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The changes in the nature of rRNA synthesis in the plumula of seedlings during vernalisation
Plant Science Letters, 30 (1983) 69--75
69
Elsevier Scientific Publishers Ireland Ltd.
THE CHANGES IN THE NATURE OF rRNA SYNTHESIS IN THE P L U M U L A O F SEEDLINGS D U R I N G V E R N A L I S A T I O N
EMIL PALDI and M A R T A
DI~VAY
Department of Plant Physiology and Biochemistry, Agricultural Research Institute, Hungarian Academy of Sciences,H-2462 Martonvasdr (Hungary)
(Received May 1st,1982) (Revision received October 20th, 1982) (Accepted October 22nd, 1982)
SUMMARY
The nature of r R N A synthesis in the plumula in the course of vernalisation at low temperature (3°C) was examined in four winter wheat varieties with different cold requirements (Triticum aestivum L. var. Bezostaya 1, Rannyaya 12, Mironovskaya 808 and B~nkuti 1201) using the pulse~hase technique. The presence of an r R N A synthesised at low temperature but decomposing at high temperature (25°C) was detected through its decomposition products, which have molecular weights of 1.05 X 106 and 0.57 X 106. It was found that the synthesis of this r R N A was dependent on the degree of vernalisation. It appears likely that the r R N A detected takes part in the specific metabolism of vernalisation.
Key words: r R N A synthesis -- Plumula -- Seedlings -- Vernalisation
INTRODUCTION
Plants will n o t flower, nor in m a n y cases respond to the environmental stimuli which ensure subsequent flowering, until they have reached a 'ripeness to flower'. Of exceptional interest in this request is the clarification of the process concerning the accelerated induction of 'ripeness to flower' by exposure to cold in cold-requiring plants such as winter cereals. This process, which ranks w i t h o u t d o u b t as one of the most interesting problems of mot-
Abbreviations: Pi, potassium orthophosphate. 0304-4211/83/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
70 phogenesis, is known as vernalisation. Vernalisation is essentially the acceleration of plant development due to cold effect. It is a prerequisite for flower formation and makes the plants 'capable of responding to the photoperiod'. In winter cereals the temperature most effective in vernalisation is at or near 0°C and the duration of the treatment for maximum acceleration of flower initiation ranges according to the variety from 1 week to 2 months. This time is called the cold requirement. The lower limit for the active vernalisation temperature is generally - 3 ° C and the upper limit 10°C [ 1 ]. Vernalisation can be reversed b y a period of high (25°--40°C) temperature immediately following vernalisation. This p h e n o m e n o n is called devernalisation. Devernalisation is n o t able to produce a permanent state. This is confirmed b y the renewed vernalisability at low temperature of plants devernalised by heat. This renewed vernalisation of devernalised plants is known as revernalisation [ 2,3 ]. The temperature range 10°--20°C is neutral, exhibiting no vernalisation or devernalisation effect. It is a significant paradox of vernalisation that an environmental condition, low temperature, which generally reduces the intensity of biochemical processes, exerts an accelerating effect on the biochemical processes forming part of vernalisation. Melchers [4] drew attention to the contradiction between the flower-inducing role of low temperature and the c o m m o n l y known temperature-dependence of biochemical reactions. He tried to remove the paradox by postulating a 3-fold reaction with various temperature dependences. In his opinion the vernalisation process can be described by the following formula: Q, olI
ql0 I'
precursor
: intermediate product
~ end p r o d u c t
I Q~oIII damage caused b y high temperature to the intermediate products Q10III > Q10II > Q10I A rough approximation of the value of Qlo was calculated from the temperature-dependences of vernalisation, devernalisation and revernalisation: Ql0III = 4.5, Q~0 = 2.5 and Ql0 I= 2.3 [5]. It must be assumed that vernalisation consists of numerous competing processes. All these processes, w i t h o u t exception, are of biochemical character, differing solely in their characteristic temperatures and in Q10 ' As a result of these temperature-dependences, the reactions taking place at low temperatures, active for vernalisation, are primarily those leading to flower formation, whereas at higher temperatures the conditions are more suitable for inhibitory or decomposing processes.
71 The end-product of vernalisation m a y include the existence of a certain 'flowering hormone', or of its precursor, or of a group of enzymes, which is able to synthesize the substances that make plants 'capable of responding' to the photoperiod. In analysing the role played b y nucleic acids in vernalisation, the tempera° ture-dependence of vernalisation, dissolved above, was used to examine the nature of r R N A synthesis as active, neutral and devernalisation temperatures. In our previous paper it was reported that vernalisation is preceded by a cold-induced increase in r R N A synthesis [6]. It is also reported that the R N A synthesis in winter wheat varieties at neutral and devernalisation temperatures is different from that at temperatures active for vernalisation. It was also confirmed that at temperatures active for vernalisation a type of r R N A was synthesized in the plumula which was n o t stable at high devernalisation temperatures. The decomposition of this rRNA could be traced by means of specific decomposition products with molecular weights of 1.05 X 106 and 0.57 X 106. The quantity of these decomposition products diminished due to the effect of revernalisation, i.e. renewed low temperature and within a few hours they could no longer be detected. The present paper is aimed at reporting results which give evidence of a connection between the above mentioned change in the nature of r R N A synthesis and the degree of vernalisation. MATERIALS AND METHODS
Chemicals 32Pi was purchased from the Institute of the Hungarian Academy of Sciences; sucrose from Merck; acrylamide and N,N,N',N'-methylene bisacrylamide from Serva. All other chemicals were of analytical grade and obtained from BDH. Plan t material Seedlings of Mironovskaya 808, B~nkuti 1201, Bezostaya 1 and Rannyaya 12 winter wheat (Triticum aestivum L.) were used in the experiment. The sterilised seeds were germinated at 20°C for 48 h in darkness on 1% agar-agar containing 2% sucrose and then vernalised at 0°C in darkness. At this temperature vernalis~/tion takes place at a normal rate, b u t cell division ceases [ 7]. The endosperm was removed after different vernalisation periods and the isolated seedlings were used for the examination of r R N A synthesis. Analyses were carried o u t weekly for the entire course of the process, in accordance with the cold requirement of the varieties. Different varieties have different cold requirements. For Mironovskaya 808, for example, the vernalisation requirement is 60 days, that of B~inkuti and Bezostaya 1 is 48 days, while that of R a n n y a y a 12 is 30 days at 0°C. Variants which received no cold treatments were considered to be unvernalised. Variants which were exposed to low temperatures for half the number of days needed to satisfy the cold requirement characteristic of the variety
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Fig. 1. rRNA synthesis in B~inkuti 1201 winter wheat seedlings during vernalisation. Experimental material: seedlings germinated in the dark for 48 h. Pulse incubation (A,C,E): 3 h at 25°C with 0.1 ml incubating solution (1000 × diluted Knop-solution + 2% sucrose + 100 ~Ci 32Pi) for each seedling. Chase incubation (B,D,F): 48 h at 3°C with 0.1 ml incubating solution containing 3 x Pi + 2% sucrose) per seedling. The fractionation of rRNAs took place on a 2.4% polyacrylamide gel (50 V, 6 mA per tube, 3.25 h). The evaluation was carried out with a Chromoscan microdensitometer at 265 nm. The histogram shows the radioactivity of the l-ram gel slices. Numbers above the peaks indicate molecular weights in millions of daltons.
.O
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1,3
E
C~
74 were considered as half-vernalised, while the fully vernalised variants were those kept at low temperature long enough to completely satisfy the cold requirement. Data on the degree of vernalisation of all three variants are presented in the paper.
Evaluation of the nature o f rRNA synthesis The pulse and chase labelling procedure was applied for the examination of r R N A synthesis. For the pulse period 0.1 ml 1000× diluted Knop-solution per plant was used, containing 100 pCi radioactive potassium orthophosphate (Pi } and 2% sucrose per millilitre. The incubation was carried o u t for 3 h at 20°C. After the pulse period, half the seedlings were processed immediately, while the other half were washed with distilled water and 10 -3 M phosphate buffer and were thereafter transferred to Knop-solution containing 2% sucrose and incubated at 3°C for 48 h for use in the chase experiment. The nucleic acids were extracted at the end of the pulse and chase periods using a modified phenol m e t h o d [ 8]. Loening's m e t h o d of polyacrylamide gel electrophoresis [ 9] was used to separate the nucleic acids. Gels containing 2.4% acrylamide, 0.06% bis-acrylamide and 0.25% agarose were used in the electrophoresis, which ran for 3.25 h, using a current of 5--6 mA per tube. The nucleic acid c o n t e n t of the gels was evaluated at 265 nm in a Joyce-Loebl 'Chromoscan' microdensitometer. The radioactivity of the 1-mm gel slices, which were frozen with solid CO2, was determined using the liquid scintillation method. In order to identify the nucleic acids their molecular weights were determined using Loening's m e t h o d [ 10]. RESULTS AND DISCUSSION The data for unvernalised (A,B), half-vernalised (C,D) and fully vernalised {E,F) seedlings of B~nkuti 1201 winter wheat are presented in Fig. 1. The results obtained for seedlings of Mironovskaya 808, Bezostaya 1 and Rannyaya 12 winter wheats are n o t presented in a separate figure, since they are identical with those obtained for B~inkuti 1201. It may be seen from Fig. 1 that changes occur in the nature of r R N A synthesis during the course of vernalisation. During the initial and middle stages of vernalisation, pulse incubation (A,C} at the lower limit of the devernalisation temperature (25°C) results in t w o separate peaks in every case, from both heavy and light rRNAs. There is no absorption peak corresponding to the second peak either for heavy or for light rRNAs, which indicates that these fractions cannot be present in significant quantities. The molecular weights of the t w o specific fractions were found to be 1.05 X 106 and 0.57 X 106. In the determinations the r R N A of Escherichia coli was taken as the molecular weight standard [6]. These t w o specific fractions are considered to be the decomposition products of r R N A which is synthesised at low temperature (3°C) b u t decomposes at high temperature (25°C). The 1.05 X 106 and 0.57 X 106 dalton r R N A varieties disappear due to the
75 e f f e c t o f chase i n c u b a t i o n (B,D) carried o u t o n unvernalised and half-vernalised seedlings at t e m p e r a t u r e s active f o r vernalisation. H o w e v e r , t h e y are easily discernable a f t e r vernalisation in b o t h t h e pulse (E) a n d t h e chase (F) experiments. T h e a p p e a r a n c e o f 1.05 X 106 a n d 0.57 X 106 d a l t o n r R N A s in pulse e x p e r i m e n t s at t h e devernalisation t e m p e r a t u r e (A,C,E) and their disappearance d u r i n g chase e x p e r i m e n t s (B,D) carried o u t at t e m p e r a t u r e active for vernalisation shows a close c o r r e l a t i o n w i t h t h e c o l d t r e a t m e n t in all f o u r w i n t e r w h e a t s investigated a n d also p o i n t s t o a c h a n g e in t h e r R N A metabolites. F u r t h e r analysis o f t h e origin a n d role o f t h e 1.05 X 106 a n d 0.57 X 106 d a l t o n r R N A varieties is n o w in progress. REFERENCES 1 2 3 4 5 6 7 8 9 10
V.I. Razumov, Izd. Selskohoz. Lit., Leningrad-Moscow, 1961, p. 62. F.G. Gregory and O.N. Purvis, Nature (Lond.), 155 (1945) 113. F.G. Gregory and O.N. Purvis, Nature (Lond.), 161 (1948) 859. G. Melchers, The Physiology of Flower Initiation, Dokumentumstelle der M.P.G., GSttingen, 1952. D.J.C. Friend and O.N. Purvis, Ann. Bot., 27 (1963) 553. E. P~ldi and M. D~vay, Biochem. Physiol.Pflanzen., 171 (1977) 249. M. Ddvay, Biomedical Processes in Vernalisation. I, in: Symposium on Genetics and Wheat Breeding, S. Rajki (Ed.), Martonv~s~r, Hungary, 1962, p. 17. 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.
69
Elsevier Scientific Publishers Ireland Ltd.
THE CHANGES IN THE NATURE OF rRNA SYNTHESIS IN THE P L U M U L A O F SEEDLINGS D U R I N G V E R N A L I S A T I O N
EMIL PALDI and M A R T A
DI~VAY
Department of Plant Physiology and Biochemistry, Agricultural Research Institute, Hungarian Academy of Sciences,H-2462 Martonvasdr (Hungary)
(Received May 1st,1982) (Revision received October 20th, 1982) (Accepted October 22nd, 1982)
SUMMARY
The nature of r R N A synthesis in the plumula in the course of vernalisation at low temperature (3°C) was examined in four winter wheat varieties with different cold requirements (Triticum aestivum L. var. Bezostaya 1, Rannyaya 12, Mironovskaya 808 and B~nkuti 1201) using the pulse~hase technique. The presence of an r R N A synthesised at low temperature but decomposing at high temperature (25°C) was detected through its decomposition products, which have molecular weights of 1.05 X 106 and 0.57 X 106. It was found that the synthesis of this r R N A was dependent on the degree of vernalisation. It appears likely that the r R N A detected takes part in the specific metabolism of vernalisation.
Key words: r R N A synthesis -- Plumula -- Seedlings -- Vernalisation
INTRODUCTION
Plants will n o t flower, nor in m a n y cases respond to the environmental stimuli which ensure subsequent flowering, until they have reached a 'ripeness to flower'. Of exceptional interest in this request is the clarification of the process concerning the accelerated induction of 'ripeness to flower' by exposure to cold in cold-requiring plants such as winter cereals. This process, which ranks w i t h o u t d o u b t as one of the most interesting problems of mot-
Abbreviations: Pi, potassium orthophosphate. 0304-4211/83/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
70 phogenesis, is known as vernalisation. Vernalisation is essentially the acceleration of plant development due to cold effect. It is a prerequisite for flower formation and makes the plants 'capable of responding to the photoperiod'. In winter cereals the temperature most effective in vernalisation is at or near 0°C and the duration of the treatment for maximum acceleration of flower initiation ranges according to the variety from 1 week to 2 months. This time is called the cold requirement. The lower limit for the active vernalisation temperature is generally - 3 ° C and the upper limit 10°C [ 1 ]. Vernalisation can be reversed b y a period of high (25°--40°C) temperature immediately following vernalisation. This p h e n o m e n o n is called devernalisation. Devernalisation is n o t able to produce a permanent state. This is confirmed b y the renewed vernalisability at low temperature of plants devernalised by heat. This renewed vernalisation of devernalised plants is known as revernalisation [ 2,3 ]. The temperature range 10°--20°C is neutral, exhibiting no vernalisation or devernalisation effect. It is a significant paradox of vernalisation that an environmental condition, low temperature, which generally reduces the intensity of biochemical processes, exerts an accelerating effect on the biochemical processes forming part of vernalisation. Melchers [4] drew attention to the contradiction between the flower-inducing role of low temperature and the c o m m o n l y known temperature-dependence of biochemical reactions. He tried to remove the paradox by postulating a 3-fold reaction with various temperature dependences. In his opinion the vernalisation process can be described by the following formula: Q, olI
ql0 I'
precursor
: intermediate product
~ end p r o d u c t
I Q~oIII damage caused b y high temperature to the intermediate products Q10III > Q10II > Q10I A rough approximation of the value of Qlo was calculated from the temperature-dependences of vernalisation, devernalisation and revernalisation: Ql0III = 4.5, Q~0 = 2.5 and Ql0 I= 2.3 [5]. It must be assumed that vernalisation consists of numerous competing processes. All these processes, w i t h o u t exception, are of biochemical character, differing solely in their characteristic temperatures and in Q10 ' As a result of these temperature-dependences, the reactions taking place at low temperatures, active for vernalisation, are primarily those leading to flower formation, whereas at higher temperatures the conditions are more suitable for inhibitory or decomposing processes.
71 The end-product of vernalisation m a y include the existence of a certain 'flowering hormone', or of its precursor, or of a group of enzymes, which is able to synthesize the substances that make plants 'capable of responding' to the photoperiod. In analysing the role played b y nucleic acids in vernalisation, the tempera° ture-dependence of vernalisation, dissolved above, was used to examine the nature of r R N A synthesis as active, neutral and devernalisation temperatures. In our previous paper it was reported that vernalisation is preceded by a cold-induced increase in r R N A synthesis [6]. It is also reported that the R N A synthesis in winter wheat varieties at neutral and devernalisation temperatures is different from that at temperatures active for vernalisation. It was also confirmed that at temperatures active for vernalisation a type of r R N A was synthesized in the plumula which was n o t stable at high devernalisation temperatures. The decomposition of this rRNA could be traced by means of specific decomposition products with molecular weights of 1.05 X 106 and 0.57 X 106. The quantity of these decomposition products diminished due to the effect of revernalisation, i.e. renewed low temperature and within a few hours they could no longer be detected. The present paper is aimed at reporting results which give evidence of a connection between the above mentioned change in the nature of r R N A synthesis and the degree of vernalisation. MATERIALS AND METHODS
Chemicals 32Pi was purchased from the Institute of the Hungarian Academy of Sciences; sucrose from Merck; acrylamide and N,N,N',N'-methylene bisacrylamide from Serva. All other chemicals were of analytical grade and obtained from BDH. Plan t material Seedlings of Mironovskaya 808, B~nkuti 1201, Bezostaya 1 and Rannyaya 12 winter wheat (Triticum aestivum L.) were used in the experiment. The sterilised seeds were germinated at 20°C for 48 h in darkness on 1% agar-agar containing 2% sucrose and then vernalised at 0°C in darkness. At this temperature vernalis~/tion takes place at a normal rate, b u t cell division ceases [ 7]. The endosperm was removed after different vernalisation periods and the isolated seedlings were used for the examination of r R N A synthesis. Analyses were carried o u t weekly for the entire course of the process, in accordance with the cold requirement of the varieties. Different varieties have different cold requirements. For Mironovskaya 808, for example, the vernalisation requirement is 60 days, that of B~inkuti and Bezostaya 1 is 48 days, while that of R a n n y a y a 12 is 30 days at 0°C. Variants which received no cold treatments were considered to be unvernalised. Variants which were exposed to low temperatures for half the number of days needed to satisfy the cold requirement characteristic of the variety
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Fig. 1. rRNA synthesis in B~inkuti 1201 winter wheat seedlings during vernalisation. Experimental material: seedlings germinated in the dark for 48 h. Pulse incubation (A,C,E): 3 h at 25°C with 0.1 ml incubating solution (1000 × diluted Knop-solution + 2% sucrose + 100 ~Ci 32Pi) for each seedling. Chase incubation (B,D,F): 48 h at 3°C with 0.1 ml incubating solution containing 3 x Pi + 2% sucrose) per seedling. The fractionation of rRNAs took place on a 2.4% polyacrylamide gel (50 V, 6 mA per tube, 3.25 h). The evaluation was carried out with a Chromoscan microdensitometer at 265 nm. The histogram shows the radioactivity of the l-ram gel slices. Numbers above the peaks indicate molecular weights in millions of daltons.
.O
>
1,3
E
C~
74 were considered as half-vernalised, while the fully vernalised variants were those kept at low temperature long enough to completely satisfy the cold requirement. Data on the degree of vernalisation of all three variants are presented in the paper.
Evaluation of the nature o f rRNA synthesis The pulse and chase labelling procedure was applied for the examination of r R N A synthesis. For the pulse period 0.1 ml 1000× diluted Knop-solution per plant was used, containing 100 pCi radioactive potassium orthophosphate (Pi } and 2% sucrose per millilitre. The incubation was carried o u t for 3 h at 20°C. After the pulse period, half the seedlings were processed immediately, while the other half were washed with distilled water and 10 -3 M phosphate buffer and were thereafter transferred to Knop-solution containing 2% sucrose and incubated at 3°C for 48 h for use in the chase experiment. The nucleic acids were extracted at the end of the pulse and chase periods using a modified phenol m e t h o d [ 8]. Loening's m e t h o d of polyacrylamide gel electrophoresis [ 9] was used to separate the nucleic acids. Gels containing 2.4% acrylamide, 0.06% bis-acrylamide and 0.25% agarose were used in the electrophoresis, which ran for 3.25 h, using a current of 5--6 mA per tube. The nucleic acid c o n t e n t of the gels was evaluated at 265 nm in a Joyce-Loebl 'Chromoscan' microdensitometer. The radioactivity of the 1-mm gel slices, which were frozen with solid CO2, was determined using the liquid scintillation method. In order to identify the nucleic acids their molecular weights were determined using Loening's m e t h o d [ 10]. RESULTS AND DISCUSSION The data for unvernalised (A,B), half-vernalised (C,D) and fully vernalised {E,F) seedlings of B~nkuti 1201 winter wheat are presented in Fig. 1. The results obtained for seedlings of Mironovskaya 808, Bezostaya 1 and Rannyaya 12 winter wheats are n o t presented in a separate figure, since they are identical with those obtained for B~inkuti 1201. It may be seen from Fig. 1 that changes occur in the nature of r R N A synthesis during the course of vernalisation. During the initial and middle stages of vernalisation, pulse incubation (A,C} at the lower limit of the devernalisation temperature (25°C) results in t w o separate peaks in every case, from both heavy and light rRNAs. There is no absorption peak corresponding to the second peak either for heavy or for light rRNAs, which indicates that these fractions cannot be present in significant quantities. The molecular weights of the t w o specific fractions were found to be 1.05 X 106 and 0.57 X 106. In the determinations the r R N A of Escherichia coli was taken as the molecular weight standard [6]. These t w o specific fractions are considered to be the decomposition products of r R N A which is synthesised at low temperature (3°C) b u t decomposes at high temperature (25°C). The 1.05 X 106 and 0.57 X 106 dalton r R N A varieties disappear due to the
75 e f f e c t o f chase i n c u b a t i o n (B,D) carried o u t o n unvernalised and half-vernalised seedlings at t e m p e r a t u r e s active f o r vernalisation. H o w e v e r , t h e y are easily discernable a f t e r vernalisation in b o t h t h e pulse (E) a n d t h e chase (F) experiments. T h e a p p e a r a n c e o f 1.05 X 106 a n d 0.57 X 106 d a l t o n r R N A s in pulse e x p e r i m e n t s at t h e devernalisation t e m p e r a t u r e (A,C,E) and their disappearance d u r i n g chase e x p e r i m e n t s (B,D) carried o u t at t e m p e r a t u r e active for vernalisation shows a close c o r r e l a t i o n w i t h t h e c o l d t r e a t m e n t in all f o u r w i n t e r w h e a t s investigated a n d also p o i n t s t o a c h a n g e in t h e r R N A metabolites. F u r t h e r analysis o f t h e origin a n d role o f t h e 1.05 X 106 a n d 0.57 X 106 d a l t o n r R N A varieties is n o w in progress. REFERENCES 1 2 3 4 5 6 7 8 9 10
V.I. Razumov, Izd. Selskohoz. Lit., Leningrad-Moscow, 1961, p. 62. F.G. Gregory and O.N. Purvis, Nature (Lond.), 155 (1945) 113. F.G. Gregory and O.N. Purvis, Nature (Lond.), 161 (1948) 859. G. Melchers, The Physiology of Flower Initiation, Dokumentumstelle der M.P.G., GSttingen, 1952. D.J.C. Friend and O.N. Purvis, Ann. Bot., 27 (1963) 553. E. P~ldi and M. D~vay, Biochem. Physiol.Pflanzen., 171 (1977) 249. M. Ddvay, Biomedical Processes in Vernalisation. I, in: Symposium on Genetics and Wheat Breeding, S. Rajki (Ed.), Martonv~s~r, Hungary, 1962, p. 17. 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.