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Phosphoglycerate mutase activity and mRNA levels during germination of maize embryos
Plant Science, 89 (1993) 147-151 Elsevier Scientific Publishers Ireland Ltd.
147
Phosphoglycerate mutase activity and mRNA levels during germination of maize embryos X a v i e r G r a f m , C r i s t i n a Broceflo, J u d i t G a r r i g a , P a b l o P6rez de la O s s a a n d F e r n a n d o Climent Unitat de Bioquimica, Facultat de Medicina, Universitat de Barcelona (Spain) (Received July 9th, 1992; revision received December 24th, 1992; accepted December 24th, 1992)
Phosphoglycerate mutase activity and mRNA levels during maize embryo germination were studied. Enzyme levels, activity and mRNA were found to be markedly increased during germination although in dry embryos phosphoglycerate mutase was detected in the absence of mRNA. The results here reported demonstrate that the enzyme was present in dry embryos but new synthesis of the phosphoglycerate mutase was required to permit the progress of germination.
Key words: phosphoglycerate mutase; Zea mays; germination; mRNA
Introduction
Seed germination and early seedling growth involve an increase in several metabolic processes including, among others, oxygen consumption, ATP synthesis and storage mobilization. These processes are concomitant with an increase in the activity of several enzymes which begins with the imbibition of dry seeds. The transition of seeds from a dormant state to germination is associated with both an increase in respiratory enzymes and an increase in the activity of the enzymes involved in reserve mobilization (for review, see Ref. 1). Phosphoglycerate mutase (PGAM, EC 5.4.2.1) is a glycolytic enzyme which catalyses the interconversion of glycerate 3-phosphate and glycerate 2-phosphate. This enzyme is found in two different types: one that requires 2,3-bisphosphoglycerate Correspondence to: Dr. Fernando Climent, Unitat de Bioquimica, Facultat de Medicina, Universitat de Barcelona, Casanovas 143, 08036 Barcelona, Spain. Abbreviations." PGAM, phosphoglycerate mutase; SSC, 0.15 M NaC1, 0.015 M sodium citrate (pH 7.0); SDS, sodium dodecyl sulfate
as a cofactor and the other which does not. The cofactor-dependent enzyme is present in all vertebrates, most invertebrates, and some fungi and bacteria. The independent enzyme is present in all plants, algae and some invertebrates, fungi and bacteria [2,3]. The two PGAM types not only differ in their distribution but also in their structure, reaction mechanism and kinetic properties (see Ref. 4 for a review). In mammals, cofactor-dependent PGAM is present as a dimer resulting from homodimeric and heterodimeric combinations of two different subunits which each possess a molecular mass of 30 kDa. The cDNA of the two subunits have been cloned and sequenced [5-9], and the human muscle specific gene has also been isolated [10,11]. Moreover, mammalian cofactor-dependent PGAM has been shown to be evolutionarily related to a family of acid phosphatases [12,13]. In contrast to cofactor-dependent PGAM, the cofactor-independent PGAM is a monomeric enzyme with a molecular mass of 62 kDa and recently we cloned its cDNA from maize. Its amino acid sequence revealed no homology with
0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
148
the cofactor-dependent type but a partial identity with alkaline phosphatase was detected which suggests a relationship with alkaline phosphatase [ 14]. The lack of homology between cofactor-dependent and -independent PGAM strongly suggests that both enzymes are evolutionarily unrelated and arose from two independent ancestral genes. While seeking a good source for PGAM purification, we found a great increase in the specific activity of this enzyme in maize seed embryos during germination and similar results were obtained by Botha and Dennis on the endosperm of developing and germinating Ricinus communis seeds [15]. However, whether PGAM activity is due to activation of pre-existing PGAM in dry seeds or to enzyme synthesis remains unknown. We had previously purified maize PGAM and its antibodies [16]. With the aid of both and with maize PGAM cDNA, levels of PGAM and PGAM mRNA in maize embryos during germination and early seedling growth were studied. We conclude that both PGAM activation and synthesis occur in the first days of maize seed germination. Material and Methods
Enzymes, substrates and cofactors were purchased from Boehringer Manheim, Sigma or Pharmacia and [ot-32p]CTP from Amersham. Purified specific antibodies against maize seed PGAM were obtained in our laboratory as previously described [16]. Peroxidase-conjugated swine anti-rabbit IgG was from Dakopatt and Diaminobenzidine was from Sigma. All other reagents were analytical grade.
Plant material Maize seeds (Z. mays L. var. W64A) were germinated at 30°C in a humid chamber in the dark. During germination, seeds were collected at different times (0-72 h) and their embryos excised and frozen in liquid nitrogen. The frozen embryos were powdered in a steel mortar and approx. 100 mg of the frozen material were homogenized in a potter with 1 ml of 20 mM phosphate buffer (pH 7.5) for protein measurements or in l ml of Z6 buffer (pH 7) (8 M guanidinium hydrochloride, 20 mM MES and 20 mM EDTA) for RNA extrac-
tion. After centrifugation the supernatant was removed, kept on ice, and immediately processed.
Enzyme assays and protein determinations PGAM activity was assayed by coupling glycerate-2-phosphate formation to NADH oxidation as described previously [16]. One unit of enzyme is defined as the catalytic activity which converts 1/zmol substrate/min under assay conditions. Protein concentration was determined as described by Bradford [17] using bovine serum albumin as a standard. Isozyme electrophoresis in acetate cellulose was performed according to Andr6s et al. [18] using 1.5 mU of each PGAM sample loaded in each lane. Western blot experiments were performed as previously reported [19]. Fifty #g of protein extracts were used in all experiments. Northern blot analysis Total RNA was prepared essentially according to Logemann et al. [20]. Twenty micrograms of total RNA were electrophoresed in a 1.5%denaturing formaldehyde agarose gel, transferred to a Hybond-N nylon sheet in the presence of 10 x SSC for 24 h and hybridized with [a32p]CTP-radiolabelled maize PGAM cDNA. The filters were washed with 0.1 x SSC, 0.1% SDS at 60°C as reported previously [14]. Densitometry analysis Densitometry analyses of nitrocellulose sheets, acetate cellulose strips and X-ray films were performed with a Shimadzu densitometer model CS-9000. Results and Discussion
Increased enzyme activity during germination could be due to activation of pre-existing enzymes or to enzyme synthesis. With the use of PGAM antibodies and PGAM cDNA the two processes were studied to ascertain whether either or both are involved in the PGAM activity increase. Moreover, possible translation of pre-existing PGAM transcripts due to the activation of protein synthesis machinery was also investigated. Levels of PGAM specific activity during the
149
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Fig. 1. Changes in PGAM, P G A M activity and P G A M m R N A levels during maize germination. I , P G A M activity; 0, densitometry analysis of Western blot experiments for P G A M immuno-detennination; &, densitometry analysis of Northern blot experiments for PGAM m R N A determination.
early days of germination (Fig. 1) show that PGAM activity is detected in dry embryos and, following weak declination, activity increases twofold after 72 h of germination. Western blot experiments showed similar results in different independent experiments. Densitometric analysis of an immunoblot experiment (Fig. 1) shows a significant increase in the intensity of PGAM from 24
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to 72 h. The similar profiles of PGAM activity and Western blot experiments suggest that changes in PGAM activity detected during germination are a consequence of increased PGAM concentration. The weak declination of PGAM activity before 24 h of germination is not surprising since an increase in the ratio between total protein and PGAM caused by faster synthesis of other proteins
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Fig. 2. Acetate cellulose electrophoresis of maize P G A M isozymes during germination. Numbers correspond to the hours of germination.
150
could occur, although inactivation of pre-existing enzymes cannot be ruled out. As increased enzyme concentration is usually a consequence of increased gene expression, we carfled out Northern blot analyses to determine PGAM mRNA levels during germination. Figure 1 shows the densitometric profiles of the Northern blots. In dry embryos, PGAM mRNA was not detected and after 12 h of germination, levels sharply increased and later decreased. Etidium bromide-stained RNA before transference shows the presence of similar amounts of total RNA in each lane (data not shown). Comparison among PGAM activity, PGAM concentration measurements and PGAM mRNA levels (Fig. 1) shows that PGAM activity induction occurred after PGAM mRNA increase and that during the first day of germination transcription takes place without increase in either PGAM activity or PGAM concentration. As shown in Fig. 2, maize PGAM is present in two enzymatic forms, probably isozymes [16], which can be separated by acetate cellulose electrophoresis. These forms were found in all maize tissues studied (coleoptile, root, endosperm and dry embryos) and the proportion between both electrophoretic bands was always very similar in the four tissues (data not shown). It has been reported that intracellular location of PGAM depends on the plant type. In some plants PGAM is present only in cytosol, whereas in others it appears in both plastids and cytosol [15]. Although the presence of the two maize isoforms in the same proportion in all tissues studied might suggest that both forms are cytosolic in maize, results are not conclusive since a significant proportion of plastids are disrupted during the homogenization of tissues. Purification of cellular organelles and PGAM determination in these organelles are necessary to determine the intracellular location of PGAM isozymes. A significant increase in the ratio between the higher and lower mobility forms during germination is observed. Densitometry analyses showed that this ratio varied from 1.37 in dry embryos to 2.33 at 72 h germination (data not shown), which corresponds to an increase of 70%. The PGAM isoforms cannot be separated by SDS polyacryl-
amide electrophoresis [16] and possess common antigenic determinants, since antibodies raised against purified low mobility isoform recognized only a band in Western blot and are able to immunoprecipitate PGAM activity completely [16]. These results suggest a high degree of homology between both isoforms. Although the reasons for the increase in the high-mobility PGAM isoform are difficult to discern, the presence of two groups of bands on S I protection assays previously reported [14] suggest that two different PGAM genes may be present in the maize genome. Therefore, these different genes could give rise to different PGAM mRNA species of very similar length and they could cross-hybridize with the cDNA used in the experiments. Thus, the mRNA levels here reported could represent the total contents of PGAM mRNA. Further cloning of the other PGAM genes is necessary to clarify the changes in the levels of the two PGAM isoforms. The data here reported demonstrate that the first requirements for PGAM are supplied by preexisting molecules. Pre-existing PGAM is sufficient for the first steps of germination, but new synthesis of the enzyme is necessary to permit the progress of germination and early growth. In situ hybridization studies in developing and germinating maize seeds are in progress to seek specific increases in the level of PGAM transcripts associated with some new developing tissues. Furthermore, the cloning of maize PGAM gene and analysis of its promoter might allow for study of the regulation of this gene in relation to some regulatory processes such as maturation and germination of the seed.
Acknowledgments This work has been supported by CICYT (Grant PB88-0561). We thank P. Puigdomenech for helpful discussions and critical reading of the manuscript.
References 1 J.D. Bewley and M. Black, Seeds: Physiology of Development and Germination, Plenum Press, New York, London, 1985, pp. 135-173. 2 J. Carreras, J. Mezquita, J. Bosch, R. Bartrons and G.
151
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7
8
9
10
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Pons, Phylogeny and ontogeny of the phosphoglycerate mutases-IV. Distribution of glycerate-2,3,P 2 dependent and independent phosphoglycerate mutases in algae, fungi, plants and animals. Comp. Biochem. Physiol., 71B (1982) 591-597. C.M. Johnson and N.C. Price, Do metal ions promote the re-activation of the 2,3-bisphosphoglycerate-independent phosphoglycerate mutases? Biochem. J., 252 (1988) 111-117. L. Fothergill-Gilmore and H. Watson, The phosphoglycerate mutases. Adv. Enzymol., 62 (1989) 227-313. S. Shanske, S. Sakoda, M.A. Hermodson, S. DiMauro and E.A. Schon, Isolation of a cDNA encoding the muscle-specific subunit of human phosphoglycerate mutase. J. Biol. Chem., 262 (1987) 14612-14617. J. Castella-Escol~., L. Montoliu, G. Pons, P. Puigdomenech, M. Cohen-Solal, J. Carreras, J. Rigau and F. Climent, Sequence of rat skeletal muscle phosphoglycerate mutase cDNA. Biochem. Biophys. Res. Commun., 165 (1989) 1345-1351. S. Sakoda, S. Shanske, S. DiMauro and E. Schon, Isolation of a cDNA encoding the B isozyme of human phosphoglycerate mutase (PGAM) and characterization of the PGAM gene family. J. Biol. Chem., 263 (1988) 16899-16905. K. Uchida, cDNA encoding type B subunit of rat phosphoglycerate mutase: its isolation and nucleotide sequence. Arch. Biochem. Biophys., 288 (1991) 558-561. J.M. Urena, X. Grana, L. de Lecea, P. Ruiz, J. Castell~, J. Carreras, G. Pons and F. Climent, Isolation and sequencing of a cDNA encoding the B isozyme of rat phosphoglycerate mutase. Gene, 113 (1992) 281-282. S. Tsujino, S. Sakoda, R. Mizuno, T. Kobayashi, T. Suzuki, S. Kishimoto, S. Shanske, S. DiMauro and E.A. Schon, Structure of the gene encoding the muscle-specific subunit of human phosphoglycerate mutase. J. Biol. Chem., 264 (1989) 15334-15337. J. Castell~.-Escol~i, D.M. Ojcius, P. LeBoulch, V. Joulin, Y. Blouquit, M.C. Garel, C. Valentin, R. Rosa, F. Climent-Romeo and M. Cohen-Solal, Isolation and characterization of the gene encoding the muscle-specific isozyme of human phosphoglycerate mutase. Gene, 91 (1990) 225-232.
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17
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J.F. Bazan, R.J. Fleterick and S.J. Pilkis, Evolution of a bifunctional enzyme: 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Proc. Natl. Acad. Sci. USA, 86(1989) 9642-9646. J.F. Bazan and R.J. Fletterick, Tertiary Structure Modeling of 6-phosphofructo-2-kinase/Fructose 2,6-bisphosphatase: structural, functional and evolutionary design of a bifunctional enzyme in: S.J. Pilkis (Ed.), Fructose 2,6Bisphosphate, CRC Press, Boca Raton, 1990, pp. 125-171. X. Graxaa, L. de Lecea, R. El-Maghrabi, J. Urena, C. Caellas, J. Carreras, P. Puigdomenech, S. Pilkis and F. Climent, Cloning and sequencing of a cDNA encoding 2,3-bisphosphoglycerate-independent phosphoglycerate mutase from maize. J. Biol. Chem., 267 (1992) 12797-12803. F.C. Botha and D.T. Dennis, Phosphoglycerate mutase activity and concentration in the endosperm of developing and germinating Ricinus communis seeds. Can. J. Bot., 65 (1987) 1908-1912. X. Gra~a, J.M. Urena, D. Ludevid, J. Carreras and F. Climent, Purification, characterization and immunological properties of 2,3-bisphosphoglycerate-independent phosphoglycerate mutase from maize (Zea mays) seeds. Eur. J. Biochem., 186 (1989) 149-153. M. Bradford, A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72 (1976) 248-254. V. Andres, R. Cuss6 and J. Carreras, Distribution and developmental transition of phoshoglycerate mutase and creatine phosphokinase isozymes in rat muscles of different fiber-type composition. Differentiation, 41 (1989) 72-77. J. Castell~_, J. Urena, D. Ludevid, J. Carreras and F. Climent, Immunological properties of rat phosphoglycerate mutase isozymes. Biochim. Biophys. Acta, 956 (1988) 97-102. J. Logemann, J. Schell and L. Willmitzer, Improved method for the isolation of RNA from plant tissues. Anal. Biochem., 163 (1987) 16-20.
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Phosphoglycerate mutase activity and mRNA levels during germination of maize embryos X a v i e r G r a f m , C r i s t i n a Broceflo, J u d i t G a r r i g a , P a b l o P6rez de la O s s a a n d F e r n a n d o Climent Unitat de Bioquimica, Facultat de Medicina, Universitat de Barcelona (Spain) (Received July 9th, 1992; revision received December 24th, 1992; accepted December 24th, 1992)
Phosphoglycerate mutase activity and mRNA levels during maize embryo germination were studied. Enzyme levels, activity and mRNA were found to be markedly increased during germination although in dry embryos phosphoglycerate mutase was detected in the absence of mRNA. The results here reported demonstrate that the enzyme was present in dry embryos but new synthesis of the phosphoglycerate mutase was required to permit the progress of germination.
Key words: phosphoglycerate mutase; Zea mays; germination; mRNA
Introduction
Seed germination and early seedling growth involve an increase in several metabolic processes including, among others, oxygen consumption, ATP synthesis and storage mobilization. These processes are concomitant with an increase in the activity of several enzymes which begins with the imbibition of dry seeds. The transition of seeds from a dormant state to germination is associated with both an increase in respiratory enzymes and an increase in the activity of the enzymes involved in reserve mobilization (for review, see Ref. 1). Phosphoglycerate mutase (PGAM, EC 5.4.2.1) is a glycolytic enzyme which catalyses the interconversion of glycerate 3-phosphate and glycerate 2-phosphate. This enzyme is found in two different types: one that requires 2,3-bisphosphoglycerate Correspondence to: Dr. Fernando Climent, Unitat de Bioquimica, Facultat de Medicina, Universitat de Barcelona, Casanovas 143, 08036 Barcelona, Spain. Abbreviations." PGAM, phosphoglycerate mutase; SSC, 0.15 M NaC1, 0.015 M sodium citrate (pH 7.0); SDS, sodium dodecyl sulfate
as a cofactor and the other which does not. The cofactor-dependent enzyme is present in all vertebrates, most invertebrates, and some fungi and bacteria. The independent enzyme is present in all plants, algae and some invertebrates, fungi and bacteria [2,3]. The two PGAM types not only differ in their distribution but also in their structure, reaction mechanism and kinetic properties (see Ref. 4 for a review). In mammals, cofactor-dependent PGAM is present as a dimer resulting from homodimeric and heterodimeric combinations of two different subunits which each possess a molecular mass of 30 kDa. The cDNA of the two subunits have been cloned and sequenced [5-9], and the human muscle specific gene has also been isolated [10,11]. Moreover, mammalian cofactor-dependent PGAM has been shown to be evolutionarily related to a family of acid phosphatases [12,13]. In contrast to cofactor-dependent PGAM, the cofactor-independent PGAM is a monomeric enzyme with a molecular mass of 62 kDa and recently we cloned its cDNA from maize. Its amino acid sequence revealed no homology with
0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
148
the cofactor-dependent type but a partial identity with alkaline phosphatase was detected which suggests a relationship with alkaline phosphatase [ 14]. The lack of homology between cofactor-dependent and -independent PGAM strongly suggests that both enzymes are evolutionarily unrelated and arose from two independent ancestral genes. While seeking a good source for PGAM purification, we found a great increase in the specific activity of this enzyme in maize seed embryos during germination and similar results were obtained by Botha and Dennis on the endosperm of developing and germinating Ricinus communis seeds [15]. However, whether PGAM activity is due to activation of pre-existing PGAM in dry seeds or to enzyme synthesis remains unknown. We had previously purified maize PGAM and its antibodies [16]. With the aid of both and with maize PGAM cDNA, levels of PGAM and PGAM mRNA in maize embryos during germination and early seedling growth were studied. We conclude that both PGAM activation and synthesis occur in the first days of maize seed germination. Material and Methods
Enzymes, substrates and cofactors were purchased from Boehringer Manheim, Sigma or Pharmacia and [ot-32p]CTP from Amersham. Purified specific antibodies against maize seed PGAM were obtained in our laboratory as previously described [16]. Peroxidase-conjugated swine anti-rabbit IgG was from Dakopatt and Diaminobenzidine was from Sigma. All other reagents were analytical grade.
Plant material Maize seeds (Z. mays L. var. W64A) were germinated at 30°C in a humid chamber in the dark. During germination, seeds were collected at different times (0-72 h) and their embryos excised and frozen in liquid nitrogen. The frozen embryos were powdered in a steel mortar and approx. 100 mg of the frozen material were homogenized in a potter with 1 ml of 20 mM phosphate buffer (pH 7.5) for protein measurements or in l ml of Z6 buffer (pH 7) (8 M guanidinium hydrochloride, 20 mM MES and 20 mM EDTA) for RNA extrac-
tion. After centrifugation the supernatant was removed, kept on ice, and immediately processed.
Enzyme assays and protein determinations PGAM activity was assayed by coupling glycerate-2-phosphate formation to NADH oxidation as described previously [16]. One unit of enzyme is defined as the catalytic activity which converts 1/zmol substrate/min under assay conditions. Protein concentration was determined as described by Bradford [17] using bovine serum albumin as a standard. Isozyme electrophoresis in acetate cellulose was performed according to Andr6s et al. [18] using 1.5 mU of each PGAM sample loaded in each lane. Western blot experiments were performed as previously reported [19]. Fifty #g of protein extracts were used in all experiments. Northern blot analysis Total RNA was prepared essentially according to Logemann et al. [20]. Twenty micrograms of total RNA were electrophoresed in a 1.5%denaturing formaldehyde agarose gel, transferred to a Hybond-N nylon sheet in the presence of 10 x SSC for 24 h and hybridized with [a32p]CTP-radiolabelled maize PGAM cDNA. The filters were washed with 0.1 x SSC, 0.1% SDS at 60°C as reported previously [14]. Densitometry analysis Densitometry analyses of nitrocellulose sheets, acetate cellulose strips and X-ray films were performed with a Shimadzu densitometer model CS-9000. Results and Discussion
Increased enzyme activity during germination could be due to activation of pre-existing enzymes or to enzyme synthesis. With the use of PGAM antibodies and PGAM cDNA the two processes were studied to ascertain whether either or both are involved in the PGAM activity increase. Moreover, possible translation of pre-existing PGAM transcripts due to the activation of protein synthesis machinery was also investigated. Levels of PGAM specific activity during the
149
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Fig. 1. Changes in PGAM, P G A M activity and P G A M m R N A levels during maize germination. I , P G A M activity; 0, densitometry analysis of Western blot experiments for P G A M immuno-detennination; &, densitometry analysis of Northern blot experiments for PGAM m R N A determination.
early days of germination (Fig. 1) show that PGAM activity is detected in dry embryos and, following weak declination, activity increases twofold after 72 h of germination. Western blot experiments showed similar results in different independent experiments. Densitometric analysis of an immunoblot experiment (Fig. 1) shows a significant increase in the intensity of PGAM from 24
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to 72 h. The similar profiles of PGAM activity and Western blot experiments suggest that changes in PGAM activity detected during germination are a consequence of increased PGAM concentration. The weak declination of PGAM activity before 24 h of germination is not surprising since an increase in the ratio between total protein and PGAM caused by faster synthesis of other proteins
12 • . . .~..=.=.=. : . ... ,. . . . .
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Fig. 2. Acetate cellulose electrophoresis of maize P G A M isozymes during germination. Numbers correspond to the hours of germination.
150
could occur, although inactivation of pre-existing enzymes cannot be ruled out. As increased enzyme concentration is usually a consequence of increased gene expression, we carfled out Northern blot analyses to determine PGAM mRNA levels during germination. Figure 1 shows the densitometric profiles of the Northern blots. In dry embryos, PGAM mRNA was not detected and after 12 h of germination, levels sharply increased and later decreased. Etidium bromide-stained RNA before transference shows the presence of similar amounts of total RNA in each lane (data not shown). Comparison among PGAM activity, PGAM concentration measurements and PGAM mRNA levels (Fig. 1) shows that PGAM activity induction occurred after PGAM mRNA increase and that during the first day of germination transcription takes place without increase in either PGAM activity or PGAM concentration. As shown in Fig. 2, maize PGAM is present in two enzymatic forms, probably isozymes [16], which can be separated by acetate cellulose electrophoresis. These forms were found in all maize tissues studied (coleoptile, root, endosperm and dry embryos) and the proportion between both electrophoretic bands was always very similar in the four tissues (data not shown). It has been reported that intracellular location of PGAM depends on the plant type. In some plants PGAM is present only in cytosol, whereas in others it appears in both plastids and cytosol [15]. Although the presence of the two maize isoforms in the same proportion in all tissues studied might suggest that both forms are cytosolic in maize, results are not conclusive since a significant proportion of plastids are disrupted during the homogenization of tissues. Purification of cellular organelles and PGAM determination in these organelles are necessary to determine the intracellular location of PGAM isozymes. A significant increase in the ratio between the higher and lower mobility forms during germination is observed. Densitometry analyses showed that this ratio varied from 1.37 in dry embryos to 2.33 at 72 h germination (data not shown), which corresponds to an increase of 70%. The PGAM isoforms cannot be separated by SDS polyacryl-
amide electrophoresis [16] and possess common antigenic determinants, since antibodies raised against purified low mobility isoform recognized only a band in Western blot and are able to immunoprecipitate PGAM activity completely [16]. These results suggest a high degree of homology between both isoforms. Although the reasons for the increase in the high-mobility PGAM isoform are difficult to discern, the presence of two groups of bands on S I protection assays previously reported [14] suggest that two different PGAM genes may be present in the maize genome. Therefore, these different genes could give rise to different PGAM mRNA species of very similar length and they could cross-hybridize with the cDNA used in the experiments. Thus, the mRNA levels here reported could represent the total contents of PGAM mRNA. Further cloning of the other PGAM genes is necessary to clarify the changes in the levels of the two PGAM isoforms. The data here reported demonstrate that the first requirements for PGAM are supplied by preexisting molecules. Pre-existing PGAM is sufficient for the first steps of germination, but new synthesis of the enzyme is necessary to permit the progress of germination and early growth. In situ hybridization studies in developing and germinating maize seeds are in progress to seek specific increases in the level of PGAM transcripts associated with some new developing tissues. Furthermore, the cloning of maize PGAM gene and analysis of its promoter might allow for study of the regulation of this gene in relation to some regulatory processes such as maturation and germination of the seed.
Acknowledgments This work has been supported by CICYT (Grant PB88-0561). We thank P. Puigdomenech for helpful discussions and critical reading of the manuscript.
References 1 J.D. Bewley and M. Black, Seeds: Physiology of Development and Germination, Plenum Press, New York, London, 1985, pp. 135-173. 2 J. Carreras, J. Mezquita, J. Bosch, R. Bartrons and G.
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8
9
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