (406).

As sea water is often variable (413-414), a few attempts hâve been made to grow Acetabularia in artificial media (404-415), as a set to overcome these drawbacKs. However, the later development of the alga is difficult under these conditions.

Under our conditions, for the cultivation of Acetabularia maditerranea (406), it requires about 180 days to pass from one génération to another and it consista of 10 stages in its life cycles according to Bonotto and Krichmann (416).

The cell cycle summarised in Fig. I (Chapter II) is based on recent concepts (236).

A light intensity of about 1300 lux provides good growth con­

ditions for the algae (417). Many worKershave discussed the light intensity required for culture (400-41-7-430).

The quality of light has also received attention (431).

The optimum température for Acetabularia mediterranea is around 20-21° C. Conditions influencing morphogenesis, cell metabolism, chlorophyll content and photosynthetic activity hâve been discussed by Puiseux-Dao (216)

Figure

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