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Keywords:

  • ammonium;
  • biochemical pH-stat;
  • biophysical pH-stat;
  • cytosolic pH;
  • ion transport;
  • malate;
  • nitrate;
  • nitrogen assimilation;
  • PEP carboxylase

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. THE PROTON ECONOMY AND NITROGEN ACQUISITION
  5. AN ANALYSIS OF INORGANIC CYTOCHEMISTRY AND ITS COMPARTMENTATION
  6. PHOSPHOENOLPYRUVATE CARBOXYLASE REGULATION: IS IT COMPATIBLE WITH A PH-STAT ROLE FOR THE ENZYME?
  7. CONCLUDING REMARKS
  8. ACKNOWLEDGMENTS
  9. REFERENCES
  10. Appendix

The classic biochemical pH-stat model of cytosolic pH regulation in plant cells presupposes a pH-dependent biosynthesis and degradation of organic acids, specifically malic acid, in the cytosol. This model has been used to explain the higher tissue accumulation of organic acids in nitrate (NO3)-grown, relative to ammonium (NH4+)-grown, plants, the result of proposed cytosolic alkalinization by NO3 metabolism, and acidification by NH4+ metabolism. Here, a critical examination of the model shows that its key assumptions are fundamentally problematic, particularly in the context of the effects on cellular pH of nitrogen source differences. Specifically, the model fails to account for proton transport accompanying inorganic nitrogen transport, which, if considered, renders the H+ production of combined transport and assimilation (although not the accumulation) to be equal for NO3 and NH4+ as externally provided N sources. We show that the model's evidentiary basis in total-tissue mineral ion and organic acid analysis is not directly relevant to subcellular (cytosolic) pH homeostasis, while the analysis of the ionic components of the cytosol is relevant to this process. A literature analysis further shows that the assumed greater activity of the enzyme phosphoenolpyruvate (PEP) carboxylase under nitrate nutrition, which is a key characteristic of the biochemical pH-stat model as it applies to nitrogen source, is not borne out in numerous instances. We conclude that this model is not tenable in its current state, and propose an alternative model that reaffirms the anaplerotic role of PEP carboxylase within the context of N nutrition, in the production of carbon skeletons for amino acid synthesis.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. THE PROTON ECONOMY AND NITROGEN ACQUISITION
  5. AN ANALYSIS OF INORGANIC CYTOCHEMISTRY AND ITS COMPARTMENTATION
  6. PHOSPHOENOLPYRUVATE CARBOXYLASE REGULATION: IS IT COMPATIBLE WITH A PH-STAT ROLE FOR THE ENZYME?
  7. CONCLUDING REMARKS
  8. ACKNOWLEDGMENTS
  9. REFERENCES
  10. Appendix

“Model pH-stats are real enough – at least in the mind of their originators . . . The extent to which they represent reality in the fine control of cytosolic pH remains to be established.”–Davies DD (1986), ‘The fine control of cytosolic pH’

A remarkable feature of plant cells is that they are able to homeostatically maintain the pH of the cytosolic compartment (pHcyt), which in an unstressed cell is about 7.2–7.5 (Felle 2001), corresponding to a [H+]cyt of 32–63 n m. These values are even lower than that of the cytosolic calcium pool (∼200 n m), and, like calcium homeostasis, proton homeostasis in the cytosol is sustained despite the adjacency of apoplastic and vacuolar spaces, which contain proton (and calcium) pools several orders of magnitude more concentrated than the cytosolic pool. Cytosolic pH homeostasis is a result of several processes, including the activities of intracellular open- and closed-system buffering components such as bicarbonate, phosphates, and protein buffers, and the active pumping, and channel-mediated transport, of protons between cytosol and apoplast or vacuole, in concert with the charge-compensating movement of other ions (Kurkdjian & Guern 1989; Felle 2001). The closed-system buffering capacity of the cytosol, however, appears to be limited relative to the scale of changes in cytosolic [H+] that are expected from normal metabolic reactions pertaining to growth and maintenance (Pfanz & Heber 1986; Kurkdjian & Guern 1989; Sakano 2001), while pH maintenance by the electrogenic pumping of protons across membranes bounding the cytosol may be limited by the availability of charge-compensating ion transport, which is required for maintenance of electrical homeostasis in the cell (Gerendás & Schurr 1999). Several biochemical or metabolic ‘pH stats’ have been proposed as additional mechanisms responsible for the control of pH homeostasis in the cytosol, the most widely recognized of which functions via the cytosolically localized, pH-dependent formation or destruction of carboxylic acid groups, in particular the 4-C carboxyl group of malic acid (Davies 1986; Raven 1986; Gerendás & Ratcliffe 2002). Its mechanism is thought to be regulated by the complementary pH-optima of the implicated enzymes (Fig. 1): specifically, the high-pH optimum of the carboxylation enzyme, phosphoenolpyruvate (PEP) carboxylase (in concert with the non-limiting activity, or co-regulation, of malate dehydrogenase), and the low-pH optimum of the decarboxylation enzyme, malic enzyme (Davies 1973; Mathieu 1982).

image

Figure 1. Summary of traditional concept of biochemical pH-stat. Enzymes 1 (PEP carboxylase) and 2 (malate dehydrogenase) operate when cytosolic pH is high, while enzyme 3 (malic enzyme) operates when pH is low. Note that the PEP carboxylase/malate dehydrogenase sequence does not actually release protons. The protons associated with the two carboxylic acid groups of malate were previously present; one was released in glycolysis (prior to the formation of PEP), the other produced and released in the solubilization of CO2 via carbonic anhydrase. PEP, phosphoenol pyruvate; OAA, oxaloacetate.

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Although the biochemical pH-stat concept has received some degree of criticism & revision (Smith & Bown 1981; Roberts et al. 1984; Davies 1986; Leport et al. 1996; Savchenko et al. 2000; Sakano 2001), it has become so widely accepted over the last few decades that it can be considered ‘textbook knowledge’ (Dennis & Turpin 1990; Salisbury & Ross 1992; Marschner 1995; Mengel & Kirkby 2001). In particular, cytosolic pH regulation by this mechanism is routinely implicated as a major response to the differential pH stresses proposed to result from variations in plant nitrogen nutrition (Kirkby & Mengel 1967; Raven & Smith 1974; Raven 1985; Schweizer & Erismann 1985; Allen & Smith 1986; van Beusichem, Kirkby & Bass 1988; Müller et al. 1990; Marschner 1995; Sagi et al. 1998; Pasqualini et al. 2001). In this view, nitrate (NO3) assimilation is considered to be a proton-consuming process, as the summary reaction through nitrate and nitrite reductases suggests: NO3+ 4[NAD(P)H + H+] + 2H+ [RIGHTWARDS ARROW] NH4+ + 4[NAD(P)+] + 3H2O. Ammonium (NH4+) assimilation, by contrast, is generally viewed as a proton-producing process, because NH3, the inorganic substrate for glutamine synthetase, is produced from the deprotonation of ammonium: NH4+ [RIGHTWARDS ARROW] NH3 + H+ (Kirkby & Mengel 1966; van Beusichem et al. 1988). [See note (1) in the Appendix.]

The proposed rectification of these opposite pHcyt stresses by the biochemical pH-stat is commonly used to explain the well-documented increases in malate and other organic acid anions that are observed in nitrate-grown relative to ammonium-grown plants (Kirkby & Mengel 1966; van Beusichem et al. 1988; Lüttge et al. 2000; Pasqualini et al. 2001; however, diurnal oscillations in tissue malate contents complicate this picture – see Lang & Kaiser 1994). This result is somewhat counter-intuitive, in that malate accumulation is usually associated with plant cells absorbing more cations than anions (Ulrich 1941; Kirkby & Mengel 1967; Hiatt & Hendricks 1967; van Beusichem et al. 1988). Despite taking up more anions than cations, however, NO3-grown plants accumulate substantially fewer anions, due to the reduction of nitrate to ammonium; this ‘anion deficit’ (and the associated malate buildup) is even greater than that found with NH4+-grown plants (see van Beusichem et al. 1988).

In the present paper, we raise several key questions regarding fundamental principles and assumptions of the biochemical pH-stat model in the context of nitrogen assimilation. We argue that the prevailing view is incorrect, and propose alternative explanations for the physiological differences, as a function of N source, that proponents of the existing model have sought to explain.

THE PROTON ECONOMY AND NITROGEN ACQUISITION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. THE PROTON ECONOMY AND NITROGEN ACQUISITION
  5. AN ANALYSIS OF INORGANIC CYTOCHEMISTRY AND ITS COMPARTMENTATION
  6. PHOSPHOENOLPYRUVATE CARBOXYLASE REGULATION: IS IT COMPATIBLE WITH A PH-STAT ROLE FOR THE ENZYME?
  7. CONCLUDING REMARKS
  8. ACKNOWLEDGMENTS
  9. REFERENCES
  10. Appendix

Ion transport in plant cells is intimately tied to cellular [H+] regulation, since the energy sources driving most transport events are the electrical and [H+] potential differences across cell membranes, energized by H+-pumping ATPases and pyrophosphatases (Serrano 1990; Barkla & Pantoja 1996; Sze, Li & Palmgren 1999; Martinoia, Massonneau & Frangne 2000; Palmgren 2001; Sakano 2001). The coupling of ion fluxes with co- or counter-fluxes of protons has emerged as such a ubiquitous theme in plant mineral nutrition that both active and passive ion transport can be regarded as based upon a ‘proton economy’, while the plasma membrane proton ATPase has been designated a ‘master enzyme’ (Serrano 1990; Marschner 1995), since it is the mechanism responsible for the primary establishment, and steady-state maintenance, of the plasma membrane electrical potential (Fig. 2). The quantity and direction of proton-coupled transport, however, is constrained by cellular requirements to maintain electrical homeostasis, as even a small imbalance between transported positive and negative charges across a membrane can have a substantial impact on the degree of membrane polarization (Clarkson 1974; Gerendás & Schurr 1999; Nobel 1999). For this reason, electroneutral ion fluxes must be achieved, except in the generation of the minute charge differential that brings about the membrane electrical potential. Studies of the mechanisms of isolated (unidirectional) ion fluxes indicate that ions typically cross the plasma membrane with an initial, electrogenic overshoot of positive charge, as membrane depolarization measurements upon sudden (re)supply of the ion show. In the case of nitrate, the anion assumes a cationic behaviour because the mechanism of its transport involves the cotransport of more than one proton (typically, two), and hence causes a depolarization (Meharg & Blatt 1995; Mistrik & Ullrich 1996; Fig. 2a). Similar events occur with other transported anions (Mistrik & Ullrich 1996). These depolarization events are quickly stabilized, or fully rectified, by the plasma membrane ATPase (Ullrich et al. 1984; Glass et al. 1992; Wang et al. 1994; Mistrik & Ullrich 1996; White & Broadley 2001; Sakano 2001), indicating the attainment of electroneutrality. Thus, NH4+, K+, and Ca2+ ions, which account for the great majority of cations normally taken into plant cells, must individually cross the plasma membrane in a counter-exchange with a resultant quantity of protons corresponding to the charge of the ion, while, similarly, the major anions NO3, SO42–, and H2PO4 are transported into the cell with cotransported protons, the quantity again corresponding to the charge on the transported nutrient ion (Ullrich & Novacky 1981, 1990; Serrano 1990; Mistrik & Ullrich 1996; Sze et al. 1999; Sakano 2001; also see Fig. 2a).

image

Figure 2. The role of proton transport as charge-balancer in inorganic nitrogen transport. (a) Transport of NO3 and NH4+ across the plasma membrane, rectified by proton pumping by membrane ATPase (Ullrich et al. 1984; Ullrich & Novacky 1990; Glass et al. 1992; Wang et al. 1994; Mistrik & Ullrich 1996); (b) Transport of malate ion across the tonoplast, from cytosol to vacuole, rectified by proton pumping by ATPase and/or pyrophosphatase (Gout et al. 1993; Cheffings et al. 1997).

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The proton-coupled transport processes for NO3 and for NH4+ are summarized in Fig. 2a, and the net result of proton coupling is that the chemical equivalents of nitric acid (NO3, cotranported with one H+) and ammonia (NH4+, counter-transported with one H+) in effect appear inside the cytosol following the transport and subsequent membrane repolarization events (see Fig. 2a). For this reason, their accumulation poses a challenge to mechanisms maintaining cytosolic pH, since one is a strong acid, and the other a weak base that is nevertheless expected to be approximately 99% protonated at cytosolic pH (pKa of NH4+ = 9.25). In other words, the uptake of nitrate is a cytosol-acidifying process whereas that of ammonium is a cytosol-alkalinizing one. These tendencies, rarely considered, work against the pH changes classically proposed to be involved in the subsequent metabolism of the ions taken up (see Introduction), although they agree with, if not fully explaining, the widely documented tendency for roots of nitrate-grown plants to alkalinize the external medium, and for roots of ammonium-grown plants to acidify it (Kirkby & Mengel 1966; van Beusichem et al. 1988; Magalhaes & Huber 1989; Marschner 1995; Mistrik & Ullrich 1996). Moreover, this agrees with a growing number of studies showing that a transient rise in cytosolic pH is stimulated by provision of ammonium (Kurkdjian, Leguay & Guern 1978; Roberts et al. 1982; Herrmann & Felle 1995; Giglioli-Guivarc’h et al. 1996; Yin et al. 1996; Kosegarten et al. 1997; Plieth, Sattelmacher & Knight 2000; Outlaw et al. 2002). [See note (2) in the Appendix.] NO3 transport has recently been shown to stimulate a transient lowering of pHcyt, which is enhanced by provision of the nitrate reductase inhibitor, tungstate (Espen, Nocito & Cocucci 2004). These pH changes are expected to be only transient because subsequent assimilation (see below), in addition to flux regulation processes such as efflux, allostery of transport proteins, and differential expression of genes encoding transporters, rapidly alters the sizes of the ‘HNO3’ and ‘NH3’ pools. However, such pH stresses are potentially significant when the concentration of the pool is increasing, given that unassimilated NO3 and NH4+ often accumulate to substantial concentrations (approximately 4–30 m m or higher) within the cytosol (Lee & Clarkson 1986; Devienne, Mary & Lamaze 1994; Wells & Miller 2000; Britto et al. 2001b; Britto & Kronzucker 2003).

When nitrate is assimilated, the net proton introduced to the cytosol in the nitrate transport step is effectively neutralized, as can be seen in a revised equation that integrates nitrate transport and reduction: NO3(ext) + H+(ext) + 4[NAD(P)H + H+](cyt) + H+(cyt) [RIGHTWARDS ARROW] NH4+(cyt) + 4[NAD(P)+](cyt) + 3H2O(cyt) (where ‘ext’ and ‘cyt’ refer to the extracellular or cytoplasmic location, respectively, of the substrates and products involved; also see Sakano 2001). In this equation, only a single proton is consumed in addition to that which entered in a symport step with nitrate. The present analysis differs from the traditional summary, which does not consider the transported proton, and therefore entails a net loss of two protons from the cell for every NO3 molecule taken up and assimilated. [See note (3) in the Appendix.] If this equation is added to that for ammonium deprotonation (NH4+ [RIGHTWARDS ARROW] NH3 + H+) prior to its entry into the GS-GOGAT cycle (a necessary addition, considering that very little of the NH4+ produced in nitrate reduction accumulates in plant tissues!), then the summation of both processes is completely proton neutral: NO3(ext) + H+(ext) + 4[NAD(P)H + H+](cyt) [RIGHTWARDS ARROW] NH3(cyt) + 4[NAD(P)+](cyt) + 3H2O(cyt). In the case of ammonium nutrition, the proton lost from the cell in the transport step exactly counterbalances the proton released when NH4+ enters GS-GOGAT via NH3. This also results in an integrated acquisition process that is proton-neutral, starting with uptake and continuing through to the amidation of 2-oxoglutarate, and the subsequent formation of glutamate (Fig. 3).

image

Figure 3. The proton-neutrality of ammonia assimilation. The first reaction is catalysed by glutamine synthetase (GS), and the second by glutamine 2-oxoglutarate aminotransferase (GOGAT). The protons associated with the hydrolysis of ATP and the oxidation of NAD(P)H + H+ cancel each other out (Gerendás & Ratcliffe 2000; cf. Kosegarten et al. 1997), and are not relevant in the larger context because these compounds are regenerated in the steady state, under most conditions (Reid, Loughman & Ratcliffe 1985). For proton balance associated with the provision of C skeletons for this pathway, see text.

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However, a more complete analysis of N uptake, reduction, and incorporation into amino acids should also take into account the proton balance associated with the provision of C substrates for reductant and amino acid skeletons. A step-by-step exposition then emerges as follows (also see Gerendás & Ratcliffe 2000). First, the uptake processes for NO3 and NH4+ are summarized in the following equations.

  • NO3(ext) + H+(ext) [RIGHTWARDS ARROW] NO3 (cyt) + H+(cyt)(1)
  • NH4+(ext) [RIGHTWARDS ARROW] NH3(cyt) + H+(ext)(2)

Note that these equations indicate the net result of the N transport and ATPase activities at the membrane, as depicted in Fig. 2a. Starting from glucose as a source of reductant, NO3 reduction can then be written

  • 3NO3(cyt) + 6H+(cyt) + 2C6H12O6(cyt) + 6O2(cyt)  [RIGHTWARDS ARROW] 3NH4+(cyt) + 12CO2(cyt) + 9H2O(cyt)(3)

Adding Eqns 1 and 3 yields the following

  • 3NO3(ext) + 3H+(ext) + 3H+(cyt) + 2C6H12O6(cyt) + 6O2(cyt) [RIGHTWARDS ARROW] 3NH4+(cyt) + 12CO2(cyt) + 9H2O(cyt)(4)

Equation 4 reiterates the idea that only a single cytoplasmic proton is consumed in the combined transport and reduction of NO3. The subsequent assimilation of NH4+ can also be expressed to include glucose as a source of reductant and carbon

  • 6NH4+(cyt) + 6C6H12O6(cyt) + 9O2(cyt) [RIGHTWARDS ARROW] 6C5O4H8N(cyt) + 6CO2(cyt) + 12H+(cyt) + 18H2O(cyt)(5)

When this equation is combined with Eqn 4, the net result of NO3 uptake, reduction, and assimilation is

  • 6NO3(ext) + 6H+(ext) + 10C6H12O6(cyt) + 21O2(cyt) [RIGHTWARDS ARROW] 6C5O4H8N(cyt) + 30CO2(cyt) + 6H+(cyt) + 36H2O(cyt)(6)

This summary equation shows that the overall proton balance of NO3 utilization yields one proton generated in the cell, and one proton consumed from the external medium, for every NO3 ion incorporated into glutamate. In the case of NH4+, the NH3 transported into the cell in step (2) above will be mostly in the protonated form

  • NH3(cyt) + H+(cyt) [RIGHTWARDS ARROW] NH4+(cyt)(7)

Combining Eqns 2, 5 and 7 yields the summary reaction for NH4+ uptake and assimilation

  • 6NH4+(ext) + 6C6H12O6(cyt) + 9O2(cyt) [RIGHTWARDS ARROW] 6C5O4H8N(cyt) + 6CO2(cyt) + 6H+(cyt) + 6H+(ext) + 18H2O(cyt)(8)

In this case, there is also one H+ produced in the cell for every molecule of inorganic N incorporated into glutamate, but, unlike with NO3, the assimilation of NH4+ also generates a proton in the external medium. [See note (4) in the Appendix.]

AN ANALYSIS OF INORGANIC CYTOCHEMISTRY AND ITS COMPARTMENTATION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. THE PROTON ECONOMY AND NITROGEN ACQUISITION
  5. AN ANALYSIS OF INORGANIC CYTOCHEMISTRY AND ITS COMPARTMENTATION
  6. PHOSPHOENOLPYRUVATE CARBOXYLASE REGULATION: IS IT COMPATIBLE WITH A PH-STAT ROLE FOR THE ENZYME?
  7. CONCLUDING REMARKS
  8. ACKNOWLEDGMENTS
  9. REFERENCES
  10. Appendix

It may appear that the acidification of the cytosol due to H+/NO3 symport could be offset by the higher uptake of cations (especially K+ and Ca2+) that is seen with nitrate-grown relative to ammonium-grown plants (Kirkby & Mengel 1966; van Beusichem et al. 1988, by virtue of increased H+/K+ antiport at the plasma membrane. Indeed, Espen et al. (2004) showed that nitrate-dependent cytosolic acidification was reduced when K+ was provided as the counterion. In the context of a nutritional steady state (which this cited study did not investigate), however, it is crucial that the increased net flux of K+ does not result in an increase in the cytosolic K+ pool; rather, this pool is homeostatically maintained at approximately 100 m m under most circumstances (Walker, Leigh & Miller 1996; Leigh 2001), and independently of N-source, except under conditions of extraordinarily high NH4+ supply (Kronzucker, Szczerba & Britto 2003). Therefore, the additional K+ flux results in a greater accumulation of K+ in the vacuole, and the associated H+ efflux at the plasma membrane (in the initial transport step for K+) is compensated for by equivalent proton fluxes at the tonoplast, directed in this instance from vacuole to cytosol.

The difference in pool sizes of strong cations and strong anions is considered an independent variable determining pH in living systems (Stewart 1983; Gerendás & Schurr 1999). If, as postulated in the biochemical pH-stat model, malate and other organic anions are effective agents for pHcyt homeostasis, it must be argued that these compounds variably accumulate in the cytosol to counterbalance a variable excess of cations in that compartment. Crucially, however, no evidence exists to show that nitrate-grown plants accumulate more organic anions in the cytosol than ammonium-grown plants, nor is there evidence to indicate that there is a larger accumulation of inorganic cations in the cytosol, relative to anions, in nitrate-grown plants. On the contrary, an analysis of literature values of cytosolic ion content strongly suggests that an excess of inorganic cations over inorganic anions prevails to a greater extent in ammonium-grown plants. As indicated in Table 1, the cytosolic pools of most major inorganic ions are independent of nitrogen source. This applies in particular to K+, Ca2+, Mg2+, and Pi, the cytosolic concentrations of which are held under strict homeostatic control over broad ranges of culture conditions, including nutrient supply (Lee & Ratcliffe 1993a, b; Felle 2001; Igamberdiev & Kleczkowski 2001; Leigh 2001; Kronzucker et al. 2003). Similarly, N-source independence has been shown for cytosolic Na+ and Cl pool sizes under non-salinity conditions (Speer & Kaiser 1994; Gerendás & Schurr 1999; Britto et al. 2004). In addition, variations in the cytosolic SO42– pool, while at present not well investigated, appear to be minimal, and the overall concentration of this pool, relative to potassium and inorganic nitrogen pools, is generally small (Bell, Cram & Clarkson 1994; Speer & Kaiser 1994; Buchner, Takahashi & Hawkesford 2004). Against this backdrop of relatively constant ion pools, the pools of inorganic N themselves can be superimposed, sizes of which have been shown by a variety of methods to typically fall into a 4–30 m m range, although some evidence suggests that they can be substantially higher (Britto et al. 2001b; Britto & Kronzucker 2003). The charge contribution from cytosolic N pools clearly establish a larger cytosolic excess of cations over anions in the case of NH4+ nutrition relative to NO3 nutrition. Taken together, the data in Table 1 therefore indicate that the cytosol of ammonium-grown plants is likely to accumulate a larger amount of organic anions (the identity and relative quantities of which have yet to be determined) than nitrate-grown plants, because the steady-state pH values for the cytosol of NH4+ or NO3-grown plants tend to be virtually identical (Bligny et al. 1997), within 0.2 pH units of one another (Gerendás, Ratcliffe & Sattelmacher 1990). In summary, the direction of this subcellularly localized strong ion difference directly contradicts the classical pH-stat paradigm as it applies to the cytosolic compartment.

Table 1.  Nitrogen-source effects upon the distribution of the main inorganic ions within tissue and cytosol of nitrate- and ammonium-grown plants
 Total tissueCytosolReferences
NO3NH4+NO3NH4+
  1. Inequality or equal signs indicate comparisons of ionic pool sizes, between NO3 or NH4+ growth conditions. The bottom line of the table indicates the difference between major inorganic cations and anions for each nitrogen condition. Total tissue data is widely available and fairly consistent among plant species (see, for instance, Kirkby & Mengel 1966; van Beusichem et al. 1988). Cytosolic pool size comparisons for these ions were determined using data from multiple sources: (a)Miller & Smith (1996); Britto & Kronzucker (2003); (b)Lee & Ratcliffe (1993ab); (c)Bell et al. (1994); (d)Britto et al. (2004); (e)Wang et al. (1993); Wells & Miller (2000); (f)Leigh et al. 2001; Kronzucker et al. (2003); (g)Speer & Kaiser (1994); Flowers & Hajibagheri 2001; (h)Bush (1995); (i)Igamberdiev & Kleczkowski (2001); See text for further details.

Anions
 NO3>>a
 H2PO4+ HPO42–<=b
 SO42–<=c
 Cl==d
Cations
 NH4+<<e
 K+>=f
 Na+==g
 Ca2+>=h
 Mg2+>=i
Cations – anions>< 

If no differences in H+ production or consumption occur as a result of differences in inorganic nitrogen source, then, the question remains as to why nitrate-grown plants have a pronouncedly higher accumulation of organic acid anions, particularly malate, relative to ammonium-grown plants. A straightforward explanation for this may be found in the greater tissue accumulation of cations by nitrate-grown plants (see above). For the achievement of electroneutrality by the plant, these additional positive charges require charge-balancing by strong anions, of which malate is a good candidate (Torii & Laties 1966). However, it must be emphasized that this differential, N-source-dependent accrual of inorganic ions and organic anions does not occur in the cytosol, but specifically in the vacuole. Evidence from organelle fractionation studies in spinach, barley, and potato, for instance, showed that malate concentrations in the cytosol (and the chloroplast stroma) were below detection limits, and at least an order of magnitude smaller than those in the vacuole (Winter, Robinson & Heldt 1993, 1994; Leidreiter et al. 1995; see also review by Martinoia & Rentsch 1994); by contrast, the concentrations of other metabolic intermediates, including amino acids, were almost always substantially larger in the cytosol than in the vacuole. Malate was an interesting exception in this regard because its relatively low cytosolic concentration is congruent with its role as a potent negative feedback agent upon PEP carboxylase (see below). Conversely, this pattern is inconsistent with malate's postulated role as a cytosolic proton source. This is because, mechanistically, the transport of organic anions to the vacuole is subject to the constraints of the ‘proton economy’ as described above; that is, their movement is accompanied by the flux of protons in the same direction (Fig. 2b), and therefore the cytosolic synthesis and subsequent transport to the vacuole of malic acid cannot contribute to alleviating cytosolic alkalosis. This requirement has been studied in detail for malate transport from cytosol to vacuole, in which channel-mediated malate transport is coupled to the pumping of a charge equivalence of protons into the vacuole by the tonoplast V-type ATPase and pyrophosphatase (i.e. both the malate anion and its two associated protons leave the cytosol in a symport process with overall electroneutrality; Gout et al. 1993; Lüttge et al. 1995, 2000; Cheffings et al. 1997). For this reason, the protons made available via carboxylation events in the cytosol cannot be used to neutralize any alkalinizing processes in situ, but are required as substrates for flux coupling across the tonoplast membrane; otherwise, the trans-tonoplast electrical potential would not be sustainable (see analysis by Gerendás & Schurr 1999). It is also important to point out that, while malate accumulation and transport to the vacuole are stimulated by nitrate (Lüttge et al. 2000), studies examining the relationship between malate pools and the flux through nitrate reduction do not support the proposed concerted synthesis of malate and reduction of nitrate. For instance, a 10-fold decrease in nitrate reduction resulted in no change in the malate pools of Catharanthus roseus cells (Marigo, Bouyssou & Belkoura 1985; also see Purvis, Peters & Hageman 1974). In another instance, mutant tobacco plants, growing on nitrate, but possessing no detectable root nitrate reductase activity, nevertheless accumulated more than twice as much malate in the root than wild-type plants (Stoimenova et al. 2003a).

PHOSPHOENOLPYRUVATE CARBOXYLASE REGULATION: IS IT COMPATIBLE WITH A PH-STAT ROLE FOR THE ENZYME?

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. THE PROTON ECONOMY AND NITROGEN ACQUISITION
  5. AN ANALYSIS OF INORGANIC CYTOCHEMISTRY AND ITS COMPARTMENTATION
  6. PHOSPHOENOLPYRUVATE CARBOXYLASE REGULATION: IS IT COMPATIBLE WITH A PH-STAT ROLE FOR THE ENZYME?
  7. CONCLUDING REMARKS
  8. ACKNOWLEDGMENTS
  9. REFERENCES
  10. Appendix

The pH responses of PEP carboxylase and malic enzyme, the enzymes most commonly invoked as metabolic rectifiers of cytosolic pH perturbations, are a central feature of the biochemical pH-stat model. The pH-optima studies that have led to the model's proposal, however, as well as many studies comparing N-source variations in the activities of these enzyme systems, have been conducted under in vitro conditions that do not reflect the chemical complexities of the living cell. This is a particularly important concern in the case of PEP carboxylase, as extensive work has shown it to be a multipurpose, highly regulated enzyme, subject to phosphorylation and modulation by pools of a wide range of metabolites. These modulators include light, iron, PEP, shikimate, glucose-6-phosphate, bicarbonate, citrate, malate, aspartate, asparagine, glutamate, and glutamine, in addition to protons (Davies 1979; Mathieu et al. 1982; Van Quy, Foyer & Champigny 1991; Sugiharto & Sugiyama 1992; Leport et al. 1996; Vuorinen & Kaiser 1997; Chinthapalli et al. 2000; Espen et al. 2000; Murchie et al. 2000; Parvathi et al. 2000; Pasqualini et al. 2001; Ferrario-Mery et al. 2002; Lepiniec, Thomas & Vidal 2003). Given this plethora of regulatory agents for PEP carboxylase, it is difficult to conceive how their action could be subordinated to the proposed crucial role of the enzyme in cytosolic pH regulation. The difficulty is compounded by observations that the pool sizes of many of these regulating compounds, and hence their effects on PEP carboxylase, vary considerably over time. This is in sharp contrast to the cytosolic H+ pool size, changes of which are restrained by buffering and transport systems. We are not aware of any studies that have addressed the problems associated with this aspect of the proposed pH-stat.

In addition to being modulated by multiple regulatory agents, PEP carboxylase activity can also be substrate-limited. In a particularly striking example, Gout et al. (1993) show that when cytosolic pH in sycamore protoplasts drops from 7.5 to 7.0 as a result of the external provision of 5 m m bicarbonate, malate synthesis increases significantly. The authors attribute this increase to substrate limitation of PEP carboxylase, which clearly over-rides, and indeed contradicts, the proposed direction of the enzyme's activity relative to changes in cytosolic pH.

Nevertheless, it remains possible that an in vivo pH dependence of PEP carboxylase can coexist, perhaps in a regulatory hierarchy, with these other regulatory processes. Indeed, various reports have shown that changes in PEP carboxylase activity, in apparent response to cytosolic pH changes, are consistent with the biochemical pH stat model (e.g. Gout, Bligny & Douce 1992; Rajagopalan, Gayathri & Raghavendra 1998; Sakano, Kiyota & Yazaki 1998; Gerendás & Ratcliffe 2000). However, these papers do not refer to differences in PEP carboxylase activities that result from differences in nitrogen source, which is the central subject here. Moreover, in two key studies it was concluded that the proposed pH-stat mechanism was too slow, or too low in capacity, to be of significance to pH rectification (Goutet al. 1992; Gerendás & Ratcliffe 2000), a conclusion also supported by others (Savchenko et al. 2000). In addition, there are other reports that indicate that pH stresses on the cytosol failed to modulate PEP carboxylase activity in a direction appropriate for a true pH-stat (Gout et al. 1993; Meinhard & Schnabl 2001; Outlaw et al. 2002).

Notwithstanding the regulatory complexity of PEP carboxylase, abundant evidence indicates that the enzyme's activity is especially strongly associated with the assimilatory flux of inorganic nitrogen into amino acids. In particular, changes in PEP carboxylase activity have been shown to be positively correlated with the activity of glutamine synthetase (Arnozis, Nelemans & Findenegg 1988; Vanlerberghe et al. 1990; Sugiharto & Sugiyama 1992; Sugiharto et al. 1992; Manh et al. 1993; Díaz, Lacuesta & Muñoz-Rueda 1996; Koga & Ikeda 2000), the enzyme that catalyses the entry of inorganic N (irrespective of N source) into the organic N pool (see Fig. 3). This relationship is highlighted by the potent stimulatory effects of glutamine itself, as well as the glutamine : glutamate ratio, on the activity and synthesis of PEP carboxylase (Vanlerberghe et al. 1990; Sugiharto et al. 1992; Sugiharto & Sugiyama 1992; Manh et al. 1993; Foyer et al. 1994; Díaz et al. 1996; Li, Zhang & Chollet 1996; Koga & Ikeda 2000; Murchie et al. 2000; Ferrario-Mery et al. 2002; Britto & Kronzucker 2004). The reason for this close correspondence between PEP carboxylase activity and nitrogen assimilation, irrespective of inorganic N source, appears to be clear: the carbon skeletons aminated to form amino acids in primary N assimilation are organic acids (more specifically, 2-oxo acids, especially 2-oxoglutaratic and oxaloacetic acid) that also function as intermediates in the tricarboxylic acid cycle, and therefore their depletion must be counteracted. Hence, they need to be synthesized in anaplerotic reactions such as that catalysed by PEP carboxylase (see Fig. 4). The control of PEP carboxylase by the glutamine : glutamate ratio is indicative of the enzyme's anaplerotic role, in that this ratio reflects the extent to which nitrogen has been incorporated into organic forms by drawing from organic acid pools. The role of PEP carboxylase in inorganic N assimilation is further indicated by the well-documented rise in this enzyme's activity that results from provision of either NO3 or NH4+ (Hammel, Cornwell & Bassham 1979; Müller, Baier & Kaiser 1991; Sugiharto et al. 1992; Díaz et al. 1995, 1996; Scheible et al. 1997; Esposito, Carillo & Carfagna 1998; Koga & Ikeda 2000; Murchie et al. 2000; Lepiniec et al. 2003). Interestingly, the conclusion that PEP carboxylase has a major anaplerotic function in N assimilation has been drawn in a large number of studies examining nitrate and/or ammonium nutrition, despite its incongruence with a proposed pH-stat role of PEP carboxylase in the context of N acquisition, namely the enzyme's proposed down-regulation during NH4+ assimilation (Popp & Summons 1983; Dahlbender & Strack 1986; Melzer & O’Leary 1987; Arnozis et al. 1988; Guy, Vanlerberghe & Turpin 1989; Vanlerberghe et al. 1990; Cramer, Lewis & Lips 1993; Foyer et al. 1994; Díaz et al. 1995, 1996; Gao & Lips 1997; Golombek et al. 1999; Koga & Ikeda 2000; Murchie et al. 2000; Norici, Dalsass & Giordano 2002; Rademacher et al. 2002).

image

Figure 4. Model outlining a revised role of PEP carboxylase in inorganic N assimilation. Key enzymes or enzyme groups are indicated in boxed numbers as follows: (1) PEP carboxylase; (2) malate dehydrogenase; (3) tricarboxylic acid cycle enzymes; (4) glutamine synthetase (GS); (5) aspartate aminotransferase; (6) glutamine 2-oxoglutarate aminotransferase (GOGAT). Note that (as in the legend to Fig. 1), bicarbonate (HCO3) is produced by the activity of carbonic anhydrase. Dashed lines indicate feedback activities on PEP carboxylase by metabolic intermediates. Circled plus or minus signs indicate positive or negative feedback, respectively. Thickness of dashed lines indicates intensity of feedback, due to nitrogen-source-dependent variations in pool sizes of feedback agents (N source given in parentheses).

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Perhaps the most powerful challenge to the proposed role of a PEP carboxylase-based, N-source-dependent, pH stat, however, is the large number of studies indicating that PEP carboxylase activity can be substantially higher in NH4+-grown, relative to NO3-grown, plants (Schweizer & Erismann 1985; Arnozis et al. 1988; Arnozis & Barneix 1989; Ikeda, Mizoguchi & Yamakawa 1992; Villa et al. 1992; Cramer & Lewis 1993; Cramer et al. 1993; Díaz et al. 1996; Koga & Ikeda 1997, 2000; Lasa et al. 2002; Norici et al. 2002; Viktor & Cramer 2005). This most likely reflects the higher GS activities, glutamine contents, glutamine : glutamate ratios, and higher rates of amino acid synthesis that are often observed in NH4+-grown plants (Yemm & Willis 1956; van Beusichem et al. 1988; Magalhäes & Huber 1989; Sugiharto & Sugiyama 1992; Cramer et al. 1993; Díaz et al. 1996; Claussen & Lenz 1999; Pasqualini et al. 2001; Lasa et al. 2002). Where observed, the lower PEP carboxylase activities of NO3-grown plants may be due to their much larger malate pools and the potential negative feedback exerted on the enzyme by such pools (Pasqualini et al. 2001; Rademacher et al. 2002). Although the predominant subcellular location of malate is the vacuole (see above), while PEP carboxylase is a cytosolic enzyme, the higher accumulation of malate in vacuoles of NO3-grown plants may translate into higher cytosolic malate levels relative to those of cells of NH4+-grown plants. Moreover, while NO3 nutrition has been shown to reduce malate inhibition of PEP carboxylase activity to some degree (Duff & Chollet 1995; Murchie et al. 2000), this release from inhibition appears not to generally result in an increased in vivo PEP carboxylase activity in NO3-grown plants, possibly due to the hyperaccumulation of malate to concentrations an order of magnitude greater than in NH4+-grown plants. Indeed, it has been suggested that the increased malate pools under nitrate nutrition indicate a lower demand for anaplerotic carbon fixation, in direct contradiction to the often assumed positive correlation between malate pool size and ongoing organic acid synthesis via PEP carboxylase and malate dehydrogenase (Gao & Lips 1997).

The trend of higher PEP carboxylase activity associated with NH4+ nutrition is more apparent in roots than shoots (Schweizer & Erismann 1985; Arnozis et al. 1988), reflecting the general rule that primary NH4+ assimilation takes place mostly in roots, whereas the location of primary NO3 assimilation is predominantly in shoots (Andrews 1986). In other words, PEP carboxylase activity appears to be colocalized with nitrogen assimilation and its associated anaplerotic requirement. An alternative explanation, that leaves assimilating nitrogen must use biochemical pH-stat pathways because (unlike roots) they lack the ability to dispose of protons into an extracellular medium (Raven & Smith 1976; Marschner 1995), is challenged by the observation that leaves exposed to acid stressors such as SO2 translocate resultant acids to the root, which then excretes excess protons (Thomas & Runge 1992; Kaiser, Hofler & Heber 1993). More fundamentally, it is falsified by the proton neutrality of integrated N transport and assimilation (see above).

CONCLUDING REMARKS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. THE PROTON ECONOMY AND NITROGEN ACQUISITION
  5. AN ANALYSIS OF INORGANIC CYTOCHEMISTRY AND ITS COMPARTMENTATION
  6. PHOSPHOENOLPYRUVATE CARBOXYLASE REGULATION: IS IT COMPATIBLE WITH A PH-STAT ROLE FOR THE ENZYME?
  7. CONCLUDING REMARKS
  8. ACKNOWLEDGMENTS
  9. REFERENCES
  10. Appendix

While attractive in its simplicity, the malate-based biochemical pH stat model agrees neither with current views of ion transport, nor with regulatory information about the key enzyme PEP carboxylase. This lack of agreement applies in particular to nitrogen acquisition, when the following considerations are fully taken into account: the proton involvement in nitrogen transport and metabolism, the subcellular compartmentation of ions, and the numerous exceptions to the proposed increased PEP carboxylase activity in nitrate-fed plants. Although such plants unquestionably build up higher malate concentrations than NH4+-grown plants, these pools exist almost entirely in the vacuole, where they cannot influence cytosolic pH. Rather, the high malate pools reflect the larger vacuolar excess of cations over anions, and may indeed reflect a lesser anaplerotic requirement, and therefore a lower PEP carboxylase activity, in NO3-grown plants.

Past emphases on this biochemical pH stat model may have detracted from the importance and potency of other pH maintenance and rectification systems in plants. These include systems that are analogous to those well established in microbial and animal physiology, and which are likely to act much more rapidly than metabolic processes (Savchenko et al. 2000). For instance, the operation of open-system buffers (in addition, and in contrast, to stationary buffers such as proteins) has received little or no attention in plant systems. Nevertheless, such systems, in which changes in partial pressures of CO2 and concentrations of bicarbonate (for instance) can resist pH changes very effectively in the human body (Putnam 1998), and should be expected to do so in plants, given the widespread occurrence of carbonic anhydrase, which can dramatically speed up the velocity of the equilibration reactions. Rectification of pH changes in the cytosol involving the antiport or symport of protons with ions such as K+, Na+, or Cl, across the plasma membrane, tonoplast, or plastidic membranes (Felle 1991; Siebke et al. 1992; Raghavendra, Yin & Heber 1993; Bligny et al. 1997; Venema et al. 2003; Song et al. 2004), may have also been underestimated. Recent evidence for the potency of such mechanisms is indicated by the observaion that a mutation in sodium-proton antiport at the thylakoid membrane has consequences for cytosolic pH (Song et al. 2004). Transient shifts in steady-state concentrations of nutrient ions, to compensate for proton deficits or excesses, should be readily tolerable by the plant, given that baseline proton concentrations are many orders of magnitude smaller than the concentrations of most nutrient ions. The enormous and well-documented capacity of plant cells to effectively maintain cytosolic calcium homeostasis, following the sudden, and intense, changes in cytosolic [Ca2+] that are associated with cellular signalling events, provides striking evidence of the feasibility of rapid, and highly refined, ion homeostasis based solely on membrane transport mechanisms.

The likely existence of these alternative pH-regulating mechanisms in plants, in conjunction with well-established pH stats and buffering systems, calls into question the necessity of postulating a biochemical ‘fine tuning’ mechanism. Even if such a mechanism does exist, its postulated functioning in the context of N nutrition, via differential synthesis of malic acid, is clearly contradicted by a wide range of studies. Other metabolic pH stat mechanisms may nevertheless be of importance in some contexts, such as the shift from lactic acid to ethanol production in plants undergoing extended periods of hypoxia or anaerobiosis (Roberts et al. 1984; Gerendás & Ratcliffe 2002; Stoimenova et al. 2003b), although, again, such mechanisms have not been demonstrated to occur within the context of nitrogen source differences, or even under non-adverse physiological conditions.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. THE PROTON ECONOMY AND NITROGEN ACQUISITION
  5. AN ANALYSIS OF INORGANIC CYTOCHEMISTRY AND ITS COMPARTMENTATION
  6. PHOSPHOENOLPYRUVATE CARBOXYLASE REGULATION: IS IT COMPATIBLE WITH A PH-STAT ROLE FOR THE ENZYME?
  7. CONCLUDING REMARKS
  8. ACKNOWLEDGMENTS
  9. REFERENCES
  10. Appendix

We wish to thank the Natural Sciences and Engineering Research Council (NSERC) of Canada and the University of Toronto for financial support while writing this manuscript. We also thank Professors R. George Ratcliffe and Greg C. Vanlerberghe for insightful discussion.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. THE PROTON ECONOMY AND NITROGEN ACQUISITION
  5. AN ANALYSIS OF INORGANIC CYTOCHEMISTRY AND ITS COMPARTMENTATION
  6. PHOSPHOENOLPYRUVATE CARBOXYLASE REGULATION: IS IT COMPATIBLE WITH A PH-STAT ROLE FOR THE ENZYME?
  7. CONCLUDING REMARKS
  8. ACKNOWLEDGMENTS
  9. REFERENCES
  10. Appendix
  • Allen S. & Smith J.A.C. (1986) Ammonium nutrition in Ricinus communis – its effect on plant growth and the chemical composition of the whole plant, xylem and phloem saps. Journal of Experimental Botany 37, 15991610.
  • Andrews M. (1986) The partitioning of nitrate assimilation between root and shoot of higher plants. Plant, Cell and Environment 9, 511519.
  • Arnozis P.A. & Barneix A.J. (1989) PEP carboxylase activity during ammonium assimilation in wheat plants. Journal of Plant Nutrition 12, 8594.
  • Arnozis P.A., Nelemans J.A. & Findenegg G.R. (1988) Phosphoenolpyruvate carboxylase activity in plants grown with either NO3 or NH4+ as inorganic nitrogen source. Journal of Plant Physiology 132, 2327.
  • Barkla B.J. & Pantoja O. (1996) Physiology of ion transport across the tonoplast of higher plants. Annual Review of Plant Physiology and Plant Molecular Biology 47, 159184.
  • Bell C.I., Cram W.J. & Clarkson D.T. (1994) Compartmental analysis of 35SO42− exchange kinetics in roots and leaves of a tropical legume Macroptilium atropurpureum cv Siratro. Journal of Experimental Botany 45, 879886.
  • Van Beusichem M.L., Kirkby E.A. & Baas R. (1988) Influence of nitrate and ammonium nutrition on the uptake, assimilation, and distribution of nutrients in Ricinus communis. Plant Physiology 86, 914921.
  • Bligny R., Gout E., Kaiser W., Heber U., Walker D. & Douce R. (1997) pH regulation in acid-stressed leaves of pea plants grown in the presence of nitrate or ammonium salts: studies involving 31P-NMR spectroscopy and chlorophyll fluorescence. Biochimica et Biophysica Acta – Bioenergetics 1320, 142152.
  • Britto D.T. & Kronzucker H.J. (2003) The case for cytosolic NO3 heterostasis: a critique of a recently proposed model. Plant, Cell and Environment 26, 183188.
  • Britto D.T. & Kronzucker H.J. (2004) Bioengineering N acquisition in rice: Can novel initiatives in rice genomics and physiology contribute to global food security? Bioessays 26, 683692.
  • Britto D.T., Glass A.D.M., Kronzucker H.J. & Siddiqi M.Y. (2001a) Cytosolic concentrations and transmembrane fluxes of NH4+/NH3. An examination of recent proposals. Plant Physiology 125, 523526.
  • Britto D.T., Ruth T.J., Lapi S. & Kronzucker H.J. (2004) Cellular and whole-plant chloride dynamics in barley: insights into chloride–nitrogen interactions and salinity responses. Planta 218, 615622.
  • Britto D.T., Siddiqi M.Y., Glass A.D.M. & Kronzucker H.J. (2001b) Futile transmembrane NH4+ cycling: a cellular hypothesis to explain ammonium toxicity in plants. Proceedings of the National Academy of Sciences of the USA 98, 42554258.
  • Buchner P., Takahashi H. & Hawkesford M.J. (2004) Plant sulphate transporters: co-ordination of uptake, intracellular and long-distance transport. Journal of Experimental Botany 55, 17651773.
  • Bush D.S. (1995) Calcium regulation in plant cells and its role in signaling. Annual Review of Plant Physiology and Plant Molecular Biology 46, 95122.
  • Cheffings C.M., Pantoja O., Ashcroft F.M. & Smith J.A.C. (1997) Malate transport and vacuolar ion channels in CAM plants. Journal of Experimental Botany 48, 623631.
  • Chinthapalli B., Raghavan C., Blasing O., Westhoff P. & Raghavendra A.S. (2000) Phosphoenolpyruvate carboxylase purified from leaves of C-3, C-4, and C-3-C-4 intermediate species of Alternanthera: Properties at limiting and saturating bicarbonate. Photosynthetica 38, 415419.
  • Clarkson D.T. (1974) Ion Transport and Cell Structure in Plants. McGraw-Hill, London, UK.
  • Claussen W. & Lenz F. (1999) Effect of ammonium or nitrate nutrition on net photosynthesis, growth, and activity of the enzymes nitrate reductase and glutamine synthetase in blueberry, raspberry and strawberry. Plant and Soil 208, 95102.
  • Cramer M.D. & Lewis O.A.M. (1993) The influence of NO3 and NH4+ nutrition on the gas-exchange characteristics of the roots of wheat (Triticum aestivum) and maize (Zea mays) plants. Annals of Botany 72, 3746.
  • Cramer M.D., Lewis O.A.M. & Lips S.H. (1993) Inorganic carbon fixation and metabolism in maize roots as affected by nitrate and ammonium nutrition. Physiologia Plantarum 89, 632639.
  • Dahlbender B. & Strack D. (1986) The role of malate in ammonia assimilation in cotyledons of radish (Raphanus sativus L). Planta 169, 382392.
  • Davies D.D. (1973) Control of and by pH. Symposia of the Society for Experimental Biology 27, 513530.
  • Davies D.D. (1979) Central role of phosphoenolpyruvate in plant metabolism. Annual Review of Plant Physiology 30, 131158.
  • Davies D.D. (1986) The fine control of cytosolic pH. Physiologia Plantarum 67, 702706.
  • Dennis D.T. & Turpin D.T. (1990) Plant Physiology, Biochemistry and Molecular Biology. Longman, Essex, UK.
  • Devienne F., Mary B. & Lamaze T. (1994) Nitrate transport in intact wheat roots. 1. Estimation of cellular fluxes and NO3 compartmental analysis from data of 15NO3 distribution using efflux. Journal of Experimental Botany 45, 667676.
  • Díaz A., Lacuesta M. & Muñoz-Rueda A. (1996) Comparative effects of phosphinothricin on nitrate and ammonium assimilation and on anaplerotic CO2 fixation in N-deprived barley plants. Journal of Plant Physiology 149, 913.
  • Díaz A., Maza H., Gonzalezmoro B., Lacuesta M., Gonzalezmurua C. & Muñoz-Rueda A. (1995) Phosphinothricin reverts the ammonia-dependent enhancement of phosphoenolpyruvate carboxylase activity. Journal of Plant Physiology 145, 1116.
  • Duff S.M.G. & Chollet R. (1995) In vivo regulation of wheat-leaf phosphoenolpyruvate carboxylase by reversible phosphorylation. Plant Physiology 107, 775782.
  • Espen L., Dell’Orto M. , De Nisi P. & Zocchi G. (2000) Metabolic responses in cucumber (Cucumis sativus L.) roots under Fe-deficiency: a 31P-nuclear magnetic resonance in-vivo study. Planta 210, 985992.
  • Espen L., Nocito F.F. & Cocucci M. (2004) Effect of NO3 transport and reduction on intracellular pH: an in vivo NMR study in maize roots. Journal of Experimental Botany 55, 20532061.
  • Esposito S., Carillo E. & Carfagna S. (1998) Ammonium metabolism stimulation of glucose-6-P dehydrogenase and phosphoenolpyruvate carboxylase in young barley roots. Journal of Plant Physiology 153, 6166.
  • Felle H.H. (1991) The role of the plasma membrane proton pump in short-term pH regulation in the aquatic liverwort Riccia fluitans L. Journal of Experimental Botany 42, 645652.
  • Felle H.H. (2001) pH: Signal and messenger in plant cells. Plant Biology 3, 577591.
  • Ferrario-Mery S., Hodges M., Hirel B. & Foyer C.H. (2002) Photorespiration-dependent increases in phosphoenolpyruvate carboxylase, isocitrate dehydrogenase and glutamate dehydrogenase in transformed tobacco plants deficient in ferredoxin-dependent glutamine-alpha-ketoglutarate aminotransferase. Planta 214, 877886.
  • Flowers T.J. & Hajibagheri M.A. (2001) Salinity tolerance in Hordeum vulgare: ion concentrations in root cells of cultivars differing in salt tolerance. Plant and Soil 231, 19.
  • Foyer C.H., Noctor G., Lelandais M., Lescure J.C., Valadier M.H., Boutin J.P. & Horton P. (1994) Short-term effects of nitrate, nitrite and ammonium assimilation on photosynthesis, carbon partitioning and protein phosphorylation in maize. Planta 192, 211220.
  • Gao Z.F. & Lips S.H. (1997) Effects of increasing inorganic carbon supply to roots on net nitrate uptake and assimilation in tomato seedlings. Physiologia Plantarum 101, 206212.
  • Gerendás J. & Ratcliffe R.G. (2000) Intracellular pH regulation in maize root tips exposed to ammonium at high external pH. Journal of Experimental Botany 51, 207219.
  • Gerendás J. & Ratcliffe R.G. (2002) Root pH regulation. In Plant Roots: The Hidden Half, 3rd edn (eds. Y.Waisel, A.Eshel & U.Kafkafi), pp. 553570. Marcel Dekker, New York, NY, USA.
  • Gerendás J. & Schurr U. (1999) Physicochemical aspects of ion relations and pH regulation in plants – a quantitative approach. Journal of Experimental Botany 50, 11011114.
  • Gerendás J., Ratcliffe R.G. & Sattelmacher B. (1990) 31P Nuclear magnetic resonance evidence for differences in intracellular pH in the roots of maize seedlings grown with nitrate or ammonium. Journal of Plant Physiology 137, 125128.
  • Giglioli-Guivarc’h N., Pierre J.N., Brown S., Chollet R., Vidal J. & Gadal P. (1996) The light-dependent transduction pathway controlling the regulatory phosphorylation of C4 phosphoenolpyruvate carboxylase in protoplasts from Digitaria sanguinalis. Plant Cell 8, 573586.
  • Glass A.D.M., Shaff J.E. & Kochian L.V. (1992) Studies of the uptake of nitrate in barley. IV. Electrophysiology. Plant Physiology 99, 456463.
  • Golombek S., Heim U., Horstmann C., Wobus U. & Weber H. (1999) Phosphoenolpyruvate carboxylase in developing seeds of Vicia faba L. Gene expression and metabolic regulation. Planta 208, 6672.
  • Gout E., Bligny R. & Douce R. (1992) Regulation of intracellular pH values in higher-plant cells –13C and 31P nuclear magnetic resonance studies. Journal of Biological Chemistry 267, 1390313909.
  • Gout E., Bligny R., Pascal N. & Douce R. (1993) 13C nuclear magnetic resonance studies of malate and citrate synthesis and compartmentation in higher plant cells. Journal of Biological Chemistry 268, 39863992.
  • Guy R.D., Vanlerberghe G.C. & Turpin D.H. (1989) Significance of phosphoenolpyruvate carboxylase during ammonium assimilation – Carbon isotope discrimination in photosynthesis and respiration by the N-limited green alga Selenastrum minutum. Plant Physiology 89, 11501157.
  • Hammel K.E., Cornwell K.L. & Bassham J.A. (1979) Stimulation of dark CO2 fixation by ammonia in isolated mesophyll cells of Papaver somniferum L. Plant and Cell Physiology 20, 15231529.
  • Herrmann A. & Felle H.H. (1995) Tip growth in root hair cells of Sinapis alba L – significance of internal and external Ca2+ and pH. New Phytologist 129, 523533.
  • Hiatt A.J. & Hendricks S.B. (1967) The role of CO2 fixation in accumulation of ions by barley roots. Zeitschrift für Pflanzenphysiologie 56, 220232.
  • Igamberdiev A.U. & Kleczkowski L.A. (2001) Implications of adenylate kinase-governed equilibrium of adenylates on contents of free magnesium in plant cells and compartments. Biochemical Journal 360, 225231.
  • Ikeda M., Mizoguchi K. & Yamakawa T. (1992) Stimulation of dark carbon fixation in rice and tomato roots by application of ammonium nitrogen. Soil Science and Plant Nutrition 38, 315322.
  • Kaiser W.M., Hofler M. & Heber U. (1993) Can plants exposed to SO2 excrete sulfuric acid through the roots? Physiologia Plantarum 87, 6167.
  • Kirkby E.A. & Mengel K. (1967) Ionic balance in different tissues of tomato plants in relation to nitrate, urea and ammonium nutrition. Plant Physiology 42, 614.
  • Koga N. & Ikeda M. (1997) Responses to nitrogen sources and regulatory properties of root phosphoenolpyruvate carboxylase. Soil Science and Plant Nutrition 43, 643650.
  • Koga N. & Ikeda M. (2000) Methionine sulfoximine suppressed the stimulation of dark carbon fixation by ammonium nutrition in wheat roots. Soil Science and Plant Nutrition 46, 393400.
  • Kosegarten H., Grolig F., Wieneke J., Wilson G. & Hoffmann B. (1997) Differential ammonia-elicited changes of cytosolic pH in root hair cells of rice and maize as monitored by 2′,7′-bis-(2-carboxyethyl) -5 (and -6) -carboxyfluorescein-fluorescence ratio. Plant Physiology 113, 451461.
  • Kronzucker H.J., Szczerba M.W. & Britto D.T. (2003) Cytosolic potassium homeostasis revisited: 42K tracer analysis in Hordeum vulgare L. reveals set-point variations in [K+]. Planta 217, 540546.
  • Kurkdjian A. & Guern J. (1989) Intracellular pH – measurement and importance in cell activity. Annual Review of Plant Physiology and Plant Molecular Biology 40, 271303.
  • Kurkdjian A., Leguay J.J. & Guern J. (1978) measurement of intracellular pH and aspects of its control in higher plant cells cultivated in liquid medium. Respiration Physiology 33, 7589.
  • Lang B. & Kaiser W.M. (1994) Solute content and energy status of roots of barley plants cultivated at different pH on nitrate nitrogen or ammonium nitrogen. New Phytologist 128, 451439.
  • Lasa B., Frechilla S., Aparicio-Tejo P.M. & Lamsfus C. (2002) Role of glutamate dehydrogenase and phosphoenolpyruvate carboxylase activity in ammonium nutrition tolerance in roots. Plant Physiology and Biochemistry 40, 969976.
  • Lee R.B. & Clarkson D.T. (1986) 13N studies of nitrate fluxes in barley roots.1. Compartmental analysis from measurements of 13N efflux. Journal of Experimental Botany 37, 17531767.
  • Lee R.B. & Ratcliffe R.G. (1993a) Nuclear magnetic resonance studies of the location and function of plant nutrients in vivo. Plant and Soil 156, 4555.
  • Lee R.B. & Ratcliffe R.G. (1993b) Subcellular-distribution of inorganic phosphate, and levels of nucleoside triphosphate, in mature maize roots at low external phosphate concentrations – measurements with 31P-NMR. Journal of Experimental Botany 44, 587598.
  • Leidreiter K., Kruse A., Heineke D., Robinson D.G. & Heldt H.W. (1995) Subcellular volumes and metabolite concentrations in potato (Solanum tuberosum cv. Desiree) leaves. Botanica Acta 108, 439444.
  • Leigh R.A. (2001) Potassium homeostasis and membrane transport. Journal of Plant Nutrition and Soil Science-Zeitschrift für Pflanzenernährung und Bodenkunde 164, 193198.
  • Lepiniec L., Thomas M. & Vidal J. (2003) From enzyme activity to plant biotechnology: 30 years of research on phosphoenolpyruvate carboxylase. Plant Physiology and Biochemistry 41, 533539.
  • Leport L., Kandlbinder A., Baur B. & Kaiser W.M. (1996) Diurnal modulation of phosphoenolpyruvate carboxylation in pea leaves and roots as related to tissue malate concentrations and to the nitrogen source. Planta 198, 495501.
  • Li B., Zhang X.Q. & Chollet R. (1996) Phosphoenolpyruvate carboxylase kinase in tobacco leaves is activated by light in a similar but not identical way as in maize. Plant Physiology 111, 497505.
  • Lüttge U., Fischerschliebs E., Ratajczak R., Kramer D., Berndt E. & Kluge M. (1995) Functioning of the tonoplast in vacuolar C storage and remobilization in Crassulacean acid metabolism. Journal of Experimental Botany 46, 13771388.
  • Lüttge U., Pfeifer T., Fischer-Schliebs E. & Ratajczak R. (2000) The role of vacuolar malate-transport capacity in crassulacean acid metabolism and nitrate nutrition. Higher malate-transport capacity in ice plant after crassulacean acid metabolism-induction and in tobacco under nitrate nutrition. Plant Physiology 124, 13351347.
  • Magalhaes J.R. & Huber D.M. (1989) Ammonium assimilation in different plant species as affected by nitrogen form and pH control in solution culture. Fertilizer Research 21, 16.
  • Manh C.T., Bismuth E., Boutin J.P., Provot M. & Champigny M.L. (1993) Metabolite effectors for short-term nitrogen-dependent enhancement of phosphoenolpyruvate carboxylase activity and decrease of net sucrose synthesis in wheat leaves. Physiologia Plantarum 89, 460466.
  • Marigo G., Bouyssou H. & Belkoura M. (1985) Vacuolar efflux of malate and its influence on nitrate accumulation in Catharanthus roseus cells. Plant Science 39, 97103.
  • Marschner H. (1995) Mineral Nutrition of Higher Plants, 2nd edn. Academic Press, London, UK.
  • Martinoia E. & Rentsch D. (1994) Malate compartmentation – responses to a complex metabolism. Annual Review of Plant Physiology and Plant Molecular Biology 45, 447467.
  • Martinoia E., Massonneau A. & Frangne N. (2000) Transport processes of solutes across the vacuolar membrane of higher plants. Plant and Cell Physiology 41, 11751186.
  • Mathieu Y. (1982) pH-dependence of phosphoenolpyruvate carboxylase from Acer pseudoplatanus cell suspensions. Plant Science Letters 28, 111119.
  • Meharg A.A. & Blatt M.R. (1995) NO3 transport across the plasma-membrane of Arabidopsis thaliana root hairs – kinetic control by pH and membrane voltage. Journal of Membrane Biology 145, 4966.
  • Meinhard M. & Schnabl H. (2001) Fusicoccin- and light-induced activation and in vivo phosphorylation of phosphoenolpyruvate carboxylase in Vicia guard cell protoplasts. Plant Science 160, 635646.
  • Melzer E. & O'Leary M.H. (1987) Anapleurotic CO2 fixation by phosphoenolpyruvate carboxylase in C-3 plants. Plant Physiology 84, 5860.
  • Mengel K. & Kirkby E.A. (2001) Principles of Plant Nutrition, 5th edn. Kluwer, Dordrecht, The Netherlands.
  • Miller A.J. & Smith S.J. (1996) Nitrate transport and compartmentation in cereal root cells. Journal of Experimental Botany 47, 843854.
  • Mistrik I. & Ullrich C.I. (1996) Mechanism of anion uptake in plant roots: Quantitative evaluation of H+/NO3 and H+/H2PO4 stoichiometries. Plant Physiology and Biochemistry 34, 629636.
  • Müller R., Baier M. & Kaiser W.M. (1991) Differential stimulation of PEP carboxylation in guard cells and mesophyll cells by ammonium or fusicoccin. Journal of Experimental Botany 42, 215220.
  • Müller R., Steigner W., Gimmler H. & Kaiser W.M. (1990) Effect of ammonium on dark-CO2 fixation and on cytosolic and vacuolar pH values in Eremosphaera viridis Debary (Chlorococcales). Journal of Experimental Botany 41, 441448.
  • Murchie E.H., Ferrario-Mery S., Valadier M.H. & Foyer C.H. (2000) Short-term nitrogen-induced modulation of phosphoenolpyruvate carboxylase in tobacco and maize leaves. Journal of Experimental Botany 51, 13491356.
  • Nobel. P. (1999) Physicochemical and Environmental Plant Physiology, 2nd edn. Academic Press, San Diego, CA, USA.
  • Norici A., Dalsass A. & Giordano M. (2002) Role of phosphoenolpyruvate carboxylase in anaplerosis in the green microalga Dunaliella salina cultured under different nitrogen regimes. Physiologia Plantarum 116, 186191.
  • Outlaw W.H., Du Z.R., Meng F.X., Aghoram K., Riddle K.A. & Chollet R. (2002) Requirements for activation of the signal-transduction network that leads to regulatory phosphorylation of leaf guard-cell phosphoenolpyruvate carboxylase during fusicoccin-stimulated stomatal opening. Archives of Biochemistry and Biophysics 407, 6371.
  • Palmgren M.G. (2001) Plant plasma membrane H+-ATPases: Powerhouses for nutrient uptake. Annual Review of Plant Physiology and Plant Molecular Biology 52, 817845.
  • Parvathi K., Bhagwat A.S., Ueno Y., Izui K. & Raghavendra A.S. (2000) Illumination increases the affinity of phosphoenolpyruvate carboxylase to bicarbonate in leaves of a C4 plant, Amaranthus hypochondriacus. Plant and Cell Physiology 41, 905910.
  • Pasqualini S., Ederli L., Piccioni C., Batini P., Bellucci M., Arcioni S. & Antonielli M. (2001) Metabolic regulation and gene expression of root phosphoenolpyruvate carboxylase by different nitrogen sources. Plant, Cell and Environment 24, 439447.
  • Pfanz H. & Heber U. (1986) Buffer capacities of leaves, leaf cells, and leaf cell organelles in relation to fluxes of potentially acidic gases. Plant Physiology 81, 697602.
  • Plieth C., Sattelmacher B. & Knight M.R. (2000) Ammonium uptake and cellular alkalisation in roots of Arabidopsis thaliana: The involvement of cytoplasmic calcium. Physiologia Plantarum 110, 518523.
  • Popp M. & Summons R.E. (1983) Phosphoenolpyruvate carboxylase and amino acid metabolism in roots. Physiologie Végetalé 21, 10831089.
  • Purvis A.C., Peters D.B. & Hageman R.H. (1974) Effect of carbon dioxide on nitrate accumulation and nitrate reductase induction in corn seedlings. Plant Physiology 53, 934941.
  • Putnam R.W. (1998) Intracellular pH regulation. In Cell Physiology Source Book, 2nd edn (ed. N.Sperelakis), pp. 293311. Academic Press, San Diego, CA, USA.
  • Rademacher T., Hausler R.E., Hirsch H.J., Zhang L., Lipka V., Weier D., Kreuzaler F. & Peterhansel C. (2002) An engineered phosphoenolpyruvate carboxylase redirects carbon and nitrogen flow in transgenic potato plants. Plant Journal 32, 2539.
  • Raghavendra A.S., Yin Z.H. & Heber U. (1993) Light-dependent pH changes in leaves of C-4 plants – comparison of the pH response to carbon dioxide and oxygen with that of C-3 plants. Planta 189, 278287.
  • Rajagopalan A.V., Gayathri J. & Raghavendra A.S. (1998) Modulation by weak bases or weak acids of the pH of cell sap and phosphoenolpyruvate carboxylase activity in leaf discs of C-4 plants. Physiologia Plantarum 104, 456462.
  • Raven J.A. (1985) pH regulation in plants. Science Progress 69, 495509.
  • Raven J.A. (1986) Biochemical disposal of excess H+ in growing plants. New Phytologist 104, 175206.
  • Raven J.A. & Smith F.A. (1974) Significance of hydrogen ion transport in plant cells. Canadian Journal of Botany 52, 10351048.
  • Raven J.A. & Smith F.A. (1976) Nitrogen assimilation and transport in vascular land plants in relation to intracellular pH regulation. New Phytologist 76, 415431.
  • Reid R.J., Loughman B.C. & Ratcliffe R.G. (1985) 31P NMR measurements of cytoplasmic pH changes in maize root tips. Journal of Experimental Botany 36, 889897.
  • Roberts J.K.M., Callis J., Wemmer D., Walbot V. & Jardetzky O. (1984) Mechanism of cytoplasmic pH regulation in hypoxic maize root tips and its role in survival under hypoxia. Proceedings of the National Academy of Sciences of the USA 81, 33793383.
  • Roberts J.K.M., Wemmer D., Ray P.M. & Jardetzky O. (1982) Regulation of cytoplasmic and vacuolar pH in maize root tips under different experimental conditions. Plant Physiology 69, 13441347.
  • Sagi M., Dovrat A., Kipnis T. & Lips H. (1998) Nitrate reductase, phosphoenolpyruvate carboxylase, and glutamine synthetase in annual ryegrass as affected by salinity and nitrogen. Journal of Plant Nutrition 21, 707723.
  • Sakano K. (2001) Metabolic regulation of pH in plant cells: Role of cytoplasmic pH in defense reaction and secondary metabolism. International Review of Cytology – a Survey of Cell Biology 206, 144.
  • Sakano K., Kiyota S. & Yazaki Y. (1998) Degradation of endogenous organic acids induced by Pi uptake in Catharanthus roseus cells: involvement of the biochemical pH-stat. Plant and Cell Physiology 39, 615619.
  • Salisbury F.B. & Ross C.W. (1992) Plant Physiology, 4th edn. Wadsworth, Belmont, CA, USA.
  • Savchenko G., Wiese C., Neimanis S., Hedrich R. & Heber U. (2000) pH regulation in apoplastic and cytoplasmic cell compartments of leaves. Planta 211, 246255.
  • Scheible W.R., Gonzalez-Fontes A., Lauerer M., Müller R.B., Caboche M. & Stitt M. (1997) Nitrate acts as a signal to induce organic acid metabolism and repress starch metabolism in tobacco. Plant Cell 9, 783798.
  • Schweizer P. & Erismann K.H. (1985) Effect of nitrate and ammonium nutrition of nonnodulated Phaseolus vulgaris L. on phosphoenolpyruvate carboxylase and pyruvate kinase activity. Plant Physiology 78, 455458.
  • Serrano R. (1990) Recent molecular approaches to the physiology of the plasma-membrane proton pump. Botanica Acta 103, 230234.
  • Siebke K., Yin Z.H., Raghavendra A.S. & Heber U. (1992) Vacuolar pH oscillations in mesophyll cells accompany oscillations of photosynthesis in leaves – Interdependence of cellular compartments, and regulation of electron flow in photosynthesis. Planta 186, 526531.
  • Smith C.E. & Bown A.W. (1981) The regulation of oat coleoptile phosphoenolpyruvate carboxylase and malic enzyme activities by H+ and metabolites – kinetic evidence for and against a metabolic pH-stat. Canadian Journal of Botany 59, 13971404.
  • Song C.P., Guo Y., Qiu Q.S., Lambert G., Galbraith D.W., Jagendorf A. & Zhu J.K. (2004) A probable Na+ (K+) /H+ exchanger on the chloroplast envelope functions in pH homeostasis and chloroplast development in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the USA 101, 1021110216.
  • Speer M. & Kaiser W.M. (1994) Replacement of nitrate by ammonium as the nitrogen-source increases the salt sensitivity of pea plants. II. Intercellular and intracellular solute compartmentation in leaflets. Plant, Cell and Environment 17, 12231231.
  • Stewart P.A. (1983) Modern quantitative acid-base chemistry. Canadian Journal of Physiology and Pharmacology 61, 14441461.
  • Stoimenova M., Hänsch R., Mendel R., Gimmler H. & Kaiser W.M. (2003a) The role of nitrate reduction in the anoxic metabolism of roots I. Characterization of root morphology and normoxic metabolism of wild type tobacco and a transformant lacking root nitrate reductase. Plant and Soil 253, 145153.
  • Stoimenova M., Libourel I.G.L., Ratcliffe R.G. & Kaiser W.M. (2003b) The role of nitrate reduction in the anoxic metabolism of roots II. Anoxic metabolism of tobacco roots with or without nitrate reductase activity. Plant and Soil 253, 155167.
  • Sugiharto B. & Sugiyama T. (1992) Effects of nitrate and ammonium on gene expression of phosphoenolpyruvate carboxylase and nitrogen metabolism in maize leaf tissue during recovery from nitrogen stress. Plant Physiology 98, 14031408.
  • Sugiharto B., Suzuki I., Burnell J.N. & Sugiyama T. (1992) Glutamine induces the N-dependent accumulation of messenger RNAs encoding phosphoenolpyruvate carboxylase and carbonic anhydrase in detached maize leaf tissue. Plant Physiology 100, 20662070.
  • Sze H., Li X.H. & Palmgren M.G. (1999) Energization of plant cell membranes by H+-pumping ATPases: regulation and biosynthesis. Plant Cell 11, 677689.
  • Thomas F.M. & Runge M. (1992) Proton neutralization in the leaves of English oak (Quercus robur L.) exposed to sulfur dioxide. Journal of Experimental Botany 43, 803809.
  • Torii K. & Laties G.G. (1966) Organic acid synthesis in response to excess cation absorption in vacuolate and non-vacuolate sections of corn and barley roots. Plant and Cell Physiology 7, 395403.
  • Ullrich C.I. & Novacky A.J. (1990) Extracellular and intracellular pH and membrane-potential changes induced by K+, Cl, H2PO4, and NO3 uptake and fusicoccin in root hairs of Limnobium stoloniferum. Plant Physiology 94, 15611567.
  • Ullrich W.R. & Novacky A. (1981) Nitrate-dependent membrane potential changes and their induction in Lemna gibba G1. Plant Science Letters 22, 211217.
  • Ullrich W.R., Larsson M., Larsson C.-M., Lesch S. & Novacky A. (1984) Ammonium uptake in Lemna-gibba G1 related membrane potential changes and inhibition of anion uptake. Physiologia Plantarum 61, 369376.
  • Ulrich A. (1941) Metabolism of non-volatile organic acids in excised barley roots as related to cation-anion balance during salt accumulation. American Journal of Botany 28, 526537.
  • Van Quy L., Foyer C. & Champigny M.L. (1991) Effect of light and NO3 on wheat leaf phosphoenolpyruvate carboxylase activity – evidence for covalent modulation of the C3 enzyme. Plant Physiology 97, 14761482.
  • Vanlerberghe G.C., Schuller K.A., Smith R.G., Feil R., Plaxton W.C. & Turpin D.H. (1990) Relationship between NH4+ assimilation rate and in vivo phosphoenolpyruvate carboxylase activity – regulation of anaplerotic carbon flow in the green alga Selenastrum minutum. Plant Physiology 94, 284290.
  • Venema K., Belver A., Marin-Manzano M.C., Rodriguez-Rosales M.P. & Donaire J.P. (2003) A novel intracellular K+/H+ antiporter related to Na+/H+ antiporters is important for K+ ion homeostasis in plants. Journal of Biological Chemistry 278, 2245322459.
  • Viktor A. & Cramer M.D. (2005) The influence of root assimilated inorganic carbon on nitrogen acquisition/assimilation and carbon partitioning. New Phytologist 165, 157169.
  • Villa M.S., Gonzalez G.A., Torres J.L.T. & Santelises A.A. (1992) Effect of the NH4+/NO3 ratio on GS and PEPcase activities and on dry-matter production in wheat. Journal of Plant Nutrition 15, 25452557.
  • Vuorinen A.H. & Kaiser W.M. (1997) Dark CO2 fixation by roots of willow and barley in media with a high level of inorganic carbon. Journal of Plant Physiology 151, 405408.
  • Walker D.J., Leigh R.A. & Miller A.J. (1996) Potassium homeostasis in vacuolate plant cells. Proceedings of the National Academy of Sciences of the USA 93, 1051010514.
  • Wang M.Y., Glass A.D.M., Shaff J.E. & Kochian L.V. (1994) Ammonium uptake by rice roots. III. Electrophysiology. Plant Physiology 104, 899906.
  • Wang M.Y., Siddiqi M.Y., Ruth T.J. & Glass A.D.M. (1993) Ammonium uptake by rice roots. I. Fluxes and subcellular distribution of 13NH4+. Plant Physiology 103, 12491258.
  • Wedin D.A. & Tilman D. (1996) Influence of nitrogen loading and species composition on the carbon balance of grasslands. Science 274, 17201723.
  • Wells D.M. & Miller A.J. (2000) Intracellular measurement of ammonium in Chara corallina using ion-selective microelectrodes. Plant and Soil 221, 103106.
  • White P.J. & Broadley M.R. (2001) Chloride in soils and its uptake and movement within the plant: a review. Annals of Botany 88, 967988.
  • Winter H., Robinson D.G. & Heldt H.W. (1993) Subcellular volumes and metabolite concentrations in barley leaves. Planta 191, 180190.
  • Winter H., Robinson D.G. & Heldt H.W. (1994) Subcellular volumes and metabolite concentrations in spinach leaves. Planta 193, 530535.
  • Yemm E.W. & Willis A.J. (1956) The respiration of barley plants IX. The metabolism of roots during the assimilation of nitrogen. New Phytologist 55, 229252.
  • Yin Z.H., Kaiser W.M., Heber U. & Raven J.A. (1996) Acquisition and assimilation of gaseous ammonia as revealed by intracellular pH changes in leaves of higher plants. Planta 200, 380387.

Appendix

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. THE PROTON ECONOMY AND NITROGEN ACQUISITION
  5. AN ANALYSIS OF INORGANIC CYTOCHEMISTRY AND ITS COMPARTMENTATION
  6. PHOSPHOENOLPYRUVATE CARBOXYLASE REGULATION: IS IT COMPATIBLE WITH A PH-STAT ROLE FOR THE ENZYME?
  7. CONCLUDING REMARKS
  8. ACKNOWLEDGMENTS
  9. REFERENCES
  10. Appendix

List of additional notes. Numbers refer to those given in the main text.

  • 1
    A more complicated metabolic scheme, in the form of an ‘equation for growth with ammonium as the nitrogen source’: 3NH4+ + 60(CH2O) + 15O2 [RIGHTWARDS ARROW] 15CO2 + 28H2O + C45H72N3O32+ 4H+, is sometimes used to convey the hypothetical trend of NH4+-dependent cellular acidification (Raven 1985; also cited in Marschner 1995). However, the consideration of unlikely and somewhat arbitrarily constructed compounds (‘C45H72N3O32’) in these schemes underscores their speculative character and obscures the mechanisms of N uptake and assimilation, which are at the very centre of the discussion; moreover, untenable assumptions are built into such schemes, such as the premises that C/N ratios are invariable (cf. Wedin & Tilman 1996), that all plant N is organic (cf. Kirkby & Mengel 1967), and that all carbon for N assimilation derives from carbohydrate, discounting the role of anaplerotic C fixation in this process (cf. Melzer & O’Leary 1987; also see below). While the source of C for amino acid skeletons and for reducing power is important in the context of metabolic H+ production and consumption during N assimilation, especially in the context of aerobic versus anaerobic conditions (Gerendás & Ratcliffe 2000; Stoimenova et al. 2003b; also see below), it should be noted that GS/GOGAT is the entry point for most inorganic N into the amino acid pool, irrespective of N source, and therefore the H+ balance associated with the production of carbon skeletons, and with the oxidation of carbohydrate, is also independent of N source (except insofar as NH4+-grown plants often synthesize more amino acids).
  • 2
    Although this pH rise is often attributed to NH3 diffusion through the lipid bilayer of the plasma membrane, much evidence supports the idea that NH4+ is the permeating species, especially in cases where the external pH is well below the pKa for NH3/NH4+ (e.g. pH 7 in the study by Kosegarten et al. 1997); for a further analysis of this subject, see Britto et al. 2001a. Interestingly, whether NH4+ uptake (accompanied by the associated membrane potential rectification) or NH3 uptake occurs, the consequences for cytosolic pH are identical.
  • 3
    Interestingly, as pointed out by Stoimenova et al. (2003b), the first step of NO3 reduction, catalysed by NR, localized in the cytosol, and producing nitrite (NO2), is not a proton-consuming process, as can be seen in the equation NO3+ [NAD(P)H + H+[RIGHTWARDS ARROW] NO2 + [NAD(P)+] + H2O. Conversely, the subsequent, proton-consuming, reduction step from NO2 to NH4+, is localized in the plastid; in leaf tissue, this localization should intensify the trans-thylakoid ΔpH, and should therefore have consequences for photosynthetic processes. This possibility has been rarely, if ever, considered.
  • 4
    Note that, as with all summary equations of this nature (see note (1)), while deceptively more complete and satisfying than piecemeal approaches, nevertheless fail to account for some well-known physiological observations. In particular: (1) a significant fraction of the carbon budget in amino acid anabolism derives not directly from glucose, but is introduced anaplerotically via PEP carboxylase (see below); and (2) contrary to the apparent outcomes of Eqns 6 and 8, the rates of overall respiration, and of O2 consumption and CO2 evolution, are typically significantly higher in NH4+-grown than in NO3-grown plants (see Britto et al. 2001b), indicating the importance of physiological processes superimposed upon the primary events of N acquisition. In addition to these shortcomings, and as pointed out in note (3), metabolic schemes such as the one given here do not often take into account the effects of subcellular compartmentation of metabolism.