Planta 9 Springer-Verlag 1989
Planta (1989) 178:421-424
Effects of 2,4-dinitrophenol and anoxia on the inorganic-pyrophosphate content of the spadix of Arum maculatum and the root apices of Pisum sativum Jane E. Dancer* and Tom ap Rees Botany School, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK
Abstract, This work was done to determine whether the inorganic-pyrophosphate (PPi) content of plant tissues changes when the rate of glycolysis is altered. Treatment of excised clubs of the spadix of Arum maeulatum L. and root apices of Pisum sativum L. with 2,4-dinitrophenol increased the rates of respiration but had no detectable effects on PPi contents. When the two tissues were subjected to up to 60 min anoxia, no changes in PPi were detected. Anoxia was shown to lead to a fall in ATP and concomitant rises in ADP and AMP in pea roots. It is argued (i) that variation in the rate of glycolysis was not accompanied by detectable changes in PPi content, (ii) that this observation does not favour the view that pyrophosphate fructose 6-phosphate 1-phosphotransferase mediates appreciable entry into glycolysis, and (iii) that PPi content can be maintained when respiratory-chain phosphorylation is inhibited. Key words: Anoxia Arum 2,4-Dinitrophenol Pisum (pyrophosphate) Pyrophosphate, inorganic - Root apex (PPi) content - Spadix
Introduction
We know that pyrophosphate:fructose 6-phosphate 1-phosphotransferase [PFK(PPi) : EC 2.7.1.90] is widely distributed with high activity in plants, that it catalyses a near-equilibrium reaction in vivo and is confined to the cytosol, yet we do not know the function of this enzyme (Weiner et al. 1987; see ap Rees 1988 for a review). * Present address: Botanisches Institut der Universitfit Bayreuth, Lehrstuhl Pflanzenphysiologie, Postfach 1012 51, D-8580 Bayreuth, FRG
FW=fresh weight; PFK(PPi)=pyrophosphate fructose 6-phosphate 1-phosphotransferase; PPi=inorganic pyrophosphate Abbreviations:
Suggestions that it is an important point of entry of substrate into cytosolic glycolysis continue to be made (Black et al. 1987). In general, the evidence for this view is not conclusive (see ap Rees and Dancer 1987 for a review). The best of such evidence are the demonstrations that illumination of guard-cell protoplasts is accompanied by a rise in fructose-2,6-bisphosphate and a fall in glucose 6-phosphate (Hedrich et al. 1985), and that fructose-2,6-bisphosphate rises when the metabolism of storage tissue is increased by slicing (Van Schaftingen and Hers 1983). However, these observations do not demonstrate a glycolytic role for PFK(PPi). First, hexose 6-phosphate is the substrate for the universally distributed classical glycolytic enzyme, 6-phosphofructokinase, as well as for PFK(PPi). Second, the changes in fructose-2,6-bisphosphate could be associated with increased production of inorganic pyrophosphate (PPi) by PFK(PPi), and the use of this PPi in the breakdown of sucrose via sucrose synthase and uridine 5'-diphosphoglucose pyrophosphorylase (ap Rees and Dancer 1987). We suggest that in determining whether PFK(PPi) mediates entry into glycolysis the key substrate to measure is PPi as this is not shared with 6-phosphofructokinase. Accordingly we have determined whether variation in the rate of glycolysis in the clubs of the spadix of Arum maeulatum and in the root apices of Pisum sativum is accompanied by changes in PPi content. We used the uncoupler, 2,4-dinitrophenol, and anoxia to vary the rate of glycolysis. In some experiments we also measured hexose phosphates and adenine nucleotides. Materials and methods Enzymes were from Boehringer, Lewes, Sussex, UK, except that PFK(PPi) was from Sigma (London) Chemical Co., Poole, Dorset, UK. Substrates were from Sigma except that Materials.
422
J.E. Dancer and T. ap Rees : 2,4-Dinitrophenol, anoxia and pyrophosphate content
ATP, N A D and N A D P were from Boehringer. Peas (Pisum sativum L. cv. Kelvedon Wonder; Sanders Ltd., Cambridge, UK) were grown as described by Dancer and ap Rees (1988). The apical 2 cm of the roots of 5-d-old seedlings were excised and used within 10 min. Complete inflorescences ofArum maculatum L. were collected from local natural sites. Within an hour of collection, the clubs, the swollen ends of the appendices, were excised and used at once. The stages of development (c~, /~, 7) have been defined (ap Rees et al. 1976). Measurement of gas exchange. Warburg's direct manometric method was used at 25 ~ C. Replicate samples of 20 pea root apices (0.6 g fresh weight, FW) were suspended in 2.5 ml 0.02 M KI-I2PO4, pH 5.2, or 2.5 ml 25 I~M 2,4-dinitrophenol in 0.02 M KH2PO4 adjusted to pH 5.2. For the experiments with Arum, replicate samples were made by cutting clubs into halves, or quarters, longitudinally. Each half or quarter club was stood in the centre well of a manometer flask in 0.1 ml 0.02 M KH2PO4, pH 5.2, or 0.1 ml 5 m M 2,4-dinitrophenol in 0.02 M KH2PO,, pH 5.2. The alkali was placed in the side-arm. Anoxia was achieved by gassing the flasks with nitrogen for 7-8 min. Control samples were run in each experiment. These samples were treated exactly as the experimental samples, except that 0,02 M KH2PO4 was used instead of the 2,4-dinitrophenol and flasks were gassed with air not nitrogen. Rates of the treated samples are compared with those of the control samples measured at the same time. For pea roots, comparisons were made between replicate samples of the same batch of roots. For Arum, comparisons were made between portions, almost always halves, of the same club. Tests of significance were made by the paired sample " t " test: Fisher's P values >0.05 are given as N.S. (not significant). Measurements ofmetabolites. For experiments with 2,4-dinitrophenol, samples of tissue were prepared and treated as for the manometry. Pea root apices were freeze-clamped after 30 rain in the uncoupler and the Arum clubs after 90 min. Similarly prepared samples of tissue were made for treatment with anoxia and were put in air-tight bags (10 cm long, 4 cm diameter) of dialysis tubing. Each of the latter was tied securely at one end and closed at the other end with a rubber bung fitted with an inlet and an outlet. The bag was flushed with nitrogen or air (control samples) for 7 min and then the taps in the bung were closed so that the bags remained inflated throughout the incubation, which was at 25 ~ C. The treatments were terminated by freeze-clamping the samples whilst they were still inside the bags. Thus metabolism was arrested before oxygen had access to the tissue. Control samples were used as described for manoanetry. The freeze-clamped samples were killed and extracted as described by Dancer and ap Rees (1989). Inorganic pyrophosphate was assayed with PFK(PPi) as described by Dancer and ap Rees (1989). Hexose 6-phosphates were assayed according to Michal (1974a). The initial reaction mixture, 1 ml, contained 50 mM 4-(2-hydroxyethyl)-i-piperazineethanesulphonic acid (Hepes), pH 7.6, 0.2 m M N A D P +, 4 m M MgC12 and 2.8 units glucose 6-phosphate dehydrogenase (EC 1.1.1.49). Once glucose 6-phosphate had been measured, 0.7 unit glucose-6-phosphate isomerase was added to measure fructose 6-phosphate. Fructose-l,6-bisphosphate was assayed as described by Michal (1974b) in a 1-ml reaction mixture of 75 mM Hepes, pH 7.5, 0.2 m M N A D H , 1.7 units glycerol-3phosphate dehydrogenase (EC 1.1.1.8), 5 units triosephosphate isomerase (EC 5.3.1.1) and 0.45 unit aldolase (EC 4.1.2.14). The assay of ATP was according to Trautschold et al. (1974) in a 1-ml reaction mixture of 100 m M triethanolamine, pH 7.6, 4 mM MgCI2, 2 mM glucose, 2 mM N A D P +, 2.8 units glucose6-phosphate dehydrogenase and 1.8 units hexokinase
(EC 2.7.1.1). Both ADP and A M P were measured (Jaworek and Welsch 1974) in a 1-ml reaction mixture of 0A M triethanolamine, pH 7.0, 0.94mM phosphoenolpyruvate, 33.4 m M MgSO~, 0.12 m M KC1, 0.36 mM N A D H , 24 units lactate dehydrogenase (EC 1.1.1.27) and 18 units pyruvate kinase (EC 2.7.1.40); 16 units of adenylate kinase (EC 2.7.4.3) were added subsequently to measure AMP. For each metabolite assayed we carried out recovery experiments on both the control and the experimental samples. For each test we prepared duplicate samples; for Arum these consisted of longitudinal halves of the same club. One sample was freeze-clamped, killed and extracted as we have described. The other sample was treated similarly except that measured amounts of the metabolites in question were added to the sample immediately after freeze-clamping. The amounts added were between one and two times the quantities found in the tissue sample. The percentages of the metabolites recovered were: Arum: PPi, 100; pea root: PPi, 92; fructose 6-phosphate, 83; glucose 6-phosphate, 102; ATP, 95; ADP, 96; AMP, 88.
Results
Effects of 2,4-dinitrophenol. We determined the effects of a range of concentrations of 2,4-dinitrophenol on the oxygen uptake of pea roots and Arum clubs and chose those that produced appreciable effects, i.e. a doubling of the oxygen uptake of the clubs and a 20% increase in that of pea roots (Table 1). In pea roots we showed that CO2 production was increased to a greater extent (35%) than was oxygen uptake. The marked difference in the concentration of 2,4-dinitrophenol required to produce the above effects in the two tissues is ascribed to the different ways in which the uncoupler was supplied to the two tissues. The pea roots were immersed in the 2,4-dinitrophenol whilst only the stalks of the clubs were in contact with the uncoupler. We found, for a large number of independent samples, that, although 2,4-dinitrophenol had an appreciable effect on the rate of respiration, no significant effect on PPi content could be detected (Table 1).
Effects of anoxia. Production of C O 2 by y-stage clubs of Arum maculatum was 2514_++865 gl.h -1. g - a FW. After 30 min anoxia this rate fell to 609 _+ 23: values are means__+_SE for three clubs. Extensive measurements of the PPi content of such clubs after 1, 15 and 60 min anoxia failed to reveal any effect of anoxia on PPi content (Table 2). When pea roots were made anoxic there was a fall in the rate of CO2 production but no change in PPi content was detected (Table 3). Other metabolites did change: ATP fell and there were concomitant increases in A D P and AMP. Fructose-l,6-bisphosphate increased but a fall in hexose 6-phosphates was not demonstrated conclusively.
J.E. Dancer and T. ap Rees: 2,4-Dinitrophenol, anoxia and pyrophosphate content
423
Table 1. Effect of 2,4-dinitrophenol on respiration and PPi content of e-stage clubs of Arum maculatum, and the apical 2 cm of the roots of 5-d-old pea seedlings. Clubs were treated with 2,4-dinitrophenol for 90 min and root apices for 60 min. Data are means _+SE of estimates from the number of samples shown in parenthesis. N.S., not significant Tissue
Treatment
Gas exchange (gl. h 1. g- 1 FW) 02 uptake
Arum club
Control I Dinitrophenol II Fisher's P value for I vs. II
Pea root
Control III Dinitrophenol IV Fisher's P value for III vs. IV
771_+20 (9) 1510_+ 80 (9)
Planta (1989) 178:421-424
Effects of 2,4-dinitrophenol and anoxia on the inorganic-pyrophosphate content of the spadix of Arum maculatum and the root apices of Pisum sativum Jane E. Dancer* and Tom ap Rees Botany School, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK
Abstract, This work was done to determine whether the inorganic-pyrophosphate (PPi) content of plant tissues changes when the rate of glycolysis is altered. Treatment of excised clubs of the spadix of Arum maeulatum L. and root apices of Pisum sativum L. with 2,4-dinitrophenol increased the rates of respiration but had no detectable effects on PPi contents. When the two tissues were subjected to up to 60 min anoxia, no changes in PPi were detected. Anoxia was shown to lead to a fall in ATP and concomitant rises in ADP and AMP in pea roots. It is argued (i) that variation in the rate of glycolysis was not accompanied by detectable changes in PPi content, (ii) that this observation does not favour the view that pyrophosphate fructose 6-phosphate 1-phosphotransferase mediates appreciable entry into glycolysis, and (iii) that PPi content can be maintained when respiratory-chain phosphorylation is inhibited. Key words: Anoxia Arum 2,4-Dinitrophenol Pisum (pyrophosphate) Pyrophosphate, inorganic - Root apex (PPi) content - Spadix
Introduction
We know that pyrophosphate:fructose 6-phosphate 1-phosphotransferase [PFK(PPi) : EC 2.7.1.90] is widely distributed with high activity in plants, that it catalyses a near-equilibrium reaction in vivo and is confined to the cytosol, yet we do not know the function of this enzyme (Weiner et al. 1987; see ap Rees 1988 for a review). * Present address: Botanisches Institut der Universitfit Bayreuth, Lehrstuhl Pflanzenphysiologie, Postfach 1012 51, D-8580 Bayreuth, FRG
FW=fresh weight; PFK(PPi)=pyrophosphate fructose 6-phosphate 1-phosphotransferase; PPi=inorganic pyrophosphate Abbreviations:
Suggestions that it is an important point of entry of substrate into cytosolic glycolysis continue to be made (Black et al. 1987). In general, the evidence for this view is not conclusive (see ap Rees and Dancer 1987 for a review). The best of such evidence are the demonstrations that illumination of guard-cell protoplasts is accompanied by a rise in fructose-2,6-bisphosphate and a fall in glucose 6-phosphate (Hedrich et al. 1985), and that fructose-2,6-bisphosphate rises when the metabolism of storage tissue is increased by slicing (Van Schaftingen and Hers 1983). However, these observations do not demonstrate a glycolytic role for PFK(PPi). First, hexose 6-phosphate is the substrate for the universally distributed classical glycolytic enzyme, 6-phosphofructokinase, as well as for PFK(PPi). Second, the changes in fructose-2,6-bisphosphate could be associated with increased production of inorganic pyrophosphate (PPi) by PFK(PPi), and the use of this PPi in the breakdown of sucrose via sucrose synthase and uridine 5'-diphosphoglucose pyrophosphorylase (ap Rees and Dancer 1987). We suggest that in determining whether PFK(PPi) mediates entry into glycolysis the key substrate to measure is PPi as this is not shared with 6-phosphofructokinase. Accordingly we have determined whether variation in the rate of glycolysis in the clubs of the spadix of Arum maeulatum and in the root apices of Pisum sativum is accompanied by changes in PPi content. We used the uncoupler, 2,4-dinitrophenol, and anoxia to vary the rate of glycolysis. In some experiments we also measured hexose phosphates and adenine nucleotides. Materials and methods Enzymes were from Boehringer, Lewes, Sussex, UK, except that PFK(PPi) was from Sigma (London) Chemical Co., Poole, Dorset, UK. Substrates were from Sigma except that Materials.
422
J.E. Dancer and T. ap Rees : 2,4-Dinitrophenol, anoxia and pyrophosphate content
ATP, N A D and N A D P were from Boehringer. Peas (Pisum sativum L. cv. Kelvedon Wonder; Sanders Ltd., Cambridge, UK) were grown as described by Dancer and ap Rees (1988). The apical 2 cm of the roots of 5-d-old seedlings were excised and used within 10 min. Complete inflorescences ofArum maculatum L. were collected from local natural sites. Within an hour of collection, the clubs, the swollen ends of the appendices, were excised and used at once. The stages of development (c~, /~, 7) have been defined (ap Rees et al. 1976). Measurement of gas exchange. Warburg's direct manometric method was used at 25 ~ C. Replicate samples of 20 pea root apices (0.6 g fresh weight, FW) were suspended in 2.5 ml 0.02 M KI-I2PO4, pH 5.2, or 2.5 ml 25 I~M 2,4-dinitrophenol in 0.02 M KH2PO4 adjusted to pH 5.2. For the experiments with Arum, replicate samples were made by cutting clubs into halves, or quarters, longitudinally. Each half or quarter club was stood in the centre well of a manometer flask in 0.1 ml 0.02 M KH2PO4, pH 5.2, or 0.1 ml 5 m M 2,4-dinitrophenol in 0.02 M KH2PO,, pH 5.2. The alkali was placed in the side-arm. Anoxia was achieved by gassing the flasks with nitrogen for 7-8 min. Control samples were run in each experiment. These samples were treated exactly as the experimental samples, except that 0,02 M KH2PO4 was used instead of the 2,4-dinitrophenol and flasks were gassed with air not nitrogen. Rates of the treated samples are compared with those of the control samples measured at the same time. For pea roots, comparisons were made between replicate samples of the same batch of roots. For Arum, comparisons were made between portions, almost always halves, of the same club. Tests of significance were made by the paired sample " t " test: Fisher's P values >0.05 are given as N.S. (not significant). Measurements ofmetabolites. For experiments with 2,4-dinitrophenol, samples of tissue were prepared and treated as for the manometry. Pea root apices were freeze-clamped after 30 rain in the uncoupler and the Arum clubs after 90 min. Similarly prepared samples of tissue were made for treatment with anoxia and were put in air-tight bags (10 cm long, 4 cm diameter) of dialysis tubing. Each of the latter was tied securely at one end and closed at the other end with a rubber bung fitted with an inlet and an outlet. The bag was flushed with nitrogen or air (control samples) for 7 min and then the taps in the bung were closed so that the bags remained inflated throughout the incubation, which was at 25 ~ C. The treatments were terminated by freeze-clamping the samples whilst they were still inside the bags. Thus metabolism was arrested before oxygen had access to the tissue. Control samples were used as described for manoanetry. The freeze-clamped samples were killed and extracted as described by Dancer and ap Rees (1989). Inorganic pyrophosphate was assayed with PFK(PPi) as described by Dancer and ap Rees (1989). Hexose 6-phosphates were assayed according to Michal (1974a). The initial reaction mixture, 1 ml, contained 50 mM 4-(2-hydroxyethyl)-i-piperazineethanesulphonic acid (Hepes), pH 7.6, 0.2 m M N A D P +, 4 m M MgC12 and 2.8 units glucose 6-phosphate dehydrogenase (EC 1.1.1.49). Once glucose 6-phosphate had been measured, 0.7 unit glucose-6-phosphate isomerase was added to measure fructose 6-phosphate. Fructose-l,6-bisphosphate was assayed as described by Michal (1974b) in a 1-ml reaction mixture of 75 mM Hepes, pH 7.5, 0.2 m M N A D H , 1.7 units glycerol-3phosphate dehydrogenase (EC 1.1.1.8), 5 units triosephosphate isomerase (EC 5.3.1.1) and 0.45 unit aldolase (EC 4.1.2.14). The assay of ATP was according to Trautschold et al. (1974) in a 1-ml reaction mixture of 100 m M triethanolamine, pH 7.6, 4 mM MgCI2, 2 mM glucose, 2 mM N A D P +, 2.8 units glucose6-phosphate dehydrogenase and 1.8 units hexokinase
(EC 2.7.1.1). Both ADP and A M P were measured (Jaworek and Welsch 1974) in a 1-ml reaction mixture of 0A M triethanolamine, pH 7.0, 0.94mM phosphoenolpyruvate, 33.4 m M MgSO~, 0.12 m M KC1, 0.36 mM N A D H , 24 units lactate dehydrogenase (EC 1.1.1.27) and 18 units pyruvate kinase (EC 2.7.1.40); 16 units of adenylate kinase (EC 2.7.4.3) were added subsequently to measure AMP. For each metabolite assayed we carried out recovery experiments on both the control and the experimental samples. For each test we prepared duplicate samples; for Arum these consisted of longitudinal halves of the same club. One sample was freeze-clamped, killed and extracted as we have described. The other sample was treated similarly except that measured amounts of the metabolites in question were added to the sample immediately after freeze-clamping. The amounts added were between one and two times the quantities found in the tissue sample. The percentages of the metabolites recovered were: Arum: PPi, 100; pea root: PPi, 92; fructose 6-phosphate, 83; glucose 6-phosphate, 102; ATP, 95; ADP, 96; AMP, 88.
Results
Effects of 2,4-dinitrophenol. We determined the effects of a range of concentrations of 2,4-dinitrophenol on the oxygen uptake of pea roots and Arum clubs and chose those that produced appreciable effects, i.e. a doubling of the oxygen uptake of the clubs and a 20% increase in that of pea roots (Table 1). In pea roots we showed that CO2 production was increased to a greater extent (35%) than was oxygen uptake. The marked difference in the concentration of 2,4-dinitrophenol required to produce the above effects in the two tissues is ascribed to the different ways in which the uncoupler was supplied to the two tissues. The pea roots were immersed in the 2,4-dinitrophenol whilst only the stalks of the clubs were in contact with the uncoupler. We found, for a large number of independent samples, that, although 2,4-dinitrophenol had an appreciable effect on the rate of respiration, no significant effect on PPi content could be detected (Table 1).
Effects of anoxia. Production of C O 2 by y-stage clubs of Arum maculatum was 2514_++865 gl.h -1. g - a FW. After 30 min anoxia this rate fell to 609 _+ 23: values are means__+_SE for three clubs. Extensive measurements of the PPi content of such clubs after 1, 15 and 60 min anoxia failed to reveal any effect of anoxia on PPi content (Table 2). When pea roots were made anoxic there was a fall in the rate of CO2 production but no change in PPi content was detected (Table 3). Other metabolites did change: ATP fell and there were concomitant increases in A D P and AMP. Fructose-l,6-bisphosphate increased but a fall in hexose 6-phosphates was not demonstrated conclusively.
J.E. Dancer and T. ap Rees: 2,4-Dinitrophenol, anoxia and pyrophosphate content
423
Table 1. Effect of 2,4-dinitrophenol on respiration and PPi content of e-stage clubs of Arum maculatum, and the apical 2 cm of the roots of 5-d-old pea seedlings. Clubs were treated with 2,4-dinitrophenol for 90 min and root apices for 60 min. Data are means _+SE of estimates from the number of samples shown in parenthesis. N.S., not significant Tissue
Treatment
Gas exchange (gl. h 1. g- 1 FW) 02 uptake
Arum club
Control I Dinitrophenol II Fisher's P value for I vs. II
Pea root
Control III Dinitrophenol IV Fisher's P value for III vs. IV
771_+20 (9) 1510_+ 80 (9)
![[Studies on microbodies in spadix appendices of Arum maculatum L. and Sauromatum guttatum schott].](/assets/img/hold.png)
