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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