CHAPTER 1

INTRODUCTION

1.1 Introduction to photosynthetic systems

Life on earth depends on the Sun. Higher plants,

eukaryotic algae and cyanobacteria (blue green algae) chemically

sequester light energy through photosynthesis, a light driven

process in which co2 is fixed to yield carbohydrates.

The chemical free energy stored in these substrates and

the oxygen evolved by photosynthetic organisms is what drives

practically all life processes.

1.1.1 Chloroplasts

The site of oxygenic photosynthesis in eukaryotes (algae

and higher plants) is the chloroplast. A cell of higher plants

contains typically 20 to 40 chloroplasts. Electron micr0graph of

a thin section of chloroplast shows a double layered envelope

membrane. The inner membrane surrounds stroma containing soluble

enzymes, genetic material and thylakoid membranes. The thylakoid

is probably a single highly folded vesicle although in most

organisms it appears as stacks of disk like sacs called grana,

which are interconnected by unstacked stroma lamellae.

Photosynthesis occurs in two distinct phases.

1. The 1 ight reactions, which use light energy to generate

NADPH, H+ and ATP.

2. The dark reactions, actually light independent reactions,

which use NADPH and ATP to drive the synthesis of carbohydrate

from co 2 and H2o.

1

The light reactions take place in thylakoids that contain

the li9ht absorbing pigments (antenna molecules) and the sites

where the primary photochemical reaction occurs (reaction

centres). Reaction centres generate the set of re:dox compounds

that are necessary for the overall process of photosynthesis. In

oxygen evolving organisms, two 1 ight driven photochemical

reactions, occurring at photosystem II (PSII) and photosystem I

(PSI), operate in series and drive electrons from the electron

donor H2o to the final electron acceptor NADP+. Eventually, the

Calvin cycle uses NADPH and ATP as substrates and reduces co~. L

1.1. 2 Absorption of light and the fate of absorl~d photon

Each of the photosynthetic pigments residing in the

thylakoids has a characteristic absorption spectrum when studied

in isolation outside the chloroplast i.e. in vitro. Each pigment

also has a characteristic fluorescence spectrum. 'rhe various

chlorophylls are highly conjugated molecules and strongly absorb

visible light. In fact, the peak molar extinction coefficients of

the various chlorophylls of over 10- 15 M- 1• cm- 1 , an:~ among the

highest known for organic molecules yet. The relatively small

chemical differences among the various chlorophylls greatly

affect their absorption spectra. These spectral differences are

functionally significant and ensure absorption from almost entire

visible solar spectrum. Inside the chloroplast, howE~ver, Chl b

fluorescence is not detected, even when the actinic light us,ed

is of a wavelength known to be absorbed predominantly by Chl b.

Similarly, fluorescence is never observed from the carotenoid

pigment and to only a small extent by the phycobilins in

cyanobacteria. Only Chl a fluoresces naturally inside the

chloroplast. This surprising phenomenon was explained by Duysens

(Duysens, 1952) He showed that Chl b, carotenoids and the

phycobilins act as "antennas" to harvest light energy. Their

2

energy is transferred very efficiently to Chl a. Only Chl a is

actively involved in the subsequent reactions of photosynthesis.

Absorption of light sends a molecule to the excited state. An

electronically excited molecule can dissipate its excitation

energy in one of the following ways,

1.1.2.1 Internal conversion

It is a common mode of decay in which electronic energy is

converted to the kinetic energy of molecular motion, i.e., to

heat. This proce~s is completed in ~lo-11s. Chl molecules usually

relax only to their lowest excited states. Therefore, the

photosynthetically applicable excitation energy of chlorophyll

molecule that has absorbed a photon in its short wavelength band,

which corresponds to its second excited state, is no different

than if it had absorbed a photon in its less energetic long

wavelength band.

1.1.2.2 Fluorescence

In this process an electronically excited molecule decays

to its ground state by emitting a photon. Such a process requ~res

~10-8 s, so it occurs much more slowly than internal conversion.

A photon emitted as fluorescence generally has a longer

wavelength (lower energy) than that initially absorbed.

Fluorescence accounts for the dissipation of only 3% to 6% of the

light energy absorbed by plants.

1.1.2.3 Excitation transfer (resonance energy transfer)

In this process an excited molecule directly transfers its

excitation energy to nearby molecules by resonance or Forster

transfer to neighbouring molecules.

3

1.1.2.4 Photochemical conversion

If trapped at the reaction centres, absorbed energy drives

the primary photochemical reactions. The reactions can be shown

to take place in an order as shown in fig. 1.1 (Z scheme). Every

photochemica 1 reaction involves transfer of one electron, thus

creating one positive and one negative charge. The charge

separation leaves a hole in the form of p+ 680 . This ·positive

charge is neutralized by electron donation from secondary donor

Z. z+, which has now been identified as a tyrosine residue of

the D1 protein of the PSII reaction centres (Debus et al, 1988)

in turn gets electron by oxidation of water. The process of

charge separation is very rapid (K -10 11 -10 12 s- 1 1. For

efficient photochemistry, the separated charges must be

stabilized, i.e. the probability of back reaction must be

minimized. This is achieved by spatial separation of the charges.

This process not only stabilizes the charge separation, but also

brings the reaction centre of PSII to a open state for the second

photochemical event. To make the rate of electrons transport

optimal, the quanta should arrive at the reaction cent:re at a

rate as fast as the removal of charges from the previous

photochemical event. The intensity of light which plants are

normally exposed is not sufficient. Therefore, the plants have

evolved a mechanism by which the effective cross section of

reaction centre for light absorption is considerably increased.

This is achieved by light harvest by several hundred antenna

chlorophylls as stated earlier. All the molecules absorb quanta

of different energy and transfer to reaction centre with such a

high efficiency that more than 90% of absorbed light is utilized

by photosynthesis.

4

Fig. 1.1

·------------ ----------------------------------------------------------------

-I :

-C€

-cs

-c ~ co

~ ·' -02 §

I G " :?. 0 c: '-'

·( ' 2 :> IJJ-0 < . g

<'6 0 Q

Fig. 1.1

photosynthetic

lifetimes of

"' c " 8 '? c

" ::; 0

~ c: ~

" -

An updated version of the 'Z'- scheme of

electron transport in higher plants showing the

various electron transfer reactions (After

Govindjee and Eaton-Rye, 1986).

1.2

1.2.1

1.2.1.1

organization of photosynthetic systems

Structure and function of the photosynthetic

apparatus in higher plants Cthylakoid membranes)

Structure and

lipids

distribution of the thylakoid acyl

Thylakoid membranes are made up of approximately 60%

protein, 30% lipid and 10% chlorophyll (by weight). The

predominant lipids of thylakoids are the uncharged galactolipids,

monogalactosyldiacylglycerol (MGDG) and

digalactosyldiacylglycerol {DGDG). Together they constitute about

75% of the total thylakoid lipid {50% and 25% respectively) (Webb

and Green, 1991) . The remaining 25% is comprised mostly of the

anionic lipids sulfoquinovosyldiacyl glycerol (SQDG) and

phosphatidylglycerol {PG) (Allen and Gord, 1971). Thylakoid

membranes do not contain sterols. Whether or not thylakoid

cont~fn phosphatidyl choline (PC) is debatable. There is strong

evidence (Dorne et al, 1990) that the endogenous levels of PC as

shown by Siegenthaler et al ( 1989) were contaminants from the

chloroplast envelope membranes. The remainder includes a wide

variety of lipophilic pigments, including the chlorophyll a and

Chl b, xanthophylls, B-carotene and the quinones. With the

possible exception of the specific quinones, these pigments are

non-covalently bound to the polypeptides of the pigment-protein

complexes (Murphy, 1986a; Murphy, 198Gb). A remarkable feature

of thylakoid lipids is the presence of high proportion of

polyunsaturated acyl groups, in particular that of the 18:3·

linolenic acid. However, a higher unsaturation index does not

confer a high membrane fluidity, probably because the motion of

acyl chains of lipids in the thylakoid membrane is considerably

5

ceduced by the presence of intrinsic membrane proteins. There is

no evidence for any specific lipid association with peptides

apart from the interaction of ch lorophy 11 with Chl-prote in

complexes. All chlorophyll molecules are bound to protein rather

than being 'free' in the lipid bilayer of the thylakoid membrane

(Markwell et al, 1979). The thylakoid membranes contain five

supramolecular assemblies embedded in the lipid bilayers.

1.2.1.2

1.2.1.2.1

Structure

complexes

and function of electron transJX>rt

Photosystem II (PSIIJ complex

PSII can be defined as the part of oxygenic photosynthesis

which catalyzes the photoinduced transfer of electrons from

water to plastoquinone (PQ).

The organization of all the components involved in this

reaction is shown in a pictorial model (fig. 1.2) given by

Hansson and Wydrzynski (1990). The genes of the proteins, their

molecular weight (MW) , the location of their transcri?tion and

probable function are shown in table 1.1. Biochemical

characterization of PSII suggests that around 22 polypeptides are

associated with PSII (Masojidek et al, 1987). In the hypothetical

picture cf PSII given by Hansson and Wydrzynski ( 1990), PSII is

subdivided into four functional parts.

1.2.1.2.1.1 Reaction core: The reaction core contains the primary

reactants, PSII reaction centre Chl a (P680 ), oxyqEm evolving

and plastoquinone (PQ) reducing components. Dl and 02 polypeptide

of PSII exhibit a significant degree of sequence homology with

the L and M subunits of the reaction centres of photosynthetic

6

Fig. 1.2 A schematic representation of the functior

organization of photosystem II (PSII) according to Hansson a

Wydrzynski (1990). The definition of symbols are as follows:

the primary electron donor chlorophyll (Chl) (P680); HA, t

primary electron acceptor pheophytin (Pheo); H8 , a second Phe

QA and Q8 , the first and second quinone electron acceptor

respectively; Fe, a non-heme iron atom; Y 2 , the tyrosine, fir

electron donor to P; Y0 , a second tyrosine; M, the manganes

containing component involved in oxygen evolution; cyt b55 Cytochrome b 559 heterodimer; D1 and D2, Reaction core subunit

QSP, Quinone-shielding protein; AIP, Accessory intrins

proteins; EP 33, EP 23 and EP 16, Extrinsic proteins of the

Regulatory Cap; AEP, Accessory extrinsic proteins; CP ~7 and CP

43, Chl proteins of the Proximal Antenna; ACP II, Accessory Chl

proteins of the Distal Antenna and LHC II, the Chl ajb light­

harvesting complex.

Fig. 1.2

LUMEN

Approximate Location Apparent Mo-

Gene (Eukaryotes) Product lecular Mass

psbA chloroplast D1 (PS II-A) 32,000 psbB chloroplast CP47 (PS II-B) 47,000 psbC chloroplast CP43 (PS II-C) 43,000 psbD chloroplast D2 (PS II-D) 32,000 psb£ chloroplast cyt b-559 (PS 11-E) 9,000 psbF chloroplast cyt b-559 (PS II-F) 4,000 psbH chloroplast "10,000 M, phosphoprotein" (PS II-H) 10,000 psbl chloroplast reaction center component (PS II-I) 4,800 psbf chloroplast n.d. ("PS 11-J") n.d. psbK chloroplast PS Il-K 2,400 psbL chloroplast PS Il-L 5,000 IISb!v: chloroplast PS 11-M 4,700 IISbN chloroplast PS II-N 4,700 IISbO nuclear MSP; PS 11-0 33,000 psbP nuclear 24,000-M, protein (OEE2; PS 11-P) 24,000 /ISbQ nuclear 18,000-M, protein (0EE3; PS 11-Q) 18,000 /ISbR nuclear 10,000-M.-protein (PS 11-R) 10,000 cab nuclear (family) LHC lis 21,000-29,000

Table 1.1 PSII genes and their products as listed by Erickson

and Rochaix (1992).

bacteria (Rochaix et al, 1984; Youvan et al, 1984). This has led

to speculation that 01 and 02 constitute the reaction centre in

PSII of higher organisms. Isolation of a minimal unit of PSII

which can do photochemistry and contains 01, 02 and 10 KOa (Nanba

and Satoh, 1987; Okamura et al, 1987) subunit of Cyt b559 has

supported this speculation. cyt b559 is also associated with the

reaction centre. The molecular structure of Cyt b559 was recently

established to be an inter molecular heme cross-linker dimer of

14 KOa (Cramer et al, 1986), which could be either a homodimer or

heterodimer (Tae et al, 1988). The stoichiometry of Cyt b599 and

P680 a stripped 01-02-Cyt b559 reaction centre particle is 1. 3

(Nanba and Satoh, 1987)~ suggesting that the stoichiometry could

be one Cyt b559 heme per PS II centre (Barber et al, 1987;

Gounaris et al, ·1987). There is a lot of ambiguity regarding the

functional role of Cyt b559 , which led researchers to propose

following hypothesis for its function,

1. An electron transfer intermediate on the oxidizing side of

PSII (Knaff and Arnon, 1969).

2. In a bypass or cyclic electron

(Ben-Hayyim, 1974); Arnon and Tang,

(1990) proposed that the primary role

transfer route around PSII

1988) . Canaani and Havaux

of Cyt b 559 in vivo is to

direct excess photons from linear to a cyclic electron flow at

high light intensities for protection of the 01 and 02 proteins

against photodamage. In addition to this, cyt b559 is suggested

to have a role in the deactivation of the powerful oxidant z+ in

the dark, also (Canaani and Havaux, 1990).

3. Linking the two photosystems (Ortega et al, 1989) in non-

cyclic electron transfer.

1.2.1.2.1.2 Regulatory cap: Regulatory cap of PSII contains a set

of three proteins having molecular weight of 33, 23 and 16 KDa.

All the three proteins are extrinsically bound to the luminal

surface of the thylakoid membrane. In addition to these proteins

regulatory cap may contain another extrinsic protein of 5 KDa

7

(Ljungberg et al, 1986}. The regulatory cap proteins have been

assigned a role in regulating the ionic requirement (i.e. Cl and

ca++) of o2 evolution and in controlling the exchange of

reactants with the manganese (Mn) cluster. One of the important

component associated with regulatory cap is Mn clusters. The Mn

stoichiometry has been determined by various external extraction

procedures (Yocum et al, 1981}. It is generally agreed that

stoichiometrically, four Mn ions are associated with each PSII.

'Despite extensive studies, the exact location and functional

operation of the Mn is still under considerable debate (Hansson

and Wydrzynski, 1990). Valance state changes of the Mn ions are

shown to be associated with the S state transitions. Mn ions are

believ1~d to be located within a restricted compartment toward~.

the luminal side of the membrane.

1. 2. 1. 2. 1. 3 Proximal antenna: The proximal antenna of PSII

comprises two pigment-protein complexes CP47 and CP43 (Green,.

1988). The polypeptides of these complexes have molecular weight

of 4.5-51 and 40-45 kDa, respectively. Each of these proteins

binds 20-25 Chl a and about 5 molecules of 8-carotene, but no Chl _

b or Pheo. The proximal antenna is apparently tightly coupled to

the reaction core. The CP4 7 and CP4 3 proteins are always

present in oxygen evolving preparations of PSII from higher

plants. Studies show that electron transfer from exogenous donors

to QA can take place in the absence of CP47 and CP43 (Yamaguchi

et al, 1980) but that the ability to reduce QA is lost when both

CP43 and CP47 are removed (Akabori, et al 1988}. Inactivation of

the genes for CP47 or CP43 by site directed mutagenesis prevents

or reduces, respectively, the assembly of reaction core (Vermaas

et al, 1988). Therefore, the earlier belief that these complexes

may directly bind the reaction centre and primary acceptor of

PSII (Nakatani et al, 1984; Inoue, 1983; de Vitry et al, 1984} or

have a role in the regulation of early photochemical events can

not be totally excluded.

8

1.2.1.2.1.4 Distal antenna: The distal antenna of PSII in higher

plants is light harvesting chlorophyll protein complex (LHCII).

The smallest, resolvable natural unit of LHCII is a dimer

consisting of two polypeptides with approximate molecular weights

of 25 and 27 kDa. LHCII has a Chl ajb ratio of 1.2 ± 0.2 and

contains xanthophyll as the major carotenoid. About 30% of LHCII

can be phosphorylated by a membrane bound kinase. The extent of

phosphorylation is regulated by the levels of reduced PQ.

Following phosphorylation, LHCII migrates laterally from

appressed to nonappressed regions of thylakoids, where it is

proposed to enhance the light harvesting capacity of PSI (Larsson

et al, 1987a and b; Bassi et al, 1988). LHCII is also shown to

have a role in cation dependent stacking of thylakoid membranes

to form grana (Mullet and Arntzen, 1980; Steinback et al, 1982).

PSII distal antenna also contains Chl protein complexes which are

not a part of LHCII. Among these components are CP 29 (Green,

1988), CP 26 and CP 24 (Dunahay and Staehlin, 1986; Bassi et al,

1987). The Chl ajb ratio of these complexes vary from 2 to 3.

None of these proteins are phosphorylated. According to Hansson

and Wydrzynski ( 1990) , these compl"exes may be closer to the

proximal antenna and the reaction core and may serve as 'linker'

or 'anchor' for the LHCII proteins. One suggestion has been that

these Chl proteins may participate in the dissipation of excess

excitation energy arriving at the reaction core to counter

photoinhibition (Bassi et al, 1987).

1.2.1.2.2 Heterogeneity in PSII

The notion of functionally, and possibly structurally,

distinct populations of PSII centres within the thylakoid

membranes is well established for long. The existence of two

types of primary acceptor, Q, (Q1 and Q2) was suggested on the

basis of differences in the fluorescence yield after single

9

turnover light flashes (Joliet and Joliet, 1977; 1979; 1981;

1983).

On redox considerations, QA has been shown to exist: in two

forms; low potential (QL, - 250mv) and high potential (QH, 0 mv)

(Cramer and Butler, 1969; Horton and Croze, 1979). Since the QL

and QH are believed to be associated with different reaction

centre proteins, two forms of PSII one with QL and another with

QH are expected in vivo (Vermaas and Govindjee, 1981; DinE~r and

Delosme, 1983; van Gorkom, 1985; Govindjee, 1990).

Measurements of the ability of spinach chloroplasts to reduce PQ

indicate that there are about 1. 7 m mol of active PSII centres

per mol of Chl (Graan and Ort, 1984). Measurement of the ability

of chloroplasts to oxidize water in the presence of DMQ yielded

similar results (Whitmarsh and Ort, 1984). However, estimates of

the reduction of the primary quinone electron acceptor QA (Melis

and Anderson, 1983; McCauley and Melis, 1986) suggestE~d the

number of PSII centres about 3 m mol per mol Chl. The <estimate

of active centres per mol Chl was increased to a similar number

( 3 m mol), when certain halogenated benzoquinones (e.g. DCBQ)

were used as electron acceptors (Graan and ort, 1986).

Quantitative estimates of the oxidation of the electron donor Z

also supports these results (Babcock et al, 1983). This

difference in the quantities of PSII per mol Chl as revealed by

different techniques was explained on the basis of a population

of PSII centres deficient in sustained water oxidation capacity.

The evidence regarding the heterogeneity in PSII has also

come from the interpretation of Chl a fluorescence induction in

the thylakoids. Melis and Homann (1975) analyzed the induction of

Chl a fluorescence in DCMU poisoned chloroplasts (DCMU inhibits

electron transfer from the primary acceptor QA to QB) 1n terms of

two distinct p)1ases, an initial sigmoidal phase (o<J and a slower

exponential phase (8). These two phases are assumed to be because

of two different type of PSII centres in thylakoid membranes

occurring in vivo which are designated as PSIIac and PSII 8

10

centres. The analysis method of Melis (Melis and Homann, 1975;

1976) involved calculation of the growth of complementary area,

defined by the induction curve and the line of the maximum

fluorescence level (FM), with time and the semilogarithmic plot

of such an analysis. However, since it is difficult to determine

asymptotic level of FM , it is always difficult to determine the

precise value of complementary area. Assuming that the last part

of the fluorescence induction curve is exponential, Hsu et al

( 1989) determined FM using mathematical analysis and curve

fitting. They analyzed the Chl a fluorescence induction in DCMU

poisoned chloroplasts into three phases, a major rapid sigmoidal

~phase followed by two minor slower exponential phases B and t.

The kinetic analysis both in the case of Melis and Homann (1975;

1976) and Hsu et al (1989) showed that rate constant of the oC

phase is light intensity dependent and is affected by presence of Mg++.

Chylla et al (1987) reported that certain PSII centers are

slow in the reduction of Q8 and take around 1.7 s before they are

ready for a second photochemical act. The authors designated them

as slow turning over PSII centres (inactive PSII) centres in ,,

comparison· to normal PSII units (active). They further showed

that around 44 % of PSII centres exist in inactive state (Chylla

et al, 1987; Chylla and Whitmarsh, 1989). Cao and Govindj ee

(1989) suggested that Fo to F1 rise seen in the Chl a

fluorescence induction occurs due to inactive centres. Hsu et al

(1989) studied the kinetics of this rise (F0 to F1 rise). They

found this rise to be monoexponential. Kinetic parameters show

that rate constant and response of this phase towards Mg++

depletion is very similar to oC phase in DCMU poisoned

chloroplasts (F0 to F1 rise was studied in absence of DCMU).

Depending on these observations, these authors suggested that F0 ·

to F1 rise is due to PSII ~ centres, a pool of which are inactive

in PQ reduction. In their analysis they found out that

complementary area of growth phase due to F0 to F1 rise is 10% to

11

the total area occupied by oe. phase. This suggests that only 10%

out of total pool of PSII centres are inactive which is contrary

to Chylla and Whitmarsh (1987) who suggested this number to be

40%. Hsu and Lee (1991) tried to explain the reason for this

discrepancy. They suggested that a large portion of the inactive

PSII centres are not detectable in fluorescence rise from F0 to

FI. They also mention that probably these left over centres,

which escape detection in their experiments represent inactive

PSII centres with low fluorescence yield. They further suggested

that the population of the inactive PSII centres is likely to be

heterogeneous. So, methods qther than fluorescence induction are

required to clarify this suggestion. In a recent study, Chylla

and Whitmarsh (1990) using light saturation curves of flash

induced absorbance change at 518 nm have shown that inactive PSII

centres require twice as many photons as the active: reaction

centres to achieve the same yield. They suggested that the

inactive PSII centres have a smaller effective absorption cross

section, due to a smaller antenna size, or due to a lower quantum

yield for photochemistry.

These studies thus suggest that PSII population in

thylakoids is heterogenous. The heterogeneity has been shown to

exist in terms of antenna size (oe, B heterogeneity; Melis and

Homann, 1975; Anderson and Melis, 1983; Melis, 1985; Guenther et

al, 1988). In addition to~' B heterogeneity, PSII centres also

display heterogeneity on the reducing side of QA with respect to

electron flow to the plastoquinone pool. several investigators

have shown that a number of PSII centres, though photochemically

competent are unable to transfer electrons from QA to QB

(Thielen and van Gorkom, 1981; Lavergne, 1982; Melis, 1985; Graan

and Ort, 1986; Greene et al, 1988; Guenther et al, 1988).

In Lavergne's nomenclature ( 1982) these centres are termed as

PSII Q8-non reducing centres.

12

-

1.2.1.2.3 Plastoquinone ~

Plastoquinone acts as a mobile electron carrier between

QB (PSII) and Cyt b 6/f

Barber, 1984; Whitmarsh,

molecule and consists of

and an isoprenoid side

complex (Anderson,

1986). It is a

a quinone ring with

chain attached to

1980; Millner and

relatively small

two methyl groups

it. The size of

plastoquinone pool has been estimated to be between i (in the

cyanobacterium Anabaena var.:i,.abilis and 40 (in chloroplasts and

Anacystis nidulans) molecules per P700 (Hauska and Hurt, 1982).

Thus, this component provides a large pool of electron acceptors

near PSII. Each quinone molecule transfers two electrons during

a reduction-oxidation reaction, which must be accompanied by the

uptake of two H+ ions (Jagendorf, 1977). The flux of electrons

through the plastoquinone pool seems to be the rate-limiting step

in photosynthetic electron transport. Oxidation of PQH2 by the

Cyt b 6/f complex is linked to translocation of protons from the

stromal phase into the lumen, generating a proton gradient across

the thylakoid membranes (Velthuys, 1978; Whitmarsh, 1986).

Plastoquinone also plays a role in the regulation of

excitation energy distribution between PS II and PSI under

changing environmental conditions. Reduced state of

plastoquinone is reported to activate a kinase (Bennett et al,

1980; Howarth et al,. 1982; Allen, 1992) , which phosphorylates

LHCII. Phosphorylated pool of LHCII molecules decouple themselves

energetically (functionally) and migrate to non-appressed

membrane region where they are known to associate with the PSI

(Fork and Satoh, 1986; Bennett, 1991; Allen, 1992).

1.2.1.2.4

The cytochrome b 6-f complex mediates electron flow between

the plastoquinone generated by PSII activity and the diffusible

plastocyanin carrier. In this way, it links the electron

13

transport chains of PSII and PSI. The complex is made up of four

polypeptides of M.W. higher than 15 KDa; a 19-23 KDa (P'et c gene

product), which binds one high potential (2Fe-2S)' Rieske' group

(Widger and Cramer, 1991), a Cyt f (pet A gene product, =32 KDa),

Cyt b 6 (pet B gene product,=25 KDa), subunit IV (pet D gene

product, -18 KDa). In addition to this, two to three small

polypeptides of molecular weight less than 6 KDa have also been

reported as a part of purified Cyt b 6 /f complex (W'idger and

Cramer, 1991). This complex transports protons as: well as

electrons from the outside to the inside of the thylakoid

membrane. It has been suggested that much of the electrochemical

proton gradient needed for the synthesis of ATP is qenerated

because of electron transport activity of Cyt b 6-f complex. This

complex is shown to be uniformly distributed in the grana and

stromal membranes (Cox and Andersson, 1981; Anderson, 1982;

Peters et al, 1983) . However, a model has also been proposed

according to which this complex is placed in the region around

the grana. (Barber, 1983; Ghirardi and Melis, 1984; Webber et al,

1988).

1.2.1.2.5 Photosystem L (PSIJ complex

PSI acts as a plastocyanin ferredoxin photoxidoreductase,

which transfers the electron from plastocyanin (located on the

luminal side of the membrane) through a series of five or six

electron carriers to ferredoxin (towards the stromal side of the

membrane). Figure 1.3 represents the diagrammatic picture of PSI

as suggested by Golbeck (1992). The genes of proteins associated,

their molecular weight (MW) , the location of their transcription

and probable function are shown in table 1. 2. PSI is a

multiprotein complex and can be divided into two structu1~al units

based on functional considerations.

14

Fig. 1.3

Fig. 1·3

STI{0;\1A

I

LUMEN

!'sa:\ 1\3.2-ki>a

The architecture

proposed by Golbeck ( 1992). The

l'saB S2.5-kDa

l'saF I'I;L\IOC)'allin Dock in~-:

for the

symbols

PSI

are

1.2 and also in section 1.2.1.2.5.

PsaL

reactio.n centre as

described in table

Protein Site Residues· Mass Cofactors Function 'Pr0J.H:>rt ie:' (kDa)•

Hydrophobic PSI core proteins

PsaA c 750 83 Antenn:1e chloro--100Chla

phyll and photo· 12- 15 {1-Carotene

chemical charge s;ep-2 Ph,·Iloquinone

a ration (Vit K 1)

1 ]-lfe-4S]

PsaB c 734 82 Antennae and pho-

toprotN'tion

Char[!<' >'tahi!tzat ion

Ch.arg,· ;tahiliz:nion

PsaF N 154 17.3 None Pla;otocyantn· dock-

ing protein

p,aG" N 98 10.8 !'one

Psal c 36 Tran:-:.nH·mhrant' )

h<>!tx

P<aJ c 44 5 Tran~mt.•mhr;1nt· ,.

helix

P<aK N !;I 1:'.4 !\on~ lntrin:::.ic mt·n:br;tllt'

protein

PsaL N 169 18 None lntrin~ir mt·n:hrant.·

prot(~in

Psa~l· c 30 32 :1.5 ~one lntrin~ic nwmhrane

protein

P,-aN· C? -50-{;() Vi None Intrinsic nH'mhr;1nt'

protein

PsaO•' N -90 9.0 None lntrin~ic nwmhrttne

prote1n

H~·drophilic PSI peripheral prot .. ins

P,;aC c 81 8.9 2]4Fe-4S] Terminal el<·ctron

acct-ptors

PsaD N 162 1/.9 None Ferreooxin-duck in~ protC'in·p,,.c l11nd-

ing

PsaE N 91 9.7 !'lone Cyclic ele\tron

flow .'ft·rn·doxin-

dockinr:?

PsaH" N 95 10.2 None LHC I Jink(•r pro-

tein?

Hydrophobic Chi a/b binding proteins (eukaryotes only)

Cab-6Ai6B ~ 201-2 ~2 Chi a. Chi b. Xan- Ant~nna: LHCI-~:lO:

thophylls T,p.-1 C-'.1:!' Cab-7 ~ 209-10 24.9 Chi a. Chi b. Xan- Antt·nna: LHCI-6i<O:

thophylls T,·p~ II CAB'

Cab-8 s 2-l0-241 26.1 Chi a. Chi b. Xan- Antenna: LHCI-6:'0:

thophylls • Type Ill CAB' Cab-11/12 ~ 200 0•? Chi a. Chi b, Xan- Antt-nna: LHCI ~:lO:

thophylls Typ<· l\" C.-\ B'

Table 1. 2 PSI genes and their products (Golbeck, 1992).

1. 2. 1. 2. 5. 1 PSI core complex Two high molecular weight Chl

binding subunit proteins of about 83 KDa and 82 KDa (psa A and

psa B gene products) comprise the PSI core complex. The electron

transfer components associated with the PSI core complex include,

a) A primary electron donor P700 , possibly a dimer of Chl a

molecules (Philipson et al, 1972; Bengis and Nelson, 1977; Mullet

et al, 1980).

b) A0 , a Chl a monomer which acts as primary electron acceptor

(Shuvalov et al, 1986).

c) A1 , a quinone (probably phylloquinone) (Biggins and Mathis,

1988)

d) Three non-sulfur centres, Fx, F8 , and FA, which are probably

associated with two low molecular weight proteins ( 15 and 18-19

KDa, Bonnergea et al, 1985), or 12 KDa polypeptide (Lagoutte et

al, 1984) of the PSI complex.

Recently, Krauss et al, ( 1993). determined the three­

dimensional structure of PSI trimers isolated from Synechococcus

sp. at 6A resolution which has provided further information

regarding the arrangement of 4Fe-4S clusters, oC-helices of

reaction centre protein and Chl a in reaction centre.

1. 2. 1. 2. 5. 2 Chl a/b light harvesting complex (LHCI) : This

constitutes a peripheral antenna of PSI reaction centre. LHCI

contains three polypeptides with apparent molecular weights of

17-26 kDa (Haworth et al, 1983) and one of about 10 kDa. The Chl

ajb ratio of LHCI is 3.5 to 4.0. The major function of LHCI is to

hold pigment molecules in the right orientation for efficient

light harvesting and subsequent transfer of excitation energy to

the reaction centre. However, Ikegami and Katoh (1991) have shown

that Chl a of antenna has a structural role in stabilizing the

functional conformation of P700 chlorophyll -protein complexes.

The LHCI contains a long wavelength absorbing Chl a species

(Chl 705 ) which is responsible for the 735 fluorescence band of

PSI at 77K (Mullet et al, 1980).

15

1.2.1.2.6 Heterogeneity in.PSI

Rurainski (1981), using isolated broken chloroplasts with

ferredoxin and NADP as substrates, showed that the total pool of

P700 ban be divided into at least two smaller pools. These pools

differ in their relaxation times and magnitudes and are proposed

to have different functions in photosynthetic electron transport.

This showed a heterogeneity in the reaction centre of PSI. In

contrast to PSII, the antenna size of PSI has previously been

thought to b~ fairly homogeneous. However, recent studies have

shown that there exists a heterogeneity in PSI as well with

respect to antenna size. (Andereasson et al, 1988) . · Under green

light illumination, the antenna size of PSI of the oC vesicles

(PSioe) was 40% larger than that of PSI of the B (PSI8 ) vesicles

(Svensson et al, 1991) . The oe and .B vesicle were suggested to

originate from grana and stroma lamellae, respectively

(Andereasson et al, 1991). A heterogeneity was also reported in

their localization; PSioC being located in the periphery of grana

and PS t 8 in the stroma lamellae (Svensson et al, 1991).

1.2.1.2.7 Plastocyanin

Plastocyanin, a copper containing peripheral membrane

protein is localized on the luminal ~urface of thylakoids (Katoh,

1977; Haehnel et al, 1981). The monomeric form of this protein

has a molecular weight of about 10.5 KDa (Katoh, 1977; Boger,.

1978). It is a flattened barrel like structure with dimensions of

4. Ox3. 2x2. 8 nm (Coleman et al, 197 8) . The X-ray structure of

plastocyanin shows that the Cu atom is coordinated with distorted

tetrahedral geometry by one Cys, one Met and two His residues.

Plastocyanin mediates the electron transport between Cyt b6-f

complex and PSI.

16

1.2.1.2.8 ATPase-ATP synthase

This complex comprises intrinsic protonopore CF0 , and an

extrinsic enzyme CF1 , \\·~ i ~h catalyzes ATP synthesis and

hydrolysis. CF 1 also serves as a plus to prevent uncontrolled

leakage of protons through CF0 . Chloroplast '-'l'P synthase is

located in the unstacked portions of the thylakoid memb.c;;;:,,c_:_ Cr'0 has four sub- units, I (18-19 KDa), II (16 KDa), III (8 KDa) and

IV (27 KDa). (Hudson and Mason, 1988). All subunits are coded by

the chloroplast genome exc~pt subunit II, which is coded by

nuclear genome ( Ne'J,:Son et al, 1980; Westhoff et al, 1985) .

Subunit III forms hexamer, which appears as rods in the eiectron

microscope. (Fromme et al, 1987). It also forms a proton channel

and contains the binding sites of DCCD. ( Sigrist-Nelson et al,

1978; Sebald and Hoppe, 1981). It is suggested that proton pore

made by subunit III is stabilized by the interaction of one of

the alpha-helix cf the subunit II polypeptides. The carboxy

tcr~~n~~ ~f subunit I, which protrudes into the stroma, provides

an anchor for CF1 attachment (Otto and Berzbom, 1989). Thus CF0 serves a dual purpose; very rapid and specific proton conductance

through the membrane and CF1 binding. CF1 is comprised of five

subunits, designated oe, B, i , b and € , present in the ratio of

3:3:1:1:1 ·(Stratmann and Bickel-Sand Kotter, 1984). The

~subunit has a molecular mass of 55-56 kDa. This subunit contains

adenylate binding site, which may occur at some part of the~/B

interface. B subunit has a molecular weight of 52-54 kDa. Both

~and B are encoded in chloroplasts. The isubunit has a molecular

mass of about 3 7 KDa and encoded by nuclear genome. The most

clearly defined function of t is in regulation of ATP synthesis

activity. This is achieved by oxidation and reduction of Cys

residues. The b subunit ranges from 21 to 25 kDa and is nuclear

genome encoded. The function of the <£ subunit has been only

partly elucidated. Studies show that this subunit is required to

block the leakage of protons. Thus, it lS required for

17

photophosphorylation. However, removal of this subunit has no or

little effect on ATPase activity of CF1 . The € subunit, a 14-

kDa polypeptide, is a chloroplast- _encoded protein. This subunit . ~-· ......

has been attributed a :.o;.e . in the regulation of activity of

ATPase. Remov:'· ' c-,: S subunit causes a marked increase in the

A.TPase · a.::ti. "~'t.Y. The CF 1 forms a more or less globular head,

which is bound to the CF0 by weak electrostatic fofces (Sigrist­

Nelson et al, 1978) on the thylakoid membrane surface.

1. 3. Chl a fluorescence analysis

Chlorophyll in organic solvents emits fluorescence with a

quantum yield of around 30-40 % (Latimer et al,

since the yield of photochemistry is very high

1956). However,

in vivo (around

90%}, the yield of fluorescence is only around 3-4% (Weber,

1960}. The light reactions saturate at high light intensities.

When added, the inhibitors of photosynthetic electron transport

block the flow of electrons at specific sites. Under both of

these conditions, photochemical yield decreases and the decreased

yield is accompanied by a complementary increase in the

fluorescence yield. As stated earlier Duysens and Sweer {1963}

showed that yield of fluorescence is regulated by the redox state

of PSII acceptor, Q (quencher of fluorescence in its oxidized

state}. The fluorescence yield and concentration of Q are not

linearly related. This is reflected in a sigmoidal rise of Chl a

fluorescence in presence of DCMU which blocks the flow of

electrons from QA to Q8 . The sigmoidicity arises as a result of

migration of excitation energy between PSII units (Joliot and

Joliot, 1964).

The relationship of Chl a fluorescence emission and redox

state of Q in vivo has been extensively studied and both the

kinetics and steady state measurements of Chl a fluorescence

18

have been used to get information regarding photochemical events

and other reactions.

The rate of fluorescence emission , F, is given by

(1.1}

Absorbed light

KF, Rate constant of fluorescence

K1 , Sum of rate constants of all competing reactions

that result in the return of the Chl molecule to ground state.

The most important of these deactivation rates are,

photochemical reaction (Kp), thermal deactivation (K0 ) and

excitation energy transfer to non-fluorescent pigments (PSI)

(KT) ' The fluorescence yield may, therefore, be expressed as,

Yield of photochemical conversion of energy is maximum

when KP>> Kp+K0+KT. For example when primary stable electron

acceptor of PSII (QA) is fully reduced (i.e. all PSII reaction

c~ntres are in the state, z+ P680 Pheo QA-), excitation of P680 can not result in stable charge separation (Kp=O). This state of

PSII reaction centres is known as closed state against an open

state where all the QA molecules are in oxidized state. Under

the closed state of PSII reaction centres, the yield of

deexcitation via fluorescence becomes maximum, (FM). The yield

under these conditions is around 3 to 5 fold of the minimal

fluorescence yield obtained when all the PSII reaction centres.

are open ( F 0 ) . The increase from F 0 (open state of reaction

centres) to FM (closed state of reaction centre) represents

variable fluorescence (Fv) and reflects the reduced state of QA,

i.e. (QA-) at any particular time.

19

1. 3.1 Chl g_ fluorescence emission characteristics

1.3.1.1 Room temperature measurements

Characteristics of Chl a fluorescence emission spectra

vary with the state and nature of photosynthetic apparatus.

Isolated thylakoids, intact chloroplasts and intact leaves at

room temperature show a characteristic predominant band with a

peak at about 685 nm and a shoulder at 735 nm (fig. 1.4A). As

stated earlier, this emission emanates mostly from PSII while the

contribution from PSI is quite small.

1.3.1.2 Low temperature measurements

At 77 K, the thylakoid membrane from higher plants and

green algae show three emission bands with peaks at about 685 nm

(F685), 695 nm (F695), and a predominant 715 to 740 nm band

((fig., 1.4b) (Govindjee, 1963; Bose, 1982)). Second-derivative

spectroscopy and curve fitting methods reveal four additional

bands at about 680 nm (F680), 705 nm (F705), 715-725 nm (F720)

and 735-745 nm (F735). F680 has been attributed to LHCII

(Vernotte et al, 1976; Rijersberg et al, 1979) whereas F685 is

assigned to core antenna of PSII (Gasanov et al, 1979). F695

arises as

recombine

luminescence when

(Briantais et al,

the separated charges

1986). Igekami and Ke

in PSII

(1984)

proposed that F705 originates from charge recombination in PSI

reaction centre (P700+ A1-). Core antenna and peripheral antenna

(LHCI) of PSI produce F720 and F735, respectively (Butler and

Kitajima, 1975a and b; Bose, 1982; Briantais et al, 1986; Siffel

and Sestak, 1988).

20

Fig. 1. 4 Typical fluorescence emission spectra of higher

plants thylakoids under unstacked conditions, A. Room

temperature Ch 1 a fluorescence emission spectrum, B. Low

temperature (77 K) Chl a fluorescence emission spectrum.

Fig. 1.5 Kinetics of Chl a fluorescence emission at room

temperature, A) Fast fluorescence induction transients in

isolated thylakoids. Broken line represents the transients

obtained in the DCMU poisoned thylakoids. B) Slow fluorescence

transients (in intact leaves); F0 , 'Minimal' level of

fluorescence; FM, 'Maximal' level of fluorescence; I, Inflection

point (intermediate level); D; Dip level; Sl and S2 represent,

Steady states levels 1 and 2 and Ml and M2 denote, Relative

maxima 1 and 2 M2; T, Terminal level.

lfl c ClJ ....... c

QJ

u c QJ

u V'l (lJ L

0 ::J

I.J...

>--iii c ~

c

i1J u c i1J u V'l i1J .._ 0 ::J -

LL

Fig. 1.4

600

Fig.1.5

® ~ON

I

-682 ® ®

( PS II) 695

(PS II I

685 ...

( PS I ) 735

700 800 700 800 '

Wave length ( nm)

® t OFF J ON

Fm p --- -- - - - --- -~

~ / p / M· I

M2 i I 1S1 I I I Fcj 52

L ~ 200 ms

OFF j

T

1. 3. 2 Kinetics of Chl ~ fluorescence emission

1.3.2.1 Fast fluorescence induction kinetics

Kautsky and Hirsch (1931), showed that when dark adapted

photosynthetic cells were illuminated with light, th~ yield of

Chl a fluorescence follows a particular time course, These

transient changes in the fluorescence yield are known as Kautsky

phenomenon. At physiological temperatures Chl fluorescence is

emitted· mostly from Chl associated with PSII with weak

contributions from PSI (Duysens and Sweers, 1963; Mathis and

Paillotin, 1981). Figure 1. SA represents a typical fast

fluorescence Chl a induction curve. The various symbols represent

the levels of fluorescence attained after a given time period of

illumination. The .F0 represents the basal level of fluorescence

emissions, originating from light harvesting antenna (LHCII)

when all PSII re~ction centres are in the open state (Telfer et

al, 1983). There are a variety of technical problems in measuring

F0 , precisely. For the determination of F0 , rapid transients

should be captured using a device such as a digital Oscilloscope,

transient recorder or a microcomputer attached with an analogue­

to-digital signal converter. An electronic shutter with fast

opening time of the order of 2 ms is usually used. The data

points captured by the detector at the end of opening of shutter

is an operational measure of F0 . The intensity of excitation

light within this time period is assumed to be too weak to induce

photochemistry. A data sampling rate of 10 KHz (i.e. 1 data point

per 100 us) may permit measurement of a point which may be taken

as a reliable measure of F0 . This point is the meeting point of

extrapolated lines of the initial variable fluorescence rise and

of F0 rise. However, it is difficult to consider it to be the

true F0 . This problem may be overcome, at least, in principle, by

the use of modulated fluorometers where fluorescence yield can be

measured by exciting with a modulated light of low intensity

21

which is assumed to be too weak to induce any· photochemical

event. In these instruments also, sensitivity of detection puts a

limit for choosing a light of sufficiently weak intensity for

such purpose. Thus most instruments measure a ''so called " F0 , a

value that closely approximates the true F0 . It is termed as Fi

(for F initial). Fi can be taken as F0 if its yield does not

depend .on the intensity of exciting light (Munday and Govindjee,

1969; Lavorel and Etienne, 1977) and is not quenched by added ...

electron acceptors like bCBQ (Cao and Govindjee, 1989). 'I'·~

represents fluorescence intensity at the first inflection point.

Melis (1985) suggested that 0 to I rise represent an increase in

the variable fluorescence yield due to PSIIB centres. According

to Cao and Govindjee (1989), the oro phase of the fluorescence

transients is contributed by the inactive PSII centres i.e.

centres which are slow in the reduction of Q8 . Fluorescence

decrease from I to D is indicative of the oxidation of QA or

electron transfer from QA to Q8 . D to P rise reflects reducti6n

of QA to QA-. Majority of photosynthetic systems can not attain a

maximum value of fluorescence (FM) even at a high intensity of

light so as P level is often lower than FM, which is attained by ~

the addition of DCMU to the sample (Arnold and Sherwood (1957).

1.3.2.2 Slow fluorescence induction kinetics

After reaching the level P, Chl a fluorescence level

decreases slowly through points S and M to a steady state

terminal level T ((fig. 1.5b) (Papageorgiou, 1975; Mohanty and

Govindjee, 1974; Briantais et al, 1986)). Since these transient

points are attained in a time scale of several seconds after

illumination, the fluorescence induction is termed to be slow. P

to T decline of Chl a fluorescence has been shown to result due ~

to two independent quenching mechanisms; photochemical and non­

photochemical.

22

1.3.2.2.1 Photochemical quenching ~ of Chl ~ fluorescence

This quenching occurs due to oxidation of QA which in turn

results from a high rate of non-cyclic e~ectron transport.

Modulated fluorometers permit measurement of slow fluorescence

induction of Chl a with repetitive application of saturation

light pulses. These saturation pulses imposed during the course

of fluorescence (Kautsky transients) make it possible to resolve

the photochemical and non-photochemical components of

fluorescence ·quenching (Schreiber et al, 1986).

a typical slow induction curve of Chl a

application of saturation pulses. The dark

Figure ·1. 6 shows

with repetitive

adapted state is

characterized by absence of non-photochemical quenching and

maximal photochemical quenching as all PSII reaction centers are

open in dark. At this state, fluorescence yield is defined as F0 .

When a brief saturation pulse (duration -500 ms of intensity,

2000 fE.m- 2 .s-1 ) is given at this stage, all PSI! reaction

centres become closed and system attains a state of maximum

fluorescence which in fig. 1. 6 is shown by FM. The point FM

represents the maximal yield of variable fluorescence when all QA

molecule are in the reduced state. The yield of FM, therefore,

consists of maximum yield

fluorescence (F0 ) and variable

period, following the flash,

stage if the actini6 light

of two components constant

fluorescence (Fv>· During the dark

reduced QA are oxidized. At this

is switched on for fluorescence

induction, the system attains a level of variable fluorescence,

(Fv), the yield of which depends on the intensity of actinic

light used. At any given intensity, Fv is suppressed compared to

the ma~ima 1 variable fluorescence ( Fv) M, by the amount of

fluorescence quenching.

The quenching is caused by two independent mechanisms, the

23

coefficients of which consist of two parts, qQ and qE. To

differentiate between these two, saturation flashes are given

during the Chl · a fluorescence induction. Following each

saturating pulse, fluorescence is pushed to (Fv>s because qQ is

eliminated. (Fv}s is, therefore, expressed as,

(Fv} s 'is less than (Fv}M, because of among other

factors, the generation of a proton gradient and its associated

quenching (qE} so that,

Using this technique, Schreiber et al (1986}, derived

equations to determine the value of qQ and qE at any point of

induction curve for variable fluorescence (Fv).

qQ = [ <Fv> s- <Fv> l I <Fv> s

qE = [(FV)M-(Fv)g]/(Fv)M

Thus qQ and qE bear a direct relationship to _ Chl a

fluorescence so that qE follows the lowering of· fluorescence

yield by the development of the proton gradient and qQ reflects

the degree of redox state of QA.

1.3.2.2.2 Non-photochemical

fluorescence

quenching

qE arises because of an increase in the trans-thylakoid

proton gradient (Govindjee et al, 1967; Murata and sugahara,

1969}. It is formed in the light and relaxes in the dark within

2-10 minutes (Horton and Hague, 1988; Quick and Stitt, 1989). qE

24

Fig.

Fig. 1.6

F.--+ m --------------

0

J

\.

'""--....... _

-----~~------------

' saturating _ e_ul~e- __ _

t measuring

beam

1--20 s --1

t actinic

beam

qE ·(F)m

----qa· (~~

Fv

-------+

·(F)m

(F)s

1.6 Definition of quenching coefficients etc. by

Schreiber et al ( 1986). F0 : Fluorescence displayed by a dark­

adapted leaf in very weak modulated light, (Fv)M: Maximum

variable fluorescence first seen when a pulse of saturating

actinic light is applied, (Fv): Variable fluorescence induced due

to continuous actinic light, (Fv) 5 : Peak fluorescence attained

when pulses of saturating light is applied in presence of

continuous actinic light, qE: Non-photochemical quenching

coefficient, qQ: Photochemical quenching coefficient.

has been assigned a role both in the protection of PSII apparatus

from excess light (Krause et al, 1982) and in the dynamic control

over the rate of electron transport through PSII (Weis and Berry,

1987; Genty et al, 1989).

Photochemical and non-photochemical quenching are the two

main mechanisms which cause the decline of Chl a fluorescence

yield from P to T. However, other than these two, depletion of

cation levels, phosphorylation of LHCII and generation of reduced

pheophytin has also been shown to cause Chl a fluorescence

quenching.

1.4 THERMOLUMINESCENCE

Thermoluminescence results due to the emission of light

induced by heating of preilluminated photosynthetic materials

Arnold and Sherwood, 1957). When illuminated, photosystem

reaction centres undergo charge separation. These charges are

quickly removed away, to other neighbouring species. This

process, known as charge stabilization, ensures that the charges

do not recombine . .If same sample is frozen quickly just after or

during the illumination, the system gets frozen in the charge

separated state. If the sample is then warmed slowly, opposite

charges come near to each other and recombine.

provides the necessary energy for this process

Temperature

(Arnold and

Sherwood, 1957). The recombination of charges produces an

excited state of Chl a which finally deexcites to produce

luminescence. Light emission from various photosynthetic

materials have been observed to have at least 13 bands in the

range of temperature from 77 K to 373 K (Sane and Rutherford,

1986). A lot of variation has been reported in the position and

number of thermoluminescence (TL) peaks in the glow curves (Sane

and Rutherford, 1986). The characteristics of different TL peaks

25

are summarized in table 1.3.

1. 4.1 Effects of electron transport inhibitors which act at

the acceptor side of PSII

pattern

transport on thermoluminescence

All the herbicides which interrupt electron transport

between QA and Q8 (like DCMU) modify the glow pattern (Arnold and

Azzi, 1968; shuvalov and Litvin, 1969). Since DCMU can be taken·

as an ·ideal representative of this class of herbicides, the

effect of DCMU on TL has extensively been studied. Treatment of

chloroplasts with other herbicides which have site of action

similar to DCMU, also cause

curve pattern (Droppa et al,

et al ( 1982) , found out

characteristic changes in the glow

1981; Horvath et al, 1986). Demeter

that in presence of DCMU, band B

disappears arid a new band (Q band) emerges at a temperature lower

than B. They also found that peak position and activation energy

of this band vary depending on the group of herbicide used.

Treatment with urea, pyridazinone, phenyl carbazide and DSPD

shifts the Q band at +6°c whereas triazines and hydroxyquinone

give a band at o0c. Phenolic herbicides (hydroxybenzonitrite and

dinitrophenol) shift the Q bani to -14°C (Droppa et al, 1981).

All the above mentioned groups of herbicides inhibit electron

transport at the same site i.e. between QA and Q8 (Pfister and

Arntzen, 1979; Demeter et al, 1979). Thus, the source of negative

charge needed for recombination to generate thermoluminescence is

QA in all the cases. On the other hand, it is generally

considered that activation energy and peak positions are defined

by the redox span between the donor ( +ve) and acceptor ( -ve)

molecules participating in the charge recombination (Crofts et

al, 1971; Vass et al, 1981). To explain this discrepancy, three

explanations have been put forward.

1. Other than inhibiting at donor side of PSII, herbicides also

26

Table 1.3 Thermoluminescence in Plants

Peak

Approximate emission temperature (°C) Origin

z

I (A)

II

-160

-70 (variable)

-20

-0.0 (D or Q)

III 10

IV(B) 25

V(C) 48

No. Author

?

+ -Z Q8 (?) and/

+ -or Z QA (?)

Signal II-slow

1. Sane et al, 1974 2. Arnold and Azzi, 1968 3. Vass et al, 1984 4. Inoue et al, 1977 5. Inoue and Shibata, 1978 6. Rutherford and Inoue, 1984 7. Demeter et al, 1982

Characterstics

Emission max. 740 nm (1); excitation max. blue light (2)

Oscillates with preflash number; maxima coincide with s 1 (3) Insensitive to Tris (4) and NH20H (3)

Oscillates with preflash number maxima coincide with s3 (5)

Insensitive to Tris (6)

Oscillates with flash number when diuron is added after excitation· maxima coincide with s 2 and s 3 (7,8) Sensitive to Tris and tetranitro methane treatment (9)

Formed in leaves or in the _presence of DCCD (9,10) Sensitive to Tris and NH 2oH (11)

Oscillates with flash number maxima coincide with s 2 and s 3 (5) Sensitive to Tris and NH 20H treatment ( 4, 12)

Oscillates with flash number when diuron is added after excitation; maxima coincide with s 0 and s 1 (13) Insensitive to Tris treatment (14)

No. Author

8. Demeter, 1982 9. Sane et al, 1983b 10. Sane et al 1 1977 11. Sane et al, 1983a 12. Rozsa and Demeter, 1982 13. Demeter et al, 1984 14. Inoue et al, 1977

shift the redox potential of S states.

2 . There may be one intermediary species existing in between

P680 and QB and herbicides of above mentioned three groups

inhibit electron flow at the site of these intermediary

acceptors.

3. The three group of herbicides bind to different position in

the QB binding protein and thus cause different extent of shifts

in the redox state of QA. This difference in shift in the redox

potential of primary acceptor presumably produces difference in

the peak position and redox potential.

1.5 stoichiometry of

photosynthesis

reaction centres in oxygenic

As recently as 1980, it was widely accepted that PSII and

PSI centres existed in 1:1 ratio in the thylakoid membranes.

Experimental data obtained from the measurements of the

electrochromic band-shift at 518 nm supported this assertion

(Schliephake et al, 1968).

In recent years, development of many techniques such as

sensitive absorbance difference spectrophotometry (Melis and

Brown, 1980), measurement of QB/herbicide binding sites in the

thylakoid membranes (Vermaas and Arntzen, 1983; Jursinic and

Stemler, 1983), the Cyt b559 content (Lam et al, 1983; Murata et

al, 1984) and amounts of Mn and Z (Babcock et al, 1983; Yocum et

al, 1981), have permitted direct quantitation of integrate

components in the reaction centre of PSI! (QA, P680 , Phe) and PSI

(P760 ). Repetitive flash illumination of photosynthetic material

in vivo and in vitro leads to oxidation of water. Oxygen yield

per flash, measured from the experimental data of such

experiments have also been used to quantitate the number of PSI!

units (Whitmarsh and Ort, 1984; Myers et al, 1980; Kawamura et

27

al, 1979; Chow et al, 1990) . Quantitative measurements with

thylakoids from spinach and from other sun-adapted plants showed

the presence of one PSII reaction centre per 350 Chl (a+b)

molecules and the presence of one PSI reaction centre per about

600 Chl (a+b) molecules (Melis, 1991). This shows a photosystem

stoichiometry of PSII: PSI, 1.7: l.O (Melis and Anderson, 1983;

Melis and Brown, 1980; Melis and Harvey, 1981). Moreover, the

measurement regarding the ratio of PSII/PSI varied in diverse

photosynthetic species.

The information as available regarding the organization of

thylakoid membranes thus suggest that photosynthetic organisms

have the ability to regulate the photosystem stoichiometry in the

thylakoid membrane and, thereby, to adjust and optimize the

process of light absorption and linear electron transport {Melis

and Brown, 1980; Whitmarsh and Ort, 1984; Jursinic and

Dennenberg, 1989; Melis, 1985; Ort and Whitmarsh, 1990).

1.6 Dynamics of thylakoid structure and function

1. 6.1 stater-state!! transition

Light energy absorbed by the antenna molecules is

distributed between the two photosystems. Since photosystems

absorb at different region of the spectrum of incident radiation,

any change in the quality of light causes an imbalance in the

distribution of excitation energy between PSII and PSI. Murata

{1969) and Bonaventura and Myers {1969) independently showed that

thylakoid membranes have an in vivo regulatory mechanism, which

corrects for any imbalance in the excitation of photosystem II

reaction centres. This process by which antenna molecules can

reorient themselves to direct excitation energy to either of the

28

photosystem reaction centres is known as state change. There is

now evidence (Bennett, 1979b; Horton and Black, 1980; Allen et

al, 1981; Barber, 1982) that such regulatory mechanism (state

change) which compensates for the imbalance in excitation energy

distribution involves phosphorylation/dephosphorylation dependent

reorganisation of thylakoid membranes. The phosphorylation and

dephosphorylation takes place in the thylakoid membranes by a

membrane bound kinase and phosphatase, respectively in a

reversible fashion (Bennett, 1979a). The preferred substrate for

these reactions is the surface exposed domain of LHCII. The

activity of kinase is regulated by the redox state of PQ pool

(Bennett, 1979a}. How many kinase(s) are involved in LHCII­

phosphorylation is not certain. There are observations which

support ·a single thylakoid protein

et al, 1981; Coughlan and Hind,

multiple kinase (Lin et al, 1982;

1991). Under the conditions

kinase (Berinett, 1979b; Horton

1987; Bennett et al, 1988) or

Farchaus et al; 1985; Bennett,

when PSII mediated electron

transport is high· and PQ pool is reduced, phosphorylation of

LHCII polypeptides takes place. Under the conditions when PSI

activity is higher or there. is no PSII · electron transport,

depletion of reduced PQ pool takes place which leads to kinase

inactivation. As a consequence, dephosphorylation of LHCII

occurs. Phosphorylation of LHCII

residues on the outer surface

polypeptide occurs at threonine

near the N-terminus. This

increases the net negative charge on the surface of LHCII

complex. A negative surface charge destabilizes the aggregation

of these complex in appressed regions of thylakoid membranes

(Barber, 1980; Kyle and Arntzen, 1983) and has been assumed to

trigger the lateral migration of LHCII from grana lamellae to

stroma lamellae (Andersson et al, 1982; Kyle et al, 1983). Under

these conditions excitation energy is preferentially directed to

PSI and thylakoids are said to be in stater. Under Stateii

condition, LHCII is dephosphorylated and functions as antenna

of PSII reaction centre. Thus, the stater - stateii conversion is

29

regulated by redox level of PQ, which in turn depends, as well as

decides, the turnover rate of the two reaction "centres through

phosphorylation and dephosphorylation. It is proposed that this

is the regulating mechanism by which plants adapt to changing

wavelengths of light (Allen et al, 1981; Bennett et al, 1991;

Allen, 1992).

1.7 Review of Literature

1. 7.1 Pyridazinones as photosynthetic herbicides

Figure above Rl­

common

. ;

.. '

structure for pyridazinone

derivatives. Depending on the various substitutions, R1, R2 and

R3, more than forty derivatives of pyridazinone have been.

synthesized and tested for their interaction with plant systems

(St. John et al, 1979). Though some of the derivatives like

pyrazon ( 5-amino-4-chloro-2-phenyl pyridazin-3-one) are used in

agriculture for weed control, most of the pyridazinone

derivatives are used as experimental photosynthetic herbicides

because they can affect structure or function of photosynthetic

apparatus in many ways. In general effects of pyridazinone

herbicides on photosynthesis can be classified as follows,

1. Inhibition of photosynthetic electron transport

2. Alteration in the fatty acid composition of thylakoid

membranes lipid.

3. Inhibition of pigment formation

4. Changes in the 'structure-function' relationship of

thylakoids.

30

1.7.1.1 Inhibition of photosynthetic electron transport

Many of the substituted pyridazinone compounds have been

shown to inhibit Hill reaction in isolated chloroplasts. (Hilton

et al 1969; Tischer and Strotmann, 1977; Mannan and Bose,

1985a). They are also known to inhibit light induced o2 evolution

from isolated leaf mesophyll cells (Hilton et al, 1969) and

intact algae cells (Herczeg et al, 1979; Karapetyan et al, 1981).

However, there is a large variation in the pyridazinone

derivatives in their efficiency to inhibit photosynthetic

electron transport in isolated chloroplast. The concentration of

SAN 6706 to obtain 50% inhibition of electron flow (I 50 ) in

isolated chloroplast was 5 pM while for SAN 9789 it was 90 pM.

The r 50 value reported for SAN 97_85 were 14 p.M (Hilton et al,

1969) and 20 fM (Mannan and Bose, 1985a). Thus,, pyridazinone

compounds are 100-150 fold weaker inhibitors of photosynthetic

electron transport as compared to DCMU and atrazine. This is

attributed to their 100-150 fold lower binding constant as

compared to DCMU (Tischer and Strotmann, 1977). The competition

experiments have shown that pyridazinones and DCMU inhibit

electron transport by interaction with the same component (QB

protein); the mode of action being similar as well (Tischer and

Strotmann, 1977).

The substructures as mentioned below have been identified

as features common to all inhibitors which inhibit PSII dependent

electron transport (Buchel, 1972; Trebst and Harth, 1974)

Pyridazinone compounds fulfill these requirements,

1. the group >N-C-CX, Where X = 0 or N, but not S and

2. a hydrophobic residue in close vicinity to the above

mentioned group

A model which encompasses biochemical, biophysical and

structure-activity considerations for PSII herbicides considers

31

that all these compounds are non-reducible analogues of

plastoquinone or its semiquinone anion (Gardner, 1989). The model

suggests that sp2-carbon of the essential element of the

herbicide corresponds to the sp2 carbon of the quinone carbonyls,

and thus there are two possible sites to which this element may

bind. The essential features of Gardner's model are,

1. Plastoquinone molecule is asymmetric and, therefore, receptor

should also have asymmetry.

2. A hydrophobic domain in the vicinity of essential element of ~

the herbicide should be present, which corresponds to isoprenoid

tail of the plastoquinone. A hypothetical picture depicting how

one of the PSII inhibitors (atrazine) supposedly acts on PSII

shown in fig. 1.7.

is

1.7.1.2 Alteration in the fatty acid composition of thylakoid

membrane lipids

Substituted pyridazinone compounds e.g. SAN 9785 cause an

increase in the linoleic acid content of thylakoid membrane

lipids (St. John, 1976; Frosch et al, 1979; Ashworth et al, 1981

and Mannan and Bose, 1984), when the plants are grown in their

presence. In vitro studies using 14c acetate and leaf discs

have shown that this specific increase in the linoleic acid was

due to the inhibition of desaturation of linolenic acid to form

linoleic acid (Murphy et al, 1980; Willemot et al, 1982). Out

of forty-four substituted pyridazinones tested for their ability

to induce a specific decrease in 1 inolenic acid content, SAN

9785 was found to be the most efficient (St.John, 1979).

1.7.1.3 Inhibition of pigment formation

Depending on the nature of substitution in pyridazinone

32

\

.....

Fig. 1.7

Fig. 1.7 Schematic figure of the plastoquinone/herbicide binding

pocket of the 01 protein. Dashed lines represent hydrogen bonds;

dotted lines represent hydrophobic interactions, A.

Plastoquinone binds to the Dl protein, accepts two electrons and

two protons, and is released as plastohydroquinone. B. Atrazine

binds to the 01 protein and prevents the binding of

plastoquinone. Adopted from Fuerst and Norman (1991).

compounds, these herbicides cause bleaching of photosynthetic

pigments to varying degree in higher plants and algal cells

(Kleudgen, 1979; Kummel and Grimme, 1975) . Urbach et al ( 1976)

have proposed certain features in the structure of the herbicide

which are necessary for efficient bleaching activity. SAN 9785 is

expected not to have any bleaching activity on this basis. In

fact SAN 9785 did not cause any significant bleaching in many

plant species upto a concentration of 100 fM (Urbach, 1976; St.

John, 1976; Mannan and Bose, 1985b). Therefore, SAN 9785 is

considered to be a non-bleaching or weakly bleaching herbicide

(St. John, 1976). The bleaching activity of pyridazinone

compounds is highly dependent on light intensity. When seeds ~ere

germinated and seedlings were raised in presence of 100 pM SAN

9789, carotenoid biosynthesis was inhibited by 99% (Lehoczki et

al, 1982). Therefore, under these conditions the plants can grow

only if the light intensity is kept extremely low and thus avoids

Chl bleaching. The mechanism of bleaching of photosynthetic

pigments by pyridazinone compounds is still not certain

(Karapetyan, 1993). However, three hypothesis have been proposed

to explain the bleaching phenomenon.

1. Su.bsti tuted pyr idazinone compounds inhibit carotenoid

biosynthesis, directly. Sandmann et al ( 1980) showed that SAN

9789 inhibits phytoene synthetase. Ben- Aziz and Koren ( 1974)

noted that SAN 6706 interfered with the cyclization reactions in

the biosynthesis of carotenoids whereas Bartels and McCullough

(1972) and Bartels and Watson (1978) found that this compound

inhibits dehydrogenation reactions. Carotenoids are shown to have

a protective role against chlorophyll bleaching (Frosch et al,

1979) . Since coloured carotenoids are decreased in presence of

SAN 6706, chlorophyll molecules get photodegraded.

2. According to the second hypothesis, pyridazinone derivatives

do inhibit carotenoid biosynthesis. However, this hypothesis does

not consider that carotenoids have a protective role against the

bleaching of chlorophylls. Instead, the hypothesis considers that

33

carotenoid biosynthesis is highly coordinated with chlorophyll

biosynthesis. Though this type of control mechanism was seen in

Euglena gracilis by Vaisberg and Schiff ( 1976) it was not found

in higher plants in the studies of Frosch et al (1979) and Pardo

and Schiff (1980).

3. The third hypothesis considers a more generalized effect of

pyridazinones. According to this hypothesis, pyridazinone

derivatives cause a decrease in the content of 70 S ribosomes.

This in turn causes a decrease in the synthesis of enzymes which

are responsible for carotenoid and chlorophyll biosynthesis

(Lichtenthaler and Kleudgen, 1977). Direct evidence in support of

this hypothesis is, however, still lacking.

1.7.1.4 Changes in the structure-function

thylakoids

relationship of

The structural and functiona 1 changes rn the thylakoids

induced in response to substituted pyridazinone tre:atment vary

to a great extent and depend upon the derivative of pyridazinone

used. Herczeg et al (1980) reported an increase in the light

induced oxygen evolution in Chlorella cells grown in presence of

sublethal concentrations of SAN 6706, SAN 9785 and SAN 9789.

Plants treated or grown in presence of SAN 9785 exhibit several

a 1 terations in the structure and function of their thylakoids.

Plants grown in presence of SAN 9785 show an increased stacking

(Khan et al, 1979) whereas those grown in presence of SAN 6706

and SAN 9789 show a disorganization of thylakoid membrane

structure (Laskay et al, 1983; Karapetyan, 1993). Many

investigators have reported an increased emission of Chl a

fluorescence and a higher rate of PSII catalyzed electron

transport in thylakoids isolated from SAN 9785 grown plants in

comparison to thylakoids of control plants on Chl basis (Bose et

al, 1984; Leech et al, 1985; Mannan and Bose, 1985; Laskay and

34

Lehoczki, 1986). It was suggested that such increase in rate of

PSII electron transport rate in SAN 9785 grown wheat seedlings

occurred due to a preferential synthesis of PSII units in these

plants (Bose et al, 1984). These results suggested that PSI! to

PSI ratio increased in thylakoids of SAN 9785 grown plants (Bose

et al, 1984). Such an increase in the ratio of PSII/PSI explains

the increase in the relative area occupied by appressed membranes

in SAN 9785 grown plants as revealed by ultrastructural studies

done using electron microscopic studies (Khan et al, 1979; Leech

et al, 1985; Bose et al, 1992). However, this effect seems to be

specific to SAN 9785 as SAN 9789 (norflurazon} treatment have

been reported to result in a drastic decrease in PSI! to PSI

ratio in the thylakoids of barley plants (Lehoczki et al, 1982;

Karapetyan, 1993}.

In general, the alterations in the thylakoid membrane

structure and function in the plants grown in presence of

·sublethal concentrations of one of the pyridazinone derivatives,

SAN 9785 can be summarized-in the following way,

1.

2 •

3 •

4.

5.

Characteristic

Parameters

Chl ajb

CPII/CPI

PSI to total

Chlorophyll

QA/PQ

F0 and Fv/Fo

35

Response Reference

Decreased ( 1} , (2)

Increased ( 1) , (2)

Decreased ( 3)

Decreased ( 3)

Decreased ( 3)

~I

I '

Number

(1)

(2)

(3)

Reference

Leech et al, 1985

Mannan and Bose, 1985a and b

Bose et al, 1984

Based on the energy flux theory for biomembranes

(Strasser, 1978), Graf et al (1986) have proposed a model of the

modified organisation of the photosynthetic system in the leaves

of SAN 9785 treated plants. According to model the t:hylakoid

membranes of treated plants exhibit following changes compared to

thylakoids of control plants.

1. A decrease in the excitation energy transfer (:spillover)

from PSII to PSI

2. A decrease in co-operativity (grouping) between PSII units.

3. Inhibition of LHCII phosphorylation induced changes in

electron transport.

4. Decrease in the antenna s1ze of PSII

Above mentioned changes in functional organizat.ion of

thylakoids of SAN 9785 treated plants were speculated to occur

because of an altered lipid matrix in these plants. However,

these conclusions

temperature (77K)

biochemical evidence.

were based on room temperature and low

fluorescence studies only, and lacked

SAN 9785 grown plants also show an inhibition of cation

mediated modulation of structure-function relationship of

thylakoid membranes. Bose et al (1992) showed an inhib~tion of

cation regulation of room temperature Chl a fluorescence emission

and absorption properties of thylakoids in SAN 9785 grown plants.

Based on these observations Bose et al ( 1992) proposed that

cation induced changes in the excitation energy distribution were

inhibited in the thylakoids isolated from Pisum sativum plants

grown in the sublethal concentration of SAN 9785. Simu11:aneous

measurement of PSII and PSI fluorescence emission kinetics at 77K

36

~------------------------ -~~------------~

showed that the ability of cations to regulate excitation energy

spillover from PSII to PSI was also inhibited in the thylakoids

of SAN 9785 grown. The authors correlated these functional

changes with the ultrastructural changes in the thylakoid

membranes of SAN 9785 grown plants and concluded that the

observed inhibition of cation induced spillover changes occurs

due to loss in the ability of thylakoids to undergo unstacking in

low salt medium.

1.8 Adaptive

membranes

herbicides

changes in

of plants

the chloroplasts

grown/treated

thylakoid

with PSII

Plants grown or treated with sublethal concentrations of

herbicide have a partially inhibited PSII electron transport

capacity. This generates a significant imbalance of electron

transport between PSII and PSI. Such imbalance in electron

transport between PSII and PSI is suggested to induce an

adaptive change in the structure-function relationship of

thylakoids (Melis et al, 1985; Melis, 1991} as a result of which

thylakoid structure in the plants ·grown in presence of sublethal

concentration of PSII herbicides and strong light appears very

much similar to shade (low intensity} grown plants (Leong and

Anderson, 1984; Davies et al, 1986; De la Torre and Burkey,

1990). The physiological changes induced in herbicide grown

plants are, therefore, commonly said to produce a shade

phenotype.

Various studies have been conducted to observe the

herbicide induced physiological changes in the thylakoid

structure and function. Lichtenthaler (1979), studied the effect

of two biocides, bentazon and triadimefon on the growth response

of raddish seedlings (Raphanus sativus L.}. Plants grown in

37

presence of fungicide, triadimefon (10- 5M) induced strong light

growth response and the formation of sun-type chloroplasts. The

total chlorophyll content, ratio of Chl ajb and content of prenyl

quinones all increased in triadimefon treated plants. On the

contrary, PSII herbicide, bentazon induced a shade type

adaptation, when the plants were grown under a regime of strong

light intensity. The content of chlorophyll, carotenoids and

ratio of Chl ajb, all decreased in bentazon treated plants

relative to control plants. However, the ratio of Chl a f prenyl

quinones and xanthophyll/carotenoides increased in bentazon

treated plants. Such effects of bentazon were strongly dependent

on the light intensity under which the plants were grown as

chloroplast composition and organization in the plants grown at

low light intensity was affected very little (Lichtenthaler,

1979). Shade type adaptation in bentazon treated plants were

also characterized by increased formation of chloroplast lamellae

and grana stacks. Lichtenthaler et al ( 1982) latE!I' showed that

bentazon treated plants grown under strong light conditions show

low levels of CPI+CPia in comparison to that shown by control

plants. Fedtke (1979) obtained similar results when plants were

treated with metabenzthiazuron (MBT) instead of bentazon.

Chloroplasts of MBT-treated plants exhibited a higher degree of

stacking of thylakoids (Fedtke et al, 1977) as well as lower Chl

aj b ratios, lower amounts of b-carotene and prenylquinones

(Kleudgen, 1978). It was later shown that a shade type

adaptation similar to that induced by bentazon and MBT can also

be induced by DCMU (Lichtenthaler et al, 1980).

Similar kinds of results were

treated with atrazine. Matteo et

obtained when plants

al (1984b) showed

were

that

cul ti vat ion of Spirodela oligorrhiza on a sublethal dose of

atrazine induces a shade type of response. Chloroplast structure

of plants grown in presence of sublethal concentration of

herbicide was very much similar to that in triazine resistant

plants. Vaughn and Duke ( 1984) found out that chloroplasts of

38

atrazine susceptible (S) biotypes could be modified to

ultrastructural phenocopies of those in resistant (R) biotypes by

treatment with sublethal levels of the PSII inhibiting

herbicides, bentazon, diuron and prometon. In a recent study, De

la Torre and Burkey (1992) reported the physiological effects of

sublethal (0.07 mM) concentrations of atrazine on thylakoid

membrane activity and composition of barley. Thylakoids from

plants treated with sublethal concentrations of atrazine showed

an increased level of the LHCII and lower levels of CPI in

comparison to that shown by thylakoids of control plants. The

number of PSII and PSI,reaction centre and Cyt b 6 /f.complex per

unit Chl were not a·ffected by sublethal concentrations. of

atrazine. This suggests an overall effect of atrazine treatment

on redistribution of chlorophyll associated with PSI to PSII with

no effect on the number of thylakoid membrane protein complexes

associated with electron transport (De ld Torre and Burkey,

1992).

The shade tY.pe adaptations of thylakoids by sublethal

concentrations of herbicides is also shown to be induced . in

cyanobacteria. Koenig, (1987 a,b) showed that shade .type·

appearance can be induced· in Anacystis nidulans in high light

intensity in the presence of sublethal conc;:entrations of DCMU

type inhibitors. The cell types grown in presence of sublethal

concentrations of herbicides· were characterized by higher

concentrations of both Chl a and phycocyanin , (PC) per cell and,

in addition, by a higher ratio of PC to Chl ,as compared to the

corresponding control cells (Sun-type).

Various workers have tried to explain: the physiological

significance of adaptive changes induced in 'the thylakoids of

plants grown in presence of sublethal concentration of herbicide.

Mel is et al ( 1985) proposed that such changes are regulatory

mechanism of the plants to balance the electron transport between

PSII and PSI. Fedtke (1977; 1979) assumed that the adap~ive

changes are produced due to the lower level of soluble sugars.and

39

other photosynthetic products, as a result of the strongly

inhibited photosynthesis. From the observations that exogenous

cytokinins induce the formation of sun-type chloroplasts and the

, plants grown at high light intensities contain more cytokinins

t:han shade-plants (Kohler, 1977), Lichtenthaler et al, 1980

assumed that PSII-herbicides decreases the endogenous cytokinin

level. Lichtenthaler (1979) interpreted the increased formation

of chloroplast lamellae and grown stacks in bentazon treated

plants as a part of an inactivation mechanism for the herbicide

through providing more binding sites within a chloroplast. Mattoo

et al (1984b) proposed that adaptive reorganization of thylakoid

components in plants grown in presence of sublethal concentration

of PSII herbicide (atrazine) may be a compensatory mechanism for

maintenance of a functional interaction of the proteins and

lipids of the PSI! complex.

As discussed above, the physiological changes produced in

the thylakoids of plants grown in presence of sublethal

concentrations of herbicides are generally believed to arise as a

result of an in situ inhibited PSII activity which generates an

imbalance of electron transport between PSI! and PSI (Melis,

1985; Melis, 1991). However, Koenig (1990) showed that the shade­

type adaptation grown under Anacystis nidulans under strong

light in the presence of sublethal concentrations of DCMU-type

inhibitors is not due to an imbalance of electron transport

because such changes can not be prevented or reverted by using a

electron donor like thiosulphate. Since thiosulphate is known

to feed electrons at the Cyt b6 /f complex (Utkilen, 1976;

Peschek, 1978; Koenig, 1990), it is expected to compensate for

any imbalance between PSII and PSI created by in situ inhibition

of PSII electron transport by herbicide.

40

1.9 About this investigation

Upon reviewing the SAN 9785 action on photosynthetic

system of higher plants suggests that SAN 9785 is an effective

inhibitor of electron transport in isolated thylakoids in vitro;

the site and mode of action of this inhibitor is, however, not

well elucidated. The in vivo action of SAN 9785 on photosynthesis

has also been investigated by various workers (Bose et al, 1984;

Mannan and Bose, 1985b; Leech et al, 1985; Karapetyan, 1993; Bose

et al,. 1992). Thus, SAN 9785 becomes an useful tool to study the

structural and functional changes produced in the thylakoid

membranes in . response to in situ partial inhibition o,f PSII

mediated electron transport. However, among the pyridazinone

derivatives this compound has maximum ability of increasing the

ratio of saturated to unsaturated fatty acids with no or ~ittle

changes in the pigment content in plants grown in presence of

pyridazinone compounds (St. John et al, 1979). Thus, any

alteration in the structure and function of thylakoid membranes

in SAN 9785 grown plants may be considered to arise as a result

of partial inhibition of PSII mediated electron transport or \due

to changes in the lipid environment of the thylakoid membranes.

It was shown by other workers (Murphy · et al, 1985) that

alterations in the fatty acid composition of thylakoid membranes

in response to SAN 97"85 varied depending upon the plant species

used and P. sativum shown to be resistant against such changes·.

In spite of this, P. sati vum remains highly susceptible to SAN

9785 dependent photosynthetic changes .. Thus, P. sativum as an

experimental system, offers a special advantage where the';

structural and functional changes induced in the thylakoid

membranes in response to SAN 9785 treatment can be considered to

arise only due to the ability of SAN 9785 to cause in situ.

partial inhibition of PSII electron transport and not due to the

SAN 9785 mediated fatty acid changes.

The subject matter reviewed in relation to adaptive

41

- '

capacity of plants to the stress given in the form of a sub­

lethal concentration of PSII herbicide suggests that treated

plants undergo an adaptive change in the organizat~ion of their

thylakoids so that structural organization of thylakoids of

treated plant become very similar to the thylakoids of shade

grown plants. Though the information is available regarding the

nature of herbicide induced changes, not much information is

available regarding the structure-function ?relationship of

thylakoids in the plants grown or treated with herbicide and

associated alterations in the excitation energy distribution

between photosystems.

In view of this, we were encouraged to focus attention on

adaptive capability of thylakoids of P. sativum plants treated

with SAN 9785, especially on the potential for change in

composition and function (long term changes) and the capacity to

undergo short term changes. This essentially involved,

1. Studies regarding further characterization of site of action

of SAN 9785, the PSII inhibitor used in this study.

2. Measurement of the extent of in situ inhibition of PSII

electron transport rate

the extent of stress.

in SAN 9785 treated plants to evaluate

·3. Characterization of structure-function relationship of

thylakoids isolated from plants treated with SAN 9785.

4. To determine the dynamic ability of thylakoids of SAN 9785

treated plants to undergo state-transition and phosphorylation

mediated energy distribution.

Our experimental protocol involved transferring either 11

day old seedlings of P. sativum or 41 d old seedlings of

Chenopodium album to a 125 fM solution of SAN 9785 in 0.5 S

Hoagland solution, where plants were allowed to grow for further

72-84 h. This type of treatment is different from those. of

previous investigations (Leech et al, 1985; Bose et al, 1984;

Mann an

plants

and Bose, 1985a and b;

were grown in presence

42

Bose et al, 1992) , where the

of SA.N 9785 from the onset of

germination. In the latter treatments, it was extremely difficult

to identify the sites of action responsible for changes in the

photosynthetic apparatus. For instance, certain developmental

changes that occur in the chloroplasts during maturation may

overlap with the adaptive changes that occur due to partial

inhibition of PSII. Further, presence of SAN 9785 may affect the

normal process of development, itself. H~nce, in the present

investigation, normally grown seedlings were treated with SAN

9785 as mentioned above. Since the possibility of developmental

changes being incorporated in the photosynthetic apparatus is

excluded, adaptive structural and functional changes in the

thylakoids of SAN 9785 treated plants can be considered to arise

due to an in situ partially inhibited PSII mediated electron

transport.

43