Powles, 1984; Anderson and. Osmond ... ter (Kyle et al., 1984; Powles, 1984). We have ..... JOHN, M. K.: Cadmium uptake by eight food crops as influenced by.
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© 1998 by Gustav Fischer Verlag, Jena
A Cadmium-tolerant Chlamydomonas Mutant Strain Impaired in Photosystem II Activity 1 ]URGEN VOIGT ,2, 1
KLAus
1 NAGEL ,3,
and DAGMAR WRANN 1
Botanisches Institut derTechnischen Universitat Braunschweig, Mendelssohnstra& 4, D-38092 Braunschweig, Germany
2
Physiologisch-Chemisches Institut der Universitat Tiibingen, Hoppe-Seyler-Stra«e 4, D-72076 Tiibingen, Germany
3
Institut fur Ostseeforschung, Seestra«e 15, D-18119 Warnemiinde, Germany
Received December 12, 1997 . Accepted March 25, 1998
Summary
Growth of a cadmium-tolerant mutant strain of the unicellular green alga Chlamydomonas reinhardtii was found to be impaired under photoautotrophic conditions, but not in the presence of acetate. Comparative investigations of wild-type and mutant cells regarding cyclic and linear phosphorylation as well as different photochemical reactions revealed that photosystem II is exclusively affected in this particular mutant strain. This conclusion was corroborated by differential effects of the photosystem II inhibitor DCMU on chlorophyll fluorescence induction in wild-type and mutant cells. Furthermore, sensitivity towards photoinhibition was found to be considerably decreased in the case of the cadmium-tolerant mutant strain.
Key words: Chlamydomonas reinhardtii, cadmium-tolerant mutant, photosynthesis, photosystem II defect. Abbreviations: DCMU =3-(3,4-dichlorophenyl)-I,I-dimethylurea. Introduction
Heavy metal-binding peptides (methallothioneins and phytochelatins, respectively) are assumed to play an important role in the detoxification of Cd2 + and in the tolerance towards it (Grill and Zenk, 1985; Fowler et al., 1987; Kligi et al., 1987; Gekeler et al., 1988; Grill et al., 1988; Howe and Merchant, 1992; Reddy and Prasad, 1993). Plants and green algae respond to heavy-metal stress with the induction of phytochelatins, a class of peptides consisting of repeating units of y-glutarnylcystein followed by a C-terminal glycine (Kondo et al., 1983; Grill and Zenk, 1985; Jackson et al., 1987; Reese and Wagner, 1987; Howe and Merchant, 1992), which are derived from glutathione by the action of phytochelatin Syt1thase (Rennenberg, 1987). Cadmium-sensitive mutants of Arabidopsis thaliana have been described that are deficient in phytochelatin Syt1thase (Howden et al., 1995 a) or glutathione Syt1thesis (Howden et al., 1995 b). Phytochelatins seem to be ubiquitous in higher plants and algae (Gekeler et
J Plant PhysioL VOL 153. pp. 566-573 (1998)
al., 1988, 1989; Howe and Merchant, 1992). In the unicellular green alga Chlamydomonas reinhardtii, phytochelatins have been found both in the cytosol and in the chloroplast (Nagel et al., 1996). A previously isolated cadmium-tolerant mutant strain of the unicellular green alga Chlamydomonas reinhardtii was shown to be not affected with respect to uptake and sequestering of Cd2 + by phytochelatins (Nagel and Voigt, 1995). Photosynthesis, however, was found to be impaired in the case of this particular mutant (Nagel and Voigt, 1995), as revealed by a considerably decreased growth rate under photoautotrophic conditions, a decreased, but Cd2+ -resistant photoSyt1thetic oxygen evolution and altered chlorophyll fluorescence induction in dark-adapted cells. On the other hand, inhibition of photosynthesis by sublethal Cd2 + concentrations has been already described for Nostoc linckia (Husaini and Rai, 1991), Anabena (Bolanos et al., 1992) Euglena gracilis (De Filippis et al., 1981 a, b; De Filippis and Ziegler, 1993), a grassland moss (Wells and Brown, 1995), and
A Cd2+ -tolerant Mutant with Impaired PS II Activity
higher plants (Woszny et al., 1990; Malik et al., 1992; Vasilev et al., 1995; Fodor et al., 1996). Cadmium tolerance in a metal-contaminated population of the grassland moss Rhytidiadelphus squa"OSUS was found to be accom;anied by a decreased sensitivity of photosynthesis to Cd2 (Wells and Brown, 1995). In the literature, two functional sites of the photosynthetic apparatus have been reported to be particularly sensitive to Cd2+ ions: photosystem II (Van Duijvendijk-Matteoli and Desmet, 1975) and the chloroplast ATP synthase/ATPase (Teige et al., 1990). Whether photosynthetic ATP formation or r,hotosystem II activity is more sensitive to non-lethal Cd + concentrations is obviously dependent on the organism investigated (Van Duijvendijk-Matteoli and Desmet, 1975; De Filippis et al., 1981 a, b; Teige et al., 1990; Husaini and Rai, 1991; De Filippis and Ziegler, 1993). Therefore, we have comparatively analysed cadmium-tolerant and cadmium-sensitive Chlamydomonas cells with respect to cyclic and linear photophosphorylation, different photochemical reactions and photoinhibition. As shown in the present communication, photosystem II, but not photosystem I or ATP synthase, is affected in the cadmium-tolerant mutant strain Cdr 125.
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Leitz, Germany) equipped with a halogen lamp (Xenophot, Osram, Germany).
ATPase assay ATPase activities of isolated thylakoids were measured as described by Lien and Racker (1971).
Photochemical reactions The photochemical reaction of whole cells and chloroplast fragments was measured according to Chua et al. (1975). The p-benzoquinone-Hill reactions were carried out with suspensions of whole cells (corresponding to 1511g chlorophyll/mL) in phosphate buffer containing 2 mmol/L p-benzoquinone. The ferticyanide-Hill and the methylviologen-Hill reactions were performed with suspensions of thylakoids (3011g chiorophyll/mL). The photoreduction of memylviologen (0.2 mmol/L) with DPIP-ascorbate couple (0.1 mmol/L and 3 mmollL, respectivdy) as the dectron donor was measured with suspensions of fragments (7.5-15 I1g chiorophyll/mL). The samples (4 mL) were incubated at 24'C in a plexiglas cuvette and laterally irridiated using a slide projector (Prado, Leitz, Germany) equipped with a halogen lamp (Xenophot, Osram, Germany). Photosynthetic production of O 2 and Orconsumption were monitored polarographically using an oxygen dectrode (ySI modd 53; Yellow Springs Instrument Co., Yellow Springs, Ohio, USA).
Materials and Methods
Strains and growth conditions The Cd2+ -sensitive, cell wall-deficient strain Chlamydomonas reinhardtii CW15 (Davies and Plaskitt, 1971) was obtained from the Sammlung von Algenkulturen at the University of Gottingen. The mutant strain Cd'125 was isolated from the Cd2+ -tolerant population CW15-Cd', which was derived from Cd2+ -sensitive, cell walldeficient strain Chlamydomonas reinhardtii CW15 (Davies and Plaskitt, 1971) by cultivation under mixotrophic growth conditions in the presence of successively increasing concentrations of CdCh (Nagd and Voigt, 1989, 1995). Unless otherwise stated, cells were grown at 21 'c and a photon fluence rate of 20 I1mollm2 s in a highsalt medium with or without 0.2 % (w/v) sodium acetate as described previously (Voigt et al., 1989).
Chlorophyll Chlorophyll concentrations were determined according to Arnon (1949).
ATP levels ATP levds were measured by luminescence using the luciferase assay (Lundin and Thore, 1975).
Cyclic and linear photophosphorylation Cyclic and linear photophosphorylation of ADP by isolated thylakoid membranes (preilluminated for 2 min at a photon fluence rate of 40 I1mollm2 s in the presence of DTE) were measured by incorporation of 32p; and subsequent determination of incorporated 32p (Piccioni et al., 1981). Linear photophosphorylation was measured using p-benzoquinone (1 mmollL) as dectron acceptor. Cyclic photophosphorylation was determined in the presence of phenazine methosulfate (0.5 mmol/L) and sodium ascorbate (1 mmollL). The samples were incubated at 21 ·C and laterally irridiated with a photon fluence rate of 120 I1mol/m2 s using a slide projector (Prado,
Photoinhibition Cell suspensions at a concentration of 30 I1g chlorophyll/mL (corresponding to 1 X 107 cells/mL) were exposed in a water bath at 24 'c to white light supplied by a tungsten/halogen lamp providing 1800 ).Lmol/m2s at the surface of the cell suspension (Schuster et al., 1988). Rates of photosystem-II-dependent electron flow were recorded as the (DCMU-sensitive) reduction of 2,6-dichloroindophenol (DPIP) by thylakoids using H 20 as dectron donor.
Chlorophyll-jluorescence induction The kinetics of chlorophyll-fluorescence induction in darkadapted cultures were investigated prior to and 30 min afrer addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU; 10 I1mollL) using a Hitachi modd F-3000 spectrofluorometer (excitation at 480 nm; emission measured at 685 nm).
Statistics Statistical testing of the obtained data was performed by one way ANOVA (F-test) and Student's Hest, respectivdy.
Results
ATP kvels and photophosphorylation As compared with wild-type strains, cell propagation of a previously isolated cadmium-tolerant mutant strain of the unicellular green alga Chlamydomonas reinhardtii was found to be decreased under photoautotrophic conditions, but not in the presence of acetate (Nagel and Voigt, 1995). Photoautotrophically grown cells of the cadmium-tolerant mutant strain Cdr125 contained considerably lower ATP levels than cadmium-sensitive cw-15 cells when illuminated at photon fluence rates of 20 J.lmollm2 s (P
