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Changes in adenylate energy pool and energy charge of developing and germinated rice embryos
Plant Science Letters, 21 (1981) 357--360 © Elsevier/North-Holland Scientific Publishers Ltd.
357
CHANGES IN ADENYLATE ENERGY POOL AND ENERGY CHARGE OF DEVELOPING AND GERMINATED RICE EMBRYOS
K. BARUA, M.M. CHOUDHURI and B. GHOSH*
Department of Botany, Plant Physiology Laboratory, Bose Institute, Calcutta 700009 West Bengal (India) (Received September 11th, 1980) (Revision received November 24th, 1980) (Accepted February 9th, 1981)
SUMMARY
A study of the energy state and the pattern of adenosine phosphates was conducted in the developing and germinating rice embryos. After 16 days of pollination, a peak value of adenylate energy pool was obtained which then showed a gradual decline and the energy charge (EC) varied between 0.7 and 0.8. Total energy pool was m a x i m u m after 24-h germination of embryos and ATP level increased 14-fold compared to the initial value of the dry embryo. But the m a x i m u m value of ADP and AMP was obtained after 72 h of germination and EC value fluctuated from 0.5 to 0.9 during the germination period.
INTRODUCTION
Developing seeds require a tremendous supply of ATP for the biosynthesis of various seed components and particularly for the accumulation of RNA with the cell proliferation [1]. The content of ATP or adenylate energy supply and energy charge often control metabolic activity and growth [2--4]. As the synthesis of proteins and nucleic acids are the major events in embryonic tissue, ATP is also in great demand during germination [ 5,6]. In addition to ATP, levels of ADP and AMP may also play a key role. Both of these nucleotides decrease in wheat embryo after the first 4 h of imbibition, at a time when protein synthesis is increasing markedly [6]. Similarly, in both pea and lettuce seeds, there is depletion of AMP coincident with ATP synthesis and ADP level remaining constant [3,7]. But the knowledge regarding the nucleotide pool in developing seeds is rather limited. This
*To whom reprint requests should be made.
358
paper reports the changes in the pattern of adenylate nucleotides during development and germination of rice seeds. MATERIALS AND METHODS Winter variety of rice (Oryza sativa L.), cv Rupsail, was grown during the Kharif season in the field of Bose Institute, Calcutta. Collection of the developing seeds was started just after pollination and continued at 4
Germination o f seed After surface sterilization with 0.1% HgC12, the seeds were allowed to germinate in dark at 37 -+ 1°C on moistened filter paper in petri dish and adenylate nucleotides have been determined in embryonic axis at 0--5 days after germination.
Determination of adenyl nucleotides Adenosine phosphates were extracted according to the method of Ching et al. [1]. One gram of developing seeds of rice embryos were homogenized with 0.25 N chilledperchloricacid, centrifugedat 10 000 × g for 10 min and the supernatant collected.The process was repeated twice to ensure complete extractioh of nucleotides.Supernatant was then neutralized with ( N ) K O H and centrifugedto remove precipitate.From the supernatant ATP, A D P and A M P were measured spectrofluorometrically(excitation 340 nm, emission 465 nm) using hexokinase, pyruvate kinase and myokinase respectivelyaccording to the method of Williamson and Corkey [8]. Energy charge was calculatedaccording to the definitionof Atkinson [2]. RESULTS AND DISCUSSIONS
The data in Fig. 1 show that the totalenergy pool as measured by total adenosine phosphates increased gradually during the development of seeds, having a peak value after 16 days of pollination (milky stage) and then gradually decreased. After 4 days of pollinationan average of 107 nmol of ATP, 8.6 nmol of A D P and 13.3 nmol of A M P (nmol/g wet wt.) were obtained. At the milky stage these values became 193 nmol (ATP), 82 nmol (ADP) and 6.8 nmol AMP, respectively,which signify that A T P level is found to be doubled, A D P remains nearly constant and A M P levelreduces to half of the initial value at this particular stage. After 16 days both ATP and ADP levels reduced whereas AMP remained the same and the total energy pool also followed the s ~ pattern. The pattern of rise and fall is correlated with the cellular activities of the seeds which was also observed in developing wheat [9] and rape seed [1,9]. Atkinson [ 2] proposed the concept of energy charge (EC) as an overall measure of the energy state of the cell. EC modulates the activity of various
359
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=o
300 I
~,.>, ,., ,-, 2001
¢0"8
z~
Iso
0"6
P,~ ioc
~0"4 0"2
E
=
~ 4
0
8
~ 12
16
20
24
NO. OF DAYS AFTER POLLINATION Fig. 1. Changes in adenylate energy pool and energy charge during development of rice seeds. Total adenylate phosphates (s), ATP (o), ADP (~), AMP ([]), E C (×).
S ~2oo ~ ~ iooo 0"8
800
(0.6
~ 400
0"4
ifoo
0"2 I
24 48 72 96 HOURS OF GERMINATION
120
Fig. 2. Changes in adenylate energy pool and energy charge during germination of rice seeds. Total adenylate phosphates (s), ATP (o), ADP (4), AMP (o), EC (×). °
360
metabolic sequences related to energy utilization and regeneration. Generally when EC is greater than 0.5 ATP utilizing systems increase their activities. Multiplying and growing bacterial cells maintain a high EC o f around 0.8, senescent cells have a low EC o f 0.5 [4]. High EC value of 0.7--0.8 in developing rice seeds supports the fact that the energy level is directly correlated with protein synthesis and cellular processes [10]. In rice seeds energy pool is directly related with the germination process (Fig. 2). In dry e m b r y o s 227 nmol of total adenosine phosphates were observed of which ATP was 76 nmol, ADP 107 nmol and AMP 44 nmol. During 24 h germination period the ATP level increased b y 14-fold {1050 nmol), ADP showed no significant change b u t AMP reduced to 13 nmol and EC was remarkably increased to 0.9. High value o f EC is related to increased synthesis o f mitochondria in the newly formed cells and respiration [ 11 ]. Despite the large increase in the ATP c o n t e n t of the seeds, the ADP showed no p r o f o u n d change and it implies the existence of a mechanism for replacing ADP lost by phosphorylation to ATP. Present c o n c e p t implicating ADP concentration as the controlling factor of tissue respiration [12] suggests that this mechanism b y preventing depletion of ADP is a key factor in maintaining the rise o f respiratory rate during initiation of germination. The close correlation b e t w e e n the decreasing c o n t e n t of AMP and the formation of ATP justifies the mechanism of the phosphorylation o f AMP. After 48 h of germination, ATP level gradually decreased by ADP showed an increase which declined after 72 h. AMP level increased 2-fold (90 nmol) at the f o r t y ~ i g h t h hour and decreased after 72 h. The increase of ADP and AMP obviously signifies the metabolism o f synthesised ATP. After 96 h of germination with decrease of EC value from 0.9 to 0.6, the different nucleotide values also reduced which proves that the activities of all cellular processes begin to decline during the period. REFERENCES 1 T.M. Ching, J.M. Crane and D.L. Stamp, Plant Physiol., 54 (1974) 748. 2 D.E. Atkinson, Ann. Rev. Microbiol., 23 (1969) 47. 3 A. Pradet, A. Naryanan and J. Vermeersch., Bull. Soc. Fr. Physiol. Veg., 14 (1968) 107. 4 A.G. Chapman, L. Fall and D.E. Atkinson, J. Bactariol., 108 (1971) 1072. 5 T.M. Ching and K.K. Chin$, Plant Physiol., 50 (1972) 536. 6 A. Marcus, Syrup. Soc. Exp. Biol., 23 (1969) 143. 7 E.G. Brown, Biochem. J., 95 (1965) 509. 8 J.R. Williamson and B.E. Corkey, Methods Enzymol., 13 (1969) 434. 9 C.F. Jenner, Plant Physiol., 43 (1968) 41. 10 D2. Nierlich, Science, 184 (1974) 1043. 11 W. Stiles, in: W. Ruhlanch (Ed.), Encyclopaedia of Plant Physiol., Vol. 2, part 2, Springer Verlag, Berlin, 1960, p. 465. 12 B. Chance and G.R. Williams, Adv. Enzymol., 17 (1954) 65.
357
CHANGES IN ADENYLATE ENERGY POOL AND ENERGY CHARGE OF DEVELOPING AND GERMINATED RICE EMBRYOS
K. BARUA, M.M. CHOUDHURI and B. GHOSH*
Department of Botany, Plant Physiology Laboratory, Bose Institute, Calcutta 700009 West Bengal (India) (Received September 11th, 1980) (Revision received November 24th, 1980) (Accepted February 9th, 1981)
SUMMARY
A study of the energy state and the pattern of adenosine phosphates was conducted in the developing and germinating rice embryos. After 16 days of pollination, a peak value of adenylate energy pool was obtained which then showed a gradual decline and the energy charge (EC) varied between 0.7 and 0.8. Total energy pool was m a x i m u m after 24-h germination of embryos and ATP level increased 14-fold compared to the initial value of the dry embryo. But the m a x i m u m value of ADP and AMP was obtained after 72 h of germination and EC value fluctuated from 0.5 to 0.9 during the germination period.
INTRODUCTION
Developing seeds require a tremendous supply of ATP for the biosynthesis of various seed components and particularly for the accumulation of RNA with the cell proliferation [1]. The content of ATP or adenylate energy supply and energy charge often control metabolic activity and growth [2--4]. As the synthesis of proteins and nucleic acids are the major events in embryonic tissue, ATP is also in great demand during germination [ 5,6]. In addition to ATP, levels of ADP and AMP may also play a key role. Both of these nucleotides decrease in wheat embryo after the first 4 h of imbibition, at a time when protein synthesis is increasing markedly [6]. Similarly, in both pea and lettuce seeds, there is depletion of AMP coincident with ATP synthesis and ADP level remaining constant [3,7]. But the knowledge regarding the nucleotide pool in developing seeds is rather limited. This
*To whom reprint requests should be made.
358
paper reports the changes in the pattern of adenylate nucleotides during development and germination of rice seeds. MATERIALS AND METHODS Winter variety of rice (Oryza sativa L.), cv Rupsail, was grown during the Kharif season in the field of Bose Institute, Calcutta. Collection of the developing seeds was started just after pollination and continued at 4
Germination o f seed After surface sterilization with 0.1% HgC12, the seeds were allowed to germinate in dark at 37 -+ 1°C on moistened filter paper in petri dish and adenylate nucleotides have been determined in embryonic axis at 0--5 days after germination.
Determination of adenyl nucleotides Adenosine phosphates were extracted according to the method of Ching et al. [1]. One gram of developing seeds of rice embryos were homogenized with 0.25 N chilledperchloricacid, centrifugedat 10 000 × g for 10 min and the supernatant collected.The process was repeated twice to ensure complete extractioh of nucleotides.Supernatant was then neutralized with ( N ) K O H and centrifugedto remove precipitate.From the supernatant ATP, A D P and A M P were measured spectrofluorometrically(excitation 340 nm, emission 465 nm) using hexokinase, pyruvate kinase and myokinase respectivelyaccording to the method of Williamson and Corkey [8]. Energy charge was calculatedaccording to the definitionof Atkinson [2]. RESULTS AND DISCUSSIONS
The data in Fig. 1 show that the totalenergy pool as measured by total adenosine phosphates increased gradually during the development of seeds, having a peak value after 16 days of pollination (milky stage) and then gradually decreased. After 4 days of pollinationan average of 107 nmol of ATP, 8.6 nmol of A D P and 13.3 nmol of A M P (nmol/g wet wt.) were obtained. At the milky stage these values became 193 nmol (ATP), 82 nmol (ADP) and 6.8 nmol AMP, respectively,which signify that A T P level is found to be doubled, A D P remains nearly constant and A M P levelreduces to half of the initial value at this particular stage. After 16 days both ATP and ADP levels reduced whereas AMP remained the same and the total energy pool also followed the s ~ pattern. The pattern of rise and fall is correlated with the cellular activities of the seeds which was also observed in developing wheat [9] and rape seed [1,9]. Atkinson [ 2] proposed the concept of energy charge (EC) as an overall measure of the energy state of the cell. EC modulates the activity of various
359
¢/)
=o
300 I
~,.>, ,., ,-, 2001
¢0"8
z~
Iso
0"6
P,~ ioc
~0"4 0"2
E
=
~ 4
0
8
~ 12
16
20
24
NO. OF DAYS AFTER POLLINATION Fig. 1. Changes in adenylate energy pool and energy charge during development of rice seeds. Total adenylate phosphates (s), ATP (o), ADP (~), AMP ([]), E C (×).
S ~2oo ~ ~ iooo 0"8
800
(0.6
~ 400
0"4
ifoo
0"2 I
24 48 72 96 HOURS OF GERMINATION
120
Fig. 2. Changes in adenylate energy pool and energy charge during germination of rice seeds. Total adenylate phosphates (s), ATP (o), ADP (4), AMP (o), EC (×). °
360
metabolic sequences related to energy utilization and regeneration. Generally when EC is greater than 0.5 ATP utilizing systems increase their activities. Multiplying and growing bacterial cells maintain a high EC o f around 0.8, senescent cells have a low EC o f 0.5 [4]. High EC value of 0.7--0.8 in developing rice seeds supports the fact that the energy level is directly correlated with protein synthesis and cellular processes [10]. In rice seeds energy pool is directly related with the germination process (Fig. 2). In dry e m b r y o s 227 nmol of total adenosine phosphates were observed of which ATP was 76 nmol, ADP 107 nmol and AMP 44 nmol. During 24 h germination period the ATP level increased b y 14-fold {1050 nmol), ADP showed no significant change b u t AMP reduced to 13 nmol and EC was remarkably increased to 0.9. High value o f EC is related to increased synthesis o f mitochondria in the newly formed cells and respiration [ 11 ]. Despite the large increase in the ATP c o n t e n t of the seeds, the ADP showed no p r o f o u n d change and it implies the existence of a mechanism for replacing ADP lost by phosphorylation to ATP. Present c o n c e p t implicating ADP concentration as the controlling factor of tissue respiration [12] suggests that this mechanism b y preventing depletion of ADP is a key factor in maintaining the rise o f respiratory rate during initiation of germination. The close correlation b e t w e e n the decreasing c o n t e n t of AMP and the formation of ATP justifies the mechanism of the phosphorylation o f AMP. After 48 h of germination, ATP level gradually decreased by ADP showed an increase which declined after 72 h. AMP level increased 2-fold (90 nmol) at the f o r t y ~ i g h t h hour and decreased after 72 h. The increase of ADP and AMP obviously signifies the metabolism o f synthesised ATP. After 96 h of germination with decrease of EC value from 0.9 to 0.6, the different nucleotide values also reduced which proves that the activities of all cellular processes begin to decline during the period. REFERENCES 1 T.M. Ching, J.M. Crane and D.L. Stamp, Plant Physiol., 54 (1974) 748. 2 D.E. Atkinson, Ann. Rev. Microbiol., 23 (1969) 47. 3 A. Pradet, A. Naryanan and J. Vermeersch., Bull. Soc. Fr. Physiol. Veg., 14 (1968) 107. 4 A.G. Chapman, L. Fall and D.E. Atkinson, J. Bactariol., 108 (1971) 1072. 5 T.M. Ching and K.K. Chin$, Plant Physiol., 50 (1972) 536. 6 A. Marcus, Syrup. Soc. Exp. Biol., 23 (1969) 143. 7 E.G. Brown, Biochem. J., 95 (1965) 509. 8 J.R. Williamson and B.E. Corkey, Methods Enzymol., 13 (1969) 434. 9 C.F. Jenner, Plant Physiol., 43 (1968) 41. 10 D2. Nierlich, Science, 184 (1974) 1043. 11 W. Stiles, in: W. Ruhlanch (Ed.), Encyclopaedia of Plant Physiol., Vol. 2, part 2, Springer Verlag, Berlin, 1960, p. 465. 12 B. Chance and G.R. Williams, Adv. Enzymol., 17 (1954) 65.