Journal of Experimental Botany, Vol. 51, No. 352, pp. 1903-1909,
November 1, 2000
© 2000 Oxford University Press
Original Papers |
Phosphoenolpyruvate carboxylase in cucumber (Cucumis sativus L.) roots under iron deficiency: activity and kinetic characterization
Dipartimento di Produzione Vegetale, University of Milan, Via Celoria 2, I-20133 Milano, Italy
Received 11 February 2000; Accepted 19 June 2000
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Abstract |
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Phosphoenolpyruvate carboxylase (PEPCase) activity was investigated in cucumber roots grown under iron starvation. The enzyme extracted from plants grown in the presence and in the absence of Fe was characterized both kinetically and biochemically. Extractable PEPCase activity was increased by 4-fold in the absence of Fe. This increase began about 5 d after Fe starvation. Western blot analysis revealed the presence of two polypeptides with apparent molecular masses of 103 and 108 kDa. At the beginning both the polypeptides were equally present in the control and in the Fe-deficient roots. After 10 d of Fe starvation the increase was clearly evident and concerned, in particular, the polypeptide of 103 kDa whose enhancement was around 3-fold with respect to the control. Re-supply of iron to Fe-starved roots decreased both the activity and the concentration of the enzyme to the control values. Determination of kinetic parameters revealed that the Km values for the substrates were the same, while the Vmax was increased by four times for the enzyme extracted from Fe-deficient roots. Also the responses to pH changes and to the allosteric modulators malate and glucose-6-phosphate were different. The kinetic data, the increase in PEPCase specific activity and in the PEPCase polypeptides concentration seem to indicate that under Fe deficiency the enzyme regulation might be, in part, exerted at the transcriptional level.
Key words: Cucumis sativus, Fe-deficiency, phosphoenol-pyruvate carboxylase.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Plants growing under iron deficiency are able to develop several adaptive mechanisms for the mobilization and acquisition of this element. Two major mechanisms (strategies) have been proposed (Römheld and Marschner, 1986
; Marschner and Römheld, 1994; Schmidt, 1999): strategy I, to which dicots and non-Gramineae monocts belong, and strategy II, to which the Gramineae belong. Strategy II is a very simple mechanism and is based on the release by plant roots of small molecules with chelating properties in the rhizosphere, called siderophores. The Fe-siderophore complex is subsequently taken up by plant roots.
In plants belonging to strategy I, iron deficiency causes peculiar biochemical, physiological and morphological responses in order to mobilize and increase Fe availability for root uptake. Fe3+ reduction (since only Fe2+ is taken up), acidification of the rhizosphere and organic acids synthesis are common responses shared by these plants. These activities have been well characterized recently (Römheld and Marschner, 1986; Schmidt, 1999) and some metabolic implications have also been determined (Bienfait, 1996; Schmidt, 1999). As a consequence of increased proton extrusion and organic acid synthesis, an interesting correlation was shown among Fe-deficiency and phosphoenolpyruvate carboxylase (PEPCase) activity (Landsberg, 1986; Bienfait et al., 1989; Rabotti et al., 1995). In previous work (Rabotti et al., 1995), it was shown that under iron starvation the activity of PEPCase was enhanced several times and that the activation of H+ extrusion and the synthesis of organic acids were supported by a major CO2 dark fixation and by an increase in the activity of the PEPCase itself. Active proton extrusion seen under Fe-deficiency (Zocchi and Cocucci, 1990, and references therein) will, in fact, lead to the alkalinization of cytoplasm (Felle, 1988) and the activation of PEPCase seems to be an obligatory step to balance the change in pHc according to the pH-stat theory (Davies, 1973; Sakano, 1998).
PEPCase (EC 4.1.1.31) is a ubiquitous enzyme in plants which catalyses the bicarbonate fixation to phosphoenolpyruvate to produce oxalacetate and release Pi. It plays an important fixation role in C4 and CAM plants in the photosynthetic carbon assimilation (O'Leary, 1982; Chollet et al., 1996). In C3 plants and in non-photosynthetic tissues PEPCase is active in several anaplerotic reactions to replenish the intermediate of the Krebs cycle, to provide carbon skeletons to sustain the synthesis of amino acids during 
assimilation (Melzer and O'Leary, 1987; Schuller et al., 1990), and for the regulation of cytoplasmic pH (Davies, 1973; Latzko and Kelly, 1983). For all of these reasons this enzyme has received much attention and it has been widely studied and characterized for its role in photosynthetic tissues from C4 and CAM plants (for a complete review see Lepiniec et al., 1994; Chollet et al., 1996; Vidal and Chollet, 1997) and for its important contribution in providing the carbon units during the amino acids biosynthesis in developing and germinating seeds (Huppe and Turpin, 1994; Gonzalez et al., 1998; Golombek et al., 1999).
In this work an attempt has been made to characterize the activity of PEPCase in a strategy I plant known to respond to Fe starvation by enhancing the proton extrusion linked to the activity of the plasmalemma H+-ATPase (Rabotti and Zocchi, 1994). The activation of the enzyme during Fe-deficiency has been confirmed. The increase in the PEPCase activity seen in Fe-starved conditions seems to be a direct consequence of an over-production of the enzyme in the roots. The data obtained in this work give further support to the key role that this enzyme also plays in non-photosynthetic tissues and support its importance in the biochemical responses of strategy I plants when grown under Fe-deficiency.
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Materials and methods |
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Plant material and growth conditions
Cucumber seeds (Cucumis sativus L. cv. Marketer from F.lli Ingegnoli, Milan, Italy) were allowed to germinate in Agriperlite watered with 0.1 mM CaSO4 for 4 d in the dark at 26 °C. Seedlings were transferred to 20 l tanks for hydroponic culture as already reported (Zocchi and Cocucci, 1990
; Rabotti and Zocchi, 1994). Iron, when present, was supplied at 0.1 mM final concentration as Fe3+-EDTA. Plants were grown in a growth chamber with a day/night regime of 16/8 h and a PPFD of 200 µmol m-2 s-1 at the plant level. The temperature was 18 °C in the dark with an RH of 60% and 24 °C in the light with an RH of 80%.
PEPCase assay
Roots of plants grown in the presence or in the absence of Fe were harvested, rinsed in distilled H2O and homogenized in a buffer containing 50 mM TRIS-HCl (pH 7.5), 10 mM MgCl2, 10% (v/v) glycerol, 1 mM EDTA, and 14 mM ß-mercaptoethanol. 1 mM PMSF and 10 µg ml-1 leupeptin were added to avoid or minimize proteolysis (according to Gonzales et al., 1998). A ratio of 1 ml of buffer for 1 g of roots was used to keep the initial volume low. The homogenate was centrifuged at 13 000 g for 15 min and the supernatant was again centrifuged at 100 000 g for 30 min. The soluble extracted proteins were dialysed against the same homogenization buffer and used for PEPCase activity assays directly or after storing in liquid N2. PEPCase was determined by coupling its activity to malate dehydrogenase-catalysed NADH oxidation in 1.5 ml final volume of a standard buffer containing 100 mM TRIS-HCl (pH 8.0), 5 mM MgCl2, 2.5 mM PEP, 0.2 mM NADH, 10 mM NaHCO3, and 15 µg ml-1 MDH (Boehringer-Mannheim). NADH oxidation was determined at 340 nm in a model V550 spectrophotometer (Jasco) at 25 °C. Assays were initiated by adding aliquots of the protein extracts. Protein was determined by the Bradford procedure using 
-globulin as the standard (Bradford, 1976).
Determination of kinetic properties
Kinetic analysis was performed in the standard buffer by varying each time the parameter considered. Concentration range for HCO-3, PEP and Mg2+ were from 0.05–10 mM for 
and PEP and from 0.5–10 mM for Mg2+, respectively. Decarbonated water was used for the determination of kinetic. Vmax and Km values for , PEP and Mg2+ were calculated using the Eadie-Hofstee plot.
PEPCase immunocharacterization
Soluble protein extracted from roots of plants grown in the presence and in the absence of Fe was loaded on a discontinuous SDS-polyacrylamide gel (3.75% [w/v] acrylamide stacking gel, and typically 9% [w/v] acrylamide separating gel) (according to the method of Laemmli, 1970).
After SDS-PAGE proteins were electrophoretically transferred to PVDF membrane filters (Sigma) using a semi-dry blotting system with a buffer containing 10 mM 3-cyclohexylamino-1-propane sulphonic acid (pH 11 with NaOH) and 10% (v/v) methanol for 1.5 h at room temperature at a current intensity of 0.8 mA cm-2. Polyclonal antibodies raised against a PEPCase isoform of sorghum were used (a kind gift from Dr J Vidal, Université de Paris-Sud). Antiserum was diluted 1:1000 in TBS-T buffer [20 mM TRIS-HCl (pH 7.5), 200 mM NaCl, 0.05% (w/v) Tween 20] and incubation was carried out overnight at 4 °C. After rinsing with TBS-T, PVDF membranes were incubated at room temperature for 2 h with a 1:25 000 diluted secondary antibody (alkaline phosphatase-conjugated anti-rabbit IgG, Sigma). After rinsing in TBS-T the filters were incubated in 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (FAST BCIP/NBT, Sigma). The blots were scanned and the PEPCase immunoreactive bands were quantified by using a Panasonic videocamera linked to a personal computer. Images were processed with a CREAM software (KEM-EN-TEC, Copenhagen, Denmark).
In vitro phosphorylation
Aliquots of dialysed soluble proteins extracted from roots of plants grown in the presence and in the absence of Fe were incubated in 100 µl final volume of a phosphorylation buffer containing 50 mM TRIS-MES (pH 8.0), 5 mM MgCl2, 10% glycerol, 0.050 mM CaCl2, 1 mM DTT, and 10 µg ml-1 leupeptin. The reaction was initiated by addition of 2.5 µCi of [-32P]ATP. After 1 h incubation at 30 °C the reaction was stopped by adding 100 µl of a double-strength SDS-PAGE sample buffer (Laemmli, 1970) and heated for 5 min at 90 °C. SDS-PAGE and immunodetection analysis were performed as specified above. The patterns of 32P phosphorylated proteins were visualized by autoradiography using a Kodak X-Omat AR film.
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Results |
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It was previously shown that cucumber plants subjected to Fe starvation were able to increase the synthesis of organic acids, CO2 dark fixation and the activity of the extractable PEPCase, and that these activities were strictly correlated to the increase in proton extrusion from the roots (Rabotti et al., 1995
). PEPCase is present in different isoforms which show different kinetic and regulatory characteristics (Chollet et al., 1996). While these properties are well characterized for the photosynthetic forms, little is known about the forms of non-photosynthetic tissues, particularly for the root forms.
Kinetic characterization of PEPCase activity from cucumber roots
The effect of the pH on the PEPCase activity extracted from roots of cucumbers grown in the presence and in the absence of Fe and assayed under optimal substrate conditions (i.e. 10 mM , 2.5 mM PEP and 5 mM Mg2+) is reported in Fig. 1. The pH pattern showed a broad optimum above pH 8.0 for both the extracted enzymes. A difference was, however, present: until 7.0 the two pH profiles were similar, but at 7.5 a marked increase in that of Fe-deficient enzyme was evident. In fact, while for the control the activity was increased by about four times from pH 7.0 to pH 8.0, for the Fe-deficient enzyme the increase was about 10 times in the same range of pH, showing a different responsiveness to the pH changes.
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Table 1
shows the kinetic parameters for the two substrates and PEP and for Mg2+, which is required for the enzyme activation (O'Leary, 1982). The Km values did not show any significant difference; on the contrary the calculated values for the Vmax were always greater (around four times) for the enzyme extracted from Fe-deficient roots with respect to the control.
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PEPCase activity was measured under non-limiting substrate conditions in extracts from roots grown in the presence and in the absence of Fe and in the presence of the inhibitor malate and of the allosteric activator glucose-6-phosphate (Fig. 2
) at different pH values. The enzyme activity was almost unaffected at pH 8.0 for both the control and the Fe-deficient conditions (Fig. 2A, D). At pH 7.5 (Fig. 2B) and to a lesser extent at pH 7.0 (Fig. 2C), malate exhibited its inhibitory effect at 1 mM concentration. The negative effect of malate was greater on the activity extracted from Fe-deficient roots at all the pH values considered and particularly at pH 7.5 where, at 1 mM, the inhibition already reached 50% while at 5 mM it was around 75%. The PEPCase activity was strongly activated by glucose-6-phosphate and the effect was greater, as for malate, at pH 7.5 (Fig. 2E) and 7.0 (Fig. 2F). In this case, it was the enzyme extracted from roots grown under Fe deficiency that exhibited greater activation by glucose-6-phosphate.
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Activity and immunodetection analysis of PEPCase
Polyclonal antibodies raised against the C4 isoenzyme of sorghum were used to assess the presence of the enzyme in these preparations and to evaluate possible differences in the expression of the enzyme under Fe starvation. Western blot analysis of proteins obtained from roots of cucumbers grown in the presence and in the absence of Fe for 10 d and from leaves (photosynthetic forms) and roots (non-photosynthetic forms) of maize seedlings are compared in Fig. 3B. In the lanes with the proteins extracted from cucumber roots the antibody reacts against two polypeptides with apparent molecular masses of 103 and 108 kDa, in agreement with those reported in the literature (Osuna et al., 1999, and references therein). In the Fe-deficient conditions (Fig. 3B) both polypeptides were enhanced, but this increase was particularly evident for the polypeptide of 103 kDa. This increase was already detectable in the SDS-PAGE (Fig. 3A arrows). Figure 4 shows the time-course of the PEPCase activity (Fig. 4A) and compares the Western blot analysis of the proteins extracted at different plant age (Fig. 4B). At days 3 and 5 a small difference was present between the control and the Fe-deficient conditions. No appreciable difference in the amount of protein present, as determined by the immunodetection analysis, was evident at these stages of growth. After about 5 d of Fe starvation a rapid increase occurred in the enzymatic activity that was further increased at day 10. At this stage the activity of Fe-starved roots was around four times greater than that of control roots. The comparison of the immunoblots (Fig. 4B) clearly shows that the enhancement in the PEPCase activity was accompanied by an increase in the amount of the enzyme present in the extract. Changes in the band composition occurred at 10 d and Fig. 4C shows the relative intensities of the bands. In particular, this increase seems mainly to involve the form at 103 kDa, which is effectively enhanced by about three times; the form at 108 kDa was increased, but to a lesser extent (less than 50%). Further administration of Fe to deficient plants (dotted line in Fig. 4A) reduced the enzyme activity to the level seen at the beginning. When Fe-deficient roots were resupplied with iron not only the enzymatic activity was decreased (Fig. 4A) but also the bands decreased their intensities to the same value as seen in the control (Fig. 4B, C).
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Phosphorylation assay
Phosphorylation of PEPCase was examined by 32P in vitro incorporation. After the SDS-PAGE, proteins were analysed by Western blotting and autoradiography. As shown in Fig. 5 there is an important difference in the results. In fact, only in the Fe-sufficient condition were the two polypeptides both phosphorylated (Fig. 5B) while in the Fe-deficient condition only the band corresponding to the 103 kDa was phosphorylated. The level of phosphorylation seems to be greater in the +Fe condition if the amount of protein present in the immunoblotting assay is compared (Fig. 5A).
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Discussion |
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Strategy I plants are known to respond to Fe deficiency in quite a complex way that involves not only the classical responses of this category and which are mainly directed to the mobilization and acquisition of the ion (Schmidt, 1999
). Increased reductase activity, proton extrusion and synthesis of organic compounds (organic acids and in most cases phenols) require the enhancement of a catabolic flux necessary (a) to produce NAD(P)H and ATP that are the substrates of the NAD(P)H:Fe3+ reductase and of the H+-ATPase localized on the plasmalemma and primarily involved in the reduction-based iron uptake and (b) for the synthesis of the organic compounds necessary to sustain other correlated activities. It has been shown that under Fe deficiency some cytosolic dehydrogenases (Rabotti et al., 1995) as well as some glycolytic enzymes and the respiration rate (Espen et al., 2000) were enhanced. Moreover, in the same deficient conditions increases in CO2 dark fixation and organic acid synthesis were demonstrated (Landsberg, 1986; Bienfait et al., 1989; Rabotti et al., 1995). PEPCase was hypothesized to be strictly linked to these processes but no attempts were made to determine the factor(s) responsible for its activity increase (Rabotti et al., 1995).
In this work an attempt has been made to compare and characterize, both kinetically and biochemically, PEPCase activities extracted from cucumber roots grown in the presence and in the absence of iron. Both are characterized by a broad optimum pH above 8.0 (Fig. 1
) and showed more or less the same Km values for , PEP and Mg2+ (Table 1). By contrast, Vmax was increased by about four times for the enzyme extracted from Fe-deficient roots when compared to the control (Table 1) confirming and extending the previous results (Rabotti et al., 1995). Increase in the Vmax was not the only difference between the control and the Fe-deficient extracted enzymes. A further significant difference concerned the pH response. In fact, for the Fe-deficient form the shift from pH 7.0 to pH 8.0 resulted in a 10-fold increase well above that of the control (only 4-fold). Second, the sensitivity to the inhibitor malate and to the allosteric activator glucose-6-phosphate was greater in the Fe-deficient form (Fig. 2). PEPCase activity was shown to increase by four times in the Fe-deficient conditions from 5–10 d after Fe starvation (Fig. 4A); this period coincided with the activation in the roots of the adaptive responses, i.e. increase in the reductase and proton extrusion activities, CO2 dark fixation and organic acid synthesis (Rabotti and Zocchi, 1994; Rabotti et al., 1995).
Different forms of PEPCase are encoded by a small multigene family and are involved in specific physiological functions (Lepiniec et al., 1994). Western blot analysis revealed that the anti-sorghum C4 PEPCase polyclonal antibody immunodecorated two polypeptides with apparent molecular masses of 103 and 108 kDa (Fig. 4B). The same result occurred in germinating barley (Osuna et al., 1999), wheat (Osuna et al., 1996; Gonzalez et al., 1998) and castor oil seeds (Sangwan et al., 1992). Both the polypeptides were increased by Fe deficiency when compared to the control, but, while for the 108 kDa it was around 50%, that for the 103 kDa was increased by almost three times (Fig. 4C). The 103 kDa polypeptide has been referred to as the constitutive form, while the 108 kDa was considered an inducible form in developing and germinating seeds (Sangwan et al., 1992; Osuna et al., 1996, 1999; Gonzalez et al., 1998). Also in proteoid roots of Lupinus albus, phosphorus deficiency caused the de novo synthesis of PEPCase. In this latter case only one polypeptide was revealed by immunoblot analysis (Johnson et al., 1996). Under Fe deficiency the situation does not exactly coincide with the data reported in the literature for developing and germinating seeds and for phosphorus-starved white lupin roots. In fact, the two forms seem to be equally expressed in the cucumber roots both in the control and at the beginning of Fe starvation (Fig. 4B, days 3 and 5), but with the proceeding of Fe deficiency the increase was evident mainly at the level of the 103 kDa form (Fig. 4B, day 10 and Fig. 4C). It should seem at this point that Fe deficiency simply induced an increase in the synthesis of the enzymatic forms already present. Both the control and the Fe-deficient extracts shown the same Km values for the substrates and the increase in the Vmax is more likely due to the increased concentration of the enzyme(s). This is also supported by the kinetic analysis in the presence of different amounts of MgCl2 where, again, the Km was the same and the Vmax increased. The different sensitivity to pH, malate and glucose-6-phosphate might be related to the different ratio of the two PEPCase isoforms. The 103 kDa expressed under Fe deficiency could be more sensitive to the biochemical changes occurring under this condition. The increase in PEPCase specific activity and in PEPCase enzyme indicates that this protein, in Fe-deficient plants, is in part under transcriptional regulation.
The greater effect of glucose-6-phosphate could be explained as an allosteric push to increase the rate of glycolysis to produce enough PEP to satisfy the change in the pHc but also to sustain the other activities that are increased under Fe deficiency. In fact, it was found (Espen et al., 2000) that the level of glucose-6-phosphate was higher in Fe-deficient cucumber roots and that both glycolytic enzymes and respiration rate were increased in this condition. Enhanced inhibition by malate on the enzyme from Fe-deficient roots might be less physiologically understandable; in fact a major activity of the PEPCase should result in a greater production of malate via oxalacetate reduction. However, two major facts could be invoked to explain this contradictory result. First, malate could be rapidly removed by transport into mitochondria to replenish the Krebs cycle in order to sustain the increased demand of ATP, so its cytosolic concentration is kept below its inhibitory concentration or that the presence of a higher concentration of glucose-6-phosphate was able to antagonize its negative effect (Chollet et al., 1996). The second is the phosphorylation status of PEPCase. Phosphorylation of the photosynthetic form of enzyme is known to decrease the sensibility to the inhibitor malate (Chollet et al., 1996), but little is known about the regulation of the non-photosynthetic forms even if it has been shown that the enzyme could be phosphorylated in vivo (Duff and Chollet, 1995; Zhang et al., 1995; Osuna et al., 1996). Also in cucumber roots the enzyme is phosphorylated (this work); however, under Fe deficiency it seems that the PEPCase is less phosphorylated with respect to the control. Only the polypeptide at 103 kDa was phosphorylated (but to a lower level), while the 108 kDa was not (Fig. 5) and this could explain why in the –Fe condition the inhibition by malate was greater (Fig. 2). Further work will be necessary to separate the two isoforms of PEPCase and to asses their involvement in the root metabolism and which effect is exerted by the phosphorylation on PEPCase extracted from Fe-starved roots.
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Acknowledgments |
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This work was supported by a grant from MURST (Cofin 98) to GZ. The authors thank Dr J Vidal for the generous gift of the sorghum PEPCase antibodies.
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Notes |
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1 To whom correspondence should be addressed. Fax: +39 02 2663057. E-mail: graziano.zocchi{at}unimi.it
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Abbreviations |
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pHc, cytosolic pH; PEPCase, phosphoenolpyruvate carboxylase; PMSF, phenylmethylsulphonyl fluoride; PPFD, photosynthetic photon flux density; PVDF, polyvinylidene difluoride; RH, relative humidity.
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