Journal of Experimental Botany, Vol. 51, No. 349, pp. 1349-1356,
August 2000
© 2000 Oxford University Press
Original Papers |
Short-term nitrogen-induced modulation of phosphoenolpyruvate carboxylase in tobacco and maize leaves
1 Laboratoire du Métabolisme et de la Nutrition des Plantes, INRA, Route de St Cyr, 78026 Versailles Cedex, France
2 Department of Biochemistry and Physiology, IACR-Rothamstead, Harpenden, Hertfordshire AL5 2JQ, UK
Received 1 February 2000; Accepted 12 April 2000
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Abstract |
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Untransformed maize and tobacco plants and tobacco plants constitutively expressing nitrate reductase were grown with sufficient
to support maximal growth. Four days prior to treatment the tobacco plants were deprived of nitrogen. Excised maize leaves and tobacco leaf discs were fed with either 40 mM KNO3 or 40 mM KCl (control) in the light. Phosphoenolpyruvate (PEP) carboxylase (Case) activity was measured at 0.3 mM and 3 mM PEP. The light- induced increase in PEPCase Vmax was greater in maize than tobacco. Furthermore light decreased malate sensitivity in maize (which was N-replete) but not in N-deficient tobacco. treatment increased PEPCase Vmax values in both species and decreased the sensitivity to inhibition by malate, but effects of were much more pronounced in tobacco than maize. PEPCase kinase activity was, however, greater in maize leaves fed than in the Cl--treated controls, suggesting that it is responsive to leaf nitrogen supply. A correlation between foliar glutamine content and PEPCase activity was observed. It is concluded that PEPCase is sensitive to N metabolites which favour increased flow through the anapleurotic pathway in both C3 and C4 plants. Key words: Anapleurotic pathway, nitrogen assimilation, protein kinase, C3 PEPCase, C4 PEPCase.
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Introduction |
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PEPCase (EC 4.1.1.31) catalyses the carboxylation of PEP to form OAA utilizing atmospheric CO2 (as
). It is a ubiquitous cytosolic enzyme amongst algae, bacteria and higher plants and has multiple roles, including the photosynthetic fixation of CO2 in C4 and CAM plants (O'Leary, 1982
; Muller and Kluge, 1983). In the anapleurotic pathway it replenishes OAA in the TCA cycle during inorganic nitrogen assimilation (Melzer and O'Leary, 1987; Guy et al., 1989). C3, CAM and C4-type PEPCase are sensitive to allosteric inhibitors and effectors, in particular malate is a powerful inhibitor (Wedding et al., 1990; Schuller and Werner, 1993). In C4 plants the strong light/dark modulation of PEPCase activity has been attributed to protein phosphorylation which is suggested to be regulated directly by photosynthesis. This results in an increase in maximal activity and a decrease in the sensitivity of the enzyme to inhibition by L-malate and other inhibitors such as shikimate (Jiao and Chollet, 1991, 1992; Bakrim et al., 1993, 1998; Gao and Woo, 1996; Colombo et al., 1998).
The phosphorylation state of PEPCase is largely determined by the action of a specific Ca2+-independent protein kinase (Carter et al., 1991; Li and Chollet, 1994) although regulation by other Ca2+-dependent protein kinases has also been demonstrated (Osuna et al., 1999; Ogawa et al., 1992, 1998; Karibe et al., 1996; Zhang and Chollet, 1997). The transcription of the PEPCase kinase gene and the abundance of PEPCase kinase mRNA responds to photosynthesis in C3 and C4 plants (Hartwell et al., 1996) and to metabolic triggers in CAM plants (Borland et al., 1999) and in maize (Hartwell et al., 1999). It follows, therefore, that PEPCase kinase activity is dictated by relevant metabolic cues, particularly those arising from carbon and nitrogen metabolism.
Controversy still exists concerning the nature of metabolic regulation of PEPCase activity in C3 plants. Light/dark modulation of PEPCase activity has been clearly demonstrated in some cases (Van Quy et al., 1991a; Rajagopalan et al., 1993; Duff and Chollet, 1995), but is less evident in others (Chastain and Chollet, 1989; Leport et al., 1996). Feeding to either C3 or C4 leaves in the light causes an increase in the activity of PEPCase, an increased flow of carbon into amino acid synthesis and a concomitant reduction of sucrose synthesis via regulation of SPS activity (Van Quy et al., 1991b; Sugiharto and Sugiyama, 1992; Foyer et al., 1994). The precise mechanisms of control by nitrogen assimilation are uncertain, although changes in the activities of kinases have been implicated (Champigny and Foyer, 1992; Duff and Chollet, 1995). Important observations were made using the C3 plant wheat (Manh et al., 1993; Duff and Chollet, 1995) where the feeding of to illuminated leaves caused a several-fold greater induction of PEPCase kinase activity than light alone. This suggests that nitrogen may modulate the light-induced activation process. The nature of the interaction between the photosynthetic carbon and nitrogen assimilation pathways in the regulation of PEPCase in C4 plants has not, to date, been fully characterized. Here it is shown that supplying to the transpiration stream in the light modifies the kinetic properties of PEPCase in a C3 species, tobacco, and also affects the kinetic properties of PEPCase through changes in PEPCase kinase activity in the C4 species, maize.
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Materials and methods |
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Plant material
Maize (Zea mays), untransformed tobacco (Nicotiana plumbaginifolia) and transformed tobacco constitutively expressing nitrate reductase under the control of the 35S promoter (Foyer et al., 1994
; Ferrario-Méry et al., 1997), were grown on sand in pots 12 cm in diameter for 15 d prior to use at a light intensity of 200 µmol quanta m-2 s-1, relative humidity (RH) of 85% and on a nutrient solution. N was supplied as 0.8 mM KNO3 for maize and 10 mM KNO3 and 2 mM 
for tobacco. Other nutrients were 1.0 mM KH2PO4, 0.2 mM NaCl, 0.1 mM K2HPO4 and either 0.7 mM K2SO4, 0.4 mM MgSO4 or 1.4 mM CaCl2 in addition to micronutrients (Foyer et al., 1994). N was supplied in the nutrient solution (pH 6.0) by automatic delivery at a rate of 100 ml every 30 min during the light period and every 2 h during the night. For tobacco, the concentration of was lowered to 0.8 mM 8 d before use and finally to 0.05 mM 4 d before use.
Treatments and sampling
Mature third leaves were cut from maize plants submerged in nutrient medium and incubated for 1 h at a light intensity of 400 µmol quanta m-2 s-1 in tubes containing nutrient medium. Leaves were then transferred to tubes containing nutrient medium supplemented with either KNO3 or KCl, both at a final concentration of 40 mM and incubated for a further 3 h. Tobacco leaf discs were cut from the two young fully expanded leaves of 20 plants and floated on nutrient solution at a light intensity of 200 µmol quanta m-2 s-1. After 1 h, they were transferred to nutrient solution containing 40 mM KNO3 or 40 mM KCl. Discs were sampled over a 4 h time course (18 discs per time point) and immediately frozen in liquid nitrogen and stored at -80 °C. All experiments were repeated two or three times.
Assays of amino acids and
Total amino acids were extracted as described for the carbohydrates and determined by a colorimetric method (Rosen, 1957). For the determination of amino acid composition, amino acids were extracted from the lyophilized powder with 2% 5-sulphosalicylic acid (10 mg dry weight ml-1). The crude extracts were centrifuged at 12 000 g for 5 min, and an aliquot of the supernatant was analysed by ion-exchange chromatography (model LC5001 analyser, Biotronics, Lowell, MA; Rochat and Boutin, 1989); the physiological program was run with lithium citrate buffers and detection at A570 and A440 after post-column derivatization with ninhydrin (Rochat and Boutin, 1989). content was analysed in the supernatant from the leaf extracts for NR activity according to the method of Cataldo et al. (Cataldo et al., 1975).
Assays of PEPCase
Leaf tissue was ground in liquid nitrogen in a medium containing 100 mM TRIS/HCl (pH 8.0), 10 mM MgCl2, 20% glycerol, 5 mM DTT or ß-mercaptoethanol, 5 mM NaF, 1 mM PMSF, 1 µM microcystin, 10 µg ml-1 leupeptin, 10 µg ml-1 chymostatin, and 2% (w/v) PVPP. Extracts were centrifuged at 4 °C for 15 min at 15 000 rpm and desalted rapidly twice on Sephadex G-25 spin columns. The reaction medium consisted of 50 mM HEPES/KOH (pH 7.3 (maize) and pH 7.5 (tobacco)), 5 mM MgCl2, 1 mM NaHCO3, 5 mM NaF, 0.2 mM NADH, 10 units of malate dehydrogenase and either 3 mM (maize) or 0.3 mM (tobacco) PEP in a final volume of 1 ml. The reaction was followed at 340 nm in cuvettes maintained at 30 °C. Control cuvettes were without PEP. Malate sensitivity was determined by the addition of 0–0.4 mM malate (for tobacco) and 0–3 mM malate (for maize) to both the sample and control cuvettes. IC50 L-malate values were determined according to Job et al. (Job et al., 1978).
In vitro phosphorylation of PEPCase
The phosphorylation assay contained 0.14 mM [
-32P]ATP (specific activity 0.56 mCi mmol-1), a volume of extract corresponding to 200 µg protein in a total volume of 160 ml. In addition, 2 U of purified sorghum PEPCase was added to the reaction medium. Phosphorylation was given 20 min at 25 °C and then arrested by the addition of sufficient polyclonal antibody (kindly provided by Dr Jean Vidal, Orsay, France) to immunoprecipitate 0.05 units of PEPCase. This was incubated on ice for 1 h and then centrifuged for 5 min at 13 000 rpm. The pellet was washed three times in 50 mM TRIS (pH 7.5) and 50 mM NaCl. PEPCase-antibody complexes were dissociated and denatured in solution containing 5% SDS and analysed by SDS-PAGE (with coomassie staining) and autoradiography.
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Results |
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Effects of N-feeding on
and amino acid pools of untransformed and transformed tobacco leavesPrior to feeding the
content of the leaves was below the level of detection. Following the incubation of tobacco leaf discs with KNO3 the and amino acid contents increased rapidly (Table 1) indicating that the was quickly taken up and assimilated by the leaf. The increase in content observed in leaves incubated in KCl was small and transient. The N-induced increases in were followed by large time-dependent increases in the Gln (30–40 fold) Asp (4–7 fold) and Asn (4–10 fold over 2–4 h) pools. The quantities of individual amino acids given in Table 1 were determined by HPLC while the total amino acid pool was determined by the Rosen method. The sum of the individual amino acids determined by HPLC represents about 70–80% of the total amino acid pool as determined by the Rosen method, but the latter also includes ammonium. An increase in NR activity was observed in the untransformed lines following N-feeding but not in the transformed line (data not shown). The accumulation of Gln, Asp and Asn was greater in the transformed line as consistent with the higher NR activities of these plants. The pools of Ser and Ala were also increased by N-feeding whereas the Gly pool decreased. A reduction in the levels of Glu is consistent with the increase in Gln. Data showing changes in content of individual amino acids of maize leaves following N-feeding has been shown by Foyer et al. (Foyer et al., 1994).
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Effect of N-feeding on in vivo PEPCase activity from maize and tobacco leaves
An increase in PEPCase activity (Fig. 1A) and a corresponding decrease in the sensitivity of the enzyme to the inhibitor malate (Fig. 1B) was observed in maize leaves within the first hour following the transition from darkness to light. No further increases in PEPCase activity or decreases in sensitivity to malate were observed in maize leaves supplied with KCl (Fig. 1). When was added in place of KCl an increase in activity was observed compared to the KCl-treated controls (Fig. 1A) with a corresponding small decrease in malate sensitivity becoming apparent 3 h after the onset of -feeding (Fig. 1B).
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). (A) PEPCase activity expressed as a percentage of that in leaves exposed to 1 h light. (B) IC50 L-malate (see text for details). Light was applied at 0 h. KNO3 or KCl applied where shown by arrow. Values are means±SE of three independent experiments. PEPCase activity at 1 h was typically 0.2–0.25 mmol h-1 mg-1 Chl. For this figure, if error bars are not shown they are smaller than the symbols.In illuminated tobacco leaves incubated with subsaturating concentrations of PEP (0.3 mM) an increase in activity and a decrease in the IC50 L-malate were observed following addition of
(Fig. 2A, B) but not KCl (Fig. 2B). Light activation of PEPCase was therefore not apparent in tobacco leaves in the absence of . The addition of was required to increase PEPCase activity and to decrease IC50 L-malate in the light at sub-saturating (0.3 mM) and saturating (3 mM) concentrations (Fig. 2). PEPCase activity increased between 25% and 50% in the presence of while the IC50 L-malate was doubled at both PEP concentrations.
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The transformed tobacco line constitutively expressing NR contained slightly higher foliar PEPCase activitites than the untransformed controls (Fig. 3A
). As found with untransformed tobacco plants, no light-dependent effects on Vmax were observed in leaves supplied with KCl (Fig. 3B). In contrast, increased foliar PEPCase Vmax activity in the light in both transformed and untransformed plants.
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Effects of on PEPCase phosphorylation
To investigate potential changes in the phosphorylation status of PEPCase induced by , maize leaf extracts were used in an in vitro assay, carried out following extraction in the presence of a cocktail of protease and phosphatase inhibitors. To ensure that enough dephosphorylated PEPCase was present in the reaction medium to act as a substrate for the kinases, purified sorghum PEPCase was also added to the reaction medium. This was successfully immunoprecipitated by the maize-directed antibodies (Fig. 4). The polyclonal antibody resulted in precipitation of equal amounts of protein in each extract (Fig. 4A) and Western blotting of SDS-PAGE gels revealed the band at 100 kDa to be PEPCase. Figure 4B shows that all PEPCase activity was removed from the extract by immunoprecipitation and centrifugation. No PEPCase protein remained in the supernatant following centrifugation (data not shown).
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After 4 h incubation leaves from the
-treated plants contained more radioactivity in PEPCase than the KCl-treated controls (Fig. 5A). This was found to be the case in three independent experiments. To quantify the differences in phosphorylation state, PEPCase bands were excised from the gel and the amount of radioactivity in each determined. Data from three experiments were pooled and the visible differences were quantitatively confirmed (Fig. 5B). The induces changes in PEPCase activity and IC50 L-malate closely followed changes in kinase activity. There was no change in the amount of PEPCase in maize leaves over the 4 h (Fig. 4
). Figure 5A shows a darkening of the first band on the blot (dark, 0 h), although when cpms from three experiments were pooled the dark level was lower (Fig. 5B).
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Relationships between foliar glutamine and PEPCase activity in tobaco leaves
The increase in PEPCase Vmax activity observed following N feeding was related to the foliar Gln content (Fig. 6A), as was the increase in IC50 L-malate. Higher foliar Gln contents were correlated with higher IC50 L-malate and higher PEPCase Vmax activities (Fig. 6).
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), transformed tobacco incubated with KCl (
), untransformed tobacco incubated with KNO3 (
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Discussion |
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It is now well-established that nitrate increases the expression of genes encoding enzymes of N metabolism and leads also to a stimulation of organic acid biosynthesis (Scheible et al., 1997
; Morcuende et al., 1998; Lancien et al., 1999). The observations of nitrogen modulation of tobacco leaf PEPCase activity and kinetic properties are consistent with results of previous studies on the phosphorylation of wheat leaf PEPCase (Duff and Chollet, 1995; Van Quy et al., 1991) and soybean nodule PEPCase (Zhang et al., 1995). Phosphorylation altered the kinetic properties of the soybean enzyme in vitro (Schuller and Werner, 1993) in a manner similar to that observed in the present study on tobacco. Since the illumination of tobacco leaves induced an increase in PEPCase activity in the presence of (but not in its absence) both and light were required for the modification of IC50 L-malate. It is concluded that light and nitrogen exert interactive effects upon the kinetic properties of PEPCase in tobacco leaves.
Changes in Vmax and IC50 L-malate occur concomitantly with phosphorylation in maize PEPCase (Jiao and Chollet, 1991, 1992; Bakrim et al., 1993) though an increased PEPCase kinase activity in barley leaf protoplasts did not necessarily coincide with increased IC50 L-malate (Smith et al., 1996). In nitrogen-replete tobacco leaves, light induced simultaneous changes in both IC50 L-malate and Vmax (Li et al., 1996). The data presented here suggest that the effects of covalent regulation on the kinetic properties of PEPCase in C3 and C4 plants is dependent on the N status of the tissues. Neither PEPCase kinase activity nor IC50 L-malate were changed in the presence of photosynthesis inhibitors (Smith et al., 1996; Li et al., 1996). Conversely, inhibition of Gln synthesis in tobacco leaves in the light caused an inhibition of PEPCase kinase activity which was partially reversed by feeding Gln (Li et al., 1996). However, maize leaf PEPCase did not show the same sensitivity to inhibitors of nitrogen assimilation (Li et al., 1996). The results presented in this study suggest that in maize leaves grown on 0.8 mM , PEPCase activity was only affected to a small extent by feeding. It is important to note that the plants used in this experiment were not nitrogen deficient; growth of maize on 0.8 mM results in maximal growth rates but little or no free is present in the leaf tissues (Foyer et al., 1994). It is possible that the maize leaves used in these experiments already contained sufficient concentrations of Gln that effects of Gln on PEPCase activity were already maximal. PEPCase in maize is regulated by signals associated with photosynthesis (Jiao and Chollet, 1992). In maize, however, light activation of PEPCase kinase was unimpaired in a mutant defective in ribulose-1,5-bisphosphate carboxylase-oxygenase and lacking an operative Calvin cycle (Smith et al., 1998) indicating that metabolism, rather than photosynthesis per se, regulates PEPCase kinase. Since nitrogen assimilation in leaves is dependent on photosynthesis, for carbon skeletons and reducing power, it follows that there may be a sensitive balance between the relative ‘strengths’ of these two processes, and it is this balance which regulates PEPCase. In other words, the magnitude of the light-associated response would be dependent on the sizes of the pools of C and N metabolites and would hence respond to signals from the nitrogen assimilation pathway.
For the first time in a C4 plant, an increase of PEPCase kinase activity in response to a supplemented leaf nitrogen supply has been shown directly. PEPCase exists as a gene family in both C3 and C4 plants and the latter contains both the C3 and C4 forms in leaves (Kawamura et al., 1992). The clear increases in PEPCase kinase activity in maize seen in this study could refer to alterations in the C3 form, the C4 form, or both. Recently, high levels of expression of a maize PEPCase in transformed rice was found to result in changes in photosynthesis but no changes in organic acid metabolism resulting from this transformation have been reported to date (Ku et al., 1999). Transformed potato plants expressing a PEPCase from Corynebacterium glutamicum that is not modulated by protein phosphorylation had increased rates of respiration in the dark and the light (Hausler et al., 1999). Total foliar amino acids were only slightly increased in these transformants but malate, sucrose and starch were greatly increased. These results are consistent with an enhanced flux of photoassimilate into glycolysis and subsequently into the citric acid cycle. Whilst nitrogen assimilation in maize requires PEPCase kinase activity, the situation is complicated by the dominance of PEPCase activity in photosynthesis. It is, therefore, impossible to distinguish between the roles of the C3 and C4 isoenzymes in the present experiments.
Glutamine has previously been suggested to be an effector of PEPCase activity (Manh et al., 1993). This may also depend upon other factors such as carbohydrate accumulation (Sawada et al., 1999), a condition which was not observed in the current study. The mode of action of Gln is uncertain though may be indirect (Gao and Woo, 1996). Certain other amino acids may also be involved in the regulation of PEPCase (Golombek et al., 1999). The data presented here shows a correlation between the foliar Gln content and the activity of PEPCase. Changes in IC50 L-malate in the presence of high Gln are consistent with the hypothesis that Gln is involved in the activation of PEPCase in tobacco (Fig. 6). Wheat leaf PEPCase-kinase activity was increased several-fold more in response to nitrogen supply in the light than to light alone (Duff and Chollet, 1995). These results confirm that PEPCase regulation is sensitive to signals derived from N-metabolism, particularly Gln, in both C3 and C4 plants.
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Acknowledgments |
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We thank Dr Jean Vidal (Orsay) for the kind gift of maize PEPCase antibody and sorghum PEPCase. This work was funded by the European Economic Community and was a project of the Technical Priority Network D—Nitrogen Utilization and Efficiency.
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Notes |
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3 Current address: Robert Hill Institute, Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, UK.
4 To whom correspondence should be addressed. Fax: +44 1582 763010. E-mail:christine.foyer{at}bbsrc.ac.uk
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Abbreviations |
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PEPCase, phosphoenolpyruvate carboxylase; PEP, phosphoenolpyruvate; OAA, oxaloacetic acid; CAM, Crassulacean acid metabolism; DTT, 1,4-dithiothreitol; TRIS, tri-(hydroxymethyl)aminomethane; PMSF, phenylmethylsulphonylfluoride; HEPES, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulphonic acid; PVPP, polyvinylpolypyrrolidone; SDS-PAGE, sodium dodecylsulphate-polyacrylamide gel electrophoresis; TCA, tricarboxylic acid..
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