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Changes in phosphoenolpyruvate carboxylase and ribulose-biphosphate carboxylase activities during the photoheterotrophic growth of Nicotiana tabacum (CV xanthi) cell suspensions
Plant Science Letters, 13 (1978) 49--56 © Elsevier/North-Holland Scientific Publishers Ltd.
49
CHANGES IN PHOSPHOENOLPYRUVATE CARBOXYLASE AND RIBULOSE-BIPHOSPHATE CARBOXYLASE ACTIVITIES DURING THE PHOTOHETEROTROPHIC GROWTH OF N I C O T I A N A T A B A C U M (CV XANTHI) CELL SUSPENSIONS
A. NATO and Y. MATHIEU Laboratoire de Physiologic Cellulaire V~g~tale, Associ~ au CNRS (LA 40) d~pendant de l'Universit~ de Paris-Sud, 91405 Orsay Cedex (France) (Received December 9th, 1977) (Accepted March 14th, 1978)
SUMMARY
The capacities (in vitro* measurements) of phosphoenolpyruvate carboxylase (PEPCase) and ribulose-biphosphate carboxylase (RuBPCase) have been studied in photoheterotrophically growing cell suspensions ofNicotiana tabacum L. (cv Xanthi). The most significant observation was the different behaviour of the two carboxylases. The PEPCase capacity expressed on a g dry wt. basis rises from an initial day 0 value of 200--300/lmol of CO2 fixed to 600--800 gmol during the exponential phase and then returns to the initial value during the post~exponential and stationary phases. The RuBPCase capacity was nearly constant (90--140 pmol of CO2 fixed) throughout the growth cycle. The peak of respiration and the high PEPCase capacity during the exponential growth phase were concomitant with an active process of soluble protein synthesis. After electrophoresis of cell-free extracts, two bands of PEPCase activity (forms I and II with Rm values of 0.23 and 0.38 respectively) and one band of RuBPCase activity (Rm of 0.27) were localized on polyacrylamide gels. The densitometric profiles of soluble proteins showed a marked enrichment of PEPCase form II band during the active phase of cell division as compared to * in vitro experiments related in this report are enzymatic capacities measurements performed with cell-free preparations studied in optimal conditions of pH and in excess of enzyme substrate. Abbreviations: DTT, dithiothreitol; Fast violet B, 6-benzamido-4-methoxy-m-toluidine diazonium chloride; MDH, malic dehydrogenase; PEP, PEPCase, phosphoenolpyruvate, PEPCarboxylase; Rm, electrophoretic mobility relative to mobility of tracking dye; RuBP, RuBPCase, ribulose-biphosphate, RuBPCarboxylase ; TEMED, N,N,N',N'-tetramethylethylenediamine.
50
that of cells in the stationary phase of growth. Moreover, the 2-fold increase of the specific activity (pmol CO2 fixed/h and/mg of soluble proteins) of the PEPCase during the exponential phase suggested that the enhancement of the catalytic power of this enzyme could be due to de novo protein synthesis. The biosynthetic pattern of PEPCase was essentially that of peak enzyme in contrast with RuBPCase which showed only slight variations over the growth cycle.
INTRODUCTION
Our previous studies [1] have reported that marked changes occur in the photosynthetic capacity and in respiratory pathways of Nicotiana tabacum (cv Xanthi) green cells in batch suspension culture. During the exponential phase of growth, in vivo* capacities measurements revealed a high photosynthetic ratio (O2/CO2 near 2) concomitant with a rapid 02 uptake. At the same time, there was a rapid nitrogen uptake by cells from the culture medium, reflecting an important energetic requirement for the nitrogen metabolism. Analysis of label distribution among the products of '4CO2 fixation showed that both CO2 assimilation pathways, linked to the ribulose-biphosphate carboxylase (the autotrophic pathway) and to phosphoenolpyruvate carboxylase (the non-autotrophic) were operative in cells with an important increase, from 10--50% of the total fixation capacity of the latter during the exponential growth phase. The stimulation of the non-autrotrophic CO2 fixation can be explained both by a high rate of enzyme substrate supply by the glycolytic pathway and/or by increase in the endogenous enzyme level. This suggestion stimulated the present investigation into the quantitative and qualitative changes of soluble proteins during the growth cycle of cells. From the electrophoretic profiles of soluble proteins, PEPCase and RuBPCase were localized. Their qualitative variations were related to the measurements of their in vitro capacities from the cell-free preparations. MATERIALS AND METHODS
The origin of the Nicotiana tabacum (cv Xanthi) culture, subculture procedure and growth conditions of cell suspensions were the same as previously described [ 1 ]. Cell-free preparation Cells at different growth day intervals were collected on nylon bolting cloth (50 p m diameter), washed with distilled H20 and weighed. *in vivo experiments related in this report axe capacities measurements performed with entire cells studies in optimal conditions of pH and light, different to those of growth conditions.
51 5--10 g of fresh cells were suspended in 20--40 ml of 0.1 M Tris--HC1 pH 7.8, 10 mM MgC12,5 mM mercaptoethanol or DTT, 0.25 mM EDTA and Polyclar AT (1 g/g dry wt.). Cell-free extract was obtained b y disrupting the cells with a needle valve pressure (French press). The brei was filtered through a nylon net and the filtrate was centrifuged at 30 000 g for 10 min. The pellet along with the cell debris left on the nylon net was resuspended and washed once with the same buffer solution. After centrifugation, the two supernatants were combined and used for the measurements of enzymatic capacities and analysis of soluble proteins. All operations were carried o u t at 4°C.
E n z y m e assays. The reaction mixture in a total volume of 0.5 ml consists of: 0.1 M Tris--HC1 pH 7.6, 10 mM MgC12, 5 mM DTT, 0.25 mM EDTA and an enzymatic extract (50 or 100 pl). PEPCase capacity was determined b y measuring the incorporation of 20 mM NaH14CO3 (2 Ci/mol) into acid stable [14C]malate after addition of 6 mM PEP, 1 mM NADH2 and commercial MDH in excess. RuBPCase capacity was measured by addition to the reaction mixture of 20 mM NaH~4CO3 (2 Ci/mol) and 2 mM RuBP. A blank w i t h o u t substrate (PEP or RuBP) was run. In all cases, reactions were carried o u t at 28°C and the kinetics o f ~4CO2 fixation was followed during 30 min : an aliquot (100 ul) of the reaction mixture was taken up every 10 min and acidified b y addition of 50 pl 3N HCL. Radioactivity was counted on 50/21 samples, after drying, with a Nuclear Chicago gas flow counter. Soluble protein determination. Proteins were determined b y the m e t h o d of L o w r y et al. [2] on trichloracetic acid precipitated samples, using bovine serum albumin as the standard. Gel electrophoresis. Gels containing 5% acrylamide and 0.13% methylenebisacrylamide in a buffer solution consisting of 0.25 mM Tris--HCl (pH 8.3), 0.2 M glycine, 0.12% TEMED and 0.16% ammonium persulfate were polymerized in 6 × 70 mm glass tube. Gels were pre-electrophoresed for 10 min at 3 mA/gel with 25 mM Tris-HC1 pH 8.3 and 0.2 M glycine in the electrode vessels. Samples {50--200 ul) were then applied and electrophoresis was performed at 3mA/gel in a 4°C r o o m until the b r o m o p h e n o l blue tracker d y e reached the end o f the gel. After electrophoresis, some gels were stained with Coomassie blue and scanned at 590 nm using the gel scanning attachment of a Unicam SP 1809 spectrophotometer. E n z y m e activities on gels. The remaining unstained gels were used for localization o f carboxylases activities according to t w o methods.
52
(a) method using [14C] bicarbonate. Slices of I mm thickness were cut out from the gel using a Joyce Loebl gel slicer. Each section was incubated (20 min, 28°C) firstly in a reaction mixture appropriate for PEPCase activity detection and secondly, after washing with the reaction buffer, transferred into a reaction mixture for RuBPCase activity localization. In both cases, after removing the gel slices, the reaction mixtures were acidified by adding 50 pl of 3 N HC1 to eliminate the excess of [14C] bicarbonate. Profiles of 14CO2 fixed permitted localization of the protein-enzyme migration. (b ) method using the colorimetric staining of oxaloacetate. PEPCase activity was localized by incubating gels in an assay system described by Scrutton and Fatebne [3]. After incubation at 28°C for 20 min, the gels were transferred to a solution containing fast Violet B (3 mg/ml). The colored gels were scanned at 530 nm in order to detect the appearance of the red bands. A blank without PEP in the assay system was performed. RESULTS
Changes in soluble protein content. A rapid rate of soluble protein synthesis occurs during the early exponential phase of growth (Fig. 1 A). After this period, interruption of net protein synthesis leads to a sharp fall in cell soluble protein, followed, during the stationary phase, by a return of the initial level (Fig. 1 B).
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Fig. 1. (left) A semi-logarithmic plot o f the relative change in soluble proteins (A) and changes in the soluble protein content (B) during the growth cycle of tobacco ceils. Fig. 2. (right) A semi-logarithmic plot of the relative change in dry wt. (o o) and changes of PEPCase (~ z ) and RuBPCase (x x) capacities measured in vitro during the growth cycle of tobacco cells.
53
The respiratory measurements already reported [1] show quite similar variations as these of soluble proteins content. The quotient (~mol 02 consumed per mg soluble protein) is quite constant (near 0.9) during the growth cycle. Apparently, the peak respiration rate is correlated with an active soluble protein synthesis process in cells at the beginning of rapid phase of division. Similar observations were reported by Givan and Collin [4] with a suspension culture of Acer pseudoplatanus and by Nash and Davies [5] with cultures of Rosa sp.
Changes in carboxylases capacities PEPCase capacity (Fig. 2) rises to a maximum (600--800 umol of fixed CO2/h and/g dry wt.) which is reached between the 4th and 8th day, then the capacity decays to the initial day 0 value (200--300 pmol) by the 12th day. In contrast, RuBPCase capacities show only slight variations (90--140/~mol of fixed CO2 ) during the growth cycle. The twofold increase in PEPCase specific activities of the crude extract (Table I) occurs during the exponential growth phase, followed by a return of the initial activity during the stationary phase. This result suggests that the enzyme is synthetized at a much higher rate than the average protein synthesis rate during the active phase of cell division. The twofold decrease in RuBPCase specific activity of crude extract (Table I) during the first 10 days of culture indicates that the rate of enzyme synthesis is slower than the average protein synthesis rate. After this period, the twofold increase of the specific activity is mainly due to a decrease in the average soluble protein content (Fig. 1). During exponential growth phase, the PEPCase/RuBPCase ratio is about 7, whereas in the cells at the stationary phase, this ratio is near 2 (Table I). The developmental patterns observed for the two carboxylases capacities measured in vitro correlate with those obtained with in vivo experiments. However, with the in vitro systems, in contrast to the in vivo experiments, RuBPCase never appears as the main carboxylating enzyme. Codd and Merret [6], with synchronized Euglena culture, observed that PEPCase capacity measured in vitro higher than RuBPCase capacity was not associated with a significant in vivo CO2 fixation via the C4 dicarboxylic acids. TABLE I V A R I A T I O N S O F PEPCase A N D RuBPCase S P E C I F I C A C T I V I T I E S IN C R U D E E X T R A C T O F T O B A C C O C E L L S D U R I N G T H E G R O W T H CYCLE. S t a n d a r d e r r o r "was c a l c u l a t e d at n -- 7.
E x p o . Phase 3--10 d a y s Stat. Phase 10--21 d a y s
PEPCase RuBPCase #tool CO~/rag sol. prot.
PEPCase RuBPCase
6.84 + 0.56
1.01 -+ 0.18
6.77
3.47 +- 0.86
1.9 + 0.2
1.8
54
In addition, low activity of pyruvate kinase as already reported by Kisaki et al. [7] with young tobacco leaves might be responsible of the low rate of in vivo CO2 fixation via the C4 dicarboxylic acid pathway.
Studies of carboxylases with polyacrylamide gel electrophoresis. Two peaks of PEPCase activity (Rm of 0.23 and 0.38) and one peak of RuBPCase activity (Rm of 0.27) were located among the peaks of soluble proteins (Fig. 3A and Fig. 3B). The two peaks of PEPCase activity were confirmed by the oxaloacetic specific staining procedure with fast Violet B (Fig. 4). Several reports have already demonstrated the existence of multiple forms of PEPCase in the leaf tissue of C3 plant [8,9] or C4 plant species [10,11]. The analysis in polyacrylamide gels of soluble proteins of tobacco cells in any phase of growth revealed the presence of two forms of PEPCase proteins and one band of RuBPCase but their relative importance varies during the growth cycle. Thus, gel profiles of soluble proteins from cells in the stationary
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Fig. 3.--A. Densitometric scans at 590 nm o f electrophoretic gels of soluble protein (50 #g) extracts from tobacco cells in stationary phase o f growth (RuBPCase ~ ). B. Localization of PEPCase (forms I and II) and RuBPCase activities after Polyacrylamide gel electrophoresis.
55
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Fig. 4 (left) Densitometric scans at 530 n m of an electrophoretic polyacrylamide gel stained with Fast Violet B for detection of PEPCase (forms I and II) activity. Soluble protein extract (100 ug) f r o m cells in exponential phase (5 days) was performed. Fig. 5 (right) Densitometric scans at 590 n m of etectrophoretic gels of soluble protein extracts (50 ug) from tobacco cells in stationary (21 days) or in exponential (5 days) phases
of growth.
phase (21 days) show that RuBPCase appears to be the major protein band compared to the slight appearance o f the peaks of PEPCase forms I and II (Fig. 5). However during the exponential growth phase (5 days) form II of PEPCase appears to be the major soluble protein compared to the slight appearance of the peak of RuBPCase. In addition, the form I o f PEPCase appears to be more distinct than at the 21st day (Fig. 5). This result suggests a rapid PEPCase synthesis and a slow rate o f synthesis o f RuBPCase during the phase of active cell division. CONCLUSION
During the photoheterotrophic growth of tobacco cell suspensions, the biosynthetic pattern of PEPCase is essentially that of a peak enzyme, i.e. an enzyme which is synthetized during only one part of the cycle, namely exponential
56
phase. In contrast, RuBPCase is synthetized at a lower rate than the average rate of protein synthesis during the exponential phase, the RuBPCase content per cell being nearly constant all over the growth cycle. Increase in PEPCase capacity occurs when both energy and carbon chain demand for protein synthesis is high and may represent a process which is limiting the loss of carbon as CO2 at a time where the drainage of carbon chains out of the tricarboxylic cycle is maximum [12,13]. The developmental patterns of the two carboxylases, suggest, in agreement with other reports [7,14--17], that plants which are normally thought to be photosynthesizing by a C3 type metabolism may, during the early stage of their development, exhibit an active C4 dicarboxylic type of CO2 uptake. ACKNOWLEDGEMENTS:
The authors are grateful to Mr. J.M. Dreuillaux for his skilful assistance in culturing tobacco cells. Discussions and comments on the manuscript with Prof. A. Moyse are acknowledged. We also thank Dr R. R~my for advice on the use of the gel electrophoresis technique and for helpful discussions in the course of this study. This research was partly supported by the Centre National de la Recherche Scientifique ATP n 2681. REFERENCES 1 A. Nato, S. Bazetoux and Y. Mathieu, Physiol. Plant., 41 (1977) 116. 2 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. 3 M.C. Scrutton and F. Fatebne, Anal. Biochem. 69 (1975) 247. 4 C.V. Givan and H.A. Collin, J. Expt. Bot., 18 (1967) 34. 5 D.T. Nash and M.E. Davies, J. Expt. Bot., 23 (1973) 75. 6 G.A. Codd and M.J. Merrett, Planta, 100 (1971) 124. 7 T. Kisaki, S. Hirabayashi and N. Yano, Plant Cell Physiol., 14 (1973) 505. 8 S.K. Mukerji and I.P. Ting, Arch. Biochem. Biophys., 143 (1971) 297. 9 J. Vidal, G. Cavali~ and P. Gadal, Plant Sci. Lett., 7 (1976) 265. 10 M.D. Hatch, C.B. Osmond, J.H. Troughton and O. BjSrkmann, Carnegie Inst. Washington. Yearb., 71 (1972) 135. 11 S.K. Mukerji, Arch. Bioch. Biophys., 182 (1977) 343. 12 L. Hunt and J.S. Fletcher, Plant Physiol., 57 (1976) 304. 13 K.K. Nesius and J.S. Fletcher, Plant Physiol., 55 (1975) 643. 14 C.L. Hedley and A.O. Rowland, Plant Sci. Lett. 5 (1975) 119. 15 R.M. Smillie, Plant Physiol., 37 (1962) 716. 16 M.L. Salin and P.H. Homann, Plant Physiol., 48 (1971) 193. 17 D.I. Dickmann, Plant Physiol., 48 (1971) 143.
49
CHANGES IN PHOSPHOENOLPYRUVATE CARBOXYLASE AND RIBULOSE-BIPHOSPHATE CARBOXYLASE ACTIVITIES DURING THE PHOTOHETEROTROPHIC GROWTH OF N I C O T I A N A T A B A C U M (CV XANTHI) CELL SUSPENSIONS
A. NATO and Y. MATHIEU Laboratoire de Physiologic Cellulaire V~g~tale, Associ~ au CNRS (LA 40) d~pendant de l'Universit~ de Paris-Sud, 91405 Orsay Cedex (France) (Received December 9th, 1977) (Accepted March 14th, 1978)
SUMMARY
The capacities (in vitro* measurements) of phosphoenolpyruvate carboxylase (PEPCase) and ribulose-biphosphate carboxylase (RuBPCase) have been studied in photoheterotrophically growing cell suspensions ofNicotiana tabacum L. (cv Xanthi). The most significant observation was the different behaviour of the two carboxylases. The PEPCase capacity expressed on a g dry wt. basis rises from an initial day 0 value of 200--300/lmol of CO2 fixed to 600--800 gmol during the exponential phase and then returns to the initial value during the post~exponential and stationary phases. The RuBPCase capacity was nearly constant (90--140 pmol of CO2 fixed) throughout the growth cycle. The peak of respiration and the high PEPCase capacity during the exponential growth phase were concomitant with an active process of soluble protein synthesis. After electrophoresis of cell-free extracts, two bands of PEPCase activity (forms I and II with Rm values of 0.23 and 0.38 respectively) and one band of RuBPCase activity (Rm of 0.27) were localized on polyacrylamide gels. The densitometric profiles of soluble proteins showed a marked enrichment of PEPCase form II band during the active phase of cell division as compared to * in vitro experiments related in this report are enzymatic capacities measurements performed with cell-free preparations studied in optimal conditions of pH and in excess of enzyme substrate. Abbreviations: DTT, dithiothreitol; Fast violet B, 6-benzamido-4-methoxy-m-toluidine diazonium chloride; MDH, malic dehydrogenase; PEP, PEPCase, phosphoenolpyruvate, PEPCarboxylase; Rm, electrophoretic mobility relative to mobility of tracking dye; RuBP, RuBPCase, ribulose-biphosphate, RuBPCarboxylase ; TEMED, N,N,N',N'-tetramethylethylenediamine.
50
that of cells in the stationary phase of growth. Moreover, the 2-fold increase of the specific activity (pmol CO2 fixed/h and/mg of soluble proteins) of the PEPCase during the exponential phase suggested that the enhancement of the catalytic power of this enzyme could be due to de novo protein synthesis. The biosynthetic pattern of PEPCase was essentially that of peak enzyme in contrast with RuBPCase which showed only slight variations over the growth cycle.
INTRODUCTION
Our previous studies [1] have reported that marked changes occur in the photosynthetic capacity and in respiratory pathways of Nicotiana tabacum (cv Xanthi) green cells in batch suspension culture. During the exponential phase of growth, in vivo* capacities measurements revealed a high photosynthetic ratio (O2/CO2 near 2) concomitant with a rapid 02 uptake. At the same time, there was a rapid nitrogen uptake by cells from the culture medium, reflecting an important energetic requirement for the nitrogen metabolism. Analysis of label distribution among the products of '4CO2 fixation showed that both CO2 assimilation pathways, linked to the ribulose-biphosphate carboxylase (the autotrophic pathway) and to phosphoenolpyruvate carboxylase (the non-autotrophic) were operative in cells with an important increase, from 10--50% of the total fixation capacity of the latter during the exponential growth phase. The stimulation of the non-autrotrophic CO2 fixation can be explained both by a high rate of enzyme substrate supply by the glycolytic pathway and/or by increase in the endogenous enzyme level. This suggestion stimulated the present investigation into the quantitative and qualitative changes of soluble proteins during the growth cycle of cells. From the electrophoretic profiles of soluble proteins, PEPCase and RuBPCase were localized. Their qualitative variations were related to the measurements of their in vitro capacities from the cell-free preparations. MATERIALS AND METHODS
The origin of the Nicotiana tabacum (cv Xanthi) culture, subculture procedure and growth conditions of cell suspensions were the same as previously described [ 1 ]. Cell-free preparation Cells at different growth day intervals were collected on nylon bolting cloth (50 p m diameter), washed with distilled H20 and weighed. *in vivo experiments related in this report axe capacities measurements performed with entire cells studies in optimal conditions of pH and light, different to those of growth conditions.
51 5--10 g of fresh cells were suspended in 20--40 ml of 0.1 M Tris--HC1 pH 7.8, 10 mM MgC12,5 mM mercaptoethanol or DTT, 0.25 mM EDTA and Polyclar AT (1 g/g dry wt.). Cell-free extract was obtained b y disrupting the cells with a needle valve pressure (French press). The brei was filtered through a nylon net and the filtrate was centrifuged at 30 000 g for 10 min. The pellet along with the cell debris left on the nylon net was resuspended and washed once with the same buffer solution. After centrifugation, the two supernatants were combined and used for the measurements of enzymatic capacities and analysis of soluble proteins. All operations were carried o u t at 4°C.
E n z y m e assays. The reaction mixture in a total volume of 0.5 ml consists of: 0.1 M Tris--HC1 pH 7.6, 10 mM MgC12, 5 mM DTT, 0.25 mM EDTA and an enzymatic extract (50 or 100 pl). PEPCase capacity was determined b y measuring the incorporation of 20 mM NaH14CO3 (2 Ci/mol) into acid stable [14C]malate after addition of 6 mM PEP, 1 mM NADH2 and commercial MDH in excess. RuBPCase capacity was measured by addition to the reaction mixture of 20 mM NaH~4CO3 (2 Ci/mol) and 2 mM RuBP. A blank w i t h o u t substrate (PEP or RuBP) was run. In all cases, reactions were carried o u t at 28°C and the kinetics o f ~4CO2 fixation was followed during 30 min : an aliquot (100 ul) of the reaction mixture was taken up every 10 min and acidified b y addition of 50 pl 3N HCL. Radioactivity was counted on 50/21 samples, after drying, with a Nuclear Chicago gas flow counter. Soluble protein determination. Proteins were determined b y the m e t h o d of L o w r y et al. [2] on trichloracetic acid precipitated samples, using bovine serum albumin as the standard. Gel electrophoresis. Gels containing 5% acrylamide and 0.13% methylenebisacrylamide in a buffer solution consisting of 0.25 mM Tris--HCl (pH 8.3), 0.2 M glycine, 0.12% TEMED and 0.16% ammonium persulfate were polymerized in 6 × 70 mm glass tube. Gels were pre-electrophoresed for 10 min at 3 mA/gel with 25 mM Tris-HC1 pH 8.3 and 0.2 M glycine in the electrode vessels. Samples {50--200 ul) were then applied and electrophoresis was performed at 3mA/gel in a 4°C r o o m until the b r o m o p h e n o l blue tracker d y e reached the end o f the gel. After electrophoresis, some gels were stained with Coomassie blue and scanned at 590 nm using the gel scanning attachment of a Unicam SP 1809 spectrophotometer. E n z y m e activities on gels. The remaining unstained gels were used for localization o f carboxylases activities according to t w o methods.
52
(a) method using [14C] bicarbonate. Slices of I mm thickness were cut out from the gel using a Joyce Loebl gel slicer. Each section was incubated (20 min, 28°C) firstly in a reaction mixture appropriate for PEPCase activity detection and secondly, after washing with the reaction buffer, transferred into a reaction mixture for RuBPCase activity localization. In both cases, after removing the gel slices, the reaction mixtures were acidified by adding 50 pl of 3 N HC1 to eliminate the excess of [14C] bicarbonate. Profiles of 14CO2 fixed permitted localization of the protein-enzyme migration. (b ) method using the colorimetric staining of oxaloacetate. PEPCase activity was localized by incubating gels in an assay system described by Scrutton and Fatebne [3]. After incubation at 28°C for 20 min, the gels were transferred to a solution containing fast Violet B (3 mg/ml). The colored gels were scanned at 530 nm in order to detect the appearance of the red bands. A blank without PEP in the assay system was performed. RESULTS
Changes in soluble protein content. A rapid rate of soluble protein synthesis occurs during the early exponential phase of growth (Fig. 1 A). After this period, interruption of net protein synthesis leads to a sharp fall in cell soluble protein, followed, during the stationary phase, by a return of the initial level (Fig. 1 B).
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. 21
Fig. 1. (left) A semi-logarithmic plot o f the relative change in soluble proteins (A) and changes in the soluble protein content (B) during the growth cycle of tobacco ceils. Fig. 2. (right) A semi-logarithmic plot of the relative change in dry wt. (o o) and changes of PEPCase (~ z ) and RuBPCase (x x) capacities measured in vitro during the growth cycle of tobacco cells.
53
The respiratory measurements already reported [1] show quite similar variations as these of soluble proteins content. The quotient (~mol 02 consumed per mg soluble protein) is quite constant (near 0.9) during the growth cycle. Apparently, the peak respiration rate is correlated with an active soluble protein synthesis process in cells at the beginning of rapid phase of division. Similar observations were reported by Givan and Collin [4] with a suspension culture of Acer pseudoplatanus and by Nash and Davies [5] with cultures of Rosa sp.
Changes in carboxylases capacities PEPCase capacity (Fig. 2) rises to a maximum (600--800 umol of fixed CO2/h and/g dry wt.) which is reached between the 4th and 8th day, then the capacity decays to the initial day 0 value (200--300 pmol) by the 12th day. In contrast, RuBPCase capacities show only slight variations (90--140/~mol of fixed CO2 ) during the growth cycle. The twofold increase in PEPCase specific activities of the crude extract (Table I) occurs during the exponential growth phase, followed by a return of the initial activity during the stationary phase. This result suggests that the enzyme is synthetized at a much higher rate than the average protein synthesis rate during the active phase of cell division. The twofold decrease in RuBPCase specific activity of crude extract (Table I) during the first 10 days of culture indicates that the rate of enzyme synthesis is slower than the average protein synthesis rate. After this period, the twofold increase of the specific activity is mainly due to a decrease in the average soluble protein content (Fig. 1). During exponential growth phase, the PEPCase/RuBPCase ratio is about 7, whereas in the cells at the stationary phase, this ratio is near 2 (Table I). The developmental patterns observed for the two carboxylases capacities measured in vitro correlate with those obtained with in vivo experiments. However, with the in vitro systems, in contrast to the in vivo experiments, RuBPCase never appears as the main carboxylating enzyme. Codd and Merret [6], with synchronized Euglena culture, observed that PEPCase capacity measured in vitro higher than RuBPCase capacity was not associated with a significant in vivo CO2 fixation via the C4 dicarboxylic acids. TABLE I V A R I A T I O N S O F PEPCase A N D RuBPCase S P E C I F I C A C T I V I T I E S IN C R U D E E X T R A C T O F T O B A C C O C E L L S D U R I N G T H E G R O W T H CYCLE. S t a n d a r d e r r o r "was c a l c u l a t e d at n -- 7.
E x p o . Phase 3--10 d a y s Stat. Phase 10--21 d a y s
PEPCase RuBPCase #tool CO~/rag sol. prot.
PEPCase RuBPCase
6.84 + 0.56
1.01 -+ 0.18
6.77
3.47 +- 0.86
1.9 + 0.2
1.8
54
In addition, low activity of pyruvate kinase as already reported by Kisaki et al. [7] with young tobacco leaves might be responsible of the low rate of in vivo CO2 fixation via the C4 dicarboxylic acid pathway.
Studies of carboxylases with polyacrylamide gel electrophoresis. Two peaks of PEPCase activity (Rm of 0.23 and 0.38) and one peak of RuBPCase activity (Rm of 0.27) were located among the peaks of soluble proteins (Fig. 3A and Fig. 3B). The two peaks of PEPCase activity were confirmed by the oxaloacetic specific staining procedure with fast Violet B (Fig. 4). Several reports have already demonstrated the existence of multiple forms of PEPCase in the leaf tissue of C3 plant [8,9] or C4 plant species [10,11]. The analysis in polyacrylamide gels of soluble proteins of tobacco cells in any phase of growth revealed the presence of two forms of PEPCase proteins and one band of RuBPCase but their relative importance varies during the growth cycle. Thus, gel profiles of soluble proteins from cells in the stationary
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Fig. 3.--A. Densitometric scans at 590 nm o f electrophoretic gels of soluble protein (50 #g) extracts from tobacco cells in stationary phase o f growth (RuBPCase ~ ). B. Localization of PEPCase (forms I and II) and RuBPCase activities after Polyacrylamide gel electrophoresis.
55
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Fig. 4 (left) Densitometric scans at 530 n m of an electrophoretic polyacrylamide gel stained with Fast Violet B for detection of PEPCase (forms I and II) activity. Soluble protein extract (100 ug) f r o m cells in exponential phase (5 days) was performed. Fig. 5 (right) Densitometric scans at 590 n m of etectrophoretic gels of soluble protein extracts (50 ug) from tobacco cells in stationary (21 days) or in exponential (5 days) phases
of growth.
phase (21 days) show that RuBPCase appears to be the major protein band compared to the slight appearance o f the peaks of PEPCase forms I and II (Fig. 5). However during the exponential growth phase (5 days) form II of PEPCase appears to be the major soluble protein compared to the slight appearance of the peak of RuBPCase. In addition, the form I o f PEPCase appears to be more distinct than at the 21st day (Fig. 5). This result suggests a rapid PEPCase synthesis and a slow rate o f synthesis o f RuBPCase during the phase of active cell division. CONCLUSION
During the photoheterotrophic growth of tobacco cell suspensions, the biosynthetic pattern of PEPCase is essentially that of a peak enzyme, i.e. an enzyme which is synthetized during only one part of the cycle, namely exponential
56
phase. In contrast, RuBPCase is synthetized at a lower rate than the average rate of protein synthesis during the exponential phase, the RuBPCase content per cell being nearly constant all over the growth cycle. Increase in PEPCase capacity occurs when both energy and carbon chain demand for protein synthesis is high and may represent a process which is limiting the loss of carbon as CO2 at a time where the drainage of carbon chains out of the tricarboxylic cycle is maximum [12,13]. The developmental patterns of the two carboxylases, suggest, in agreement with other reports [7,14--17], that plants which are normally thought to be photosynthesizing by a C3 type metabolism may, during the early stage of their development, exhibit an active C4 dicarboxylic type of CO2 uptake. ACKNOWLEDGEMENTS:
The authors are grateful to Mr. J.M. Dreuillaux for his skilful assistance in culturing tobacco cells. Discussions and comments on the manuscript with Prof. A. Moyse are acknowledged. We also thank Dr R. R~my for advice on the use of the gel electrophoresis technique and for helpful discussions in the course of this study. This research was partly supported by the Centre National de la Recherche Scientifique ATP n 2681. REFERENCES 1 A. Nato, S. Bazetoux and Y. Mathieu, Physiol. Plant., 41 (1977) 116. 2 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. 3 M.C. Scrutton and F. Fatebne, Anal. Biochem. 69 (1975) 247. 4 C.V. Givan and H.A. Collin, J. Expt. Bot., 18 (1967) 34. 5 D.T. Nash and M.E. Davies, J. Expt. Bot., 23 (1973) 75. 6 G.A. Codd and M.J. Merrett, Planta, 100 (1971) 124. 7 T. Kisaki, S. Hirabayashi and N. Yano, Plant Cell Physiol., 14 (1973) 505. 8 S.K. Mukerji and I.P. Ting, Arch. Biochem. Biophys., 143 (1971) 297. 9 J. Vidal, G. Cavali~ and P. Gadal, Plant Sci. Lett., 7 (1976) 265. 10 M.D. Hatch, C.B. Osmond, J.H. Troughton and O. BjSrkmann, Carnegie Inst. Washington. Yearb., 71 (1972) 135. 11 S.K. Mukerji, Arch. Bioch. Biophys., 182 (1977) 343. 12 L. Hunt and J.S. Fletcher, Plant Physiol., 57 (1976) 304. 13 K.K. Nesius and J.S. Fletcher, Plant Physiol., 55 (1975) 643. 14 C.L. Hedley and A.O. Rowland, Plant Sci. Lett. 5 (1975) 119. 15 R.M. Smillie, Plant Physiol., 37 (1962) 716. 16 M.L. Salin and P.H. Homann, Plant Physiol., 48 (1971) 193. 17 D.I. Dickmann, Plant Physiol., 48 (1971) 143.