The donor side of photosystem II is impaired in a Cd ... - Science Direct

J. Plant Physiol. 159. 941 – 950 (2002)  Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp

The donor side of photosystem II is impaired in a Cd2 + -tolerant mutant strain of the unicellular green alga Chlamydomonas reinhardtii Jürgen Voigt1 *, Klaus Nagel2 1

Physiologisch-chemisches Institut der Universität Tübingen, Hoppe-Seyler-Straße 4, D-72076 Tübingen, Germany

2

Institut für Ostseeforschung, Seestraße 15, D-18119 Warnemünde, Germany

Received November 2, 2001 · Accepted April 22, 2002

Summary Growth of a cadmium-tolerant mutant strain of the unicellular green alga Chlamydomonas reinhardtii was found to be impaired under photoautrotrophic, but not under mixotrophic conditions. As compared to wild-type cells, oxygen evolution by the photoautotrophically grown mutant was considerably decreased and higher photon fluence rates were required both for light compensation of oxygen consumption/production and maximal oxygen evolution. The capability for oxygen production was decreased in Chlamydomonas reinhardtii cells when grown in the presence of acetate without aeration. Wild-type cells grown under these conditions showed a rather low but significant oxygen evolution immediately after transfer to photoautotrophic conditions. This residual oxygen production was completely suppressed in the presence of acetate, obviously due to acetate inhibition of the watersplitting complex. In the case of our cadmium-tolerant mutant strain, however, residual oxgen production was measured even in the presence of acetate. After removal of acetate, oxygen evolution by the cadmium-tolerant mutant strain was increased to higher rates than measured for wild-type cells, but considerably higher photon fluence rates were required both for light compensation of oxygen consumption/production and maximal oxygen evolution. The conclusion that the donor side of photosystem II is affected in our cadmium-tolerant mutant strain was further corroborated by a stronger decrease of the fluorescence level caused by hydroxylamine. Key words: Chlamydomonas reinhardtii – acetate inhibition – cadmium-tolerant mutant – dark respiration – photosynthesis – photosystem II

Introduction Plants and green algae respond to heavy metal stress with the induction of phytochelatins, a class of peptides consisting of repeating units of γ-glutamylcystein followed by a C-terminal glycine (Grill and Zenk 1985, Jackson et al. 1987, Reese * E-mail corresponding author: [email protected]

and Wagner 1987, Howe and Merchant 1992, Cobbett 2000), which are derived from glutathione by the action of phytochelatine synthase (Rennenberg 1987). Cadmium-sensitive mutants of Arabidopsis thaliana have been described that are deficient in phytochelatine synthase (Howden et al. 1995 a) or glutathione synthesis (Howden et al. 1995 b). Phytochelatins seem to be ubiquitous in higher plants and algae (Gekeler et al. 1988, 1989, Howe and Merchant 1992). In the unicellular 0176-1617/02/159/09-941 $ 15.00/0

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Jürgen Voigt, Klaus Nagel

green alga Chlamydomonas reinhardtii, phytochelatins have been found both in the cytosol and in the chloroplast (Nagel et al. 1996). Inhibition of photosynthesis by sublethal Cd2 + concentrations has been described for Euglena gracilis (De Filippis et al. 1981), Nostoc linckia (Husaini and Rai 1991), Anabena (Bolanos et al. 1992), a grassland moss (Wells and Brown 1995), and higher plants (Malik et al. 1992, Vassilev et al. 1995). Cadmium tolerance in a metal-contaminated population of the grassland moss Rhytidiadelphus squarrosus was found to be accompanied by a decreased sensitivity of photosynthesis to Cd2 + (Wells and Brown 1995). A cadmium-tolerant population of the unicellular green alga Chlamydomonas reinhardtii was selected from the cadmium-sensitive, cell wall deficient strain cw-15 under mixotrophic conditions by successively increasing Cd2 + concentrations within a period of 9 months (Nagel and Voigt 1989). The predominant mutant in this cadmium-tolerant population was shown not to be affected with respect to uptake and sequestering of Cd2 + by phytochelatins (Nagel and Voigt 1995), in contrast to other Cd2 + -tolerant Chlamydomonas mutant strains isolated by Collard and Matagne (1994), which showed a slower accumulation of the metal. In the case of our mutant strain, however, photosynthesis was found to be impaired (Nagel and Voigt 1995, Voigt et al. 1998). When grown under photoautotrophic conditions, this mutant showed a decreased, but cadmium-tolerant photosynthetic oxygen evolution (Nagel and Voigt 1995). As previously reported (Voigt et al. 1998), the cellular ATP level in the cadmium-tolerant mutant, Cdr125, was substantially lower than in the parent strain, cw15, when grown in the presence of acetate at a photon fluence rate of 20 µmol m – 2 s –1. This difference in the ATP levels could be overcome by increasing the photon fluence rate to 100 µmol m – 2 s –1 (Voigt et al. 1998), indicating that an increased photon fluence rate is required for photosynthesis in the case of our particular cadmium-tolerant mutant strain. On the other hand, the uptake of [14C]acetate was found to be increased in the cadmium-tolerant mutant Cdr125 as compared to the parent strain cw15 (Nagel and Voigt 1995), indicating that there is a metabolic co-operation between chloroplast and mitochondria in C. reinhardtii as previously reported by Lemaire et al. (1988). Therefore, we have investigated the effects of increasing photon fluence rates on oxgen evolution and oxygen consumption by the cadmium-tolerant mutant Cdr125 and its cadmium-sensitive parent strains grown under photoautotrophic and mixotrophic conditions, respectively. Furthermore, we have comparatively analysed the polypeptide patterns of the thylakoid membranes and the chlorophyll content.

Materials and Methods Strains and growth conditions Chlamydomonas reinhardtii 137 ˚C mt + (a wild-type strain from the collection of W. T. Ebersold) was provided by Professor Dr. D. Mer-

genhagen (Institut für Allgemeine Botanik, Universität Hamburg, Germany). The Cd2 + -sensitve, cell wall-deficient Chlamydomonas reinhardtii strain, CW15 (Davies and Plaskitt 1971), was obtained from the Sammlung von Algenkulturen at the University of Göttingen. The mutant strain Cdr125 was isolated from the Cd2 + -tolerant population, CW15-Cdr, which was derived from the Cd2 + -sensitive, cell wall-deficient strain Chlamydomonas reinhardtii CW15 (Davies and Plaskitt 1971) by cultivation under mixotrophic growth conditions in the presence of successively increasing concentrations of CdCl2 (Nagel and Voigt 1989). Cells were grown at 21˚C and at a photon fluence rate of 20 µmol m – 2 s –1 in a high-salt medium with or without 0.2 % (w/v) sodium acetate as described previously (Voigt and Münzner 1987). Cell densities were determined by hemocytometer counting.

Measurements of O2-production and O2-consumption Cells were washed and resuspended in either 0.5 % (w/v) sodium bicarbonate buffer or acetate-containing culture medium to obtain a chlorophyll concentration of 0.2 mg mL –1. The samples (4 mL) were incubated at 21 ˚C in a plexiglas cuvette and laterally irradiated using slide projectors (Prado, Leitz, Germany) equipped with halogen lamps (Xenophot, Osram, Germany). Photon fluence rates were measured with a quantum photometer (Li 185-A; Lambda Instruments Corp., Lincoln, NE, U.S.A.). Photosynthetic production of O2 and O2-consumption were monitored polarographically using an oxygen electrode (YSI model 53; Yellow Springs Instrument Co., Yellow Springs, Ohio, USA).

Isolation of thylakoid membranes Thylakoid membranes were purified from cell-free homogenates by the flotation procedure as described by Chua et al. (1975).

Pigment analysis Pigments were extracted from intact cells or thylakoid membranes with 80 % acetone. Chlorophyll a, b and total Chl content were estimated using the method of Arnon (1949). The spectra were measured against 80 % acetone using a PMQ2 uv/vis spectrophotometer (Carl Zeiss, Oberkochem, Germany).

Measurement of chlorophyll fluorescence induction Chlorophyll fluorescence induction was investigated using a Hitachi model F-3000 spectrofluorometer (excitation at 480 nm; emission measured at 685 nm).

Enzyme activities Cytochrom-c-oxidase activity (EC 1.9.3.1) was determined photometrically as described by Wharton and Tzagoloff (1967) and Yonetani (1967). The reaction was started by addition of C. reinhardtii homogenate obtained by ultrasonication of 108 cells. NADP + -dependent glyceraldehyde-3-phosphate dehydrogenase activity (EC 1.2.1.12) was measured photometrically at 340 nm as described by Latzko and Gibbs (1969) and Müller et al. (1969) using ho-

A Cd2 + -tolerant Chlamydomonas mutant impaired in PS II activity

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mogenates prepared by ultrasonication of 1– 5 × 107 cells. The reaction was started by addition of 3-phosphoglycerate.

Determination of protein Quantitation of protein was performed by the method of Bradford (1976) using bovine IgG as standard.

Electrophoresis Aliquots of purified thylakoid membranes corresponding to 80 µg of protein were subjected to SDS-PAGE according to Laemmli (1970) using gel slabs (15 × 12.5 × 0.15 cm) containing 15 % (w/v) acrylamide. The gels were stained for proteins with Coomassie brillant blue G-250.

Results Cell growth The cadmium-tolerant C. reinhardtii mutant strain Cdr125 can be propagated under photoautotrophic conditions. However, the growth rate was found to be considerably decreased as compared to the wild type 137 C( + ) and to the cell wall-defi-

Figure 2. Absorbance spectra of acetone extracts from photoautotrophically grown Cdr125 and cw-15 cells. The same number of cells (1 × 108) of both strains were extracted with 80 % (v/v) acetone and analysed as described under ‹Materials and Methods›.

cient, Cd2 + -sensitive strain, cw-15, at a photon fluence rate of 20 µmol m – 2 s –1 (Fig. 1 A). This defect in cell growth could be overcome by addition of acetate which is metabolised by C. reinhardtii cells (Sager and Granick 1953). When cultivated in the presence of acetate without aeration (Voigt and Münzner 1987), the same growth rates were observed for the cadmium-tolerant mutant strain Cdr125 and the cadmium-sensitive strains (Fig. 1B).

Pigment composition, protein content and dry weight

Figure 1. Cell growth in the absence of Cd2 + of the Cd2 + -tolerant mutant Cdr125 and the Cd2 + -sensitive strains cw-15 and 137(= wild type) under photoautotrophic (A) and mixotrophic conditions (B).

No difference between the Cd2 + -tolerant Chlamydomonas mutant strain Cdr125 and the Cd2 + -sensitive strain cw-15 was observed with respect to the absorption spectra between 350 and 800 nm of the corresponding acetone extracts of photoautotrophically grown cells (Fig. 2) or isolated thylakoid membranes (data not shown), indicating that the pigment composition is not changed in our particular Cd2 + -tolerant mutant strain. Furthermore, the chl a : chl b ratio was not changed in our particular cadmium-tolerant mutant strain (data not shown). However, the absorbance of the acetone extracts of Cdr125 cells was generally lower than in the case of cw-15 (Fig. 2) or wild-type cells. Indeed, the chlorophyll level was found to be decreased by about 30 % in photoautotrophically grown Cdr125 cells (Table 1). Protein level and dry weight were decreased also in these Cd2 + -tolerant mutant cells (Table 1). As revealed by microscopic inspection, the Cdr125 cells were generally smaller than cw-15 and wild-type cells (data not shown). These differences were less pronounced in the case of cells grown in the presence of acetate (Table 1).

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Table 1. Chlorophyll and protein contents of Cd2 + -sensitive and Cd2 + -tolerant Chlamydomonas reinhardtii strains. photoautotrophically grown culturesa

micotrophically grown culturesb

Strain

dry weightc

proteinc

chlorophyllc

dry weightc

proteinc

chlorophyllc

wt cw-15 Cdr125

62 ± 8.7 60 ± 9.1 44 ± 6.3

14.1 ± 1.9 13.7 ± 2.1 9.2 ± 1.7

11.2 ± 1.4 10.8 ± 1.2 6.5 ± 0.8

68 ± 6.3 66 ± 6.5 57 ± 6.1

16.5 ± 1.4 16.8 ± 1.7 13.3 ± 1.5

11.0 ± 0.9 10.6 ± 0.7 7.5 ± 0.6

a b c

Cells were grown under continuous light (photon fluence rate: 20 µmol m – 2 s – 1) in the absence of acetate and bubbled filtered air. Cells were grown without aeration in the presence of sodium acetate under continuous light at a photon fluence rate of 20 µmol m – 2 s – 1. pg per cell ± SD.

Table 2. Effect of growth conditions on oxygen production/consumption by illuminated Cd2 + -sensitive and Cd2 + -tolerant Chlamydomonas reinhardtii strains. Rate of reactionb (µmol O2 evolved or consumed/mg chlorophyll per hour)c

Straina

wt cw-15 Cdr 125 a

b c d

photoautotrophically grown cultures aeratedd

micotrophically grown cultures aeratedd

mixotrophically grown cultures not aerated

86.2 ± 17.4 78.0 ± 22.7 13.5 ± 5.9

14.2 ± 1.9 12.5 ± 2.4 13.0 ± 3.1

– 1.0 ± 0.18 – 1.7 ± 0.23 – 2.6 ± 0.25

Cells were grown under continuous light (photon fluence rate: 20 µmol m – 2 s – 1) in the absence (photoautotrophic conditions) or in the presence of sodium acetate (mixotrophic conditions). Oxygen consumption/production was measured after transfer to fresh medium at 24 C at a photon fluence rate of 20 µmol m – 2 s – 1. Cdr 125 cells contained lower chlorophyll levels than cw-15 and wild-type cells (80%). Cultures were aerated by bubbling with filtered air.

Light-dependence of oxygen consumption/production When grown under photoautotrophic conditions at a photon fluence rate of 20 µmol m – 2 s –1, Cdr125 cells produced considerably lower amounts of oxygen than cw-15 and wild-type cells (13.4 versus 78.0 and 86.2 µmol O2/mg chlorophyll, respectively; Table 2). This finding is in agreement with the decreased growth rate of this particular mutant strain under photoautotrophic conditions, (Fig. 1A). When cultivated in the presence of acetate with aeration at the same photon fluence rate, oxygen production by cw-15 and wild-type cells was strongly decreased as compared to photoautotrophic conditions, whereas oxygen evolution by Cdr125 cells was not decreased under these conditions (Table 2). About 13 µmol O2/mg chlorophyll were produced by all these strains (Table 2). Since, however, Cdr125 cells contain lower chlorophyll levels than cells of the other strains (Table 1), oxygen production per Cdr125 cell was lower than oxgen production by cw-15 or wild-type cells under these conditions. When the strains were cultivated in the presence of acetate at the same photon fluence rate but without aeration, no oxygen production, but a slow oxygen consumption was measured after transfer of the cells to fresh culture medium (Table 2). The latter findings, which were not expected, induced us to comparatively investigate the light-saturation of oxygen consumption/production

by the Cdr125 mutant and its parent strains grown under photoautotrophic (Fig. 3) and mixotrophic conditions (Fig. 4), respectively.

Figure 3. The photon fluence rate-response curves of oxygen consumption and photosynthetic oxygen evolution by the Cd2 + -tolerant mutant strain Cdr125 and the Cd2 + -sensitive strains CW15 and 137 C( + ) under photoautotrophic conditions. Given values are means ± SD from four independent experiments.


Figure 4. The photon fluence rate-response curves of oxygen consumption and photosynthetic oxygen evolution by the Cd2 + -tolerant mutant strain Cdr125 and the Cd2 + -sensitive strains CW15 and 137C( + ) grown under mixotrophic conditions (in the presence of acetate). Given values are means ± SD from four independent experiments.

When grown under photoautotrophic conditions, a considerably decreased photosynthetic oxygen production by our Cd2 + -tolerant mutant strain, as compared to Cd2 + -sensitive cells, was measured even at light saturation (photon fluence rates above 300 µmol m – 2 s –1; Fig. 3). Increased photon fluence rates, were required for both maximal oxygen evolution and light compensation of oxygen consumption/production in the case of our particular Cd2 + -tolerant mutant strain (Fig. 3). However, no difference between the Cd2 + -tolerant Cdr125 cells and the Cd2 + -sensitive strains was observed with respect to dark respiration (about 30 µmol O2 per h and mg chlorophyll). In the case of cultures grown without aeration in the presence of acetate (= mixotrophic conditions), no oxygen production was observed for the Cd2 + -sensitive cw-15 and wildtype cells at any photon fluence rate (Fig. 4). However, a rather low, but significant, residual photosynthetic oxygen evolution was measured when mixotrophically grown Cdr125 cells were illuminated at photon fluence rates of more than 150 µmol m – 2 s –1 (Fig. 4). At light saturation (at photon fluence rates > 300 µmol m – 2 s –1), this residual O2 production (2.5 µmol O2 per hour and mg chlorophyll) corresponded to 2 % of the maximal O2-production by photoautotrophically grown Cdr125 cells (Figs. 3 and 4). In the dark and at rather low photon fluence rates, oxygen consumption by the Cdr125 cells and the cadmium-sensitive strains was decreased markedly in the case of cultures grown under mixotrophic conditions without aeration (Fig. 4) as compared to photoautotrophically grown cultures (Fig. 3). Acetate was found to interfere with photosynthesis in two different ways:

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1. In unicellular green algae, acetate causes a decreased synthesis of components of the photosynthetic apparatus (Boege et al. 1981, Polle et al. 2000). 2. Acetate is an inhibitor of photosynthetic oxygen-production, which directly interacts with the water splitting complex (Sinclair 1984, Kuhne et al. 1999 and references cited by these authors). Therefore, we have measured the light-saturation of oxygen consumption/production by the different strains immediately after removal of acetate (Fig. 5). As in the experiment shown in Figure 4, the cells were grown under mixotrophic conditions, collected by centrifugation and washed and subsequently resuspended in sodium bicarbonate buffer. A rather low, but significant, photosynthetic oxygen production was observed for wild-type and cw-15 cells at photon fluence rates above 20 µmol m – 2 s –1, light saturation was reached at photon fluence rates above 50 µmol m – 2 s –1 (Fig. 5). Even under these conditions, photosynthetic oxygen production by the mutant strain Cdr125 was found to be increased at photon fluence rates of more than 100 µmol m – 2 s –1 (Fig. 5). At light saturation, the rate of photosynthetic oxygen evolution by the Cd2 + -tolerant Cdr125 cells was found to be increased by 100 % after removal of acetate (Figs. 4 and 5). Considerably higher photon fluence rates were required for both light-compensation and and light-saturation of photosynthetic oxygen evolution in the case of our particular mutant strain (Fig. 5). As compared to photoautotrophically grown cultures (Fig. 3), both oxygen production in the light and oxygen consumption

Figure 5. The photon fluence rate-response curves of oxygen consumption and photosynthetic oxygen evolution by mixotrophically grown cells of the Cd2 + -tolerant mutant Cdr125 and the Cd2 + -sensitive CW15 and 137C( + ) strains after removal of acetate. Cells were grown under mixotrophic conditions (in the presence of acetate) and subsequently washed with and resuspended in sodium bicarbonate buffer (without acetate) before measuring oxygen consumption/production. Given values are means ± SD from four independent experiments.

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in the dark were strongly decreased in mixotrophically grown cells even after removal of acetate indicating that de novo synthesis of components of the photosynthesis apparatus and of other polypeptides is required for adaptation to photoautotrophic growth.

Polypeptide pattern of the thylakoid membranes The polypeptide patterns of thylakoid membranes isolated from Cd2 + -tolerant Cdr125 and its Cd2 + -sensitive parent strains grown under either photoautotrophic or mixotrophic conditions were compared by SDS-PAGE (Fig. 6). The polypeptide patterns of the thylakoid membranes were found to be dependent on the growth conditions as recently reported (Heifetz et al. 2000, Polle et al. 2000). In all strains, polypeptides with apparent molecular masses of 29 kDa and 22 kDa were predominant in thylakoid membranes isolated from photoautotrophically grown cells (Fig. 6, lanes 1– 3). These components are obviously LHC proteins. Decreased levels of these polypeptides were found in the thylakoids of mixotrophically grown cells (Fig. 6, lanes 5–7). Accordingly, the relative amounts of the other polypeptide constituents were higher in the thylakoid membranes of mixotrophically grown cells than in thylakoids from photoautotrophically grown cells. Neither

Figure 6. Comparative SDS-PAGE analysis of the polypeptide patterns of the thylakoids of Cdr125 and cw-15 cells grown under photoautotrophic and mixotrophic conditions, respectively. Purified thylakoid membranes corresponding to 80 µg of protein were subjected to SDS-PAGE as described in the ‹Materials and Methods› section. Lane 1 = Cdr125 cells grown under photoautotrophic conditions; lane 2 = cw-15 cells grown under photoautotrophic conditions; lane 3 = wild-type cells grown under photoautotrophic conditions; lane 5 = Cdr125 cells grown under mixotrophic conditions; lane 6 = cw-15 cells grown under mixotrophic conditions; lane 7 = wild-type cells grown under mixotrophic conditions; lanes 4 and 8 = protein molecular weight standard: α2-macroglobuline (Mr 170 000), β-galactosidase (Mr 116 350), fructose-6-phosphate kinase (Mr 85 200), glutamate dehydrogenase (Mr 55 600), aldolase (Mr 39 200), triosephosphate isomerase (Mr 26 650), trypsin inhibitor (Mr 20 100), lysozyme (Mr 14 300).

under photoautotrophic (Fig. 6, lanes 1– 3) nor under mixotrophic conditions (Fig. 6, lanes 5–7) were differences observed between the polypeptide patterns of the thylakoid membranes from Cdr125, cw-15 and wild-type cells with the exception of a 38 kDa component, the level of which was found to be decreased in mixotrophically grown Cdr125 cells (Fig. 6, lane 5) as compared to cw-15 and wild-type cells (Fig. 6, lanes 6 and 7).

Effects of hydroxylamine on chlorophyll fluorescence induction As previously reported, our Cd2 + -tolerant mutant strain, Cdr125, is impaired in photosystem II activity (Voigt et al. 1998). It is well established that oxygen evolution by photosystem II is inhibited by NH2OH via a release of manganese ions from the water splitting complex (Sharp and Yocum 1981, Robinson et al. 1981, Guiles et al. 1990) and that this compound diminishes the fluorescence yield (Den Haan et al. 1974, 1976, Joliot 1977) because it is an electron donor of photosystem II (Bennoun and Joliot 1969, Bouges-Bocquet et al. 1973, Messinger et al. 1991). Therefore, we have compared the effects of NH2OH on chlorophyll fluorescence induction in autotrophically grown cells of the Cd2 + -tolerant mutant strain, Cdr125 (Fig. 7 A), and its Cd2 + -sensitive parent strain cw-15 (Fig. 7 B). As previously reported (Nagel and Voigt 1995), the kinetics of chlorophyll fluorescence induction is strongly affected in our Cd2 + -tolerant mutant strain (Fig. 7A, B). Addition of NH2OH caused a decrease in chlorophyll fluorescence in both strains (Fig. 7 A, B). However, this effect was considerably more pronounced in the Cd2 + -tolerant mutant Cdr125 (Fig. 7 A) than in its Cd2 + -sensitive parent strain cw-15 cells

Figure 7. Effect of hydroxylamine on the kinetics of chlorophyll fluorescence induction in dark-adapted cells of the Cd2 + -tolerant mutant Cdr125 (A) and the Cd2 + -sensitive strain cw-15 (B). Cells were grown under photoautotrophic conditions and subsequently incubated in the dark for 10 h. (a) Untreated cells; (b) cells incubated for 30 min in the presence of 10 – 5 mol/L hydroxylamine; (c) cells incubated for 30 min in the presence of 10 – 4 mol/L hydroxylamine.


Figure 8. Light-dependent up-regulation of cytochrome c oxidase activity in Cdr125 and cw-15 cells in the absence and presence of Cd2 + . Cells were grown in the presence of acetate in aerated cultures and subsequently incubated in the dark for 10 h with or without addition of Cd2 + (final concentrations: 30 µmol/L for cw-15; 100 µmol/L for Cdr125). Aliquots were harvested and analysed for cytochrome c oxidase activities before and during onset of illumination. Given values are means ± SD from four independent experiments.

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Figure 9. Light-dependent up-regulation of NADP + -dependent glyceraldehyde-3-P dehydrogenase activity in Cdr125 and cw-15 cells in the absence and presence of Cd2 + . Cells were grown in the presence of acetate in aerated cultures and subsequently incubated in the dark for 10 h with or without addition of Cd2 + (final concentrations: 30 µmol/ L for cw-15; 100 µmol/L for Cdr125). Aliquots were harvested and analysed for NADP-dependent glyceraldehyde-3-P dehydrogenase activities before and during onset of illumination. Given values are means ± SD from four independent experiments.

(Fig. 7 B), indicating that the donor side of photosystem II is affected in our particular Cd2 + -tolerant mutant strain.

of the NAD + -dependent glyceraldehyde-3-P dehydrogenase was not affected in our particular mutant strain (data not shown).

Cytochrome oxidase and NADP + -dependent glyceraldehyde-3-P dehydrogenase activities

Discussion

Since our Cd2 + -tolerant mutant strain, Cdr125, is impaired in photosystem II activity (Fig. 7; ref. Voigt et al. 1998), question arose as to why, under mixotrophic conditions, acetate consumption by this particular mutant strain is increased as compared to its parent strain, cw-15 (Nagel and Voigt 1995). Therefore, we have compared the activities of cytochrome oxidase (Fig. 8) and of NADP + -dependent glyceraldehyde-3P dehydrogenase (Fig. 9) in both strains. In dark-adapted Cdr125 and cw-15 cells, no difference in cytochrome oxidase activity was detected (Fig. 8). However, the activity of this mitochondrial enzyme was found to be increased by 100 % in Cd2 + -treated cells (Fig. 8). In both strains, an increase of cytochrome oxidase activity was observed during illumination of dark-adapted cells both in the absence and presence of Cd2 + , maximal activities being reached after 5 hours (Fig. 8). The NADP + -dependent glyceraldehyde-3-P dehydrogenase activity of dark-adapted cells was increased in the Cd2 + tolerant mutant strain Cdr125, both in the absence and presence of Cd2 + (Fig. 9). Upon illumination, an increase in glyceraldehyde-3-P dehydrogenase activity was observed in both the Cd2 + -tolerant mutant strain, and in its parent strain, cw-15 (Fig. 9). Addition of Cd2 + caused a further increase of glyceraldehyde-3-P dehydrogenase activity in the Cd2 + tolerant mutant strain, but not in its parent strain, cw-15 (Fig. 9). In contrast to the NADP + -dependent enzyme, the activity

A Cd2 + -tolerant mutant strain of the unicellular green alga C. reinhardtii was previously found not to be affected with respect to Cd2 + -uptake or detoxification, but impaired in photosynthetic oxygen production (Nagel and Voigt 1995). Since we have shown that a considerable proportion of Cd2 + ions taken up by Chlamydomonas cells is accumulated in the chloroplast (Nagel et al. 1996), a selection of mutants with impaired chloroplast functions by successively increasing Cd2 + concentrations is not astonishing. Recently, we have shown that our particular Cd2 + -tolerant mutant strain, Cdr125, is affected in the activity of photosystem II (Voigt et al. 1998). On the other hand, sensitivity of photosynthetic oxygen production to low Cd2 + -concentrations is decreased in the Cd2 + tolerant mutant strain (Nagel and Voigt 1995) as also observed for a heavy metal-contaminated population of the grassland moss Rhytidiadelphus squarrosus (Wells and Brown 1995). When grown in the light at a photon fluence rate of 20 µmol m – 2 s –1, cell growth and photosynthetic oxygen evolution of the Cd2 + -tolerant mutant, Cdr125, and the Cd2 + sensitive strains were found to be affected markedly by the growth conditions (Table 2). Under photoautotrophic conditions, both cell growth (Fig. 1A) and oxygen production (Table 2) of the Cd2 + -tolerant mutant Cdr125 were considerably decreased as compared to the Cd2 + -sensitive strains cw-15 and 137 ˚C. The slowed growth rate of our mutant strain, Cdr125,

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observed under photoautotrophic conditions (Fig. 1 A), could be overcome by addition of acetate (Fig. 1 B), which is metabolized by C. reinhardtii (Sager and Granick 1953). However, oxygen production decreased markedly under these conditions (Table 2). Furthermore, cells grown in the presence of acetate without aeration showed oxygen consumption instead of oxygen production after transfer to fresh culture medium (Table 2). These findings induced us to comparatively study the effects of increasing photon fluence rate on oxygen consumption/production by the Cd2 + -tolerant mutant, Cdr125, and the Cd2 + -sensitive strains grown under different conditions (Figs. 3 – 5). When grown under photoautotrophic conditions, photosynthetic oxygen evolution by the Cd2 + -tolerant mutant was markedly decreased as compared to cw-15 and wild-type cells even at light saturation and light compensation of oxygen consumption/production was observed at higher photon fluence rates (Fig. 3). When grown under mixotrophic conditions without aeration (Voigt and Münzner 1987), no oxygen evolution was measured for cw-15 and wild-type cells (Fig. 4). Immediately after removal of acetate, a rather low but significant oxygen production by cw-15 and wild-type cells was observed at light saturation (Fig. 5), which was, however, considerably lower than oxygen evolution by photoautotrophically grown cells (Fig. 3). Therefore, adaption of mixotrophically grown cells to photoautotrophic conditions requires longer time periods and de novo synthesis of proteins as corroborated by the different polypeptide patterns of thylakoids from photoautotrophically and mixotrophically grown cells (Fig. 6). The latter findings are in agreement with results previously published by other authors (Boege et al. 1981, Polle et al. 2000). The low, but significant, oxygen production of mixotrophically grown cw-15 and wild-type cells measured immediately after removal of acetate (Fig. 5) is assumed as the water-splitting capacity of these cells. This residual oxygen production is suppressed (Fig. 4), obviously by acetate inhibition of the water-splitting complex (Sinclair 1984, Bock et al. 1988, Kuhne et al. 1999, Boussac et al. 2000 and references cited by these authors). Our finding that, in contrast to its Cd2 + -sensitive parent strains, our Cd2 + -tolerant mutant, Cdr125, is able to produce oxygen even in the presence of acetate (Fig. 4) indicates that the water-splitting complex is affected in this particular mutant strain. This conclusion was further corroborated by the differential effects of hydroxylamine on the kinetics of chlorophyll fluorescence induction in Cdr125 and cw-15 cells (Fig. 7 A, B). Hydroxylamine is known to inhibit photosystem II activity via interactions with the water-splitting complex (Sharp and Yocum 1981, Robinson et al. 1981, Guiles et al. 1990) and to cause a decrease in chlorophyll fluorescence (Den Haan et al. 1974, 1976, Joliot 1977) because it is an electron donor of photosystem II (Bennoun and Joliot 1969, Bouges-Bocquet et al. 1973, Messinger et al. 1991). However, the water-splitting complex of our Cd2 + tolerant mutant is not insensitive to acetate ions. Photosynthetic oxygen production by this mutant strain was increased

immediately after substitution of the acetate-containing medium by sodium bicarbonate buffer (compare Figs. 4 and 5). No striking difference could be observed between the polypeptide patterns of the thylakoid membranes isolated from the Cd2 + -tolerant mutant and its Cd2 + -sensitive parent strains grown either under photoautotrophic (Fig. 6, lanes 1– 3) or under mixotrophic conditions (Fig. 6, lanes 5–7). Therefore, we conclude that Cd2 + -tolerance and impaired photosynthetic water-splitting of our particular mutant is caused by mutation of a polypeptide constituent of the donor side of photosystem II, which essentially does not affect the polypeptide composition of photosystem II, but causes altered interactions of the water-splitting complex with Cd2 + ions, acetate and hydroxylamine. Therefore, identification and characterisation of the protein altered in this Cd2 + -tolerant mutant is expected to give insights both into the effect of Cd2 + on photosystem II and into the mechanism of water splitting. On the other hand, question arose as to why our Cd2 + -tolerant mutant, Cdr125, exhibits an increased acetate consumption (Nagel and Voigt 1995). As shown in the present communication, acetate not only affects photosynthetic oxygen evolution, but also oxygen consumption by C. reinhardtii strains (Figs. 4 and 5, Table 2). In all the C. reinhardtii strains investigated, oxygen consumption in the dark and at low photon fluence rates decreased markedly in cells grown in the presence of acetate without aeration (Fig. 4) as compared to photoautotrophically grown cells (Fig. 3). This finding cannot be explained on the basis of the published data. The residual capability for oxygen consumption increased in the case of our Cd2 + -tolerant mutant Cdr125 (Figs. 4 and 5). In dark-adapted cells, the activity of cytochrome c oxidase was not changed in our Cd2 + -tolerant mutant strain (Fig. 8). Both in the Cd2 + -tolerant Cdr125 strain and in the Cd2 + -sensitive strain cw-15, the activities of this mitochondrial marker enzyme were found to be increased in the presence of Cd2 + (Fig. 8). Furthermore, the cytochrome c oxidase activity differentially increased in the Cd2 + -tolerant strain Cdr125 and in the Cd2 + -sensitive strain cw-15 after onset of illumination both in the absence and presence of Cd2 + (Fig. 8). The activity of the NADP + -dependent glyceraldehyde-3-P dehydrogenase, however, increased in our Cd2 + tolerant strain, Cdr125, under all experimental conditions (Fig. 9). Again, the activity of this enzyme increased after onset of illumination (Fig. 9). These findings demonstrate that there is an interrelationship between metabolic activities of chloroplasts and mitochondria in the case of C. reinhardtii as previously reported (Lemaire et al. 1988). At least in the case of the Cd2 + -tolerant strain Cdr125, the NADP + -dependent glyceraldehyde-3-P dehydrogenase might be a key enzyme in this process.

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