- Email: [email protected]
The effect of stratification on in vitro protein synthesis in seeds of Pinus lambertiana
Life Sciences Vol. 14, pp. 653-658, 1974. Printed in Great Britain
Pergamon Press
THE EFFECTOF STRATIFICATIONON IN VITRO PROTEINSYNTHESIS IN SEEDSOF PINUS LAMBERTIANA Lewis B. Barnett, Robert E. Adams, and James A. Ramsey Department of Biochemistry and Nutrition and Department of Forestry and Forest Products Virginia Polytechnic Institute and State University Blacksburg, Virginia
24061
(Received 27 August 1973; in final form B January 1974)
Incorporation of 14C-phenylalanine in in vitro systems from sugar pine (Pinus lambertiana) seeds was stuBTe~mbryo ribosomes from both dry and stratified seeds supported incorporation (431 and 326 pmoles, respectively, of phenylalanine per mg ribosome) when combined with an embryo pH 5 fraction from stratified seeds. Female gametophyte ribosomes from dry seeds were active (302 pmoles phenylalanine incorporated per mg ribosome) but lost 61 percent of their capacity to support protein synthesis after 35 hours' stratification. The pH 5 fraction from embryos increased in capacity to support incorporation as stratification progressed up to 60 days (398 pmoles phenylalanine per m9 ribosome when ribosomes were from gO-day stratified embryos) while the pH 5 fraction from female gametophytes was never active. Research in this laboratory has involved biochemistry and physiology of gemination of sugar pine (Pinus lam~rtiana).
A unique feature of this mate-
r i a l , as contrasted with several widely studied seeds such as wheat, cotton, peas, etc., is that sugar pine seeds are dormant when shed from the mother tree and require an extensive period of moist, cold (4° ) conditions ("stratification") to break dormancy of the embryo ( I ) .
Thus, the time between imbibition
( i n i t i a l , rapid uptake of moisture by the dry seed) and gemination is separated by many weeks, and processes occurring as dormancy is broken may be studied. We have previously characterized a ce11-free, amino acid incorporating system prepared from embryos of sugar pine (2).
This communication describes
the effect of different lengths of stratification upon activity of both 653
654
Stratification Effect on Protein Synthesis
Vol. 14, No. 4
ribosomes and pH 5 fractions in the in vitro system from sugar pine embryos and female gametophytes. Methods and Materials The methods and materials were the same as those employed by Adams, Shih and Barnett (2) with minor variations.
The female gametophyte tissue from lO0
seeds was homogenized for 2 min in a Sorvall omni-mixer in 50 ml of 15 mM KCI, 20mM MgCl2, 0.4 M sucrose, 6 mM B-mercaptoethanol, 50 mMTris-HCl, pH 8.0, and 0.2% sodium deoxycholate. This homogenatewas thee treated in the same manner as the embryo tissue (2) to obtain a ribosomal fraction and a pH 5 fraction. Stratification was accomplished by storing seeds in moist, sterile sand at 4° in the dark. The incorporation of 14C-phenylalanine into hot trichloroacetic acid insoluble material, which will be referred to as protein synthesis in this paper, was determined using the f i l t e r disc method of Mans and Novelli (3) and is expressed as pmoles of phenylalanine incorporated per mg of ribosome. Each incubation tube contained 20 mMTris-HCl (pH 8.0), 15 mM KCI, 7.5 mM MgCl2, 6 mM B-mercaptoethanol, l mM phosphoenol pyruvate, I mM ATP, O.l mMGTP and 5 pg pyruvate kinase (EC 2.7.1.40), 0.23 ug 14C-phenylalanine (360 mCi/mmol), 50 ug polyuridylic acid, 0.5 mg ribosomes and 0.5 to l mg pH 5-fraction protein in a total volume of 0.5 ml.
Incubation was terminated after 45 min. Results and Discussion
Protein-synthetic activities of ribosomal and pH 5 fractions from dry and stratified seeds are shown in Table I.
The pH 5 fraction is used in these
studies because we have been unable to obtain an active fraction by other means Unsuccessful preparations include the 30,000 x 9. supernatant (dialyzed and undialyzed) and the I00,000 x 9_ supernatant which was untreated, dialyzed, membrane-fi I tered, or gel - f i I tered. The in vitro system containing pH 5 and ribosome fractions from embryos of stratified seeds had higher activity than systems from female gametophytes or
Vol. 14, No. 4
Stratification Effect on Pfoteln Synthesis
655
TABLE I Protein Synthetic Activity* of Ribosomal and pH 5 Fractions from Embryo and Female Gametophyte Tissues of Dry Seeds and of 90-Day Stratified Seeds
Source of pH 5 Fraction
Embryo (dry)
Embryo (dry)
24 ± 12
Embryo (stratified)
341 + 54
Source of Ribosomal Fractions Female Embryo gametophyte (stratified) (dry) 56 ± 26 326 + l l
Fema]e gametophyte (dry)
2±
l
10± 4
Female gametophyte (stratified)
I ± l
4 ± I
Femal• gametophyte (stratified)
48 ± 30
4 + 3
302 ± 82
13 + 5
2±
1
2 _+ 2
4±I 8 ±l
* p moles 14C-phenylalantne incorporated per mg ribosome ± Standard Error of the Mean embryos of unstratified seeds.
However, comparison of incorporation by
ribosomes from embryos of dry and s t r a t i f i e d seeds u t i l i z i n g each of the four pH 5 fraction sources (embryos and female gametophytes of both dry and s t r a t i fied seeds) shows that a c t i v i t y of embryo ribosomes was not affected by stratification.
Ribosomes from female gametophytes of dry seeds were capable
of supporting protein synthesis while those from s t r a t i f i e d seeds did not.
The
pH 5 fraction from embryos of s t r a t i f i e d seeds had greater a c t i v i t y than that from dry seeds, the l a t t e r producing only about 17% as much incorporation when systems u t i l i z i n g ribosomes from s t r a t i f i e d embryos are compared.
In the
female gametophyte, the pH S fraction showed no a b i l i t y to support protein synthesis regardless of whether the source was from dry or 90-day s t r a t i f i e d seeds. In order to determine the effect of duration of s t r a t i f i c a t i o n on the a b i l i t y of the ribosomes of female gametophytes to support protein synthesis, seeds were s t r a t i f i e d various times.
Results in Table II indicate the loss of
biological a c t i v i t y of female gametophyte ribosomes was very rapid - 6]% of the
656
Stratification Effect on Protein Synthesis
Vol. 14, No. 4
TABLE II Effect of Stratification on the Protein Synthetic Activity of Female Gametophyte Ribosomes* and of Embryo pH 5 Fraction** No. of days of stratification
0
1.5
10
30
60 25+
Activity of female gametophyte ribosomes* 302 ± 82
118 ± 33
20~
17@
Activity of embryo pH 5 fraction**
110 ± 35
154@
159@
56 ± 26
90 13 ±
5
398@ 326 ± 11
* The pH 5 fraction in each instance was from the embryos of 90-day stratified seeds. ** The ribosomal fraction in each instance was from the embryos of 90-day stratified seeds. Average of two samples ± Standard Error of the Mean activity was lost after 36 hours and 93% was lost after lO days of stratification.
During this i n i t i a l 36 hours of stratification there is a rapid uptake
of water (imbibition) by the female gametophyte accompanied by development of polyribosomes from monoribosomes and preformed messenger-RNA (4). Sturani (5) observed that ribosomes from endosperm of dry castor beans had only about 6% of the activity in an in vitro system of those from germinated seeds. Furthermore, ribosomes from dry endosperms inhibited polyphenylalanine synthesis by ribosomes from endosperm of germinated seeds apparently by binding polyuridylic acid into an inactive complex. She concluded that as the castor bean matures, which is accompanied by breakdown of ribosomes (6), damage occurs at the functional level to the ribosome before disappearance of ribosome structure.
The observed loss in the a b i l i t y of the ribosome to support polyphenyl-
alanine synthesis during the f i r s t 36 hours of stratification may involve a continuation of a ripening process similar to that in castor bean endosperm. Kobayashi (7) also observed that ribosomes from spores of Bacillus cereus strain T were inactive, apparently due to removal of proteins from the ribosomes in vegetative cells.
He speculated that this may occur because of physico-
Vol. 14, No. 4
Stratification Effect on Protein Synthesis
657
chemical structural changes in the ribosome which a11owed the observed dissociation of protein. The loss of ribosome function in the present study, as well as in castor beans and spores, may involve a reversal of the ribosome maturation process that has recently been reported for several plant systems during germination (8-11).
Experiments are continuing to determine the cause of inactivation of
female gametophyte ribosomes during stratification. Table II also shows that the pH 5 fraction from the embryo increases during stratification in its ability to support protein synthesis from a value of 56 pmoles of phenylalanine per mg of ribosome to a value of 326. No explanation can be offered at this time regarding the specific site of the observed increase of activity.
However, Sturani (5) observed that the pH 5
fraction from the dry endosperm of castor bean seeds had much less "leucinesRNA synthesizing" activity than did the pH 5 fraction from endosperm of germinated seeds. This problem is currently under investigation. We have been unable to observe any appreciable support of protein synthesis by either the ribosomal fraction or the pH 5 fraction of stratified female gametophytes. Deoxycholate is added to the female gametophyte during homogenization to solubilize the lipid components and thereby increase the yield of ribosomes. However, ribosomes and pH 5 fractions isolated from stratified seeds in the absence of deoxycholate do not support protein synthesis either. We have also used the 100,000 x ~supernatant of the female gametophyte homogenized without deoxycholate as well as after filtration or dialysis and found all of these fractions to be inactive.
The pH 5 fraction from the female
gametophyte tissue had no inhibiting effect on protein synthesis by the embryo ce11-free systems. References I.
T. A. VILLIERS, in Seed Biol I I , T. T. Kozlowski, ed., pp. 219-291, Academic Press, N. Y. (1972).
658
Stratification Effect on Protein Synthesis
Vol. 14, No. 4
2.
R. E. ADAMS, D. S. SHIH and L. B. BARNETT, Forest Sci. 16, 212-218 (1970).
3.
R. J. MANSand G. D. NOVELLI, Arch. Biochem. Bioph~/s. 94, 48-53 (1961).
4.
R. E. ADAMS, Synthesis of Nucleic Acids and Po1¥ribosomes durin 9 S t r a t i f i cation and Germination i n Sugar Pine Female Gametophytes, I07 pp., Ph.D. thesis, State Univ. of N. Y. Col1. of Environmental Sci. and Forestry
(1972). 5.
E. STURANI, Life Sciences 7, 527-537 (1968).
6.
E. STURANIand S. COCUCCl, Life Sciences 4, 1937-1944 (1965).
7.
Y. KOBAYASHI, in Spores V, H. O. Halvorson, R. Hanson and L. L. Campbell, eds., pp. 269-276. Amer. Soc. for Microbiology, Ann Arbor, Mich. (1972).
8.
E. SILVERSTEIN, Biochemistry 12, 951-958 (1973).
9.
D. CHEN, G. SCHULTZand E. KATCHALSKI, Nature New Biol. 231, 69-72 (1971).
10. A. CHAKRAVORTY,Biochim. Biophys. Acta 179, 83-96 (1969). If.
K. HORIKOSHI and Y. IKEDA, Biochim. Bioph~s. Acta 166, 505-511 (1968).
Pergamon Press
THE EFFECTOF STRATIFICATIONON IN VITRO PROTEINSYNTHESIS IN SEEDSOF PINUS LAMBERTIANA Lewis B. Barnett, Robert E. Adams, and James A. Ramsey Department of Biochemistry and Nutrition and Department of Forestry and Forest Products Virginia Polytechnic Institute and State University Blacksburg, Virginia
24061
(Received 27 August 1973; in final form B January 1974)
Incorporation of 14C-phenylalanine in in vitro systems from sugar pine (Pinus lambertiana) seeds was stuBTe~mbryo ribosomes from both dry and stratified seeds supported incorporation (431 and 326 pmoles, respectively, of phenylalanine per mg ribosome) when combined with an embryo pH 5 fraction from stratified seeds. Female gametophyte ribosomes from dry seeds were active (302 pmoles phenylalanine incorporated per mg ribosome) but lost 61 percent of their capacity to support protein synthesis after 35 hours' stratification. The pH 5 fraction from embryos increased in capacity to support incorporation as stratification progressed up to 60 days (398 pmoles phenylalanine per m9 ribosome when ribosomes were from gO-day stratified embryos) while the pH 5 fraction from female gametophytes was never active. Research in this laboratory has involved biochemistry and physiology of gemination of sugar pine (Pinus lam~rtiana).
A unique feature of this mate-
r i a l , as contrasted with several widely studied seeds such as wheat, cotton, peas, etc., is that sugar pine seeds are dormant when shed from the mother tree and require an extensive period of moist, cold (4° ) conditions ("stratification") to break dormancy of the embryo ( I ) .
Thus, the time between imbibition
( i n i t i a l , rapid uptake of moisture by the dry seed) and gemination is separated by many weeks, and processes occurring as dormancy is broken may be studied. We have previously characterized a ce11-free, amino acid incorporating system prepared from embryos of sugar pine (2).
This communication describes
the effect of different lengths of stratification upon activity of both 653
654
Stratification Effect on Protein Synthesis
Vol. 14, No. 4
ribosomes and pH 5 fractions in the in vitro system from sugar pine embryos and female gametophytes. Methods and Materials The methods and materials were the same as those employed by Adams, Shih and Barnett (2) with minor variations.
The female gametophyte tissue from lO0
seeds was homogenized for 2 min in a Sorvall omni-mixer in 50 ml of 15 mM KCI, 20mM MgCl2, 0.4 M sucrose, 6 mM B-mercaptoethanol, 50 mMTris-HCl, pH 8.0, and 0.2% sodium deoxycholate. This homogenatewas thee treated in the same manner as the embryo tissue (2) to obtain a ribosomal fraction and a pH 5 fraction. Stratification was accomplished by storing seeds in moist, sterile sand at 4° in the dark. The incorporation of 14C-phenylalanine into hot trichloroacetic acid insoluble material, which will be referred to as protein synthesis in this paper, was determined using the f i l t e r disc method of Mans and Novelli (3) and is expressed as pmoles of phenylalanine incorporated per mg of ribosome. Each incubation tube contained 20 mMTris-HCl (pH 8.0), 15 mM KCI, 7.5 mM MgCl2, 6 mM B-mercaptoethanol, l mM phosphoenol pyruvate, I mM ATP, O.l mMGTP and 5 pg pyruvate kinase (EC 2.7.1.40), 0.23 ug 14C-phenylalanine (360 mCi/mmol), 50 ug polyuridylic acid, 0.5 mg ribosomes and 0.5 to l mg pH 5-fraction protein in a total volume of 0.5 ml.
Incubation was terminated after 45 min. Results and Discussion
Protein-synthetic activities of ribosomal and pH 5 fractions from dry and stratified seeds are shown in Table I.
The pH 5 fraction is used in these
studies because we have been unable to obtain an active fraction by other means Unsuccessful preparations include the 30,000 x 9. supernatant (dialyzed and undialyzed) and the I00,000 x 9_ supernatant which was untreated, dialyzed, membrane-fi I tered, or gel - f i I tered. The in vitro system containing pH 5 and ribosome fractions from embryos of stratified seeds had higher activity than systems from female gametophytes or
Vol. 14, No. 4
Stratification Effect on Pfoteln Synthesis
655
TABLE I Protein Synthetic Activity* of Ribosomal and pH 5 Fractions from Embryo and Female Gametophyte Tissues of Dry Seeds and of 90-Day Stratified Seeds
Source of pH 5 Fraction
Embryo (dry)
Embryo (dry)
24 ± 12
Embryo (stratified)
341 + 54
Source of Ribosomal Fractions Female Embryo gametophyte (stratified) (dry) 56 ± 26 326 + l l
Fema]e gametophyte (dry)
2±
l
10± 4
Female gametophyte (stratified)
I ± l
4 ± I
Femal• gametophyte (stratified)
48 ± 30
4 + 3
302 ± 82
13 + 5
2±
1
2 _+ 2
4±I 8 ±l
* p moles 14C-phenylalantne incorporated per mg ribosome ± Standard Error of the Mean embryos of unstratified seeds.
However, comparison of incorporation by
ribosomes from embryos of dry and s t r a t i f i e d seeds u t i l i z i n g each of the four pH 5 fraction sources (embryos and female gametophytes of both dry and s t r a t i fied seeds) shows that a c t i v i t y of embryo ribosomes was not affected by stratification.
Ribosomes from female gametophytes of dry seeds were capable
of supporting protein synthesis while those from s t r a t i f i e d seeds did not.
The
pH 5 fraction from embryos of s t r a t i f i e d seeds had greater a c t i v i t y than that from dry seeds, the l a t t e r producing only about 17% as much incorporation when systems u t i l i z i n g ribosomes from s t r a t i f i e d embryos are compared.
In the
female gametophyte, the pH S fraction showed no a b i l i t y to support protein synthesis regardless of whether the source was from dry or 90-day s t r a t i f i e d seeds. In order to determine the effect of duration of s t r a t i f i c a t i o n on the a b i l i t y of the ribosomes of female gametophytes to support protein synthesis, seeds were s t r a t i f i e d various times.
Results in Table II indicate the loss of
biological a c t i v i t y of female gametophyte ribosomes was very rapid - 6]% of the
656
Stratification Effect on Protein Synthesis
Vol. 14, No. 4
TABLE II Effect of Stratification on the Protein Synthetic Activity of Female Gametophyte Ribosomes* and of Embryo pH 5 Fraction** No. of days of stratification
0
1.5
10
30
60 25+
Activity of female gametophyte ribosomes* 302 ± 82
118 ± 33
20~
17@
Activity of embryo pH 5 fraction**
110 ± 35
154@
159@
56 ± 26
90 13 ±
5
398@ 326 ± 11
* The pH 5 fraction in each instance was from the embryos of 90-day stratified seeds. ** The ribosomal fraction in each instance was from the embryos of 90-day stratified seeds. Average of two samples ± Standard Error of the Mean activity was lost after 36 hours and 93% was lost after lO days of stratification.
During this i n i t i a l 36 hours of stratification there is a rapid uptake
of water (imbibition) by the female gametophyte accompanied by development of polyribosomes from monoribosomes and preformed messenger-RNA (4). Sturani (5) observed that ribosomes from endosperm of dry castor beans had only about 6% of the activity in an in vitro system of those from germinated seeds. Furthermore, ribosomes from dry endosperms inhibited polyphenylalanine synthesis by ribosomes from endosperm of germinated seeds apparently by binding polyuridylic acid into an inactive complex. She concluded that as the castor bean matures, which is accompanied by breakdown of ribosomes (6), damage occurs at the functional level to the ribosome before disappearance of ribosome structure.
The observed loss in the a b i l i t y of the ribosome to support polyphenyl-
alanine synthesis during the f i r s t 36 hours of stratification may involve a continuation of a ripening process similar to that in castor bean endosperm. Kobayashi (7) also observed that ribosomes from spores of Bacillus cereus strain T were inactive, apparently due to removal of proteins from the ribosomes in vegetative cells.
He speculated that this may occur because of physico-
Vol. 14, No. 4
Stratification Effect on Protein Synthesis
657
chemical structural changes in the ribosome which a11owed the observed dissociation of protein. The loss of ribosome function in the present study, as well as in castor beans and spores, may involve a reversal of the ribosome maturation process that has recently been reported for several plant systems during germination (8-11).
Experiments are continuing to determine the cause of inactivation of
female gametophyte ribosomes during stratification. Table II also shows that the pH 5 fraction from the embryo increases during stratification in its ability to support protein synthesis from a value of 56 pmoles of phenylalanine per mg of ribosome to a value of 326. No explanation can be offered at this time regarding the specific site of the observed increase of activity.
However, Sturani (5) observed that the pH 5
fraction from the dry endosperm of castor bean seeds had much less "leucinesRNA synthesizing" activity than did the pH 5 fraction from endosperm of germinated seeds. This problem is currently under investigation. We have been unable to observe any appreciable support of protein synthesis by either the ribosomal fraction or the pH 5 fraction of stratified female gametophytes. Deoxycholate is added to the female gametophyte during homogenization to solubilize the lipid components and thereby increase the yield of ribosomes. However, ribosomes and pH 5 fractions isolated from stratified seeds in the absence of deoxycholate do not support protein synthesis either. We have also used the 100,000 x ~supernatant of the female gametophyte homogenized without deoxycholate as well as after filtration or dialysis and found all of these fractions to be inactive.
The pH 5 fraction from the female
gametophyte tissue had no inhibiting effect on protein synthesis by the embryo ce11-free systems. References I.
T. A. VILLIERS, in Seed Biol I I , T. T. Kozlowski, ed., pp. 219-291, Academic Press, N. Y. (1972).
658
Stratification Effect on Protein Synthesis
Vol. 14, No. 4
2.
R. E. ADAMS, D. S. SHIH and L. B. BARNETT, Forest Sci. 16, 212-218 (1970).
3.
R. J. MANSand G. D. NOVELLI, Arch. Biochem. Bioph~/s. 94, 48-53 (1961).
4.
R. E. ADAMS, Synthesis of Nucleic Acids and Po1¥ribosomes durin 9 S t r a t i f i cation and Germination i n Sugar Pine Female Gametophytes, I07 pp., Ph.D. thesis, State Univ. of N. Y. Col1. of Environmental Sci. and Forestry
(1972). 5.
E. STURANI, Life Sciences 7, 527-537 (1968).
6.
E. STURANIand S. COCUCCl, Life Sciences 4, 1937-1944 (1965).
7.
Y. KOBAYASHI, in Spores V, H. O. Halvorson, R. Hanson and L. L. Campbell, eds., pp. 269-276. Amer. Soc. for Microbiology, Ann Arbor, Mich. (1972).
8.
E. SILVERSTEIN, Biochemistry 12, 951-958 (1973).
9.
D. CHEN, G. SCHULTZand E. KATCHALSKI, Nature New Biol. 231, 69-72 (1971).
10. A. CHAKRAVORTY,Biochim. Biophys. Acta 179, 83-96 (1969). If.
K. HORIKOSHI and Y. IKEDA, Biochim. Bioph~s. Acta 166, 505-511 (1968).