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Changes in the activity of several enzymes during root differentiation in cultured cells of Atropa belladonna
Department of Botany, University of Helsinki, Finland
Changes in the Activity of Several Enzymes during Root Differentiation in Cultured Cells of Atropa belladonna LIISA KAARINA SIMOLA With 3 Figures Received July 28 and September 5,1972
Summary Roots were effectively formed by root callus of Atropa belladonna in suspension culture in media containing i. a-naphthoxyacetic acid (NOA 4 ppm) and a-naphthaleneacetic acid (NAA 0.5 ppm) ii. NAA (0.5 ppm). Several enzyme activities estimated separately from roots, from clumps with the roots detached, and from undifferentiated clumps were compared with the activity in an undifferentiated suspension (NAA 2 ppm) of the same age. Corresponding differences in levels of enzyme activity were found in different parts of the cultures irrespective of the type of hormonal induction. For example, roots had higher phosphatase, perioxidase and ribonuclease activities than clumps but glutamate: oxaloacetate transaminase and alanine aminopeptidase activities were higher in clumps with roots than in other parts.
Introduction Many chemical constituents, such as proteins, amino acids and alkaloids, have been estimated from callus tissue (KRIKORIAN and STEWARD, 1969). These studies have indicated that callus loses many of the secondary chemical features of the tissue in which it arises and the enzymes connected with their biosynthesis. But the activities of several degrading enzymes are higher in rapidly proliferating tumours or callus tissue than in healthy normal tissue (MACIEJEWSKA-POTAPCZYK, 1960; GAINOR and CRISLEY, 1961; CHEN and VENKETESWARAN, 1966). Differentiation is regulated by the balance of growth regulators and these compounds are known both to stimulate and to depress enzyme synthesis or activity. Atropa belladonna callus forms effectively roots in culture if exogenous auxin is omitted or if the medium contains tropic acid or a-naphthoxyacetic acid (THOMAS and STREET, 1970). In a previous study SIMOLA and SOPANEN (1971) showed that the curves of enzyme activities were very similar during different stages of growth after treatments resulting root differentiation, but these trends were different from those obtained with an undifferentiated callus suspension. The present paper reports the differences in enzyme activities between roots and undifferentiated parts within cultures and the results of tests on whether these differences are determined by the conditions under which differentiation occurs or are characteristic of the tissues themselves (e. g. reots or cell clumps) irrespective of the kind of growth regulator inducing root differentiation. Z. Pflanzenphysiol. Ed. 68. S. 373-378. 1973.
374
L. K. SIMOLA
Material and Methods Initial suspension: Root callus of Atropa belladonna cultivar lutea Doll cultivated on WOOD and BRAUN'S medium containing 2 ppm of a-naphthaleneacetic acid (NAA), and 0.5 ppm of kinetin, and 2 per cent sucrose was transferred to corresponding liquid medium (d. BHANDARY et aI., 1969; THOMAS and STREET, 1970, SIMOLA and SOPANEN, 1971). When the initial suspensions were 20 days old the fine cell suspension was discarded and only the small undifferentiated clumps were transferred with a spatula to different media (see below). In this way material of about the same physiological stage was obtained and the transference of old medium was avoided. Material Jor enzyme assays: 1. NAA (2 ppm), kinetin (0.1 ppm); no differentiation after 9 days. 2. NAA (0.5 ppm), kinetin (0.1 ppm); effective root differentiation after 10 days (d. Fig. 1). Roots and clumps from which the roots had been detached were analysed separately; there was too little undifferentiated material for analysis. 3. NOA (4 ppm), NAA (0.5 ppm) and kinetin (0.1 ppm); effective root differentiation after 9-10 days. Roots, clumps from which the roots were detached, and undifferentiated clumps analysed separately. Samples were prepared (roots, clumps from which the roots were detached, and undifferentiated clumps), washed with distilled water and lyophilized. Corresponding parts from parallel experiments were combined in order to obtain enough material for duplicate enzyme extractions.
~I\)
Fig. 1. Effective root formation in a cell clump suspension culture of Atropa belladonna grown in a medium containing 0.5 ppm of NAA. Enzyme extraction and estimation: 10 ml of cold Tris-HCl buffer (0.05 M contaInIng 0.06 M sodium thioglycollate, pH 8.0) was used for extraction of 200 mg of lyophilized material. The material was ground twice with a pestel in a chilled mortar as described earlier
z. PJlanzenphysiol. Bd. 68. S. 373-378. 1973.
Enzymes during Root Differentiation
375
(SIMOLA and SOPANEN, 1970). The combined extracts were centrifuged (13000 >< g) and a portion of the supernatant was dialysed for protein determinations because sodium thioglycollate interferes with protein determinations. The crude extracts for enzyme assays were stored at -20 0 C until used for analyses. The following enzymes were assayed by methods described in detail previously (SIMOLA and SOPANEN, 1970, 1971): aldolase, glutamate :oxaloacetate transaminase (GOT), alanine aminopeptidase (but incubation time 1 hour instead of 2), peroxidase (guaiacol as substrate, a method described by MAEHLY and CHANCE 1954, p. 387), acid phosphatase (p-nitrophenylphosphate as substrate) and ribonuclease. The protein content of the extrac ts was estimated by the method of LOWR Y et al. (1951 cit. by BAILEY, 1962). The protein content of the extracts was relatively similar and the activity patterns per mg dry weight and per mg protein have about the same form. Therefore the va lues in the graphs are expressed as specific activities.
Results and Discussion The differences in protein content of the extracts were usually small (Fig. 2 A). The undifferentiated parts of the clumps with roots usually had a higher protein concentration per mg dry weight than the roots growing out from the clumps. In earlier experiments higher protein content was found in cells grown with NAA than in those cultured with NOA or without added auxin (SIMOLA and SOPANEN, 1971). RIBONUCLEASE
8 PROTEIN )II!/MG D.W.T.
A
120
4
80
40
UD
RC R
C KC R
UD
RC R
C RC R
2 ppm NAA 0.5 ppmNAA 0.5 ppm NAA 4 ppmNOA
Fig. 2. Protein content per mg dry weight (A) and specific ribonuclease activity (B) in
Atropa belladonna cultured in different media. Undifferentiated culture (UD); undifferentiated cell clumps (C), clumps which rOOts had been detached (RC) and roots (R) were analysed separately; duplicate extracts.
The levels of specific activities were usually about the same in the corresponding parts in different treatments (Figs. 2 and 3). The most substantial difference was seen in alanine aminopeptidase activity of roots (higher activity if grown with 0.5 ppm of NAA) . But in both treatments that produced roots, those had a lower alanine aminopeptidase activity than the corresponding clumps. Glutamate :oxaloacetate transZ. PJlanzenphysiol. Bd. 68. S. 373-378. 1973.
376
L. K.
SIMOLA
aminase (GOT) was also present in the roots but was clearly or much (2-4 times) higher in the clumps from which they grew. This indicates effective turnover of amino acids and proteins in the undifferentiated parts of the clumps (especially treatments with NOA). The activity of GOT in roots was not affected by the growth substances but there were clear differences between differentiated and undifferentiated tissue within a culture. NAA (2 ppm), however, stimulated this activity as it did at the different stages of the growth curve of Atropa, but in this medium the cells did not differentiate (SIMOLA and SOPANEN, 1971). The activity of GOT was always relatively much lower in cultures in which differentiation occurred. PEROXIDASE
ACID PHOSPHATASE BL UNITS
6 10
UD
RC
R
UD
C RC R
2ppmNAA O.SppmNAA O.SppmNAA 4 ppmNOA
RC
R
C RC R
GLUTAMATE: OXALOACETATE TRANSAMINASE WROBL UNITS
ALANINE AMINOPEPTIDASE
200
150
100
so
UD
RC R
C RC R
UD
RC R
C RC R
Fig. 3. Specific enzyme activities in suspension cultures of Atropa belladonna cells; for explanations see Fig. 1.
z. Pjlanzenphysiol. Bd. 68. S. 373-)78.1973.
Enzymes during Root Differentiation
377
Ribonuclease activity (Fig. 2 B) was much higher in roots than in the undifferentiated parts of the cultures, and cultures grown with NAA (2 ppm) displayed very low activity. High ribonuclease activity was found in maturing leaf tissue and this activity paralleled the amount of RNA (PHILLIPS and FLETCHER, 1969; PHILLIPS et al., 1969). Moreover, older cultures of Atropa belladonna and Acer pseudoplatanus possessed higher specific activities than younger cultures with higher mitotic rates (SIMOLA and SOPANEN, 1970, 1971). The roots were found to represent a maturing, differentiating tissue. The root axis mainly consisted of highly vacuolated cells (SIMOLA, in press). The peroxidase and acid phosphatase activity patterns were very similar (Fig. 3). The roots displayed much higher activities than the clumps. These enzymes usually have higher activities in ageing cell cultures (DE JONG et al., 1967; SIMOLA and SOPANEN, 1970). The difference in enzyme activities in differentiating Atropa cultures were quantitative, not qualitative, in these experiments and analyses. Different plant organs usually have characteristic isoenzyme patterns, and changes occurring during the development are well known (SCHWARTZ et al., 1964; HESS, 1967; CHEN et al., 1970). Growth regulators are also able to induce and depress the synthesis of some isoenzymes (OCKERSE et al., 1966; RITZERT and TURIN, 1970). These experiments with Atropa callus show that the pattern of enzyme activity depends more on the differentiation of the tissue than on the growth regulator inducing differentiation. Thus all root extracts have clear similarities, whereas undifferentiated clumps have other features in common, and these differences are found irrespective of the composition of the culture medium.
Acknowledgements My thanks are due to Mr. TuoMAs SOPANEN, Miss TEIJA MIKKILA, M. Sc., and Miss MAIJALnsA SALONEN, B. Sc., for kind technical assistance. The work has been supported by grants from the State Council for Natural Sciences and Jenny and Antti Wihuri's Foundation.
References BAILEY, ]. 1.: Techniques in protein chemistry. Elsevier, Amsterdam, London, New York
(1962). BHANDARY, S. B. R., H. A. COLLIN, E. THOMAS, and H. E. STREET: Ann. Bot. 33, 647-656
(1969). CHEN, S.-1., 1. R. TOWILL, and]. R. LOEWENBERG: Physio!. Plant 23,434-443 (1970). CHEN, P. K., and S. VENKETESVARAN: Plant Physio!. 41, 842-846 (1966). DE JONG, D. W., E. F. JANSEN, and A. COLSON: Exp. Cel!. Res. 47,139-156 (1967). GAINOR, C, and F. D. CRISLEY: Nature 190,1031-1032 (1961). HESS, D.: Z. Pflanzenphysio!. 56, 295-298 (1967). KRIKORIAN, A. D., and F. C STEWARD: Plant physiology. Steward, F. C, ed. New York and London, Academic Press, pp. 227-326 (1969). MACIEJEWSKA-POTAPCZYK, W.:]. expo Bot. 11,98-103 (1960). MAEHLY, A. C, and B. CHANCE: Methods of biochemical analysis. Click, D., ed. New York, Interscience Publishers, Vo!' 1, pp. 357-424 (1954). OCKERSE, R., B. Z. SIEGEL, and A. W. GALSTON: Science 151,452-453 (1966).
Z. PJlanzenphysiol. Bd. 68. S. 373-378. 1973.
378
L. K. SIMOLA
PHILLIPS, D. R., R. F. HORTON, and R. A. FLETCHER: Physio!. Plant. 22, 1050-1054 (1969). PHILLIPS, D. R., and R. A. FLETCHER: Physio!. Plant. 22, 764-768 (1969). RITZERT, R. W., and B. A. TURIN: Phytochem. 9, 1701-1705 (1970). SCHWARTZ, H. M., S. r. BIEDRON, M. M. VON HOLDT and S. REHM: Phytochem. 3, 189-200 (1964). SIMOLA, L. K.: Z. Pflanzenphysiol. In print. SIMOLA, L. K., and T. SOPANEN: Physio!. Plant. 23,1212-1222 (1970). SIMOLA, L. K., and T. SOPANEN: Physio!. Plant. 25, 8-15 (1971). THOMAS, E., and H. E. STREET: Ann. Bot. 34, 657-669 (1970). LnSA KAARINA SIMOLA, Department of Botany, University of Helsinki, Unioninkatu 44, SF-00170 Helsinki 17, Suomi-Finland.
z.
PJlanzenphysiol. Bd. 68. S. 373-378. 1973.
Changes in the Activity of Several Enzymes during Root Differentiation in Cultured Cells of Atropa belladonna LIISA KAARINA SIMOLA With 3 Figures Received July 28 and September 5,1972
Summary Roots were effectively formed by root callus of Atropa belladonna in suspension culture in media containing i. a-naphthoxyacetic acid (NOA 4 ppm) and a-naphthaleneacetic acid (NAA 0.5 ppm) ii. NAA (0.5 ppm). Several enzyme activities estimated separately from roots, from clumps with the roots detached, and from undifferentiated clumps were compared with the activity in an undifferentiated suspension (NAA 2 ppm) of the same age. Corresponding differences in levels of enzyme activity were found in different parts of the cultures irrespective of the type of hormonal induction. For example, roots had higher phosphatase, perioxidase and ribonuclease activities than clumps but glutamate: oxaloacetate transaminase and alanine aminopeptidase activities were higher in clumps with roots than in other parts.
Introduction Many chemical constituents, such as proteins, amino acids and alkaloids, have been estimated from callus tissue (KRIKORIAN and STEWARD, 1969). These studies have indicated that callus loses many of the secondary chemical features of the tissue in which it arises and the enzymes connected with their biosynthesis. But the activities of several degrading enzymes are higher in rapidly proliferating tumours or callus tissue than in healthy normal tissue (MACIEJEWSKA-POTAPCZYK, 1960; GAINOR and CRISLEY, 1961; CHEN and VENKETESWARAN, 1966). Differentiation is regulated by the balance of growth regulators and these compounds are known both to stimulate and to depress enzyme synthesis or activity. Atropa belladonna callus forms effectively roots in culture if exogenous auxin is omitted or if the medium contains tropic acid or a-naphthoxyacetic acid (THOMAS and STREET, 1970). In a previous study SIMOLA and SOPANEN (1971) showed that the curves of enzyme activities were very similar during different stages of growth after treatments resulting root differentiation, but these trends were different from those obtained with an undifferentiated callus suspension. The present paper reports the differences in enzyme activities between roots and undifferentiated parts within cultures and the results of tests on whether these differences are determined by the conditions under which differentiation occurs or are characteristic of the tissues themselves (e. g. reots or cell clumps) irrespective of the kind of growth regulator inducing root differentiation. Z. Pflanzenphysiol. Ed. 68. S. 373-378. 1973.
374
L. K. SIMOLA
Material and Methods Initial suspension: Root callus of Atropa belladonna cultivar lutea Doll cultivated on WOOD and BRAUN'S medium containing 2 ppm of a-naphthaleneacetic acid (NAA), and 0.5 ppm of kinetin, and 2 per cent sucrose was transferred to corresponding liquid medium (d. BHANDARY et aI., 1969; THOMAS and STREET, 1970, SIMOLA and SOPANEN, 1971). When the initial suspensions were 20 days old the fine cell suspension was discarded and only the small undifferentiated clumps were transferred with a spatula to different media (see below). In this way material of about the same physiological stage was obtained and the transference of old medium was avoided. Material Jor enzyme assays: 1. NAA (2 ppm), kinetin (0.1 ppm); no differentiation after 9 days. 2. NAA (0.5 ppm), kinetin (0.1 ppm); effective root differentiation after 10 days (d. Fig. 1). Roots and clumps from which the roots had been detached were analysed separately; there was too little undifferentiated material for analysis. 3. NOA (4 ppm), NAA (0.5 ppm) and kinetin (0.1 ppm); effective root differentiation after 9-10 days. Roots, clumps from which the roots were detached, and undifferentiated clumps analysed separately. Samples were prepared (roots, clumps from which the roots were detached, and undifferentiated clumps), washed with distilled water and lyophilized. Corresponding parts from parallel experiments were combined in order to obtain enough material for duplicate enzyme extractions.
~I\)
Fig. 1. Effective root formation in a cell clump suspension culture of Atropa belladonna grown in a medium containing 0.5 ppm of NAA. Enzyme extraction and estimation: 10 ml of cold Tris-HCl buffer (0.05 M contaInIng 0.06 M sodium thioglycollate, pH 8.0) was used for extraction of 200 mg of lyophilized material. The material was ground twice with a pestel in a chilled mortar as described earlier
z. PJlanzenphysiol. Bd. 68. S. 373-378. 1973.
Enzymes during Root Differentiation
375
(SIMOLA and SOPANEN, 1970). The combined extracts were centrifuged (13000 >< g) and a portion of the supernatant was dialysed for protein determinations because sodium thioglycollate interferes with protein determinations. The crude extracts for enzyme assays were stored at -20 0 C until used for analyses. The following enzymes were assayed by methods described in detail previously (SIMOLA and SOPANEN, 1970, 1971): aldolase, glutamate :oxaloacetate transaminase (GOT), alanine aminopeptidase (but incubation time 1 hour instead of 2), peroxidase (guaiacol as substrate, a method described by MAEHLY and CHANCE 1954, p. 387), acid phosphatase (p-nitrophenylphosphate as substrate) and ribonuclease. The protein content of the extrac ts was estimated by the method of LOWR Y et al. (1951 cit. by BAILEY, 1962). The protein content of the extracts was relatively similar and the activity patterns per mg dry weight and per mg protein have about the same form. Therefore the va lues in the graphs are expressed as specific activities.
Results and Discussion The differences in protein content of the extracts were usually small (Fig. 2 A). The undifferentiated parts of the clumps with roots usually had a higher protein concentration per mg dry weight than the roots growing out from the clumps. In earlier experiments higher protein content was found in cells grown with NAA than in those cultured with NOA or without added auxin (SIMOLA and SOPANEN, 1971). RIBONUCLEASE
8 PROTEIN )II!/MG D.W.T.
A
120
4
80
40
UD
RC R
C KC R
UD
RC R
C RC R
2 ppm NAA 0.5 ppmNAA 0.5 ppm NAA 4 ppmNOA
Fig. 2. Protein content per mg dry weight (A) and specific ribonuclease activity (B) in
Atropa belladonna cultured in different media. Undifferentiated culture (UD); undifferentiated cell clumps (C), clumps which rOOts had been detached (RC) and roots (R) were analysed separately; duplicate extracts.
The levels of specific activities were usually about the same in the corresponding parts in different treatments (Figs. 2 and 3). The most substantial difference was seen in alanine aminopeptidase activity of roots (higher activity if grown with 0.5 ppm of NAA) . But in both treatments that produced roots, those had a lower alanine aminopeptidase activity than the corresponding clumps. Glutamate :oxaloacetate transZ. PJlanzenphysiol. Bd. 68. S. 373-378. 1973.
376
L. K.
SIMOLA
aminase (GOT) was also present in the roots but was clearly or much (2-4 times) higher in the clumps from which they grew. This indicates effective turnover of amino acids and proteins in the undifferentiated parts of the clumps (especially treatments with NOA). The activity of GOT in roots was not affected by the growth substances but there were clear differences between differentiated and undifferentiated tissue within a culture. NAA (2 ppm), however, stimulated this activity as it did at the different stages of the growth curve of Atropa, but in this medium the cells did not differentiate (SIMOLA and SOPANEN, 1971). The activity of GOT was always relatively much lower in cultures in which differentiation occurred. PEROXIDASE
ACID PHOSPHATASE BL UNITS
6 10
UD
RC
R
UD
C RC R
2ppmNAA O.SppmNAA O.SppmNAA 4 ppmNOA
RC
R
C RC R
GLUTAMATE: OXALOACETATE TRANSAMINASE WROBL UNITS
ALANINE AMINOPEPTIDASE
200
150
100
so
UD
RC R
C RC R
UD
RC R
C RC R
Fig. 3. Specific enzyme activities in suspension cultures of Atropa belladonna cells; for explanations see Fig. 1.
z. Pjlanzenphysiol. Bd. 68. S. 373-)78.1973.
Enzymes during Root Differentiation
377
Ribonuclease activity (Fig. 2 B) was much higher in roots than in the undifferentiated parts of the cultures, and cultures grown with NAA (2 ppm) displayed very low activity. High ribonuclease activity was found in maturing leaf tissue and this activity paralleled the amount of RNA (PHILLIPS and FLETCHER, 1969; PHILLIPS et al., 1969). Moreover, older cultures of Atropa belladonna and Acer pseudoplatanus possessed higher specific activities than younger cultures with higher mitotic rates (SIMOLA and SOPANEN, 1970, 1971). The roots were found to represent a maturing, differentiating tissue. The root axis mainly consisted of highly vacuolated cells (SIMOLA, in press). The peroxidase and acid phosphatase activity patterns were very similar (Fig. 3). The roots displayed much higher activities than the clumps. These enzymes usually have higher activities in ageing cell cultures (DE JONG et al., 1967; SIMOLA and SOPANEN, 1970). The difference in enzyme activities in differentiating Atropa cultures were quantitative, not qualitative, in these experiments and analyses. Different plant organs usually have characteristic isoenzyme patterns, and changes occurring during the development are well known (SCHWARTZ et al., 1964; HESS, 1967; CHEN et al., 1970). Growth regulators are also able to induce and depress the synthesis of some isoenzymes (OCKERSE et al., 1966; RITZERT and TURIN, 1970). These experiments with Atropa callus show that the pattern of enzyme activity depends more on the differentiation of the tissue than on the growth regulator inducing differentiation. Thus all root extracts have clear similarities, whereas undifferentiated clumps have other features in common, and these differences are found irrespective of the composition of the culture medium.
Acknowledgements My thanks are due to Mr. TuoMAs SOPANEN, Miss TEIJA MIKKILA, M. Sc., and Miss MAIJALnsA SALONEN, B. Sc., for kind technical assistance. The work has been supported by grants from the State Council for Natural Sciences and Jenny and Antti Wihuri's Foundation.
References BAILEY, ]. 1.: Techniques in protein chemistry. Elsevier, Amsterdam, London, New York
(1962). BHANDARY, S. B. R., H. A. COLLIN, E. THOMAS, and H. E. STREET: Ann. Bot. 33, 647-656
(1969). CHEN, S.-1., 1. R. TOWILL, and]. R. LOEWENBERG: Physio!. Plant 23,434-443 (1970). CHEN, P. K., and S. VENKETESVARAN: Plant Physio!. 41, 842-846 (1966). DE JONG, D. W., E. F. JANSEN, and A. COLSON: Exp. Cel!. Res. 47,139-156 (1967). GAINOR, C, and F. D. CRISLEY: Nature 190,1031-1032 (1961). HESS, D.: Z. Pflanzenphysio!. 56, 295-298 (1967). KRIKORIAN, A. D., and F. C STEWARD: Plant physiology. Steward, F. C, ed. New York and London, Academic Press, pp. 227-326 (1969). MACIEJEWSKA-POTAPCZYK, W.:]. expo Bot. 11,98-103 (1960). MAEHLY, A. C, and B. CHANCE: Methods of biochemical analysis. Click, D., ed. New York, Interscience Publishers, Vo!' 1, pp. 357-424 (1954). OCKERSE, R., B. Z. SIEGEL, and A. W. GALSTON: Science 151,452-453 (1966).
Z. PJlanzenphysiol. Bd. 68. S. 373-378. 1973.
378
L. K. SIMOLA
PHILLIPS, D. R., R. F. HORTON, and R. A. FLETCHER: Physio!. Plant. 22, 1050-1054 (1969). PHILLIPS, D. R., and R. A. FLETCHER: Physio!. Plant. 22, 764-768 (1969). RITZERT, R. W., and B. A. TURIN: Phytochem. 9, 1701-1705 (1970). SCHWARTZ, H. M., S. r. BIEDRON, M. M. VON HOLDT and S. REHM: Phytochem. 3, 189-200 (1964). SIMOLA, L. K.: Z. Pflanzenphysiol. In print. SIMOLA, L. K., and T. SOPANEN: Physio!. Plant. 23,1212-1222 (1970). SIMOLA, L. K., and T. SOPANEN: Physio!. Plant. 25, 8-15 (1971). THOMAS, E., and H. E. STREET: Ann. Bot. 34, 657-669 (1970). LnSA KAARINA SIMOLA, Department of Botany, University of Helsinki, Unioninkatu 44, SF-00170 Helsinki 17, Suomi-Finland.
z.
PJlanzenphysiol. Bd. 68. S. 373-378. 1973.