Planta (1981)152:336 340
P I ~ I J H ' ~ 9 Springer-Verlag 1981
Uptake of [2-14C]abscisic acid and distribution of 14C in apple embryos P. Barthe and C. Bulard Laboratoire de Physiologic V6g+tale, Universit6 de Nice, 28 Avenue Valrose, F-06034 NICE Cedex, France
Abstract. Pyrus malus L. var. Golden delicious em-
bryos were incubated with (+)-[2-14C]abscisic acid (ABA) (10- 5 M, 355 kBq pmol- 1). After incubations of various durations, the radioactivity was measured in whole embryos, cotyledons, and embryonic axes. With either 48-h or 16-d incubation periods, the uptake of [14C]ABA depended upon the mode of culture used. The lowest values corresponded to the absorption by the embryonic axis, the highest to the absorption by the distal parts of the two cotyledons. The cotyledons accumulated the main part of the radioactivity under all conditions. Dormant and almost completely after-ripened embryos cultivated for 4 d showed no significant differences in the radioactivity uptake for identical modes of culture. There was a linear relationship between exogenous ABA concentrations (0.5 to 3.10 -5 M) and ABA uptake for embryos cultivated for 4 d with the distal parts of the cotyledons immersed in the medium. Key words: Abscisic acid - Cotyledons - Embryonic axis - Embryo culture Pyrus.
Introduction
Apple embryos have often been used as a model in experiments dealing with embryo dormancy, on the one hand to define more precisely the role played by abscisic acid (ABA) and on the other hand to reach a better understanding of the nature of the interacAbbreviations: A B A = a b s c i s i c acid. R M , R M +, C/2 M, and C M are different modes of embryo cultures : embryonic axis immersed alone (RM), together with the proximal parts of the cotyledons ( R M +); distal parts of the cotyledons immersed alone (CM); embryo flat on the medium, the root and the external surface of one cotyledon being in contact with the medium (C/2 M). P P = proximal parts of the cotyledons; D P = d i s t a l parts of the cotyledons
0032-0935/81/0152/0336/$01.00
tions demonstrated between embryonic axis and cotyledons. The supply of exogenous ABA to partially or completely after-ripened embryos cultivated in vitro induced the inhibition of germination. The longer the after-ripening treatment, the higher must be the ABA concentration applied to induce a secondary dormancy (Rudnicki 1969; Kaminski et al. 1971; Rudnicki et al. 1971). In 1975, Durand et al. proved the existence of complex interactions between embryonic axes and cotyledons in after-ripened embryos induced into dormancy by ABA. In the present work, using dormant as well as almost completely after-ripened apple embryos, we analyzed the results concerning the uptake kinetics of [14C]ABA and the distribution of the radioactivity between cotyledons and embryonic axis. We took into account the results previously obtained with in vitro cultures of apple embryos showing the important influence of the mode of culture on the further behavior of the embryos (Thevenot and C6me 1971 ; Thevenot and C6me 1973 a and b; Le Page-Degivry and Bulard 1979; Thevenot 1980). Our study showed that cotyledons and embryonic axis offered very different potentialities as regards the uptake of [14C]ABA as well as the accumulation of the radioactivity. Material and methods We used Pyrus malus L. var. Golden delicious embryos. The treatments applied to the fruits before the isolation of the embryos were: - 12 m o n t h s at 4 ~ (lot A and lot D, respectively 1978 and 1979 harvests) - or 4 m o n t h s at 14-18 ~ C (lot B, 1979 harvest). Lot C embryos were isolated from the fruits at harvest time (1980 harvest). Figure 1, which shows the curves of germination of the isolated embryos cultivated on water at 23 ~ C, allows us to appreciate comparatively the depth of dormancy of the different lots. The germination curve of lot E, for completely after-ripened embryos, is given as a reference (the seeds isolated from the fruits at harvest time were treated for three m o n t h s at 4 ~ C in wet vermiculite).
P. Barthe and C. Bulard: Abscisic acid in apple embryos
100
~
E
~
A quenching correction curve was established by counting a series of standard samples of known activity and varying degrees of quenching. This curve was used for correcting counting efficiencbs of samples. Each point represents the mean of six (lots A and B) and of ten samples (lots C and D).
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Results and discussion
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Fig. 1. Germination percentages of apple embryos (C/2 M) cultivated on filter paper imbibed with water at 23~ C under fluorescent light (16 h a day, 4,000 lx at the embryos level)9 The embryos were isolated: - from fruits which had been maintained 12 months at 4~ (lot A, 1978 harvest, lot D, 1979 harvest) or 4 months at 14 18~ (lot B, 1979 harvest), from seeds which had been after-ripened 3 months at 4~ C (lot E, 1978 harvest). Lot C embryos were isolated from the fruits at harvest time (1980 harvest)
After having extracted the seeds from the fruits, the embryos were immmediately isolated with no previous imbibition of the seeds. As soon as each embryo was isolated from the seed, it was introduced into a polyethylene tube (65.10 ram) containing 0.2 ml of water agar (60/00 agar) and (+)-[2-i4C]cis abscisic acid (specific activity 355 kBq gmol-1) purchased from Radiochemical, Amersham, England_ The contact between embryo and medium was obtained in four different ways: only the root was immersed (RM), the root as well as the proximal part of the two cotyledons - about one-third of their total length , were immersed (RM +), - the embryo was laid flat on the medium, the root and the whole of the external surface of one cotyledon being in contact with the medium (C/2 M), the distal part of both cotyledons were immersed in the medium to one-third their total lengths (CM). In this last case we studied the influence of inverting the position of the tubes during the experiment9 That is to say we compared embryos in upright and inverted positions. We used whole embryos in all experiments but one. In this particular case, only embryonic axes which had been isolated from the cotyledons were used. To study the time course of uptake the chosen concentration of [14C]ABAwas 10-5 M. In special four-day experiments on CM embryos we studied the [14C]ABAuptake in relation to its concentration in the medium (0.5 to 3.10 5 M). At the temperature chosen (23~ C), these ABA treatments inhibited completely the germination of the embryos, whatever their origin, at least till the end of our experiments. All the culture tubes, which had been plugged with cotton wool, were maintained in darkness at 23~ C. B, C, and D cultures were maintained in a water-saturated atmosphere. After culture, the embryos were removed from the tubes and dried on filter paper. RM, RM +, and CM embryos were immediately divided into embryonic axes and cotyledons. In some experiments the cotyledons were divided into two parts : proximal, near the root (PP), and distal, far from the root (DP). The material was crushed in the presence of sand and 80% ethanol (twice 0.5 ml) in a polyethylene tube (65-10 mm). The supernatants, 1 ml, were counted in 3 ml scintillation fluid (Bray) using a SL 32 Intertechnique Scintillation Liquid Spectrometer.
[i4C]ABA uptake by whole embryos. The present study b r o u g h t to evidence i m p o r t a n t q u a n t i t a t i v e differences c o n c e r n i n g [14C]ABA u p t a k e by the whole e m b r y o in relation to the m o d e of culture used (Fig. 2 lot A, 48 h uptake). The lowest values c o r r e s p o n d e d to the a b s o r p t i o n b y the e m b r y o n i c axis alone ( R M ) , whereas the highest c o r r e s p o n d e d to the a b s o r p t i o n by the distal parts of the cotyledons (CM), C/2 M a p p e a r i n g as an intermediate. O n the other h a n d , there was n o difference between C M cultures m a i n tained in u p r i g h t or in inverted positions. W e noticed b u t little difference between R M a n d R M +, the latter showing slightly higher values. The proximal parts of the cotyledons were then far less efficient t h a n the distal parts, as far as [ ' 4 C ] A B A u p t a k e was concerned. I n l o n g - t e r m studies (Table 1 lot B, 8 a n d 16 d uptake), the differences between the three m o d e s of culture ( R M , C/2 M a n d C M ) increased considerably. In the course of these l o n g - t e r m experiments the possibility of a partial m e t a b o l i s m of the A B A in the m e d i u m c a n n o t be excluded. A c o m p a r a t i v e study, c o n d u c t e d with d o r m a n t (lot C) a n d with almost completely after-ripened (lot D) embryos, showed no differences in the [14C]ABA u p t a k e after 4 days culture, in C M as well in R M modes of culture (Table 2). There was a linear relationship between exogenous [I~C]ABA c o n c e n t r a t i o n s (0.5 to 3 . 1 0 - 5 M ) a n d [i4C]ABA u p t a k e by whole e m b r y o s cultivated 4 days in C M m o d e of culture (lot D). The slope of the regression line was 0.87 with a value of 0.99 for the regression coefficient. The e q u a t i o n of the regression line, for the c o n c e n t r a t i o n s used, was Y = 0 . 8 7 X - 5 . 2 1 , where X represented the l o g a r i t h m of the ABA concentration. Radioactivity distribution between organs Embryonic axes. In short-term experiments (48 h, lot A, Fig. 3), as well as in l o n g - t e r m (8 a n d 16 d, lot B, T a b l e 1), the a m o u n t of radioactivity a c c u m u l a t e d in the axes was always very low c o m p a r e d to that of the whole embryos. The a m o u n t of [x'~C]ABA accum u l a t e d was however sufficient to induce a n d m a i n tain the growth i n h i b i t i o n of the roots in lot A embryos which had been almost completely after-ripened. To u n d e r s t a n d the reason for this, one m u s t
338
P. Barthe and C. Bulard: Abscisic acid in apple embryos
E ~0~ 0
5 E 30C O. 0
3,ooc m
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P I ~ I J H ' ~ 9 Springer-Verlag 1981
Uptake of [2-14C]abscisic acid and distribution of 14C in apple embryos P. Barthe and C. Bulard Laboratoire de Physiologic V6g+tale, Universit6 de Nice, 28 Avenue Valrose, F-06034 NICE Cedex, France
Abstract. Pyrus malus L. var. Golden delicious em-
bryos were incubated with (+)-[2-14C]abscisic acid (ABA) (10- 5 M, 355 kBq pmol- 1). After incubations of various durations, the radioactivity was measured in whole embryos, cotyledons, and embryonic axes. With either 48-h or 16-d incubation periods, the uptake of [14C]ABA depended upon the mode of culture used. The lowest values corresponded to the absorption by the embryonic axis, the highest to the absorption by the distal parts of the two cotyledons. The cotyledons accumulated the main part of the radioactivity under all conditions. Dormant and almost completely after-ripened embryos cultivated for 4 d showed no significant differences in the radioactivity uptake for identical modes of culture. There was a linear relationship between exogenous ABA concentrations (0.5 to 3.10 -5 M) and ABA uptake for embryos cultivated for 4 d with the distal parts of the cotyledons immersed in the medium. Key words: Abscisic acid - Cotyledons - Embryonic axis - Embryo culture Pyrus.
Introduction
Apple embryos have often been used as a model in experiments dealing with embryo dormancy, on the one hand to define more precisely the role played by abscisic acid (ABA) and on the other hand to reach a better understanding of the nature of the interacAbbreviations: A B A = a b s c i s i c acid. R M , R M +, C/2 M, and C M are different modes of embryo cultures : embryonic axis immersed alone (RM), together with the proximal parts of the cotyledons ( R M +); distal parts of the cotyledons immersed alone (CM); embryo flat on the medium, the root and the external surface of one cotyledon being in contact with the medium (C/2 M). P P = proximal parts of the cotyledons; D P = d i s t a l parts of the cotyledons
0032-0935/81/0152/0336/$01.00
tions demonstrated between embryonic axis and cotyledons. The supply of exogenous ABA to partially or completely after-ripened embryos cultivated in vitro induced the inhibition of germination. The longer the after-ripening treatment, the higher must be the ABA concentration applied to induce a secondary dormancy (Rudnicki 1969; Kaminski et al. 1971; Rudnicki et al. 1971). In 1975, Durand et al. proved the existence of complex interactions between embryonic axes and cotyledons in after-ripened embryos induced into dormancy by ABA. In the present work, using dormant as well as almost completely after-ripened apple embryos, we analyzed the results concerning the uptake kinetics of [14C]ABA and the distribution of the radioactivity between cotyledons and embryonic axis. We took into account the results previously obtained with in vitro cultures of apple embryos showing the important influence of the mode of culture on the further behavior of the embryos (Thevenot and C6me 1971 ; Thevenot and C6me 1973 a and b; Le Page-Degivry and Bulard 1979; Thevenot 1980). Our study showed that cotyledons and embryonic axis offered very different potentialities as regards the uptake of [14C]ABA as well as the accumulation of the radioactivity. Material and methods We used Pyrus malus L. var. Golden delicious embryos. The treatments applied to the fruits before the isolation of the embryos were: - 12 m o n t h s at 4 ~ (lot A and lot D, respectively 1978 and 1979 harvests) - or 4 m o n t h s at 14-18 ~ C (lot B, 1979 harvest). Lot C embryos were isolated from the fruits at harvest time (1980 harvest). Figure 1, which shows the curves of germination of the isolated embryos cultivated on water at 23 ~ C, allows us to appreciate comparatively the depth of dormancy of the different lots. The germination curve of lot E, for completely after-ripened embryos, is given as a reference (the seeds isolated from the fruits at harvest time were treated for three m o n t h s at 4 ~ C in wet vermiculite).
P. Barthe and C. Bulard: Abscisic acid in apple embryos
100
~
E
~
A quenching correction curve was established by counting a series of standard samples of known activity and varying degrees of quenching. This curve was used for correcting counting efficiencbs of samples. Each point represents the mean of six (lots A and B) and of ten samples (lots C and D).
9 D
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Results and discussion
(5
9
o
5
i
10
.
.
.
.
i
15
.
.
.
.
i
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.
.
.
20
i
25 Days
Fig. 1. Germination percentages of apple embryos (C/2 M) cultivated on filter paper imbibed with water at 23~ C under fluorescent light (16 h a day, 4,000 lx at the embryos level)9 The embryos were isolated: - from fruits which had been maintained 12 months at 4~ (lot A, 1978 harvest, lot D, 1979 harvest) or 4 months at 14 18~ (lot B, 1979 harvest), from seeds which had been after-ripened 3 months at 4~ C (lot E, 1978 harvest). Lot C embryos were isolated from the fruits at harvest time (1980 harvest)
After having extracted the seeds from the fruits, the embryos were immmediately isolated with no previous imbibition of the seeds. As soon as each embryo was isolated from the seed, it was introduced into a polyethylene tube (65.10 ram) containing 0.2 ml of water agar (60/00 agar) and (+)-[2-i4C]cis abscisic acid (specific activity 355 kBq gmol-1) purchased from Radiochemical, Amersham, England_ The contact between embryo and medium was obtained in four different ways: only the root was immersed (RM), the root as well as the proximal part of the two cotyledons - about one-third of their total length , were immersed (RM +), - the embryo was laid flat on the medium, the root and the whole of the external surface of one cotyledon being in contact with the medium (C/2 M), the distal part of both cotyledons were immersed in the medium to one-third their total lengths (CM). In this last case we studied the influence of inverting the position of the tubes during the experiment9 That is to say we compared embryos in upright and inverted positions. We used whole embryos in all experiments but one. In this particular case, only embryonic axes which had been isolated from the cotyledons were used. To study the time course of uptake the chosen concentration of [14C]ABAwas 10-5 M. In special four-day experiments on CM embryos we studied the [14C]ABAuptake in relation to its concentration in the medium (0.5 to 3.10 5 M). At the temperature chosen (23~ C), these ABA treatments inhibited completely the germination of the embryos, whatever their origin, at least till the end of our experiments. All the culture tubes, which had been plugged with cotton wool, were maintained in darkness at 23~ C. B, C, and D cultures were maintained in a water-saturated atmosphere. After culture, the embryos were removed from the tubes and dried on filter paper. RM, RM +, and CM embryos were immediately divided into embryonic axes and cotyledons. In some experiments the cotyledons were divided into two parts : proximal, near the root (PP), and distal, far from the root (DP). The material was crushed in the presence of sand and 80% ethanol (twice 0.5 ml) in a polyethylene tube (65-10 mm). The supernatants, 1 ml, were counted in 3 ml scintillation fluid (Bray) using a SL 32 Intertechnique Scintillation Liquid Spectrometer.
[i4C]ABA uptake by whole embryos. The present study b r o u g h t to evidence i m p o r t a n t q u a n t i t a t i v e differences c o n c e r n i n g [14C]ABA u p t a k e by the whole e m b r y o in relation to the m o d e of culture used (Fig. 2 lot A, 48 h uptake). The lowest values c o r r e s p o n d e d to the a b s o r p t i o n b y the e m b r y o n i c axis alone ( R M ) , whereas the highest c o r r e s p o n d e d to the a b s o r p t i o n by the distal parts of the cotyledons (CM), C/2 M a p p e a r i n g as an intermediate. O n the other h a n d , there was n o difference between C M cultures m a i n tained in u p r i g h t or in inverted positions. W e noticed b u t little difference between R M a n d R M +, the latter showing slightly higher values. The proximal parts of the cotyledons were then far less efficient t h a n the distal parts, as far as [ ' 4 C ] A B A u p t a k e was concerned. I n l o n g - t e r m studies (Table 1 lot B, 8 a n d 16 d uptake), the differences between the three m o d e s of culture ( R M , C/2 M a n d C M ) increased considerably. In the course of these l o n g - t e r m experiments the possibility of a partial m e t a b o l i s m of the A B A in the m e d i u m c a n n o t be excluded. A c o m p a r a t i v e study, c o n d u c t e d with d o r m a n t (lot C) a n d with almost completely after-ripened (lot D) embryos, showed no differences in the [14C]ABA u p t a k e after 4 days culture, in C M as well in R M modes of culture (Table 2). There was a linear relationship between exogenous [I~C]ABA c o n c e n t r a t i o n s (0.5 to 3 . 1 0 - 5 M ) a n d [i4C]ABA u p t a k e by whole e m b r y o s cultivated 4 days in C M m o d e of culture (lot D). The slope of the regression line was 0.87 with a value of 0.99 for the regression coefficient. The e q u a t i o n of the regression line, for the c o n c e n t r a t i o n s used, was Y = 0 . 8 7 X - 5 . 2 1 , where X represented the l o g a r i t h m of the ABA concentration. Radioactivity distribution between organs Embryonic axes. In short-term experiments (48 h, lot A, Fig. 3), as well as in l o n g - t e r m (8 a n d 16 d, lot B, T a b l e 1), the a m o u n t of radioactivity a c c u m u l a t e d in the axes was always very low c o m p a r e d to that of the whole embryos. The a m o u n t of [x'~C]ABA accum u l a t e d was however sufficient to induce a n d m a i n tain the growth i n h i b i t i o n of the roots in lot A embryos which had been almost completely after-ripened. To u n d e r s t a n d the reason for this, one m u s t
338
P. Barthe and C. Bulard: Abscisic acid in apple embryos
E ~0~ 0
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