Chapter I

Introduction

Contents Page No.

General introduction to photosynthesis

Thylakoid membranes and its intrinsic protein complexes

Functional and structural aspects of the PS II complex

Water oxidation and ‘S’ state cycle

Role of proteins and cofactors in water oxidation

PS II reaction center complex

Chlorophyll protein 43 and chlorophyll protein 47 (Core

Antenna)

Electron transport in PS II

Cytochrome b 559

Cytochrome b6f complex (The Q Cycle)

Plastocyanin

Structural and functional aspects of PS I complex

Polypeptides of PS I complex

P700, The Primary electron donor of PS I

Electron transport in photosystem I

Ferredoxin and Ferredoxin-NADP+ reductase

ATP synthase complex

Thylakoid membrane lipids and their role

Cyanobacterial photosynthesis

Cyanobacterial photosynthetic pigments

Phycobiliproteins-subunit composition and spectral

characteristics

Types of biliproteins and their occurrence

Structure and spectral properties of the biliproteins

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4

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8

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10

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14

15

15

17

19

19

20

21

23

23

26

26

28

30

30

The molecular architecture of cyanobacterial phycobilisomes

Linker polypeptides – their role in structural organization

Functional aspects of the phycobilisomes

Environmental factors affect the energy transfer in

cyanobacteria

Chromatic adaptation

Photosynthetic and respiratory electron transport interaction in

cyanobacteria

Spectral characteristics of the oxygenic photosynthetic

systems

Spectral characteristics

Fluorescence induction as an indicator of thylakoid membrane

alterations

Partial photochemical reactions

Review of literature

Aim and objectives of present investigation

Reasons for selecting cyanobacterial systems

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41

43

46

50

50

1

 

General introduction to photosynthesis:

Photosynthesis is the most important physiological process on which the

existence of life on planet earth depends. Higher plants and algae through this

process convert solar energy into chemically energy rich compounds, which

are necessary for their growth. In addition to this, molecular oxygen is released

as a result of an early event of photosynthesis. Thus life on our planet is

dependent on this important biological process.

In higher plants and green algae the entire process of photosynthesis

takes place in chloroplast, whereas in cyanobacteria the same process occurs in

the intact cells. In the intact cells, the thylakoid membranes or thylakoids of

chloroplast converts solar energy in to chemical energy. This process is known

as "light reaction" of photosynthesis. The pigment protein complexes on the

thylakoid membranes help in the harvesting of light energy and its conversion

to chemical form. The energy rich products formed in the "light reaction" are

utilized for the fixation of carbon dioxide (Fig.1). The process of carbon

fixation and its conversion to the sugars is known as the "dark reaction".

Enzymes present in the cytosol take care of the process of carbon fixation.

The thylakoid membranes possess two distinct pigment protein beds

namely, photosystem (PS) I and II. The chlorophylls of the photosystems are

driven to higher energy state with the help of light energy. This gained energy

could be utilized in different ways such as photochemistry, energy transfer

between two photosystems, light emission and heat dissipation.

2

 

Fig 1: Representation of light and dark reactions of photosynthesis

3

 

In the two photosystems, the PS II and PS I, two specialized chlorophyll

(Chl) molecules namely P700 belonging to PS I and P680 belonging to PS II, are

capable of undergoing light induced charge separation and subsequent electron

transfer. These specialized Chl a molecules act as reaction centers. These two

photosystems act in series. Chl antennae of PS II get excited with PS II

wavelength light and oxidizes the reaction center Chl a, P680. The electron then

gets transferred to the plastoquinone (PQ) pool. The electron whole left on P680

is filled ultimately, by the electrons resulting from the oxidation of water. The

photolysis of water not only evolves oxygen but also releases protons to the

inside of the thylakoids. Electrons from reduced PQ pool are transferred with

the help of carriers ultimately to the PS I.

Mainly by absorbing infra red light, the reaction center P700 of PS I gets

oxidized. After excitation, an electron leaves P700 of PS I to NADP+, the

terminal electron acceptor via several intermediate electron carriers. Electron

coming from PQ pool fills the electron hole created on P700 via (cytochrome)

Cyt b6f and plastocyanin (PCy). Protons pumped from outside to inside of the

thylakoid membranes during the oxidation of water and PQ shuttling largely

account for a proton motive force (PMF) needed for the synthesis of ATP from

ADP and Pi. The ATP synthesis occurs by the coupling factor [ATP synthase

(CF0-CF1)] of the intact cells or chloroplast. The products of the light reaction

namely NADPH + H+ and ATP are used for the reduction of CO2 to

carbohydrates through the catalysis of a number of enzymes present in the

cytosol of microorganisms or in the stroma of chloroplast.

4

 

Thylakoid membranes and its intrinsic protein complexes:

Thylakoid membranes comprises of four multi protein complexes to

perform photosynthetic electron transport in the thylakoid membranes (Fig.2).

They are PS II complex, Cyt b6f complex, PS I complex and ATP synthase

complex. The protein complexes are linked by mobile electron carriers such as

PQ, PCy and ferredoxin (Fd). Both of the photosystems possess additional light

harvesting complexes (LHC) which help in the transfer of absorbed light to the

reaction center (RC) (Murphy, 1986; Anderson, 1987; Nixon and Mullineaux,

2001; Anderson, 2002; Stephan and Karin, 2005; Dekker and Boekema, 2005).

Functional and structural aspects of the PS II complex:

PS II functions as a light-driven PQ reductase (Zouni et al., 2001;

Barber and Nield, 2002). Two dissimilar functional units namely water

oxidation complex (WOC) and PS II reaction center complex are involved in

this process (Hankamer et al., 1996; Barber, 2002; Wang et al., 2002;

Coleman, 2005: Watanabe et al. 2009; Dimitrios et al.2009.) (Fig.3). The

WOC needs to accumulate four positive charges for oxidizing H2O to O2. The

redox state of WOC is denoted by the "S" state where So, S1, S2, S3 & S4 denote

the oxidizing equivalents on the WOC (Kok et al., 1970; Bricker and

Ghanotakis, 1996). In addition to these, several polypeptides cooperate in the

water splitting process (Miayo and Murata, 1989).

5

 

Fig 2: Organization of pigment proteins and electron transport components in

thylakoid membrane

6

 

Fig 3: Structural organization of photosystem II complex in cyanobacteria

7

 

Water oxidation and "S" state cycle:

The photosynthetic oxygen evolution from photosynthesis of water is

linked to PS II activity. Kok et al. (1970) formulated the generally accepted

"S-state scheme" for evolution of O2. Oxygen evolving complex undergoes a

successive series of increasing oxygen states from S0 to S4 on excitation of its

associated PS II reaction centre.

After the enzyme reaches to S4 it releases O2 and returns to the S0 state.

Each of the light induced transitions from S1 to S2 and S2 to S3 results in

release of two protons. A physiological donor Z is the primary physiological

electron donor to P680. Z has shown to be a tyrosine molecule. Manganese is a

critical cofactor in oxygen evolution and there is general agreement that water

oxidation activity requires four metal atoms per center (Cheniae and Martin,

1970; Debus, 1992). The redox state of native Mn of PS II is also correlated

with the "S" states (Amesz, 1983; Dismukes, 1986; Babcock, 1987; Dekker

and Van Gorkom, 1987; Andreasson and Vanngard, 1988; Ghanotakis and

Yocum, 1990; Hankamer et al., 1997) Mn valency changes during the "S"

state cycle have been revealed from EPR (Dismukes and Siderer, 1981). Thus

Mn stores the oxidizing equivalents during water oxidation. Mn2+ also

functions as the substrate binding site of O2 evolution centre and water

analogues bind to Mn2+ (Beck et al., 1986; Hansson et al., 1986).

8

 

Role of proteins and cofactors in water oxidation:

Three extrinsic water soluble polypeptides are necessary for the

function of the O2 evolution i.e, 33, 24 and 18 kDa (Table. 1) (Murata and

Miyao, 1987; Anderson, 1987; Homann, 1987 and 1988; Bricker and

Ghanotakis, 1996; Enami et al., 1997; Hankamer et al., 1997; Nijafpour,

2006.). The 33 kDa protein helps in the Mn stabilization and relieving the

higher chloride requirements. It also accelerates the S3 - So transition. The 24

kDa polypeptide is considered as a Ca2+ trapper and also relieves high

requirement of cl-. Miyao and Murata (1985) have shown that the 17 kDa

protein is involved in the retention of Cl- at the active site of water oxidation.

The Cl- requirement is necessary for the optimal O2 evolution (Coleman

and Govindjee, 1987; Homann, 1987). It suggested that Cl- acts as ligand to

Mn and stabilizes the higher "S" oxidation states. Calcium ions mimic for the

function of 23 and 17 kDa polypeptides, which are necessary for oxygen

evolution (Ghanotakis et al., 1985; Homann, 1988; Ort and Yocum, 1996;

Hankamer et al., 1997).

PS II reaction center complex:

PS II reaction center comprises of the D1 (34 kDa) and D2 (32 kDa) are

intrinsic membrane protein components present in the thylakoid membrane

(Tang et al., 1990; Bricker and Ghanotakis, 1996; Kruse et al., 1997;

Hankamer et al., 1997; Aro et al., 2005). Nanba and Satoh (1987) have

isolated PS II complex which comprises of D1 and D2 is similar in structure

and appear to form a heterodimer that exhibits two fold symmetry (Mitchell et

al., 1988; Anbudurai et al., 1994; Zouni et al., 2001). This D1 and D2 hetero

9

 

Table 1: Important cofactors, pigments of photosystem II and water oxidation

complex

Cofactor Steriochemistry Function

Mn 4 Water oxidation

Ca2+ 2 – 3 Regulation of Mn function in H2O oxidation

Cl 4 – 5 Water oxidation

Tyr D+ 1 Oxidation of S0 to S 1

Tyr Z+ 1 Oxidation of Mn/ reduction of P680+

Chl a 50 Photochemistry / antenna

Pheo a 2 Charge separation / electron acceptor

PQ 2 Electron acceptors: QA and QB

Non-heme Fe 1 Regulation of QA/QB electron transfer

Heme Fe 2(b559) Stabilization of D1 and D2

(Taken from Ghanotakis and Yocum, 1990)

10

 

-dimer binds to a variety of cofactors such as P680, primary electron acceptor

pheophytin (Pheo) QA, the non heam iron and the Tyr Z+ and Tyr D+ (Bricker

and Ghanotakis, 1996; Rick De wizn et al., 2002). In addition to this PS II

reaction center complex consists of four Chl a molecules, two non

photochemical Pheo molecules, one or two molecules of β carotene and one

Cyt b559 molecule (Table 2). These proteins contribute ligands to the

manganese, calcium and chloride ions that are associated with the WOC. QA is

known to bind with the pocket of D2 where QB is known to bind with pocket of

D1 (Xing et al., 1996). The functions of the D1, D2 are studied well but the

exact role of b559 is unknown. The isolated PS II reaction center complex is

highly active in its functions (Barber et al., 1987; Chapman et al., 1998).

Chlorophyll protein 43 and chlorophyll protein 47 (core antenna):

P680 core is tightly coupled with two pigment protein complexes called

chlorophyll protein (CP) 47 and CP 43. These CP complexes are integral

protein components of PS II (Jansson, 1994; Foder et al., 1995; Tsiotis et al.,

1996; Hankamer et al., 1997). Both these CP complexes contain Chl a and β-

carotene bound to the apoproteins, but not Chl b (Eijckelhoff et al., 1997). The

excitation energy captured by light harvesting pigment protein (LHCP)

complex is transformed to RC by these CP (Horton and Ruban, 2005). The

LHCP II contains covalent bound Chl a, Chl b and xanthophylls. Each CP

consists of six trans membrane spanning regions with a large loop of 190

amino acids in CP 47 and 140 amino acids resides in CP 43 and interact with

other components of WOC system (Vermass, 1993; Enami et al., 1997;

Whitelegge, 2005).

11

 

Table 2: Polypeptides present in photosystem II

(Compiled from Ghanotakis and Yocum 1990; Jennings et al., 1996)

Nature Polypeptide Molecular

weight kDa

Gene Function

Intrinsic

D1 32 Psb A TyrZ+ and binds P680, QB

D2 34 Psb D TyrD+ and binds P680, QA

CP 47 47 Psb B Excitation, energy transfer , binds 33 kDa

CP 43 43 Psb C Excitation, energy transfer , binds 33 kDa

CP 29 29 Ihcb 4 Excitation, energy transfer and dissipation

CP 26 26 Ihcb 5 Excitation, energy transfer and dissipation

CP 24 24 Ihcb 6 Excitation, energy transfer and dissipation

a Cyt b559 9 Ihcb E Binds heme, photoprotection

Cyt b559 4.5 Psb F Binds heme, photoprotection

Extrinsic

33 kDa protein 33 Psb O Stabilizes Mn cluster, Ca2+

and Cl- binding 22 kDa protein 22 Psb P Ca2+ and Cl- binding

17 kDa protein 17 Psb Q Ca2+ and Cl- binding

LHCb 1 25 Ihcb 1 Light harvesting

LHCb 2 25 Ihcb 2 Light harvesting

LHCb 3 25 Ihcb 3 Light harvesting

H protein 7.5 Psb H Photoprotection

K protein 4 Psb K PA II assembly, PS II stability

R protein 10 Psb R Donor and acceptor side function

X protein 4 Psb X QA function

12

 

Electron transport in PS II:

After absorption of light, which is captured by the antenna is transferred

to PS II reaction center of P680 which goes to excitation state, the primary

charge transfer takes place from excited P680 (Jennings et al., 1996; Connelly et

al., 1997; Dekker and Van Grondelle, 2000; Diner and Rappaport, 2002). This

entire reaction takes place in 3 pico seconds. As a consequence of charge

separation takes place from pheo, the electrons will be transferred to quinones

(acceptor of PS II) (Haehnal, 1984). Removal of an electron from the reaction

center creates a hole which will be filled by the electron coming from break

down of water molecule from WOC through Z (Lancaster et al., 1996; Asada,

1999). "Z" is a tyrosine molecule of the Dl polypeptide (Diner and Babcock,

1996) and it can be measured by EPR signal.

Existing of intermediates between P680 and Q suggest that pheo could be

the primary electron acceptor of PS II (Klimov et al., 1977; Diner, 1986). This

has been confirmed by optical absorption spectroscopy (Fajer et al., 1980) EPR

(Schuvalov et al., 1986) and ENDOR studies (Rodriguez et al., 1987). The

reduced pheo is oxidized by an electron acceptor QA (Petrouleas and Diner,

1986). In green plants Fe2+ is closely associated with QA and helps in the

stabilization of semiquinone forms QA and QB (Klimov et al., 1981; Petrouleas

and Diner, 1986; Diner and Babcock, 1996). QA is the secondary electron

acceptor and it binds to 32 kDa of Dl polypeptide (Mc Pherson et al., 1994)

(Fig 4).

13

 

Fig 4: Electron transport in photosystem II

14

 

The reduced electron acceptor QA- oxidized by the QB

- yields the

semiquinone QB- . The loss of the electron returns QA- to QA. A second electron

is then transferred from QA- to QB

- to produce a reduced Qb2- molecule. Qb

2-

accepts two protons and forms quinol (QBH2) functioning as two protons and

two electrons gate. This quinol is easily displaced from its binding site by fully

oxidized quinones .Several herbicides which are structurally different include

DCMU and atrazine etc. acts on the photosynthetic electron transport and

inhibit the electron flow from QA to QB by binding site of Dl Polypeptide

(Velthuys,1981; Trebst, 1986). PQ is a vital electron transport intermediate

which acts as diffusible electron carrier (Haehnel, 1980) between QB and Cyt

b6f. PQ has been suggested as mobile electron carrier between PS II and Cyt b6f

complex (Haehnel, 1980; Anderson, 1981).

In addition to electron transport, PQ plays a key role in the generation of

proton gradient across the membrane (Ort and Yocum, 1996) and helps in the

phosphorylation of LHCP through activation of a kinase. This phosphorylation

regulates energy distribution between PS I and PS II (Fork and Satoh, 1986;

Allen, 2004).

Cytochrome b 559:

This protein is closely related with the reaction center since it is purified

with D1 and D2 (Nanba and Satoh, 1987; Barber et al., 1987). The PS II

complex contains two protein subunits of 9 and 4 kDa (Cramer et al., 1986). It

has been suggested that Cyt b559 may function in photoactivation (Cramer et

15

 

al., 1986) or stabilization of D1/D2 complex and protecting reaction center

against photoinhibition (Whitmarsh and Parkrashi, 1996; Kaminskaya et al.,

1999).

Cytochrome b6f complex (The Q cycle):

Cytochrome b6f complex can be considered as a plastoquinol-plastocyanin

oxidoreductase. Plastoquinone is an important electron carrier between QB and

Cyt b6 f. It contains four major polypeptides, which are Cyt f (31 kDa), Cyt b6

(22.5 kDa) (Fig 5), the Rieske FeS protein (22 kDa) and 16.5 kDa protein of

unknown function as major proteins; two minor proteins of 9 kDa are

associated with this complex (Hauska, 1986). The stoichiometry of Cyt f/ Cyt

b6 16.5 kDa polypeptides was estimated as 1:2:1, whereas the Rieske FeS

protein was present in sub stoichiometric amounts. Cytochrome b6f complex

operates electron transfer in the cyclic process known as Q cycle (Mitchell,

1976; Baniolis et al., 2008). This cycle helps in the transfer of protons across

the membrane, the oxidation of quinol and the reduction of the plastocyanin.

DBMIB is an inhibitor of the Q cycle.

Plastocyanin:

Plastocyanin (PCy) serves as a mobile carrier between Cyt b6f to P700+

(Haehnel, 1984). PCy is a copper containing peripheral membrane protein

(10.5 kDa) which is located on the luminal side of the thylakoid membrane

(Katoh, 1997; Sigfridsson et al., 1997).

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Fig 5: Structure of cytochrome b6f complex

17

 

It accepts electrons from Cyt f and form a pool of electrons, from here it

can be passed to PS I (Drepper, 1996; Sigfridson, 1997). In some algae and

cyanobacteria the biosynthesis of plastocyanin is controlled by availability of

Cu in the growth medium. In the absence of Cu, these cells are able to

accumulate plastocyanin and instead synthesize a C-type cytochrome,

cytochrome C553, which is functionally interchangeable with plastocyanin.

Structural and functional aspects of PS I complex:

PS I is a membrane bound protein complex consists of four

multisubunit complexes of thylakoid membranes (Brettel and Liebl, 2001;

Jordan et al., 2001; Chitnis, 2001) which helps in the transfer of electrons from

water to NADP+. It is mainly situated in the non stacked, stromal lamellae

regions of the thylakoid membrane and functions of PCy- Fd oxidoreductase

(Chitnis and Nelson, 1991; Golbeck, 1992; Golbeck, 1994; Nishushtai et al.,

1996; Jordan et al., 2001). Cyanobacterial PS I can exist in photosynthetic

membrane in both trimeric and monomeric forms. The trimeric form is the

prominent oligomeric state varies with the environmental conditions such as

light intensity and nutrient supply and also shows differences between

individual species of cyanobacteria. The monomeric unit of PS I consists of 12

protein subunits to which 127 cofactors are non covalently bound (Fig 6). The

PS I complex is having the reaction center chlorophyll P700 which acts as

primary electron donor and it donates the electron to A0. A0 is special Chl a

that acts as primary electron acceptor, A1 a quinone intermediate electron

acceptor (Gantt et al., 2003) and Fx, FA and FB are iron-sulfur centers.

18

 

Fig 6: Structure of photosystem I complex

19

 

centers. PS I core complex is surrounded by the LHC I (Ben-shen et al., 2003:

Melkozernov and Blankenship, 2005; Kouril. et al., 2005; Veih and Buchel,

2007). This LHC I help in the harvesting of light and funneling its excited

energy in to the reaction center P 700 and A0 .The electrons then pass through

intermediate acceptor A1 and FX to terminal electron acceptor FB/ FA (Golbeck,

1987).

Polypeptides of PS I complex:

PS I complex contain 15-20 polypeptides, these polypeptides are

categorized in to three groups. 68-70 kDa polypeptides are associated with P700

(RC) (Golbeck, 1992). The iron-sulfur centers X, A1 and B (15-19 kDa),

which are electron transport components are associated with PS I (Golbeck,

1987; Malkin, 1987; Nelson, 1987; Golbeck, 1992). The iron sulfur centers FA

and FB are also associated with 8-9 kDa polypeptides (Lagoutte et al., 1984).

The FX is considered to be associated with the two core polypeptides (Golbeck

and Cornelius, 1986; Golbeck, 1992). In addition to the RC, the PS I complex

contain photosynthetic pigments and all electron carriers required to carry out

the electron transfer (Nechushtai et al., 1996).

P700, the primary electron donor of PS I:

P700 is the primary electron donor of PS I Kok (1957) observed the

reversible absorption decrease at 700 nm which was due to the photo

bleaching of the pigment present in the PS I and named it as P700. Its midpoint

20

 

potential is +130 mV. It has a light and chemically- induced differential

spectrum characteristic of Chl a and in oxidized state generates an ESR signal

at g = 2.0025, ΔH = 7.29 gauss and exhibits signal I, typical of an organic

radical (Beinert et al., 1962; Warden et al., 1974). The P700 is believed to be

dimeric in nature (Mathis and Rutherford, 1987; Andreasson and Vanngard,

1988).

Electron transport in photosystem I

(i) A0 and A1:

A0 and A1 are the primary and intermediate electron acceptor

components of PS I (Chitnis, 1996; Malkin, 1996; Jordan et al., 2001). Ao is a

special form of Chl a monomer with absorption maximum at 670 nm attached

to the RC of PS I (Ikegami and Ke, 1984; Malkin, 1996). A1 was found to be a

phylloquinone from analytical, optical and EPR studies. These electron

acceptors function in a sequential order.

(ii) Iron sulfur centers:

Iron sulfur centers (FX, FA and FB) are the PS I electron transport components at

the acceptor side (Malkin, 1996; Ishikawa et al., 1999; Grotjohann, and

Fromme, 2005). The component FX was identified by new EPR signal as

unusual iron sulfur center (Parrett et al., 1987). Iron sulfur centers accept

electrons from PS I complex (Chamarovsky and Cammack, 1982). A secondary

acceptor has been found in the form of ESR signals characteristic of 2Fe-2 S or

4Fe - 4 S centers is an 18 kDa protein (Lagoutte et al., 1984; Ciurli and

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Musiani, 2005). FA and FB interact closely with one another and transfers

electrons from X to A (Malkin, 1996; Lakshmi et al., 1999) (Fig 7). A charge

separation occurs in PS I complex, starts with the photo oxidation of pigments

P700 (Norris et al., 1971). Then the electron move through AO and A1 (Vitamin

Kl molecule) to the first iron sulfur center FX, from FX the electron is

transferred to two other iron sulfur clusters (Scheller et al., 1989) and finally

reaches Fd.

Ferredoxin and Ferredoxin-NADP+ reductase:

The electrons transfer between the PS I RC and NADP+ is mediated by

extrinsic iron sulfur protein Fd and the flavin containing proteins ferredoxin

NADP oxidoreductase (FNR). These are the final electron acceptors from PS I.

Under iron limiting conditions, a special type of flavin containing

protein present is flavodoxin (Tollin and Edmonson, 1980). It serves as mobile

electron carrier to shuttle the electrons from FA/FB to the site where ferredoxin,

NADP+reductase (FNR) is bound to the membrane (Forti and Grubas, 1985).

PS I RC and FNR are the two independent sites of Fd on the thylakoid

membrane (Merati and Zanetti, 1987). FNR is a 33-38 kDa iron sulfur

containing protein with FAD as its sole prosthetic group (Carrillo and Vallejos,

1987; Knaff, 1996). This protein is attached to the thylakoid membrane near

PS I clusters and ATP synthase complex.

22

 

Fig 7: Electron transport in photosystem I

23

 

ATP synthase complex:

The transport of electrons from PS II to PS I will liberate the protons

(H+) from stroma to the lumen of the thylakoids. The protons are also released

in to the lumen due to oxidation of H2O by PS II (Mills, 1996). This electro

chemical gradient is responsible for the synthesis of ATP from ADP and Pi.

ATP synthase Complex consists of the proton core CF0, which is embedded in

the thylakoid membrane and CF1 the extrinsic enzyme that catalyzes ATP

synthesis and hydrolysis (Fig 8). The molecular weight of CF1 is 320 kDa. It

consists of five subunits of molecular weight 53.4 kDa, 51.6 kDa, 36 kDa, 21.1

kDa and 14.7 kDa respectively (Boekema and Luken, 1996). This occurs in a

symmetrical ring of six alternating x and p subunits with a hole in α subunits.

The CF0 is oligomeric in nature and comprises four different protein subunits in

both green algae and higher plants. It is self assembled in the membrane bilayer

to form a proton conducting “core” as well as the site to which CF0 binds

(Richter and Mills, 1996; McCarty, 2000).

Thylakoid membrane lipids and their role:

The thylakiod membrane is unique in plant cell in having a high

proportion of glyceroglycolipids and a high proportion of phospholipids. The

glycerolipids are monogalactosyl diacylglycerol (MGDG) and digalactosyl

diacylglycerol (DGDG) and sulpholipids (SQDG) and they account for 40-

50%, 20-30% and 5-10%, respectively.

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Fig 8: Structure of ATP synthase complex

25

 

The phospholipid in the thylakoid membrane is phosphotidylglycerol

(PG), which accounts for 10-20% of the total lipids. The specific binding of

glycerolipids to protein complexes from the thylakoid membranes has been

well studied, specifically the association of SQDG and DGDG with the ATP

synthase (Pick et al., 1985) that of phospholipids, PG with Cyt b6f complex

(Doyle and Yu, 1985) and that of PG with LHCP complex (Tremolieres et al.,

1981). It has been shown that the activity of ATP synthase is stimulated by

MGDG that contains poly unsaturated fatty acids (Pick et al., 1987).

The Cyt b6f activity i.e. the plastoquinol/plastocyanin oxidoreductase

activity was stimulated by DGDG, PG, and PC, but not MGDG and SQDG

(Chain, 1985). Lipase treatment studies (Siegenthaler and Rawyler, 1986) and

catalytic hydrogenation studies (Horvath et al., 1987) indicated that lipid to

protein ratio and/or the extent of lipid unsaturation determine the fluidity which

is essential for thylakiod membrane stability and function (Gounaries et al.,

1983). PG is necessary for oligomeric organization of LHCP and its

monomeric form (Murphy, 1986). Lipids are also shown to be essential for a

stable charge separation between P680+ and QA (Akabori et al., 1988). Recently

Murata et al. (1990) reported that the presence of the phospholipids vary with

the nature of PS II complex preparation. One MGDG molecule, containing

high saturated fatty acids is associated with active PS II reaction centre

complex which performs only the charge separation. This is essential for the

proper confirmation of the PS II reaction centre complex. About ten lipid

molecules, including MGDG, DGDG and PG are associated with active PS II

core complex which perform both charge separation and O2 evolution

suggestion the participation of lipids in photosynthetic oxygen evolution.

26

 

Cyanobacterial photosynthesis:

Cyanobacteria are oxygenic photosynthetic prokaryotes whose

photosynthetic apparatus shows resemblance to those of higher plants. Plants

and cyanobacteria contain similar PS II and PS I reaction center complex (Fig

9). Phycobilisomes are major light harvesting complex proteins in PS II of

cyanobacteria and transfer the energy to photochemical reaction center to

perform photosynthesis. In addition to this, some of the cyanobacteria are able

to fix nitrogen. Hence they are known as ‘biofertilizers’.

Cyanobacterial photosynthetic pigments:

Chlorophyll a:

All cyanobacteria possess Chl a as the major pigment. It is believed that

Chl a occurs in vivo in several spectroscopic forms. Chl a 660, Chl a 670, Chl

a 680, Chl a 685, Chl a 690 and Chl a 700-720. The number indicates their red

absorption maximum of each of the spectral forms (Rabinowitch and

Govindjee, 1961; French, 1966). The strongly fluorescing short wavelength

forms of Chl a are mainly present in PS II. The weakly fluorescing long

wavelengths forms are mostly present in PS I. In cyanobacteria Chl b is absent.

Carotenoids and Xanthophylls:

Almost all cyanobacteria contain the yellow and orange pigments called

carotenoids and xanthophylls respectively, which act as accessory pigments in

photosynthesis. The action spectrum of photosynthesis demonstrates that light

energy absorbed by carotenoids is utilized with varying degrees of efficiency in

photosynthesis.

27

 

Fig 9: Organization of polypeptides in cyanobacterial thylakoid membrane

28

 

The light energy absorbed by the carotenoids is not used directly but

transferred to Chl a where it is efficiently used in the photosynthetic process

(Clayton, 1962; Cogdell and Gardiner, 1993; Wilson, 2006).

Phycobilins:

In cyanobacteria, the PS II contains only small fraction of Chl a. The

major light harvesting pigments are phycobilins. There are three types of

phycobilins e.g phycocyanobilin, phycoerythrobilin and phycourobilin. The

structures of these chromophores are shown in Fig.10. These are linear tetra

phyrrole rings which are attached to the cysteine amino acid of the apoprotein

by thioether linkages. In cyanobacteria only phycoerythrobilin and

phycocyanobilin are present. In red algae in addition to phycoerythrobilin, a

second chromophore group is present which is known as phycourobilin.

Phycobiliproteins-subunit composition and spectral characteristics:

Cyanobacteria, red algae and cryptomonads contains unique light

harvesting pigment proteins called phycobiliproteins (PBP) which are absent

in higher plants. Unlike higher plant light harvesting chlorophyll proteins,

these PBPs are packed in multimeric pigment protein complexes called

phycobilisomes (PBsomes) located on the stromal surface of the thylakiod

membrane. Several workers have reviewed the various aspects of PBsome

structure and function (Gantt, 1981; Cohen - Bazire and Bryant, 1982; Glazer,

1982; MacColl, 1982; Tandeau de marsac, 1983; Glazer, 1984; Glazer, 1985;

Zuber, 1985; Zilinskas and, Grossman et al., 1993; Sidler, 1994; Mullineaux,

2008).

29

 

Fig 10: Structures of protein-bound phycobilins

30

 

Types of biliproteins and their occurrence:

The major components of PBsomes are the bilin-containing proteins;

phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC). The last

two pigment proteins are universally present in all cyanobacteria and red algae

(Bryant et al., 1979; Gantt et al., 1979), while PE is a variable component and

its presence is regulated by the available quality of light (Bogorad, 1975;

Tandeau de Marsac, 1977; Bryant, 1982). In cyanobacteria another pigment

protein called phycoerythrocyanin (PEC) replaces the PE whose synthesis is

regulated only by light quantity not by quality (Bryant, 1982, Sarah, 2005).

These phycobiliproteins collectively absorb light in green, orange and red

region of the spectrum allowing these organisms, which contain them to carry

out photosynthesis.

Structure and spectral properties of the biliproteins:

All the biliproteins are composed of an apoprotein portion to which

linear tetrapyrrole structures are linked by cysteine thioether bonds (Brown et

al., 1979). The spectral properties and subunit composition of the individual

phycobiliproteins are listed in Table 3. The diversity of spectral properties of

PBPs results largely from the environment of chromophore conferred by the

apoprotein rather than the structural properties of the chromophore itself. In

addition to this, spectral properties of the biliproteins will be determined by

the participation of the specific polypeptides in the formation of higher

aggregate state (Lundell et al., 1981; Yamazaki et al., 1984).

31

 

Table: 3 Spectral properties and polypeptide composition of various

phycobiliproteins in cyanobacteria

Biliproteins Source

Absorption peak /

shoulders (nm)

Flurescence maxima

(nm)

Subunit composition

and possible

aggregation stages

Phycobilin chromophore

type and number per submit

C – PE Cyanobacteria 540,575 577 ( ) ( )3 6αβ αβ α β γ

R – PE Red algae 567,538,498 578 ( )6αβ γ 2

PEB 3

PEB -

b – PE Red algae 545,563 570 ( )nαβ 2

PEB

2 PEB & 1 PUB

1 PUB & 1 PUB

B – PE Red algae 545,563,498 575 ( )6αβ γ 2

PEB 3

PEB -

PEC Cyanobacteria 568,590 610 ( )3αβ 2

PCB 3

PEB

2 PUB & 2 PUB

C – PC Cyanobacteria 620 642 ( ) ( )3 6αβ αβ 2

PEB 2

PCB -

R – PC Red algae 617,555 636 ( )3αβ

1 PCB

& PEB

2 PEB -

APC Cyanobacteria Red algae 650,620 660 ( )3

αβ 1 PCB

1 PCB & 1 PCB

-

APC Cyanobacteria Red algae 654,610 680 ( )3

αβ γ 1 PCB

1 PCB -

APC B Cyanobacteria Red algae 671,618 680 ( ) ( )*

2αβ αβ 1

PCB 1

PCB1

PCB

32

 

The protein portion of the PBPs consists of two dissimilar polypeptides

designated as α, β which occur in 1: l ratio in all PBPs (Liu et al., 2005).

Additional polypeptides are also present B-PE and R-PE (Glazer, 1980). The

building block for PBPs is the monomer (αβ). It generally exists either as

trimer (αβ)3 or hexamer (αβ)6 which are common aggregation states. Amino

acids sequences for the α and β subunits of biliproteins from the

cyanobacterum Mastigocladus Iaminosus revealed that there is a 64%

homology between subunits of PC whereas it shows 26% homology with α

subunit of APC. The β subunit of PEC exhibits 67% homology with this in C-

PC and only 37% homology with the β subunit of APC (Frank et al., 1978;

Anderson and Toole, 1998). These results suggest that the degree of sequence

conservation strongly control the functional properties of these biliprotein

subunits.

The molecular architecture of cyanobacterial phycobilisomes:

The most commonly occurring structure called hemidiscoidal is

found in both red algae (Koller et al., 1979) and cyanobacteria (Bryant et al.,

1979). The model for this type of PBsomes is made of two distinct domains; a

core made up of in Synechococcus 6301; (Glazer et al., 1979; Anderson and

Toole, 1998) or three (all other cyanobacteria) cylindrical objects which

contain APC from which six rods made up of stacked discs. Other

phycobiliproteins extend in a hemidiscoidal array (Fig.11). The discs

proximal to core contain PC, whereas discs distal to the core contain PE.

33

 

Fig 11: Structure of phycobilisome in cyanobacteria

34

 

The structure and intactness of the PBsome will be maintained by certain

colourless polypeptides which are known as linker polypeptides in addition to

the phycobiliproteins (Tandeau de Marsac and Cohen Bazire, 1977; Glazer,

1984; Li et al., 2003; Liu et al., 2005).

Linker polypeptides - their role in structural organization:

The structure of PBsome is maintained both by hydrophobic

interactions between components in this biliprotein aggregate (Rigbiet et al.,

1980; Zilinskas, 1982) and by several linker polypeptides which are first

described by Tandeau de Marsac and Cohen- Bazire (1977). The linker

polypeptides have been divided into three groups depending on their

functions and molecular weights (Tandeau de Marsac and Cohen-Bazire,

1977). The group I polypeptides vary in the molecular weights depending on

the organism used and isolation procedure followed. These types of

polypeptides are involved in the attachment of PBsomes to the thylakoid

membrane. The group I polypeptides from three organisms: Synechococcus

6301 (Lundell et al., 1981) Prophyridium cruentum (Redlinger and Gantt,

1981) Nostoc sp (Ziiinskas, 1982) Spirulina platensis (Murthy and Mohanty,

1991) has molecular weights 75, 95 and 95 kDa respectively. It was also

shown that phycocyanobilin chromophore is presenting these polypeptides.

The structural properties of these pigments suggested that they act as terminal

35

 

acceptor of excitation energy transfer in the PBsome (Cohen-Bazire et al.,

1977; Dagen et al., 1986; Grossman et al., 1993). Group II polypeptides have

the molecular weights ranging from 30 to 70 kDa. It varies in different

organisms from two in Synechococcus 6301 (Yamanaka et al., 1978) to six in

Prophyridium cruentum (Redlinger and Gantt, 1981). The function of these

polypeptides is to maintain rod structures in both PE-PC rods. The number of

the group II polypeptides varies depending on the light under which the

organisms are grown (Gingrich et al., 1982; Zilinskas and Howell, 1983).

Glazer (1982) has suggested that group II polypeptides have dual functions of

linking' two trimers to form hexamers and joining separate hexamers to form

rods.

Group III polypeptides which are having molecular weights of 25 to 30

kDa (Tandeau de Marsac and Cohen-Bazire, 1977) are involved in attaching

rods to the APC core. They may also join two trimers of PC to form hexamer

which is directly linked to the APC core (Glazer, 1982). Reconstitution

studies in Nostoc sp provided the direct evidence for the involvement of group

III polypeptides to attach PC hexamers to APC core (Glick and Zilinskas,

1983). In addition to the above mentioned linker polypeptides (10 and 19

kDa) have been isolated from the PBsomes of Nostoc sp (Zilinskas and

Howell, 1983). They are most likely to be the core components of APC which

are isolated with APC.

36

 

Functional aspects of the Phycobilisomes:

Energy transfer

The molecular architecture of the PBsome is such that the excitation

energy absorbed by these phycobiliproteins is transferred to the PS II reaction

center with an efficiency of approximately 80-90%. In earlier studies to

deduce the sequence of the energy transfer, the controlled dissociation in

reduced ionic strength buffer was used. These studies indicated that there is a

stepwise energy transfer of excitation energy as shown in the following

sequence (Gantt, 1975; Gantt et al., 1976; Glazer, 1989).

Later Porter's group have applied picosecond time-resolved

spectroscopy to study the sequential energy transfer in higher plant Chl

antenna (Tredwell et al., 1978) as well as in PBsome containing organisms

(Porter et al., 1978). These studies allowed direct measurement and

confirmation of proposed sequential energy transfer. Picosecond (ps) time-

resolved energy transfer studies by Wendler et al., (1984) using a laser dye

are also in agreement with the findings of Porter's group supporting the fact

that the energy transfer occurs from PE to PC to APC. Since the initial studies

of Porter's group with red algae, These picosecond time-resolved

measurements have been extended to several related systems such as the

intact cells of several species of cyanobacteria (Brody et al., 1981; Brody et

al., 1981b; Mimuro et al., 1984; Yamazaki et al., 1984; Bruce et al., 1985;

Glazer et al., 1985; Mimuro et al., 1985), red algae (Porter et al., 1978;

Brody et al., 1981 b; Karukstis and Sauer, 1984; Mimuro et al., 1984; Bruce

37

 

et al., 1985), isolated PBsomes (Searle et al., 1978; Pelligrino et al., 1981;

Holzworth et al., 1982; Gillbro et al., 1983; Wendler et al., 1984; Glazer et

al., 1985) and PBsome components (Holzworth et al., 1983; Switalski and

Sauer, 1984; Dagen et al., 1986). The data obtained through the use of time-

resolved fluorescence and absorption spectroscopy supports an arrangement

of chromophores ordered within the PBsome such that homotransfer is

minimized and the flow of energy is polar, directed towards the PBsome

terminal emitter. This directional energy transfer in PBsome is possible by

several structural features 1). The biliproteins of each type contains both s and

f (sensitizing and fluorescing) chromophores (Dale and Teale, 1970). The f

chromophores of PE become sensitizers in the transfer of excitation energy to

PC in PE-PC heteroaggregates or PBsomes. Transfer is more rapid among

heteroaggregates than among homoaggregates (Yamazaki et al., 1984;

Mimuro et al., 1985). Glazer et al., (1985) suggested that rate limiting step in

PBsome is the transfer of excitation energy from one disc to new disc within

the PBsome rod; 2). The rod sub-structure of PBsome creates six separate

domains so that inter-rod transfer of energy is prohibited by distance

constraints; 3) Transfer within the rod is directional. Energy difference

between different rod elements and the core minimize reverse energy transfer

and allow the long wavelength absorbing core to serve as efficient traps. This

molecular architecture of PBsome described ensures that random walking is

minimized and they exists the directional energy transfer to the final emitter in

the core of the PBsome.

38

 

Environmental factors affect the energy transfer (phycocyanin to

chlorophyll a) in cyanobacteria:

The light energy absorbed by PBsome is transferred to the reaction

centre (RC) of PS II through the antenna chlorophylls (Clement-Metral and

Gantt, 1983; Fork and Mohanty, 1986). A variety of environment factors are

known to affect the efficiency of energy transfer from PC to Chl a by affecting

the pigment protein interaction i.e. heat treatment (Singhal et al., 1981),

nitrogen stress (Yamanaka and Glazer, 1980), low temperature (Schreiber,

1979) and mercury (heavy metal) stress (Fujimori, 1964; Pecci and Fujimori,

1967; Sacina et al., 2001).

Chromatic adaptation:

Variations in the pigmentation of cyanobacteria, that result from

different illumination conditions is known as complementary chromatic

adaptation (Bogorad, 1975; Tandeau de Marsac, 1977, 1983; Grossman et al.,

1993). Cells grown in red light appear to be blue green in colour due the

presence of only PC in their PBsome rods. Transfer of these cells to green or

cool white fluorescent light leads to the development of a brown pigmentation

indicating the presence of both PE and PC. Thus, PBsomes are helpful to the

organism to adapt to different qualities of light.

Photosynthetic and respiratory electron transport interaction in

cyanobacteria:

Cyanobacteria are photosynthetic prokaryotes which can release oxygen.

Their main difference with other algae is the lack of particular organelles such

39

 

as chloroplasts or mitochondria, but has different types of membranes. These

cyanobacteria contain Cyt- C553 in the place of PCy which not only takes

participation in photosynthetic electron transport but also acts as a good

electron source to cytochrome oxidase (Lockau, 1981) (Fig 12). Reconstitution

experiments of the complete photosynthetic and respiratory electron transport

chains of Nostoc muscorum provided evidence that Cyt C553 is involved in both

the pathways (Sturzl et al., 1982). Recently, location and interaction between

respiratory and photosynthetic electron transport was reviewed by Sandmann et

al., (1984).

Spectral characteristics of the oxygenic photosynthetic systems:

The spectral characteristics of the different photosynthetic systems in

vivo originate as a result of different interaction of the chromophore with

proteins and lipids (Thornber, 1975) and/or water and other chromophores

(Katz and Morris, 1973).

Spectral characteristics:

The absorption spectrum of intact cells of green algae, Chlorella or

chloroplasts of the higher plant at room temperature exhibits two main peaks at

680 nm and 440 nm and two shoulders at 650 nm and 480 nm (Goedheer,

1968). The absorption peak at 680 nm indicates the presence of Chl a and peak

at 480 nm indicates the absorption of carotenoids; while the shoulder at 650 nm

being to be an indicative of the presence of Chl b (Cho and Govindjee, 1970; Li

et al., 2004).

40

 

Fig 12: Photosynthetic and respiratory electron transport interaction in

cyanobacteria

41

 

In the absorption spectrum of cyanobacterium a special peak at 620 nm

is due to the presence of PC present in the PBsomes. The peak at 440 nm

indicates the presence of Chl a and Chl b is absent in cyanobacteria (Fork and

Mohanty, 1986; Li et al., 2003) (Table 4).

Fluorescence induction as an indicator of thylakoid membrane

alterations:

Fluorescence can be used as probe to understand the basic

photochemistry of photosystem and it can be used as a reliable tool to

understand stress induced alterations in the photosystems. Several

workers have used fluorescence for selecting tolerant and stress sensitive

species.

Fluorescence emission spectral characteristics:

The room temperature fluorescence emission spectrums of intact cells

or PBS exhibit various emission bands at different wavelengths depending on

the excitation wavelength. When we excite the intact Spirulina at 440 nm, a

main emission peak at 685 nm will be observed from Chl a. When we excite

the cells in the PC absorbing region at 540 nm, an emission peak is observed

mainly at 655 nm due to PC (Fork and Mohanty, 1986; Li et al., 2003; Li et

al., 2004), and also a hump is observed at 680-683 nm due to Chl a emission.

The fluorescence emitted from PS I is always weak at room temperature.

Intact cells or thylakoids at low temperature (77K) exhibit three emission

peaks.

42

 

Table 6: Spectral characteristics of photosynthetic pigments of thylakoid membranes.

Absorption Fluorescence Emission System Pigment

protein Peak

position (nm)

System Excitation wavelength

(nm)

Peak position (nm) Pigment protein

At room

temperatureAt low

temperature (77K)

Green algae chloroplast

Chl a

Chl b Carotenoids Chl a soret

band

680

650 480 440

Green algae chloroplast/

Cyanobacteria

440 685 685

695

735

Chl a (PS II)

Chl a (PS II)

Chl a (PS I)

Cyanobacteria Chl a PC

Carotenoids Chl a soret

band

680 630 480 440

Cyanobacteria 545 650 650 685 695 715

PC APC-B (PS II) Chl a (PS II) Chl a (PS I)

(Compiled from Singhal et al., 1981; Fork and Mohanty, 1986)

43

 

The emission peak at 685 nm and 695 nm are contributed by PS II, whereas

the peak at 715 nm is contributed by PS I and the peak at 650 nm is due to the

presence of PC (Goedheer, 1968; Fork and Mohanty, 1986).

Partial photochemical reactions:

Generally partial photochemical reactions are assayed by the help of artificial

exogenously added electron donors and electron acceptors and by the use of electron

transport inhibitors. Their sites of electron donation, acceptance and inhibition are

shown in the Fig 13. These exogenous donors and acceptors are used to evaluate the

photochemical activities of thylakiod membranes as well as photochemical reactions

catalyzed by PS I and PS II separately or together (Trebst, 1974; Izawa, 1980;

Kleczkowski, 1994). These partial reactions are valuable tools in assessing the

photochemical potential of chloroplast and intact cells. Hydroxylamine (Bennoun and

Jolit, 1969), hydrazobenzine (Haveman et al., 1972) ascorbate and manganese (Trebst,

1974) have however been used to unravel the path of electron transfer at oxidizing site

of photosystem II. To measure photosystem II activites of chloroplast, cyanobacteria,

the interception of electron transfer from photosystem II to photosystem I have been

made by using Hill oxidants. Various electron acceptors like FerricCyanide, 2, 6-di

chlorophenol indophenols (DCPIP) (hydrophilic acceptors from QB) (Lien and

Bannister, 1971) benzoquinone, phenylenediamines and other substituted

benzoquninones (Trebst, 1974; Velthuys, 1980) are being used as Hill oxidants while

44

 

Acceptors: a1: Silicomolybdic acid; a2: Phenylenediamine, p-Benzoquinone; 2,5-dimethyl p-benzoquinone, and 2,5-

dichloro-p-benzoquinone; a3 Methylviologen,Anthroquinone, Ferricyanide Donors: d1: Catechol; Ascorbate; H2O, Diphenylcarbazide, NH2OH; d2: Duroquinol; d3: Diaminodurene;

Dichlorophenol indophenol; Tetramethyl phenyl durene. All are reduced by ascorbate. Inhibitors: In1: NH2OH; In2: diuron; In3: Dibromothymoquinone; In4: KCN and HgCl2; In5: DSPD

Figure 9: Commonly used artificial electron acceptors, donors and inhibitors of electron transport chain (compiled by Trebst, 1974; Hauska, 1977; Izawa, 1980)

H2O→Z → P680 → Pheo → QA → QB → PQ → Cyt b6f → PCy → P700→X → NADP+

in1

↑↑ d1

↑↑ a1

↑↑ a2

↑↑ d2 ↑↑d3

In2 In3 In4 ↑↑a3 In5

45

 

photosystem I activity has been assayed using NADP (Vernon and Zaugy, 1960) and

auto oxidisable viologens (Mehler, 1951) as electron acceptor and reduced 2,6-

dichlorophenol indophenols (Vernon and Zaugg, 1960), phenylenediamine,

diaminobenzidine and duroquinol, as electron donor to photosystem. 2,6-

dichlorophenol is suggested to have two donor sites one close to P700 and other close

to plastoquinone (Gould, 1975). Inhibitors with specific site of action have been

identified ( Trebst et al ., 1974; Izawa, 1977, 1980 ; Jurisnic and Stemler, 1983) and

are being used to characterize the electron transport components under light varying

conditions (Murthy et al., 1995; Prakash et al., 1998; Haddy et al., 1999) 3-(3,4-

dichlorophenyl)-1,1-dimethyl urea (DCMU) is a quinone analogue and blocks the

electron transfer from QA to QB, thus separating photosystem II from photosystem I.

2,5-dibromo-3-methyl-6-isoproply-p-benzoquinone (DBMIB) is also a structural

analogue of plastoquinone and stops electron transfer from plastoquinone to

cytochome f (Trebst et al., 1974). Atrazine prevents the binding of QB to D1 protein

and thus causing inhibition at the level of QB (Juisnic and Stemler, 1983).

Phosphoadenosine diphosphate ribose, an analogue of NADP+ reductase. Further,

spectral characteristics of photosynthetic membranes are known to provide

information on structure and function of the membranes.

46

 

Review of the Literature:

Nitrogen stress induced alterations in primary reaction of photosynthesis:

The availability of inorganic nutrients influences and regulates the plant

growth and development. Depending upon their necessity to plant growth, they are

classified in to two types namely macronutrients which are required in higher

quantities like K, Ca, Mg and N and micro nutrients which are required in small

quantities like Cu, Mn, Fe and Co. Nitrogen is a quantitatively important bioelement,

which is incorporated into the biosphere through assimilation process carried out by

the microorganisms and plants. Different organisms can use numerous nitrogen

containing compounds as source of nitrogen. These include for instance inorganic

ions like nitrate or ammonium or simple organic compounds like urea, amino acids

etc. Additionally many bacteria and cyanobacteria are able to fix nitrogen. In a

variety of ecosystems the combined nitrogen supply limits growth and physiology of

cyanobacteria. The diazotropic strains of cyanobacteria are able to fix nitrogen;

thereby escaping from nitrogen depletion. In contrast, non-diazotropic cyanobacteria

respond to the lack of nitrogen source by a process called bleaching of

Photosynthetic pigments (Allen and Smith, 1969). This results in the change of

colour of cultures from blue-green to yellow, a process known as chlorosis (Lau et

al., 1977). Even chlorosis also occurs upon starvation for other nutrients, which

shows differences at cellular level depending on the nature of nutrient limitations

(Warner et al., 1986; Collier and Grossman, 1994). There is a suggestion that

47

 

nitrogen starvation induces chlorosis and maintains low level of photosynthesis

during nitrogen limitation (Sauer et al., 2001; Allen et al., 1990).

Of the different chlorotic reactions, that include by nitrogen starvation has

been most extensively studied. When cells were shifted to nitrogen-deprived medium

by filtration, a decline in photosynthetic O2 evolution was observed within few hours.

PS II activity decreased to undetectable values with in 120 h of nitrogen starvation.

But PS I activity declined much slowly and reached undetective value only after

350h. From this study it is clear that compared to Chl a, phycocyanin is a storage

protein i.e., phycobiliprotein in cyanobacteria, which seems to decrease in rapid

manner due to chlorosis. Hence compare to PS II, PS I seems to be resistant to

nitrogen starvation. Since PS II of cyanobacteria contain phycobiliproteins as light

harvesting complex (Duke et at., 1989), nitrogen stress can induce alterations in the

LHC of cyanobacteria.

The photosynthetic apparatus of cyanobacteria is similar to that of green plants

(Bryant, 1991), except that light harvested by the accessing pigments called

phycobiliproteins. They are nitrogen rich water-soluble multi protein complexes,

which are attached to cytoplasmic surface of thylakoid membranes (Grossman et al.,

1993a; Bryant, 1986). As mentioned above PBsomes can be readily degraded by

proteases under nitrogen-starved conditions. The pattern of degradation in

Synechococcus 6301 consists of two phases (Yamanaka and Glazer, 1980; Collier

and Grossman 1992). First the PBsomes lose the PC hexamers and linkers located

48

 

most distal to the core during trimming process which reduces 50% of the PBsome

size. But under nitrogen stress both trimming of rods and PBsome core degradation

also occurs in cyanobacteria (Duke et al., 1989; Grossman et al., 1993).

PBsome degradation requires energy, protein synthesis and unstable

proteolytic activity. A serine type protease capable of degrading PBPs as well as

other protein has been reported by several workers (Locke et al., 1988: Maldener et

al., 1991). A second type of proteolytic activity containing enzyme has been shown

namely phycocyaninase involved in the degradation of phycobiliproteins under

nitrogen deprivation (Baussiba and Richmond, 1980). Nitrogen starvation of

Synechococcus 6301 causes the decrease of PC and linker polypeptides like 75, 33,

and 30 kDa and affect the energy transfer from phycobilisomes to the P680, RC of PS

II. The second phase of response to nitrogen starvation is gradual loss of Chl a and in

third phase the cells become depigmented and reside in dormant state. From this state

they will be re- entering into growth within few days, by the addition of nitrogen

source. The material released by protein degradation may provide substances for the

synthesis of new polypeptides required for acclimation to new nitrogen source (Allen

and Smith, 1969). The degradation of phycobiliproteins was correlated with the

expression of nbl A cluster genes. Majority of the studies made by several workers on

the ultrastructure and morphology of Synechococcus 6301 and Agmendllum

quadruplicatum (Warner et al., 1986). Sauer et al., (1999) suggested the involvement

49

 

of glutamine synthase and NtcA in phycobiliprotein degradation and survival of

Synechococcus PCC 7942. Similar studies made the above workers regarding the

recovery from nitrogen starvation showed that glnN product glutamine synthase III

helps to recover from the prolonged nitrogen chlorosis is in Synechococcus 7942. In

cyanobacteria the status of nitrogen can be detected by measuring the intracellular 2-

oxoglutarate levels. Recently Sauer et al., (1999) studied the changes in the

physiological aspects of the cyanobacterium Oscillatoria willei. Carotenoids seems to

be sensitive to nitrogen stress when compare to the D1 protein of PS II in

cyanobacteria (Biswal, 1994). Alanine dehydrogenase activity is required for the

progression of PBsome degradation in nitrogen stress of Synechococcus sp. Changes

in the nitrogen source affect the excitation energy transfer in Phormidium laminosum.

Duke and Allen (1990) suggested that rubisco is very sensitive to nitrogen stress and

affects CO2 fixation capacity during photosynthesis. Up to now complete and critical

studies related to the affect of nitrogen stress on non diazotropic cyanobacteria is

scanty. Therefore we have made an attempt to study the effect of nitrogen depletion on

electron transport properties and energy transfer process by using cyanobacterial

system with the following aim and objectives.

Aim and objectives of present study

Photosynthesis is fundamental and essential process, which determines the

plant productivity. Higher plants and algae convert solar energy in to chemical

energy through this process. In addition to this molecular oxygen is also released as

50

 

an early event of photosynthesis. In this way, life on our planet is dependent on this

important biological process. In higher plant and green algae this entire processes

takes place in chloroplast where as in cyanobacteria the same process occurs in intact

cells. The cyanobacteria resemble higher plants in performing oxygenic

photosynthesis. In intact cell or in chloroplast thylakoid membranes are involved in

the conversion of solar energy in to chemical energy.

Reasons for selecting cyanobacterial systems:

Being oxygenic and prokaryotic photosynthesizes in nature, cyanobacteria

provide several advantages to study the mechanism of photosynthesis. The result

obtained from these studies can be easily comparable to that of higher plants, since

cyanobacteria are oxygenic autotraphs. Cyanobacteria have unique ability to adapt

themselves easily to the fluctuations in the environment conditions such as light,

salinity, metal toxicity and temperature. Thus cyanobacteria offer great promise to

study the altered photosynthetic functions under various stress conditions.

Synechococcus 6301 is an unicellular cyanobacterium which has been extensively

used for biochemical, biophysical and genetic manipulation studies. The effect of

nitrogen starvation on the abundance of pigment molecules in several cyanobacteria

has been well documented. This nitrogen starvation leads to decrease in the Chl and

phyciobilisome content and leads to dramatic change in colour from greenish blue to

yellowish green. This is known as bleaching or chlorosis. Phycobilisomes constitute

51

 

up to 70% of total cellular protein, which is progressively, rapidly and completely

degraded under nitrogen starvation. Due to this there will be alteration in

photosynthetic apparatus of PS II in cyanobacteria. By keeping the above points in

mind this cyanobacterium Synechococcus 6301 has been chosen as an experimental

model in the present investigation with the following objectives.

1. Characterization of nitrogen stress induced alterations on photosynthetic electron

transport properties in the intact cells as well as the spheroplasts from

Synechococcus 6301 cells.

2. Analysis of the nitrogen deprivation caused changes in the energy transfer process

of phycobilisomes and spectral properties of other pigment proteins in the above

cyanobacterium.

3. To study the effect of nitrogen starvation on the lipid and protein organization in

the thylakoid membranes of Synechococcus 6301 cells.