Quantification of protein was performed by the method of Brad- ford (1976) using bovine IgG as ..... ROBINSON: Poly (y-glutamylcysteinyl) glycine: its role in cad-.
J. Plant Physiol. Wll. 149. pp. 86-90 (1996)
Subcellular Distribution of Cadmium in the Unicellular Green Alga Chlamydomonas reinhardtii K. 1
NAGEL 1,2,3,
u. AnELMEIER\ and J. VOIGT 1,2,*
Institut fur Biochemie und Lebensmittelchemie, Universit:h Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany
2
Botanisches Institut, Technische Universitat Braunschweig, MendeissohnstraBe 4, D-38092 Braunschweig, Germany
3
Institut fur Ostseeforschung, Sektion Meereschemie, SeestraBe 15, D-18119 Warnemiinde, Germany
Received July 27, 1995 . Accepted November 1, 1995
Summary
As previously reported, cadmium strongly inhibits photosynthesis in the unicellular green alga Chlamydomonas reinhardtii. A cadmium-tolerant mutant with impaired ghotosynthesis has been isolated recently. Therefore, we have investigated the subcellular distribution of 1 9Cd2 + in the cell-wall deficient C. reinhardtii mutant strain CW15. The cytosol only contained about 10 % of the incorporated \09Cd2 +. The predominant proportion of Cd2 +, however, was found in the purified chloroplasts (more than 50 %), although a considerable part of the chloro~lasts was destroyed during the disrupture of the cells. Fractionation of (poly)peptide-bound and free 1 9Cd2 + by Sephadex G75 chromatography revealed essentially the same distribution for the lysates of purified chloroplasts as for the cytosol. A considerable proportion of the incorporated '09Cd2 + was sequestered by complex formation with oligopeptides both in the cytosol and in the chloroplasts.
Key words: Chlamydomonas reinhardtii, cadmium, chloroplast, phytochelatim, subcellular distribution. Introduction
Sequestering of Cd2 + by cadmium-binding peptides (metallothioneins and phytochelatins, respectively) is assumed to play an important role in the detoxification of this environmental pollutant and in the tolerance towards it (Grill and Zenk, 1985; Fowler et al., 1987; Kagi et al., 1987; Gekeler et al., 1988; Grill et al., 1988; Howe and Merchant, 1992; Reddy and Prasad, 1993). Plants respond to heavy-metal stress with the induction of phytochelatins, a class of peptides consisting of repeating units of y-glutamylcystein followed by a C-terminal glycine (Kondo et al., 1983; Grill and Zenk, 1985; Jackson et al., 1987; Reese and Wagner, 1987), which are derived from glutathione by the action of phytochelatin synthase (Rennenberg, 1987). Phytochelatins seem to be ubiquitous in higher plants (Gekeler et al., 1989) and have been also found in algae (Gekeler et al., 1988) and in some yeasts, particularly Schizosaccharomyces pombe, in which they
* Correspondence. © 1996 by Gustav Fischer Verlag, Stuttgart
were first discovered (Murasugi et al., 1981). Recently, cadmium-sensitive mutants of Arabidopsis thaliana have been described which are deficient in phytochelatin synthase (Howden et al., 1995 a) or glutathione synthesis (Howden et aI., 1995 b). Photosynthetic oxygen evolution is strongly inhibited by low cadmium concentrations as shown for algae and isolated chloroplasts of higher plants (van Duijvendijk-Matteoli and Desmet, 1975; De Filippis et al., 1981; Nagel and Voigt, 1989, 1995). Recently, a mutant strain with impaired photosynthesis has been isolated from a cadmium-tolerant population of the unicellular green alga Chlamydomonas reinhardtii (Nagel and Voigt, 1989, 1995). When homo~enates of C. reinhardtii cells incubated in the presence of o9Cd2 + were subjected to differential centrifugation at 30,000 gn, the predominant proportion of the incorporated cadmium was found in the pellet fraction both in the case of cadmium-sensitive and cadmium-tolerant cells (Nagel and Voigt, 1995). Therefore, we have investigated the subcelullar distribution of the incorporated cadmium in Chlamydomonas cells. Fur-
Subcellular distribution ofCd2+ in Chlamydomonas
thermore, we have comparatively analysed the relative proportions of free and peptide-bound cadmium in the cytosol and in the lysates of isolated chloroplasts. Materials and Methods
Straim and growth conditiom The Cd2+ -sensitve, cell wall-deficient strain Chlamydomonas reinhardtii CW15 (Davies and Plaskitt, 1971) was obtained from the
Sammlung von Algenkulturen at the University of Gottingen. Cells were grown at 21°C and 20,000 Ix in CTM medium (cadmium test medium) consisting of 0.5 mmol/L MgC12' 0.5 mmol/L CaCI 2, 7.5 mmol/L (NH4) H 2P04, 7.5 mmol/L KCi, 5 mmol/L PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)) (pH 6.3), and trace elements according to Starr (1971) supplemented with 0.2 % (w/v) sodium acetate as previously described (Nagel and Voigt, 1989, 1995). Cell concentrations were determined by duplicate hemocytometer counting.
Uptake and subcellular distribution of ctJ2 + Mixotrophically ~rowing cultures (1 litre) were adjusted to a cell density of 2 x 10- cells/mL and to a CdCh-concentration of 3011mol/L. After addition of 2.5 MBq of 109CdCh per litre, the cultures were incubated under a 12 h light-12 h dark regime. After 48 h, the final cell densities were determined and the cultures harvested by centrifugation at 6,000 Kn for 10 min. All the subsequent steps were performed at 0-4 0c. The cells were resuspended to a final density of 0.5-1 x 109 cells mL -I in ice-cold homogenization buffer containing 250 mmol/L sorbitol, 1 mmol/L MnClb 1 mmol/L MgC12' 2 mmol/L EDTA, 20 mmol/L KCl and 50 mmollL HepesNaOH (pH 7.5) and disrupted using a Potter-Elvejham homogenizer. The homogenates were fractionated by subsequent centrifugations at 500 Kn (10 min), 3,000 Kn (10 min), 20,000 Kn (30 min) and 100,000 Kn (60 min) and the pellets resuspended in homogenization buffer. The different fractions (100,000 Kn supernatant and the resuspended pellets were measured for 109Cd using an Auto-GammaScintillation Spectrometer (Packard Instruments).
Isolation ofchloroplasts and mitochondria Chloroplasts were prepared by a procedure similar to the method of Klein et al. (1983). Cells were harvested, resuspended in homoge-
87
nization buffer and disruped by use of a Potter-Elvejham homogenizer as described above. The homogenates were centrifuged at 500 Kn for 10 min. Aliquots of the supernatants (2 mL) were layered on top of a two-step Percoll gradient (5 mL of 30 % Percoll and 3 mL of 50 % Percoll in homogenization buffer in 15 mL Corex tubes) and centrifuged at 10,000 l{n) (4°C) for 20 min on a swinging bucket rotor. For analytical purpose, the gradients were fractionated. Fractions of 0.2 mL were collected and assayed for radioactivity, chlorophyll and enzyme activities. Intact chloroplasts banding at the 30/ 50 % Percoll interface were harvested and washed with homogenization buffer. Mitochondria were found at the 0/30 % Percoll interface, diluted with homogenization buffer, collected by centrifugation at 20,000 Kn and subsequently washed with homogenization buffer.
Separation offree and peptide-bound J09 Cd Separation of free and peptide-bound 109Cd was performed by chromatography on Sephadex G-75 fine columns (2.5 cm x 90 cm) equilibrated and eluated with N 2-saturated buffer containing 10 mmol/L Tris-HCl (pH 8.0) and 1 mmol/L 2-mercaptoethanol and classified as described by Hart and Bertram (1980). Aliquots of the different fractions were analysed for radioactivity using an AutoGamma-Scintillation Spectrometer (Packard Instruments).
Determinatiom ofchlorophyll and protein Quantification of protein was performed by the method of Bradford (1976) using bovine IgG as standard. Chlorophyll was measured according to Arnon (1949).
Enzyme activities The activities of the marker enzymes cytochrome c oxidase for the mitochondria and NADP-dependent glyceraldehyde-3P dehydrogenase for the chloroplast (Klein et al., 1983) were measured with a Gilford recording spectrophotometer (model 250) according to the methods of Wharton and Tzagoloff (1967) and Latzko and Gibbs (1969), respectively. The reaction mixtures included Triton X-IOO at a final concentration of 0.1 % (v/v) to lyse completely the protoplasts and organelles and make the enzymes more accessible to added cofactors and substrates.
Table 1: Distribution of 109Cd in subcellular fractions of Chlamydomonas reinhardtii'. 109Cd
Subcellular fraction
Homogenate PO.5 P3 P20 P 100 S 100 Gradient-purified chloroplasts Gradient-purified mitochondria
Glyceraldehyde-3P dehydrogenase (NADP)
[10 3 cpm/10 9 cells)
[%)
[mg/ 109 cells)
[%)
[nkatll 09 cells)
[%)
Cytochrome c oxidase 9 [nkat/10 cells) [%)
200±25 30±4.5 128±15 14.4± 1.9 6.8±1.2 20.8±3.1
100 15 64 7.2 3.4 10.4
12± 1.5 1.9±0.22 8.2±0.9 1.1±0.1 0.8±0.1 0
100 16 68 9.4 6.6 0
194±11 29±3.4 118± 13 6.8±0.9 1.4±0.3 27±4.6
100 15 61 3.5 0.7 13.8
17.6±1.3 2.3±0.4 4.2±0.6 8.4± 1.1 1.3±0.4 0.1 ±0.04
101±12
50.5
6.5±0.8
54
101 ± 16
52
0.4±0.2
7±2.1
3.5
0.9±0.24
Chlorophyll
7.3
3.7± 1.1
1.9
6.4±1.2
100 13 24 48 7.4 0.6 2.2 36
• Determined afrer 48 h of incubation in the presence of 30 11M CdCl2 and 0.25 MBq of I09CdCh. The subcellular fractions were prepared and assayed for chlorophyll, enzyme activities and readioactivity as described in the ,Materials and Methods. section. P 0.5 = 500 gn pellet; P 3 =3,000 gn pellet; P 20 = 20,000 gn pellet; P 100 = 100,000 gn pellet; S 100 = 100,000 gn supernatant. Values are means ± SD.
88
K.
NAGEL,
U. AnELMEIER, and]. VOIGT
Results
400~----------------------------------"
The subcellular distribution of I09Cd2+ was investigated in cells of the wall-deficient C. reinhardtii strain CW15 grown in the presence of 30 IlmollL Cd2+ which caused a decrease of cell growth by 50 % (Nagel and Voigt, 1989). To obtain the subcellular fractions, crude homogenates were subjected to differential centrifugation at 500 g." 3,000 g." 20,000 g., and 100,000 g.,. The predominant proportion of the incorporated I09Cd2+ was found in the 3,000 g., pellet (64 %; Table 1). Considerable amounts of radioactivity were also present in the 100,000 g., supernatant (10.4 %), in the 20,000 g., pellet (7.2 %) and in the 500 g., pellet (15 %). The relatively high proportion of 109Cd2+ found in the 500 g., pellet was due to the undestroyed cells observed in this fraction by phase contrast microscopy. The 3,000 g., pellet largely consisted of intact chloroplasts as revealed by phase contrast microscopy I... E! and further corroborated by measuring the distributions of chlorophyll and of the chloroplast-specific NADP-dependent ~ 1 GAP-DH activity (Table 1). Chloroplast fragments were pre= sent in the 20,000 g., pellet and in the 100,000 g., pellet as indicated by the relatively high amounts of chlorophyll and the rather low activities of NADP-dependent GAP-DH (Table 1). Mitochondria were present in the 3,000 g., pellet and in the 20,000 g., pellet as revealed by the distribution of cytochrome c oxidase activity. Purified mitchondria contained 36 % of total cytochrome c oxidase activity of the crude homogenate, but only 3.5 % of the incorporated I09Cd2+ (Table 1). Taking into account that a considerable proportion of the 1 2 3 4 G 8 7 8 9 10ml chloroplasts was more or less destroyed during disrupture of Top Bottom the cells as revealed by the distributions of chlorophyll and NADP-dependent GAP-DH (Table 1), the findin~ de- Fig. 1: Co-sedimention of 109Cd with chloroplast during Percoll gra- . scribed above indicate that most of the incorporated I Cd2+ dient centrifugation. Cultures of the wall-deficient C reinhardtii was largely accumulated in the chloroplast. This conclusion mutant strain CW15 were incubated for 48 h in the presence of was further corroborated by the observation that the predo- 2.5 MBq I09Cd2 + and 30 IJ.mollL CdCl2 • The cells were harvested minant proportion of I09Cd2+ co-sedimented with chloro- by centrifugation, resuspended in homogenization buffer and disphyll and the chloroplast-specific, NADP-dependent GAP- ruped by use of a Potter-Elvejham homogenizer as described in the DH during Percoll-gradient centrifugation (Fig. 1). The gra- section. The homogenates were centrifuged dient-purified intact chloro~lasts still contained more than at 500 ~ for 10 min. Aliquots of the supernatants (2 mL) were layered on top of two-step Percoll gradients (5 mL of 30 % Percoll 50% of total incorporated 1 Cd2+ (Table1). and 3 mL of 50 % Percoll in homogenization buffer in 15 mL Corex Since it has been shown that in C. reinhardtii, like in other tubes), centrifuged at 10,000 ~ (4°C) for 20 min on a swinging algae and .higher plants, cadmium is sequestered by complex- bucket rotor and fractionated. Fractions of 0.2 mL were collected formation with phytochelatins (Gekeler et al., 1988, 1989), and assayed for radioactivity, chlorophyll and enzyme activities as we have investigated the proportions of free and peptide- described in the section. bound I09Cd2+ in lysates of gradient purified chloroplasts. As shown in Fig. 2 A, Sephadex G 75 chromatography of lysates of gradient-purified chloroplasts revealed three peaks of were found to be very similar for the lysates of purified radioactivity: polypeptide-bound (peak I), oligo-peptide- chloroplasts and cytosol, respectively (Table 2). The data bound (peak II) and free cadmium (peak III). The same shown in Fig. 2 and Table 2 indicate that cadmium is sequespeaks were observed when the cytosol fraction (= 100,000 g., tered by cadmium-binding oligopeptides both in the chlorosupernatant) was subjected to Sephadex G 75 chromatogra- plasts and in the cytosol. phy (Fig. 2 B). However, in the case of the cytosol fractions (Fig. 2 B), elution of the oligo-peptide-bound I09Cd2+ (peak Discussion II) was found to be considerably delayed as compared to the lysates of gradient-purified chloroplasts (Fig. 2A). This findLow concentrations of Cd2+ strongly affect photosynthesis ing indicates that the cytosolic cadmium-oligopeptide complexes have lower molecular weights than the cadmium-pep- in the unicellular green alga C. reinhardtii (Nagel and Voigt, tide complexes present in the chloroplast. The proportions of 1989, 1995). Indeed, the predominant proportion of the inpolypeptide-bound, oligopeptide-bound and free cadmium corporated cadmium is accumulated by the chloroplast as
.
Subcellular distribution of Cd2+ in Chlamydomonas
89
Table 2: Peptide-bound and free I09Cd in the chloroplast and in the cytosol of Chlamydomonas reinhardtii cells'. Total 109Cd
Chloroplast lysate Cytosol
Peptide-bound 109Cd
free I09Cd
[cpmJ
109Cd bound to high-mol-we compounds [cpmJ
[cpmJ
[cpmJ
100,000 ± 8,00 (100%) 20,000±2,000 (100%)
16,300 ± 1,200 (16.3%) 1,600±180 (8.0%)
59,800±5,300 (59.8%) 12,800 ± 1,000 (64.0%)
24,000± 1,900 (24.9%) 5,500±6oo (27.5%)
Determined after 48 h of incubation in the presence of 30 I1mol/L CdCl 2 and 0.25 MBq of I09CdCI2. Lysates of gradient purified chloroplasts and cytosol (= 100,000 gn supernatant), respectively, were fractionated by Sephadex G 75 chromatography as described in the ,Materials and Methods> section. Values are means ± SO. Numbers in parentheses indicate percent of total radioactivity applied to the column.
a
1~000
A
,....
n
A
II
~
0
'-' 10
.
~
GOOO
w
~ 0
,....
r\
Iq,
+
~
to>
m
r
¥\
B
11
3000
II
~
0 '-'
1t
+ 01
."
to>
§
10
so
30
Fraotion
40
~
80
70
80
90 100
number
Fig. 2: Separation of free and peptide-bound 109Cd2+ by Sephadex G75 chromatography of lysates of gradient-purified chloroplasts (A) and cytosolic fractions (= 100,000 b'.> supernatant; B). The 100,000 b'.> supernatant and the gradient-purified chloroplasts were prepared from the homogenates of C reinhardtii CW15 cells incubated for 48 h in the presence of 2.5 MBq 109Cd2+ and 30l1moi/L CdCI2. The purified chloroplasts were disrupted by addition of Triton X-100 to a final concentration of 0.2 % (v/v). The chloroplast lysates (A) and the 100,000 b'.> supernatants (B) were applied to Sephadex G 75 fine columns (2.5 cm x 90 cm) equilibrated and eluated with N 2-saturated buffer containing 10 mmol/L TrisHCl (pH 8.0) and 1 mmol/L 2-mercaptoethanol. Aliquots of the different fractions were analysed for radioactivity using an AutoGamma-Scintillation Spectrometer (Packard Instruments). The peaks were classified as described by Hart and Bertram (1980).
shown by the present communication. When cells of the wall-deficient strain CW15 incubated in the presence of
30 IlmollL 109Cd2+ were disrupted under mild conditions, more than 50 % of the radioactivity was present in the intact, gradient-purified chloroplasts, but only 10 % in the cytosol fraction. Disruption of the cells under harsher condition increased the proportion of cadmium in the 100,000 ~ supernatant to a level of 30 % (Nagel and Voigt, 1989, 1995), obviously due to a leakage from the destroyed chloroplasts. Chlamydomonas cells contain a single cup-shaped chloroplast which occupies more than 50 % of the cell volume. Therefore, Chlamydomonas chloroplasts are damaged to a considerable degree during homogenization even under mild conditions as shown by the distributions of chlorophyll and the chloroplast-specific, NADP-dependent GAP-DH (Table 1). For this reason, the proportion of cadmium found in the chloroplasts was obviously underestimated, whereas the cytoplasmic level was presumably overestimated. When lysates of gradient-purified chloroplasts were subjected to gel exclusion chromatography on Sephadex G 75 columns, three peaks of radioactivity were observed (Fig. 2 A): polypeptide-bound (peak I), oligopeptide-bound (peak II) and free 109Cd2+ (peak III). The proportion of peptidebound cadmium (peak II; 60%) was found to be very similar as in the cytosol (Fig. 2; Table 2). The Cd2+-binding oligopep tides of C. reinhardtii (peak II) were shown to be phytochelatins (Gekeler et aI., 1988). Phytochelatins are heavy metal-binding peptides possessing the unusual structure (y-GluCys)n-Gly which are formed by plant cells in response to heavy metal stress (Rennenberg, 1987; Steffens, 1990). In Chlamydomonas reinhardtii, phytochelatins with n = 2 - 5 were detected (Gekeler et aI., 1988). Since elution of the cytosolic phytochelatins (peak II) from the Sephadex G75 columns (Fig. 2 B) was considerably delayed as compared to the oligopeptide-bound 109Cd2+ present in the lysates of gradient-purified chloroplasts (Fig. 2A), we assume that the cytosol predominantly contained phytochelatins with n = 2-3 whereas larger phytochelatins (n=3-5) occured in the chloroplast. From the data described above, we conclude that in C. reinhardtii, the predominant proportion of phytochelatins must be localized in the chloroplast. For other plants, phytochelatin synthesis is presumed to be cytoplasmatic and cadmium-phytochelatin complexes have been found in the cytosol and in the vacuol (Steffens, 1990). However, sequestering of heavy metals in the chloroplasts has not been studied so far.
90
K. NAGEL, U. AnELMEIER, and J. VOIGT
Acknowledgements
This work was supported by granrs from the Deutsche Forschungsgemeinschaft (Na 146/1 and Vo 327/3).
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