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The Auxin-induced Growth of Tobacco Callus Tissue
Biochem. Physiol. Pflanzen (BPP), Bd. 165, S. 114-118 (1974)
Department of Botany, University of Veterinary Sciences, Budapest
The Auxin-induced Growth of Tobacco Callus Tissue I. Correlation between Growth and RNase Activity
By JANOS VETTER (Received June 6, 1973)
Summary The author analysed the steril tissue, isolated from the stem of tobacco (Nicotiana tabacum), grown 34 days on synthetic nutrient medium, supplemented by different amount of auxin. On the basis of data of fresh weight, dry matter, cell number and RNase activity after 34 days and after a short term auxin treatment in relation of RNase activity, it can be stated: 1. The growth of tissue is changed - in dependence with the auxin concentration applied according to an optimum curve.
2. A reverse proportionality was found between the growth and the RNase activity. On the other hand: the short term (2 or 4 hours) auxin treatment caused a remerkable inhibition in activity at the growth-promoting concentrations and vice varsa. The experimental series indicated the probable central role of nucleic acid metabolism and the nucleases in the auxin-action. On the other hand the technique of steril cultivation is a usefu I method in the examinations of the plant growth.
Introduction
To our kr.owledge the effect of growth regulating substance indol-3-acetic acid (IAA) has to do with the processes of nucleic acid- (BENDANA et al. 1965, BRIQUET et al. 1967) and protein metabolism (NOODEN and THIMANN 1963). The mechanism of the auxin-induced cell enlargement and growth is in connection with some other level and processes of plant metabolism too (cell wall metabolism, regulation of photosynthesis and respiration etc.). In the auxin action - on the basis of new observations (as has been demonstrated by TRUELSEN 1967; CALDOGNO et al. 1968: PrLET and BRAUN ]970; LONTAI 1971) - the possibility of the direct effect of nucleasc cEzymes exists. In the experiments - presented in this paper - a steril tissue technique was applied to establishe the change of growth parameters (the fresh wcight, dry matter content, the }lumber of cells and the RNase activity) and to showe the occasional relations between the growth and the RNase activity.
115
The Auxin-induced Growth of Tobacco Callus Tissue
Material and methods Isolated steril callus tissue from the stem of tobacco (Nicotiana tabacum L.) was the object of the examinations. The tissues were grown on a modified variant of the medium MURASHIGE and SKOOG (1962); precise composition of the applied medium was described in the other paper (VETTER 1973). The nutrient was supplemented with several auxin concentrations: 0.25; 1.0; 2.0; 8.0; and 16.0 mg per liter, respectively. In each flask one tissue fragment (200-200 mg fresh weight) was held. The experimental variants were incubated for 34 days at 18-22 °C, in the diurnal changes of light. The author analysed the weight increase, the changes of dry matter, the number of cells, the RNase-, activity of tobacco callus. The weight increase was characterised by the daily growth (mg per day). The evaluation of dry matter content was carried out by earlier method (VETTER and MAROTI 1971) and was calculated in per cent of fresh weight. Method of BROWN and RICKLESS (1950) was used to determine the cell number from 25 mg tissue. The data of determination were expressed as cell number per g fresh weight. From these dah the fresh weight of cells was calculated (x 10-4 mg per cell). The determination of RNase activity was performed according to our earlier method (VETTER 1972) from 250 mg tissue. The optical density was measured at 260 m,u, caused by the decomposed substrate (yeast RNA) only. In the short term incubation experiments the tissue fragments were placed on filtere paper, moistened with auxin solutions and after the exposition (2 or 4 hours) was characterised the RNase activity with the above described method; the activities were compared to the untreated (control) tissue.
Resnlts
The data of experimental series are listed in table 1. GROWTH. The growth parameters showed a strong stimulation at 0.25 mg per liter auxin concentration, but the 1 and 2 mg per liter caused a remerkable stimulation. in the daily growth too. The change of weight at the high concentrations (8 and 16 mg per liter) is greater than the Table 1
Effect of IAA on growth parameters and RNase activity of tobacco callus tissue (incubation time: 34 days), x: arithmetical mean; s: error Concentration of IAA (mg/l) Control
0.25
1.0
2.0
8.0
16.0
7.2 0.9
5.8 2.7
Growth (mg/day)
x
2.2 0.9
Dry matter (in per cent of fresh weight)
x
8.75 0.73
5.41 0.59
6.42 0.76
5.64 D.30
8.17 1.12
9.82 0.49
Cell number (x 10 6 /g fresh weight)
x
1.48 0.29
1.21 0.17
1.81 0.27
1.90 0.19
2.19 0.34
2.60 0.24
The fresh weight of cells ( x 10-4 mg/cell)
x
6.7 1.3
8.2 1.1
5.5 0.8
5.2 1.0
4.5 0.7
3.8 0.6
RNase activity (OD 26o )
x
0.618 0.065
0.386 0.012
0.383 0.060
0.358 0.008
0.401 0.025
0.483 0.030
8*
s
224 39
97 36
45 26
116
J.
VETTER
control, but - in comparison to the strongly significant stimulation - one can make this to an inhibitory effect. It is remerkable to note, that the rate of daily growth - probably - is not uniform in the incubation period, thus these values are relative, but they capable to the comparison. DRY MATTER. In the dry matter content nf the tissue a decrease was established at the tissue of stimulated growth, however no significant difference to the control was found at the 8 and 16 mg per liter concentrations. The results indicate, that the auxin induced growth-stimulation correlates with the increased water uptake, while the water uptake was smaller at the other variants. CELL NUMBER. It seems, that the increasing auxin concentration caused a promotion in the cell number, this increase was maximum + 45 and + 75 %. In comparison to the growth values it ean be stated, that the stimulated tissue has a decreased cell number. The parameters of fresh weight suggest, that in the case of a greater intensity, one can observe an increase of these parameters. The stimulated tissue has less cells, with a greater water content, and - subsequently - greater size and volume, than the other. At the higher concentrations - between 2 and 16 mg per liter - the fresh weight of cells decrease in comparison to the stimulated variants and to the control too. There seems a significant effect in the change of RNase activities of callus tissue. The homogenates obtained from the tissue showed a remerkable decrease of enzyme activity (except the 16 mg per liter variant). It can be concluded, that the presence of IAA in the medium - someway - plays a role in the regulation of the RNase activity. It was examined the short term auxin-effect on the RNase activity of tobacco tissue too. In the first experimental series (table 2) the activity was measured after two hours incubabation and the data were compared to the control, incubated with Table 2
Effect of IAA on RNase activity of tobacco callus tissue in short term experiments (incubation time: 2 and 4 hours) IAA concentration RNase activity (in per cent of the control) (mg/l) Incubation time 2 hours
Incubation time 4 hours
control
100,0
100,0
1 2 8 16 32 64 128
120.0 100.9 89.3 73.3
102.1 75.8 79.0 70.3 68.8
151.2 181.8
The Auxin-induced Growth of Tobacco Callus Tissue
117
distilled water. The applied wide concentration interval (0,01-64 mg per liter) caused a heterogenous effect: the 16 mg/l concentration induced a decrease, the highest concentration (64 mg per liter) induced a great increase in activity, while the other treatments were unaffected. In the second series (incubation time: 4 hours) the variants between 2-32 mg per liter caused a decrease of activity (- 25 %, - 30 %) while the highest (128 mg per liter) concentration strongly stimulated the activity (181 % of the control). Discussion
The callus culture of tobacco was applied for establishing new details of the mode of action exerted by auxin in the regulation of growth. It is generally agreed, that the process of plant growth has a complex character. In these examinations it could be established an intensive promotion of weight increase of tissue, as a lightly measurable quantitative parameter of the growth. A stimulation caused by 0,25-1,0 mg per liter concentration interval, was found and a relative inhibition at the higher concentrations. On the basis of data it can be stated, that one of the consequences of auxin effect is the increasing water uptake and the enlargement of cell volume is arise from this too. The earlier investigations have revealed, that the nucleic acid metabolism has to do with the auxin effect (BRIQUET et al. 1967), and an interaction of IAA with RNA and growth has been deduced (NooDEN and THIMANN 1963). It was previously noted, that the RNA levels may be controlled by the auxin via RNase activity. An inverse proportionality was found between RNA content and RNase activity (PILET and BRAUN 1967; TRUELSEN 1967). In our experiments a reduced RNase activity was established in the auxin treated tobacco tissue, after 34 days on nutrient medium. This effect depends upon the concentration applied, while the great - that is the growth-inhibiting - auxin concentrations caused a remerkable stimulation of activity. By reducing the incubation time (2 and 4 hours) the author found a similar tendency, while the changes in RNase activity seems have to do with the presence of several amounts of exogenous auxin. Possible, that various kinds of nuclease enzymes in tissue plays a different and important role in the processes of plant growth. References BENDANA, F. E., GALSTON, A. W., KARSAWHNEY, R, and PENNY, P. J., Recovery of Labelled Ribonucleic Acid Following Administration of Labeled Auxin to Green Stem Sections. Plant Physiol. 40, 977 -983 (1965). BRIQUET, M. V., DECALLONE, J. R, LAMBERT, R R, and WIAUX, A. L., DMPA and Avena Coleoptiles RNA Turnover. Physiol. plantarum 20, 337 -341 (1967). BROWN, R, RICKLEss, P., A new method for the study of cell division and cell extension with preliminary observation on the effect of temperature and nutrient. Proc. Roy. Soc. B. 136, 110-125 (1950).
118
J. VETTER, The Auxin-induced Growth of Tobacco Callus Tissue
CALDOGNO, F. R., LADo, P., and PE;\[NACCHIONI, A., SuI rapporto tra crescita peer distensione dello sviluppo dell' attivita RNasica in internodi di pisello trattati con auxina e benziladenina. Instituto Lombardo (Rend. Sci.) B. 102, 285-291 (1968). LONTAI, I., A nukleazok szerepe a navcnyi anyagcsere folyamatokban (On the function of the nucleases in the plant metabolism). Bot. Kozl., 58, 239-241 (1971). MURASHIGE, L. D., and SKOOG, F., A revised medium for rapid growth and bioassays with Tobacco Tissue Cultures, Physiol. plantarum 15, 473-479 (1962). NOODEN, L. D., and THIl\IANN, K. V., Evidence for a requirement for protein synthesis for auxin-induced cell enlargement. Proc. Nat. Acad. Sci. USA 50, 194-200 (1963). PILET, P. E., and BRAUN, R., Ribonuclease Activity and auxin effects in the Lens Root. Physiol. plant. 23, 245-250 (1970). TRUELSEN, T. A., Indolacetic Acid Induced decrease of the Ribonuclease Activity in vivo. Physiol. plant. 20, 1112 -1119 (1967). VETTER, J., and MAROTI, M., Effect of auxin and kinetin on the increase in materia al of the mycelial culture in Amanita pantherina. Acta Bot. Acad. Sci. Hung. 17, 259-271 (1971). Auxin-induced changes in the growth, peroxidase- and ribonuclease activity of maize seedlings. Annal. Univ. Sci. Budapestinensis 14, 73-81 (1972). A cink hatasa a doMny kallusz tenyeszet novekedesere (Effect of zinc on the growth of tobacco callus culture) Bot. Kozl., in press (1973). Author's address: Dr. J. VETTER, Department of Botany, University of Veterinary Sciences, Budapest, 1400 Pf 2 (Hungary).
Department of Botany, University of Veterinary Sciences, Budapest
The Auxin-induced Growth of Tobacco Callus Tissue I. Correlation between Growth and RNase Activity
By JANOS VETTER (Received June 6, 1973)
Summary The author analysed the steril tissue, isolated from the stem of tobacco (Nicotiana tabacum), grown 34 days on synthetic nutrient medium, supplemented by different amount of auxin. On the basis of data of fresh weight, dry matter, cell number and RNase activity after 34 days and after a short term auxin treatment in relation of RNase activity, it can be stated: 1. The growth of tissue is changed - in dependence with the auxin concentration applied according to an optimum curve.
2. A reverse proportionality was found between the growth and the RNase activity. On the other hand: the short term (2 or 4 hours) auxin treatment caused a remerkable inhibition in activity at the growth-promoting concentrations and vice varsa. The experimental series indicated the probable central role of nucleic acid metabolism and the nucleases in the auxin-action. On the other hand the technique of steril cultivation is a usefu I method in the examinations of the plant growth.
Introduction
To our kr.owledge the effect of growth regulating substance indol-3-acetic acid (IAA) has to do with the processes of nucleic acid- (BENDANA et al. 1965, BRIQUET et al. 1967) and protein metabolism (NOODEN and THIMANN 1963). The mechanism of the auxin-induced cell enlargement and growth is in connection with some other level and processes of plant metabolism too (cell wall metabolism, regulation of photosynthesis and respiration etc.). In the auxin action - on the basis of new observations (as has been demonstrated by TRUELSEN 1967; CALDOGNO et al. 1968: PrLET and BRAUN ]970; LONTAI 1971) - the possibility of the direct effect of nucleasc cEzymes exists. In the experiments - presented in this paper - a steril tissue technique was applied to establishe the change of growth parameters (the fresh wcight, dry matter content, the }lumber of cells and the RNase activity) and to showe the occasional relations between the growth and the RNase activity.
115
The Auxin-induced Growth of Tobacco Callus Tissue
Material and methods Isolated steril callus tissue from the stem of tobacco (Nicotiana tabacum L.) was the object of the examinations. The tissues were grown on a modified variant of the medium MURASHIGE and SKOOG (1962); precise composition of the applied medium was described in the other paper (VETTER 1973). The nutrient was supplemented with several auxin concentrations: 0.25; 1.0; 2.0; 8.0; and 16.0 mg per liter, respectively. In each flask one tissue fragment (200-200 mg fresh weight) was held. The experimental variants were incubated for 34 days at 18-22 °C, in the diurnal changes of light. The author analysed the weight increase, the changes of dry matter, the number of cells, the RNase-, activity of tobacco callus. The weight increase was characterised by the daily growth (mg per day). The evaluation of dry matter content was carried out by earlier method (VETTER and MAROTI 1971) and was calculated in per cent of fresh weight. Method of BROWN and RICKLESS (1950) was used to determine the cell number from 25 mg tissue. The data of determination were expressed as cell number per g fresh weight. From these dah the fresh weight of cells was calculated (x 10-4 mg per cell). The determination of RNase activity was performed according to our earlier method (VETTER 1972) from 250 mg tissue. The optical density was measured at 260 m,u, caused by the decomposed substrate (yeast RNA) only. In the short term incubation experiments the tissue fragments were placed on filtere paper, moistened with auxin solutions and after the exposition (2 or 4 hours) was characterised the RNase activity with the above described method; the activities were compared to the untreated (control) tissue.
Resnlts
The data of experimental series are listed in table 1. GROWTH. The growth parameters showed a strong stimulation at 0.25 mg per liter auxin concentration, but the 1 and 2 mg per liter caused a remerkable stimulation. in the daily growth too. The change of weight at the high concentrations (8 and 16 mg per liter) is greater than the Table 1
Effect of IAA on growth parameters and RNase activity of tobacco callus tissue (incubation time: 34 days), x: arithmetical mean; s: error Concentration of IAA (mg/l) Control
0.25
1.0
2.0
8.0
16.0
7.2 0.9
5.8 2.7
Growth (mg/day)
x
2.2 0.9
Dry matter (in per cent of fresh weight)
x
8.75 0.73
5.41 0.59
6.42 0.76
5.64 D.30
8.17 1.12
9.82 0.49
Cell number (x 10 6 /g fresh weight)
x
1.48 0.29
1.21 0.17
1.81 0.27
1.90 0.19
2.19 0.34
2.60 0.24
The fresh weight of cells ( x 10-4 mg/cell)
x
6.7 1.3
8.2 1.1
5.5 0.8
5.2 1.0
4.5 0.7
3.8 0.6
RNase activity (OD 26o )
x
0.618 0.065
0.386 0.012
0.383 0.060
0.358 0.008
0.401 0.025
0.483 0.030
8*
s
224 39
97 36
45 26
116
J.
VETTER
control, but - in comparison to the strongly significant stimulation - one can make this to an inhibitory effect. It is remerkable to note, that the rate of daily growth - probably - is not uniform in the incubation period, thus these values are relative, but they capable to the comparison. DRY MATTER. In the dry matter content nf the tissue a decrease was established at the tissue of stimulated growth, however no significant difference to the control was found at the 8 and 16 mg per liter concentrations. The results indicate, that the auxin induced growth-stimulation correlates with the increased water uptake, while the water uptake was smaller at the other variants. CELL NUMBER. It seems, that the increasing auxin concentration caused a promotion in the cell number, this increase was maximum + 45 and + 75 %. In comparison to the growth values it ean be stated, that the stimulated tissue has a decreased cell number. The parameters of fresh weight suggest, that in the case of a greater intensity, one can observe an increase of these parameters. The stimulated tissue has less cells, with a greater water content, and - subsequently - greater size and volume, than the other. At the higher concentrations - between 2 and 16 mg per liter - the fresh weight of cells decrease in comparison to the stimulated variants and to the control too. There seems a significant effect in the change of RNase activities of callus tissue. The homogenates obtained from the tissue showed a remerkable decrease of enzyme activity (except the 16 mg per liter variant). It can be concluded, that the presence of IAA in the medium - someway - plays a role in the regulation of the RNase activity. It was examined the short term auxin-effect on the RNase activity of tobacco tissue too. In the first experimental series (table 2) the activity was measured after two hours incubabation and the data were compared to the control, incubated with Table 2
Effect of IAA on RNase activity of tobacco callus tissue in short term experiments (incubation time: 2 and 4 hours) IAA concentration RNase activity (in per cent of the control) (mg/l) Incubation time 2 hours
Incubation time 4 hours
control
100,0
100,0
1 2 8 16 32 64 128
120.0 100.9 89.3 73.3
102.1 75.8 79.0 70.3 68.8
151.2 181.8
The Auxin-induced Growth of Tobacco Callus Tissue
117
distilled water. The applied wide concentration interval (0,01-64 mg per liter) caused a heterogenous effect: the 16 mg/l concentration induced a decrease, the highest concentration (64 mg per liter) induced a great increase in activity, while the other treatments were unaffected. In the second series (incubation time: 4 hours) the variants between 2-32 mg per liter caused a decrease of activity (- 25 %, - 30 %) while the highest (128 mg per liter) concentration strongly stimulated the activity (181 % of the control). Discussion
The callus culture of tobacco was applied for establishing new details of the mode of action exerted by auxin in the regulation of growth. It is generally agreed, that the process of plant growth has a complex character. In these examinations it could be established an intensive promotion of weight increase of tissue, as a lightly measurable quantitative parameter of the growth. A stimulation caused by 0,25-1,0 mg per liter concentration interval, was found and a relative inhibition at the higher concentrations. On the basis of data it can be stated, that one of the consequences of auxin effect is the increasing water uptake and the enlargement of cell volume is arise from this too. The earlier investigations have revealed, that the nucleic acid metabolism has to do with the auxin effect (BRIQUET et al. 1967), and an interaction of IAA with RNA and growth has been deduced (NooDEN and THIMANN 1963). It was previously noted, that the RNA levels may be controlled by the auxin via RNase activity. An inverse proportionality was found between RNA content and RNase activity (PILET and BRAUN 1967; TRUELSEN 1967). In our experiments a reduced RNase activity was established in the auxin treated tobacco tissue, after 34 days on nutrient medium. This effect depends upon the concentration applied, while the great - that is the growth-inhibiting - auxin concentrations caused a remerkable stimulation of activity. By reducing the incubation time (2 and 4 hours) the author found a similar tendency, while the changes in RNase activity seems have to do with the presence of several amounts of exogenous auxin. Possible, that various kinds of nuclease enzymes in tissue plays a different and important role in the processes of plant growth. References BENDANA, F. E., GALSTON, A. W., KARSAWHNEY, R, and PENNY, P. J., Recovery of Labelled Ribonucleic Acid Following Administration of Labeled Auxin to Green Stem Sections. Plant Physiol. 40, 977 -983 (1965). BRIQUET, M. V., DECALLONE, J. R, LAMBERT, R R, and WIAUX, A. L., DMPA and Avena Coleoptiles RNA Turnover. Physiol. plantarum 20, 337 -341 (1967). BROWN, R, RICKLEss, P., A new method for the study of cell division and cell extension with preliminary observation on the effect of temperature and nutrient. Proc. Roy. Soc. B. 136, 110-125 (1950).
118
J. VETTER, The Auxin-induced Growth of Tobacco Callus Tissue
CALDOGNO, F. R., LADo, P., and PE;\[NACCHIONI, A., SuI rapporto tra crescita peer distensione dello sviluppo dell' attivita RNasica in internodi di pisello trattati con auxina e benziladenina. Instituto Lombardo (Rend. Sci.) B. 102, 285-291 (1968). LONTAI, I., A nukleazok szerepe a navcnyi anyagcsere folyamatokban (On the function of the nucleases in the plant metabolism). Bot. Kozl., 58, 239-241 (1971). MURASHIGE, L. D., and SKOOG, F., A revised medium for rapid growth and bioassays with Tobacco Tissue Cultures, Physiol. plantarum 15, 473-479 (1962). NOODEN, L. D., and THIl\IANN, K. V., Evidence for a requirement for protein synthesis for auxin-induced cell enlargement. Proc. Nat. Acad. Sci. USA 50, 194-200 (1963). PILET, P. E., and BRAUN, R., Ribonuclease Activity and auxin effects in the Lens Root. Physiol. plant. 23, 245-250 (1970). TRUELSEN, T. A., Indolacetic Acid Induced decrease of the Ribonuclease Activity in vivo. Physiol. plant. 20, 1112 -1119 (1967). VETTER, J., and MAROTI, M., Effect of auxin and kinetin on the increase in materia al of the mycelial culture in Amanita pantherina. Acta Bot. Acad. Sci. Hung. 17, 259-271 (1971). Auxin-induced changes in the growth, peroxidase- and ribonuclease activity of maize seedlings. Annal. Univ. Sci. Budapestinensis 14, 73-81 (1972). A cink hatasa a doMny kallusz tenyeszet novekedesere (Effect of zinc on the growth of tobacco callus culture) Bot. Kozl., in press (1973). Author's address: Dr. J. VETTER, Department of Botany, University of Veterinary Sciences, Budapest, 1400 Pf 2 (Hungary).