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Calcium Inhibition of Phosphoenolpyruvate Carboxylase: Possible Physiological Consequences for C4-Photosynthesis
Laboratory of Plant Physiology, University of Patras, Patras, Greece
Calcium Inhibition of Phosphoenolpyruvate Carboxylase: Possible Physiological Consequences for C4-Photosynthesis NIKOS A. GAVALAS and YIANNIS MANETAS With 2 figures Received March 25, 1980 . Accepted June 4, 1980
Summary Calcium is a strong inhibitor of phosphoenolpyruvate carboxylase at concentrations low enough (0.5-1.0 mM) to be of physiological significance. The inhibition is competitive in respect to magnesium and affects both C a- and Ccphosphoenolpyruvate carboxylases. The results are consistent with the hypothesis that low soluble calcium is a prerequisite for C 4 -photosynthetic carbon fixation.
Key words: phosphoenolpyruvate carboxylase, calcium inhibition, C 4-photosynthesis.
Introduction The physiological and ecological significance of Crphotosynthesis has been duly appreciated during the last decade and considerable information on anatomical. biochemical and regulatory details of this carbon-fixation pathway, has accumulated (BLACK, 1973; LAETSCH, 1974; HATCH, 1976; KLUGE, 1977; RAY and BLACK, 1979). Nevertheless, knowledge on its interrelationships with other physiological processes (e.g., mineral nutrition) is still fragmentary. Recently, we obtained evidence that C 4-plants are calciophobes, i.e., they keep soluble calcium at low levels in their leaf tissues, and we expressed the view that this property might be a prerequisite for normal functioning of the C 4 -pathway (GAVALAS and MANETAS, 1980). On the basis of this hypothesis it could be predicted that Ca 2+ plays an inhibitory role at one or more central point(s) of the pathway. Phosphoenolpyruvate carboxylase (Ee. 4.1.1.31), the catalyst of the initial CO 2 fixation, appeared to be the most prominent target for Ca 2+ inhibition. A search of the literature, however, did not reveal much information; MUKERJI (1977) presented some evidence for Ca 2+ inhibition of the residual PEP-carboxylase activity obtained without Mg2+, but a detailed investigation of this effect has not been undertaken. The objective of the present study was to ascertain that Ca 2+ is indeed inhibitory in the presence of Mg2+ and to provide some details on the characteristics of the alleged inhibition.
z.
Pflanzenphysiol. Bd. 100. S. 179-184. 1980.
180
NIKOS A. GAVALAS and YIANNIS MANETAS
Materials and Methods Plant material was collected from the field and used immediately. Suitable amounts of leaf tissue (2 g fresh weight from C 4 -plants; 4 g from C 3-plants) were thoroughly washed with deionized water and extracted, in a prechilled mortar, with 10 ml of extraction medium and a small amount of purified sea sand. The extraction medium was 0.1 M Tris-HCl, pH 7.6, 1 mM EDTA, 10 mM MgCI 2 , 10 mM mercapto2thanol, 3 Ofo w/v polyvinylpyrrolidone plus a small amount of insoluble polyvinylpyrrolidone per sample. The extract was centrifuged at 5000 g for 5 min and the supernatant desalted through Sephadex G-25, equilibrated with 0.1 M Tris-HCl, pH 7.7, 5 mM mercaptoethanol and 20 % v/v glycerol. All above steps were carried out at about 4°C. The activity of PEP carboxylase was assayed, immediately after desalting, by NADH oxidation, in a reaction coupled to malate dehydrogenase. Assays were run at 28°C in 3 ml final volume of 0.1 M Tris-HCl, pH 7.7, 0.14 mM NADH, 1 mM NaHC0 3, 5 mM MgCl 2 (unless otherwise ~pecified), 10 mM dithiothreitol, 1.82 mM PEP (unless otherwise specified), plus 4.5 units of malate dehydrogenase (pig heart, Sigma). The reaction was started with the addition of 25-200.t1i of the desalted plant extract and the oxidation of NADH measured at 340 nm, between 45-90 sec after the addition of the extract. Calcium was added as CaCI 2 • Concerning the use of dithiothreitol in the assay medium, it should be noted that it has been found necessary for stabilisation of the enzyme during the assay at low substrate (PEP) concentrations. At PEP concentrations higher than 0.6 mM the reaction rate was linear with time even in the absence of dithiothreitol.
Results and Discussion
The results of a representative experiment are shown in Figure 1. As predicted, calcium is inhibitory to PEP carboxylase of Atriplex tatarica L., a C-plant, at concentrations low enough to be of physiological significance. Similar results were obtained with PEP carboxylase from two other C 4-plants (Amaranthus viridis L., Atriplex halimus L.). Since PEP carboxylase activity was assayed by NADH oxidation, we ascertained that the coupling enzyme (malate dehydrogenase) is not inhibited by calcium at the concentrations used. An interesting effect of Ca 2+ was also observed at low substrate (PEP) concentrations, in the absence of dithiothreitol. Under these assay conditions the enzyme suffers a fast inactivation, so that measured activities are lower than true initial ones (see Materials and Methods). Calcium acts not only as an inhibitor of the enzymic reaction but also as a stabiliser of the enzyme; thus, at PEP concentrations lower than 0.6 mM, the inhibitory effect of calcium is masked by its stabilising action, when dithiothreitol is omitted. Since both effects of calcium might have a physiological significance, we are currently working on the stabilisation of the enzyme by Ca 2+ and we plan to report our results separately in the near future. Obviously, at the present state of our knowledge, any attempt to interpret the stabilising effect of Ca 2+ in physiological terms would be premature. Since Mg2+ is an essential cofactor for PEP carboxylase activity, a plausible explanation for the inhibitory effect of Ca 2+ would be a competition between these
z. Pjlanzenphysiol. Bd.
100. S. 179-184. 1980.
Calcium inhibition of PEP carboxylase
181
0.3
0.2
c: E
--'" 0
~
0
1.0 Mm
0
Co
2+
0.1
0.243
0.728
[PEP] mM
1.215
1.820
Fig. 1: Effect of 1.0 mM CaCl 2 on the activity of PEP carboxylase. Plant: Atriplex tatari-
ca L. Normal Michaelis-Menten kinetics are observed in both inhibited and uninhibited
reaction rate.
cations. Evidence that such is indeed the case was obtained in a study of PEP carboxylase activity versus Mg2+ concentration, with and without Ca 2+. A double reciprocal plot of the data invariably shows the. typical features of competitive inhibition (Fig. 2). This type of Ca 2+ inhibition opens up a useful route for future investigation into the mechanism of the enzyme-substrate-metal complex formation. The data of Figure 2 may also serve as an evidence that Ca 2+, and not Cl-, is the inhibitory ion, since the inhibition is prevented by MgCl 2 at higher concentrations. Substantial differences, concerning Km for PEP and Mg2+ and allosteric properties, have been reported between PEP carboxylases from C 4 - and Ca-plants (TING and OSMOND, 1973; RAGHAVENDRA and DAS, 1977). It was pertinent, therefore, to see whether Ca 2 + inhibition, of the same nature and magnitude, also occurs in PEP carboxy lases from Ca-plants. Using Apium graveolens L., Stella ria media (L.) VILL. and Spinacia oleracea L. as sources of PEP carboxylase, we found that Ca 2+ inhibits, competitively with respect to Mg 2 +, the Ca-enzyme as well. Comparative data on activity and inhibition characteristics of the C a and C alloenzymes are given in Table 1. Calcium appears to Z. Pflanzenphysiol. Bd. 100. S. 179-184. 1980.
182
NIKOS
A.
GAVALAS
and
YIANNIS MAN ETAS
20 r---~~----~-----------~------~--------------~
18
16
'i C
12
E . 0-
~ 10
Q
o
2..
0.5 mM Co 2 +
8
6
0.2 Q29
0.5
1 1.33 1 1/ [Mg2+] ( mMr
2
Fig. 2: Double reciprocal plots of velocity vs Mg2+ concentration for PEP carboxylase activity at three levels of Ca 2+. Plant: Atriplex tatarica L.; Calculated Km(Mg) 2.24 mM.
be a strong competitive inhibitor for both C 4 - and Ca-PEP carboxylases. Ki values with respect to Mg 2 +, in the ranges of 0.32-0.45 mM and 0.46-1.76 mM were calculated for C a- and Crplants respectively. The differences in V max inhibition (Table 1) presumably reflect the different affinities of the enzymes for Mg2+ and Ca 2+. The functional significance of the Ca 2+ inhibition of PEP carboxylase remains to be evaluated. Unequivocal data on PEP, Mg2+ and Ca 2+ concentrations at the sites of PEP carboxylation, during steady state Crphotosynthesis, are lacking in the relevant literature and rather difficult to obtain. However, taking into account that C 4 PEP carboxylases exhibit much lower affinities (higher Km) of Mg2+ than C a PEP carboxy lases (TING and OSMOND, 1973; RAGHAVENDRA and DAS, 1977), the
z. P/lanzenphysiol. Bd.
100. S. 179-184. 1980.
Calcium inhibition of PEP carboxylase
183
Table 1: Absolute values and percent inhibition of V",H of phosphoenolpyruvate carboxylase from C,and Crplants.
--------------------------------------------Plant species
Vm ,,"")
% Inhibition
/LMoles CO,/ min· g fro wt.
ofVmax
42.38
31.38
57 70
8.94
49
0.58
33 39 3-l
by 1 mM Ca 2 +
C-Plants Amaranthus viridis L. Atriplex tatarica L. Atriplex halimu5 L.
Cl-Plants ApIum graveolens L. Stellana media (L.) VIl L. SpinaCla oleracea L.
O.-lS
1.10
".J Vn" , estimated from l.inewcavcr-Burk plots. assumption can be made that in vivo available Mg2+ could be limiting for C 4-photosynthesis but not (or much less) for C a PEP carboxylation. If that were the case, the same level of Ca 2+ would be more inhibitory for C 4-photosynthesis than for the functions (anaplerotic of cation compensating) of C,l PEP carboxylase. Intersecting evidence in support of a role of Ca 2+ in Crphotosynthesis comes from data showing that C 4 -plants keep soluble calcium at low levels in their leaf tissues, either by reduced absorpionltranslocation or by formation of insoluble calcium salts (GAvALAS and MANETAs, 1980). We should also note that Ca 2+, at concentrations up to 2 mM, does not inhibit RuDP-carboxylase (data not shown here), in accordance with the view that only C 4 -photosynthesis is adversely affected by calcium. Our hypothesis could be extended into crassulacean acid metabolism and photorespiration, but a final decision on its merits should await for additional experimental evidence, which is the aim of our current effort. Acknowledgements The technical assistance of Mr S. CARAVATAS is gladly acknowledged.
References BLACK, C. c.: Photosynthetic carbon fixation in relation to net CO 2 uptake. Ann. Rev. Plant Physio!. 24, 253-286 (1973). GAVALAS, N. A. and Y. MANETAS: Calcium inhibition of pyrophosphatase in crude plant extracts: Implication of soluble calcium in C 4 -photosynthesis, Plant Physio!. 65, 860-863 (1980). HATCH, M. D.: Photosynthesis: The path of carbon. In: BONNER, J. and J. E. VARNER (Eds.): Plant Biochemistry, 3rd Ed., 794-844. Academic Press, London, 1976. Z. Pjlanzenphysiol. Bd. 100. S. 179-184. 1980.
184
NIKOS A. GAVALAS and YIANNIS MAN ETAS
KLUGE, M.: Regulation of carbon dioxide fixation in plants. In: JENNINGS, D. (Ed.): Proc. 31st Symp. Exp. Bio!.: Integration of activity in higher plants, 155-175. Cambridge University Press, Cambridge, 1977. LAETscH, W. M.: The C 4 syndrome: A structural analysis. Ann. Rev. Plant Physio!. 25, 27-52 (1974). MUKER]I, S. K.: Corn leaf phosphoenolpyruvate carboxylases. The effect of divalent cations on activity. Arch. Bioch. Biophys. 182, 352-359 (1977). RAGHAVENDRA, A. S. and V. S. R. DAs: Purification and properties of phosphoenolpyruvate and ribulose diphosphate carboxy lases from C 4 and C 3 plants. Z. Pflanzenphysio!. 82, 315-321 (1977). RAY, T. B. and C. C. BLACK: The C 4 pathway and its regulation. In: GIBBS, M. and E. LATZKO (Eds.): Photosynthesis II. Photosynthetic carbon metabolism and related processes. (Eneyc!. Plant Physio!., N.S., Vo!' 6), 77-101. Springer-Verlag, Berlin, 1979. TING, I. P. and C. B. OSMOND: Photosynthetic phosphoenolpyruvate carboxylases. CharaetCflSncs of alloenzymcs from leaves of C a and C 4 plants. Plant Physio!. 51, 439-477 (1973).
Prof. Dr. N. A. GAVALAS, Dr. Y. MANETAS, Laboratory of Plant Physiology, University of Patras, Patras, Greece.
Z. P/lanzenphysiol. Bd. 100. S. 179-184. 1980.
Calcium Inhibition of Phosphoenolpyruvate Carboxylase: Possible Physiological Consequences for C4-Photosynthesis NIKOS A. GAVALAS and YIANNIS MANETAS With 2 figures Received March 25, 1980 . Accepted June 4, 1980
Summary Calcium is a strong inhibitor of phosphoenolpyruvate carboxylase at concentrations low enough (0.5-1.0 mM) to be of physiological significance. The inhibition is competitive in respect to magnesium and affects both C a- and Ccphosphoenolpyruvate carboxylases. The results are consistent with the hypothesis that low soluble calcium is a prerequisite for C 4 -photosynthetic carbon fixation.
Key words: phosphoenolpyruvate carboxylase, calcium inhibition, C 4-photosynthesis.
Introduction The physiological and ecological significance of Crphotosynthesis has been duly appreciated during the last decade and considerable information on anatomical. biochemical and regulatory details of this carbon-fixation pathway, has accumulated (BLACK, 1973; LAETSCH, 1974; HATCH, 1976; KLUGE, 1977; RAY and BLACK, 1979). Nevertheless, knowledge on its interrelationships with other physiological processes (e.g., mineral nutrition) is still fragmentary. Recently, we obtained evidence that C 4-plants are calciophobes, i.e., they keep soluble calcium at low levels in their leaf tissues, and we expressed the view that this property might be a prerequisite for normal functioning of the C 4 -pathway (GAVALAS and MANETAS, 1980). On the basis of this hypothesis it could be predicted that Ca 2+ plays an inhibitory role at one or more central point(s) of the pathway. Phosphoenolpyruvate carboxylase (Ee. 4.1.1.31), the catalyst of the initial CO 2 fixation, appeared to be the most prominent target for Ca 2+ inhibition. A search of the literature, however, did not reveal much information; MUKERJI (1977) presented some evidence for Ca 2+ inhibition of the residual PEP-carboxylase activity obtained without Mg2+, but a detailed investigation of this effect has not been undertaken. The objective of the present study was to ascertain that Ca 2+ is indeed inhibitory in the presence of Mg2+ and to provide some details on the characteristics of the alleged inhibition.
z.
Pflanzenphysiol. Bd. 100. S. 179-184. 1980.
180
NIKOS A. GAVALAS and YIANNIS MANETAS
Materials and Methods Plant material was collected from the field and used immediately. Suitable amounts of leaf tissue (2 g fresh weight from C 4 -plants; 4 g from C 3-plants) were thoroughly washed with deionized water and extracted, in a prechilled mortar, with 10 ml of extraction medium and a small amount of purified sea sand. The extraction medium was 0.1 M Tris-HCl, pH 7.6, 1 mM EDTA, 10 mM MgCI 2 , 10 mM mercapto2thanol, 3 Ofo w/v polyvinylpyrrolidone plus a small amount of insoluble polyvinylpyrrolidone per sample. The extract was centrifuged at 5000 g for 5 min and the supernatant desalted through Sephadex G-25, equilibrated with 0.1 M Tris-HCl, pH 7.7, 5 mM mercaptoethanol and 20 % v/v glycerol. All above steps were carried out at about 4°C. The activity of PEP carboxylase was assayed, immediately after desalting, by NADH oxidation, in a reaction coupled to malate dehydrogenase. Assays were run at 28°C in 3 ml final volume of 0.1 M Tris-HCl, pH 7.7, 0.14 mM NADH, 1 mM NaHC0 3, 5 mM MgCl 2 (unless otherwise ~pecified), 10 mM dithiothreitol, 1.82 mM PEP (unless otherwise specified), plus 4.5 units of malate dehydrogenase (pig heart, Sigma). The reaction was started with the addition of 25-200.t1i of the desalted plant extract and the oxidation of NADH measured at 340 nm, between 45-90 sec after the addition of the extract. Calcium was added as CaCI 2 • Concerning the use of dithiothreitol in the assay medium, it should be noted that it has been found necessary for stabilisation of the enzyme during the assay at low substrate (PEP) concentrations. At PEP concentrations higher than 0.6 mM the reaction rate was linear with time even in the absence of dithiothreitol.
Results and Discussion
The results of a representative experiment are shown in Figure 1. As predicted, calcium is inhibitory to PEP carboxylase of Atriplex tatarica L., a C-plant, at concentrations low enough to be of physiological significance. Similar results were obtained with PEP carboxylase from two other C 4-plants (Amaranthus viridis L., Atriplex halimus L.). Since PEP carboxylase activity was assayed by NADH oxidation, we ascertained that the coupling enzyme (malate dehydrogenase) is not inhibited by calcium at the concentrations used. An interesting effect of Ca 2+ was also observed at low substrate (PEP) concentrations, in the absence of dithiothreitol. Under these assay conditions the enzyme suffers a fast inactivation, so that measured activities are lower than true initial ones (see Materials and Methods). Calcium acts not only as an inhibitor of the enzymic reaction but also as a stabiliser of the enzyme; thus, at PEP concentrations lower than 0.6 mM, the inhibitory effect of calcium is masked by its stabilising action, when dithiothreitol is omitted. Since both effects of calcium might have a physiological significance, we are currently working on the stabilisation of the enzyme by Ca 2+ and we plan to report our results separately in the near future. Obviously, at the present state of our knowledge, any attempt to interpret the stabilising effect of Ca 2+ in physiological terms would be premature. Since Mg2+ is an essential cofactor for PEP carboxylase activity, a plausible explanation for the inhibitory effect of Ca 2+ would be a competition between these
z. Pjlanzenphysiol. Bd.
100. S. 179-184. 1980.
Calcium inhibition of PEP carboxylase
181
0.3
0.2
c: E
--'" 0
~
0
1.0 Mm
0
Co
2+
0.1
0.243
0.728
[PEP] mM
1.215
1.820
Fig. 1: Effect of 1.0 mM CaCl 2 on the activity of PEP carboxylase. Plant: Atriplex tatari-
ca L. Normal Michaelis-Menten kinetics are observed in both inhibited and uninhibited
reaction rate.
cations. Evidence that such is indeed the case was obtained in a study of PEP carboxylase activity versus Mg2+ concentration, with and without Ca 2+. A double reciprocal plot of the data invariably shows the. typical features of competitive inhibition (Fig. 2). This type of Ca 2+ inhibition opens up a useful route for future investigation into the mechanism of the enzyme-substrate-metal complex formation. The data of Figure 2 may also serve as an evidence that Ca 2+, and not Cl-, is the inhibitory ion, since the inhibition is prevented by MgCl 2 at higher concentrations. Substantial differences, concerning Km for PEP and Mg2+ and allosteric properties, have been reported between PEP carboxylases from C 4 - and Ca-plants (TING and OSMOND, 1973; RAGHAVENDRA and DAS, 1977). It was pertinent, therefore, to see whether Ca 2 + inhibition, of the same nature and magnitude, also occurs in PEP carboxy lases from Ca-plants. Using Apium graveolens L., Stella ria media (L.) VILL. and Spinacia oleracea L. as sources of PEP carboxylase, we found that Ca 2+ inhibits, competitively with respect to Mg 2 +, the Ca-enzyme as well. Comparative data on activity and inhibition characteristics of the C a and C alloenzymes are given in Table 1. Calcium appears to Z. Pflanzenphysiol. Bd. 100. S. 179-184. 1980.
182
NIKOS
A.
GAVALAS
and
YIANNIS MAN ETAS
20 r---~~----~-----------~------~--------------~
18
16
'i C
12
E . 0-
~ 10
Q
o
2..
0.5 mM Co 2 +
8
6
0.2 Q29
0.5
1 1.33 1 1/ [Mg2+] ( mMr
2
Fig. 2: Double reciprocal plots of velocity vs Mg2+ concentration for PEP carboxylase activity at three levels of Ca 2+. Plant: Atriplex tatarica L.; Calculated Km(Mg) 2.24 mM.
be a strong competitive inhibitor for both C 4 - and Ca-PEP carboxylases. Ki values with respect to Mg 2 +, in the ranges of 0.32-0.45 mM and 0.46-1.76 mM were calculated for C a- and Crplants respectively. The differences in V max inhibition (Table 1) presumably reflect the different affinities of the enzymes for Mg2+ and Ca 2+. The functional significance of the Ca 2+ inhibition of PEP carboxylase remains to be evaluated. Unequivocal data on PEP, Mg2+ and Ca 2+ concentrations at the sites of PEP carboxylation, during steady state Crphotosynthesis, are lacking in the relevant literature and rather difficult to obtain. However, taking into account that C 4 PEP carboxylases exhibit much lower affinities (higher Km) of Mg2+ than C a PEP carboxy lases (TING and OSMOND, 1973; RAGHAVENDRA and DAS, 1977), the
z. P/lanzenphysiol. Bd.
100. S. 179-184. 1980.
Calcium inhibition of PEP carboxylase
183
Table 1: Absolute values and percent inhibition of V",H of phosphoenolpyruvate carboxylase from C,and Crplants.
--------------------------------------------Plant species
Vm ,,"")
% Inhibition
/LMoles CO,/ min· g fro wt.
ofVmax
42.38
31.38
57 70
8.94
49
0.58
33 39 3-l
by 1 mM Ca 2 +
C-Plants Amaranthus viridis L. Atriplex tatarica L. Atriplex halimu5 L.
Cl-Plants ApIum graveolens L. Stellana media (L.) VIl L. SpinaCla oleracea L.
O.-lS
1.10
".J Vn" , estimated from l.inewcavcr-Burk plots. assumption can be made that in vivo available Mg2+ could be limiting for C 4-photosynthesis but not (or much less) for C a PEP carboxylation. If that were the case, the same level of Ca 2+ would be more inhibitory for C 4-photosynthesis than for the functions (anaplerotic of cation compensating) of C,l PEP carboxylase. Intersecting evidence in support of a role of Ca 2+ in Crphotosynthesis comes from data showing that C 4 -plants keep soluble calcium at low levels in their leaf tissues, either by reduced absorpionltranslocation or by formation of insoluble calcium salts (GAvALAS and MANETAs, 1980). We should also note that Ca 2+, at concentrations up to 2 mM, does not inhibit RuDP-carboxylase (data not shown here), in accordance with the view that only C 4 -photosynthesis is adversely affected by calcium. Our hypothesis could be extended into crassulacean acid metabolism and photorespiration, but a final decision on its merits should await for additional experimental evidence, which is the aim of our current effort. Acknowledgements The technical assistance of Mr S. CARAVATAS is gladly acknowledged.
References BLACK, C. c.: Photosynthetic carbon fixation in relation to net CO 2 uptake. Ann. Rev. Plant Physio!. 24, 253-286 (1973). GAVALAS, N. A. and Y. MANETAS: Calcium inhibition of pyrophosphatase in crude plant extracts: Implication of soluble calcium in C 4 -photosynthesis, Plant Physio!. 65, 860-863 (1980). HATCH, M. D.: Photosynthesis: The path of carbon. In: BONNER, J. and J. E. VARNER (Eds.): Plant Biochemistry, 3rd Ed., 794-844. Academic Press, London, 1976. Z. Pjlanzenphysiol. Bd. 100. S. 179-184. 1980.
184
NIKOS A. GAVALAS and YIANNIS MAN ETAS
KLUGE, M.: Regulation of carbon dioxide fixation in plants. In: JENNINGS, D. (Ed.): Proc. 31st Symp. Exp. Bio!.: Integration of activity in higher plants, 155-175. Cambridge University Press, Cambridge, 1977. LAETscH, W. M.: The C 4 syndrome: A structural analysis. Ann. Rev. Plant Physio!. 25, 27-52 (1974). MUKER]I, S. K.: Corn leaf phosphoenolpyruvate carboxylases. The effect of divalent cations on activity. Arch. Bioch. Biophys. 182, 352-359 (1977). RAGHAVENDRA, A. S. and V. S. R. DAs: Purification and properties of phosphoenolpyruvate and ribulose diphosphate carboxy lases from C 4 and C 3 plants. Z. Pflanzenphysio!. 82, 315-321 (1977). RAY, T. B. and C. C. BLACK: The C 4 pathway and its regulation. In: GIBBS, M. and E. LATZKO (Eds.): Photosynthesis II. Photosynthetic carbon metabolism and related processes. (Eneyc!. Plant Physio!., N.S., Vo!' 6), 77-101. Springer-Verlag, Berlin, 1979. TING, I. P. and C. B. OSMOND: Photosynthetic phosphoenolpyruvate carboxylases. CharaetCflSncs of alloenzymcs from leaves of C a and C 4 plants. Plant Physio!. 51, 439-477 (1973).
Prof. Dr. N. A. GAVALAS, Dr. Y. MANETAS, Laboratory of Plant Physiology, University of Patras, Patras, Greece.
Z. P/lanzenphysiol. Bd. 100. S. 179-184. 1980.