Planta
Planta 136, 281-282 (1977)
9 by Springer-Verlag 1977
Short Communication
The Concentration of Cytochrome f and P700 in Chlorophyll-Deficient Mutants of Chlorella fusca A. Wild and K.-H. Fuldner Institut fiir Allgemeine Botanik der Universitfit, Saarstr. 21, D-6500 Mainz, Federal Republic of Germany
Abstract. The ratio of Chlorophyll : Cytochrome f and of Chlorophyll: P700 (reaction center pigment in photosystem I) is essentially lower in chlorophyll-deficient mutants than in the normal green strain. On a dry weight basis, the mutants have the same or a higher content of redox enzymes than the normal form. The size of the photosynthetic unit of the mutants is 4 to 7 times smaller than that of the normal strains, due mainly to a deficiency of the light-harvesting chlorophyll-protein complex. Key words: Chlorophyll-deficient mutants chrome f - P700 - Photosynthetic unit.
Cyto-
Yellow varieties of higher plants and chlorophyll-deficient mutants (yellow mutants) of green algae often have a high photosynthetic efficiency based on their chlorophyll content. Yellow mutants of Chlorella fusca show photosynthetic rates and Hill-reaction activities of P S I and PS II that are 4-5 times higher than those of the normal green form. The mutants also reach light saturation of photosynthesis at much higher light intensities. The higher capacities for CO2 fixation and electron transport reactions are due to the formation of smaller photosynthetic units. Thus, reduced chlorophyll synthesis is not correlated with an attendant reduction of the number of electron transfer chains. The deficiency of antenna chlorophyll, especially of chlorophyll b, is caused by a lack of light-harvesting chlorophyll a/b protein complexes (CP II). In the chlorophyll-deficient mutants of Chlorella, the thylakoid membranes have a primary molecular organization stage (Wild, 1969; Wild et al., 1971; Bauer and Wild, 1976). Abbreviations: Chl=chlorophyll; Cyt f=Cytochrome f; P700=
reaction center pigment in photosystem I ; PS = photosystem ; LH = light-harvesting.
This note reports a study of the concentration of cytochrome f and P700 in several chlorophyll-deficient mutants and normal green strains of chlorella to obtain additional results for the determination of the size of the photosynthetic unit. The normal strains Chlorella vulgaris 211-11 h and Chlorella fusca C- 1.1.10, the mutants 69 of ChlorelIa vulgaris and G 10 of Chlorella fusca, which require light for chlorophyll formation, and the yellow-green mutants G 28, G33, and G36 of Chlorella fusca were used. The cells were grown on a shaker in liquid medium supplemented with glucose (0.2%) under low-light (about 2000 lx) conditions in a 16-h light/8-h dark schedule. The culturing conditions were described previously (Bauer and Wild, 1976). Cell homogenates were prepared from 40-200 ml algae suspensions. After centrifugation the pelleted cells were resuspended in 20 ml of a buffer solution consisting of 0.05 mol. 1-1 tricine-KOH (pH 7.2), 0.015 mol. 1-1 KC1, and 0.35 mol. 1-1 sucrose. The concentrated cell suspension was transferred to a duran glass flask 3/4 filled with a mixture of glass beads (0.02 mm and 1.0 mm diameter) and then vibrated for 90 s in a homogenizer (type MSK, Braun, Melsungen) while cautiously cooling with CO2 snow. This procedure resulted in complete rupture of the algal cells. The contents of the flask were then filtered through a coarse, fritted-glass filter to remove the glass beads, which were subsequently washed with 10-20 ml additional buffer for complete recovery of broken cells. The broken cell suspension was divided in two parts: one centrifuged at 8000 g for 12 min and the other at 30,000g for 10 min, for analysis of cytochrome f and P700, respectively. Cyt f and P700 were determined by the oxidized-minus-reduced difference spectra according to Wild et al. (1973) and Bendall et al. (1971) using an Amino DW-2 spectrophotometer. The results reported and summarized in Table 1 show that the low chlorophyll content of the yellow mutants G 28, G 33 and G 36 is combined with a
A. Wild and K.-H. Fuldner: Cytochrome in Mutants of Chlorella
282
Table 1. Chlorophyll (Chl), cytochrome f (Cyt f), P700, and light-harvesting chlorophyll-protein complex contents (LH Chl in % of
total ChI; it is assumed that all of the chlorophyll b is associated with this complex) plant material
mg Chl a + b g dry weight
Chl a Chl b
Chl Cyt f
nmol Cyt f g dry weight
Chl P700
P700 Cyt f
LH Chl
Chlorella vutgaris 211-11 h
23.1
3. l
1807
14.4
90l
2.0
48.8
Mutant 69
28.0
3.4
1251
25.1
727
1.7
45.7
Chlorella fusca C-I.I.10
21.7
2.4
1469
'16.6
966
1.5
58.7
Mutant Mutant Mutant Mutant
33.8 6.8 3.4 3.8
3.5 16.9 10.0 7.9
1134 252 207 325
33.4 30.3 18.4 13.1
549 264 208 295
2.1 1.0 1.0 1.1
44.7 11.2 18.2 22.5
G G G G
10 28 33 36
very high Chl a: Chl b ratio. In the in vivo absorption spectra of these mutants, the chlorophyll b shoulder at 653 nm is missing, whereas it is well established in the normal green types. The ratio of Chl: Cyt f and of Chl: PT00 is essentially lower in the yellow mutants than in the normal green strains. Calculated on a dry weight basis the redox enzyme content of the mutants G 28, G 33, and G 36 reaches or even exceeds that of the normal form. Thus, reduced chlorophyll synthesis is not correlated with a decrease of the concentration of electron transfer enzymes. The yellow mutants have a P700: Cyt f ratio of 1:1 whereas in the wild types the ratio ranges from about 1.5:1 to 2:1. The normal green algal cells grown under low-light conditions have a higher content of P700 than of Cyt f, and are therefore similar in this respect to higher plants grown under low-light conditions (Wild et al., 1973; Grahl and Wild, 1975). In the wild types, the percentage of LH Chl is 49% or 59% of the total chlorophyll content, whereas in the yellow mutants only 11-23% of the total chlorophyll is bound in the light-harvesting chlorophyllprotein complex. The photosynthetic unit reduces the discrepancy between the light-harvesting capacity of chlorophyll and the rate capacity of the electron transport system. We therefore define the photosynthetic unit as the number of chlorophyll molecules per electron transport chain. Considering that the electron transport capacity is limited by the concentration of Cyt f, the size of the photosynthetic unit is consistent with the number of chlorophyll molecules per molecule of Cyt f. Thus in the photosynthetic membranes of the normal strain of Chlorellafusca, 1469 chlorophyll molecules are involved in light harvesting per electron
transport pathway, whereas in the yellow mutants only 207-325 chlorophyll molecules take part. The unit size of the mutants is 7.1 (G 33) to 4.5 times (G 36) smaller than that of the normal form, mainly because of a deficiency of the LH Chl complex. According to Anderson (1975) the chlorophyll-deficient mutants of Chlorella have thylakoid membranes characterized by a primary molecular organization stage. This work was supported by the Deutsche Forschungsgemeinschaft.
References Anderson, J.M. : The molecular organization of chloroplast thylakoids. Biochim. Biophys. Acta 416, 191-235 (1975) Bauer, K., Wild, A. : The effect of blue light on the photosynthetic electron transport in chlorophyll-deficient mutants of Chlorella fusca. Z. Pflanzenphysiol. 80, 443-454 (1976) Bendall, D.S., Davenport, H.E., Hill, R. : Cytochrome components in chloroplasts of the higher plants, In: Methods of enzymology, vol. 23, pt.A., pp. 327-347, San Pietro, A., ed., New YorkLondon: Academic Press 1971 Grahl, H., Wild, A.: Studies on the content of P700 and cytochromes in Sinapis alba during growth under two different light intensities. In: Environmental and biological control of photosynthesis, pp. 107-113, Marcelle, R., ed., The Hague: W. Junk 1975 Wild, A.: The size of the photosynthetic unit and its variability in yellow mutants. In : Progress in photosynthesis research, vol. II, pp. 871476, Metzner, H., ed., Tiibingen: H. Metzner 1969 Wild, A., Zickler, H.-O., Graht, H. : Weitere Untersuchungen zur Variabilitfit der photosynthetischen Einheit. Planta (Bed.) 97, 208-223 (1971) Wild, A., Ke, B., Shaw, E.R. : The effect of light intensity during growth of Sinapis alba on the electron-transport components. Z. Pflanzenphysiol. 69, 344-350 (1973)
Received 29 April; accepted 15 June 1977
Planta 136, 281-282 (1977)
9 by Springer-Verlag 1977
Short Communication
The Concentration of Cytochrome f and P700 in Chlorophyll-Deficient Mutants of Chlorella fusca A. Wild and K.-H. Fuldner Institut fiir Allgemeine Botanik der Universitfit, Saarstr. 21, D-6500 Mainz, Federal Republic of Germany
Abstract. The ratio of Chlorophyll : Cytochrome f and of Chlorophyll: P700 (reaction center pigment in photosystem I) is essentially lower in chlorophyll-deficient mutants than in the normal green strain. On a dry weight basis, the mutants have the same or a higher content of redox enzymes than the normal form. The size of the photosynthetic unit of the mutants is 4 to 7 times smaller than that of the normal strains, due mainly to a deficiency of the light-harvesting chlorophyll-protein complex. Key words: Chlorophyll-deficient mutants chrome f - P700 - Photosynthetic unit.
Cyto-
Yellow varieties of higher plants and chlorophyll-deficient mutants (yellow mutants) of green algae often have a high photosynthetic efficiency based on their chlorophyll content. Yellow mutants of Chlorella fusca show photosynthetic rates and Hill-reaction activities of P S I and PS II that are 4-5 times higher than those of the normal green form. The mutants also reach light saturation of photosynthesis at much higher light intensities. The higher capacities for CO2 fixation and electron transport reactions are due to the formation of smaller photosynthetic units. Thus, reduced chlorophyll synthesis is not correlated with an attendant reduction of the number of electron transfer chains. The deficiency of antenna chlorophyll, especially of chlorophyll b, is caused by a lack of light-harvesting chlorophyll a/b protein complexes (CP II). In the chlorophyll-deficient mutants of Chlorella, the thylakoid membranes have a primary molecular organization stage (Wild, 1969; Wild et al., 1971; Bauer and Wild, 1976). Abbreviations: Chl=chlorophyll; Cyt f=Cytochrome f; P700=
reaction center pigment in photosystem I ; PS = photosystem ; LH = light-harvesting.
This note reports a study of the concentration of cytochrome f and P700 in several chlorophyll-deficient mutants and normal green strains of chlorella to obtain additional results for the determination of the size of the photosynthetic unit. The normal strains Chlorella vulgaris 211-11 h and Chlorella fusca C- 1.1.10, the mutants 69 of ChlorelIa vulgaris and G 10 of Chlorella fusca, which require light for chlorophyll formation, and the yellow-green mutants G 28, G33, and G36 of Chlorella fusca were used. The cells were grown on a shaker in liquid medium supplemented with glucose (0.2%) under low-light (about 2000 lx) conditions in a 16-h light/8-h dark schedule. The culturing conditions were described previously (Bauer and Wild, 1976). Cell homogenates were prepared from 40-200 ml algae suspensions. After centrifugation the pelleted cells were resuspended in 20 ml of a buffer solution consisting of 0.05 mol. 1-1 tricine-KOH (pH 7.2), 0.015 mol. 1-1 KC1, and 0.35 mol. 1-1 sucrose. The concentrated cell suspension was transferred to a duran glass flask 3/4 filled with a mixture of glass beads (0.02 mm and 1.0 mm diameter) and then vibrated for 90 s in a homogenizer (type MSK, Braun, Melsungen) while cautiously cooling with CO2 snow. This procedure resulted in complete rupture of the algal cells. The contents of the flask were then filtered through a coarse, fritted-glass filter to remove the glass beads, which were subsequently washed with 10-20 ml additional buffer for complete recovery of broken cells. The broken cell suspension was divided in two parts: one centrifuged at 8000 g for 12 min and the other at 30,000g for 10 min, for analysis of cytochrome f and P700, respectively. Cyt f and P700 were determined by the oxidized-minus-reduced difference spectra according to Wild et al. (1973) and Bendall et al. (1971) using an Amino DW-2 spectrophotometer. The results reported and summarized in Table 1 show that the low chlorophyll content of the yellow mutants G 28, G 33 and G 36 is combined with a
A. Wild and K.-H. Fuldner: Cytochrome in Mutants of Chlorella
282
Table 1. Chlorophyll (Chl), cytochrome f (Cyt f), P700, and light-harvesting chlorophyll-protein complex contents (LH Chl in % of
total ChI; it is assumed that all of the chlorophyll b is associated with this complex) plant material
mg Chl a + b g dry weight
Chl a Chl b
Chl Cyt f
nmol Cyt f g dry weight
Chl P700
P700 Cyt f
LH Chl
Chlorella vutgaris 211-11 h
23.1
3. l
1807
14.4
90l
2.0
48.8
Mutant 69
28.0
3.4
1251
25.1
727
1.7
45.7
Chlorella fusca C-I.I.10
21.7
2.4
1469
'16.6
966
1.5
58.7
Mutant Mutant Mutant Mutant
33.8 6.8 3.4 3.8
3.5 16.9 10.0 7.9
1134 252 207 325
33.4 30.3 18.4 13.1
549 264 208 295
2.1 1.0 1.0 1.1
44.7 11.2 18.2 22.5
G G G G
10 28 33 36
very high Chl a: Chl b ratio. In the in vivo absorption spectra of these mutants, the chlorophyll b shoulder at 653 nm is missing, whereas it is well established in the normal green types. The ratio of Chl: Cyt f and of Chl: PT00 is essentially lower in the yellow mutants than in the normal green strains. Calculated on a dry weight basis the redox enzyme content of the mutants G 28, G 33, and G 36 reaches or even exceeds that of the normal form. Thus, reduced chlorophyll synthesis is not correlated with a decrease of the concentration of electron transfer enzymes. The yellow mutants have a P700: Cyt f ratio of 1:1 whereas in the wild types the ratio ranges from about 1.5:1 to 2:1. The normal green algal cells grown under low-light conditions have a higher content of P700 than of Cyt f, and are therefore similar in this respect to higher plants grown under low-light conditions (Wild et al., 1973; Grahl and Wild, 1975). In the wild types, the percentage of LH Chl is 49% or 59% of the total chlorophyll content, whereas in the yellow mutants only 11-23% of the total chlorophyll is bound in the light-harvesting chlorophyllprotein complex. The photosynthetic unit reduces the discrepancy between the light-harvesting capacity of chlorophyll and the rate capacity of the electron transport system. We therefore define the photosynthetic unit as the number of chlorophyll molecules per electron transport chain. Considering that the electron transport capacity is limited by the concentration of Cyt f, the size of the photosynthetic unit is consistent with the number of chlorophyll molecules per molecule of Cyt f. Thus in the photosynthetic membranes of the normal strain of Chlorellafusca, 1469 chlorophyll molecules are involved in light harvesting per electron
transport pathway, whereas in the yellow mutants only 207-325 chlorophyll molecules take part. The unit size of the mutants is 7.1 (G 33) to 4.5 times (G 36) smaller than that of the normal form, mainly because of a deficiency of the LH Chl complex. According to Anderson (1975) the chlorophyll-deficient mutants of Chlorella have thylakoid membranes characterized by a primary molecular organization stage. This work was supported by the Deutsche Forschungsgemeinschaft.
References Anderson, J.M. : The molecular organization of chloroplast thylakoids. Biochim. Biophys. Acta 416, 191-235 (1975) Bauer, K., Wild, A. : The effect of blue light on the photosynthetic electron transport in chlorophyll-deficient mutants of Chlorella fusca. Z. Pflanzenphysiol. 80, 443-454 (1976) Bendall, D.S., Davenport, H.E., Hill, R. : Cytochrome components in chloroplasts of the higher plants, In: Methods of enzymology, vol. 23, pt.A., pp. 327-347, San Pietro, A., ed., New YorkLondon: Academic Press 1971 Grahl, H., Wild, A.: Studies on the content of P700 and cytochromes in Sinapis alba during growth under two different light intensities. In: Environmental and biological control of photosynthesis, pp. 107-113, Marcelle, R., ed., The Hague: W. Junk 1975 Wild, A.: The size of the photosynthetic unit and its variability in yellow mutants. In : Progress in photosynthesis research, vol. II, pp. 871476, Metzner, H., ed., Tiibingen: H. Metzner 1969 Wild, A., Zickler, H.-O., Graht, H. : Weitere Untersuchungen zur Variabilitfit der photosynthetischen Einheit. Planta (Bed.) 97, 208-223 (1971) Wild, A., Ke, B., Shaw, E.R. : The effect of light intensity during growth of Sinapis alba on the electron-transport components. Z. Pflanzenphysiol. 69, 344-350 (1973)
Received 29 April; accepted 15 June 1977
