not by benzidine and MnCI2. These observations suggest that Zn ..... MnCl2 nor benzidine could alleviate the Zn inhibition. DPC partially (30%) restores the ...
Plant Physiol. (1980) 66, 1174-1178 0032-0889/80/66/1 174/05/$00.50/0
Zinc-inhibited Electron Transport of Photosynthesis in Isolated Barley Chloroplasts' Received for publication January 22, 1980 and in revised form June 9, 1980
BAISHNAB C. TRIPATHY AND PRASANNA MOHANTY School of Environmental Sciences and School of Life Sciences, Jawaharlal Nehru University, New Delhi110067, India ABSTRACT In isolated barley chloroplasts, the presence of 2 millimolar ZnS04 inhibits the electron transport activity of photosystem II, as measured by photoreduction of dichlorophenolindophenol, 02 evolution, and chlorophyll a fluorescence. The inhibition of photosystem II activity can be restored by the addition of the electron donor hydroxylamine or diphenylcarbazide, but not by benzidine and MnCI2. These observations suggest that Zn inhibits electron flow at the oxidizing side of photosystem II at a site prior to the electron donating site(s) of hydroxylamine and diphenylcarbazide. No inhibition of photosystem I-dependent electron transport by 3 millimolar ZnSO4 is observed. However, with concentrations of ZnSO4 above 5 millimolar, photosystem I activity is partially inactivated. Washing Zn 2+_ treated chloroplasts partially restores the 02-evolving activity.
It is well-established that monovalent and divalent cations, particularly, Na+, K+, Ca2+, and Mg2", regulate the electron transport and excitation energy transfer between the two photosystems of photosynthesis (24). The effect of these cations on the primary processes of photosynthesis has been investigated in some detail (19, 20). Similarly, because of environmental concern, the effect of heavy metal ions, such as Pb2+, Hg2+, and Cd2+, on photosynthesis has been studied by several workers in recent years (3, 4, 15-18). However, only limited work has been carried out on the effect of those heavy metal ions which are essential as micronutrients and which also affect plant growth and metabolism, when they are present in the environment at toxic levels. Zn is an essential micronutrient and is required as prosthetic group for many enzymes (10). Zn accumulates to a toxic level in H20 and soil through various emission sources, such as mines and smelters (5). It has been shown that Zn inhibits growth and dry matter production in barley (1). It is also preferentially accumulated by green algal blooms in waste water (14). Recently, Davies and Sleep (8) reported that Zn, even at a relatively low concentration, inhibits CO2 fixation in marine phytoplanktons. However, there is no report on the nature of inhibition of photosynthesis by Zn. The work presented here aims at characterizing the mode of inhibition of photosynthesis by Zn salts in isolated barley chloroplasts. The results show that Zn, when present in moderately high concentrations, limits electron transport at the oxidizing (H20-splitting) side of PSII. Zn toxicity, therefore, appears to
'This work was supported by Department of Science and Technology Grant 8(1) SERC/78 (to P.M.) and a University Grants Commission fellowship (to B.C.T.).
interfere with the primary photoelectron transport activities of chloroplasts.
MATERIALS AND METHODS
Plant Material. Barley (Hordeum vulgare L. cv. lB 65) plants were grown on Petri plates fitted with H20-soaked filter sheets at 25 C, under continuous illumination. Fully expanded leaves of 11to 13-day-old healthy seedlings were selected for isolation of chloroplasts. Chloroplast Isolation. Chloroplasts were isolated from barley leaves by homogenizing in a grinding medium consisting of 0.5
M sucrose, 10 mM NaCl, and 20 mm Hepes NaOH buffer (pH 7.6). The same grinding medium was also used for suspending chloroplasts. Chl was determined according to Arnon (2). Assays of Chloroplast Reactions. The electron transport activities of chloroplasts were determined by three basic assays. First, electron transport through PSII was determined by photoreduction of DCIP measured spectrophotometrically and the DCIPsupported 02 evolution monitored polarographically. For the spectrophotometric assay, 3 ml assay medium consisted of 50 mm Hepes NaOH buffer (pH 7.0), 10 mm NaCl, 2 mM MgCl2, 30 tiM DCIP. Chloroplasts were added to the above reaction mixture at a concentration of 15 ug Chl/ml. DCIP-supported 02 evolution was measured polarographically by YSI model 53 Clark-type 02 electrode connected to a recorder. The 3-ml reaction mixture for 02 evolution consisted of 3 iM MgCl2, 10 mm NaCl, 400 tIM DCIP, and 50 mm Hepes-NaOH buffer (pH 7.0). Chloroplasts were added to the above reaction mixture to a final concentration of 100 ,tg Chl. The assay mixture was irradiated with saturating white light (1.0 cal/cm2- min) that had been filtered through 1O cm of H20. In the second type of assay, the electron transport through the whole chain of photosynthesis, i.e. from H20 to MV was measured polarographically as 02 uptake. Assay medium, 3 ml, contained 50 mM Hepes, 10 mM NaCl, 2 mM NH4CI, 3 mM MgCl2, 1.0 mM sodium azide, and 0.5 mm MV adjusted at pH 7.5. Chl was added to the above reaction mixture to a final concentration of 100 ,ug. The light intensity used for irradiation was as above. In the third type of assay, the electron transport through PSI was also measured polarographically as above except that reduced DCIP was used as the electron donor. Assay conditions were identical to these of whole chain assay except that 10 /LM DCMU, I mm sodium ascorbate, and 100 ,.M DCIP were added to the above reaction mixture. Chi a Fluorescence Measurement. Chl a fluorescence intensity and emission spectra were measured at room temperature (24 C). The excitation light (436 nm) was obtained through a monochro-
2Abbreviations: DCIP, dichlorophenol indophenol; MV, methylviologen; DPC, diphenylcarbazide. 1174
|\*
Plant Physiol. Vol. 66, 1980
ZINC INHIBITION OF PHOTOSYNTHESIS
mator with a mercury lamp as the exciting source. The fluorescence intensity and spectra were measured at 900 to the direction of exciting light through another monochromator. The chloroplasts were suspended in 30 mm Hepes-NaOH buffer (pH 7.0) at a Chl concentration of 4 ,ug/ml. The fluorescence data have been presented as the relative fluorescence intensity in arbitrary units. Chemicals. Analytical grade of ZnSO4 and ZnCl2 were purchased from British Drug House, DPC was purchased from E. *Merck. Hydroxylamine was obtained from Sigma. Other details are given in the legends to the figures.
RESULTS Whole Chain Electron Transports. The entire electron transport chain activity of barley chloroplasts incubated with various concentrations of ZnSO4 has been assayed and the degree ofinhibition with the time of incubation with ZnSO4 has been measured. Figure 1 shows the effect of Zn treatment on the whole chain electron transport system as measured by photoreduction of MV with H20 as electron donor. MV photoreduction-associated 02 uptake was significantly inhibited by treatment of barley chloroplasts with low concentrations (0.5-1 mM) of ZnSO4; the degree of inhibition increased with time of incubation. At relatively high concentrations (3 mM) of ZnSO4, the extent of inhibition was fully expressed after only 5 min incubation. No difference in the degree of inhibition of electron transport was observed when chloroplasts were incubated with Zn, either in light or dark. The extent and kinetics of inhibition of electron transport by Zn2+ remained the same when ZnCl2 was substituted for ZnSO4 (data not shown). Also, 5 mm K2SO4 did not affect the whole chain electron transport, which indicated that S042- played no role in the inhibition of electron transport observed here. PSII-supported Partial Chloroplast Reactions. The assay of PSII-dependent partial chloroplast reaction was carried out with DCIP as an electron acceptor. DCIP accepted electrons from the plastoquinone pool of the electron transport chain and its photoreduction is considered primarily a PSII reaction (12, 21). The DCIP-supported 02 evolution exhibited an extent and time course of inhibition similar to that of whole chain electron transport (H20 -. MV) (Fig. 1) by Zn2+ treatment (data not shown). There was a rapid loss of 02 evolution to the extent of 85% of the initial
j 0
80
activity after 5 min of incubation of chloroplasts with 3 mm ZnSO4, and the extent of inhibition increased with the increase in the time of incubation. Similar results were obtained when DCIP photoreduction was directly measured spectrophotometrically (data not shown). As DCIP, unlike ferricyanide, does not interact with Zn, this Hill oxidant has been used in most of the studies by
the authors. It was also noted that incubation of barley chloroplasts with Zn salts at the concentration used did not alter the absorption spectrum of chloroplasts (not shown), suggesting that the Zn inhibition was limited to electron transport chain of photosynthesis and not to any alterations of pigments. PSI-supported Electron Transport. Figure 2 shows the effect of Zn2+ on the PSI-mediated electron transport from reduced DCIP to MV monitored polarographically by 02 uptake. Unlike PSIIsupported partial reaction, the PSI-mediated reaction of barley chloroplasts was not inhibited with 3 mm ZnSO4. Only at a concentration greater than 5 mm 20 to 40% of inhibition of 02 uptake was observed after 20 to 30 min incubation. To compare the relative sensitivity of the PSII and PSI reaction to Zn2+ treatments, chloroplasts were treated with various concentrations of ZnSO4, and whole chain electron transport (H20 -- MV), PSIIcatalyzed 02 evolution (H20 -- DCIP), and PSI-supported electron flow from DCIP. H2 -- MV in the presence of DCMU were monitored. Figure 3 shows that both whole chain electron transport (H20 -- MV) and DCIP-supported 02 evolution (H20 -DCIP) were inhibited by 55% of the control rate with a low concentration (0.5 mM) of ZnSO4, and both the reactions vere completely inhibited by 4 mm ZnSO4. On the other hand, the PSIcatalyzed (DCIP. H2 -* MV) 02 uptake was not at all affected at these concentrations. However, at a concentration of ZnSO4 exceeding 4 mm, a slow and gradual loss in PSI activity was seen (Fig. 3). Figure 3 clearly demonstrates that PSII-supported electron transport is more susceptible to Zn2+ inhibition than is PSIdependent electron flow. Effect of Zn on the Exogenous Donor-supported DCIP Photoreduction. The studies presented here show that the Zn inhibition of photosynthetic electron transport is in the PSII portion of the electron transport chain. To locate precisely the site of Zn inhibition, the DCIP photoreduction, supported by various exogenous donors which provide electrons to PSII, has been measured. Several such electron donors are known to provide electrons to PSII, possibly at different sites on the H20-donating side of the electron transport chain. Table I shows the effect of some exogenous donors on the DCIP photoreduction by barley chloroplasts
100X,
o-oO.5mM
0
2.0 mM
0
U
D\-c \3.0mM 60
80-
0
-j 0
z
z 15J40-
0
60
o
w
LAo O
a.
U
0
z
40
z w 0
X
a
5
0.
3.0mM
o~0- 5 .0 m M
20-
10 15 20 25 30 INCUBATION TIME, MIN. FIG. 1. Time course of inhibition of whole chain photoelectron transport (H20 -' MV) in various concentrations of Zn-treated barley chloroplasts. Zn was added as ZnS04. Control rate of 02 uptake: 50 umol/mg
Chl. h.
1175
*
-
7.0mM *lO.OM
__ 5
10
I5
20
25
30
INCUBATION TIME, MIN.
FIG. 2. Time course of inhibition of PSI reaction in Zn-treated barley chloroplasts. Zn was added as ZnSO4. Control rate of 02 uptake: 400 tLmol/mg Chl -h.
Plant Physiol. Vol. 66, 1980
TRIPATHY AND MOHANTY
1176
Table I1. Effect of Exogenous Donors in PS II Activity of Heat-treated Chloroplasts Incubated with ZnSO4 Heat treatment of chloroplasts was carried out at 50 C for 5 min. Other
100 0j 80 0 ° 60 z 0 I. 60
O~
H2 0
A-6
H20
conditions were as in Table I.
MV -D CIP
ZnSO4
U.
Concn.
0
Exogenous Elec- Concentration of Dotron Donor nor
duction
mM
pUmol/mg
0.5
Chl.h 110
0.5 5.0 5.0
30 25 20
40
wi
0
mM
z
A_
W
a
CI
D
P
.H2M
20
U
O
wx
IL.
ul
v
0
1
I
I
2
3
4
a
I
a
I
a
s
6
7
8
9
[ZinSO4] ,mM
10
DPC None DPC
2 2
0
NH20H NH20H
2
FIG. 3. Effect of various concentrations of ZnSO4 on whole chain electron transport (H20 - MV), electron transport through PSII (H20 -*
Rate of
DCIP Re-
Inhibition %
100 72
20
DCIP), and electron transport through PSI (DCIP. H2 -* MV). Chlo-
roplasts were incubated with ZnSO4 for 10 min. Control rate of H20 -* MV reaction: 50 ,umol 02 uptake/mg Chl. h; control rate for H20 -. DCIP reaction: 85 ymol DCIP reduced/mg Chlh; and control rate for DCIP. H2 -* MV reaction: 400 ,umol 02 uptake/mg Chl -h. Table I. Effect of Exogenous Electron Donors on PS II Activity of Zntreated Chloroplasts Exogenous electron donor was added after 15 min incubation of ZnSO4 with barley chloroplasts. Reaction mixture of DCIP reduction was as described under "Materials and Methods."
ZnSO4 Concn.
Exogenous Electron Donor
mM
0 2
mM
MnCl2
2
MnCl2
0
Benzidine Benzidine DPC DPC NH20H NH20H
0 2
0 2
c-J
DCIon duction
,umol/mg Chl
11 0.3 0.3 0.5 0.5 0.5 0.5 5.0 5.0
85 11 85 11 100 42 36 30
C-) zw LLI
Rate of
UJ
%
h 85
None None
0 2
Donor Concn.
w z
z (-)
0
88
88
5
10 15 20 25 INCUBATION TIME, MIN.
30
FIG. 4. Time course of inhibition of steady state Chl a fluorescence intensity (Fw) measured at room temperature in arbitrary units in Zntreated chloroplasts. Zn was added as ZnSO4.
88 58
16
in the presence of 2 mm ZnSO4 after 15 min dark incubation. As DPC forms a red color when ZnSO4 exceeds 2 mM, the electrondonating ability of all other donors has also been tested in chloroplasts treated with 2 mM ZnSO4. Table I shows that neither MnCl2 nor benzidine could alleviate the Zn inhibition. DPC partially (30%) restores the electron transport in Zn2+-treated chloroplasts. Similar results were obtained with heat-inactivated chloroplasts treated with Zn, where DPC was able to restore DCIP photoreduction by 28% (Table II). On the other hand, hydroxylamine, an immediate electron donor to the reaction center of PSII, could photoreduce DCIP effectively in the presence of Zn2+, both in normal and heat-treated chloroplasts. As indicated in Table I, hydroxylamine (5 mM) brought about partial inactivation of PSII activity in untreated chloroplasts and, therefore, the degree of inhibition of hydroxylamine-supported DCIP photoreduction by Zn2+ was calculated from the above inactivated rate. Although 5 mm hydroxylamine itself inactivated 02 evolution, it also acted as a donor to the PSII reaction center and not as an inhibitor as observed only in the low concentration range of hydroxylamine (11, 12). Effect of Zn on Chi a Fluorescence. Chl a fluorescence at room temperature is associated with PSII activity (21). Any inhibition of electron flow to the PSII reaction center from the donor side of
PSII lowers the Chl a fluorescence yield, whereas a block on the acceptor side increases the fluorescence yield (6). Figure 4 shows that incubation of chloroplasts with ZnSO4 lowers the steady-state level of Chl a fluorescence intensity measured at 686 nm (F,86). Treatment of chloroplasts with 3 mm ZnSO4 brought about an approximately 60 to 65% lowering of Chl a fluorescence intensity. Although the steady-state fluorescence (Fmax) level was measured it is believed that this decrease of Chl a fluorescence was due mostly to the loss of variable fluorescence and not the constant background, the so-called Fo level. Figure 4 shows that 40% of the total fluorescence remained unquenched even after a long period (30 min) of incubation with Zn. The lowering of Chl a fluorescence, therefore, seems to be due to a block of electron flow from the donor side of PSII. Subsequent addition of DCMU to Zn-treated chloroplasts brought about only a slight increase in fluorescence intensity. The effect of exogenous electron donors, like DPC, MnCl2, and NH2OH, on the steady-state fluorescence level of Zn-treated chloroplasts has also been studied. The addition of either MnCl2 or DPC slightly relieved the lowering of Chl a fluorescence by Zn, whereas NH20H almost completely restored the Chl a fluorescence intensity (Fig. 5). Effect of Washing Zn-treated Chloroplasts. To ascertain if Zn inhibition of photoelectron transport in barley chloroplasts was reversible, the Zn-treated chloroplasts were washed. Zn incubation was carried out both in light and dark for 30 min and then the chloroplasts were washed with isolation buffer and their activity was measured for 02 evolution with DCIP as an electron acceptor.
Plant Physiol. Vol. 66, 1980
ZINC INHIBITION OF PHOTOSYNTHESIS
0
z
0
60 0
w
z W
4040o-o No addition
Mn Cl2 D P C
oL
s
*
*NNH;OH
a 3
2
0
[ZnS04]
, mM
FIG. 5. Effect of exogenous electron donors to PSII on Chl a fluorescence intensity in Zn-treated chloroplasts. Zn was added as ZnSO4. Chloroplasts were incubated with ZnSO4 for 10 min. Exogenous electron donor concentrations were: MnCl2, 0.3 mM; DPC, 0.5 mM; and NH20H, 10 mm. Control fluorescence level in the presence of donors did not vary more than 2 to 3%.
1001
O-O Unwashed
NO ADDITION
A--A
Unwashed
MnCI2
Washed
NO ADDITION
Washed
Mn Cl2
\
o-o
800
z
0
uA.
60-
0 w
z w
40-
w
20-
203 2
3
[Zn SO4] mM FIG. 6. Effect of washing of Zn-treated chloroplasts with isolation buffer in the presence or absence of MnCl2 on 02 evolution. Chloroplasts were incubated with ZnSO4 for 30 min. Control rate of 02 evolution: 50 ymol/mg Chl-h; MnCl2 0.3 mM. =
Figure 6 shows that, at
a
low concentration (I mM) of ZnSO4,
washing treated chloroplasts relieved the Zn inhibition to an appreciable level (76%), whereas at a high concentration (3 mM) of Zn salts, the degree of alleviation of PSII activity by washing decreased considerably. Addition of MnCl2 to unwashed or washed chloroplasts did not further enhance the PSII activity.
DISCUSSION The data presented here clearly show that ZnSO4
or
ZnCl2
inhibits DCIP-supported Hill reaction which is mediated by PSII. A possibility to be examined is that Zn2+ accepts electrons from PSII prior to the coupling site of DCIP since a similar observation in relation to Hg2' has been reported previously (18), where Hg2+
1177
is shown to accept electrons from PSII prior to accepting electrons from DCIP. The similar extents of inhibition of 02 evolution, as well as the photoreduction of DCIP by Zn2+, indicate that Zn2+ actually inhibits electron transport through PSII (Fig. 3). Zn2+ inhibited electron flow from H20 through both photosystems to MV (Fig. 1). On the other hand, if electron flow is only through PSI, such as from reduced DCIP to MV, then there is no inhibition of PSI activity with a low concentration of ZnSO4. However, at Zn concentrations greater than 5 mm, the PSI activity is also partially affected (Fig. 2). As shown by several workers, hydroxylamine donates electrons very close to the primary electron donor to the PSII reaction center, DPC donates at a distant site, and MnCl2 donates at a site still farther from the reaction center of PSII (I 1, 12). As shown in Tables I and II, hydroxylamine causes 70 to 80% restoration of PSII activity. Similarly, DPC bypasses the Zn2+ inhibition approximately 30 to 40%o. Thus, the results presented here indicate that Zn2+ inhibits PSII activity at the oxidizing side of PSII, prior to the hydroxylamine donating site, and perhaps close to the DPC donating site of the electron transport chain. A clear assignment of the Zn effect on PSII comes from the measurement of Chl a fluorescence intensity of chloroplasts. If Zn can inhibit at the acceptor side of PSII, an increase in fluorescence intensity is expected (10). If, however, Zn limits electron transport at the oxidizing side of the reaction center, the fluorescence intensity should be decreased, as in the case of Tris-washed chloroplasts (14). The observations presented here show that the steady-state fluorescence level was reduced in the presence of 2 mM ZnSO4 or an equimolar amount of ZnCl2 (Fig. 4), whereas 5 mM K2SO4 had no influence on fluorescence intensity (data not shown). This suggests that So42- has no effect on chloroplast fluorescence at the above concentration. The extent of restoration of fluorescence intensity by hydroxylamine was high (90%), whereas DPC and MnCl2 were able to restore the fluorescence intensity partially (Fig. 5). This confirms that Zn limits electron transport from H20 to PSII prior to the hydroxylamine donating site. This suggests that DPC and MnCl2 donating sites are further from the reaction center II than the NH20H donating site. A similar observation was made by Honeycutt and Krogmann (11) who have shown that inhibition of PSII activity by phenylmercuric acetate is overcome by NH20H but not by DPC or MnCl2. Haberman (9) has observed that the exogenous Mn2+ reduced the degrees of inhibition of several types of chloroplast-mediated reaction by Cu2+. However, in the investigation here, the Chl a fluorescence level and DCIP-supported Hill reaction are not fully restored after addition of MnCl2 to the Zn-treated chloroplast. Also, the inability of MnCl2 to enhance further the 02-evolving activity of Zn-treated and washed chloroplasts (Fig. 6) indicates that the Zn2+ inhibition of chloroplast function may not be due to loss of chloroplast Mn2' and inactivation or loss of Mn-containing, H20-splitting enzyme. Cedeno-Maldonado et al. (7) have reported a higher degree of inhibition of 02 evolution when chloroplasts are incubated with Cu2+ in light than when incubated in darkness. It has also been reported that Cu2' does not cause any damage to the photosynthetic apparatus of whole algal cells, unless the cells are illuminated during the entire exposure period (22). However, in the investigation here, Zn2+ did not cause enhanced extent of inhibition of 02 evolution when incubation was carried out in light. In contrast to the washing of Hg2+-treated chloroplasts (15), washing of chloroplasts treated with 1 mm ZnSO4 for 30 min restored the 02-evolving activity by 76% (Fig. 6). Izawa and Good (13) have observed that the Hill activity of DCMU-treated chloroplasts can be restored after washing. Similarly, Zn2+ inhibition of PSII activity can also be relieved upon removal of Zn2+ by washings.
1178
s
TRIPATHY AN]D MOHANTY NH2OH
DPC
MnCl2 °2
I# 1,~~~~~~~~~~~~~~~~~~~~~~~~\ PS PS I ,_R oZ /I Lo'h --4
"H
I
X-
-Q
~ ~ ~ ~\/?
\ /
Z inc
(5mM )
FIG. 7. Proposed schematic diagram of sites of Zn inhibition of photoelectron transport. The major site of inhibition of PSII at low concentration of Zn2+ is in the neighborhood of NH20H and DPC electron-donating sites.
True and Gies (23) have reported that toxic effect of Zn'2 on
barley plants can be considerably reduced in the presence of Ca salts. Therefore, the effect of Ca on Zn inhibition of photoelectron transport has been tested. However, CaCl2 marginally (12%) restored the 02-evolving activity of Zn-treated chloroplasts (data not shown). This indicates that Ca2+ may have some other role in protecting Zn toxicity of barley plants. ZnSO4 partially inhibited the PSI reaction at concentrations greater than 5 mm. Similar inhibition of PSI reactions by a high concentration of Pb2+ has also been observed (25). Further inves2
tigations need to be carried out to elucidate the mode of Zn
2
inhibition of PSI reaction at a high concentration. It is concluded that Zn2+ blocks electron transport at the oxidizing side of PSII, at a site prior to the site of electron donation by hydroxylamine and DPC to the PSII reaction center (Fig. 7). Zn also inhibits PSI reaction at a high concentration. Acknowledgments-The authors thank Prof. B. Bhatia of the School of Environmental Sciences for his support, the Dean of the School of Life Sciences for providing experimental facilities, and Prof. Govindjee and Dr. George Papageorgiou for critically reading the manuscript. LITERATURE CITED 1. AGRAWALA SC, SS BISHIT, CP SHARMA 1977 Relative effectiveness of some heavy metals in producing toxicity and symptoms of iron deficiency in barley. Can J Bot 55:1299-1307 2. ARNON DI 1949 Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol 24:1-15 3. BAZZAZ MB, GOVINDJEE 1974 Effect of lead chloride on chloroplast reactions. Environ Lett 6:175-191 4. BAZZAZ MB, GOVINDJEE 1974 Effect of cadmium nitrate on the spectral characteristic and light reactions of chloroplasts. Environ Lett 6:1-12
Plant Physiol. Vol. 66, 1980
5. BUC HAAJER MJ 1973 Contamination of soil and vegetation near a zinc smelter by zinc, cadmium, copper. and lead. Environ Sci Technol 7:131-135 6. BUTLER WL 1977 Chlorophyll fluorescence as a probe for electron transfer and energy transter. In A. Trebst and N. Avron. eds. Encyclopedia Plant Physiology New Series Vol 5. Springer-Verlag, Berlin, pp 149-167 7. CDLENO-MALDONADo A. JA S\ ADLR, RL HEATH 1972 The cupric ion as an inhibition of' photosynthetic electron transport in isolated chloroplasts. Plant Physiol 50:698 701 8. DAVIES AG, JA StLLP 1979 Photosynthesis in some British coastal water may be inhibited by zinc pollution. Nature 277:292-293 9. HABERMAN HM 1969 Reversal of copper inhibition in chloroplast reaction by manganese. Plant Physiol 44:33 1-336 1(. Hi\wir EJ 1958 The role of mineral elements in the activity of plant enzyme systems. In W. Ruhland. ed, Encyclopedia Plant Physiology, Vol 4. SpringerVerlag, Berlin pp 27-48 11. HONLYC trr RC. D KROGMANN 1972 Inhibition of chloroplast reactions with phenylmercuric acetate. Plant Physiol 49:376-380 12. IZAWA S 1970 Photoreduction of 2,6-dichlorophenol indophenol by chloroplasts with exogenous Mn ' as electron donor. Biochim Biophys Acta 197:328-331 13. IZA\sws S. NE Goor) 1965 The number of sites sensitive to 3-(3,4 dichlorophenyl)I.l-dimethyl urea. 3-(4-chlorophenyl)-1.1-dimethyl urea, and 2-chloro-4-(2propylamino)-6-ethylamino-co-triazine in isolated chloroplasts. Biochim Biophys Acta 102:20-38 14. JACKSON TA 1978 The biogeochemistry of heavy metals in polluted lake streams at Flrn. Flon, Canada, and a proposed method for limiting heavy metal pollution of natural waters. Environ Biol 2:173-189 15. KIMUIMURA M, S KAsroH 1972 Studies on electron transport associated with photosystem-l. 1. Functional site of plastocyanin, inhibitory effects of HgCl2 on electron transport, and plastocyanin in chloroplasts. Biochim Biophys Acta 283:279-292 16. Li EH, CD MILES 1975 Effect of cadmium on photoreaction-ll of chloroplasts. Plant Sci Lett 5:33-40 17. MiLEs CD, JR BRANDLE. DJ DANIEL. 0 CHU-DER, PD SCHNARE. DJ UHLIK 1972 Inhibition of photosystem-11 in isolated chloroplasts by lead. Plant Physiol 49:820-825 18. MILES D, P BOLEN, S FARAG, R GOODIN. J LUTz, A MOUSLAFA. B RODRIC,iUL., C WEIL 1973 Hg2 -a DCMU-independent electron acceptor of photosystem11. Biochem Biophys Res Commun 50:1113-1119 19. MOHANTN' P. BZ BRAUN, X GOVINDJEE 1973 Light-induced slow changes in chlorophyll a fluorescence in isolated chloroplasts: effects of magnesium and phenazine methosulfate. Biochim Biophys Acta 292:459-476 20. MURATA N 1971 Control of excitation transfer in photosynthesis. V. Correlation of membrane structure to regulation of excitation transfer between two pigment systems in isolated spinach chloroplasts. Biochim Biophys Acta 245:365-372 21. PAPAGEORGIOU G 1975 Chlorophyll fluorescence: an intrinsic probe of photosynthesis. In Govindjee, ed, Bioenergetics of Photosynthesis. Academic Press, New York, pp 319-371 22. STEEMANN-NIELSEN EN. L KAMP-NIELSEN. S Wius-ANDLRSON 1969 Influence of deleterious concentration of copper ion on the photosynthesis of Chlorella pvrenoidosa. Physiol Plant 22:1121-1131 23. TRUE RH, WJ GIES 1903 On the physiological action of some of the heavy metals in mixed solutions. Bull Torrey Bot Club 30:390-402 24. WILLtI AM WP 1977 The two photosystems and their interactions. In J. Barber, ed, The Primary Processes of Photosynthesis. Elsevier, Amsterdam, pp 99-147 25. WONG D. GOVINDJEE 1976 Effect of lead ions on photosystem-I in isolated chloroplasts. Studies on the reaction center P-700. Photosynthetica 10:241-254
