- Email: [email protected]
Photosynthetic Apparatus of Spinach Exposed to Excess Copper
Photosynthetic Apparatus of Spinach Exposed to Excess Copper T. BASZYNSKI I/" M. D. WOLINSKA )
KROLl),
Z.
KRUPA I),
M.
2 RUSZKOWSKA ),
u. WOJCIESKA2) and
1) Department of Plant Physiology, Maria Curie-Sklodowska University, Lublin, Poland 2) Institute of Soil Science and Cultivation of Plants, Pulawy, Poland Received July 19, 1982 . Accepted October 4,1982
Summary The photosynthetic apparatus of spinach (Spinacia oleracea L. var. Matador) grown in sandy soil containing excess amounts of copper was investigated. In chloroplasts isolated from the leaves of copper-treated spinach the synthesis of plastid pigments and lipoquinones was slightly higher than in the controls, relative to the increase of copper content in the soil. Under experimental conditions the growth rate of roots was decreased but that of leaves and stems was almost unaffected. Photosystem II activity as measured by 2,6-dichlorophenolindophenol photoreduction was not influenced, but photosystem I activity measured in terms of O 2 uptake and NADP photoreduction were slightly enhanced due to copper concentration. This increase was related to plastocyanin synthesis, the content of which was also dependent, to some extent, on the amounts of copper in the growth medium of the spinach. Among chlorophyll-protein complexes only CP1 depended on excess copper. Results obtained are discussed with regard to the tolerance of spinach exposed to high concentrations of the metal.
Key words: Spinacia oleracea L., copper, copper tolerance, lipoquinones, plastid pigments, plas· tocyanin, photosystems.
Introduction
Heavy metals are toxic in most plant species. Plants grown in an environment polluted with heavy metals, however, are often able to evolve metal-tolerant ecotypes. Various mechanisms have been suggested to account for metal tolerance in plants. Metal exclusion, a mechanism suggested by Antonovics et al. (1971), is considered to be a rare case of tolerance, because the heavy metal tolerant plants have been found to restrict metal uptake (Foster, 1977). Another mechanism has been recognized in the binding of metals to thiol groups (De Filippis and Pallaghy, 1976). Plant tolerance to toxic concentrations of eu in the environment is relatively high. Abbreviations: Chi = chlorophyll, CP1 = P700-chlorophyll a - protein, LHCP = light harvesting chlorophyll alb protein, CPa = chlorophyll a protein, DCIP = 2,6-dichlorophenolindophenol, DCMU = (3,4-dichlorophenyl)-1,1-dimethylurea, DPC = 1,S-diphenyl-carbohydrazide, FC = free chlorophyll, MV = methyl viologen, PC = plastocyanin, PS I = photosystem I, PS II = photosystem II, SDS = sodium dodecyl sulphate, TMPD = N,N,N',N'-tetramethyl-p-phenylene diamine, Tricine = N-tris (hydroxymethyl)-methylglycine.
z. Pjlanzenphysiol. Rd. 108. S. 385-395. 1982.
386
T. BASZYNSKI, M. KROL, Z. KRUPA, M. RUSZKOWSKA, U. WOJCIESKA and D. WOLINSKA
Some species or varieties of plants show a special adaptation and accumulate large amounts of Cu in their tissues Gowett, 1958; Ashida et aI., 1963; Curvetto and Rauser, 1979; Hogan and Rauser, 1981}. Comparing the growth and photosynthesis rate of Cu-tolerant Agrostis genus with spinach plants we have found that the latter, in pot experiments without an adaptation period, can be tolerant to Cu (unpublished). This study was undertaken to obtain a better understanding of the photosynthetic electron transport chain activity and pigment and lipoquinones systems in the leaves of spinach tolerant to excess Cu in the growth medium.
Material and Methods Spinach (Spinacia oleracea L., c.v. Matador) was cultivated in a sandy soil (7 kg) in Mitscherlich pots in a greenhouse. The following amounts of mineral components were applied in mg per pot: 1050 N, 1050 K, 370 P, 42 Mg [in the form of salts Ca(N0 3)2, KN0 3, N~N03, KH 2 P04 and MgS04]. Copper sulphate was applied as an aqueous solution of different amounts, resulting in a final metal content in the pot of 1, 50, 450 and 900 mg. In the last two cases Cu was applied in doses of 150 mg per pot at intervals of a few days to avoid too high a concentration of this component in the root zone. The last dose of Cu was applied not later than 17 days before harvesting. During plant growth distilled water was added if necessary to reach maximum water capacity of the soil equal 50 %. After addition of the nutrients pH of the soil was 6.5. Experiments were carried out in four independent replicates. In each experiment 30-45 plants were used. The rosette phase was utilized for the analyses and activity measurements. Activity measurements. Class II chloroplasts were prepared from spinach leaves according to Sane et al. (1970) and resuspended in 0.05 M potassium phosphate buffer (pH 7.6) containing 0.15 MKCI. The electron transport over Photosystem II was measured spectrophotometrically at 600 nm by photoreduction of DCIP as the oxidant, and water as electron donor in the following reaction mixture (in Itmol): Tricine-NaOH buffer (pH 7.0),150; DCIP, 0.125; chloroplasts containing 30 Itg of ChI; final volume 3 ml. Where indicated, 1.5 Itmol DPC was used as an alternative electron donor. The absorbance of the reaction medium was measured before and after illumination with 1.5 x 104 ItW· cm- 2 red light (Balzer K6 filter). Photosystem I-dependent MV reduction was measured with a Clark type oxygen electrode at 24°C using sodium ascorbate and TMPD as an electron donor system. The reaction mixture for measuring O 2 uptake contained the following components (in Itmol): Tricine-NaOH buffer (pH 8.0), 150; DCMU, 0.03; sodium ascorbate, 50; TMPD, 0.2; MV, 0.4; chloroplasts equivalent to 15 Itg of ChI; final volume 1.9 ml. The assay mixture was transferred to a glass reaction cuvette and incubated at 24°C with a red light flux of 2.5 x 104 ItW . cm -2. A red filter (Balzer K6) was placed between the actinic light source and the reaction chamber. NADP photoreduction was measured spectrophotometrically by following the change in absorbance at 340 nm caused by illumination with red light (Balzer K6 filter) of an intensity equal to 1.5 x 104 ItW . cm -2. The reaction mixture contained the following components (in Itmol): Tricine-NaOH buffer (pH 8.0), 150; DCMU, 0.03; sodium ascorbate, 50; TMPD, 0.2; NADP+, 2.5; saturating amounts of ferredoxin, and chloroplasts containing 30 Itg ChI. Determination 0/ copper. The harvested plants were dried at 105°C and analysed for copper content on Pye-Unicam SP-9 atomic absorption spectrophotometer after the digestion of plant material in HN0 3 (85 %) and perchloric acid (15 %) mixture. Analysis of chloroplast components. ChI concentrations were measured using the method of Arnon (1949). Carotenoids were chromatographed and estimated by the method of Hager and
Z. Pjlanzenphysiol. Bd. 108. S. 385-395. 1982.
Photosynthesis of Cu-treated spinach
387
Bertenrath (1962). Plastid quinones were separated on silica gel layers in a mixture of light petroleum and diethyl ether (7 : 1, v/v) according to Lichtenthaler (1969). The amounts of Jipoquinones were determined spectrophotometrically according to Lichtenthaler (1968), and vitamin KJ according to Lichtenthaler and Tevini (1969). Enzyme assays. Plastocyanin activity was measured enzymatically as described by Hauska et al. (1971). Light subchloroplast particles required for the estimation of plastocyanin were obtained according to Anderson and Boardman's (1966) digitonin technique. RuBP carboxylase activity was measured according to Bowes et al. (1972). Polyacrylamide gel electrophoresis. The polypeptides were separated in polyacrylamide gel electrophoresis as described by Laemmli (1970). The electrophoresis was carried out on 10-20 % acrylamide gradient. The length of the separation gel was 13 cm. Molecular weights were obtained by comparison of separated polypeptides with Pharmacia Fine Chemicals HMW and LMW protein kits according to Weber and Osborn (1969). Isolation of ChI-protein complexes was carried out by polyacrylamide gel electrophoresis in the presence of SDS according to Wild et al. (1980). ChI alb ratio in ChI-protein complexes was estimated using the method of Wild et al. (1980). Electron microscopy. Plant segments were fixed with 2 % glutaraldehyde + 4 % paraformaldehyde in 0,05 M cacodylate buffer (pH 7.4) at room temperature for 3 h, and after washing with 0.22 M sucrose at 4°C for 12 h they were postfixed for 2 h with 2 % OS04 in the same buffer. The samples were dehydrated by 2,2-dimethoxypropane at room temperature for 2 h and embedded in Spurra. Thin sections were cut with a Reichert Om U3 ultramicrotome, stained with 8 % uranyl acetate in 0.5 % acetic acid for 30 min and poststained with 0.03 % lead citrate for 12 min. The sections were examined with a Tesla BS-500 electron microscope.
Results
eu-tolerant individuals of spinach chosen for assays could easily be distinguished by their continued growth. Non-tolerant individuals died with the increase of eu level in the soil. The percentage of surviving eu-treated plants was very high even at very high eu concentrations. The surviving plants in all eu treatments were fresh green and in good physiological condition. The results give ample evidence that spinach, under the growth conditions described, can be tolerant to eu. Table 1: The growth of spinach organs in relation to excess copper in the soil. mgCu/pot
Organs leaves stems roots whole leaves + stemsl roots leaves stems roots
50 1.988±0.276 0.641±0.106 0.152±0.031 2.781 17.30 8.26±0.58 5.56±0.64 10.63± 1.03
450
dry matter of plant organ in g 2.328 ±0.271 2.093 ± 0.398 0.820±0.067 0.601 ±0.090 0.185±0.020 0.137 ±0.020 3.333 2.831 17.02 19.66 dry matter in % 8.10±0.78 7.59±0.49 5.20±0.26 5.01 ±0.67 10.66±0.62 9.70±0.18
900 2.157 ±0.336 0.602±0.136 0.121 ±0.040 2.880 22.80 8.40±0.88 5.18 ±0.35 10.05±0.53
Values represent the mean ± SE of three replicates.
Z. Pjlanzenphysiol. Ed. 108. S. 385-395. 1982.
388
T. BASZTIISIU, M. KROL, Z. KRUPA, M. RUSZKOWSKA, U. WOJCIESKA and D. WOLINSKA
Organ growth and dry matter production were optimal when Cu concent, in the pot was 50 mg (Table 1). The dry matter of leaves and stems appears to be insignificantly affected by a higher concent of Cu in the soil (450 and 900 mg Cu/pot), but the growth rate of roots was found to be depressed under these conditions. Accordingly, the shoot to root ratio increased. The percentage of dry matter in all organs examined was not essentially different under Cu excess. Table 2: The concentration and content of copper in spinach organs related to excess copper in the soil. Organs leaves stems roots
mg Culpot 450
50 9.80± 1.39 8.32± 1.31 12.33±2.64
leaves stems roots leaves + stemslroots
19.484 5.332 1.877 13.22
900
JLg Cui g dry matter 16.35±0.86 31.66±0.81 12.67± 1.24 35.90±3.33 21.44± 1.16 56.36±2.28 JLg Cui organ 38.063 66.274 10.384 21.583 3.975 7.744 12.19 11.35
149.16± 16.07 253.05± 7.80 474.19±13.30 321.723 152.437 57.282 8.28
Values represent the mean ± SE of three replicates. To detect possible difference in Cu uptake and translocation, spinach organs were analysed for Cu concentration (Table 2). Spinach organs can accumulate high amounts of Cu. The metal uptake on a dry matter basis increased in relation to Cu Table 3: The content of plastid pigments and lipoquinones in chloroplasts isolated from leaves of Cu-treated spinach (in JLg per g of fresh weight). Components Chlorophyll a + b Carotenoids J3-carotene lutein neoxanthin violaxanthin Benzoquinones plastoquinone A a-tocopherol a-tocopherylquinone VitaminK 1 Carotenoidsl chlorophyll Benzoquinoneslchlorophyll
mg Culpot 450
50 1310 ±70 183.1 63.2± 2.7 94.0± 3.5 8.2± 0.6 17.7± 1.4 67.9 29.0± 1.3 28.0± 1.1 10.7 ± 0.7 10.4± 2.0 0.14 0.05
1480 ±30 190.2 69.0± 2.3 102.3± 3.2 4.4± 0.7 14.5± 1.0 91.9 40.4± 2.7 38.1 ± 1.3 13.4± 0.9 20.7 ± 1.4 0.13 0.06
900
1420 ±80 207.6 67.5± 1.4 117.2± 4.3 7.8± 0.5 IS.1± 1.1 87.3 34.8± 2.5 37.4± 1.6 15.1 ± 1.8 15.9± 0.9 0.15 0.06
Each value is the mean ± SE based on three replicates of three independent series.
Z. Pjlanzenphysiol. Bd. 108. S.385-395. 1982.
1510 ±70 215.1 72.9± 2.4 120 ± 3.2 7.6± 0.3 14.3± 0.9 85.9 37.6± 1.3 33.8 ± 1.1 14.5± 1.5 12.9± 0.3 0.14 0.06
389
Photosynthesis of Cu-treated spinach
level in pots. The highest amounts of accumulated Cu were found in roots. The rate of Cu concentration in these organs was 1.26-3.18 times higher than in leaves. When the total Cu was expressed per plant organ the highest amounts of the metal were observed in leaves. Table 3 summarizes the chemical components of chloroplasts isolated from spinach grown on excess Cu. The chloroplasts isolated from Cu-treated spinach do not differ significantly with respect to their Chi (a + b) content per fresh weight. A slight increase in Chi (a + b) content occurs due to Cu concentration in the medium in comparison with control. A similar tendency to that of Chi is seen in the content of total carotenoids under all experimental combinations. Thus the ratio of carotenoids to Chi was almost constant. A Cu-induced increase of lipoquinones was also observed. Table 4: Photosynthetic electron transport activities of chloroplasts isolated from leaves of Cutreated spinach plants. mg Cu/pot
Assay /Lmol equivalent per mg Chl·h TMPD-MV TMPD-NADP H 20-DCIP
,+Mn 2 + ,+DPC ,+N~Cl
1069±23 146± 5.6 49± 4 55± 2 51± 3 102± 7
50
450
900
1121±45 151± 5.6 48± 1 52± 1 53± 2 103± 2
1417±60 170± 3.7 52± 2 54± 0.4 55± 4 112± 6
1317±58 190± 3.4 47± 3 62± 0.7 50± 5 98± 4
Values represent the mean ± SE of five replicates. Table 5: Plastocyanin and RuBP carboxylase activity of spinach chloroplasts isolated from Cutreated spinach. mg Cu/pot
1
50 450 900
Plastocyanin nmol/mg Cu
Molar ratio ChI/PC
1,5 RuBP carboxylase activity counts! g . min
5.37 ±0.62 S.76±0.6S 8.96±0.82 S.80±0.56
210 127 127 127
1504± 153 1263± 102 1370±112 1485± 161
Values represent the mean ± SE of three replicates.
PS I activity measured by MV as well as NADP photoreduction of chloroplasts ~so lated from spinach was higher in plants grown in soil containing high concentrations of Cu than in the control (Table 4). This phenomenon could be related to plastocyanin synthesis enhanced at external Cu concentration. However, plastocyanin synthesis reached its maximum at exactly 50 mg Cu/pot, and a further increase of Cu supply did not enhance synthesis (Table 5). PS II activity did not vary in relation to Cu concentration in the medium. Also, an Z. Pjlanzenphysiol. Bd. 108. S. 385-395. 1982.
390
T. BASZYNSKI, M. KROL, Z. KRUPA, M. RUSZKOWSKA, U. WOJCIESKA and D. WOLINSKA
addition of Mn 2 + to the reaction mixture did not noticeably change the DCIP photoreduction rate of chloroplasts, except at 900 mg Cu/pot. DPC, an artificial electron donor for PS II, similarly caused an insignificantly slight increase of DCIP photoreduction regardless of the Cu level. However, Nf-4 + as an uncoupler strongly increased PS II activity in all combinations of the experiment. It is worth noting that the activity of RuBP carboxylase does not appear to be related to Cu concentration in the soil. Thus the association of CU with RuBP carboxylase may be controversial (Table 5). Figure 1 shows polypeptides separated by the method of Laemmli. A comparison of the electrophoretic densitograms showed that the new chloroplast proteins did not occur in Cu-treated plants. Our preliminary studies indicate, however, an increased Cu content in some protein fractions of chloroplasts. Detailed studies on Cu-binding protems are m progress. Table 6: Relative proportion of Chi-protein complexes and Chi alb ratio in chloroplasts isolated from spinach grown on sandy soil contained 1 and 450 mg Cu/pot. mg Culpot
450
%Chl Chi alb %Chl ChI alb
CP1a
CP1
LHCP2
CPa
LHCP 3
FC
1.8
20.8 5.5 27.0 4.5
2.4 1.6 2.6 1.5
6.1 2.6 6.3 2.0
40.0 1.5 36.8
28.9 3.5 25.1 3.0
2.1
-
112
-
9'
-
79 74
6
-
6' 59 52
1:
-
'6
- u
-
-
900
450
34
33
1.1
~
.211 COl
~
...
.2 ::J
u
28
~
26 23
~
0
50
mg Cu/pot Fig. 1: Polypeptide patterns of chloroplasts isolated from spinach leaves grown on sandy soil containing 1, 50, 450 and 900 mg Culpot, respectively. Z. Pjlanzenphysiol. Ed. 108. S. 385-395. 1982.
Photosynthesis of eu-treated spinach
391
The separation of ChI-protein complexes indicates that the relative content of ChI in CP1 of plants grown in soil with higher Cu doses increases from 20.8 % to 27 % of the total amount of ChI used for separation of the complexes (Table 6). The relative amounts of ChI in CPa and LHCP 3 complexes did not undergo greater changes and were approximately the same as those occurring in the corresponding complexes of control plants. However, the relative percentage content of ChI occurring in the CPa complex was lower than the values given so far (Anderson, 1978). In examining the effect of excess Cu we did not observe changes in the inner structure of chloroplasts. In the micrographs (Fig. 2), however, some deposits can be seen
Fig. 2: Electron micrographs of the ultrastructure of spinach chloroplasts: a, chloroplast of leaves from eu-treated plants, mag. x 18,000; b, control chloroplast from leaves; x 18,000; c, chloroplast from stem of eu-treated plants; x 10,000, deposits on the surface of the chloroplast envelope are seen; d, chloroplast from stem of control plants; x 10,000.
Z. Pjlanzenphysiol. Bd. 108. S. 385-395. 1982.
392
T.
BASZYNSKI,
M. KROL, Z.
KRUPA,
M.
RUSZKOWSKA,
U. WOJCIESKA and D.
WOLINSKA
inside cells, as well as on the surface of the chloroplast envelope, suggesting the presence of Cu. Such structures did not occur in control plants. Discussion
The occurrence of Cu-tolerant plants in normal populations grown on excess Cu is generally known and has been recognized in the rapidly occurring genetic changes by the excess Cu (Walley et aI., 1974). This tolerance depends on the total concentration of the metal in the soil. Even as much as 900 mg Cu/pot, when applied in six doses of 150 mg each, over several days, did not produce a decrease in growth rates of the aerial parts of plants. Fertilization with excess Cu decreased the root mass, although only to a degree that had no negative effect on the crop of leaves and stems in the course of the experiments. The maximum spinach crop obtained at 50 mg Cu/pot shows that such amount seems to be optimal for this species. As expected, a remarkable accumulation of Cu, as with other heavy metals, was observed in roots. This depended on Cu content in the medium, although it was not directly proportional. The physiological parameters studied, such as the chemical composition of the photosynthetic apparatus in control plants and those treated with high Cu doses, did not differ essentially. Increased Cu doses per pot caused only a slight stimulation of the synthesis of ChI, carotenoid pigments (particularly lutein) and lipoquinones. No toxic effect of Cu on the activity of the photosystems of chloroplast isolated from Cu-treated spinach leaves was found either. This is surprising in view of the toxic effect of Cu ions on the photosynthetic electron system of isolated chloroplasts or intact green algae (Macdowall and Haberman, 1969). It was shown that Cu ions block the photosynthetic electron transport at both the oxidizing side of PS II and the reducing side of PS I (Shioi et aI., 1978 a, b). According to Vierke and Struckmeier (1977, 1978) eu ions bind to a membrane protein which is not involved in electron transport itself but which leads to structural changes of the photosynthetic membrane with subsequent inhibition of electron transport. On the other hand, Sandmann and Boger (1980 a) have shown that free Cu ions inhibit electron transport in both photosystems leading to lipoxygenase activity and consequently the acceleration of peroxidative degradation of the lipids of the chloroplast membranes. However, in our experiments, when chloroplasts were isolated from plants provided with excess Cu some increase of PS I activity above the controls was observed. The simplest explanation would be a Cu-induced synthesis of plastocyanin, which influenced PS I activity. This would be in agreement with the earlier observation of Boger et al. (1980 b) that plastocyanin synthesis in some species of algae is strictly dependent on the Cu level in the growth medium and that Cu deficiency blocks this synthesis. Unfortunately, as mentioned above, plastocyanin synthesis in chloroplasts of spinach is not induced by very high concentrations of Cu in the soil. Z. Pjlanzenphysiol. Bd. 108. S. 385-395. 1982.
Photosynthesis of eu-treated spinach
393
PS II activity does not depend on Cu level in the growth medium of spinach. Assays with addition of Mn 2+ or DPC show that high amounts of Cu in soil do not influence the oxidizing side of PS II in chloroplasts. The increase of DCIP photoreduction rate after the addition of Nfu + to the reaction mixture indicates that all chloroplasts examined are coupled, among others those isolated from Cu-treated plants. This observation corresponds to an earlier finding (Vierke and Struckmeier, 1978) that photophosphorylation is not affected by Cu ions, but is in contrast to Samuelsson and Oquist (1980), who have shown Cu ions to be an uncoupler of photophosphorylation. Both observations were made, however, on in vitro experiments. The lack of a toxic effect of Cu on photosystems in chloroplasts isolated from spinach provided with high Cu doses, found in our studies, may result from the absence or small amounts of free Cu 2 + ions, known as the most toxic form of the metal, in chloroplasts. It is possible that during the growth of Cu-treated spinach Cu-binding proteins are formed which, as suggested by Sandmann and Boger (1980 b) and Hogan and Rauser (1981), might chemically inactivate the Cu ions taken up by the leaves. The precipitates which are visible in the micrographs and localized mainly in leaf cells and sporadically on the surface of the chloroplast envelope of stem cells, could prevent Cu from entering more sensitive sites of cell metabolism, among others those inside the chloroplasts. According to Ashida et al. (1963) the precipitation may be due to the formation of CuS via methionine. The rapid destruction of ChI-protein complexes in isolated chloroplasts due to the influence of Cu ions added to the reaction mixture, as found by Samuelsson and Oquist (1980) was not observed in our studies. The increase of the relative content of ChI in CP1 of chloroplasts isolated from Cu-treated spinach is coupled to the concomitant increase of PS I activities. Such a relationship was found earlier (Anderson, 1978). The relative content of ChI in CPa, which is probably associated with the centre of PS II reaction (Anderson, 1978) in control chloroplasts, and those isolated from Cu-treated spinach is very similar. The characteristics of the photosynthetic apparatus of spinach leaves growing in soil containing high Cu doses does not differ much from that of control plants. This indicates apparently high tolerance of Spinacia oleracea L. var. Matador to Cu added to the growth medium. Without discussing the mechanism of tolerance of spinach to Cu, it can be assumed that spinach may adapt quickly to high external concentrations of Cu, insignificantly affecting plant growth and photosynthetic activity. Acknowledgements This study was carried out under the project MR II/7 coordinated by the Institute of Plant Physiology, Polish Academy of Sciences.
References ANDERSON, J. M. and N. K. BOARDMAN: Fractionation of the photochemical systems of photosynthesis. Biochim. Biophys. Acta 112, 403-412 (1966). Z. Pjlanzenphysiol. Ed. 108. S. 385-395. 1982.
394
T. BASZYNSKI, M. KROL, Z. KRuPA, M. RUSZKOWSKA, U. WOJCIESKA and D. WOLINSKA
ANDERSON, J. M., J. C. WALDRON, and S. W. THORNE: Chlorophyll-protein complexes of spinach and barley thylakoids. Spectral characterization of six complexes resolved by an improved electrophoretic procedure. FEBS Lett. 92, 227-233 (1978). ANDERSON, J. M.: P-700 content and polypeptide profile of chlorophyll-protein complexes of spinach and barley thylakoids. Biochim. Biophys. Acta 591, 113-126 (1980). ANTONOVICS, J., A. D. BRADSHAW, and R. G. TURNER: Heavy metal tolerance in plants. Adv. Ecol. Res. 7, 1-85 (1971). ARNON, D. I.: Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1-15 (1949). ASHIDA, J., N. HIGASHI, and T. KIKUCHI: An electron microscopic study on copper precipitation by copper-resistant yeast cells. Protoplasm a 57, 27-32 (1963). BOHNER, H., H. MERKLE, P. KRONECK, and P. BOGER: High variability of the electron carrier plastocyanin in microalgae. Eur. J. Biochem. 195, 603-609 (1980). BOWES, G., W. L. OGREN, and R. H. HAGEMAN: Light saturation, photosynthesis rate, RuDP carboxylase activity and specific leaf weight in soybeans grown under different light intensities. Crop Sci. 1, 77-79 (1972). CURVETTO, N. R. and W. F. RAUSER: Isolation and characterization of copper-binding proteins from roots of Agrostis gigantea tolerant to excess copper. Plant Physiol. Suppl. 63, 59 (1979). DE FILIPPIS, L. F. and C. K. PALLAGHY: The effect of sub-lethal concentrations of mercury and zinc on Chlorella. III. Development and possible mechanisms of resistance to metals. Z. Pflanzenphysiol. 79, 323-335 (1976). FOSTER, P. L.: Copper exclusion as a mechanism of heavy metal tolerance in a green alga. Nature (London) 269, 322-323 (1977). HAGER, A. und T. BERTENRATH: Verteilungschromatographische Trennung von Chlorophyllen und Karotenoiden griiner Pflanzen an Diinnschichten. Planta (Berl.) 58,564-568 (1962). HABERMANN, H. M.: Reversal of copper inhibition in chloroplast reactions by manganese. Plant Physiol. 44, 331-336 (1969). HAUSKA, G. A., R. E. MCCARTY, R. J. BERZBORN, and E. RACKER: Partial resolution of the enzymes catalyzing photophosphorylation. VII. The function of plastocyanin and its interaction with a specific antibody. J. BioI. Chern. 246, 3524-3531 (1971). HOGAN, G. D. and W. E. RAUSER: Role of copper binding, absorption, and translocation in copper tolerance of Agrostis gigantea Roth. J. Exp. Bot. 32,27-36 (1981). JOWETT, D.: Populations of Agrostis spp. tolerant of heavy metals. Nature (London) 182, 816-817 (1958). LAEMMLI, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680-685 (1970). LICHTENTHALER, H.-K.: Verbreitung des lipophilen Plastidenchinone im nicht-griinen Pflanzengewebe. Z. Pflanzenphysiol. 59, 195-210 (1968). - Zur Synthese der lipophilen Plastidenchinone und Sekun&irkarotenoide wahrend der Chromoplastenentwicklung. Ber. dtsch. bot. Ges. 82, 483--497 (1969). LICHTENTHALER, H.-K. und M. TEVINI: Die Wirkung von UV-Strahlen auf die Lipochinon-Pigment-Zusammensetzung isolierter Spinatchloroplasten. Z. Naturforsch. 24 b, 764-769 (1969). MACDOWAL, F. D. H.: The effects of some inhibitors of photosynthesis upon the photochemical reduction of a dye by isolated chloroplasts. Plant Physiol. 24, 462-480 (1949). SAMUELSSON, G. and G. OQUIST: Effects of copper chloride on photosynthetic electron transport and chlorophyll-protein complexes of Spinacia oleracea. Plant and Cell Physiol. 21, 445-454 (1980). SANE, P. V., D. J. GOODCHILD, and R. B. PARK: Characterization of chloroplast photosystem 1 and 2 separated by a non-detergent method. Biochim. Biophys. Acta 216, 162-178 (1970).
2. Pjlanzenphysiol. Bd. 108. S. 385-395. 1982.
Photosynthesis of Cu-treated spinach
395
SANDMANN, G. and P. BOGER: Copper-mediated lipid peroxidation processes in photosynthetic membranes. Plant Physio!. 66, 797-800 (1980 a). - - Copper deficiency and toxicity in Scenedesmus. Z. Pflanzenphysio!. 98, 53-59 (1980 b). SHIOI, Y., H. TAMAI, and T. SASA: Inhibition of photosystem II in the green alga Ankistrodesmus /alcatus by copper. Physio!. Plant 44, 434-438 (1978 a). - - - Effects of copper on photosynthetic electron transport systems in spinach chloroplasts. Plant and Cell Physio!. 19,203-209 (1978 b). VIERKE, G. and P. STRUCKMEIER: Binding of copper II to proteins of the photosynthetic membrane and its correlation with inhibition of electron transport in class II chloroplasts of spinach. Z. Naturforsch. 32 c, 605-610 (1977). - - Inhibition of millisecond luminescence by copper II in spinach chloroplasts. Z. Naturforsch. 33 c, 266-270 (1978). WALLEY, K. A., M. S. 1. KHAN, and A. D. BRADSHAW: The potential for evolution of heavy metal tolerance in plants. Heredity 32,309-319 (1974). WEBER, K. and M. OSBORN: The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Bio!. Chern. 244, 4406-4412 (1969). WILD, A. and B. URSCHEL: Methodical studies in the separation of chlorophyll-protein complexes by polyacrylamide gel electrophoresis. Z. Naturforsch. 35 c, 627-637 (1980).
Z. Pjlanzenphysiol. Bd. 108. S.385-395. 1982.
KROLl),
Z.
KRUPA I),
M.
2 RUSZKOWSKA ),
u. WOJCIESKA2) and
1) Department of Plant Physiology, Maria Curie-Sklodowska University, Lublin, Poland 2) Institute of Soil Science and Cultivation of Plants, Pulawy, Poland Received July 19, 1982 . Accepted October 4,1982
Summary The photosynthetic apparatus of spinach (Spinacia oleracea L. var. Matador) grown in sandy soil containing excess amounts of copper was investigated. In chloroplasts isolated from the leaves of copper-treated spinach the synthesis of plastid pigments and lipoquinones was slightly higher than in the controls, relative to the increase of copper content in the soil. Under experimental conditions the growth rate of roots was decreased but that of leaves and stems was almost unaffected. Photosystem II activity as measured by 2,6-dichlorophenolindophenol photoreduction was not influenced, but photosystem I activity measured in terms of O 2 uptake and NADP photoreduction were slightly enhanced due to copper concentration. This increase was related to plastocyanin synthesis, the content of which was also dependent, to some extent, on the amounts of copper in the growth medium of the spinach. Among chlorophyll-protein complexes only CP1 depended on excess copper. Results obtained are discussed with regard to the tolerance of spinach exposed to high concentrations of the metal.
Key words: Spinacia oleracea L., copper, copper tolerance, lipoquinones, plastid pigments, plas· tocyanin, photosystems.
Introduction
Heavy metals are toxic in most plant species. Plants grown in an environment polluted with heavy metals, however, are often able to evolve metal-tolerant ecotypes. Various mechanisms have been suggested to account for metal tolerance in plants. Metal exclusion, a mechanism suggested by Antonovics et al. (1971), is considered to be a rare case of tolerance, because the heavy metal tolerant plants have been found to restrict metal uptake (Foster, 1977). Another mechanism has been recognized in the binding of metals to thiol groups (De Filippis and Pallaghy, 1976). Plant tolerance to toxic concentrations of eu in the environment is relatively high. Abbreviations: Chi = chlorophyll, CP1 = P700-chlorophyll a - protein, LHCP = light harvesting chlorophyll alb protein, CPa = chlorophyll a protein, DCIP = 2,6-dichlorophenolindophenol, DCMU = (3,4-dichlorophenyl)-1,1-dimethylurea, DPC = 1,S-diphenyl-carbohydrazide, FC = free chlorophyll, MV = methyl viologen, PC = plastocyanin, PS I = photosystem I, PS II = photosystem II, SDS = sodium dodecyl sulphate, TMPD = N,N,N',N'-tetramethyl-p-phenylene diamine, Tricine = N-tris (hydroxymethyl)-methylglycine.
z. Pjlanzenphysiol. Rd. 108. S. 385-395. 1982.
386
T. BASZYNSKI, M. KROL, Z. KRUPA, M. RUSZKOWSKA, U. WOJCIESKA and D. WOLINSKA
Some species or varieties of plants show a special adaptation and accumulate large amounts of Cu in their tissues Gowett, 1958; Ashida et aI., 1963; Curvetto and Rauser, 1979; Hogan and Rauser, 1981}. Comparing the growth and photosynthesis rate of Cu-tolerant Agrostis genus with spinach plants we have found that the latter, in pot experiments without an adaptation period, can be tolerant to Cu (unpublished). This study was undertaken to obtain a better understanding of the photosynthetic electron transport chain activity and pigment and lipoquinones systems in the leaves of spinach tolerant to excess Cu in the growth medium.
Material and Methods Spinach (Spinacia oleracea L., c.v. Matador) was cultivated in a sandy soil (7 kg) in Mitscherlich pots in a greenhouse. The following amounts of mineral components were applied in mg per pot: 1050 N, 1050 K, 370 P, 42 Mg [in the form of salts Ca(N0 3)2, KN0 3, N~N03, KH 2 P04 and MgS04]. Copper sulphate was applied as an aqueous solution of different amounts, resulting in a final metal content in the pot of 1, 50, 450 and 900 mg. In the last two cases Cu was applied in doses of 150 mg per pot at intervals of a few days to avoid too high a concentration of this component in the root zone. The last dose of Cu was applied not later than 17 days before harvesting. During plant growth distilled water was added if necessary to reach maximum water capacity of the soil equal 50 %. After addition of the nutrients pH of the soil was 6.5. Experiments were carried out in four independent replicates. In each experiment 30-45 plants were used. The rosette phase was utilized for the analyses and activity measurements. Activity measurements. Class II chloroplasts were prepared from spinach leaves according to Sane et al. (1970) and resuspended in 0.05 M potassium phosphate buffer (pH 7.6) containing 0.15 MKCI. The electron transport over Photosystem II was measured spectrophotometrically at 600 nm by photoreduction of DCIP as the oxidant, and water as electron donor in the following reaction mixture (in Itmol): Tricine-NaOH buffer (pH 7.0),150; DCIP, 0.125; chloroplasts containing 30 Itg of ChI; final volume 3 ml. Where indicated, 1.5 Itmol DPC was used as an alternative electron donor. The absorbance of the reaction medium was measured before and after illumination with 1.5 x 104 ItW· cm- 2 red light (Balzer K6 filter). Photosystem I-dependent MV reduction was measured with a Clark type oxygen electrode at 24°C using sodium ascorbate and TMPD as an electron donor system. The reaction mixture for measuring O 2 uptake contained the following components (in Itmol): Tricine-NaOH buffer (pH 8.0), 150; DCMU, 0.03; sodium ascorbate, 50; TMPD, 0.2; MV, 0.4; chloroplasts equivalent to 15 Itg of ChI; final volume 1.9 ml. The assay mixture was transferred to a glass reaction cuvette and incubated at 24°C with a red light flux of 2.5 x 104 ItW . cm -2. A red filter (Balzer K6) was placed between the actinic light source and the reaction chamber. NADP photoreduction was measured spectrophotometrically by following the change in absorbance at 340 nm caused by illumination with red light (Balzer K6 filter) of an intensity equal to 1.5 x 104 ItW . cm -2. The reaction mixture contained the following components (in Itmol): Tricine-NaOH buffer (pH 8.0), 150; DCMU, 0.03; sodium ascorbate, 50; TMPD, 0.2; NADP+, 2.5; saturating amounts of ferredoxin, and chloroplasts containing 30 Itg ChI. Determination 0/ copper. The harvested plants were dried at 105°C and analysed for copper content on Pye-Unicam SP-9 atomic absorption spectrophotometer after the digestion of plant material in HN0 3 (85 %) and perchloric acid (15 %) mixture. Analysis of chloroplast components. ChI concentrations were measured using the method of Arnon (1949). Carotenoids were chromatographed and estimated by the method of Hager and
Z. Pjlanzenphysiol. Bd. 108. S. 385-395. 1982.
Photosynthesis of Cu-treated spinach
387
Bertenrath (1962). Plastid quinones were separated on silica gel layers in a mixture of light petroleum and diethyl ether (7 : 1, v/v) according to Lichtenthaler (1969). The amounts of Jipoquinones were determined spectrophotometrically according to Lichtenthaler (1968), and vitamin KJ according to Lichtenthaler and Tevini (1969). Enzyme assays. Plastocyanin activity was measured enzymatically as described by Hauska et al. (1971). Light subchloroplast particles required for the estimation of plastocyanin were obtained according to Anderson and Boardman's (1966) digitonin technique. RuBP carboxylase activity was measured according to Bowes et al. (1972). Polyacrylamide gel electrophoresis. The polypeptides were separated in polyacrylamide gel electrophoresis as described by Laemmli (1970). The electrophoresis was carried out on 10-20 % acrylamide gradient. The length of the separation gel was 13 cm. Molecular weights were obtained by comparison of separated polypeptides with Pharmacia Fine Chemicals HMW and LMW protein kits according to Weber and Osborn (1969). Isolation of ChI-protein complexes was carried out by polyacrylamide gel electrophoresis in the presence of SDS according to Wild et al. (1980). ChI alb ratio in ChI-protein complexes was estimated using the method of Wild et al. (1980). Electron microscopy. Plant segments were fixed with 2 % glutaraldehyde + 4 % paraformaldehyde in 0,05 M cacodylate buffer (pH 7.4) at room temperature for 3 h, and after washing with 0.22 M sucrose at 4°C for 12 h they were postfixed for 2 h with 2 % OS04 in the same buffer. The samples were dehydrated by 2,2-dimethoxypropane at room temperature for 2 h and embedded in Spurra. Thin sections were cut with a Reichert Om U3 ultramicrotome, stained with 8 % uranyl acetate in 0.5 % acetic acid for 30 min and poststained with 0.03 % lead citrate for 12 min. The sections were examined with a Tesla BS-500 electron microscope.
Results
eu-tolerant individuals of spinach chosen for assays could easily be distinguished by their continued growth. Non-tolerant individuals died with the increase of eu level in the soil. The percentage of surviving eu-treated plants was very high even at very high eu concentrations. The surviving plants in all eu treatments were fresh green and in good physiological condition. The results give ample evidence that spinach, under the growth conditions described, can be tolerant to eu. Table 1: The growth of spinach organs in relation to excess copper in the soil. mgCu/pot
Organs leaves stems roots whole leaves + stemsl roots leaves stems roots
50 1.988±0.276 0.641±0.106 0.152±0.031 2.781 17.30 8.26±0.58 5.56±0.64 10.63± 1.03
450
dry matter of plant organ in g 2.328 ±0.271 2.093 ± 0.398 0.820±0.067 0.601 ±0.090 0.185±0.020 0.137 ±0.020 3.333 2.831 17.02 19.66 dry matter in % 8.10±0.78 7.59±0.49 5.20±0.26 5.01 ±0.67 10.66±0.62 9.70±0.18
900 2.157 ±0.336 0.602±0.136 0.121 ±0.040 2.880 22.80 8.40±0.88 5.18 ±0.35 10.05±0.53
Values represent the mean ± SE of three replicates.
Z. Pjlanzenphysiol. Ed. 108. S. 385-395. 1982.
388
T. BASZTIISIU, M. KROL, Z. KRUPA, M. RUSZKOWSKA, U. WOJCIESKA and D. WOLINSKA
Organ growth and dry matter production were optimal when Cu concent, in the pot was 50 mg (Table 1). The dry matter of leaves and stems appears to be insignificantly affected by a higher concent of Cu in the soil (450 and 900 mg Cu/pot), but the growth rate of roots was found to be depressed under these conditions. Accordingly, the shoot to root ratio increased. The percentage of dry matter in all organs examined was not essentially different under Cu excess. Table 2: The concentration and content of copper in spinach organs related to excess copper in the soil. Organs leaves stems roots
mg Culpot 450
50 9.80± 1.39 8.32± 1.31 12.33±2.64
leaves stems roots leaves + stemslroots
19.484 5.332 1.877 13.22
900
JLg Cui g dry matter 16.35±0.86 31.66±0.81 12.67± 1.24 35.90±3.33 21.44± 1.16 56.36±2.28 JLg Cui organ 38.063 66.274 10.384 21.583 3.975 7.744 12.19 11.35
149.16± 16.07 253.05± 7.80 474.19±13.30 321.723 152.437 57.282 8.28
Values represent the mean ± SE of three replicates. To detect possible difference in Cu uptake and translocation, spinach organs were analysed for Cu concentration (Table 2). Spinach organs can accumulate high amounts of Cu. The metal uptake on a dry matter basis increased in relation to Cu Table 3: The content of plastid pigments and lipoquinones in chloroplasts isolated from leaves of Cu-treated spinach (in JLg per g of fresh weight). Components Chlorophyll a + b Carotenoids J3-carotene lutein neoxanthin violaxanthin Benzoquinones plastoquinone A a-tocopherol a-tocopherylquinone VitaminK 1 Carotenoidsl chlorophyll Benzoquinoneslchlorophyll
mg Culpot 450
50 1310 ±70 183.1 63.2± 2.7 94.0± 3.5 8.2± 0.6 17.7± 1.4 67.9 29.0± 1.3 28.0± 1.1 10.7 ± 0.7 10.4± 2.0 0.14 0.05
1480 ±30 190.2 69.0± 2.3 102.3± 3.2 4.4± 0.7 14.5± 1.0 91.9 40.4± 2.7 38.1 ± 1.3 13.4± 0.9 20.7 ± 1.4 0.13 0.06
900
1420 ±80 207.6 67.5± 1.4 117.2± 4.3 7.8± 0.5 IS.1± 1.1 87.3 34.8± 2.5 37.4± 1.6 15.1 ± 1.8 15.9± 0.9 0.15 0.06
Each value is the mean ± SE based on three replicates of three independent series.
Z. Pjlanzenphysiol. Bd. 108. S.385-395. 1982.
1510 ±70 215.1 72.9± 2.4 120 ± 3.2 7.6± 0.3 14.3± 0.9 85.9 37.6± 1.3 33.8 ± 1.1 14.5± 1.5 12.9± 0.3 0.14 0.06
389
Photosynthesis of Cu-treated spinach
level in pots. The highest amounts of accumulated Cu were found in roots. The rate of Cu concentration in these organs was 1.26-3.18 times higher than in leaves. When the total Cu was expressed per plant organ the highest amounts of the metal were observed in leaves. Table 3 summarizes the chemical components of chloroplasts isolated from spinach grown on excess Cu. The chloroplasts isolated from Cu-treated spinach do not differ significantly with respect to their Chi (a + b) content per fresh weight. A slight increase in Chi (a + b) content occurs due to Cu concentration in the medium in comparison with control. A similar tendency to that of Chi is seen in the content of total carotenoids under all experimental combinations. Thus the ratio of carotenoids to Chi was almost constant. A Cu-induced increase of lipoquinones was also observed. Table 4: Photosynthetic electron transport activities of chloroplasts isolated from leaves of Cutreated spinach plants. mg Cu/pot
Assay /Lmol equivalent per mg Chl·h TMPD-MV TMPD-NADP H 20-DCIP
,+Mn 2 + ,+DPC ,+N~Cl
1069±23 146± 5.6 49± 4 55± 2 51± 3 102± 7
50
450
900
1121±45 151± 5.6 48± 1 52± 1 53± 2 103± 2
1417±60 170± 3.7 52± 2 54± 0.4 55± 4 112± 6
1317±58 190± 3.4 47± 3 62± 0.7 50± 5 98± 4
Values represent the mean ± SE of five replicates. Table 5: Plastocyanin and RuBP carboxylase activity of spinach chloroplasts isolated from Cutreated spinach. mg Cu/pot
1
50 450 900
Plastocyanin nmol/mg Cu
Molar ratio ChI/PC
1,5 RuBP carboxylase activity counts! g . min
5.37 ±0.62 S.76±0.6S 8.96±0.82 S.80±0.56
210 127 127 127
1504± 153 1263± 102 1370±112 1485± 161
Values represent the mean ± SE of three replicates.
PS I activity measured by MV as well as NADP photoreduction of chloroplasts ~so lated from spinach was higher in plants grown in soil containing high concentrations of Cu than in the control (Table 4). This phenomenon could be related to plastocyanin synthesis enhanced at external Cu concentration. However, plastocyanin synthesis reached its maximum at exactly 50 mg Cu/pot, and a further increase of Cu supply did not enhance synthesis (Table 5). PS II activity did not vary in relation to Cu concentration in the medium. Also, an Z. Pjlanzenphysiol. Bd. 108. S. 385-395. 1982.
390
T. BASZYNSKI, M. KROL, Z. KRUPA, M. RUSZKOWSKA, U. WOJCIESKA and D. WOLINSKA
addition of Mn 2 + to the reaction mixture did not noticeably change the DCIP photoreduction rate of chloroplasts, except at 900 mg Cu/pot. DPC, an artificial electron donor for PS II, similarly caused an insignificantly slight increase of DCIP photoreduction regardless of the Cu level. However, Nf-4 + as an uncoupler strongly increased PS II activity in all combinations of the experiment. It is worth noting that the activity of RuBP carboxylase does not appear to be related to Cu concentration in the soil. Thus the association of CU with RuBP carboxylase may be controversial (Table 5). Figure 1 shows polypeptides separated by the method of Laemmli. A comparison of the electrophoretic densitograms showed that the new chloroplast proteins did not occur in Cu-treated plants. Our preliminary studies indicate, however, an increased Cu content in some protein fractions of chloroplasts. Detailed studies on Cu-binding protems are m progress. Table 6: Relative proportion of Chi-protein complexes and Chi alb ratio in chloroplasts isolated from spinach grown on sandy soil contained 1 and 450 mg Cu/pot. mg Culpot
450
%Chl Chi alb %Chl ChI alb
CP1a
CP1
LHCP2
CPa
LHCP 3
FC
1.8
20.8 5.5 27.0 4.5
2.4 1.6 2.6 1.5
6.1 2.6 6.3 2.0
40.0 1.5 36.8
28.9 3.5 25.1 3.0
2.1
-
112
-
9'
-
79 74
6
-
6' 59 52
1:
-
'6
- u
-
-
900
450
34
33
1.1
~
.211 COl
~
...
.2 ::J
u
28
~
26 23
~
0
50
mg Cu/pot Fig. 1: Polypeptide patterns of chloroplasts isolated from spinach leaves grown on sandy soil containing 1, 50, 450 and 900 mg Culpot, respectively. Z. Pjlanzenphysiol. Ed. 108. S. 385-395. 1982.
Photosynthesis of eu-treated spinach
391
The separation of ChI-protein complexes indicates that the relative content of ChI in CP1 of plants grown in soil with higher Cu doses increases from 20.8 % to 27 % of the total amount of ChI used for separation of the complexes (Table 6). The relative amounts of ChI in CPa and LHCP 3 complexes did not undergo greater changes and were approximately the same as those occurring in the corresponding complexes of control plants. However, the relative percentage content of ChI occurring in the CPa complex was lower than the values given so far (Anderson, 1978). In examining the effect of excess Cu we did not observe changes in the inner structure of chloroplasts. In the micrographs (Fig. 2), however, some deposits can be seen
Fig. 2: Electron micrographs of the ultrastructure of spinach chloroplasts: a, chloroplast of leaves from eu-treated plants, mag. x 18,000; b, control chloroplast from leaves; x 18,000; c, chloroplast from stem of eu-treated plants; x 10,000, deposits on the surface of the chloroplast envelope are seen; d, chloroplast from stem of control plants; x 10,000.
Z. Pjlanzenphysiol. Bd. 108. S. 385-395. 1982.
392
T.
BASZYNSKI,
M. KROL, Z.
KRUPA,
M.
RUSZKOWSKA,
U. WOJCIESKA and D.
WOLINSKA
inside cells, as well as on the surface of the chloroplast envelope, suggesting the presence of Cu. Such structures did not occur in control plants. Discussion
The occurrence of Cu-tolerant plants in normal populations grown on excess Cu is generally known and has been recognized in the rapidly occurring genetic changes by the excess Cu (Walley et aI., 1974). This tolerance depends on the total concentration of the metal in the soil. Even as much as 900 mg Cu/pot, when applied in six doses of 150 mg each, over several days, did not produce a decrease in growth rates of the aerial parts of plants. Fertilization with excess Cu decreased the root mass, although only to a degree that had no negative effect on the crop of leaves and stems in the course of the experiments. The maximum spinach crop obtained at 50 mg Cu/pot shows that such amount seems to be optimal for this species. As expected, a remarkable accumulation of Cu, as with other heavy metals, was observed in roots. This depended on Cu content in the medium, although it was not directly proportional. The physiological parameters studied, such as the chemical composition of the photosynthetic apparatus in control plants and those treated with high Cu doses, did not differ essentially. Increased Cu doses per pot caused only a slight stimulation of the synthesis of ChI, carotenoid pigments (particularly lutein) and lipoquinones. No toxic effect of Cu on the activity of the photosystems of chloroplast isolated from Cu-treated spinach leaves was found either. This is surprising in view of the toxic effect of Cu ions on the photosynthetic electron system of isolated chloroplasts or intact green algae (Macdowall and Haberman, 1969). It was shown that Cu ions block the photosynthetic electron transport at both the oxidizing side of PS II and the reducing side of PS I (Shioi et aI., 1978 a, b). According to Vierke and Struckmeier (1977, 1978) eu ions bind to a membrane protein which is not involved in electron transport itself but which leads to structural changes of the photosynthetic membrane with subsequent inhibition of electron transport. On the other hand, Sandmann and Boger (1980 a) have shown that free Cu ions inhibit electron transport in both photosystems leading to lipoxygenase activity and consequently the acceleration of peroxidative degradation of the lipids of the chloroplast membranes. However, in our experiments, when chloroplasts were isolated from plants provided with excess Cu some increase of PS I activity above the controls was observed. The simplest explanation would be a Cu-induced synthesis of plastocyanin, which influenced PS I activity. This would be in agreement with the earlier observation of Boger et al. (1980 b) that plastocyanin synthesis in some species of algae is strictly dependent on the Cu level in the growth medium and that Cu deficiency blocks this synthesis. Unfortunately, as mentioned above, plastocyanin synthesis in chloroplasts of spinach is not induced by very high concentrations of Cu in the soil. Z. Pjlanzenphysiol. Bd. 108. S. 385-395. 1982.
Photosynthesis of eu-treated spinach
393
PS II activity does not depend on Cu level in the growth medium of spinach. Assays with addition of Mn 2+ or DPC show that high amounts of Cu in soil do not influence the oxidizing side of PS II in chloroplasts. The increase of DCIP photoreduction rate after the addition of Nfu + to the reaction mixture indicates that all chloroplasts examined are coupled, among others those isolated from Cu-treated plants. This observation corresponds to an earlier finding (Vierke and Struckmeier, 1978) that photophosphorylation is not affected by Cu ions, but is in contrast to Samuelsson and Oquist (1980), who have shown Cu ions to be an uncoupler of photophosphorylation. Both observations were made, however, on in vitro experiments. The lack of a toxic effect of Cu on photosystems in chloroplasts isolated from spinach provided with high Cu doses, found in our studies, may result from the absence or small amounts of free Cu 2 + ions, known as the most toxic form of the metal, in chloroplasts. It is possible that during the growth of Cu-treated spinach Cu-binding proteins are formed which, as suggested by Sandmann and Boger (1980 b) and Hogan and Rauser (1981), might chemically inactivate the Cu ions taken up by the leaves. The precipitates which are visible in the micrographs and localized mainly in leaf cells and sporadically on the surface of the chloroplast envelope of stem cells, could prevent Cu from entering more sensitive sites of cell metabolism, among others those inside the chloroplasts. According to Ashida et al. (1963) the precipitation may be due to the formation of CuS via methionine. The rapid destruction of ChI-protein complexes in isolated chloroplasts due to the influence of Cu ions added to the reaction mixture, as found by Samuelsson and Oquist (1980) was not observed in our studies. The increase of the relative content of ChI in CP1 of chloroplasts isolated from Cu-treated spinach is coupled to the concomitant increase of PS I activities. Such a relationship was found earlier (Anderson, 1978). The relative content of ChI in CPa, which is probably associated with the centre of PS II reaction (Anderson, 1978) in control chloroplasts, and those isolated from Cu-treated spinach is very similar. The characteristics of the photosynthetic apparatus of spinach leaves growing in soil containing high Cu doses does not differ much from that of control plants. This indicates apparently high tolerance of Spinacia oleracea L. var. Matador to Cu added to the growth medium. Without discussing the mechanism of tolerance of spinach to Cu, it can be assumed that spinach may adapt quickly to high external concentrations of Cu, insignificantly affecting plant growth and photosynthetic activity. Acknowledgements This study was carried out under the project MR II/7 coordinated by the Institute of Plant Physiology, Polish Academy of Sciences.
References ANDERSON, J. M. and N. K. BOARDMAN: Fractionation of the photochemical systems of photosynthesis. Biochim. Biophys. Acta 112, 403-412 (1966). Z. Pjlanzenphysiol. Ed. 108. S. 385-395. 1982.
394
T. BASZYNSKI, M. KROL, Z. KRuPA, M. RUSZKOWSKA, U. WOJCIESKA and D. WOLINSKA
ANDERSON, J. M., J. C. WALDRON, and S. W. THORNE: Chlorophyll-protein complexes of spinach and barley thylakoids. Spectral characterization of six complexes resolved by an improved electrophoretic procedure. FEBS Lett. 92, 227-233 (1978). ANDERSON, J. M.: P-700 content and polypeptide profile of chlorophyll-protein complexes of spinach and barley thylakoids. Biochim. Biophys. Acta 591, 113-126 (1980). ANTONOVICS, J., A. D. BRADSHAW, and R. G. TURNER: Heavy metal tolerance in plants. Adv. Ecol. Res. 7, 1-85 (1971). ARNON, D. I.: Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1-15 (1949). ASHIDA, J., N. HIGASHI, and T. KIKUCHI: An electron microscopic study on copper precipitation by copper-resistant yeast cells. Protoplasm a 57, 27-32 (1963). BOHNER, H., H. MERKLE, P. KRONECK, and P. BOGER: High variability of the electron carrier plastocyanin in microalgae. Eur. J. Biochem. 195, 603-609 (1980). BOWES, G., W. L. OGREN, and R. H. HAGEMAN: Light saturation, photosynthesis rate, RuDP carboxylase activity and specific leaf weight in soybeans grown under different light intensities. Crop Sci. 1, 77-79 (1972). CURVETTO, N. R. and W. F. RAUSER: Isolation and characterization of copper-binding proteins from roots of Agrostis gigantea tolerant to excess copper. Plant Physiol. Suppl. 63, 59 (1979). DE FILIPPIS, L. F. and C. K. PALLAGHY: The effect of sub-lethal concentrations of mercury and zinc on Chlorella. III. Development and possible mechanisms of resistance to metals. Z. Pflanzenphysiol. 79, 323-335 (1976). FOSTER, P. L.: Copper exclusion as a mechanism of heavy metal tolerance in a green alga. Nature (London) 269, 322-323 (1977). HAGER, A. und T. BERTENRATH: Verteilungschromatographische Trennung von Chlorophyllen und Karotenoiden griiner Pflanzen an Diinnschichten. Planta (Berl.) 58,564-568 (1962). HABERMANN, H. M.: Reversal of copper inhibition in chloroplast reactions by manganese. Plant Physiol. 44, 331-336 (1969). HAUSKA, G. A., R. E. MCCARTY, R. J. BERZBORN, and E. RACKER: Partial resolution of the enzymes catalyzing photophosphorylation. VII. The function of plastocyanin and its interaction with a specific antibody. J. BioI. Chern. 246, 3524-3531 (1971). HOGAN, G. D. and W. E. RAUSER: Role of copper binding, absorption, and translocation in copper tolerance of Agrostis gigantea Roth. J. Exp. Bot. 32,27-36 (1981). JOWETT, D.: Populations of Agrostis spp. tolerant of heavy metals. Nature (London) 182, 816-817 (1958). LAEMMLI, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680-685 (1970). LICHTENTHALER, H.-K.: Verbreitung des lipophilen Plastidenchinone im nicht-griinen Pflanzengewebe. Z. Pflanzenphysiol. 59, 195-210 (1968). - Zur Synthese der lipophilen Plastidenchinone und Sekun&irkarotenoide wahrend der Chromoplastenentwicklung. Ber. dtsch. bot. Ges. 82, 483--497 (1969). LICHTENTHALER, H.-K. und M. TEVINI: Die Wirkung von UV-Strahlen auf die Lipochinon-Pigment-Zusammensetzung isolierter Spinatchloroplasten. Z. Naturforsch. 24 b, 764-769 (1969). MACDOWAL, F. D. H.: The effects of some inhibitors of photosynthesis upon the photochemical reduction of a dye by isolated chloroplasts. Plant Physiol. 24, 462-480 (1949). SAMUELSSON, G. and G. OQUIST: Effects of copper chloride on photosynthetic electron transport and chlorophyll-protein complexes of Spinacia oleracea. Plant and Cell Physiol. 21, 445-454 (1980). SANE, P. V., D. J. GOODCHILD, and R. B. PARK: Characterization of chloroplast photosystem 1 and 2 separated by a non-detergent method. Biochim. Biophys. Acta 216, 162-178 (1970).
2. Pjlanzenphysiol. Bd. 108. S. 385-395. 1982.
Photosynthesis of Cu-treated spinach
395
SANDMANN, G. and P. BOGER: Copper-mediated lipid peroxidation processes in photosynthetic membranes. Plant Physio!. 66, 797-800 (1980 a). - - Copper deficiency and toxicity in Scenedesmus. Z. Pflanzenphysio!. 98, 53-59 (1980 b). SHIOI, Y., H. TAMAI, and T. SASA: Inhibition of photosystem II in the green alga Ankistrodesmus /alcatus by copper. Physio!. Plant 44, 434-438 (1978 a). - - - Effects of copper on photosynthetic electron transport systems in spinach chloroplasts. Plant and Cell Physio!. 19,203-209 (1978 b). VIERKE, G. and P. STRUCKMEIER: Binding of copper II to proteins of the photosynthetic membrane and its correlation with inhibition of electron transport in class II chloroplasts of spinach. Z. Naturforsch. 32 c, 605-610 (1977). - - Inhibition of millisecond luminescence by copper II in spinach chloroplasts. Z. Naturforsch. 33 c, 266-270 (1978). WALLEY, K. A., M. S. 1. KHAN, and A. D. BRADSHAW: The potential for evolution of heavy metal tolerance in plants. Heredity 32,309-319 (1974). WEBER, K. and M. OSBORN: The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Bio!. Chern. 244, 4406-4412 (1969). WILD, A. and B. URSCHEL: Methodical studies in the separation of chlorophyll-protein complexes by polyacrylamide gel electrophoresis. Z. Naturforsch. 35 c, 627-637 (1980).
Z. Pjlanzenphysiol. Bd. 108. S.385-395. 1982.