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Brief High Temperature Exposure to Release Dormancy Affects Soluble and Membrane-Bound Protein Composition in Echinochloa crus-galli (L.) Beauv. seeds
J.PlantPhysiol. Vol. 135.pp. 117-121 {1989}
Short Comnlunication
Brief High Temperature Exposure to Release Dormancy Affects Soluble and Membrane-Bound Protein Composition in Echinochloa crus-galli (L.) Beauv. seeds LILIANA DI NOLA!
and RAY B. TAYLORSON
U.S. Department of Agriculture, Agriculture Research Service, Weed Science Laboratory, Beltsville, Maryland 20705 USA Received January 24,1989 . Accepted April 16, 1989
Summary A brief exposure of dormant Echinochloa crus-galli (L.) Beauv. seeds to a high temperature (46°q, which stimulates subsequent germination at favorable temperatures, affects the composition of soluble and membrane-bound proteins in the cells. The changes occur during the transition of the seeds from a dormant to a non-dormant state and are reversed when the seeds are exposed to the high temperature for prolonged periods of time, which inhibits subsequent germination. Also, the formation of new proteins is induced during the prolonged heat treatment. These results indicate that upon exposure of the seeds to a high temperature during imbibition some metabolic changes occur in the soluble and membrane-bound proteins of the cells.
Key words: Echinochloa crus-galli (L.) Beauv.; donnancy; gennination; membranes; protein; seeds; temperature. Abbreviations: PMSF, Phenylmethyl-Sulfonyl Fluoride; PVP, polyvinylpyrrolidone. Introduction
Echinochloa crus-galli (barnyardgrass) is a widespread annual weed grass, whose seeds can persist in a dormant state in the field for prolonged periods of time. In the laboratory, germination of these seeds can be artificially stimulated by a brief exposure to high temperature (46 0q during imbibition, followed by transfer to suitable thermic conditions for germination [Taylorson and Di Nola, In Press]. Prolonged exposure to elevated temperature inhibits germination [Taylorson and Di Nola, In Press]. Despite many reports on the effect of elevated temperatures on germination of various seeds [Huang and Hsiao, 1987; Probert et al., 1986; Roberts and Totterdell, 1981], very little is known about the 1 Present address: Center for Biologics Evaluation and Research, Food and Drug Administration, Bldg. 29, Room 523, 8800 Rockville Pike, Bethesda, MD 20892.
© 1989 by Gustav Fischer Verlag, Stuttgart
biochemical mechanisms which regulate the response of the seeds to elevated temperature by triggering germination. In the last few years, it has been shown that vascular plants respond to thermic changes by initiating the synthesis of specific proteins. The increase in freezing tolerance during cold acclimation, as well as the response to heat shock in the acquisition of thermic tolerance have been associated with synthesis of new proteins in the plant tissue [Guy and Haskell, 1987; Kimpel and Key, 1985; Meza-Basso et al., 1986; Neumann et al., 1987]. The transition from dormancy to non-dormancy in seeds is characterized by an increase in metabolic activities during the early phases of imbibition [Di Nola and Mayer, 1985, 1986a, 1986b, and 1987; Mayer and Marbach, 1981], by the hydrolysis of storage proteins [Basha and Beevers, 1975; Youle and Huang, 1976] and by synthesis of new molecules of mRNA [Delseny et al., 1980] and DNA [Osborne et al., 1980]. In this paper we report on a study we made to determine the effect of a brief exposure to high temperature on the composition of soluble and membrane-
118
LILIANA DI NOLA and RAy B. TAYLORSON
bound proteins in barnyardgrass seeds during the transition from dormancy to germination.
mension 10% acrylamide gels and subjected to electrophoresis at 150 V for 8 h. Two-dimension diagonal gels were silver stained. All the experiments were repeated three times for each treatment. Each repetition was performed on a different protein preparation.
Materials and Methods Treatment ofseeds and protein preparations Echinochloa crus-galli (L.) Beauv. seeds (10 g dry weight) were imbibed for 96 h, beginning at 35°C, in darkness. During the fourth day of imbibition the seeds were transferred to 46°C for 1,4, 12, 16, or 20 h prior to homogenization of the tissues. The seeds were then washed and homogenized as previously described (Di Nola et al., 1989). After centrifugation at 13,OOOg, the supernatant was collected and proteins were concentrated by precipitation with TCA [7.5 % (v: v) final concentration] at 4°C. The pellet was washed 4 times with diethyl ether and finally resuspended in 25 mM MES buffer (pH 6.5), containing 30% (w/v) glycerol, 5mM Na azide, 5mM Na-molybdate, 2.5mM EDTA, and 250/LM PMSF. Protein was determined according to Lowry et al. [1951]. Analysis ofproteins Aliquots equivalent to 30/Lg protein were diluted with SDSsample buffer [Laemmli, 1970]. Proteins were separated by electrophoresis on 10 % SDS-acrylamide gels at 150 V for 8 h. The bands were stained first with Coomassie brilliant blue and then with silver staining [Wray et al., 1981]. For two-dimensional diagonal separation of seed proteins, aliquots equivalent to 200 JLg protein were diluted with SDS-sample buffer and first run on 10% polyacrylamide gels as described. Firstdimension gels were sliced vertically into segments 2 cm thick, each containing proteins from a different sample. The slices were placed transversely, without equilibration, directly on top of the second-di-
Results
E. crus-galli seeds have a high protein content, since their cells are very rich in protein bodies [Di Nola and Taylorson, In Press], which are mainly storage proteins. In our study, protein bodies were eliminated by centrifugation of the homogenate at 13,OOOg and discarding the pellet. We therefore studied the effect of a brief exposure of the seeds to 46 °e only in a fraction enriched in soluble and membrane-bound proteins. Fig. 1 shows the effect of an exposure of the seeds to 46 °e for various times on the composition of their soluble and membrane-bound proteins. The content of 58 kD, 55 kD, 52 kD, 44 kD, 41 kD, 38 kD, 36 kD, 29 kD, 26 kD, 24 kD, 23 kD, and 22 kD proteins, already present in seeds imbibed at 35°e, increased after 1 h at 46°e (arrows, column b). These changes were reversed in seeds exposed to the higher temperature for longer times. The 22 kD protein decreased only after 16 h at 46°C. The 38 kD protein was not present in seeds exposed to the high temperature for 20 h. In seeds exposed to the higher temperature for prolonged periods (12, 16, and 20 h), the formation of new 30 kD, 32 kD, and 37 kD proteins was induced. Also, the appearance of a band just below the 44 kD protein was evident in these seeds (double arrows, columns, d, e, and f).
Fig. 1: SDS-polyacrylamide silver-stained gel of soluble and membrane-bound proteins extracted from Echinochloa crus-galli seeds after: a) 4 day imbibition at 35°C; b) exposure of imbibed seeds to 46 °C for 1 h; c) for 4 h; d) for 12 h; e) for 16 h; f) for 20 h. Thirty JLg protein was applied per lane. Protein Mr standards are in the right outside lane. Thick arrows indicate protein bands whose content is increased by the heat treatment. Double arrows indicate appearance of new proteins.
119
Seed proteins
45-
a
36-
29-
24-
22-
45-
b
3829-
24-
2245-
c
3629Fig. 2: Two dimensional diagonal silverstained gels of soluble and membranebound proteins extracted from Echinochloa crus.galli seeds after: a) 4 day imbibition at 35°C; b) exposure of imbibed seeds to 46°C for 1 h ; c) for 4 h. Two hundred J.lg protein was applied per lane in the first dimension. Molecular weight in kD is indicated numerically adjacent to the gels.
24-
22-
The effect of a high temperature of imbibition on the composition of soluble and membrane-bound proteins was also studied by separation of proteins on two-dimensional diag-
onal gels. Results are shown only for proteins extracted from seeds imbibed at 35°C (Fig. 2 A) and from seeds exposed to 46°C for 1 h (Fig. 2 B) or for 4 h (Fig. 2 C) after imbibition at
120
LILIANA DI NOLA and RAY B. TAYLORSON
35°C. Exposure of the seed to 46 °C for 1 h caused evident increases in the content of the 44 kD, 41 kD, 38 kD, 36 kD, 29 kD, 24 kD, 23 kD, and 22 kD proteins. These changes were reversed in seeds exposed to 46°C for 4 h.
Discussion Germination of fully imbibed dormant E. crus-galli seeds can be stimulated by a brief exposure to high temperature (46°C) followed by transfer of the seeds to a suitable temperature for germination (20/30°C, 16 and 8 h, respectively). The stimulation is maximal after short heat treatments (1 to 2 h); the stimulation decreases with exposure of the seeds to 46°C for 4 h, and is prevented after longer ejtposures to the higher temperature [Taylorson and Di Nola, In Press]. Treatment of the seeds at high temperature and length of the exposure appear therefore to act as a signal for regulation of subsequent germination. Previous studies showed that a brief exposure to a low temperature (5°C) during imbibition strongly affected metabolism in membranes and ultrastructural changes in the embryonic axis cells of Pisum sativum 1. (pea) seeds, ultimately delaying subsequent germination at a favorable temperature [Di Nola and Mayer, 1985, 1986a, 1986 b, 1987; Hodson et aI., 1987]. These experiments also showed that the cold temperature induced the formation of several membrane proteins and inhibited the disappearance of others. These changes were still evident, even after prolonged exposure of the seed to a suitable temperature for germination [Di Nola and Mayer, 1987]. High temperature treatments affecting germination may have an effect on the composition of soluble and membrane-bound proteins in cells of E. crus-galli seeds. We found that the transition from dormancy to non-dormancy during a brief exposure to high temperature is accompanied in E. crus-galli seeds by a remarkable increase in content of soluble and membrane-bound proteins already present after imbibition at 35°C. These changes are, however, gradually reversed by exposing the seeds to the higher temperature for longer periods of time. Prolonged treatment at 46 °C induced formation of new proteins. These results suggest that the transition from a dormant to a non-dormant state preceeding germination involves some metabolic changes in soluble and membrane-bound proteins in the cells of E. crus-galli seeds. These changes are in fact no longer evident when the stimulatory effect turns into an inhibitory one. The inhibitory effect of a prolonged exposure to high temperature could be due to changes in the turnover of proteins, combined with changes in the moisture content of the seeds. It is possible that some of the proteins induced by the prolonged heat treatment have a role in promoting dormancy and preventing germination during subsequent exposure to the favorable temperature. Further studies are required in order to determine the metabolic changes which occur in soluble and membrane-bound proteins, respectively, in the cells upon exposure to high temperature during imbibition. These changes could regulate the metabolism of the seed cells, determining their response to external factors and, ultimately, their germination behavior.
References BASHA, S. M. M. and L. BEEVERS: The development of proteolytic activity and protein degradation during germination. Planta 124, 77 -87 (1975). DELSENY, M., L. ASPART, and R. COOKE: Studies on ribonucleic acids during radish seed germination: a search for potential regulatory mechanisms. Isr.J. Bot. 29, 246-258 (1980/81). DI NOLA, L., E. BERLIN, and R. B. TAYLORSON: Thermotropic properties of cellular membranes in dormant and non-dormant barnyardgrass [Echinochloa crus-galli (L.) Beauv.] seeds. Submitted to Plant Physiol. DI NOLA, L. and A. M. MAYER: Effect of temperature of imbibition on phospholipid metabolism in pea embryonic axes. Phytochemistry 24,2549-2554 (1985). - - Effect of temperature on glycerol metabolism in membranes and on phospholipases C and D of germinating pea embryos. Phytochemistry 25,2255-2259 (1986 a}. - - The effect of temperature of imbibition and germination on the lipid and fatty acid content and composition in phospholipids of embryonic axes from pea seeds. Phytochemistry 25, 2725-2731 (1986 b). - - Ethanolamine incorporation into the membranes of pea embryonic axes during germination. Phytochemistry 26, 1591-1593 (1987). DI NOLA, L. and R. B. TAYLORSON: Ultrastructural changes in radicles of barnyardgrass [Echinochloa crus-galli (L.) Beauv.] seeds during the transition from dormancy to germination. Isr. J. Bot. (In Press). GUY, C. L. and D. HASKELL: Induction of freezing tolerance in spinach is associated with the synthesis of cold acclimation induced proteins. Plant Physiol. 84, 872-878 (1987). HODSON, M. J., L. DI NOLA, and A. M. MAYER: The effect of changing temperatures during imbibition on ultrastructure in germinating pea embryonic radicles. J. Exp. Bot. 38, 525-534 (1987). HUANG, W. Z. and A. I. HSIAO: Factors affecting seed dormancy and germination of Johnsongrass, Sorghum halepense (L.) Pers. Weed Res. 27, 1-12 (1987). KIMPEL, J. A. and J. L. KEY: Heat shock in plants. Trends Biochem. Sci. 10, 353-357 (1985). LAEMMLI, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 (1970). LOWRY, O. H., N. J. ROSEBROUGH, L. A. FARIl, and R. J. RANDALL: Protein measurement with the folin reagent. J. BioI. Chem. 193, 265-275 (1951). MAYER, A. M. and I. MARBACH: Biochemistry of the transition from resting to germinating state in seeds. Progress in Phytochem. 7, 95-136 (1981). MEZA-BASSO, L., M. ALBERDI, M. RAYNAL, M. FERJlERo-CADINANOS, and M. DELSNEY: Changes in protein synthesis in rapeseed (Brassica napus) seedlings during a low temperature treatment. Plant Physiol. 82, 733-738 {1986}. NEUMANN, D., U. NIEDEN, R. MANTEUFFEL, G. WALTER, K.-D. SCHARF, and L. NOVER: Intracellular localization of heat shock proteins in tomato cell cultures. Eur. J. Cell. BioI. 43, 71-81 (1987). OSBORNE, D. J., R. SHARON, and R. BEN-ISHAI: Studies on DNA integrity and DNA repair in germinating embryos of rye (Secale cereale). Isr. J. Bot. 29, 259-272 (1980/81). PROBERT, R. J., R. D. SMITH, and P. BIRCH: Germination responses to light and alternating temperatures in european populations of Dactylis glomerata L. V. The principle components of the alternating temperature requirement. New Phytol. 102, 133 -142 (1986).
Seed proteins ROBERTS, E. H. and S. TOTIERDELL: Seed dormancy in Rumex sp. in response to environmental factors. Plant Cell Environ. 4, 97 -106 (1981). TAYLORSON, R. B. and L. DI NOLA: Increased phytochrome responsiveness and a high temperature transition in barnyardgrass (Echinochloa crus·galli) seed dormancy. Weed Sci. (In Press).
121
WRAY, W., T. BOULIKAS, V. P. WRAY, and R. HANCOCK: Silver staining of proteins in polyacrylamide gels. Anal. Biochem. 118, 197 -203 (1981). YOULE, R. J. and A. H. C. HUANG: Protein bodies from the endosperm of castor bean. Plant Physiol. 58, 703 -709 (1976).
Short Comnlunication
Brief High Temperature Exposure to Release Dormancy Affects Soluble and Membrane-Bound Protein Composition in Echinochloa crus-galli (L.) Beauv. seeds LILIANA DI NOLA!
and RAY B. TAYLORSON
U.S. Department of Agriculture, Agriculture Research Service, Weed Science Laboratory, Beltsville, Maryland 20705 USA Received January 24,1989 . Accepted April 16, 1989
Summary A brief exposure of dormant Echinochloa crus-galli (L.) Beauv. seeds to a high temperature (46°q, which stimulates subsequent germination at favorable temperatures, affects the composition of soluble and membrane-bound proteins in the cells. The changes occur during the transition of the seeds from a dormant to a non-dormant state and are reversed when the seeds are exposed to the high temperature for prolonged periods of time, which inhibits subsequent germination. Also, the formation of new proteins is induced during the prolonged heat treatment. These results indicate that upon exposure of the seeds to a high temperature during imbibition some metabolic changes occur in the soluble and membrane-bound proteins of the cells.
Key words: Echinochloa crus-galli (L.) Beauv.; donnancy; gennination; membranes; protein; seeds; temperature. Abbreviations: PMSF, Phenylmethyl-Sulfonyl Fluoride; PVP, polyvinylpyrrolidone. Introduction
Echinochloa crus-galli (barnyardgrass) is a widespread annual weed grass, whose seeds can persist in a dormant state in the field for prolonged periods of time. In the laboratory, germination of these seeds can be artificially stimulated by a brief exposure to high temperature (46 0q during imbibition, followed by transfer to suitable thermic conditions for germination [Taylorson and Di Nola, In Press]. Prolonged exposure to elevated temperature inhibits germination [Taylorson and Di Nola, In Press]. Despite many reports on the effect of elevated temperatures on germination of various seeds [Huang and Hsiao, 1987; Probert et al., 1986; Roberts and Totterdell, 1981], very little is known about the 1 Present address: Center for Biologics Evaluation and Research, Food and Drug Administration, Bldg. 29, Room 523, 8800 Rockville Pike, Bethesda, MD 20892.
© 1989 by Gustav Fischer Verlag, Stuttgart
biochemical mechanisms which regulate the response of the seeds to elevated temperature by triggering germination. In the last few years, it has been shown that vascular plants respond to thermic changes by initiating the synthesis of specific proteins. The increase in freezing tolerance during cold acclimation, as well as the response to heat shock in the acquisition of thermic tolerance have been associated with synthesis of new proteins in the plant tissue [Guy and Haskell, 1987; Kimpel and Key, 1985; Meza-Basso et al., 1986; Neumann et al., 1987]. The transition from dormancy to non-dormancy in seeds is characterized by an increase in metabolic activities during the early phases of imbibition [Di Nola and Mayer, 1985, 1986a, 1986b, and 1987; Mayer and Marbach, 1981], by the hydrolysis of storage proteins [Basha and Beevers, 1975; Youle and Huang, 1976] and by synthesis of new molecules of mRNA [Delseny et al., 1980] and DNA [Osborne et al., 1980]. In this paper we report on a study we made to determine the effect of a brief exposure to high temperature on the composition of soluble and membrane-
118
LILIANA DI NOLA and RAy B. TAYLORSON
bound proteins in barnyardgrass seeds during the transition from dormancy to germination.
mension 10% acrylamide gels and subjected to electrophoresis at 150 V for 8 h. Two-dimension diagonal gels were silver stained. All the experiments were repeated three times for each treatment. Each repetition was performed on a different protein preparation.
Materials and Methods Treatment ofseeds and protein preparations Echinochloa crus-galli (L.) Beauv. seeds (10 g dry weight) were imbibed for 96 h, beginning at 35°C, in darkness. During the fourth day of imbibition the seeds were transferred to 46°C for 1,4, 12, 16, or 20 h prior to homogenization of the tissues. The seeds were then washed and homogenized as previously described (Di Nola et al., 1989). After centrifugation at 13,OOOg, the supernatant was collected and proteins were concentrated by precipitation with TCA [7.5 % (v: v) final concentration] at 4°C. The pellet was washed 4 times with diethyl ether and finally resuspended in 25 mM MES buffer (pH 6.5), containing 30% (w/v) glycerol, 5mM Na azide, 5mM Na-molybdate, 2.5mM EDTA, and 250/LM PMSF. Protein was determined according to Lowry et al. [1951]. Analysis ofproteins Aliquots equivalent to 30/Lg protein were diluted with SDSsample buffer [Laemmli, 1970]. Proteins were separated by electrophoresis on 10 % SDS-acrylamide gels at 150 V for 8 h. The bands were stained first with Coomassie brilliant blue and then with silver staining [Wray et al., 1981]. For two-dimensional diagonal separation of seed proteins, aliquots equivalent to 200 JLg protein were diluted with SDS-sample buffer and first run on 10% polyacrylamide gels as described. Firstdimension gels were sliced vertically into segments 2 cm thick, each containing proteins from a different sample. The slices were placed transversely, without equilibration, directly on top of the second-di-
Results
E. crus-galli seeds have a high protein content, since their cells are very rich in protein bodies [Di Nola and Taylorson, In Press], which are mainly storage proteins. In our study, protein bodies were eliminated by centrifugation of the homogenate at 13,OOOg and discarding the pellet. We therefore studied the effect of a brief exposure of the seeds to 46 °e only in a fraction enriched in soluble and membrane-bound proteins. Fig. 1 shows the effect of an exposure of the seeds to 46 °e for various times on the composition of their soluble and membrane-bound proteins. The content of 58 kD, 55 kD, 52 kD, 44 kD, 41 kD, 38 kD, 36 kD, 29 kD, 26 kD, 24 kD, 23 kD, and 22 kD proteins, already present in seeds imbibed at 35°e, increased after 1 h at 46°e (arrows, column b). These changes were reversed in seeds exposed to the higher temperature for longer times. The 22 kD protein decreased only after 16 h at 46°C. The 38 kD protein was not present in seeds exposed to the high temperature for 20 h. In seeds exposed to the higher temperature for prolonged periods (12, 16, and 20 h), the formation of new 30 kD, 32 kD, and 37 kD proteins was induced. Also, the appearance of a band just below the 44 kD protein was evident in these seeds (double arrows, columns, d, e, and f).
Fig. 1: SDS-polyacrylamide silver-stained gel of soluble and membrane-bound proteins extracted from Echinochloa crus-galli seeds after: a) 4 day imbibition at 35°C; b) exposure of imbibed seeds to 46 °C for 1 h; c) for 4 h; d) for 12 h; e) for 16 h; f) for 20 h. Thirty JLg protein was applied per lane. Protein Mr standards are in the right outside lane. Thick arrows indicate protein bands whose content is increased by the heat treatment. Double arrows indicate appearance of new proteins.
119
Seed proteins
45-
a
36-
29-
24-
22-
45-
b
3829-
24-
2245-
c
3629Fig. 2: Two dimensional diagonal silverstained gels of soluble and membranebound proteins extracted from Echinochloa crus.galli seeds after: a) 4 day imbibition at 35°C; b) exposure of imbibed seeds to 46°C for 1 h ; c) for 4 h. Two hundred J.lg protein was applied per lane in the first dimension. Molecular weight in kD is indicated numerically adjacent to the gels.
24-
22-
The effect of a high temperature of imbibition on the composition of soluble and membrane-bound proteins was also studied by separation of proteins on two-dimensional diag-
onal gels. Results are shown only for proteins extracted from seeds imbibed at 35°C (Fig. 2 A) and from seeds exposed to 46°C for 1 h (Fig. 2 B) or for 4 h (Fig. 2 C) after imbibition at
120
LILIANA DI NOLA and RAY B. TAYLORSON
35°C. Exposure of the seed to 46 °C for 1 h caused evident increases in the content of the 44 kD, 41 kD, 38 kD, 36 kD, 29 kD, 24 kD, 23 kD, and 22 kD proteins. These changes were reversed in seeds exposed to 46°C for 4 h.
Discussion Germination of fully imbibed dormant E. crus-galli seeds can be stimulated by a brief exposure to high temperature (46°C) followed by transfer of the seeds to a suitable temperature for germination (20/30°C, 16 and 8 h, respectively). The stimulation is maximal after short heat treatments (1 to 2 h); the stimulation decreases with exposure of the seeds to 46°C for 4 h, and is prevented after longer ejtposures to the higher temperature [Taylorson and Di Nola, In Press]. Treatment of the seeds at high temperature and length of the exposure appear therefore to act as a signal for regulation of subsequent germination. Previous studies showed that a brief exposure to a low temperature (5°C) during imbibition strongly affected metabolism in membranes and ultrastructural changes in the embryonic axis cells of Pisum sativum 1. (pea) seeds, ultimately delaying subsequent germination at a favorable temperature [Di Nola and Mayer, 1985, 1986a, 1986 b, 1987; Hodson et aI., 1987]. These experiments also showed that the cold temperature induced the formation of several membrane proteins and inhibited the disappearance of others. These changes were still evident, even after prolonged exposure of the seed to a suitable temperature for germination [Di Nola and Mayer, 1987]. High temperature treatments affecting germination may have an effect on the composition of soluble and membrane-bound proteins in cells of E. crus-galli seeds. We found that the transition from dormancy to non-dormancy during a brief exposure to high temperature is accompanied in E. crus-galli seeds by a remarkable increase in content of soluble and membrane-bound proteins already present after imbibition at 35°C. These changes are, however, gradually reversed by exposing the seeds to the higher temperature for longer periods of time. Prolonged treatment at 46 °C induced formation of new proteins. These results suggest that the transition from a dormant to a non-dormant state preceeding germination involves some metabolic changes in soluble and membrane-bound proteins in the cells of E. crus-galli seeds. These changes are in fact no longer evident when the stimulatory effect turns into an inhibitory one. The inhibitory effect of a prolonged exposure to high temperature could be due to changes in the turnover of proteins, combined with changes in the moisture content of the seeds. It is possible that some of the proteins induced by the prolonged heat treatment have a role in promoting dormancy and preventing germination during subsequent exposure to the favorable temperature. Further studies are required in order to determine the metabolic changes which occur in soluble and membrane-bound proteins, respectively, in the cells upon exposure to high temperature during imbibition. These changes could regulate the metabolism of the seed cells, determining their response to external factors and, ultimately, their germination behavior.
References BASHA, S. M. M. and L. BEEVERS: The development of proteolytic activity and protein degradation during germination. Planta 124, 77 -87 (1975). DELSENY, M., L. ASPART, and R. COOKE: Studies on ribonucleic acids during radish seed germination: a search for potential regulatory mechanisms. Isr.J. Bot. 29, 246-258 (1980/81). DI NOLA, L., E. BERLIN, and R. B. TAYLORSON: Thermotropic properties of cellular membranes in dormant and non-dormant barnyardgrass [Echinochloa crus-galli (L.) Beauv.] seeds. Submitted to Plant Physiol. DI NOLA, L. and A. M. MAYER: Effect of temperature of imbibition on phospholipid metabolism in pea embryonic axes. Phytochemistry 24,2549-2554 (1985). - - Effect of temperature on glycerol metabolism in membranes and on phospholipases C and D of germinating pea embryos. Phytochemistry 25,2255-2259 (1986 a}. - - The effect of temperature of imbibition and germination on the lipid and fatty acid content and composition in phospholipids of embryonic axes from pea seeds. Phytochemistry 25, 2725-2731 (1986 b). - - Ethanolamine incorporation into the membranes of pea embryonic axes during germination. Phytochemistry 26, 1591-1593 (1987). DI NOLA, L. and R. B. TAYLORSON: Ultrastructural changes in radicles of barnyardgrass [Echinochloa crus-galli (L.) Beauv.] seeds during the transition from dormancy to germination. Isr. J. Bot. (In Press). GUY, C. L. and D. HASKELL: Induction of freezing tolerance in spinach is associated with the synthesis of cold acclimation induced proteins. Plant Physiol. 84, 872-878 (1987). HODSON, M. J., L. DI NOLA, and A. M. MAYER: The effect of changing temperatures during imbibition on ultrastructure in germinating pea embryonic radicles. J. Exp. Bot. 38, 525-534 (1987). HUANG, W. Z. and A. I. HSIAO: Factors affecting seed dormancy and germination of Johnsongrass, Sorghum halepense (L.) Pers. Weed Res. 27, 1-12 (1987). KIMPEL, J. A. and J. L. KEY: Heat shock in plants. Trends Biochem. Sci. 10, 353-357 (1985). LAEMMLI, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 (1970). LOWRY, O. H., N. J. ROSEBROUGH, L. A. FARIl, and R. J. RANDALL: Protein measurement with the folin reagent. J. BioI. Chem. 193, 265-275 (1951). MAYER, A. M. and I. MARBACH: Biochemistry of the transition from resting to germinating state in seeds. Progress in Phytochem. 7, 95-136 (1981). MEZA-BASSO, L., M. ALBERDI, M. RAYNAL, M. FERJlERo-CADINANOS, and M. DELSNEY: Changes in protein synthesis in rapeseed (Brassica napus) seedlings during a low temperature treatment. Plant Physiol. 82, 733-738 {1986}. NEUMANN, D., U. NIEDEN, R. MANTEUFFEL, G. WALTER, K.-D. SCHARF, and L. NOVER: Intracellular localization of heat shock proteins in tomato cell cultures. Eur. J. Cell. BioI. 43, 71-81 (1987). OSBORNE, D. J., R. SHARON, and R. BEN-ISHAI: Studies on DNA integrity and DNA repair in germinating embryos of rye (Secale cereale). Isr. J. Bot. 29, 259-272 (1980/81). PROBERT, R. J., R. D. SMITH, and P. BIRCH: Germination responses to light and alternating temperatures in european populations of Dactylis glomerata L. V. The principle components of the alternating temperature requirement. New Phytol. 102, 133 -142 (1986).
Seed proteins ROBERTS, E. H. and S. TOTIERDELL: Seed dormancy in Rumex sp. in response to environmental factors. Plant Cell Environ. 4, 97 -106 (1981). TAYLORSON, R. B. and L. DI NOLA: Increased phytochrome responsiveness and a high temperature transition in barnyardgrass (Echinochloa crus·galli) seed dormancy. Weed Sci. (In Press).
121
WRAY, W., T. BOULIKAS, V. P. WRAY, and R. HANCOCK: Silver staining of proteins in polyacrylamide gels. Anal. Biochem. 118, 197 -203 (1981). YOULE, R. J. and A. H. C. HUANG: Protein bodies from the endosperm of castor bean. Plant Physiol. 58, 703 -709 (1976).