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Stabilization by glutaraldehyde fixation of chloroplast membrane structure and function against heavy metal ion induced damage
Plant Science Letters, 253--261
253
© Ekevier/North-HollandScientific Publishers Ltd.
STABILIZATION BY GLUTARALDEHYDE FIXATION OF CHLOROPLAST MEMBRANE STRUCTURE AND FUNCTION AGAINST HEAVY METAL ION INDUCED DAMAGE
B.C. TRIPATHY and PRASANNAMOHANTYa'* School of Environmental Sciences and aSchool of Life Sciences, Jawaharial Nehru University, New Deihi-110067 (India)
(Received July 8th, 1980) (Revision received January 20th, 1981) (Accepted February 27th, 1981)
SUMMARY Stabilization by glutaraldehyde fixation of chloroplast membrane structure and function against the damage by heavy metal ions has been investigated. Zn 2+, Co 2÷ and Pb 2÷ ions inhibit dichlorophenol indophenol supported oxygen evolution in isolated chloroplasts, as well as enhance the magnitude of 90°-light scattering by chloroplasts. Fixation of chloroplasts with glutaraldehyde protects the thylakoid membranes against the damage caused by Cd 2+ and Co 2÷ ions to photosystem II (PS II) dependent oxygen evolution. On the other hand, Zn 2+ and Pb 2÷ inactivate chloroplasts oxygen evolving system even in fixed chloroplasts. Fixation of chloroplasts with glutaraldehyde reduces the extent of enhancement of 90°-light scattering of chloroplasts caused by the addition of salts. Thus the fixation of chloroplast membranes with glutaraldehyde does not completely arrest the increase in light scattering induced by metal ions which suggests that some microconformational changes still persist even after glutaraldehyde fixation. Our results indicate that Zn 2+ and Pb 2+ cause damage to the electron transport system of chloroplasts and that glutaraldehyde fixation does not provide protection against Zn 2÷- and Pb2+-induced damage of chloroplast function. Co 2+ and Cd 2+ appear to bring about a loss in chloroplast function..probably by alteration of its structure and glutaraldehyde fixation arrests such ion induced damage of PS II mediated oxygen evolution.
*To w h o m eorrespondence should be sent.
Abbreviations: Chl, chlorophyll; DCIP, diehlorophenol indophenol; DPC diphenylearbazide; GA, glutsmidchyde; PS I, photnsystem I; PS II, photosystem II,
254
INTRODUCTION Many photosynthetic electron transport intermediates are proteins. Therefore, cross-linking of thylakoid membrane proteins has a profound effect on rotational and intramolecular rearrangement of the carriers involved in the electron transport processes. The characteristic features of chloroplast membranes immobilized by bifunctional glutaraldehyde (GA) are: resistance to treatment with acetone or Triton X-100 [ 1,2], loss of volume changes in an osmotic environment [1,3], light-induced proton uptake but no translocation of NH~ ions [4], lack of light induced scattering changes [3], absence of light induced phenazine methosulfate mediated quenching of chlorophyll a fluorescence [ 5--7] and absence of spill over of excitation energy from PS II to P S I [5]. GA-fixation not only blocks chloroplast reactions at specific sites [8] but also brings about some alteration of structure of both the photosystems [7,9]. However, it is proposed that the treatments by chemical reagents, which usually cause large structural changes in unfixed chloroplast membrane, do not bring about such large configurational or structural changes in GA-fixed chloroplasts, although some microconformational changes may still occur, even after fixation of chloroplast structures [ 3,6,7]. Our investigations (unpublished) as well as those of others have shown that heavy metal cations like Zn 2÷, Co 2÷, Pb 2÷ and Cd 2÷ induce structural and functional alteration of chloroplast membranes and cause damage to O2 evolution in isolated chloroplasts [10--13]. In the present communication, we report the effect of selected heavy metal ions (Zn 2÷, Co 2÷, Pb 2÷ and Cd 2÷) on PS II
Plant materials Barley (Hordeum vulgare, L. CVJB 65) plants were grown on petri plates fitted with water soaked filter sheets at 25 -+ I°C under continuous illumination as previously described [14,15]. Fully expanded leaves of ll--13
Chloroplast isolation Chloroplasts were isolated as described previously [14]. The grinding medium consisted of 500 mM sucrose, l 0 mM NaCI and 20 mM Hepes NaOH buffer (pH 7.6). The same grinding medium was used for suspending the chloroplasts. Chlorophyll (Chl) was determined according to Arnon [ 16].
Glutaraldehyde fixation GA-fixation was carried out after Zilinskas and Govindjee [6] and Papa-
255 georgiou [7]. Chloroplasts were washed twice in the homogenization buffer without sucrose to remove stroma proteins and were suspended in the above medium at a concentration of 150/~g Chl/ml. GA was then added to the washed chloroplasts to a final concentration of 1%. This suspension was stirred at 0°C in the dark for 5 min and then centrifuged at 2000 × g for 10 min. The pellet was washed twice to remove excess of GA and finally resuspended in the chloroplast isolation medium. The degree of fixation was checked by osmotic shock as described by Zilinskas and Govindjee [6].
Measurement of oxygen evolution DCIP supported O2 evolution was measured polarographically using YSI O2 probe as described elsewhere [17]. The reaction mixture (3 ml) consisted of 3 mM MgC12, 10 mM NaCl, 400 ~M DCIP and 50 mM Hepes--NaOH buffer (pH 7.0). Chloroplasts were added to the above reaction mixture to a final concentration of 100/~g of Chl. Each experiment was repeated at least three times.
Measurement of 90°-light scattering For measurements of 90°-light scattering, the excitation light (546 nm) was obtained through a monochromator, from a mercury lamp as the exciting source. Light scattering was measured at 90 ° to the direction of the exciting light through another monochromator at the same wave length of 546 nm. Details of the experimental set, up have been described elsewhere [15]. The enhancement in light scattering was calculated as percentage increase in absorbance, due to salt addition, at 546 nm over a chloroplast sample containing the same amount of Chl but without the addition of salt.
Chemicals The chemicals used were of the highest purity grade, commercially available. Zinc was used as ZnSO4, cobalt as CoSO4, cadmium as Cd(CH3COO)2 and lead as Pb (NO3)2. GA was obtained from Ladd Research Laboratory, U.S.A. RESULTS
Figure 1A shows that ZnSO4 (3 m M ) inhibitsoxygen-evolving activityof PS II by 80--90%, both in unfixed and GA-fixed chloroplasts.GA-fixation slightlyreduces the loss in oxygen-evolving activityby Zn 2÷ and this small protecting ability of GA-fixation remains almost constant with increasing concentrations of zinc salt (1--3 mM). Figure 1B demonstrates that the addition of various concentrations of ZnSO4 enhances the extent of 90°-light scattering by unfixed chloroplasts. Enhancement of light scattering by addition of Zn 2÷ almost increases linearly with increase in the concentrations of ZnSO4 up to 3 mM and then it tends to saturate. The addition of ZnSO4 (3 mM) enhances the 90°-tight scattering maximally by 90~o in unfixed chloroplasts, and 23% in GA-fixed chloroplasts, i Similarly, Pb 2÷ inhibits oxygen evolution both in unfixed and fixed
256
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Fig. 1. A: loss in the rate of 02 evolution as a function of ZnS04 in both GA-fixed and unfixed chloroplasts. The reaction mixture (3 ml) consists of 30 mM Hepes buffer (pH 7.0), 2 mM MgCI 2, 300 . m DCIP and 100 ~,g of Chl. The control rates of both fixed and unfixed chloroplasts were normalized to 1. Saturating rates of O2 evolution in fixed chloroplast was o f the order of 25--30 . t o o l O2/mg Chl/h and unfixed chloroplasts 6 0 - - 8 0 . mol O J m g Chl/h. The inhibition o f O= evolution b y the addition o f salt is expressed as fraction of the rate without the salt. B: effect of ZnS04 on the 90°-light scattering of GA-fixed and unfixed chloroplasts. Chloroplasts were suspended in 3 ml of 30 mM methylethanesulphonate buffer (pH 6.0). The enhancement was calculated as percentage increase over control ( - s a l t ) .
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unfixed c h l o r o p l a ~ . Other experimental conditions as in case o f Fig. 1A. B: enhancement of 9(F-light ~ a t t e r i n g b y a d d i t i o m o f variotm concentrations o f Pb (NO3)2 in GA-fixed and unfixed chloroplasts. Other experimental conditions are as in Fig. lB.
257
chloroplasts (Fig. 2A). The presence of low concentration of Pb(NO3)2 (0.1--0.5 raM) inhibits oxygen evolution by 8C 05% both in unfixed as well as GA-fixed chloroplasts. Thus, as in the case of Zn 2+, GA-fixation also fails to protect against the loss of oxygen-evolving activity of PS II induced by Pb 2+. Figure 2B shows the effect of addition of Pb 2+ on the 90°-light scat~ tering of GA-fixed and unfixed chloroplasts. Pb(NO3)2 (0.1 raM) induces about 55% enhancement of the extent of light scattering in unfixed chloroplasts, and with 0.2 mM it increases to 110%. The degree of enhancement of light scattering by addition of Pb(NO3)2 is maximal at 0.2 mM. On the other hand, in GA-fixed chloroplasts, 2 mM of Pb(NO3)2 causes the light scattering to increase only by 50% as against 110% in unfixed samples. The enhancement of light scattering upon addition of Pb(NO3)2 also saturates at 0.2 mM in GA-fixed chloroplasts. Figure 3A similarly demonstrates the effect of Cd 2+ ions on PS II mediated oxygen evolution in both unfixed and GA-fixed chloroplasts. Cadmium acetate (0.5--2.0 raM) brings about 80--90% inhibition of oxygen evolution. However, contrary to our observations with Zn ~+ and Pb 2+, GA-fLxation provides protection against the Cd 2+-induced loss in oxygen-evolving activity to the extent of 75--80%. There is only a slight decline in the rate of O2 evolution in GA-fixed chloroplasts with the increasing concentration of cadmium acetate from 0.5--2.0 raM. The effect of Cd 2+ on 90°-light scattering of unfixed and GA-fixed chloroplasts is shown in Fig. 3B; Cd 2÷ induces almost 100% enhancement of light scattering over control (without Cd 2÷) in unfixed chloroplasts. A somewhat linear increase in the magnitude
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Fig. 8. A: effect of . u ~ eonmMrations of Cd acetate on O= evolution of GA fixed and unfixed chloropkea. Other ~ condit/c~ m stmilsr in Fil. 1A. B: effeet of various e o n e s ~ o f Cd s e s t ~ on 90"-l/sht seatteri~ of GA-fixed and unf=ed chloroplasts. Other ezpeekMntai e o n d i t k ~ m s s in Fig. lB.
258 of enhancement of light scattering is observed upon addition of cadmium acetate up to a concentration of 1 mM and this enhancement tends to saturate at 2 raM. On the other hand, in GA-fixed chloroplasts, low concentration (0.5 mM) o f cadmium acetate fails to bring about any enhancement of 90°-light scattering. However, at higher concentrations of cadmium acetate (1--4 mM), only a small (10--20%) enhancement of 90°-light scattering is observed. Figure 4A shows the results of a similar experiment on the effect of Co ~÷ on PS II mediated oxygen evolution in unfixed and GA-fixed chloroplasts. 1.0
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259
With high concentration of CoSO4 (3--10 mM), the DCIP supported oxygen evolution is inhibited by 60--85% in unfixed chloroplasts and GA-fixation protects this inhibition of oxygen evolution by 80-85%. There is only a slight loss in oxygen evolution capacity in fixed chloroplasts with increasing concentration of CoSO4 from 3 mM to 10 raM. The changes in 90°-light scattering induced by CoSO4 addition to GA-fixed and unfixed chloroplasts are shown in Fig. 4B. High concentrations of CoSO4 (10 mM) enhances the light scattering of unfixed chloroplasts by 55%. However, in GAfixed chloroplasts, the addition of low concentrations of CoSO4 (2--4 raM) induces no significant enhancement of 90°-light scattering and even at higher concentrations (14 mM) the extent of 90°-ligh t scattering increases only about 4% over the control. DISCUSSION Heavy metal ions like Zn 2+, Pb 2+, Co 2+ and Cd 2+ when added to chloroplast membranes inactivate oxygen evolution with DCIP as oxidant. However, the manner in which these metal ions bring about the above inactivation seems to be differentfrom one another. W e have previously shown that Zn 2+ blocks electron flow at the water splittingside of PS II and this inhibition of the PS II reaction can by relieved by addition of an exogenous electron donor like N H 2 O H which donates electrons to the reaction centre of PS II [17]. Zn 2÷ inhibition of oxygen evolution can also be alleviated by washing the chloroplasts with isolation buffer [17]. These observations indicate that Zn 2+ simply blocks electron flow from water to the reaction centre of PS II and it does not grossly affect the chloroplast structure or the reaction centre complexes [17]. Pb 2+ ions at very low concentrations, also inhibit oxygen evolution by blocking the electron flow at the oxidizing side [11]. The Pb :÷ inhibition of PS II reaction, as in the case of Zn 2+, can also be bypassed by addition of an exogenous electron donor like N H 2 O H [11]. Again, these observations show that Pb 2÷ inhibits oxygen evolution by introducing a block on the oxidizing side of PS II and it perhaps also does not destroy the chloroplast structure or pigment complexes. On the other hand, Cd 2÷inhibition of PS II activity cannot be restored by addition of any of the exogenous electron donors like MnC12, diphenylcarbazide (DPC) or NH2OH [10] although Bazaz and Govindjee have shown that DPC partially overcomes Cd 2+ inhibition [12]. It appears that Cd 2+ directly inactivates the reaction centre-pigment complex of PS II [10]. Similarly, our work (unpublished) shows that Co2+-inhibition of PS II mediated reactions cannot be reversed upon addition of exogenous electron donors like MnC12, DPC or NH2OH. These observations suggest that Co 2+ like Cd 2+ appear to damage membrane structure and inactivate reaction centre-pigment complexes of PS II. Our present observations show that Zn 2÷ and Pb 2÷ inhibition of PS II activity cannot be protected by GA-fixation of chloroplast membranes
260
(Figs. 1A and 2A), whereas Cd 2÷- and Co2÷-inhibition can be considerably protected b y the GA-fixation (Figs. 3A and 4A). The above results can be explained as follows. Lack of inhibition of PS II-dependent 02 evolution in GA-fixed chloroplasts (Fig. 3A) by the presence of Cd 2÷ and Co 2÷ indicates that GA-fixation protects the chloroplast membranes against these heavy metal ion induced damage. These results also suggest that Cd 2÷ and Co 2÷ (Figs. 3A and 4A) affect or alter the pigment complexes and/or the structure of the thylakoid membrane resulting in the loss of light-induced electron flow and oxygen evolution. Unlike Zn 2÷ or Pb 2÷, the Cd 2÷ and Co 2÷ do n o t seem to block the electron flow from water to the PS II centres. This seems logical as GA-fixation offers no significant protection to the loss in PS IIdependent 02 evolution activity brought a b o u t by Zn 2÷ or Pb 2÷ treatment. Therefore, the failure of GA-fixation to protect the PS II activity against damage caused by Zn 2÷ and Pb 2÷ indicates that in GA-fixed chloroplasts the electron flow remains sensitive to inhibition by these t w o metal ions as it remains sensitive to the herbicide DCMU [7]. Analysis of 90°-light scattering changes caused by the above mentioned metal ions indicate that all the four metal ions tested enhance the magnitude of light scattering of unfixed chloroplasts by 50--110% (see Figs. 1B, 2B, 3B and 4B). These results suggest that addition of these metal ions cause structural alteration o f the chloroplast membranes causing volume changes [7]. In GA-fixed chloroplasts the extent of enhancement of 90°-light scattering brought a b o u t b y the additions o f various concentrations o f the heavy metal ions are significantly low. At low concentrations of metal ions, the ion induced enhancement of light scattering is small with the possible exception of Pb 2+. In Co2+-treated GA-fixed chloroplasts the 90°-ligh t scattering change almost vanishes except at high concentration of the salt (Fig. 4B). However, a reduced extent of enhancement of light scattering by the addition of metal ions persisted in GA-fixed chloroplasts which suggests that some microconformational changes can still occur even in chloroplasts fixed with GA. In summary, our results clearly show that Zn 2÷ and Pb 2÷ bring a b o u t damage to the chloroplast function mostly due to their ability to inhibit electron flow from water to PS II, while Co 2+ and Cd 2+ seem to destroy the chloroplast function by primarily damaging the chloroplast membrane structure and integrity and perhaps the reaction centre complex. The latter can be prevented by fixation with bifunctional agent such as GA. ACKNOWLEDGEMENTS
We thank Professor B. Bhatia for help and support and the Dean, School of Life Sciences, for providing facilities. We are also thankful to Professor Govindjee and Dr. George C. Papageorgiou for many helpful suggestions. The work is partly supported b y a grant from Government of India, DSTSERC 8{1)/78 to P.M. BCT is a recipient o f UGC fellowship.
261 REFERENCES 1 R.B. Park, J. Kelley, S. Drury and K. Sauer, Proc. Natl. Acad. Sci. U.S.A., 25 (1966) 1056. 2 P.V. Sane and R.B.Park, Plant Physiol., 46 (1970) 852. 3 S. Murakami and L. Packer, J. Cell Biol., 47 (1970) 332. 4 L. Packer, J.M. Allen and M. Starks, Arch. Biochem. Biophys., 128 (1968) 142. 5 P. Mohanty, B.Z. Braun and Govindjee, Biocbim. Biophys Acta, 292 (1973) 459. 6 B. Zilinskas and Govindjee, Z. Pflanzenphysiol., 77 (1976) 302. 7 G.C. Papageorgiou, Molecular and functional aspects of immobilized chloroplast membranes, in: Photosynthesis in Relation to Model Systems, J. Barber (Ed.), Elsevier, Amsterdam, 1979, p. 211. 8 H. Hardt and B. Kok, Plant Physiol., 60 (1977) 225. 9 T. Oku, K. Sugahara and G. Tomita, Plant Cell Physiol., 14 (1973) 385. 10 E.H. Li and C.D. Miles, Plant Sci. Left., 5 (1975) 33. 11 C.D. Miles, J.R. Brandle, D.J. Daniel, O. Cbu-Der, P.D. Schnare and D.J. Uhlik, Plant Physiol., 49 (1972) 820. 12 M. Bazaz and Govindjee, Environ. Lett., 6 (1974) 1. 13 M. Bazaz and Govindjee, Environ. Lett., 6 (1974) 175. 14 U.C. Biswal and P. Mohanty, Plant Cell Physiol., 17 (1976) 323. 15 U.C. Biswal, G.S. Singhal and P. Mohanty, Indian J. Exp. Biol., 17 (1979) 262. 16 D.I. Arnon, Plant Physiol., 24 (1949) 1. 17 B.C. Tripathy and P. Mohanty, Plant Physiol., 66 (1981) 1174.
253
© Ekevier/North-HollandScientific Publishers Ltd.
STABILIZATION BY GLUTARALDEHYDE FIXATION OF CHLOROPLAST MEMBRANE STRUCTURE AND FUNCTION AGAINST HEAVY METAL ION INDUCED DAMAGE
B.C. TRIPATHY and PRASANNAMOHANTYa'* School of Environmental Sciences and aSchool of Life Sciences, Jawaharial Nehru University, New Deihi-110067 (India)
(Received July 8th, 1980) (Revision received January 20th, 1981) (Accepted February 27th, 1981)
SUMMARY Stabilization by glutaraldehyde fixation of chloroplast membrane structure and function against the damage by heavy metal ions has been investigated. Zn 2+, Co 2÷ and Pb 2÷ ions inhibit dichlorophenol indophenol supported oxygen evolution in isolated chloroplasts, as well as enhance the magnitude of 90°-light scattering by chloroplasts. Fixation of chloroplasts with glutaraldehyde protects the thylakoid membranes against the damage caused by Cd 2+ and Co 2÷ ions to photosystem II (PS II) dependent oxygen evolution. On the other hand, Zn 2+ and Pb 2÷ inactivate chloroplasts oxygen evolving system even in fixed chloroplasts. Fixation of chloroplasts with glutaraldehyde reduces the extent of enhancement of 90°-light scattering of chloroplasts caused by the addition of salts. Thus the fixation of chloroplast membranes with glutaraldehyde does not completely arrest the increase in light scattering induced by metal ions which suggests that some microconformational changes still persist even after glutaraldehyde fixation. Our results indicate that Zn 2+ and Pb 2+ cause damage to the electron transport system of chloroplasts and that glutaraldehyde fixation does not provide protection against Zn 2÷- and Pb2+-induced damage of chloroplast function. Co 2+ and Cd 2+ appear to bring about a loss in chloroplast function..probably by alteration of its structure and glutaraldehyde fixation arrests such ion induced damage of PS II mediated oxygen evolution.
*To w h o m eorrespondence should be sent.
Abbreviations: Chl, chlorophyll; DCIP, diehlorophenol indophenol; DPC diphenylearbazide; GA, glutsmidchyde; PS I, photnsystem I; PS II, photosystem II,
254
INTRODUCTION Many photosynthetic electron transport intermediates are proteins. Therefore, cross-linking of thylakoid membrane proteins has a profound effect on rotational and intramolecular rearrangement of the carriers involved in the electron transport processes. The characteristic features of chloroplast membranes immobilized by bifunctional glutaraldehyde (GA) are: resistance to treatment with acetone or Triton X-100 [ 1,2], loss of volume changes in an osmotic environment [1,3], light-induced proton uptake but no translocation of NH~ ions [4], lack of light induced scattering changes [3], absence of light induced phenazine methosulfate mediated quenching of chlorophyll a fluorescence [ 5--7] and absence of spill over of excitation energy from PS II to P S I [5]. GA-fixation not only blocks chloroplast reactions at specific sites [8] but also brings about some alteration of structure of both the photosystems [7,9]. However, it is proposed that the treatments by chemical reagents, which usually cause large structural changes in unfixed chloroplast membrane, do not bring about such large configurational or structural changes in GA-fixed chloroplasts, although some microconformational changes may still occur, even after fixation of chloroplast structures [ 3,6,7]. Our investigations (unpublished) as well as those of others have shown that heavy metal cations like Zn 2÷, Co 2÷, Pb 2÷ and Cd 2÷ induce structural and functional alteration of chloroplast membranes and cause damage to O2 evolution in isolated chloroplasts [10--13]. In the present communication, we report the effect of selected heavy metal ions (Zn 2÷, Co 2÷, Pb 2÷ and Cd 2÷) on PS II
Plant materials Barley (Hordeum vulgare, L. CVJB 65) plants were grown on petri plates fitted with water soaked filter sheets at 25 -+ I°C under continuous illumination as previously described [14,15]. Fully expanded leaves of ll--13
Chloroplast isolation Chloroplasts were isolated as described previously [14]. The grinding medium consisted of 500 mM sucrose, l 0 mM NaCI and 20 mM Hepes NaOH buffer (pH 7.6). The same grinding medium was used for suspending the chloroplasts. Chlorophyll (Chl) was determined according to Arnon [ 16].
Glutaraldehyde fixation GA-fixation was carried out after Zilinskas and Govindjee [6] and Papa-
255 georgiou [7]. Chloroplasts were washed twice in the homogenization buffer without sucrose to remove stroma proteins and were suspended in the above medium at a concentration of 150/~g Chl/ml. GA was then added to the washed chloroplasts to a final concentration of 1%. This suspension was stirred at 0°C in the dark for 5 min and then centrifuged at 2000 × g for 10 min. The pellet was washed twice to remove excess of GA and finally resuspended in the chloroplast isolation medium. The degree of fixation was checked by osmotic shock as described by Zilinskas and Govindjee [6].
Measurement of oxygen evolution DCIP supported O2 evolution was measured polarographically using YSI O2 probe as described elsewhere [17]. The reaction mixture (3 ml) consisted of 3 mM MgC12, 10 mM NaCl, 400 ~M DCIP and 50 mM Hepes--NaOH buffer (pH 7.0). Chloroplasts were added to the above reaction mixture to a final concentration of 100/~g of Chl. Each experiment was repeated at least three times.
Measurement of 90°-light scattering For measurements of 90°-light scattering, the excitation light (546 nm) was obtained through a monochromator, from a mercury lamp as the exciting source. Light scattering was measured at 90 ° to the direction of the exciting light through another monochromator at the same wave length of 546 nm. Details of the experimental set, up have been described elsewhere [15]. The enhancement in light scattering was calculated as percentage increase in absorbance, due to salt addition, at 546 nm over a chloroplast sample containing the same amount of Chl but without the addition of salt.
Chemicals The chemicals used were of the highest purity grade, commercially available. Zinc was used as ZnSO4, cobalt as CoSO4, cadmium as Cd(CH3COO)2 and lead as Pb (NO3)2. GA was obtained from Ladd Research Laboratory, U.S.A. RESULTS
Figure 1A shows that ZnSO4 (3 m M ) inhibitsoxygen-evolving activityof PS II by 80--90%, both in unfixed and GA-fixed chloroplasts.GA-fixation slightlyreduces the loss in oxygen-evolving activityby Zn 2÷ and this small protecting ability of GA-fixation remains almost constant with increasing concentrations of zinc salt (1--3 mM). Figure 1B demonstrates that the addition of various concentrations of ZnSO4 enhances the extent of 90°-light scattering by unfixed chloroplasts. Enhancement of light scattering by addition of Zn 2÷ almost increases linearly with increase in the concentrations of ZnSO4 up to 3 mM and then it tends to saturate. The addition of ZnSO4 (3 mM) enhances the 90°-tight scattering maximally by 90~o in unfixed chloroplasts, and 23% in GA-fixed chloroplasts, i Similarly, Pb 2÷ inhibits oxygen evolution both in unfixed and fixed
256
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ZnS04, mM
4 6 8 ZnS04 ,mM
Fig. 1. A: loss in the rate of 02 evolution as a function of ZnS04 in both GA-fixed and unfixed chloroplasts. The reaction mixture (3 ml) consists of 30 mM Hepes buffer (pH 7.0), 2 mM MgCI 2, 300 . m DCIP and 100 ~,g of Chl. The control rates of both fixed and unfixed chloroplasts were normalized to 1. Saturating rates of O2 evolution in fixed chloroplast was o f the order of 25--30 . t o o l O2/mg Chl/h and unfixed chloroplasts 6 0 - - 8 0 . mol O J m g Chl/h. The inhibition o f O= evolution b y the addition o f salt is expressed as fraction of the rate without the salt. B: effect of ZnS04 on the 90°-light scattering of GA-fixed and unfixed chloroplasts. Chloroplasts were suspended in 3 ml of 30 mM methylethanesulphonate buffer (pH 6.0). The enhancement was calculated as percentage increase over control ( - s a l t ) .
10
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0.5
1.0 1.5 ~Pb(NO3)2,mM
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Fig. 2. A : effect of WL~OUS eoncenbrations of Pb (NO~)= on O2 evolution in OA-fixed and
unfixed c h l o r o p l a ~ . Other experimental conditions as in case o f Fig. 1A. B: enhancement of 9(F-light ~ a t t e r i n g b y a d d i t i o m o f variotm concentrations o f Pb (NO3)2 in GA-fixed and unfixed chloroplasts. Other experimental conditions are as in Fig. lB.
257
chloroplasts (Fig. 2A). The presence of low concentration of Pb(NO3)2 (0.1--0.5 raM) inhibits oxygen evolution by 8C 05% both in unfixed as well as GA-fixed chloroplasts. Thus, as in the case of Zn 2+, GA-fixation also fails to protect against the loss of oxygen-evolving activity of PS II induced by Pb 2+. Figure 2B shows the effect of addition of Pb 2+ on the 90°-light scat~ tering of GA-fixed and unfixed chloroplasts. Pb(NO3)2 (0.1 raM) induces about 55% enhancement of the extent of light scattering in unfixed chloroplasts, and with 0.2 mM it increases to 110%. The degree of enhancement of light scattering by addition of Pb(NO3)2 is maximal at 0.2 mM. On the other hand, in GA-fixed chloroplasts, 2 mM of Pb(NO3)2 causes the light scattering to increase only by 50% as against 110% in unfixed samples. The enhancement of light scattering upon addition of Pb(NO3)2 also saturates at 0.2 mM in GA-fixed chloroplasts. Figure 3A similarly demonstrates the effect of Cd 2+ ions on PS II mediated oxygen evolution in both unfixed and GA-fixed chloroplasts. Cadmium acetate (0.5--2.0 raM) brings about 80--90% inhibition of oxygen evolution. However, contrary to our observations with Zn ~+ and Pb 2+, GA-fLxation provides protection against the Cd 2+-induced loss in oxygen-evolving activity to the extent of 75--80%. There is only a slight decline in the rate of O2 evolution in GA-fixed chloroplasts with the increasing concentration of cadmium acetate from 0.5--2.0 raM. The effect of Cd 2+ on 90°-light scattering of unfixed and GA-fixed chloroplasts is shown in Fig. 3B; Cd 2÷ induces almost 100% enhancement of light scattering over control (without Cd 2÷) in unfixed chloroplasts. A somewhat linear increase in the magnitude
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Fig. 8. A: effect of . u ~ eonmMrations of Cd acetate on O= evolution of GA fixed and unfixed chloropkea. Other ~ condit/c~ m stmilsr in Fil. 1A. B: effeet of various e o n e s ~ o f Cd s e s t ~ on 90"-l/sht seatteri~ of GA-fixed and unf=ed chloroplasts. Other ezpeekMntai e o n d i t k ~ m s s in Fig. lB.
258 of enhancement of light scattering is observed upon addition of cadmium acetate up to a concentration of 1 mM and this enhancement tends to saturate at 2 raM. On the other hand, in GA-fixed chloroplasts, low concentration (0.5 mM) o f cadmium acetate fails to bring about any enhancement of 90°-light scattering. However, at higher concentrations of cadmium acetate (1--4 mM), only a small (10--20%) enhancement of 90°-light scattering is observed. Figure 4A shows the results of a similar experiment on the effect of Co ~÷ on PS II mediated oxygen evolution in unfixed and GA-fixed chloroplasts. 1.0
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CoSO4mM Fig. 4. A: effect o f various concentrations o f CoSO 4 o n O~ evolution o f GA-fixed and unfixed chloroplasts. Other experimental conditions are similar to Fig. 1A. B: Effect o f various concentrations of C0804 on 90°-light scattering o f GA-fixed and unfixed chloro. plasts. Other experimental conditions are as in Fig. lB.
259
With high concentration of CoSO4 (3--10 mM), the DCIP supported oxygen evolution is inhibited by 60--85% in unfixed chloroplasts and GA-fixation protects this inhibition of oxygen evolution by 80-85%. There is only a slight loss in oxygen evolution capacity in fixed chloroplasts with increasing concentration of CoSO4 from 3 mM to 10 raM. The changes in 90°-light scattering induced by CoSO4 addition to GA-fixed and unfixed chloroplasts are shown in Fig. 4B. High concentrations of CoSO4 (10 mM) enhances the light scattering of unfixed chloroplasts by 55%. However, in GAfixed chloroplasts, the addition of low concentrations of CoSO4 (2--4 raM) induces no significant enhancement of 90°-light scattering and even at higher concentrations (14 mM) the extent of 90°-ligh t scattering increases only about 4% over the control. DISCUSSION Heavy metal ions like Zn 2+, Pb 2+, Co 2+ and Cd 2+ when added to chloroplast membranes inactivate oxygen evolution with DCIP as oxidant. However, the manner in which these metal ions bring about the above inactivation seems to be differentfrom one another. W e have previously shown that Zn 2+ blocks electron flow at the water splittingside of PS II and this inhibition of the PS II reaction can by relieved by addition of an exogenous electron donor like N H 2 O H which donates electrons to the reaction centre of PS II [17]. Zn 2÷ inhibition of oxygen evolution can also be alleviated by washing the chloroplasts with isolation buffer [17]. These observations indicate that Zn 2+ simply blocks electron flow from water to the reaction centre of PS II and it does not grossly affect the chloroplast structure or the reaction centre complexes [17]. Pb 2+ ions at very low concentrations, also inhibit oxygen evolution by blocking the electron flow at the oxidizing side [11]. The Pb :÷ inhibition of PS II reaction, as in the case of Zn 2+, can also be bypassed by addition of an exogenous electron donor like N H 2 O H [11]. Again, these observations show that Pb 2÷ inhibits oxygen evolution by introducing a block on the oxidizing side of PS II and it perhaps also does not destroy the chloroplast structure or pigment complexes. On the other hand, Cd 2÷inhibition of PS II activity cannot be restored by addition of any of the exogenous electron donors like MnC12, diphenylcarbazide (DPC) or NH2OH [10] although Bazaz and Govindjee have shown that DPC partially overcomes Cd 2+ inhibition [12]. It appears that Cd 2+ directly inactivates the reaction centre-pigment complex of PS II [10]. Similarly, our work (unpublished) shows that Co2+-inhibition of PS II mediated reactions cannot be reversed upon addition of exogenous electron donors like MnC12, DPC or NH2OH. These observations suggest that Co 2+ like Cd 2+ appear to damage membrane structure and inactivate reaction centre-pigment complexes of PS II. Our present observations show that Zn 2÷ and Pb 2÷ inhibition of PS II activity cannot be protected by GA-fixation of chloroplast membranes
260
(Figs. 1A and 2A), whereas Cd 2÷- and Co2÷-inhibition can be considerably protected b y the GA-fixation (Figs. 3A and 4A). The above results can be explained as follows. Lack of inhibition of PS II-dependent 02 evolution in GA-fixed chloroplasts (Fig. 3A) by the presence of Cd 2÷ and Co 2÷ indicates that GA-fixation protects the chloroplast membranes against these heavy metal ion induced damage. These results also suggest that Cd 2÷ and Co 2÷ (Figs. 3A and 4A) affect or alter the pigment complexes and/or the structure of the thylakoid membrane resulting in the loss of light-induced electron flow and oxygen evolution. Unlike Zn 2÷ or Pb 2÷, the Cd 2÷ and Co 2÷ do n o t seem to block the electron flow from water to the PS II centres. This seems logical as GA-fixation offers no significant protection to the loss in PS IIdependent 02 evolution activity brought a b o u t by Zn 2÷ or Pb 2÷ treatment. Therefore, the failure of GA-fixation to protect the PS II activity against damage caused by Zn 2÷ and Pb 2÷ indicates that in GA-fixed chloroplasts the electron flow remains sensitive to inhibition by these t w o metal ions as it remains sensitive to the herbicide DCMU [7]. Analysis of 90°-light scattering changes caused by the above mentioned metal ions indicate that all the four metal ions tested enhance the magnitude of light scattering of unfixed chloroplasts by 50--110% (see Figs. 1B, 2B, 3B and 4B). These results suggest that addition of these metal ions cause structural alteration o f the chloroplast membranes causing volume changes [7]. In GA-fixed chloroplasts the extent of enhancement of 90°-light scattering brought a b o u t b y the additions o f various concentrations o f the heavy metal ions are significantly low. At low concentrations of metal ions, the ion induced enhancement of light scattering is small with the possible exception of Pb 2+. In Co2+-treated GA-fixed chloroplasts the 90°-ligh t scattering change almost vanishes except at high concentration of the salt (Fig. 4B). However, a reduced extent of enhancement of light scattering by the addition of metal ions persisted in GA-fixed chloroplasts which suggests that some microconformational changes can still occur even in chloroplasts fixed with GA. In summary, our results clearly show that Zn 2÷ and Pb 2÷ bring a b o u t damage to the chloroplast function mostly due to their ability to inhibit electron flow from water to PS II, while Co 2+ and Cd 2+ seem to destroy the chloroplast function by primarily damaging the chloroplast membrane structure and integrity and perhaps the reaction centre complex. The latter can be prevented by fixation with bifunctional agent such as GA. ACKNOWLEDGEMENTS
We thank Professor B. Bhatia for help and support and the Dean, School of Life Sciences, for providing facilities. We are also thankful to Professor Govindjee and Dr. George C. Papageorgiou for many helpful suggestions. The work is partly supported b y a grant from Government of India, DSTSERC 8{1)/78 to P.M. BCT is a recipient o f UGC fellowship.
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