Planta
Planta (1989) 177:359-366
9 Springer-Verlag1989
Nitrite reduction and carbohydrate metabolism in plastids purified from roots of Pisum sativum L. C.G. Bowsher 1, D.P. Hucklesby 2 and M.J. Emes 1 * 1 Plant Science and Cytogenetics, Williamson Building, Department of Cell and Structural Biology, University of Manchester, Manchester, MI3 9PL, and 2 Department of Molecular Biology, Institute of Arable Crops Research, Long Ashton Research Station, Long Ashton, Bristol BS18 9AF, UK
Abstract. Intact preparations of plastids from pea
(Pisum sativum L.) roots have been used to investigate the metabolism of glucose-6-phosphate and reduction of inorganic nitrite within these organelles. The ability of hexose-phosphates to support nitrite reduction was dependent on the integrity of the preparation and was barely measurable in broken organelles. In intact plastids, nitrite was reduced most effectively in the presence of glucose6-phosphate (Glc6P), fructose-6-phosphate and ribose-5-phosphate and to a lesser extent glucose-lphosphate. The K m (Glc6P) of plastid-located Glc6P dehydrogenase (EC 1.1.1.49) and Glc6P-dependent nitrite reduction were virtually identical (0.68 and 0.66 m M respectively) and a similar relationship was observed between fructose-6-phosphate, bexose-phosphate isomerase (EC5.3.1.9) and nitrite reduction. The pattern of release of CO2 from different carbon atoms of Glc6P supplied to root plastids, indicates the operation of both glycolysis and the oxidative pentose-phosphate pathway with some recycling in the latter. During nitrite reduction the evolution of CO2 from carbon atom 1 of Glc6P was stimulated but not from carbon atoms 2, 3, 4, or 6. The importance of these results with regard to the regulation of the pathways of carbohydrate oxidation and nitrogen assimilation within root plastids is discussed. Key words: Glucose-6-phosphate metabolism - Ni-
trite reduction - Pisum (root plastids) Plastid (glucose-6-phosphate, nitrite) - Root plastid
Introduction
It is well established that in non-photosynthetic tissues the assimilation of nitrate is dependent * To whom correspondence should be addressed Abbreviation: Glc6P = glucose-6-phosphate
upon intermediates produced by the oxidation of carbohydrates (Willis and Yemm 1955). In particular, there is an increasing body of literature supporting the hypothesis that the oxidative pentosephosphate pathway (PPP) is the initial source of reductant for nitrite reductase in such cells (Sarkissian and Fowler 1974) and that this interaction takes place in non-chlorophyllous plastids (Emes and Fowler 1979a, b; Washitani and Sato 1977). However, whilst a number of reports have demonstrated that the oxidation of glucose-6-phosphate (Glc6P) in root plastids is capable of sustaining nitrite reduction (Emes and Fowler 1983; Miflin 1974; Dry et al. 1981 ; Oji et al. 1985) there is little information on the regulation and interdependence of these two processes. Using highly intact preparations of pea root plastids we have therefore attempted to determine (i) the ability of different carbohydrate substrates to support nitrite reduction and to characterise this process kinetically, and (ii) the impact that nitrite reduction has on Glc6P oxidation by measuring the rate and pattern of CO2 release from different carbon atoms under nitrite-assimilating and non-assimilating conditions, and under assimilating conditions where the rate of nitrite reduction is varied. Materials and methods Chemicals. All reagents were of ' A R ' grade where possible. Co-factors, enzymes and substrates were purchased from BDH, Poole, Dorset, (UK), Boehringer Mannheim, (FRG) or Sigma, Poole, Dorset, (UK). Percoll was purchased from Pharmacia, Milton Keynes, (UK) [1-14C]-, [2-14C]-, [3,4-14C]-, and [614C]Glucose were from New England Nuclear, Southampton,
(UK). Plant material. Pea (Pisum sativum L., cv. Kelvedon Wonder) seeds purchased from Asmer, Leicester, (UK), were germinated in the dark at 25~ C for 5 d as described previously (Emes and England 1986). The enzymes of nitrate assimilation were induced according to the same paper.
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C.G. Bowsher et al. : Nitrite reduction and carbon metabolism in roots
Extraction and purification of root plastids. Plastids were prepared from 60-200 g of roots, based on the method described by Emes and England (1986) and modified as described below. All procedures were carried out at 4 ~ C. Roots were homogenised in one volume of buffer A (50 m M N-[2-hydroxy-l,l-bis (hydroxymethyl)ethyl]glycine (Tricine)-NaOH pH 7.9 containing 330 m M sorbitol, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 m M MgC12, 0.1% bovine serum albumin) using the steel blade of a Kenwood Gourmet Variomatic food processor (model A5357) at 1 500 rpm for 20 s. The brei was filtered through six layers of muslin and centrifuged at 200 g for 1 min. The supernatant was then centrifuged at 4000.g for 3 rain in a 8 x 50 ml rotor (MSE, Crawley, Sussex, UK). After resuspending the resulting pellet in buffer A (without bovine albumin) by gently drawing up and down with a pasteur pipette, 10 ml aliquots of the suspension were underlaid with 10 ml 50 mM Tricine-NaOH pH 7.9 containing 330 mM sorbitol and 10% (v/v) freshly dialysed Percoll. Centrifugation was carried out in a 4 x 50 ml swing-out windshield rotor at 4000.g for 5 min. The resulting pellet, designated the plastid fraction, was gently resuspended in a small volume of buffer A (without bovine albumin). Enzyme assays. The activities of NADH-glutamate synthase (EC 2.6.1.53) and Glc6P dehydrogenase were assayed as described previously (Emes and Fowler 1979a, b). Nitrite reductase (EC 1.6.6.4) was assayed by the method of Losada and Paneque (1971). Alcohol dehydrogenase and cytochrome oxidase (EC 1.9.3.1) were assayed as described by Macdonald and ap Rees (1983). Phosphoglucomutase ((EC 2.4.5.1) was assayed in 50 m M Tricine, pH 7.5, 10 m M MgC12, 3.3 m M glucose-iphosphate, 1 m M EDTA, 1 unit Glc6P dehydrogenase and 0.18 m M NADP. Hexose-phosphate isomerase was assayed in 250 mM glycylglycine, pH 7.5, 10 m M MgC12, 1 unit Glc6P dehydrogenase, 5 mM fructose-6-phosphate and 0.39 m M NADP. All assays were carried out in a final volume of 1 ml and rates corrected for non-specific N A D P reduction. Catalase (EC 1.11.1.6) was assayed following the evolution of oxygen from hydrogen peroxide in an oxygen electrode (van Ginkel and Brown 1978). Hexose-phosphate-dependent nitrite reduction. The assay contained 150 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris)-HC1, pH 8.0, I m M NaNO2, substrate (hexose-phosphate) and intact plastids. Aliquots (0.1 ml) were removed at known times after addition of hexose phosphate, and remaining nitrite determined by the diazo-dye coupling method (Evans and Nason 1953). Protein determination. Protein was measured using a commercial Bio-Rad (Watford, Herts., UK) protein method based on. Bradford (1976) with thyroglobulin as the protein standard. Latency of enzyme activities within root plastids. This was determined according to Emes and England (1986) in the presence or absence of 0.1% Triton X-100. All assays reagents were osmotically buffered with 330 mM sorbitol. Radiochemical experiments. Glucose-6-phosphate labelled with 14C in either the carbon-I, carbon-2, carbon-3,4 or carbon-6 position was prepared according to Emes and Fowler (1983), using glucose, ATP and hexokinase. Samples were boiled for 5 rain and denatured protein removed by centrifugation. Complete conversion of glucose to Gle6P was confirmed by spectrophotometric assay of the products. [14C]Glucose-6-phosphate (37 kBq) labelled in C-1, C-2, C-3,4 or C-6 position was added to 1 ml of 100 mM Glc6P.
Aliquots (0.05 ml) were placed in the main compartment of a Warburg flask, the centre well of which contained 0.2 ml 10% (w/v) KOH. If the assay was to be carried out in the presence of nitrite, 0.05 ml of 10 mM NaNOz was added to the main compartment. Volume was made up to 0.30 ml with 150 m M Tris-HC1 buffer (pH 8.0). At zero time, 0.20 ml of plasrids (approx. 600 pg protein) were added to the main compartment, the flasks stoppered and agitated in a water bath at 27 ~ C. After incubation for known periods of time the flasks were removed and the radioactivity in the K O H determined as described previously (Emes and Fowler 1983) with a Canberra Packard Scintillation Counter (Packard, Caversham, Berks., UK). All assays were corrected for 14CO2 release from broken plastids by carrying out the same experiments using organelles lysed either by freeze-thawing or with 0.I % (v/v) Triton X-100, and subtracting these values from those obtained with intact preparations.
Electron microscopy. Samples of root plastids were fixed, embedded, sectioned, stained and viewed as described elsewhere (Emes and England 1986).
Results
The modification of the method we have already described (Emes and England 1986) and which is reported here, allows the purification of plastids from much larger quantities of root material than previously possible. On the basis of the distribution of organelle-specific enzymes (Emes and England 1986), plastid preparations were routinely recovered as 20-25% of the original starting material with only 1% contamination by other organelles or cytoplasm (Table 1). The plastids obtained were 75-95% intact based on the latency of glutamate synthase (Emes and England 1986, and see also enzyme latency in Table 2). Electron micrographs (Fig. 1) confirm the purity of the preparation and in addition the organelles can be seen to be bound by a double membrane, not clearly visible in our previous preparations and reinforcing the view that they are highly intact. The ability of Glc6P to support nitrite reduction in these plastids is shown in Fig. 2. It is clear that the integrity of the organelles is fundamental to this process, with intact plastids giving approximately eight times the activity of plastids lysed with Triton X-100. Fructose-6-phosphate and ribose-5-phosphate were both able to support nitrite reduction at rates similar to those observed with Glc6P (Fig. 2). Glucose-l-phosphate was also able to support nitrite reduction, but to a lesser extent. At 10 raM, 6phosphogluconate, fructose-l,6-bisphosphate, glyceraldehyde-3-phosphate, dihydroxyacetone phosphate, and fructose (data not shown) and glucose were individually ineffective. The ability of metabolites to support nitrite reduction may be a func-
C.G. Bowsher et ai. : Nitrite reduction and carbon metabolism in roots
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Table 1. Distribution of organelle marker enzymes after the centrifugation of the 200.g supernatant from pea root homogenate at 4000-g for 3 rain. The resuspended pellet from this step was then centrifuged through 10% (v/v) Percoll at 4000.g for 5 min (mean value_+ SE of at least four determinations). Figures in ( ) represent activity recovered in fraction as a percentage of 200.g supernatant
200 .g 4000 .g 4000 .g 4000.g 4000 .g
supernatant supernatant pellet Percoll supernatant (post-Percoll plastid pellet
Nitrite reductase (nkat nitrite reduced, rag- 1 protein) (plastids)
Cytochrome oxidase (nkat-mg- t protein) (mitochondria)
Alcohol dehydrogenase (nkats-mg 1 protein) (cytoplasm)
Catalase (nkats. mg 1 protein) (microbodies)
0,20_+0.03 (100%) 0.12-t-0.03 (63%) 0,77_+0.12 (40%) 0.19_+0.07 (13%) 2.92_+0.50 (25%)
0.78_+0.18 (100%) 0.41_+0.12(73%) 2.52_+0.65(29%) 1.58_+0.55(25%) 1.07___0.62 (1.2%)
0.29_+0.12(100%) 0.33_+0.15 (99%) 0.02_+0.02 (1%) 0.01_+0.01 (1%) 0 (0%)
98_+25 (100%) 68_+ 8 (99%) 11_+ I (2.6%) 20_+ 5 (3.1%) 72_+41 (0.65%)
Fig. 1. Electron micrograph of purified pea root ptastids. Sections were stained in uranyl acetate and lead citrate Table 2. Specific activity and latency of enzymes of hexosephosphate metabolism associated with pea root plastids. Plastids were prepared as described in Material and methods and all assays performed in the presence of 330 mM sorbitol. LaB-I tency = - - B x 100% where I is the activity measured before, and B is the activity measured often rupturing organelles with 0.1% (v/v) Triton X-100 (mean_+ SE of three determinations) Enzyme
Specific activity (nkat-mg-1 protein)
Latency (%)
Glc6P dehydrogenase Hexose-phosphate isomerase Phosphoglucomutase
1.18 • 0.35 14.35+2.05 0.58 • 0.1
75 • 5 73 • 84 _+1
tion of the permeability of the plastid envelope but is also dependent on the presence of the necessary enzymes leading to Glc6P formation. In particular, for glucose-l-phosphate and fructose-6-
phosphate to support nitrite reduction requires that the enzymes necessary for their conversion to Glc6P be associated with the plastid preparation. The results in Table 2 demonstrate the specific activity and latency of these enzymes in relation to Glc6P dehydrogenase, an enzyme known to be latent within these organelles (Emes and Fowler 1979b). The degree of latency is similar in all cases confirming the presence of hexosephosphate isomerase and phosphoglucomutase within root plastids. The dependence of nitrite reduction in intact plastids on the concentration of hexose phosphates is shown in Fig. 3 a, b. The kinetics of fructose-6phosphate- and Glc6P-dependent nitrite reduction are presented as double-reciprocal plots along with the substrate-dependence of plastid he~ose-phosphate isomerase and Glc6P dehydrogenase. The
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C.G. Bowsher et al. : Nitrite reduction and carbon metabolism in roots
Km for fructose-6-phosphate-dependent nitrite reduction is 0.32 mM compared with a value of 0.35 mM for fructose-6-phosphate for the isomerase. The Km for Glc6P-dependent nitrite reduction is 0.66 mM compared with a Km of 0.68 mM for Glc6P for the plastid-located Glc6P dehydrogenase. Having examined the effect of hexose-phosphate concentration on the rate of nitrite reduction, the effect of nitrite reduction upon carbohydrate oxidation was investigated. This involved supplying intact plastids with Glc6P, isotopically labelled in different carbon atoms, and measuring the subsequent release of 14CO2 in the presence or absence of NO2 (Fig. 4a, b). Evolution of z4CO2 from plastids is greatest when Glc6P is labelled in carbon atoms 3 and 4 and least when labelled in carbon atom 6. The release of 14CO2 from these and [2-14C]Glc6P is unaffected by the presence of 1 mM NO2. By contrast there is a significant increase in the release of CO2 from carbon atom 1 of Glc6P when sodium nitrite is supplied simultaneously. By comparison with sodium nitrite, the addition of sodium chloride and sodium nitrate had no effect on the release of 14CO2 from [114C]Glc6P (Fig. 5). Osmotic-swelling studies have demonstrated that pea_ root plastids are freely permeable to NO2, NO 2 and C1- (data not shown)
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60
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Fig. 3a, b. Lineweaver-Burke plots of hexose-phosphatedependent nitrite reduction and enzymes of hexose-phosphate metabolism. Substrate dependence of the latter was determined by using plastids lysed with 0.1% (v/ v) Triton X-100 prior to assay of activity. (Each value is the mean of three determinations) a Glucose-6-phosphate dependence of Glc6P dehydrogenase (v; values are V ~ x 10) and nitrite reduction (m) in intact root plastids. b Fructose-6-phosphate dependence of hexose-phosphate isomerase (A; values are V-z x 100) and nitrite reduction (e; values are V - i x 1.5) in intact root plastids
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C.G. Bowsher et al. : Nitrite reduction and carbon metabolism in roots
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Fig. 4a, b. Evolution of CO2 from different carbon atoms after labelled Glc6P had been supplied to intact root plastids m the presence (closed symbols) and absence (open symbols) of 1 m M sodium nitrite, a 9 * = [1J4C]Glc6P; r~ B=[6J'~C]Glc6P. b zx, 9 = [3,4-14C]Glc6P; O, * = [2JaC]Glc6P. (Mean values • SE of three determinations)
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Fig. 5a, b. Evolution of 14CO2 from [1-t4C]Glc6P supplied to intact plastids incubated with different salts, a Plastids from tissue watered with 10 m M KNO3 24 h prior to extraction, o - - o , No additional salts; e - - e , I m M NaNOz; [] ..... n, / m M NaC1; A - - - A , 1 m M NaNO3. b Plastids from tissue deprived of external nitrate throughout the growing period. ~-----~, No additional salts; ~-----~, 1 m M NaNO2. (Mean values • SE of three determinations)
364
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C.G. B o w s h e r et al. : Nitrite r e d u c t i o n a n d c a r b o n m e t a b o l i s m in r o o t s
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ruling out the possibility that differences may be due to anion accessibility or an effect of the cation. Plastids prepared from roots in which the enzymes of nitrate assimilation had not been induced, and which possess only low amounts of nitrite reductase (Emes and Fowler 1983), did not show any significant difference in 14CO 2 release from [la4C]Glc6P in the presence or absence of sodium nitrite (Fig. 5). These results taken together indicate that nitrite reduction is directly responsible for the increase in CO2 evolution from carbon atom 1 of Glc6P. That the stimulation in the release of CO2 from carbon atom 1 of Glc6P phosphate is a function of the demand for reductant during nitrite reduction is further illustrated in Fig. 6. The stimulation of CO2 evolution from intact plastids increases with the concentration of nitrite supplied. A K m of 0.4 m M for this stimulation can be calculated from the data if the amount of CO2 released from carbon atom I in the absence of nitrite is subtracted from each value. This is close to the reported Km (NO2-) of 0.1-0.6 m M for nitrite reductase from a number of sources (Vega et al. 1980). Discussion
The preparations of root plastids used in this work appear to be suitable for detailed study of their
metabolism in vitro. The rate of Glc6P-dependent nitrite reduction (0.1 n k a t . m g -1 plastid protein) at saturating substrate concentrations is similar to that obtained by Oji et al. (1985) with barley root plastids. However, Oji et al. obtained a twofold stimulation of Glc6P-dependent nitrite reduction when N A D P § was added to their assay system whereas no increase in activity was observed in our experiments when this was added to intact pea root plastids (data not shown). It is possible that the reason for this discrepancy lies in the permeability o f the preparations to pyridine nucleotides and would indicate that the barley-root plastids are more "leaky". This view is reinforced by the observation that nitrite reduction in barley root plastids is also stimulated by 6-phosphogluconate. We have previously observed the same effect (Emes and Fowler 1983) using pea root plastids purified by sucrose-density-gradient centrifugation, but with plastids prepared by the present method this is no longer apparent. It is suggested that whilst these earlier reports regarding the location and activity of nitrite-reducing systems are valid the membrane integrity of such preparations is questionable. The data presented in Fig. 2 imply that hexose monophosphates and ribose-5-phosphate are able to cross the envelope of pea root plastids. The former are not generally regarded as being readily able to enter chloroplasts (Heldt and Rapley 1970) but there is no reason a priori why non-photosynthetic root organelles should have the same permeabilities. We have previously demonstrated the presence of a phosphate-translocator-like system in pea root plastids (Emes and Traska 1987) but the kinetics of phosphate uptake appear to differ from the chloroplast protein. This probably reflects the different roles that this process plays in photosynthetic and non-photosynthetic tissues, and it need not be supposed, therefore, that movement of other metabolites should be identical. The uptake of hexose-phosphates by root plastids clearly merits further investigation. Such kinetic studies of hexose-phosphate-dependent nitrite reduction highlight the close relationship that exists between carbohydrate metabolism and nitrite reduction in root plastids. The results strongly indicate that the rate at which nitrite reduction can proceed is a function of the rate of Glc6P oxidation and, depending on the concentration of substrates in the plastid, the latter may limit the former. We have also approached this interaction from the opposite viewpoint, by looking at the way in which the rate of nitrite reduction affects the oxida-
C.G. Bowsher et al. : Nitrite reduction and carbon metabolism in roots
tion of Glc6P and the release of CO2 from different carbon atoms. A number of points can be made about the data in Fig. 4. First of all, in the presence of nitrite, the release of relatively large amounts of CO2 from carbon atoms 3 and 4 of Glc6P, and the release from other carbon atoms in the order C1 >C2 >C6 is consistent with the operation of the oxidative pentose-phosphate pathway (PPP) and glycolysis, as far as the decarboxylation of pyruvate, within these organelles (ap Rees 1980). In this paper we have demonstrated the presence of hexose-phosphate isomerase (Table 2) and routinely use triose-phosphate isomerase as a marker enzyme for root plastids. The intracellular location of all the enzymes of glycolysis in pea roots is a matter of current investigation, but a number of papers have reported the presence of the necessary glycolytic enzymes within non-green plastids, certainly as far as the formation of triose phosphate (Macdonald and ap Rees 1983; Simcox et al. 1977; Journet and Douce 1985) but with some equivocation with regard to the lower half of the pathway. The presence of pyruvate dehydrogenase in other non-green plastids has also been demonstrated (Reid et al. 1977; Journet and Douce 1985) and our results support the contention that these organelles can oxidise Glc6P to acetate/acetyl CoA. The release of CO2 from carbon atom 2 is, in the absence of nitrite, apparently higher than that from carbon atom 1. This probably arises from the correction of rates for 14CO2release from broken organelles using deliberately lysed preparations. Release of ~4COa from 100% lysed preparations is three- to fourfold higher for [1-~4C]Glc6P compared with [2-~4C]Glc6P, and probably results in an overcompensation for breakage when using preparations which may be as much as 90% intact. Nonetheless, such a result implies only limited operation of the PPP within these organelles in the absence of nitrite reduction. Present understanding of the re-arrangement of carbon atoms which takes place in the PPP requires that, for CO2 to be evolved from carbon atom 6 of Glc6P, there must be fructose-bisphosphatase activity. The possibility of cytosolic contamination seems unlikely on the basis of data in Table 1. Although membrane adsorption of the enzyme cannot be ruled out, since fructose bisphosphate is not able to support nitrite reduction in intact plastid preparations whereas fructose-6-phosphate is, this would also seem an unlikely explanation of the data. The presence of latent fructose-bisphosphatase activity within these organelles therefore warrants further investigation. Since there is no Krebs' cycle activity to be considered, the release of CO2 from carbon atom 2 as
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well as from carbon atom 1 further indicates the possibility of limited recycling through the PPP. When nitrite is being reduced by root plastids the release of CO2 from Glc6P increases specifically and only from carbon atom 1. The data imply an increased flux of carbon through the PPP but without affecting the flow of carbon through what we presume to be glycolysis as judged by the release of CO2 from [3,4-14C]Glc6P. Glucose-6phosphate dehydrogenase, the key enzyme of the PPP is known to be regulated by the N A D P : N A D P H ratio (Ashihara and Komamine 1976). As a consequence of nitrite-reductase activity and the demand for reductant it is likely that this ratio would increase, resulting in deinhibition of the enzyme and increased activity. If three molecules of N A D P H are required per molecule of nitrite reduced this could be met, in these in-vitro experiments, by the oxidation of between 1.5 and 3 molecules of Glc6P, depending on the activity of 6phosphogluconate dehydrogenase. On the basis of the measured rate of nitrite reduction within these organelles ( 0 . 1 n k a t . m g -1 protein at 1 0 m M Glc6P) a rate of Glc6P oxidation of between 0.15 and 0.3 n k a t - m g - 1 protein would be required. The kinetic studies of the plastid enzyme (Fig. 3 a) indicate that this is only between 8 15% of the maximal rate of catalysis of 2 n k a t . m g - 1 protein, given the presence of saturating levels of substrate, implying some degree of inhibition of Glc6P dehydrogenase. This model of control a n d the release of inhibition would therefore seem feasible. The dependence of this stimulation o n the rate of nitrite reduction is emphasized in Fig. 6. As the concentration of nitrite is increased from below saturating levels, there is an increase in: the release of CO2 from carbon atom 1 of Glc6P, implying an increase in the activity of the oxidative reactions to keep pace with the demand for reductant. In conclusion the results presented in this paper demonstrate the ability of root plastids to metabolise Glc6P to CO2. The dependence of nitrite reduction, and ultimately amino-acid synthesis, on the kinetics of PPP enzymes illustrates how the flux of carbon through a pathway of carbohydrate oxidation may regulate the pathway of nitrogen assimilation. Conversely, it appears t h a t the rate of nitrite reduction, when nitrite is limiting, influences the activity of the PPP.
C.G.B. is the recipient of a Science and Engineering Research Council-Cooperative Awards in Science and Engineering studentship. The authors are grateful to Mr. O. Sayed for assistance with electron microscopy.
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References ap Rees, T. (1980) The biochemistry of plants, vol 2: Metabolism and respiration, pp. 1-29, D.D. Davies, ed. Academic Press, London Ashihara, H., Komamine, A. (1976) Characterisation and regulatory properties of glucose-6-phosphate dehydrogenase from Black Gram (Phaseolus mungo). Physiol. Plant. 36, 52-59 Bradford (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal. Biochem. 72, 248-254 Dry, I., WaUace, W., Nicholas, D.J.D. (1981) Role of ATP in nitrite reduction in roots of wheat and pea. Planta 152, 23z~238 Emes, M.J., England, S. (1986) Purification of plastids from higher-plant roots. Planta 168, 161-166 Emes, M.J., Fowler, M.W. (1979a) The intracellular location of the enzymes of nitrite assimilation in the apices of seedling pea roots. Planta 144, 24%253 Emes, M.J., Fowler, M.W. (1979b) Intracellular interactions between the pathways of carbohydrate oxidation and nitrate assimilation in pea roots. Planta 145, 282292 Emes, M.J., Fowler, M.W. (1983) The supply of reducing power for nitrite reduction in plastids of seedling pea roots (Pisum sativum L.). Planta 158, 97-102 Emes, M.J. Traska, A. (1987) Uptake of inorganic phosphate by plastids purified from the roots of Pisum sativum. L. J. Exp. Bot. 38, 1781-1788 Evans H.J., Nason, A. (1953) Pyridine nucleotide nitrate reductase from extracts of higher plants. Plant Physiol 28, 233254 Heldt, H.W., Rapley, L. (1970) Unspecific permeation and specific uptake of substances in spinach chloroplasts. FEBS Lett. 7, 139-142 Journet, E.P., Douce, R. (1985) Enzymic capacities of purified cauliflower bud plastids for lipid synthesis and carbohydrate metabolism. Plant Physiol. 79, 458-467
Losada, M., Paneque, A. (1971) Nitrite reductase. Methods Enzymol. 23, 482491 Macdonald, F.D., ap Rees, T. (1983) Enzymic properties of amyloplasts from suspension cultures of soybean. Biochim. Biophys. Acta 755, 81-89 Miflin, B.J. (1974) The location of nitrite reductase and other enzymes related to amino acid biosynthesis in the plastids of roots and leaves. Plant Physiol. 54, 550-555 Oji, Y., Watanabe, M., Wakiuchi, N., Okamoto, S. (1985) Nitrite reduction in barley root plastids: Dependence on NADPH coupled with glucose-6-phosphate and 6-phosphogluconate dehydrogenase, and possible involvement of an electron carrier and a diaphorase. Planta 165, 8540 Reid, E.E., Thompson, P., Lyttle, C.R., Dennis, D.T. (1977) Pyruvate dehydrogenase complex from higher plant mitochondria and proplastids. Plant Physiol. 59, 842-848 Sarkissian, G.S., Fowler, M.W. (1974) Interrelationship between nitrate assimilation and carbohydrate metabolism in plant roots. Planta 119, 335-349 Simcox, P.D., Reid, E.E., Canvin, D.T., Dennis, D.T. (1977) Enzymes of the glycolytic and pentose phosphate pathways in proplastids from the developing endosperm of Ricinus communis L. Plant Physiol. 59, 1123-1132 Van Ginkel, G., Brown, J.S. (1978) Endogenous catalase and superoxide dismutase activities in photosynthetic membrane. FEBS Lett. 94, 284~286 Vega, J.M., Cardenas, J., Losada, M. (1980) Ferredoxin-nitrite reductase. Methods Enzymol. 69, 255-270 Washitani, I., Sato, S. (1977) Studies on the function of proplastids in the metabolism of in vitro cultured tobacco cells. III. Source of reducing power for amino acid synthesis from nitrite. Plant Cell Physiol. 18, 1235 1241 Willis, A.J., Yemm, E.W. (1955) The respiration of barley roots: nitrogen assimilation and respiration of the root system. New Phytol. 54, 163-181 Received 13 May; accepted 23 November 1988
Planta (1989) 177:359-366
9 Springer-Verlag1989
Nitrite reduction and carbohydrate metabolism in plastids purified from roots of Pisum sativum L. C.G. Bowsher 1, D.P. Hucklesby 2 and M.J. Emes 1 * 1 Plant Science and Cytogenetics, Williamson Building, Department of Cell and Structural Biology, University of Manchester, Manchester, MI3 9PL, and 2 Department of Molecular Biology, Institute of Arable Crops Research, Long Ashton Research Station, Long Ashton, Bristol BS18 9AF, UK
Abstract. Intact preparations of plastids from pea
(Pisum sativum L.) roots have been used to investigate the metabolism of glucose-6-phosphate and reduction of inorganic nitrite within these organelles. The ability of hexose-phosphates to support nitrite reduction was dependent on the integrity of the preparation and was barely measurable in broken organelles. In intact plastids, nitrite was reduced most effectively in the presence of glucose6-phosphate (Glc6P), fructose-6-phosphate and ribose-5-phosphate and to a lesser extent glucose-lphosphate. The K m (Glc6P) of plastid-located Glc6P dehydrogenase (EC 1.1.1.49) and Glc6P-dependent nitrite reduction were virtually identical (0.68 and 0.66 m M respectively) and a similar relationship was observed between fructose-6-phosphate, bexose-phosphate isomerase (EC5.3.1.9) and nitrite reduction. The pattern of release of CO2 from different carbon atoms of Glc6P supplied to root plastids, indicates the operation of both glycolysis and the oxidative pentose-phosphate pathway with some recycling in the latter. During nitrite reduction the evolution of CO2 from carbon atom 1 of Glc6P was stimulated but not from carbon atoms 2, 3, 4, or 6. The importance of these results with regard to the regulation of the pathways of carbohydrate oxidation and nitrogen assimilation within root plastids is discussed. Key words: Glucose-6-phosphate metabolism - Ni-
trite reduction - Pisum (root plastids) Plastid (glucose-6-phosphate, nitrite) - Root plastid
Introduction
It is well established that in non-photosynthetic tissues the assimilation of nitrate is dependent * To whom correspondence should be addressed Abbreviation: Glc6P = glucose-6-phosphate
upon intermediates produced by the oxidation of carbohydrates (Willis and Yemm 1955). In particular, there is an increasing body of literature supporting the hypothesis that the oxidative pentosephosphate pathway (PPP) is the initial source of reductant for nitrite reductase in such cells (Sarkissian and Fowler 1974) and that this interaction takes place in non-chlorophyllous plastids (Emes and Fowler 1979a, b; Washitani and Sato 1977). However, whilst a number of reports have demonstrated that the oxidation of glucose-6-phosphate (Glc6P) in root plastids is capable of sustaining nitrite reduction (Emes and Fowler 1983; Miflin 1974; Dry et al. 1981 ; Oji et al. 1985) there is little information on the regulation and interdependence of these two processes. Using highly intact preparations of pea root plastids we have therefore attempted to determine (i) the ability of different carbohydrate substrates to support nitrite reduction and to characterise this process kinetically, and (ii) the impact that nitrite reduction has on Glc6P oxidation by measuring the rate and pattern of CO2 release from different carbon atoms under nitrite-assimilating and non-assimilating conditions, and under assimilating conditions where the rate of nitrite reduction is varied. Materials and methods Chemicals. All reagents were of ' A R ' grade where possible. Co-factors, enzymes and substrates were purchased from BDH, Poole, Dorset, (UK), Boehringer Mannheim, (FRG) or Sigma, Poole, Dorset, (UK). Percoll was purchased from Pharmacia, Milton Keynes, (UK) [1-14C]-, [2-14C]-, [3,4-14C]-, and [614C]Glucose were from New England Nuclear, Southampton,
(UK). Plant material. Pea (Pisum sativum L., cv. Kelvedon Wonder) seeds purchased from Asmer, Leicester, (UK), were germinated in the dark at 25~ C for 5 d as described previously (Emes and England 1986). The enzymes of nitrate assimilation were induced according to the same paper.
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C.G. Bowsher et al. : Nitrite reduction and carbon metabolism in roots
Extraction and purification of root plastids. Plastids were prepared from 60-200 g of roots, based on the method described by Emes and England (1986) and modified as described below. All procedures were carried out at 4 ~ C. Roots were homogenised in one volume of buffer A (50 m M N-[2-hydroxy-l,l-bis (hydroxymethyl)ethyl]glycine (Tricine)-NaOH pH 7.9 containing 330 m M sorbitol, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 m M MgC12, 0.1% bovine serum albumin) using the steel blade of a Kenwood Gourmet Variomatic food processor (model A5357) at 1 500 rpm for 20 s. The brei was filtered through six layers of muslin and centrifuged at 200 g for 1 min. The supernatant was then centrifuged at 4000.g for 3 rain in a 8 x 50 ml rotor (MSE, Crawley, Sussex, UK). After resuspending the resulting pellet in buffer A (without bovine albumin) by gently drawing up and down with a pasteur pipette, 10 ml aliquots of the suspension were underlaid with 10 ml 50 mM Tricine-NaOH pH 7.9 containing 330 mM sorbitol and 10% (v/v) freshly dialysed Percoll. Centrifugation was carried out in a 4 x 50 ml swing-out windshield rotor at 4000.g for 5 min. The resulting pellet, designated the plastid fraction, was gently resuspended in a small volume of buffer A (without bovine albumin). Enzyme assays. The activities of NADH-glutamate synthase (EC 2.6.1.53) and Glc6P dehydrogenase were assayed as described previously (Emes and Fowler 1979a, b). Nitrite reductase (EC 1.6.6.4) was assayed by the method of Losada and Paneque (1971). Alcohol dehydrogenase and cytochrome oxidase (EC 1.9.3.1) were assayed as described by Macdonald and ap Rees (1983). Phosphoglucomutase ((EC 2.4.5.1) was assayed in 50 m M Tricine, pH 7.5, 10 m M MgC12, 3.3 m M glucose-iphosphate, 1 m M EDTA, 1 unit Glc6P dehydrogenase and 0.18 m M NADP. Hexose-phosphate isomerase was assayed in 250 mM glycylglycine, pH 7.5, 10 m M MgC12, 1 unit Glc6P dehydrogenase, 5 mM fructose-6-phosphate and 0.39 m M NADP. All assays were carried out in a final volume of 1 ml and rates corrected for non-specific N A D P reduction. Catalase (EC 1.11.1.6) was assayed following the evolution of oxygen from hydrogen peroxide in an oxygen electrode (van Ginkel and Brown 1978). Hexose-phosphate-dependent nitrite reduction. The assay contained 150 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris)-HC1, pH 8.0, I m M NaNO2, substrate (hexose-phosphate) and intact plastids. Aliquots (0.1 ml) were removed at known times after addition of hexose phosphate, and remaining nitrite determined by the diazo-dye coupling method (Evans and Nason 1953). Protein determination. Protein was measured using a commercial Bio-Rad (Watford, Herts., UK) protein method based on. Bradford (1976) with thyroglobulin as the protein standard. Latency of enzyme activities within root plastids. This was determined according to Emes and England (1986) in the presence or absence of 0.1% Triton X-100. All assays reagents were osmotically buffered with 330 mM sorbitol. Radiochemical experiments. Glucose-6-phosphate labelled with 14C in either the carbon-I, carbon-2, carbon-3,4 or carbon-6 position was prepared according to Emes and Fowler (1983), using glucose, ATP and hexokinase. Samples were boiled for 5 rain and denatured protein removed by centrifugation. Complete conversion of glucose to Gle6P was confirmed by spectrophotometric assay of the products. [14C]Glucose-6-phosphate (37 kBq) labelled in C-1, C-2, C-3,4 or C-6 position was added to 1 ml of 100 mM Glc6P.
Aliquots (0.05 ml) were placed in the main compartment of a Warburg flask, the centre well of which contained 0.2 ml 10% (w/v) KOH. If the assay was to be carried out in the presence of nitrite, 0.05 ml of 10 mM NaNOz was added to the main compartment. Volume was made up to 0.30 ml with 150 m M Tris-HC1 buffer (pH 8.0). At zero time, 0.20 ml of plasrids (approx. 600 pg protein) were added to the main compartment, the flasks stoppered and agitated in a water bath at 27 ~ C. After incubation for known periods of time the flasks were removed and the radioactivity in the K O H determined as described previously (Emes and Fowler 1983) with a Canberra Packard Scintillation Counter (Packard, Caversham, Berks., UK). All assays were corrected for 14CO2 release from broken plastids by carrying out the same experiments using organelles lysed either by freeze-thawing or with 0.I % (v/v) Triton X-100, and subtracting these values from those obtained with intact preparations.
Electron microscopy. Samples of root plastids were fixed, embedded, sectioned, stained and viewed as described elsewhere (Emes and England 1986).
Results
The modification of the method we have already described (Emes and England 1986) and which is reported here, allows the purification of plastids from much larger quantities of root material than previously possible. On the basis of the distribution of organelle-specific enzymes (Emes and England 1986), plastid preparations were routinely recovered as 20-25% of the original starting material with only 1% contamination by other organelles or cytoplasm (Table 1). The plastids obtained were 75-95% intact based on the latency of glutamate synthase (Emes and England 1986, and see also enzyme latency in Table 2). Electron micrographs (Fig. 1) confirm the purity of the preparation and in addition the organelles can be seen to be bound by a double membrane, not clearly visible in our previous preparations and reinforcing the view that they are highly intact. The ability of Glc6P to support nitrite reduction in these plastids is shown in Fig. 2. It is clear that the integrity of the organelles is fundamental to this process, with intact plastids giving approximately eight times the activity of plastids lysed with Triton X-100. Fructose-6-phosphate and ribose-5-phosphate were both able to support nitrite reduction at rates similar to those observed with Glc6P (Fig. 2). Glucose-l-phosphate was also able to support nitrite reduction, but to a lesser extent. At 10 raM, 6phosphogluconate, fructose-l,6-bisphosphate, glyceraldehyde-3-phosphate, dihydroxyacetone phosphate, and fructose (data not shown) and glucose were individually ineffective. The ability of metabolites to support nitrite reduction may be a func-
C.G. Bowsher et ai. : Nitrite reduction and carbon metabolism in roots
361
Table 1. Distribution of organelle marker enzymes after the centrifugation of the 200.g supernatant from pea root homogenate at 4000-g for 3 rain. The resuspended pellet from this step was then centrifuged through 10% (v/v) Percoll at 4000.g for 5 min (mean value_+ SE of at least four determinations). Figures in ( ) represent activity recovered in fraction as a percentage of 200.g supernatant
200 .g 4000 .g 4000 .g 4000.g 4000 .g
supernatant supernatant pellet Percoll supernatant (post-Percoll plastid pellet
Nitrite reductase (nkat nitrite reduced, rag- 1 protein) (plastids)
Cytochrome oxidase (nkat-mg- t protein) (mitochondria)
Alcohol dehydrogenase (nkats-mg 1 protein) (cytoplasm)
Catalase (nkats. mg 1 protein) (microbodies)
0,20_+0.03 (100%) 0.12-t-0.03 (63%) 0,77_+0.12 (40%) 0.19_+0.07 (13%) 2.92_+0.50 (25%)
0.78_+0.18 (100%) 0.41_+0.12(73%) 2.52_+0.65(29%) 1.58_+0.55(25%) 1.07___0.62 (1.2%)
0.29_+0.12(100%) 0.33_+0.15 (99%) 0.02_+0.02 (1%) 0.01_+0.01 (1%) 0 (0%)
98_+25 (100%) 68_+ 8 (99%) 11_+ I (2.6%) 20_+ 5 (3.1%) 72_+41 (0.65%)
Fig. 1. Electron micrograph of purified pea root ptastids. Sections were stained in uranyl acetate and lead citrate Table 2. Specific activity and latency of enzymes of hexosephosphate metabolism associated with pea root plastids. Plastids were prepared as described in Material and methods and all assays performed in the presence of 330 mM sorbitol. LaB-I tency = - - B x 100% where I is the activity measured before, and B is the activity measured often rupturing organelles with 0.1% (v/v) Triton X-100 (mean_+ SE of three determinations) Enzyme
Specific activity (nkat-mg-1 protein)
Latency (%)
Glc6P dehydrogenase Hexose-phosphate isomerase Phosphoglucomutase
1.18 • 0.35 14.35+2.05 0.58 • 0.1
75 • 5 73 • 84 _+1
tion of the permeability of the plastid envelope but is also dependent on the presence of the necessary enzymes leading to Glc6P formation. In particular, for glucose-l-phosphate and fructose-6-
phosphate to support nitrite reduction requires that the enzymes necessary for their conversion to Glc6P be associated with the plastid preparation. The results in Table 2 demonstrate the specific activity and latency of these enzymes in relation to Glc6P dehydrogenase, an enzyme known to be latent within these organelles (Emes and Fowler 1979b). The degree of latency is similar in all cases confirming the presence of hexosephosphate isomerase and phosphoglucomutase within root plastids. The dependence of nitrite reduction in intact plastids on the concentration of hexose phosphates is shown in Fig. 3 a, b. The kinetics of fructose-6phosphate- and Glc6P-dependent nitrite reduction are presented as double-reciprocal plots along with the substrate-dependence of plastid he~ose-phosphate isomerase and Glc6P dehydrogenase. The
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C.G. Bowsher et al. : Nitrite reduction and carbon metabolism in roots
Km for fructose-6-phosphate-dependent nitrite reduction is 0.32 mM compared with a value of 0.35 mM for fructose-6-phosphate for the isomerase. The Km for Glc6P-dependent nitrite reduction is 0.66 mM compared with a Km of 0.68 mM for Glc6P for the plastid-located Glc6P dehydrogenase. Having examined the effect of hexose-phosphate concentration on the rate of nitrite reduction, the effect of nitrite reduction upon carbohydrate oxidation was investigated. This involved supplying intact plastids with Glc6P, isotopically labelled in different carbon atoms, and measuring the subsequent release of 14CO2 in the presence or absence of NO2 (Fig. 4a, b). Evolution of z4CO2 from plastids is greatest when Glc6P is labelled in carbon atoms 3 and 4 and least when labelled in carbon atom 6. The release of 14CO2 from these and [2-14C]Glc6P is unaffected by the presence of 1 mM NO2. By contrast there is a significant increase in the release of CO2 from carbon atom 1 of Glc6P when sodium nitrite is supplied simultaneously. By comparison with sodium nitrite, the addition of sodium chloride and sodium nitrate had no effect on the release of 14CO2 from [114C]Glc6P (Fig. 5). Osmotic-swelling studies have demonstrated that pea_ root plastids are freely permeable to NO2, NO 2 and C1- (data not shown)
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60
a
Fig. 3a, b. Lineweaver-Burke plots of hexose-phosphatedependent nitrite reduction and enzymes of hexose-phosphate metabolism. Substrate dependence of the latter was determined by using plastids lysed with 0.1% (v/ v) Triton X-100 prior to assay of activity. (Each value is the mean of three determinations) a Glucose-6-phosphate dependence of Glc6P dehydrogenase (v; values are V ~ x 10) and nitrite reduction (m) in intact root plastids. b Fructose-6-phosphate dependence of hexose-phosphate isomerase (A; values are V-z x 100) and nitrite reduction (e; values are V - i x 1.5) in intact root plastids
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C.G. Bowsher et al. : Nitrite reduction and carbon metabolism in roots
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Fig. 4a, b. Evolution of CO2 from different carbon atoms after labelled Glc6P had been supplied to intact root plastids m the presence (closed symbols) and absence (open symbols) of 1 m M sodium nitrite, a 9 * = [1J4C]Glc6P; r~ B=[6J'~C]Glc6P. b zx, 9 = [3,4-14C]Glc6P; O, * = [2JaC]Glc6P. (Mean values • SE of three determinations)
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Fig. 5a, b. Evolution of 14CO2 from [1-t4C]Glc6P supplied to intact plastids incubated with different salts, a Plastids from tissue watered with 10 m M KNO3 24 h prior to extraction, o - - o , No additional salts; e - - e , I m M NaNOz; [] ..... n, / m M NaC1; A - - - A , 1 m M NaNO3. b Plastids from tissue deprived of external nitrate throughout the growing period. ~-----~, No additional salts; ~-----~, 1 m M NaNO2. (Mean values • SE of three determinations)
364
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C.G. B o w s h e r et al. : Nitrite r e d u c t i o n a n d c a r b o n m e t a b o l i s m in r o o t s
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ruling out the possibility that differences may be due to anion accessibility or an effect of the cation. Plastids prepared from roots in which the enzymes of nitrate assimilation had not been induced, and which possess only low amounts of nitrite reductase (Emes and Fowler 1983), did not show any significant difference in 14CO 2 release from [la4C]Glc6P in the presence or absence of sodium nitrite (Fig. 5). These results taken together indicate that nitrite reduction is directly responsible for the increase in CO2 evolution from carbon atom 1 of Glc6P. That the stimulation in the release of CO2 from carbon atom 1 of Glc6P phosphate is a function of the demand for reductant during nitrite reduction is further illustrated in Fig. 6. The stimulation of CO2 evolution from intact plastids increases with the concentration of nitrite supplied. A K m of 0.4 m M for this stimulation can be calculated from the data if the amount of CO2 released from carbon atom I in the absence of nitrite is subtracted from each value. This is close to the reported Km (NO2-) of 0.1-0.6 m M for nitrite reductase from a number of sources (Vega et al. 1980). Discussion
The preparations of root plastids used in this work appear to be suitable for detailed study of their
metabolism in vitro. The rate of Glc6P-dependent nitrite reduction (0.1 n k a t . m g -1 plastid protein) at saturating substrate concentrations is similar to that obtained by Oji et al. (1985) with barley root plastids. However, Oji et al. obtained a twofold stimulation of Glc6P-dependent nitrite reduction when N A D P § was added to their assay system whereas no increase in activity was observed in our experiments when this was added to intact pea root plastids (data not shown). It is possible that the reason for this discrepancy lies in the permeability o f the preparations to pyridine nucleotides and would indicate that the barley-root plastids are more "leaky". This view is reinforced by the observation that nitrite reduction in barley root plastids is also stimulated by 6-phosphogluconate. We have previously observed the same effect (Emes and Fowler 1983) using pea root plastids purified by sucrose-density-gradient centrifugation, but with plastids prepared by the present method this is no longer apparent. It is suggested that whilst these earlier reports regarding the location and activity of nitrite-reducing systems are valid the membrane integrity of such preparations is questionable. The data presented in Fig. 2 imply that hexose monophosphates and ribose-5-phosphate are able to cross the envelope of pea root plastids. The former are not generally regarded as being readily able to enter chloroplasts (Heldt and Rapley 1970) but there is no reason a priori why non-photosynthetic root organelles should have the same permeabilities. We have previously demonstrated the presence of a phosphate-translocator-like system in pea root plastids (Emes and Traska 1987) but the kinetics of phosphate uptake appear to differ from the chloroplast protein. This probably reflects the different roles that this process plays in photosynthetic and non-photosynthetic tissues, and it need not be supposed, therefore, that movement of other metabolites should be identical. The uptake of hexose-phosphates by root plastids clearly merits further investigation. Such kinetic studies of hexose-phosphate-dependent nitrite reduction highlight the close relationship that exists between carbohydrate metabolism and nitrite reduction in root plastids. The results strongly indicate that the rate at which nitrite reduction can proceed is a function of the rate of Glc6P oxidation and, depending on the concentration of substrates in the plastid, the latter may limit the former. We have also approached this interaction from the opposite viewpoint, by looking at the way in which the rate of nitrite reduction affects the oxida-
C.G. Bowsher et al. : Nitrite reduction and carbon metabolism in roots
tion of Glc6P and the release of CO2 from different carbon atoms. A number of points can be made about the data in Fig. 4. First of all, in the presence of nitrite, the release of relatively large amounts of CO2 from carbon atoms 3 and 4 of Glc6P, and the release from other carbon atoms in the order C1 >C2 >C6 is consistent with the operation of the oxidative pentose-phosphate pathway (PPP) and glycolysis, as far as the decarboxylation of pyruvate, within these organelles (ap Rees 1980). In this paper we have demonstrated the presence of hexose-phosphate isomerase (Table 2) and routinely use triose-phosphate isomerase as a marker enzyme for root plastids. The intracellular location of all the enzymes of glycolysis in pea roots is a matter of current investigation, but a number of papers have reported the presence of the necessary glycolytic enzymes within non-green plastids, certainly as far as the formation of triose phosphate (Macdonald and ap Rees 1983; Simcox et al. 1977; Journet and Douce 1985) but with some equivocation with regard to the lower half of the pathway. The presence of pyruvate dehydrogenase in other non-green plastids has also been demonstrated (Reid et al. 1977; Journet and Douce 1985) and our results support the contention that these organelles can oxidise Glc6P to acetate/acetyl CoA. The release of CO2 from carbon atom 2 is, in the absence of nitrite, apparently higher than that from carbon atom 1. This probably arises from the correction of rates for 14CO2release from broken organelles using deliberately lysed preparations. Release of ~4COa from 100% lysed preparations is three- to fourfold higher for [1-~4C]Glc6P compared with [2-~4C]Glc6P, and probably results in an overcompensation for breakage when using preparations which may be as much as 90% intact. Nonetheless, such a result implies only limited operation of the PPP within these organelles in the absence of nitrite reduction. Present understanding of the re-arrangement of carbon atoms which takes place in the PPP requires that, for CO2 to be evolved from carbon atom 6 of Glc6P, there must be fructose-bisphosphatase activity. The possibility of cytosolic contamination seems unlikely on the basis of data in Table 1. Although membrane adsorption of the enzyme cannot be ruled out, since fructose bisphosphate is not able to support nitrite reduction in intact plastid preparations whereas fructose-6-phosphate is, this would also seem an unlikely explanation of the data. The presence of latent fructose-bisphosphatase activity within these organelles therefore warrants further investigation. Since there is no Krebs' cycle activity to be considered, the release of CO2 from carbon atom 2 as
365
well as from carbon atom 1 further indicates the possibility of limited recycling through the PPP. When nitrite is being reduced by root plastids the release of CO2 from Glc6P increases specifically and only from carbon atom 1. The data imply an increased flux of carbon through the PPP but without affecting the flow of carbon through what we presume to be glycolysis as judged by the release of CO2 from [3,4-14C]Glc6P. Glucose-6phosphate dehydrogenase, the key enzyme of the PPP is known to be regulated by the N A D P : N A D P H ratio (Ashihara and Komamine 1976). As a consequence of nitrite-reductase activity and the demand for reductant it is likely that this ratio would increase, resulting in deinhibition of the enzyme and increased activity. If three molecules of N A D P H are required per molecule of nitrite reduced this could be met, in these in-vitro experiments, by the oxidation of between 1.5 and 3 molecules of Glc6P, depending on the activity of 6phosphogluconate dehydrogenase. On the basis of the measured rate of nitrite reduction within these organelles ( 0 . 1 n k a t . m g -1 protein at 1 0 m M Glc6P) a rate of Glc6P oxidation of between 0.15 and 0.3 n k a t - m g - 1 protein would be required. The kinetic studies of the plastid enzyme (Fig. 3 a) indicate that this is only between 8 15% of the maximal rate of catalysis of 2 n k a t . m g - 1 protein, given the presence of saturating levels of substrate, implying some degree of inhibition of Glc6P dehydrogenase. This model of control a n d the release of inhibition would therefore seem feasible. The dependence of this stimulation o n the rate of nitrite reduction is emphasized in Fig. 6. As the concentration of nitrite is increased from below saturating levels, there is an increase in: the release of CO2 from carbon atom 1 of Glc6P, implying an increase in the activity of the oxidative reactions to keep pace with the demand for reductant. In conclusion the results presented in this paper demonstrate the ability of root plastids to metabolise Glc6P to CO2. The dependence of nitrite reduction, and ultimately amino-acid synthesis, on the kinetics of PPP enzymes illustrates how the flux of carbon through a pathway of carbohydrate oxidation may regulate the pathway of nitrogen assimilation. Conversely, it appears t h a t the rate of nitrite reduction, when nitrite is limiting, influences the activity of the PPP.
C.G.B. is the recipient of a Science and Engineering Research Council-Cooperative Awards in Science and Engineering studentship. The authors are grateful to Mr. O. Sayed for assistance with electron microscopy.
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C.G. Bowsher et al. : Nitrite reduction and carbon metabolism in roots
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