RESULTS AND DISCUSSION II (Electron Transport Activity and Spectral Properties)

6.1 EFFECT OF METALS ON ELECTRON TRANSPORT ACTIVITY

6.1.1 Anacystis

6.1.1.1 PHOTOSYSTEM II ACTIVITY IN INTACT CELLS

The activity of photosystem II was studied polarographically

as well as by measuring the chlorophyll a fluorescence.

Polarographic measurements

PS II activity was measured in the presence of pBQ, which

being lipophilic enters into the cells and accepts electrons from

plastoquinone. The intact cells of Anacystis and Nostoc were

grown in the presence of different concentrations of cu, Cd, Pb

and Tl as mentioned earlier and these treated cells were taken

for the measurements.

Copper

At 5 uM of Cu PS II activity was inhibited by 25%., whereas

at 10 uM there was a gradual decrease in photosystem II activity

and it was completely inhibited after six days of stress. At

higher concentrations (15 pM) the activity decreased by 70% after

two days of stress and on incubation for longer time the activity

was completely inhibited (Fig. 15).

Cadmium

Cadmium at all concentrations reduced the activity of PS II.

The activity of PS II was reduced by (75-80%) after two days of

stress. None of the concentrations completely inhibited the PS II

activity of the cells (Fig. 15).

Lead

Lead had no effect on Photosystem II activity. The activity

56

t 120r----------.......

Cu

2 4 6 Number of days

120~-------------------­Pb

...! • 20

0 2 4 6 I\OTOer of days

Cd

o--2 4 Number of days

Tl

2 4 Number of days

Fig. 15 Effect of heavy metals on electron transport (H2o---> pBQ) catalyzed by PS II in Anacystis cells. (O) 1 (~) 1 (IJ) 1 ( •> representC.,5 1 10 1 15 ~M concentrations of Cu 1 Cd 1 Tl and 75 1 100 1

150 )JM concentrations of Pb. The values are average of four experiments and S.D. was less than 5% in every case.

at all concentrations was same as that of control (Fig. 15).

Thallium

The activity of PS II was affected significantly, when

cells were grown in the presence of metal for a longer duration

(6 days). 5 pM of thallium inhibited the PS II activity by 10%

while at 15 fM of thallium the activity was totally inhibited

after six days of stress (Fig. 15).

Chlorophyll a fluorescence measurements

Copper

Fv/Fm ratio of copper treated cells showed a decrease with

increasing concentrations of copper. Copper treatment reduced the

Fm and this decline was due to decrease of variable fluorescence.

There was no effect on Fo i.e. the initial fluorescence of the

cells. 10 and 15 ~M of copper caused total quenching of Fv after

four days of stress (Fig. 16).

Cadmium

Fv /Fm ratio in cadmium treated cells showed a decrease at

all concentrations and this reduc~ion was due to decrease in Fv.

The decrease in the chlorophyll fluorescence of the cells

indicates that cadmium affects the donor side of PS II (Fig. 16}.

Lead

There was no effect of lead on chlorophyll fluorescence and

Fv/Fm ratio was comparable to control (Fig. 16).

Thallium

Fv/Fm ratio decreased slightly due to decrease in Fv of the

cells treated with thallium. Lower concentrations of thallium

57

Cu 1.0

Q.2

~0~--~-----+----~~ 0 2 Number of days

Pb 1.0

0.8 E ~o.6 u.>

0.

2 Number of days

Cd 1.0

~0'~--~----~----=-~ 2 4 6 Number of <tlys

T(

1.0

~0. u.> 0.4

4 6 Num~r at days

Fig. 16 Fv/Fm ratio of Anacystis cells treated with heavy metals. (0) 1 (A) 1 (o) 1 <•> representc,51 10 1 15 uM concentrations of Cu 1 Cd 1 Tl and 75 1 100 1 150 uM concentrations of Pb.

decreased the FvfFm after six days of treatment. At higher

concentrations the decrease in Fv/Fm ratio was more as compared

to lower concentrations (Fig. 16).

6.1.1.2 ELECTRON TRANSPORT ACTIVITY IN THYLAKOIDS AND

SPHEROPLASTS

As ferricyanide, methyl viologen and DCPIP do not enter into

the intact cells, spheroplasts were prepared to study PS I

catalyzed electron transport activity in the cells. For measuring

whole chain transport and water oxidation mediated electron

transport activity the thylakoids were prepared.

Anacystis cells were treated with lysozyme and the

permeability of the cells was monitored by ferricyanide-dependent

oxygen evolution. The cells became permeable within 15 minutes of

incubation with lysozyme.

water to ferricyanide

The effects of copper, cadmium, thallium and lead on the

Hill reaction of spheroplasts were measured, using ferricyanide

as electron acceptor. The Hill reaction was inhibited by 33%, 40%

and 16% with Cu, Cd and Tl respectively. Lead did not have any

effect on the Hill reaction of spheroplasts {Table 10).

Water to methyl viologen

Whole chain transport activity was inhibited only by copper

and cadmium. Inhibition by cadmium (80%) was more as compared to

Cu {68%). Higher concentrations of Tl had no affect indicating

that thallium inhibits on the photosynthetic electron transport

indirectly {Table 10).

DPC to MV

Since, DPC donates electrons to PS II, inhibition of oxygen

58

TABLE 10

Effect of various metals on photochemical activities of Anacystis nidulans

Heavy Metal

Control

Copper

Cadmium

Lead

Thallium

Concentration

140.0

1 124.0 2 109.2 5 93.6

1 84.8 2 84.8 5 82.8

2 145.0 5 154.0 10 140.0

1 148.0 2 124.0 5 117.0

78 171.6 312

56.7 38.0 24.6

15.6 140.0 335.4 15.6 129.0 342.0 15.6 129.0 340.0

74 171.6 312.0 78 171.6 322.0 76 171.6 318.0

78 171.6 323.2 78 171.6 321.0 78 171.6 310.0

evolving complex can be measured by adding DPC in the reaction

mixture. DPC bypassed the inhibition of Cd, thereby indicating

that Cd affects the oxygen evolving complex. DPC--->MV reaction

could not be measured in case of copper due to its reaction with

DPC. With Tl the activity was similar to that of control (Table

10) .

DCPIP to MV

Light induced transfer of electrons from reduced DCPIP to

methyl viologen was studied after inhibition of PS II by DCMU.

This reaction was not affected by any of the metals studied.

Consumption of oxygen in the above reaction was comparable to

control in treated spheroplasts. Since, copper reacts with Asc

(Rangnathan and Bose, 1991), PSI activity could not be measured

in case of cu treated spheroplasts and thylakoids. Cadmium showed

a slight enhancement in the PSI activity (Table 10).

6 .1. 2 Nostoc

6.1.2.1 ELECTRON TRANSPORT ACTIVITY IN INTACT CELLS

Polarographic measurements

In Nostoc the response of cells to metals was similar to that

of Anacystis.

Copper

Lower concentration (5pM) of copper did not have any effect

on PS II mediated oxygen evolution but at 15 pM the oxygen

evolution was reduced by 80% after four days of stress. Higher

concentrations of copper ( 25 and 35 pM) completely inhibited the

PS II activity within two days of'stress (Fig. 17).

59

Cu 12(} 11

0 ~6 N

040 ..,.. 20

0

~ ~ • 20

0 2 4 6 ~r of days

Cd

2 4 Number of days

2 4 Number of days

Fig. 17 Effect of heavy metals on electron transport (H2o---> pBQ) catalyzed by PS II in Nostoc cells. (O), (4), (O),C•H•> representc,s, 15, il'5,35" ~M concentrations of Cu, Cd, Tl and 75, 100, 150 IJM concentrations of Pb. The values are average of four experiments and S.D. was less than 5% in every case.

caam1um

PS II activity (H2o ---> pBQ) showed a decrease in cadmium

treated cells. The decrease in oxygen evolution was more with

higher concentrations of cadmium (Fig. 17).

Lead

PS II activity measured (H2 o-~->pBQ) was comparable to that

of control (Fig. 17).

Thallium

Lower concentrations did not inhibit PS II activity in the

cells. 25 JUM of thallium inhibi-t:ed PS II mediated oxygen

evolution by 60% after 6 days of incubation, while 35 fM of

thallium inhibited the activity by &0 % (Fig. 17).

Chlorophyll a fluorescence measurements

Copper

FvfFm ratio was not significantly affected by 5-15 ;u· concentrations of copper, but higher concentrations ( 25-35 jUM)

totally quenched the variable fluorescence of the cells (Fig.

18) •

Cadmium

The Fv/Fm ratio showed a reduction in Cd treated cells and

this reduction was more at higher cbncentrations of cadmium (Fig.

18) •

Lead

Lead did not have any effect on the Fv/Fm ratio. The ratio

60

• Cu

2 4 6 Number of days

1.21 LLE 1.0: :> OB~~§f;~E~

0.6 0.4

0~ o!:,-.-----=2.__. __ 4..,. -~·6~

Number of days

u_E

1.2[ td

~0. 0.4 02

0

1.2

02

0

2 4 6 Number ot days

Tl

2 4 6 Number of days

Fig. 18 Fv/Fm ratio of Nostoc cells treated with heavy metals. (o), (4), (tJ) 1 (•)(•)representC.,5 1 151 2.5.35 uM concentrations of cu, Cd, Tl and 75 1 100, 150 uM concentrations of Pb.

was comparable to that of control (Fig. 18}.

Thallium

Fv/Fm ratio showed a decrease with increase in concentration

of thallium. The decrease was more after six days of stress

(Fig. 18).

6.1.2.2 ELECTRON TRANSPORT ACTIVITY IN THYLAKOIDS OF NOSTOC

water to ferricyanide

Copper inhibited the PS II a~tivity as measured by water to

ferricyanide assay. At higher concentrations the inhibition was

more (52%). With cadmium the inhibition was about 42%. Lead and

thallium did not have any affect (Table 11} .

There was reduction in whole chain transport activity (H2o ---> MV) by 11% at lower concentration and this reduction was more

at higher concentration of copper (70%). The whole chain

transport activity also showed a reduction by 86% in activity at

all concentrations of cadmium. Lead and thallium did not affect

the whole chain transport (Table 11).

DPC---> MV

As DPC reacts with copper, the activity could not be

measured with copper. Addition of DPC restored the activity in

cadmium treated thylakoids thereby indicating that cadmium

affects the oxygen evolving complex (Table 11}.

DCPIP---> MV

None of the metals tested had any inhibitory effect on the

61

TABLE 11

Effect of various metals on photochemical activities of Nostoc muscorum

Heavy Concentration H2o..., Fe Metal

(CN) 6 H2o...,MV opc...,MV DCPIP...,MV

-----------------------------------------------------------------Control 135.0 84.0 171.6 382.0

Copper 1 120.0 58.6 2 110.6 45.2 5 64.6 24.6

Cadmium 1 30.8 11.6 156.0 390.6 2 80.8 11.6 156.0 385.0 5 80.8 11.6 156.0 390.0

Lead 5 140.0 78.0 171.6 380.0 10 148.6 78.0 168.0 385.2 15 148.6 78.0 168.0 382.9

Thallium 1 148.6 84.0 168.0 396.2 2 135.0 84.0 165.0 398.2 5 130.8 84.0 171.6 374.4

PSI activity (Table 11).

6.2 SPECTRAL PROPERTIES OF METAL TREATED CELLS

6.2.1 Anacystis

6.2.1.1 Absorption Spectra

The absorption spectra of intact cells treated with 15 uM

concentration of Cu, Cd, Tl and 150 pM of lead was taken by

keeping the chlorophyll concentration constant at 5 ~g/ml.

The absorption spectrum of the untreated cells showed three

peaks. The peak at 622 nm and 680 nm corresponds to the

absorption of phycobiliproteins and chlorophyll a respectively,

while the peak at 440 nm represents soret band of chlorophyll a

(Fork and Mohanty, 1986) (Fig. 19a}.

Copper

In copper treated cells the peak at 622 nm was more

affected. 5 pM of copper had very less effect on phycocyanin

peak, but at 15 p.M this peak was reduced and there occured

considerable reduction in the chlorophyll peak thereby changing

the PC/chl a ratio to 0.85 (Table 12}.

Cadmium

In cadmium treated cells (15 pM} there occured a peak shift

of 2nm towards blue region at 680 nm (Fig. 19a}, while 5 uM of Cd

caused peak shift of only 1 nm. The absorbance at 680 nm was also

reduced considerably causing the PC/chl a ratio to increase from

1.09 to 1.125 (Table 12).

62

Q6

~ o. c 0 .0 '-0 V)

~ 0.2

400

0.6

'-0

~0 <(

440 ....

500

500

622

Wavelength, nm (a)

600 Wavelength, nm

(b)

700

~0

-c -·-·Cu ---Cd

800

-c --Pb -··-TI

800·

Fig. 19 (a) Absorption spectra of intact Anacystis cells treated with 15 p.M of Cu and Cd. The spectra was taken after 4 days of stress. (b) Absorption spectra of Intact Anacystis cells treated with 150 pM of Pb and 15 pM of Tl. The spectra was taken after 4 days of stress.

TABLE 12

Effect of metals on the absorption properties of the intact cells of Anacystis. Cells were grown in the presence of 15 pM of metals for 4 days

-----------------------------------------------------------Heavy Peak 440/680 490/680 622/680 metal Position Cart. /Chl PC/Chl -----------------------------------------------------------Control 440 622 680 1. 44 1.047 1. 095

cu 440 680 1. 64 1.176 0.851

Cd 440 622 678 1. 51 1.1 1.125

Pb 440 622 680 1. 44 1. 047 1. 095

Tl 440 622 680 1.6 1.175 1. 075

Lead

Lead had no affect on the absorption spectra of the cells

(Fig. 19b).

Thallium

In thallium treated cells, both chl a and phycocyanin were

affected. The phycobiliproteins are affected to a greater extent

as compared to chlorophyll. The ratio of PC/chl did not show a

very significant change at 15 ~M of thallium {Table 12). 5 pM of

thallium did not have any effect on the absorbance spectra (Fig.

19b).

6.2.1.2 Fluorescence spectra of the cells

Emission spectra (Room temperature)

At 440 nm: Since, metals affected the absorption of phycocyanin

and chlorophyll, the fluorescence emission spectra of intact

cells were recorded to understand the nature of alterations

induced in these pigments. The fluorescene emission

characteristics of the cells were taken on equal O.D. and equal

chlorophyll basis (Fig. 20).

On excitation with 440 nm the cells showed two emission

peaks. Emission at 650 nm emanates from phycobilisomes while at

685 nm from chlorophyll. The fluorescence spectra is similar to

that reported earlier (Singhal et al., 1981; Mullineaux and

Allen, 1988).

Copper

Cells exposed to 5 pM of copper showed a 20% decrease in

emission of PC (at 650 nm) {Table 13) and a peak shift of 3 nm. At

15 pM the phycobilisome peak at 650 nm disappeared (Fig. 20).

There was large decrease in the fluorescence at 685 nm at all

63

GO

50

- 40 ~

s 1! ~ 30 iii c

.Sd c:

... ~ 20 ... ~ ~ g u:

10

---- -C ---Cu - --Cd 60

--Pb -··- Tl -c

-·- Cu --- Cd

501

I --Pb --- Tl

I

I

~J 6_?!; ,' I

I ~ I I

682 .... 658 ,. --,

I

I ,.._

>. \ ~

11 650 Ill 682

~ ~ 3') \ ~

C!J

680 u c Cll ,

I I

I

I

/

.....

\ :;; 20 /

!' 6W '. I \

\ 0 :J c

' \ 10

I

I 650 m - 65()

Wa~ngth, nm (a)

Wavelengtn, n m (b)

Fig. 20 (a)Effect of metals on fluorescence spectra of intact Anacystis cells at room temperature. The cells were excited at 440 nm (slit width for excitation 10 nm and emission 5 nm). Cells equivalent to 5 pg of chl a were suspended in 3 ml of reaction buffer. (b) Effect of metals on fluorescence spectra of intact Anacystis cells at room temperature. The cells were excited at 440 nm (slit width for excitation 10 nm and emission 5 nm ). The spectra of the cells was taken by keeping the O.D. of the cells constant at 700 nrn.

--J

TABLE 13

Effect of different concentrations of heavy metals on the emission properties of chl a at room temperature. The cells were excited at 440 nrn.

Heavy Metal

Control

Copper

Cadmium

Lead

Thallium

Concentration

5 10 15

5 10 15

75 100 150

5 10 15

Peak Position

PC Chla

650 684

647 684 645 684

680

656 682 658 680 658 680

650 684 650 684 650 684

650 684 650 682 650 682

Peak ratio 650/684

0.557

0.428 0.375 0.26

0.625 0.690 0.759

0.557 0.557 0.557

0.675 0.680 0.685

concentrations of copper, but a blue shift of 4 nm was observed

only at 15 f-M.

Cadmium

On treatment with 5 JUM of cadmium, the cells showed an

increase in fluorescence intensity of phycobiliproteins with a

red shift of 6 nm (Table 13). The chlorophyll fluorescence also

increased with a blue shift of 2 nm. At 15 pM of cadmium, the

increase in the fluorescence intensity of phycocyanin and

chlorophyll was more and the peak emanating from phycocyanin

showed a red shift of 8 nm, while that emanating from chlorophyll

showed a blue shift of 4 nm (Fig. 2 0) • The peak ratio (PC/Chl a)

also showed an increase from 0.5 to 0.7.

Lead

Pb had no affect on the fluorescence emission of the cells

significantly (Fig. 20).

Thallium

In thalliun treated cells the relative fluorescence

intensity was less and PC did not show any shift in peak. The

decrease in fluorescence intensity of phycobilisomes and

chlorophyll is more at higher concentrations of thallium. The

684 peak showed a blue shift of 2nm {Table 13}.

At 545 nm: The cells were excited at 545 nm to study the changes

in the phycocyanin fluorescence. The spectra showed a prominent

peak at 650 nm emanating from PC and a shoulder at 685 nm (Fig.

21}. The spectra is similar to that obtained by Mullineux and

Allen, (1988). 15 pM of Copper caused almost 90% decrease in

fluorescence emission intensity and the position of the peak

shifted from 650 nm to 645 nm.

On treatment with, 5 fM of cadmium, there was 16% increase

64

90

8

701

.-...

601 (./)

c .:J

~ '--'50 >-_... 'Vi c CJ - 40 c

QJ u c 8 30 g] I L

0 .:J

lL 20~

I I

10i I

600

654 , '·

' ' I \

I

I

I

\

\

I

' I

ffiO • , I

I ' I

I I • I , I

645

65') V.v':Ive.engt h, nrr1

-- Cu --- Cd -C --Pb -··-TI

' '

\ ' \

'~ '

700

Fig. 21 Fluorescence spectra of intact Anacystis cells treated with heavy metals. The cells were excited at 545 nm to specifically excite the phycobilisomes {slit width for excitation 10 nm and emission 5 nm ) . Cells equivalent to 5 pg Chl a were suspended in 3 ml of reaction buffer.

TABLE 14

Metal induced alterations in the PC fluorescence emission characteristics at different concentration of metals in intact cells of Anacystis. The cells were illuminated with 545 nm light beam to excite phycobilisomes

Heavy Concentration Metal

Control

Copper 15

Cadmium 15

Thallium 15

Peak position

650

645

654

650

Intensity

74

8

91.0

59.0

% increase(+) or decrease(-)

-89%

+23%

-20%

in fluorescence emission and a red shift of 3 nm was seen. At

higher concentration ( 15 fM) there was 23% increase in

fluorescence emission with a red shift of 4 nms. In thallium

treated cells the decrease in the intensity was found to be 20 %

with no peak shift (Table 14).

Emission spectra with addition of menadione in Cd treated cells

When 1rnM of menadione , a chlorophyll fluorescence quencher,

was added to the cells the peak at 685 nm showed a decrease,

indicating that there is a back transfer of energy from PS I to

APC-B (Fig. 22).

Excitation spectra

Since, the fluorescence at 685 nm showed an increase under

cadmium stress, the excitation s~--ectra of the cells was taken.

The excitation spectra of control cells shows a peak at 662 nm

and shoulder at 650 nm. The spectral properties are similar to

those reported by Goedheer, {1968); Fork and Mohanty, {1986) (Fig.

23). In cadmium treated cells an increase in the intensity at 662

nm was observed, when cells were excited at 685 nm. This suggests

that Chl-proteins of PS II are unable to recieve light energy

from PBSornes.

The excitation spectra with 715 nm the light energy

emmitted by PS I) also showed an increase at 662 nm in cadmium

treated cells, which shows that APC-B is recieving light energy

from PSI.

Emission spectra at 77 K

The chlorophyll fluorescence emission spectra were recorded

at low temperature to find out the specific site of action of the

metal ions. The fluorescence spectra of the cells showed four

65

50

_ -Menadone

-.- .. Menadione

650 Wavel~ngth, nm

c

700

eo

_ - t-Je nod ~one

50 -- • +Me nodi one

~ -- ... !

g20 C!J

~ LL I

'l// 600 650

Wave-length, nm

,

Fig. 22 Effect of menadione on the quenching of fluorescence emission of Anacystis nidulans cells (a) control (b) cadmium treated cells

Cd

700

Cl u c

70

60

50

~ 30 ~ g

u..

~ 2 -0 ~ a:

10

Fig. 23

F6B5

I

I

I

/ I

/

~0

I

I

I

I

I

I

I

-C - -.Cd

662 ~

I

I

,-,

1~715 sol f

~I.,Q ·c :l

~ .........,

~30, Ill c c.. c

I /

I

I I

620 W .Jv~lc-ngtn, nm Wavel~ngth1 rrn

Fluorescence excitation spectra of control and cadmium treated Anacystis cells (a} at F685 nm (b) F715 nm.

I

I

-C -- -Cd

660 ,., , \

peaks on excitation at 440 nm; emission band at 650 nm due to PC;

at 685 nm due to APC-8; 695 nm due to PS II reaction centre; and

at 715 nm due to PS I reaction centre. The spectrum is similar to

that observed by Mimuro and Fujita (1980); Singhal et al., (1981)

in Anacystis nidulans (Fig. 24).

Copper

Copper caused a considerable decrease in the fluorescence

intensity of PC and APC-8. The emission band at 695 nm is absent

(Fig. 24). Since the 695 nm band is mostly contributed by PS II

reaction centre chlorophyll protein, it suggests that copper

affects the PS II reaction center. The PC peak shifted to 645 nm

from 650 nm {Table 15).

Cadmium

In contrast to room temperature, there occured only a slight

increase in the fluorescence intensity of phycocyanin in cadmium

treated cells at low temperature (Fig. 2 4) . The emission

intensity of the peak emanating f~om APC-8 also gets reduced. The

emission band at 695 nm was totally missing. However, there was

large increase ln the fluorescence intensity of the peak

emanating from PS I. The PC emission band shifted towards red

region by 8 nm and APC-8 towards blue region by 3 nms (Table 15).

The peak shift in all these peaks with cadmium treatment suggests

that there occur structural alterations in the pigment-proteins

involved in photosynthesis.

Thallium

Thallium treated cells showed a decrease in the fluorescence

intensity at 650, 685 and 715 nm. The band at 695 nm is totally

absent as in case of other metals (Fig. 24).

66

60--------------------------------------~

J -c

VI' .... r:: ::I

~40 ..._, >­...

"iii r:: «<I

c 30 ....

I L __ 600

___.. 650

I , I

: ' \' ' , . '

\ I ' . ~. I' ....

Wavelength, rvn

' I I

I

715 I

-·-(U I

~.~-~I

I

I

'· \

' '

Fig. 24 Effect of metal treatment on low temperature (77K) fluorescence emission spectra of intact Anacystis cells. The cells were excited at 440 nm with excitation slit width 10 nm and emission slit width 5 nm. Cells equivalent to 5 pg/ml chl a were taken for recording the spectra.

TABLE 15

Effect of different concentrations of metals on the fluorescence emission properties of cells at low temperature (77K). The cells were excited at 440nm. The excitation slit width was lOnm and emission slit width Snm.

Metal Cone. PC APC B PSI I PSI 685/650 715/685 fluorescence fluorescence

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

Cont 560 685 695 715 1. 61 . 0.02 1. 17. 0.17

Cu 15 645 684 715 1. 66 . 0.04 1.33• 0.10

Cd 15 654 682 712 1.31 . 0.06 2.00· 0.02

T l 15 652 685 712 1.85 . 0.05 1. 18· 0.012

Fig. 25 Polypeptide profile of total soluble cellular proteins of Anacystis cells in SDS-PAGE.

Pb Tl Cd Cu C M

+-- 66 +- ~5

~ 36

+- 29 +- 24 r 20.1 ~ 14.3

Total proteins

SDS-PAGE analysis shows that copper and cadmium caused

degradation of 18 kDa and 20kDa protein which represent the a and

13 chain of PC. Other phycobiliproteins were not affected by

metals. Besides, copper caused degradation of proteins ranging

from 25 to 75 kDa proteins. Thallium had no effect on thylakoid

and phycobiliproteins. A polypeptide of 38 kDa was absent on

treatment with all metals (Fig. 25).

6.2.2. NOSTOC

The filaments of Nostoc were lysed by sonication and the

absorption spectra was scanned from 400 nm to 800 nm (Fig. 26a).

The changes in the pigment concentration due to effect of metals

is indicated by the intensity of the peaks. In copper treated

cells, PE and PC peaks were absent and chlorophyll peak also

showed a decrease. In Cd treated cells, the phycobiliproteins

were not affected considerably, however a decrease in the

chlorophyll peak was observed. Lead did not have any affect on

Nostoc and thallium caused a general degradation of chlorophyll

and phycobiliproteins (Fig. 26b).

Total proteins

Copper caused a general degradation of proteins between 36

to 66 kDa. A polypeptide of 33 kDa. decreased on treatment with

all metals. Phycobiliproteins were most affected by copper and

cadmium but the major effect was on PE and PC (Fig. 27).

67

w u z <( co a::· ~ (!) <t

liJ u Z ·

~ a: 0 (/)

~

2.()

0.5

Q.O 400

600 "

480 567

500 600

WAVELENGTH (nrns) (C)

700

-C - ·- cu - - - Cd

BOO

2Dr-----------------------------~ -C

... .... _ .. __

WAVELENGTH ( nms) (b)

-- Pb - ·· -TI

Fig. 26 (a)Effect of Cu and Cd on the pigments of Nostoc cells after four days of stress. (b) Effect of Pb and Tl on the pigments of Nostoc cells. The spectra was taken after lysing the cells.

Fig. 27 Polypeptide profile of total soluble cellular proteins of Nostoc cells in SDS-PAGE.

C Cu Cd T! M

\

~66

+- 45

~ 36 ~ 29 ~ 2G. ~ 20 1

r ~ 14 3 ~

DISCUSSION

6.3 ELECTRON TRANSPORT ACTIVITY

The effect of copper on the H2o --> pBQ and H2o --> MV

indicates that copper inhibited the primary PS II photochemistry.

The absence of peak at 695 nm in liquid nitrogen spectra of the

cells treated with copper further confirms that copper affects

the photosystem II. However, these results do not rule out the

possibility of copper inhibiting at oxygen evolving complex and

PS I reaction centre. These reactions could not be measured due

to reaction of copper with DPC and autooxidation of Asc in

presence of copper.

Copper also caused the quenching of chlorophyll fluorescence

as measured by transients. The quenching of Fv by copper could be

due to reduction of QA or Q8 , which leads to inhibition of both

electron transport and variable fluorescence (Mohanty et al.,

1989). But even the systems devoid of QA showed quenching of

fluorescence (Ranganathan and Bose, 1991) thereby indicating that

inhibition of electron transport activity and variable

fluorescence could be due to inhibition of charge separation

between P-680+ and Pheo or the energy of the charge separated

species is dissipated through an unknown way which competes

favourably with the fluorescence and electron transport

(Ranganathan and Bose, 1991).

Michel and Deisenhofer {1988) have suggested that the

chlorophyll molecules of P-680 and non-heme iron atoms make

ligands with histidine amino acids at positions 198 and 215, in

01 and 02 proteins. Copper by interacting with any one of these

histidines may disturb the environment of prosthetic groups,

thereby perturbing the normal functioning of the reaction centre.

Since, Ranganathan and Bose did not observe total

fluorescence quenching in PS I I of pea chloroplasts they

suggested that copper inhibits the photochemistry of only a

68

fraction of PS II centres (centres/ B-type centres), while other

fraction (B-centresj non-B type centres) are insensitive to

copper. Such type of heterogeneity has not been found in

cyanobacteria as yet and even if any heterogeneity exists, all

the centres are likely to become inactive after longer incubation

with metals thereby, leading to total quenching.

In Cd treated cells, the whole chain transport (H2o --> MV)

and H2o --> pBQ was inhibited, which indicate that cadmium

affects the photosystem II. Addition of DPC restored the activity

of treated cells, thereby showing that the major site of

inhibition is the oxygen evolving complex. Since, DPC donates

directly to p 680 , the probable site of action of Cd seems to be

before z. Mn substitution by Cd may be the cause of inhibition of

oxygen evolving complex, as Cd and Zn toxicity has been found to

be reduced by Mn (Hampp et al., 1976). The effect of cadmium on

the donor side of PS II has been shown by Van Duijvendijk-

Matteoli and Desmet (1975) in isolated chloroplasts. A decrease

in Fv/Fm ratio was also observed which further indicated that Cd

affects the donor side of the PS II (lying on the inner surface

of the thylakoid) by penetrating the thylakoid membrane.

The decrease in the fluorescence could also result from

changes in the Chl-protein interactions occuring in the PS II as

is also evident from the liquid nitrogen spectra of the intact

cells.

Lead did not have any effect on the electron transport

activity, suggesting that algae are resistant to lead.

Thallium affected the PS II activity only after longer

incubation at higher concentrations. Addition of thallium to the

thylakoids did not have any effect on the H2o-> Fe(CN) 6 , H20->MV

and DPC-->MV activity, thereby indicating that thallium affects

the electron transport activity through some indirect mechanism.

The decrease in the PS II activity could be due to its ability to

strongly bind to 'the membranes c:.t the potassium sites thereby

69

causing an injury to the membrane (Hughes and Poole, 1989}. Thus,

the toxicity of thallium appears to be due to the degradation of

the thylakoids. Fv/Fm ratio and decrease in PS II activity

indicates that thallium affects the donor side of PS II.

Nos toe

Effect of all the metals in Nostoc were similar to that of

Anacystis. H2o---> pBQ activity assayed in intact cells decreased

in a dose dependent manner. The Fv /Fm ratios obtained were

similar to Anacystis showing thnt cyanobacteria have same site

specificity towards metals. Cc.dr.1ium induced inhibition of

electron transport system of Nostoc has been reported by Husaini

et al., (1991).

6.4 Spectra of intact cells

6.4.1 Anacystis

Absorption spectra: The absence of peak at 622 nm in the

absorption spectra of the copper treated c·ells indicate that

copper caused bleaching of phycocyanin. The decrease in the

absorbance at 680 nm also indicates that copper affects the

chlorophyll biosynthesis as well. The decrease in carotenoids of

metal treated cells could result from its degradation or

inhibition of biosynthesis (Stihorova et al., 1986; Sandmann and

Boger, 1980 a, b).

From the absorption spectra of the cadmium treated cells it

is clear that cadmium did not have any effect on the absorption

of PC, but it affected the absorption of chlorophyll. The

decrease in the absorbance of the chlorophyll could be due to

inhibition of chlorophyll biosynthesis or alteration in structure

of chlorophyll. The peak at 680 nm also showed a shift of 2 nm

thereby indicating the structural alterations in PS II. It also

showed that the effect of cadmium on chlorophyll is more as

70

compared to copper.

Lead had no effect on the absorption spectra of cells.

Thallium caused a decrease in absorption of phycobilisomes as

well as chlorophyll indicating that it is affecting the

biosynthesis or causing the degradation of

proteins.

Fluorescence spectra

these pigment-

At room temperature: The fluorescence spectra of cells treated

with copper showed that copper completely quenches the

fluorescence of phycocyanin and this quenching is due to the

structural alterations induced in PC due to binding of copper to

~-chain of the phycocyanin. A blue shift of 5 nm at 650 nm

indicates that copper binds to the f3 -chain of PC and brings a

change in the conformation of the protein, which causes a

decrease in the absorption as well as fluorescence of phycocyanin

(Park and Sauer, 1991). Our results are in agreement to that

obtained by Murthy and Mohanty, (1991) on treatment of Spirulina

cells with mercury. The decrease in fluorescence of chlorophyll

and a shift of 3 nm indicated that Cu caused structural

alterations in the PS II as well.

Cadmium caused an increase in fluorescence of the cells at

650 nm and the peak showed a red shift of 8 nm. The red shift in

the peak shows that the major emission is coming from APC. This

increase in the intensity of APC over PC could be due to changes

in aggregation state of the phycobilisomes so that energy is not

efficiently transferred to PS II. Schrieber et al., 1979 also

showed a cold induced decrease in the energy transfer from

phycobilisomes to Chl a in Anacystis nidulans due to enhancement

of APC fluorescence. A red shift in the peak also indicates that

energy transfer is occurring through a spectrally distinct PC

within the rods of phycobilisomes (Yamazaki et al., 1984; Bruce

et al., 1985) . The peak at 685 nm also showed an increase in

fluorescence intensity. Since, 685 nm peak is contributed by long

71

wavelength form of APC and PS II at room temperature (Gantt,

1981), the increase in the intensity of fluorescence at 685 nm

could be due to

alteration in structure of APC so that less energy is

transferred to PS II.

alteration in the structure of PS II as indicated by the

blue shift in the peak, resulting in its inability to accept

the energy from phycobilisomes (Murthy, 1991).

back transfer of energy from chlorophyll to APC (Mohanty et

al., 1985).

To check for the possibility of back transfer of energy from

chlorophyll a to PC and APC, menadione was added to the cells,

which quenches the chlorophyll fluorescence. Addition of

menadione caused a greater decrease in fluorescence at 682 nm as

compared to fluorescence at 658 nm. This shows that there is a

back transfer of energy from Chlorophyll a to APC-B and APC. APC­

B is not considered to be directly linked in the linear energy

transfer (Yamazaki et al., 1984) . A greater back transfer of

energy to APC-B from chl a is to protect the photosystem from the

damage caused by strong illumin;]tion where APC-B acts as a

energy sink to dissipate excess excitation energy. So, the effect

of cadmium might be similar to light stress (Mohanty et al.,

1985). Similar increase of fluorescence of APC-B has been

observed by Mohanty et al., (1985) under heat stress. The

excitation spectra further confirms these results suggesting that

PC, APC and APC-B transfer energy to chl a in PS I (Cho and

Govindjee, 1970; Mohanty et al., 1985). These results also

suggest that PS I is also excited by phycobilisomes and there is

a back transfer of energy from PS I to APC-B as is evident from

excitation spectra with 685 nm.

Lead did not have any effect on the fluorescence spectra of

cells. The cells treated with thallium showed a decrease in the

intensity of phycobilisomes as well as chlorophyll, which shows

that the decrease in fluorescence could be due to less absorption

by the phycobi l isomes. A decrease in the fluorescence of

72

chlorophyll a may be due to less energy being transferred from

the phycobilsomes. The decrease in absorption as well as

fluorescence could be due to structural alterations in the

phycobilisomes and thylakoids.

At 77 K: Since at room temperature the fluorescence of the cells

is affected by many factors (Krause et al., 1983; Mullineauex and

Allen, 1990; Krause and Behrend, 1983) and PS I does not show

emission, the spectra of the cells was taken by freezing them in

liquid nitrogen.

The spectra of the cells treated with copper showed a large

decrease in fluorescence at 650 and 685 nm. The peak at 695 nm

disappeared indicating that copper affects the structure of PS

II. The peak at 695 nm is contributed by 47 kD protein which is

intimately associated with PS II photochemistry (Krey and

Govindjee, 1966; Nakatani, 1983), so the absence of peak could be

due to the degradation or change in the conformation of this

protein.

In cadmium treated cells the increase in intensity at 650 nm

and a peak shift by 6 nm at room temperature indicate that

structural alterations are occuring in the phycocyanin. However,

the decrease in fluorescence at 77 K could be due to decrease in

the back transfer of energy i.e. from chlorophyll a to APC, as

the back transfer is unlikely to occur at 77 K (Mohanty et al.,

1985) . The intensity at 685 nm, which represents the peak of APC­

B, decreases which could also be due to reduction in back

transfer of energy. The absence of peak at 695 nm and a shift of

2 nm in 685 nm peak indicated that there may be some structural

alteration in the PS I I resulting in its inability to accept

light energy. At lower concentration of cadmium there was not

much alteration in the PC to Chl a ratio and the spectra showed

that energy transfer occurs from PC to Chl. But at higher

concentrations the energy transfer was less probably due to the

da:rr.age caused by cadmium to PS II antenna thereby making it

unable to recieve energy from phycobilisomes.

73

An increase in the fluoresce~ce intensity of PS I was also

observed indicating more energy being transferred to PS I as

compared to PS II. The mechanism by which state transitions in

phycobilisome-containing organisms are controlled remains

controversial (Mullineaux and Allen, 1990; Fork and Satoh, 1986).

The proposed mechanisms are

state transitions in red algae are controlled by the redox

state of PQ (Murata, 1969; Reid and Reinhardt, 1980) similar

to the mechanism found in higher plants. The increase in redox

potential causes phosphorylation of the protein which links

the phycobilisomes to PS II, followed by association of

phycobilisomes to PS I.

increase in spillover of energy to PS I.

- functional decoupling of the phycobilisomes from PS II and

coupling to PS I.

- state transitions are induced

gradient around PS II and PS I

by localised electrochemical

(Biggins et al., 1984). The

increased turnover of PS I generates a localized change in

charge distribution which in turn leads to a small

conformational change producing the functional effects of a

state 1 transition. Similarly state transition is induced by

localised electrochemical gradient around PS II.

state transitions may also be induced by respiratory electron

transport flow into the PQ pool as well as by PS II turnover

in cyanobacteria (Mullineaux and Allen, 1986; Dominy and

Williams, 1987).

high rate of cyclic electron trasport around PS I also induces

state transitions (Satoh and Fork, 1983).

An increase in the redox pot.ential of PQ is unlikely to

increase due to inhibition of oxygen evolving complex by cadmium.

Increased spillover of energy to PS I might occur due to the

cadmium induced conformational changes in the thylakoids thereby

modifying the orientation and the distance between the pigments

(Biggins and Bruce, 1989; Bruce et al., 1985). This might involve

increased membrane fluidity, as a result, distance between PS II

74

centres increases and distance between PS I and PS II decreases

leading to greater spillover of energy to PS I (Olive et al.,

1986). Since, Mg controls the distribution of excitation energy

between PS I and PS II, the increased spillover to PS I could be

due to deficiency of magnesium induced by cadmium.

The decrease in the fluorescence of PC in thallium treated

cells could be due to structural alteration in the pigment­

proteins of phycobilisomes. The absence of peak at 695 nm

indicates that PS II is being effected by thallium. The decrease

in the PS I fluorescence is due to decrease in energy transfer

from PS II to PS I.

6.4.2 Nostoc

The cells of Nostoc are filamentous and being larger in size

have a tendency to settle down in the cuvette, thereby making it

difficult to study their absorption spectra. Therefore, the cells

were lysed by sonication and the absorbance spectra was taken to

check the effect of metals on pigments. The effects on all the

pigments were similar to that of ~acystis. PE was also effected

by all the metals and appeared to be the most sensitive pigment

in Nostoc. Being the outermost pigment in the rods of

phycobilisomes it is more vulnerable to changes in the

environment. The inhibition of growth and pigment content in

Nostoc has been reported by Asthana et al., ( 1992), with Ni

(Raizada and Rai, 1985), with Cr and Pb (Singh and Rai, 1991).

Similar inhibition has been observed in other filamentous

cyanobacteria Anabaena with Cu, Ni and Fe ( Mallick and Rai,

1990; Rai et al., 1991).

The cells of Nostoc survived even at higher concentrations

of metals used. This could be due to the presence of mucilagenous

sheath which also helps in chelating the metals. Nostoc also

actively secrete polysaccharides in the culture medium which act

as metal chelators (Kaplan, 1988) thereby limiting the

availability of metals to the cells. Increased secretion of

75

extracellular polypeptides has been reported in

under cadmium stress (Mehta and Vaidya, 1978).

Nostoc cells

Conclusion

- Both Nostoc and Anacystis have same site specificity for

metals.

Copper has inhibitory action on PS II reaction centre.

- Effect of cadmium is mainly on the oxygen evolving complex

which could be due the displacement of Mn.

Thallium affects the PSII photochemistry through some indirect

mechanism, probably by damaging the membrane structure.

- Metals inhibited growth and the order of toxicity was

Cd > Cu > Tl > Pb. The inhibition of the growth was due to

increased cell lysis.

Degradation of PBSomes occured with metal treatment and

maximum effect was found on treatment with copper.

APC was found to be more resistant to metal treatment because

of lesser exposure to environment.

Fluorescence of intact cells indicated inhibition of PS II by

all metals (except Pb). It could result from degradation or

conformational changes of 47 kD protein.

Cadmium induced transition from state 1 to state 2 in the

cells as indicated by increase in fluorescence of PS I.

An increase in 685 nm peak is due to the back transfer of

energy from Chl a to APC-B.

76