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Cold-induced rRNA synthesis in wheat cultivars during the hardening period
Plant Science Letters, 8 (1977) 191--195 © Elsevier/North-HollandScientific Publishers, Ltd.
191
COLD-INDUCED rRNA SYNTHESIS IN WHEAT CULTIVARS DURING THE HARDENING PERIOD
M A R T H A D E V A Y and E. PALDI AgriculturalResearch Instituteof the Hungarian Academy of Sciences, 2462 Martonvdsdr (Hungary) (Received April 21st, 1976) (Revision received August 26th, 1976) (Accepted September 2nd, 1976)
SUMMARY The intensity of rRNA synthesis taking place in wheat seedlings at low temperatures and its quantitative variation during the hardening process were examined. Chilling approximately doubles the intensity of rRNA synthesis in winter wheat at low temperatures, while it has no effect on non frost-resistant spring wheat. No significant change, characteristic of the hardehing period, can be observed in the percentage of DNA hybridized to rRNA due to the effect of the chilling. These results indicate that the cold-induced increase in the rRNA synthesis during the hardening period is not associated with a gross amplification of the rRNA genes.
INTRODUCTION When changes occur in the metabolic activity of plants, rRNA and other components accumulate at a rapid rate [1]. Since the increase in frost hardiness during the hardening period represents a very dramatic change in the metabolic process of the winter wheat seedlings, it is interesting to determine the intensity of rRNA synthesis taking place at hardening temperatures and its quantitative variation during chilling. MATERIALS AND METHODS
(a) Growth and labelling o f wheat (Triticum aestivum L.) seedlings. The experiments were carried out on the winter wheat cultivars Mironovskaya 808, Bezostaya 1, B~nkuti 1201 and Libellula and on the spring wheat varieties Lutescens 62, Penjamo 62, Siete Cerros and Artomovka. The wheat seeds were sterilized by washing in 0.3% bromine-water and
192
germinated under sterile conditions on 1% agar containing 2% sucrose at room temperature for 48 h in darkness. The seedlings were exposed to hardening for 5 days at 2°C in a refrigerator. The intact seedlings were incubated with their root tips resting in 5 ml 1000-fold diluted Knop solution per 50 seedlings, containing 500/~Ci [32P]orthophosphate and 2% sucrose, for 24 h at 3°C in darkness. The ribosomal RNA was extracted from the isolated seedlings. (b) Extraction and separation of rRNA. The ribosomal RNAs were prepared using the modified phenol procedure described by Ingle and Burns [2]. The nucleic acids (50 #g) were fractionated by electrophoresis on 2.4% polyacrylamide gel at 50 V (6 mA/tube) for 3.5 h [3]. After electrophoresis the gels were washed for 30 rain in distilled water and scanned at 265 nm in a Joyce-Loebl Chromoscan microdensitometer. After freezing with d r y ice gels were cut into 1 mm slices, which were dried on chromatographic filter paper (Whatman No. 1). The slices were counted in toluene with 0.5% PPO and 0.03% dimethyl-POPOP in an Intertechnique SL 31 liquid scintillation counter. The quantities of nucleic acids separated in the polyacrylamide gels were determined by measuring the area under the electrophoretogram curves. The 1.1.10 ~ dalton rRNA extracted from Escherichia coli was taken as the rRNA standard. The degree of radioactivity was calculated from the histograms, after subtracting the heterogeneous nucleic acid background. ( c) Preparation of 3H-labelled rRNA for molecular hybridization. In the plant material the number of rRNA cistrons, as well as the hybridization of rRNA is determined by the source of the DNA. The labelled rRNAs extracted from artichoke explants are commonly used in such experiments [ 1] The [3H]uridine-labelled rRNA was prepared from Jerusalem artichoke (Helianthus tuberosus vat. Bunyards Round) explants. 30 explants of 9 mg were cut from the parenchymal tissue of tubers and cultured in 5 ml complete medium [6] containing 2 mCi of [3H]uridine for 3 days at 25°C. The total RNA was prepared from these tissues using the modified phenol procedure [2] and fractionated by polyacrylamide gel electrophoresis [3]. The regions containing the two cytoplasmic ribosomal RNA peaks were cut into 0.2 mm slices. Slices from the central region of two peaks were then extracted with 6 × SSC (SSC: 0.15 M NaC1, 0.015 M sodium citrate, pH 7.2) for 2 h at 60°C. The two high molecular weight rRNAs (1.3.106 and 0.7.106 daltons) collected by centrifugation at 120 000 g for 16 h were quantitatively recovered with essentially no degradation [8]. (d) Preparation of DNA for molecular hybridization. DNA preparations were made from hardened and non-hardened wheat seedlings according to the method of Wells and Ingle [7]. The crude DNA was recovered by centrifugation at 44 000 rpm for 16 h and further purified by preparative CsC1 centrifugation. The DNA was denatured and then loaded onto Millipore filters (HAWP 01300, 0.45 ~m, 13 ram). The filters were air-dried
193 and finally dried in a vacuum oven at 80°C for 2 h immediately before use [ 9 ] . U n d e r these conditions w he n 20 ~g of DNA was loaded o n t o the filter, 19 -+ 0.5 ~g was retained and 18 +- 0.6 ~g (90%) o f t he DNA was present on t h e filter at th e end o f hybridization. (e) Molecular hybridization of rRNA to DNA. T he DNA filters were incubated with a 2:1 m i x t u r e of labelled 1.3.106 (5 ~g/ml) plus 0.7.106 (2.5 ~g/ml) daltons r t t N A as well as with 1.3.106 daltons rRN A (5 ~g/ml) alone in 6 × SSC at 70°C f or 2 h [ 9 ] . T h e DNA c o n t e n t o f t he filters was routinely m o n i t o r e d by acid hydrolysis after counting [ 1 0 ] . T h e specific activity of the rRNAs used in th e experiments was 700 000 cpm/~g. T h e values represent t he mean o f triplicate filters and are calculated f r o m triplicate experiments. RESULTS AND DISCUSSION T h e intensity o f r R N A synthesis taking place in seedlings at low temperature was examhp, ed at 3°C using t he i n c o r p o r a t i o n of [32P]orthophosphate into t h e rRNA. Th e results summarized in Table I provide a clear illustration o f changes occurring in t he intensity o f r R N A synthesis in t he course of t he hardening period. T he low t e m p e r a t u r e has t h e effect of increasing t he intensity o f r R N A synthesis in winter wheat varieties. Chilling a p p r o x i m a t e l y doubles the intensity o f r R N A synthesis in frost-resistant winter wheat varieties at low TABLE I COLD-INDUCED INCREASE OF rRNA SYNTHESIS TAKING PLACE IN WHEAT CULTIVARS DURING THE HARDENING PERIOD Experimental details are given in MATERIALS AND METHODS Cultivars
32Pi incorporated a cpm, 10-3/100 ~g rRNA
Percentage stimulation c
Non-hardened
Hardened b
11.1 11.2 11.4 16.6
23.1 27.5 33.9 27.4
208 246 297 164
21.1 15.8 11.4
22.8 16.2 12.5
108 102 109
Winter wheat Mironovskaya 808 Bezostaya 1 Bdnkuti 1201 Libellula
d d d d
Spring wheat Lutescens 62 Penjamo 62 Siete Cerros
aIncubation period: 24 h at 3°C. bHardening period: 5 days at 2° C. CNon-hardened samples = 100 %. dDifferences among hardened and non-hardened variants were significan~at the 0.05 probability level as revealed by analysis of variance and "t"-test.
194
temperature, while it has no effect on non frost-resistant spring wheat. The inductibility of rRNA synthesis depends on the winter character of the varieties, but the degree of inductibility varies from onb winter variety to another. The cold-induced increase in the rRNA synthesis during the hardening period may require the amplification of the rRNA genes or may be the consequence of the presence of rRNA cistrons which are activated at low temperature. The number of rRNA cistrons was examined in various winter and spring wheat varieties by'the molecular hybridization of rRN~A to DNA. The effect of cold treatment on the percentage of DNA hybridized to rRNA is shown in Table II. The data demonstrated that the percentage of DNA hybridized to rRNA was, however, similar, regardless of whether the DNA was prepared from embryos germinated for 48 h or from seedlings after a 5-day hardening period. No significant change characteristic of the hardening period can be observed due to the effect of chilling.These results indicated that no gross amplification of the rRNA genes is associated with this phase. The difference found in the hybridizing ability of DNA extracted from seedlings of certain wheat varieties which had not undergone cold treatment (Table II) points to a higher number of rRNA cistrons already present in the winter wheat cultivars. The differences were significant at the 0.1 probability level as revealed by analysis of variance and "t"-test calculated from the data available. It seems likely that the rise in the intensity of rRNA synthesis occurring due to the effect of cold can be traced back to the higher number of rRNA cistrons present in winter wheats. Further investigations are currently in progress in this field. TABLE II E F F E C T OF H A R D E N I N G ON r R N A GENES IN WHEAT C U L T I V A R S DNA was prepared from hardened and non-hardened seedlings. The 1.3.104 and 0.7.106 dalton rRNAs labelled with [SH]uridine were extracted f r o m artichoke explants. The DNA filters were incubated with a 2:1 m i x t u r e o f 1.3.106 (5 ~g/ml) and 0.7.106 (2.5 #g/ml) rRNAs, as well as with 1.3.106 daltons r R N A (5 #g/ml) alone in 6 X SSC at 70°C for 2 h. Source o f DNA
Mironovskaya 808 Bezostaya 1 ]~lnkuti 1201 Artomovka
% DNA hybridized to a 1 . 3 . 1 0 s dalton r R N A
1.3 + 0 . 7 . 1 0 s dalton rRNAs
Non-hardened
Hardened b
Non-hardened
Hardened b
0.0713 0.0778 0.0571 0.0491
0.0711 0.0655 0.0619 0.0564
0.0846 0.0953 0.0774 0.0719
0.1066 0.0875 0.0895 0.0770
± ± ± ±
0.0016 0.0066 0.0007 0.0001
± ± ± ±
0.0008 0.0021 0.0032 0.0010
± ± ± ±
0.0113 0.0006 0.0031 0.0021
± ± ± ±
0.0021 0.0013 0.0033 0.0026
aValues are the means o f triplicate filters and are calculated from triplicate experiments. bHardening period: 5 days at 2°C. Differences a m o n g non-hardened and har,dened variants were not significant at the 0.1 probability level and differences a m o n g spring wheat (Artomovka) and winter wheat cultivars (Mironovskaya 808, Bezostaya 1 and B~nkuti 1201) were significant at the 0.1 level as revealed by analysis of variance and " t " test.
195 ACKNOWLEDGEMENT
Thanks are due to Dr. J. Ingle (by M.D.) for introducing her to the molecular hybridization technique. This work was started in the Department of Botany of Edinburgh University, Scotland. REFERENCES
1 2 3 4 5 6 7 8 9 10
J. Ingle and J. Sinclair, Nature, 235 (1972) 30. J. Ingle and R.G. Burns, Biochem. J., 110 (1968) 605. U.E. Loening, Biochem. J., 102 (1967) 251. U.E. Loening, Biochem. J., 113 (1969) 131. M.E. Rogers, U.E. Loening and R.S.S. Fraser, J. Mol. Biol., 49 (1970) 681. R.S.S. Fraser, U.E. Loening and M.M. Yeoman, Nature, 215 (1967) 873. R. Wells and J. Ingle, Plant Physiol., 46 (1970) 178. J. Ingle, Personal communication. N.S. Scott and J. Ingle, Plant Physiol., 51 (1973) 677. D.D. Brown and C.S. Weber, J. Mol. Biol., 34 (1968) 681.
191
COLD-INDUCED rRNA SYNTHESIS IN WHEAT CULTIVARS DURING THE HARDENING PERIOD
M A R T H A D E V A Y and E. PALDI AgriculturalResearch Instituteof the Hungarian Academy of Sciences, 2462 Martonvdsdr (Hungary) (Received April 21st, 1976) (Revision received August 26th, 1976) (Accepted September 2nd, 1976)
SUMMARY The intensity of rRNA synthesis taking place in wheat seedlings at low temperatures and its quantitative variation during the hardening process were examined. Chilling approximately doubles the intensity of rRNA synthesis in winter wheat at low temperatures, while it has no effect on non frost-resistant spring wheat. No significant change, characteristic of the hardehing period, can be observed in the percentage of DNA hybridized to rRNA due to the effect of the chilling. These results indicate that the cold-induced increase in the rRNA synthesis during the hardening period is not associated with a gross amplification of the rRNA genes.
INTRODUCTION When changes occur in the metabolic activity of plants, rRNA and other components accumulate at a rapid rate [1]. Since the increase in frost hardiness during the hardening period represents a very dramatic change in the metabolic process of the winter wheat seedlings, it is interesting to determine the intensity of rRNA synthesis taking place at hardening temperatures and its quantitative variation during chilling. MATERIALS AND METHODS
(a) Growth and labelling o f wheat (Triticum aestivum L.) seedlings. The experiments were carried out on the winter wheat cultivars Mironovskaya 808, Bezostaya 1, B~nkuti 1201 and Libellula and on the spring wheat varieties Lutescens 62, Penjamo 62, Siete Cerros and Artomovka. The wheat seeds were sterilized by washing in 0.3% bromine-water and
192
germinated under sterile conditions on 1% agar containing 2% sucrose at room temperature for 48 h in darkness. The seedlings were exposed to hardening for 5 days at 2°C in a refrigerator. The intact seedlings were incubated with their root tips resting in 5 ml 1000-fold diluted Knop solution per 50 seedlings, containing 500/~Ci [32P]orthophosphate and 2% sucrose, for 24 h at 3°C in darkness. The ribosomal RNA was extracted from the isolated seedlings. (b) Extraction and separation of rRNA. The ribosomal RNAs were prepared using the modified phenol procedure described by Ingle and Burns [2]. The nucleic acids (50 #g) were fractionated by electrophoresis on 2.4% polyacrylamide gel at 50 V (6 mA/tube) for 3.5 h [3]. After electrophoresis the gels were washed for 30 rain in distilled water and scanned at 265 nm in a Joyce-Loebl Chromoscan microdensitometer. After freezing with d r y ice gels were cut into 1 mm slices, which were dried on chromatographic filter paper (Whatman No. 1). The slices were counted in toluene with 0.5% PPO and 0.03% dimethyl-POPOP in an Intertechnique SL 31 liquid scintillation counter. The quantities of nucleic acids separated in the polyacrylamide gels were determined by measuring the area under the electrophoretogram curves. The 1.1.10 ~ dalton rRNA extracted from Escherichia coli was taken as the rRNA standard. The degree of radioactivity was calculated from the histograms, after subtracting the heterogeneous nucleic acid background. ( c) Preparation of 3H-labelled rRNA for molecular hybridization. In the plant material the number of rRNA cistrons, as well as the hybridization of rRNA is determined by the source of the DNA. The labelled rRNAs extracted from artichoke explants are commonly used in such experiments [ 1] The [3H]uridine-labelled rRNA was prepared from Jerusalem artichoke (Helianthus tuberosus vat. Bunyards Round) explants. 30 explants of 9 mg were cut from the parenchymal tissue of tubers and cultured in 5 ml complete medium [6] containing 2 mCi of [3H]uridine for 3 days at 25°C. The total RNA was prepared from these tissues using the modified phenol procedure [2] and fractionated by polyacrylamide gel electrophoresis [3]. The regions containing the two cytoplasmic ribosomal RNA peaks were cut into 0.2 mm slices. Slices from the central region of two peaks were then extracted with 6 × SSC (SSC: 0.15 M NaC1, 0.015 M sodium citrate, pH 7.2) for 2 h at 60°C. The two high molecular weight rRNAs (1.3.106 and 0.7.106 daltons) collected by centrifugation at 120 000 g for 16 h were quantitatively recovered with essentially no degradation [8]. (d) Preparation of DNA for molecular hybridization. DNA preparations were made from hardened and non-hardened wheat seedlings according to the method of Wells and Ingle [7]. The crude DNA was recovered by centrifugation at 44 000 rpm for 16 h and further purified by preparative CsC1 centrifugation. The DNA was denatured and then loaded onto Millipore filters (HAWP 01300, 0.45 ~m, 13 ram). The filters were air-dried
193 and finally dried in a vacuum oven at 80°C for 2 h immediately before use [ 9 ] . U n d e r these conditions w he n 20 ~g of DNA was loaded o n t o the filter, 19 -+ 0.5 ~g was retained and 18 +- 0.6 ~g (90%) o f t he DNA was present on t h e filter at th e end o f hybridization. (e) Molecular hybridization of rRNA to DNA. T he DNA filters were incubated with a 2:1 m i x t u r e of labelled 1.3.106 (5 ~g/ml) plus 0.7.106 (2.5 ~g/ml) daltons r t t N A as well as with 1.3.106 daltons rRN A (5 ~g/ml) alone in 6 × SSC at 70°C f or 2 h [ 9 ] . T h e DNA c o n t e n t o f t he filters was routinely m o n i t o r e d by acid hydrolysis after counting [ 1 0 ] . T h e specific activity of the rRNAs used in th e experiments was 700 000 cpm/~g. T h e values represent t he mean o f triplicate filters and are calculated f r o m triplicate experiments. RESULTS AND DISCUSSION T h e intensity o f r R N A synthesis taking place in seedlings at low temperature was examhp, ed at 3°C using t he i n c o r p o r a t i o n of [32P]orthophosphate into t h e rRNA. Th e results summarized in Table I provide a clear illustration o f changes occurring in t he intensity o f r R N A synthesis in t he course of t he hardening period. T he low t e m p e r a t u r e has t h e effect of increasing t he intensity o f r R N A synthesis in winter wheat varieties. Chilling a p p r o x i m a t e l y doubles the intensity o f r R N A synthesis in frost-resistant winter wheat varieties at low TABLE I COLD-INDUCED INCREASE OF rRNA SYNTHESIS TAKING PLACE IN WHEAT CULTIVARS DURING THE HARDENING PERIOD Experimental details are given in MATERIALS AND METHODS Cultivars
32Pi incorporated a cpm, 10-3/100 ~g rRNA
Percentage stimulation c
Non-hardened
Hardened b
11.1 11.2 11.4 16.6
23.1 27.5 33.9 27.4
208 246 297 164
21.1 15.8 11.4
22.8 16.2 12.5
108 102 109
Winter wheat Mironovskaya 808 Bezostaya 1 Bdnkuti 1201 Libellula
d d d d
Spring wheat Lutescens 62 Penjamo 62 Siete Cerros
aIncubation period: 24 h at 3°C. bHardening period: 5 days at 2° C. CNon-hardened samples = 100 %. dDifferences among hardened and non-hardened variants were significan~at the 0.05 probability level as revealed by analysis of variance and "t"-test.
194
temperature, while it has no effect on non frost-resistant spring wheat. The inductibility of rRNA synthesis depends on the winter character of the varieties, but the degree of inductibility varies from onb winter variety to another. The cold-induced increase in the rRNA synthesis during the hardening period may require the amplification of the rRNA genes or may be the consequence of the presence of rRNA cistrons which are activated at low temperature. The number of rRNA cistrons was examined in various winter and spring wheat varieties by'the molecular hybridization of rRN~A to DNA. The effect of cold treatment on the percentage of DNA hybridized to rRNA is shown in Table II. The data demonstrated that the percentage of DNA hybridized to rRNA was, however, similar, regardless of whether the DNA was prepared from embryos germinated for 48 h or from seedlings after a 5-day hardening period. No significant change characteristic of the hardening period can be observed due to the effect of chilling.These results indicated that no gross amplification of the rRNA genes is associated with this phase. The difference found in the hybridizing ability of DNA extracted from seedlings of certain wheat varieties which had not undergone cold treatment (Table II) points to a higher number of rRNA cistrons already present in the winter wheat cultivars. The differences were significant at the 0.1 probability level as revealed by analysis of variance and "t"-test calculated from the data available. It seems likely that the rise in the intensity of rRNA synthesis occurring due to the effect of cold can be traced back to the higher number of rRNA cistrons present in winter wheats. Further investigations are currently in progress in this field. TABLE II E F F E C T OF H A R D E N I N G ON r R N A GENES IN WHEAT C U L T I V A R S DNA was prepared from hardened and non-hardened seedlings. The 1.3.104 and 0.7.106 dalton rRNAs labelled with [SH]uridine were extracted f r o m artichoke explants. The DNA filters were incubated with a 2:1 m i x t u r e o f 1.3.106 (5 ~g/ml) and 0.7.106 (2.5 #g/ml) rRNAs, as well as with 1.3.106 daltons r R N A (5 #g/ml) alone in 6 X SSC at 70°C for 2 h. Source o f DNA
Mironovskaya 808 Bezostaya 1 ]~lnkuti 1201 Artomovka
% DNA hybridized to a 1 . 3 . 1 0 s dalton r R N A
1.3 + 0 . 7 . 1 0 s dalton rRNAs
Non-hardened
Hardened b
Non-hardened
Hardened b
0.0713 0.0778 0.0571 0.0491
0.0711 0.0655 0.0619 0.0564
0.0846 0.0953 0.0774 0.0719
0.1066 0.0875 0.0895 0.0770
± ± ± ±
0.0016 0.0066 0.0007 0.0001
± ± ± ±
0.0008 0.0021 0.0032 0.0010
± ± ± ±
0.0113 0.0006 0.0031 0.0021
± ± ± ±
0.0021 0.0013 0.0033 0.0026
aValues are the means o f triplicate filters and are calculated from triplicate experiments. bHardening period: 5 days at 2°C. Differences a m o n g non-hardened and har,dened variants were not significant at the 0.1 probability level and differences a m o n g spring wheat (Artomovka) and winter wheat cultivars (Mironovskaya 808, Bezostaya 1 and B~nkuti 1201) were significant at the 0.1 level as revealed by analysis of variance and " t " test.
195 ACKNOWLEDGEMENT
Thanks are due to Dr. J. Ingle (by M.D.) for introducing her to the molecular hybridization technique. This work was started in the Department of Botany of Edinburgh University, Scotland. REFERENCES
1 2 3 4 5 6 7 8 9 10
J. Ingle and J. Sinclair, Nature, 235 (1972) 30. J. Ingle and R.G. Burns, Biochem. J., 110 (1968) 605. U.E. Loening, Biochem. J., 102 (1967) 251. U.E. Loening, Biochem. J., 113 (1969) 131. M.E. Rogers, U.E. Loening and R.S.S. Fraser, J. Mol. Biol., 49 (1970) 681. R.S.S. Fraser, U.E. Loening and M.M. Yeoman, Nature, 215 (1967) 873. R. Wells and J. Ingle, Plant Physiol., 46 (1970) 178. J. Ingle, Personal communication. N.S. Scott and J. Ingle, Plant Physiol., 51 (1973) 677. D.D. Brown and C.S. Weber, J. Mol. Biol., 34 (1968) 681.