Planta 9 by Springer-Verlag 1980
Planta 149, 27 33 (1980)
Polyploidy and Aspartate-Transcarbamylase Activity in Hippocrepis comosa L. Monique Guern and Guy Herv6 Laboratoire de Taxonomie Exp~rimentaleet Num6rique de la Facult6 des Sciences, associg au C.N.R.S., Bfit, 362, F-01405 Orsay, and Laboratoire d'Enzymologie,Centre National de la RechercheScientifique,F-91190 Gif-sur-Yvette, France
Abstract. The D N A content of plants which were
sampled in natural di-, tetra- and hexaploid populations of Hippocrepis comosa L. was estimated and the aspartate transcarbamylase activities of the corresponding cell-free extracts were compared. The amount of D N A is not exactly proportional to the number of genomes. The three kinds of populations do not differ in their aspartate transcarbamylase specific activity. While the enzyme properties are identical in the extracts derived from the diploid and hexaploid plants, the aspartate transcarbamylase present in the tetraploid cytotype shows a slightly lower affinity for one of its substrates and a significantly lower sensitivity to the feedback inhibitor UTP which is still observed after partial purification. These properties might be related to the previously reported greater ability of the tetraploid cytotype to adapt to a variety of biotopes. Key words: Aspartate transcarbamylase - Hippocrepis - Polyploidy.
Introduction
Polyploidy, or multiplication of the entire chromosome set, is widely observed among higher plants; numerous natural polyploid complexes are known, and experimental polyploids are increasingly used in agriculture and horticulture. The morphological, ecological, and physiological consequences of polyploidy are thus of great interest. Abbreviations: ATCase=aspartate transcarbarnylase; CAP=car-
bamylphosphate; EDTA=ethylenediaminetetraceticacid; Tris= trihydroxymethylaminomethane; AMP=adenosine monophosphate; ATP=adenosine triphosphate; CMP=cytidine monophosphate; CTP=cytidine triphosphate; UMP=uridine monophosphate ; UTP = uridine triphosphate
In the particular case of Hippocrepis comosa L., a European Papilionacea, we found three kinds of natural populations: diploids ( 2 n = 2 x = 1 4 ) , tetraploids (2n = 4x = 28), and hexaploids (2n = 6x = 42) (Guern 1969). This complex is a mature one, as defined by Stebbins (1971); that is to say that tetraploids represent the more widespread cytotype, and diploidization of the polyploids is very extensive, if not completely achieved (Guern 1977, 1978). Despite the great polymorphism observed in this taxon, no morphological variability could be correlated with polyploidy. Neither macrocharacters, such as the size of leaves, flowers and seeds, or even of the entire plant, nor microcharacters, such as the size of pollen grains or stomates (Guern 1975), allow one to distinguish one cytotype from the others. Heteroblastic development and flowering time can greatly differ from one population to another, but this is independant of the degree of ploidy (Guern 1978). Thus, the better adaptability shown by the tetraploid populations cannot be attributed to any identifiable morphological or phenological character that could provide them with superiority in inter- or intraspecific competition, however, it seems that this adaptability might result from the combination of several properties of the photosynthetic system (Guern et al. 1975). It was thus interesting to investigate the enzymatic equipment of the different cytological races of H. comosa and since these three races must synthetise different amounts of DNA, the enzymes involved in the biosynthesis of this nucleic acid were the most interesting. Aspartate transcarbamylase, first enzyme of the pyrimidine pathway, is responsible for the establishment of the pyrimidine pools in the cell, and it has been suggested that the nucleotide pools might intervene in the regulation of D N A replication (Bjursell and Reichard 1973). Consequently, the activity and the properties of ATCase in the cell-free extracts derived from various di-, tetra- and hexaploid popula-
0032-0935/80/0149/0027/$01.40
28
M. Guern and G. Hervt: Polyploidy and Aspartate-Transcarbamylase Activity
tions w e r e i n v e s t i g a t e d a n d s o m e o f t h e results o b tained were confirmed using partially purified preparations.
Materials and Methods Chemicals Carbamylphosphate and aspartate were purchased from Fluka, all nucleotides (UTP, UMP, CTP, CMP, ATP, and AMP) from P.L. Biochemicals, EDTA from Merck, [14C]aspartate from Service de Biochimie, CEN-Saclay (Catalog n o CB 51), and protamine sulphate and bovine serum albumin from Sigma.
Preparation of the Extracts One gram of buds and young leaves from H. comosa were sampled and washed with distilled water. After addition of 2 ml 0.5 M Tris acetate pH 8.5, 0.25.10-2M K2HPO4, 10mM /Lmercaptoethanol, 2mM EDTA, and 10% glycerol, the samples were ground in the presence of sand (Fontaine-bleau). A high molarity of Tris was necessary to buffer the strong acidity of the extract. After centrifugation for 20 rain at 16,000 rev min 1 in a refrigerated Serval centrifuge to remove the sand and cell debris, the supernatant was dialyzed twice against 500 ml of the extraction buffer, in order to eliminate the nucleotides and metabolites which might interfere with the enzyme activity or feedback inhibition. The presence of glycerol and KzHPO4 was necessary to ensure the stabilization of the enzyme activity. The samples were stored at -180 ~ C in liquid nitrogen; no decrease of the activity was detected after 15 d of storage under these conditions. When the samples were stored at - 18~ C or +4 ~ C, they lost 20% or 60% of their activity over the same period of time.
Enzymatic Activity a) Standard Conditions. ATCase activity was determined using the method of Porter et al. (1969), under the conditions described by Perbal et Herv6 (1972), in the presence of 50 mM Tris acetate (pH 8.5), 20 mM [l*C]aspartate (5.7.103 Bq Ismol- 1), and 5 mM carbamyl phosphate. Since the method is based on the separation of N-substituted derivatives of aspartate lacking the positive charge, it allows the accurate determination of ATCase in crude extracts which also contain the other enzymes of the pyrimidine pathway. b) Enzyme Unitand Specific Activity. One unit of ATCase is defined as the amount which catalyzes the formation of 1 nmol of carbamyl aspartate per hour under the standard conditions. The specific activity in the plant extracts in expressed in units per mg protein. The protein content of the extracts was determined according to Lowry et al. (1951), using bovine serum albumin as the standard.
DNA Content Determination The DNA content per nucleus was determined by the cytophotometric method of Orstein (1952) and Patau (1952), using a MPV Leitz microcytophotometer. The two-wavelength method (495 mn and 570 nm)was used, according to Mendelsohn (1966) and Garcia and Iorio (1966), on a series of 7 gM slices of meristematic root tissue previously stained by the Feulgen method under precisely standardized conditions (Guervin 1972),
Determination of the Dry Weight of the Plant Samples and of the Corresponding Extraets The dry weight of the plant samples was determined after drying for 48 h at 120~ in an oven. The dry weight of the extracts
was measured after drying them to constant weight in an oven at 110~ C.
Aspartate Transcarbamylase Purification To 80 ml samples of crude extract prepared as previously described, 120 mg protamine sulphate were added and the mixture was stirred for 30 mn at 4~ C. The precipitated nucleic acids were eliminated by a 20 rain centrifugation at 16,000 rev min 1 in a refrigerated Serval centrifuge. The supernatant was fractionated by ammonium sulphate and the fraction obtained between 30% and 60% of samration was redissolved in 4.5 ml of the buffer used to prepare the extract. This sample was run through a column of GI50 Sephadex (48.1.2 cm) preequilibrated with the same buffer. 2.5 ml fractions were collected and their ATCase activity was estimated as previously described using 200 gl samples. The fractions showing ATCase activity were pooled (25 ml) and dialyzed twice against one liter of 25 mM potassium phosphate buffer pH=8.5, 10 mM /Lmercaptoethanol, 2 mM EDTA, 10% glycerol. This sample was then placed on top of a A50 DEAE-sephadex column (35.2 cm) previously equilibrated with the same buffer. After elution with 200 ml, a 500 ml gradient 0.2 to 0,5 M KC1 in the same buffer was applied and 4 ml fractions were collected. A sharp peak of ATCase activity appeared in the region of 0.3 M KC1. This procedure leads to a 30-fold purification.
Results Influenee of the Degree of Ploidy on the Dry-weight Protein Content and D N A Content I n o r d e r to r e l a t e t h e A T C a s e a c t i v i t y to t h e d r y w e i g h t a n d to the p r o t e i n a n d D N A c o n t e n t s o f the s a m p l e s , t h e s e p a r a m e t e r s w e r e d e t e r m i n e d in t h e dip l o i d , t e t r a p l o i d , a n d h e x a p l o i d plants.
a) Dry Weight~Fresh Weight Ratio. T h e p e r c e n t a g e o f d r y w e i g h t o f 15 s a m p l e s o f d i f f e r e n t d e g r e e o f p l o i d y was d e t e r m i n e d . N o d i f f e r e n c e b e t w e e n the three kinds of populations could be detected. This is c o r r o b o r a t e d b y t h e f a c t t h a t t h e d r y w e i g h t o f t h e e x t r a c t s p r e p a r e d as p r e v i o u s l y d e s c r i b e d f r o m t h e c o r r e s p o n d i n g p o p u l a t i o n s d o n o t differ significantly.
b) Protein Content. T h e p r o t e i n c o n t e n t o f t h e e x t r a c t s o b t a i n e d f r o m the d i f f e r e n t p o p u l a t i o n s was i n v e s t i g a t e d as a f u n c t i o n o f the a n n u a l cycle o f t h e plant. N o s i g n i f i c a n t d i f f e r e n c e b e t w e e n the di-, t e t r a - a n d h e x a p l o i d s a m p l e s c a n b e d e t e c t e d in t e r m s o f p r o t e i n c o n t e n t w h e n t h e y are a n a l y z e d at t h e s a m e t i m e o f the year. T h e o n l y s i g n i f i c a n t d i f f e r e n c e w h i c h is detected concerns the variation of protein content as a f u n c t i o n o f time, w h a t e v e r the d e g r e e o f p l o i d y .
c) D N A Content. T h e a m o u n t o f D N A was d e t e r m i n e d b y c y t o p h o t o m e t r y , as d e s c r i b e d in " M a t e r i a l s a n d M e t h o d s " , in at least 31 m e t a p h a s i c plates o f 5 d i f f e r e n t p o p u l a t i o n s . T h e results o b t a i n e d are pres e n t e d in T a b l e 1 w h e r e it can b e seen t h a t the a m o u n t o f D N A p e r n u c l e u s i n c r e a s e s w i t h the d e g r e e o f p l o i d y . H o w e v e r , it is i n t e r e s t i n g to n o t e t h a t t h e
M. Guern and G. Herv+: Polyploidy and Aspartate-Transcarbamylase Activity Table 1. DNA content per nucleus of di-, tetra- and hexaploid plants Population
Amount of DNA/nucleus (arbitrary units)
202 209 212
518 _+16 566 _+15 592+ 17
Tetraploid
403
912 + 29
Hexaploid
606
1,190 _+25
Diploids
29
Table 2. Specific ATCase activity of the extracts prepared from di-, tetra- and hexaploid samples Population
Diploids
Tetraploids
Hexaploids
Specific activity in ATCase
106 +6.6
83 _+9
94 _+11
The specific ATCase activities were determined and are expressed in units per mg of protein, as defined in "Materials and Methods" |
The DNA content per nucleus was measured as described in "Materials and Methods". The determination were made on at least 31 metaphasic plates for each population
amount of DNA is not exactly proportional to the number of genomes, especially in the hexaploids. This discrepancy was already reported in the genus Chrysanthemum (Dowrick and E1 Bayoumi 1969).
Aspartate Transcarbamylase Activity in the Di-, Tetra- and Hexaploid Populations Since ATCase activity and its regulation are responsible for the level of the pyrimidine nucleotides pool in the cell and since this pool might contribute to the control of DNA synthesis, it was interesting to test the different populations for their ATCase activity.
a) Characteristics of the A TCase Reaction. Since the radioactive assay for ATCase which is described in "Materials and Methods", is based on the separation of aspartate and carbamylaspartate on Dowex 50, any enzymatic activity which would split or substitute the amino group ofaspartate would be detected. Consequently, it was necessary to verify that all of the activity found in the extracts was due to ATCase. This control was done by using succinate, a wellknown competitive inhibitor of ATCase activity, on one hand, and by omitting the substrate carbamylphosphate on the other hand. The presence, in the incubation mixture, of 20 mM succinate decreases the rate of the reaction by 70%. In the absence of carbamylphosphate the reaction rate is decreased by 96%. Thus, it appears that at least 96% of the enzyme activity observed is due to ATCase. In addition, the strong feedback inhibition of this activity by UTP (see below) provides additional evidence. b) Specific ATCase Activity of the Different Extracts. The ATCase activity of the extracts obtained from the different di-, tetra- and hexaploid populations was determined and the results are presented in Table 2, where it can be seen that no significant difference
'
I
i
I
1,5
"f"
'-w-" D~ x
/.f~
.m Q m 9
0, 5
/
/ I
~
I
7
~ o
I
pH
I
8
~
No
I
9
Fig. 1. pH dependence of the ATCase activity of the di-, tetraand hexaploid populations. The ATCase activity of the dialysed cell-free extracts was determined under the standard conditions described in "Materials and Methods" but in the presence of 50 mM Tris-HC1 for pH 7.5 to pH 8 ([], o , /x) and in the presence of 50 mM Ethanolamine-HC1 for pH 8.5 to 11 (n, o, A). Actually, after addition of buffer and substrates to the 200 gl of plant extract, the real pH was measured and it is the values thus obtained which are indicated. The results are expressed in nmoles of carbamyl aspartate formed per minute and per mg of protein. [ ] - - m extract prepared from the diploid population n ~ 202; o - - o extract prepared from the tetraploid population n o 456; A - - A extract prepared from the hexaploid population n ~ 608
in specific activity can be detected between these populations as a function of polyploidy.
c) pH Dependence. The pH dependence of the ATCase activity of the different extracts was investigated. For this purpose, the incubations were performed under the standard conditions described in "Materials and Methods", but in the presence of 50 mM ethanolamine HC1 for pH 8.5 to 11. Actually, after addition of buffer and substrates to the plant extract, the real pH of the mixture was measured; it is the values then obtained which are reported in Fig. 1. This figure shows that the pH dependence of the
30
M. Guern and G. Herv6: Polyploidy and Aspartate-Transcarbamylase Activity
2f
B
1/V 1 ,
9~:-'~'~. ~ ~ - ~ < ~ . / ' ~
~/,
,
,
, I
o 1/[Asp.]
. . . .
I
,
,
,
,
I
]i lJV ,I
....
I = = ~-f~l~
~, I Kj~I
o
1
,,,
,I
,~,,l
lo
1/[Carbamylphosphat~
A 2
A
0
10
20
[Aspartate] mM Fig. 2A and B. Dependence of H. cornosa ATCase activity on aspartate concentration. The ATCase activity of the cell free extracts derived from di-, tetra- and hexaploid populations were determined under the standard conditions described in "Materials and Methods" but in the presence of increasing concentrations of aspartate, m - - = diploid population n~ 212; e - - e tetraploid population n~402; A - - A hexaploid population n~ 600. The results are expressed in nmoles of carbamylaspartate per hour and per mg of protein. A ATCase activity as a function of aspartate concentration. B Corresponding Lineweaver-Burk double reciprocal plots (Lineweaver and Burk 1934)
ATCases activity of the di-, tetra- und hexaploid populations do not differ and that this activity is maximal at p H = 8.
d) Affinity of the Enzyme for the Substrates. In order to obtain some indication of the affinity of H. comosa enzyme to its substrates, saturation curves for aspartate and c a r b a m y l p h o s p h a t e were determined.
i) Aspartate. Saturation curves for aspartate of the ATCases f r o m di-, tetra- and hexaploid populations are shown in Fig. 2A. N o sigmoidicity can be detected in these curves; they are hyperbolic, indicating the absence o f allosteric cooperative interactions between the catalytic sites. F o r high concentrations of aspartate, a substrate inhibition can be observed in each case. The Lineweaver-Burk, double-reciprocal plots (Fig. 2B) stiow that the lines corresponding to the three populations intersect the abscissa at the same 1 value of - ~ - ~ , indicating that the K m of the three enzymes for aspartate is a b o u t 1.2 mM.
ii) Carbamylphosphate. The saturation curves for carb a m y l p h o s p h a t e of the ATCases f r o m di-, tetra- and hexaploid populations are presented in Fig. 3A.
I
0
~
I
2
I
1
~
I
I
I
4
[Carbamylphosphatd] mM Fig. 3A and B. Dependence of H. comosa ATCase activity on carbamylphosphate concentration. The ATCase activity of the cellfree extracts derived from di-, tetra- and hexaploid populations were determined under the standard conditions decribed in "Materials and Methods" but in the presence of increasing concentrations of carbamylphosphate. - - - m diploid population n~ 212; e - - e tetraploid population n ~402; A - - A hexaploid population n~ 600. The results are expressed in nmoles of carbamylaspartate formed per hour and per mg of protein. A ATCase activity as a function of carbamylphosphate concentration. B Corresponding Lineweaver-Burk double reciprocal plots (Lineweaver and Burk 1934)
Again, there is no detectable sigmoidicity, indicating the lack of h o m o t r o p i c cooperative interactions. The Lineweaver-Burk, double-reciprocal plots (Fig. 3B) show that the lines corresponding to the ATCases of the three kinds of populations do n o t exactly inter1 sect the abscissa at the same value of - - This Km" slight difference would indicate a K m of 0.5 m M for the tetraploid plant and 1 m M for the diploid and hexaploid plants. N o substrate inhibition is observed in this case. In the case of b o t h substrates the activity present in the extracts derived f r o m the tetraploid samples shows a lower maximal velocity. The verification of these differences would require the complete purification of these enzymes.
e) Influence of Nucleotides on the ATCase Activity. In m o s t organisms, it has been reported that A T C a s e activity is feedback inhibited by at least one of the final products of the pyrimidine pathway. This feedback inhibitor, which is C T P in E. coli (Yates and Pardee 1956) and U T P in Yeast (Kaplan et al. 1967;
M. Guern and G. Herv6: Polyploidy and Aspartate-Transcarbamylase Activity Table 3. Effect of the different nucleotides on the ATCase activity of H. comosa
31
100
Inhibition (%)
75 Populations Nucleotide 10-3 M
Diploid n ~ 214
Tetraploid n ~ 403
Hexaploid n o 606
UTP UMP CTP CMP ATP AMP
22 2 0 0 1 1
7 3 0 1 0 0
13 0 1 0 0 1
=50
9~ 2 5 8
Planta 149, 27 33 (1980)
Polyploidy and Aspartate-Transcarbamylase Activity in Hippocrepis comosa L. Monique Guern and Guy Herv6 Laboratoire de Taxonomie Exp~rimentaleet Num6rique de la Facult6 des Sciences, associg au C.N.R.S., Bfit, 362, F-01405 Orsay, and Laboratoire d'Enzymologie,Centre National de la RechercheScientifique,F-91190 Gif-sur-Yvette, France
Abstract. The D N A content of plants which were
sampled in natural di-, tetra- and hexaploid populations of Hippocrepis comosa L. was estimated and the aspartate transcarbamylase activities of the corresponding cell-free extracts were compared. The amount of D N A is not exactly proportional to the number of genomes. The three kinds of populations do not differ in their aspartate transcarbamylase specific activity. While the enzyme properties are identical in the extracts derived from the diploid and hexaploid plants, the aspartate transcarbamylase present in the tetraploid cytotype shows a slightly lower affinity for one of its substrates and a significantly lower sensitivity to the feedback inhibitor UTP which is still observed after partial purification. These properties might be related to the previously reported greater ability of the tetraploid cytotype to adapt to a variety of biotopes. Key words: Aspartate transcarbamylase - Hippocrepis - Polyploidy.
Introduction
Polyploidy, or multiplication of the entire chromosome set, is widely observed among higher plants; numerous natural polyploid complexes are known, and experimental polyploids are increasingly used in agriculture and horticulture. The morphological, ecological, and physiological consequences of polyploidy are thus of great interest. Abbreviations: ATCase=aspartate transcarbarnylase; CAP=car-
bamylphosphate; EDTA=ethylenediaminetetraceticacid; Tris= trihydroxymethylaminomethane; AMP=adenosine monophosphate; ATP=adenosine triphosphate; CMP=cytidine monophosphate; CTP=cytidine triphosphate; UMP=uridine monophosphate ; UTP = uridine triphosphate
In the particular case of Hippocrepis comosa L., a European Papilionacea, we found three kinds of natural populations: diploids ( 2 n = 2 x = 1 4 ) , tetraploids (2n = 4x = 28), and hexaploids (2n = 6x = 42) (Guern 1969). This complex is a mature one, as defined by Stebbins (1971); that is to say that tetraploids represent the more widespread cytotype, and diploidization of the polyploids is very extensive, if not completely achieved (Guern 1977, 1978). Despite the great polymorphism observed in this taxon, no morphological variability could be correlated with polyploidy. Neither macrocharacters, such as the size of leaves, flowers and seeds, or even of the entire plant, nor microcharacters, such as the size of pollen grains or stomates (Guern 1975), allow one to distinguish one cytotype from the others. Heteroblastic development and flowering time can greatly differ from one population to another, but this is independant of the degree of ploidy (Guern 1978). Thus, the better adaptability shown by the tetraploid populations cannot be attributed to any identifiable morphological or phenological character that could provide them with superiority in inter- or intraspecific competition, however, it seems that this adaptability might result from the combination of several properties of the photosynthetic system (Guern et al. 1975). It was thus interesting to investigate the enzymatic equipment of the different cytological races of H. comosa and since these three races must synthetise different amounts of DNA, the enzymes involved in the biosynthesis of this nucleic acid were the most interesting. Aspartate transcarbamylase, first enzyme of the pyrimidine pathway, is responsible for the establishment of the pyrimidine pools in the cell, and it has been suggested that the nucleotide pools might intervene in the regulation of D N A replication (Bjursell and Reichard 1973). Consequently, the activity and the properties of ATCase in the cell-free extracts derived from various di-, tetra- and hexaploid popula-
0032-0935/80/0149/0027/$01.40
28
M. Guern and G. Hervt: Polyploidy and Aspartate-Transcarbamylase Activity
tions w e r e i n v e s t i g a t e d a n d s o m e o f t h e results o b tained were confirmed using partially purified preparations.
Materials and Methods Chemicals Carbamylphosphate and aspartate were purchased from Fluka, all nucleotides (UTP, UMP, CTP, CMP, ATP, and AMP) from P.L. Biochemicals, EDTA from Merck, [14C]aspartate from Service de Biochimie, CEN-Saclay (Catalog n o CB 51), and protamine sulphate and bovine serum albumin from Sigma.
Preparation of the Extracts One gram of buds and young leaves from H. comosa were sampled and washed with distilled water. After addition of 2 ml 0.5 M Tris acetate pH 8.5, 0.25.10-2M K2HPO4, 10mM /Lmercaptoethanol, 2mM EDTA, and 10% glycerol, the samples were ground in the presence of sand (Fontaine-bleau). A high molarity of Tris was necessary to buffer the strong acidity of the extract. After centrifugation for 20 rain at 16,000 rev min 1 in a refrigerated Serval centrifuge to remove the sand and cell debris, the supernatant was dialyzed twice against 500 ml of the extraction buffer, in order to eliminate the nucleotides and metabolites which might interfere with the enzyme activity or feedback inhibition. The presence of glycerol and KzHPO4 was necessary to ensure the stabilization of the enzyme activity. The samples were stored at -180 ~ C in liquid nitrogen; no decrease of the activity was detected after 15 d of storage under these conditions. When the samples were stored at - 18~ C or +4 ~ C, they lost 20% or 60% of their activity over the same period of time.
Enzymatic Activity a) Standard Conditions. ATCase activity was determined using the method of Porter et al. (1969), under the conditions described by Perbal et Herv6 (1972), in the presence of 50 mM Tris acetate (pH 8.5), 20 mM [l*C]aspartate (5.7.103 Bq Ismol- 1), and 5 mM carbamyl phosphate. Since the method is based on the separation of N-substituted derivatives of aspartate lacking the positive charge, it allows the accurate determination of ATCase in crude extracts which also contain the other enzymes of the pyrimidine pathway. b) Enzyme Unitand Specific Activity. One unit of ATCase is defined as the amount which catalyzes the formation of 1 nmol of carbamyl aspartate per hour under the standard conditions. The specific activity in the plant extracts in expressed in units per mg protein. The protein content of the extracts was determined according to Lowry et al. (1951), using bovine serum albumin as the standard.
DNA Content Determination The DNA content per nucleus was determined by the cytophotometric method of Orstein (1952) and Patau (1952), using a MPV Leitz microcytophotometer. The two-wavelength method (495 mn and 570 nm)was used, according to Mendelsohn (1966) and Garcia and Iorio (1966), on a series of 7 gM slices of meristematic root tissue previously stained by the Feulgen method under precisely standardized conditions (Guervin 1972),
Determination of the Dry Weight of the Plant Samples and of the Corresponding Extraets The dry weight of the plant samples was determined after drying for 48 h at 120~ in an oven. The dry weight of the extracts
was measured after drying them to constant weight in an oven at 110~ C.
Aspartate Transcarbamylase Purification To 80 ml samples of crude extract prepared as previously described, 120 mg protamine sulphate were added and the mixture was stirred for 30 mn at 4~ C. The precipitated nucleic acids were eliminated by a 20 rain centrifugation at 16,000 rev min 1 in a refrigerated Serval centrifuge. The supernatant was fractionated by ammonium sulphate and the fraction obtained between 30% and 60% of samration was redissolved in 4.5 ml of the buffer used to prepare the extract. This sample was run through a column of GI50 Sephadex (48.1.2 cm) preequilibrated with the same buffer. 2.5 ml fractions were collected and their ATCase activity was estimated as previously described using 200 gl samples. The fractions showing ATCase activity were pooled (25 ml) and dialyzed twice against one liter of 25 mM potassium phosphate buffer pH=8.5, 10 mM /Lmercaptoethanol, 2 mM EDTA, 10% glycerol. This sample was then placed on top of a A50 DEAE-sephadex column (35.2 cm) previously equilibrated with the same buffer. After elution with 200 ml, a 500 ml gradient 0.2 to 0,5 M KC1 in the same buffer was applied and 4 ml fractions were collected. A sharp peak of ATCase activity appeared in the region of 0.3 M KC1. This procedure leads to a 30-fold purification.
Results Influenee of the Degree of Ploidy on the Dry-weight Protein Content and D N A Content I n o r d e r to r e l a t e t h e A T C a s e a c t i v i t y to t h e d r y w e i g h t a n d to the p r o t e i n a n d D N A c o n t e n t s o f the s a m p l e s , t h e s e p a r a m e t e r s w e r e d e t e r m i n e d in t h e dip l o i d , t e t r a p l o i d , a n d h e x a p l o i d plants.
a) Dry Weight~Fresh Weight Ratio. T h e p e r c e n t a g e o f d r y w e i g h t o f 15 s a m p l e s o f d i f f e r e n t d e g r e e o f p l o i d y was d e t e r m i n e d . N o d i f f e r e n c e b e t w e e n the three kinds of populations could be detected. This is c o r r o b o r a t e d b y t h e f a c t t h a t t h e d r y w e i g h t o f t h e e x t r a c t s p r e p a r e d as p r e v i o u s l y d e s c r i b e d f r o m t h e c o r r e s p o n d i n g p o p u l a t i o n s d o n o t differ significantly.
b) Protein Content. T h e p r o t e i n c o n t e n t o f t h e e x t r a c t s o b t a i n e d f r o m the d i f f e r e n t p o p u l a t i o n s was i n v e s t i g a t e d as a f u n c t i o n o f the a n n u a l cycle o f t h e plant. N o s i g n i f i c a n t d i f f e r e n c e b e t w e e n the di-, t e t r a - a n d h e x a p l o i d s a m p l e s c a n b e d e t e c t e d in t e r m s o f p r o t e i n c o n t e n t w h e n t h e y are a n a l y z e d at t h e s a m e t i m e o f the year. T h e o n l y s i g n i f i c a n t d i f f e r e n c e w h i c h is detected concerns the variation of protein content as a f u n c t i o n o f time, w h a t e v e r the d e g r e e o f p l o i d y .
c) D N A Content. T h e a m o u n t o f D N A was d e t e r m i n e d b y c y t o p h o t o m e t r y , as d e s c r i b e d in " M a t e r i a l s a n d M e t h o d s " , in at least 31 m e t a p h a s i c plates o f 5 d i f f e r e n t p o p u l a t i o n s . T h e results o b t a i n e d are pres e n t e d in T a b l e 1 w h e r e it can b e seen t h a t the a m o u n t o f D N A p e r n u c l e u s i n c r e a s e s w i t h the d e g r e e o f p l o i d y . H o w e v e r , it is i n t e r e s t i n g to n o t e t h a t t h e
M. Guern and G. Herv+: Polyploidy and Aspartate-Transcarbamylase Activity Table 1. DNA content per nucleus of di-, tetra- and hexaploid plants Population
Amount of DNA/nucleus (arbitrary units)
202 209 212
518 _+16 566 _+15 592+ 17
Tetraploid
403
912 + 29
Hexaploid
606
1,190 _+25
Diploids
29
Table 2. Specific ATCase activity of the extracts prepared from di-, tetra- and hexaploid samples Population
Diploids
Tetraploids
Hexaploids
Specific activity in ATCase
106 +6.6
83 _+9
94 _+11
The specific ATCase activities were determined and are expressed in units per mg of protein, as defined in "Materials and Methods" |
The DNA content per nucleus was measured as described in "Materials and Methods". The determination were made on at least 31 metaphasic plates for each population
amount of DNA is not exactly proportional to the number of genomes, especially in the hexaploids. This discrepancy was already reported in the genus Chrysanthemum (Dowrick and E1 Bayoumi 1969).
Aspartate Transcarbamylase Activity in the Di-, Tetra- and Hexaploid Populations Since ATCase activity and its regulation are responsible for the level of the pyrimidine nucleotides pool in the cell and since this pool might contribute to the control of DNA synthesis, it was interesting to test the different populations for their ATCase activity.
a) Characteristics of the A TCase Reaction. Since the radioactive assay for ATCase which is described in "Materials and Methods", is based on the separation of aspartate and carbamylaspartate on Dowex 50, any enzymatic activity which would split or substitute the amino group ofaspartate would be detected. Consequently, it was necessary to verify that all of the activity found in the extracts was due to ATCase. This control was done by using succinate, a wellknown competitive inhibitor of ATCase activity, on one hand, and by omitting the substrate carbamylphosphate on the other hand. The presence, in the incubation mixture, of 20 mM succinate decreases the rate of the reaction by 70%. In the absence of carbamylphosphate the reaction rate is decreased by 96%. Thus, it appears that at least 96% of the enzyme activity observed is due to ATCase. In addition, the strong feedback inhibition of this activity by UTP (see below) provides additional evidence. b) Specific ATCase Activity of the Different Extracts. The ATCase activity of the extracts obtained from the different di-, tetra- and hexaploid populations was determined and the results are presented in Table 2, where it can be seen that no significant difference
'
I
i
I
1,5
"f"
'-w-" D~ x
/.f~
.m Q m 9
0, 5
/
/ I
~
I
7
~ o
I
pH
I
8
~
No
I
9
Fig. 1. pH dependence of the ATCase activity of the di-, tetraand hexaploid populations. The ATCase activity of the dialysed cell-free extracts was determined under the standard conditions described in "Materials and Methods" but in the presence of 50 mM Tris-HC1 for pH 7.5 to pH 8 ([], o , /x) and in the presence of 50 mM Ethanolamine-HC1 for pH 8.5 to 11 (n, o, A). Actually, after addition of buffer and substrates to the 200 gl of plant extract, the real pH was measured and it is the values thus obtained which are indicated. The results are expressed in nmoles of carbamyl aspartate formed per minute and per mg of protein. [ ] - - m extract prepared from the diploid population n ~ 202; o - - o extract prepared from the tetraploid population n o 456; A - - A extract prepared from the hexaploid population n ~ 608
in specific activity can be detected between these populations as a function of polyploidy.
c) pH Dependence. The pH dependence of the ATCase activity of the different extracts was investigated. For this purpose, the incubations were performed under the standard conditions described in "Materials and Methods", but in the presence of 50 mM ethanolamine HC1 for pH 8.5 to 11. Actually, after addition of buffer and substrates to the plant extract, the real pH of the mixture was measured; it is the values then obtained which are reported in Fig. 1. This figure shows that the pH dependence of the
30
M. Guern and G. Herv6: Polyploidy and Aspartate-Transcarbamylase Activity
2f
B
1/V 1 ,
9~:-'~'~. ~ ~ - ~ < ~ . / ' ~
~/,
,
,
, I
o 1/[Asp.]
. . . .
I
,
,
,
,
I
]i lJV ,I
....
I = = ~-f~l~
~, I Kj~I
o
1
,,,
,I
,~,,l
lo
1/[Carbamylphosphat~
A 2
A
0
10
20
[Aspartate] mM Fig. 2A and B. Dependence of H. cornosa ATCase activity on aspartate concentration. The ATCase activity of the cell free extracts derived from di-, tetra- and hexaploid populations were determined under the standard conditions described in "Materials and Methods" but in the presence of increasing concentrations of aspartate, m - - = diploid population n~ 212; e - - e tetraploid population n~402; A - - A hexaploid population n~ 600. The results are expressed in nmoles of carbamylaspartate per hour and per mg of protein. A ATCase activity as a function of aspartate concentration. B Corresponding Lineweaver-Burk double reciprocal plots (Lineweaver and Burk 1934)
ATCases activity of the di-, tetra- und hexaploid populations do not differ and that this activity is maximal at p H = 8.
d) Affinity of the Enzyme for the Substrates. In order to obtain some indication of the affinity of H. comosa enzyme to its substrates, saturation curves for aspartate and c a r b a m y l p h o s p h a t e were determined.
i) Aspartate. Saturation curves for aspartate of the ATCases f r o m di-, tetra- and hexaploid populations are shown in Fig. 2A. N o sigmoidicity can be detected in these curves; they are hyperbolic, indicating the absence o f allosteric cooperative interactions between the catalytic sites. F o r high concentrations of aspartate, a substrate inhibition can be observed in each case. The Lineweaver-Burk, double-reciprocal plots (Fig. 2B) stiow that the lines corresponding to the three populations intersect the abscissa at the same 1 value of - ~ - ~ , indicating that the K m of the three enzymes for aspartate is a b o u t 1.2 mM.
ii) Carbamylphosphate. The saturation curves for carb a m y l p h o s p h a t e of the ATCases f r o m di-, tetra- and hexaploid populations are presented in Fig. 3A.
I
0
~
I
2
I
1
~
I
I
I
4
[Carbamylphosphatd] mM Fig. 3A and B. Dependence of H. comosa ATCase activity on carbamylphosphate concentration. The ATCase activity of the cellfree extracts derived from di-, tetra- and hexaploid populations were determined under the standard conditions decribed in "Materials and Methods" but in the presence of increasing concentrations of carbamylphosphate. - - - m diploid population n~ 212; e - - e tetraploid population n ~402; A - - A hexaploid population n~ 600. The results are expressed in nmoles of carbamylaspartate formed per hour and per mg of protein. A ATCase activity as a function of carbamylphosphate concentration. B Corresponding Lineweaver-Burk double reciprocal plots (Lineweaver and Burk 1934)
Again, there is no detectable sigmoidicity, indicating the lack of h o m o t r o p i c cooperative interactions. The Lineweaver-Burk, double-reciprocal plots (Fig. 3B) show that the lines corresponding to the ATCases of the three kinds of populations do n o t exactly inter1 sect the abscissa at the same value of - - This Km" slight difference would indicate a K m of 0.5 m M for the tetraploid plant and 1 m M for the diploid and hexaploid plants. N o substrate inhibition is observed in this case. In the case of b o t h substrates the activity present in the extracts derived f r o m the tetraploid samples shows a lower maximal velocity. The verification of these differences would require the complete purification of these enzymes.
e) Influence of Nucleotides on the ATCase Activity. In m o s t organisms, it has been reported that A T C a s e activity is feedback inhibited by at least one of the final products of the pyrimidine pathway. This feedback inhibitor, which is C T P in E. coli (Yates and Pardee 1956) and U T P in Yeast (Kaplan et al. 1967;
M. Guern and G. Herv6: Polyploidy and Aspartate-Transcarbamylase Activity Table 3. Effect of the different nucleotides on the ATCase activity of H. comosa
31
100
Inhibition (%)
75 Populations Nucleotide 10-3 M
Diploid n ~ 214
Tetraploid n ~ 403
Hexaploid n o 606
UTP UMP CTP CMP ATP AMP
22 2 0 0 1 1
7 3 0 1 0 0
13 0 1 0 0 1
=50
9~ 2 5 8
