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- Materials and Methods
The anaplerotic role of phosphoenolpyruvate carboxylase (PEPc) in root tissue is widely recognized. The dependence of this enzyme on the concentration of dissolved inorganic carbon (DIC) in the root tissue is less often considered, since it is assumed that this occurs at high concentrations as a result of respiratory activity. Unlike aerated hydroponic solutions, soils have high CO2 concentrations (c. 2000 to 200 000 p.p.m., Norstadt & Porter, 1984) mainly because of limited diffusion and the biological components of the soil. Although the overwhelming flux of CO2 is out of the root, the root tissue DIC concentrations are determined by both the rate of root respiration and the gradient of CO2 between the root tissue and the external root environment. Root-zone DIC concentration influences photosynthesis (Cramer & Richards, 1999), respiration (van der Westhuizen & Cramer, 1998), NO3− uptake (Cramer et al., 1996), partitioning of C and N to organic and amino acid synthesis (Cramer & Lewis, 1993) and growth (Cramer & Richards, 1999). Positive effects of root-zone DIC on plant growth have been reported previously (Vapaavuori & Pelkonen, 1985), particularly in plants growing under high irradiances, salinity stress or high shoot temperatures (Cramer & Richards, 1999).
Reincorporation of root-zone DIC serves an anaplerotic function by providing intermediates for the tri-carboxylic acid (TCA) cycle through the activity of PEPc (Vuorinen & Kaiser, 1997), although, this contributes little to the carbon (C) budget of the whole plant (Viktor & Cramer, 2003). Incorporation of DIC enables synthesis of organic acids, which may in turn be utilized for amino acid synthesis (Schweizer & Erismann, 1985; Ikeda et al., 1992; Cramer et al., 1993; Vuorinen & Kaiser, 1997), exudation (e.g. for chelation of aluminium (Al); Cramer & Titus, 2001) and other processes dependant on organic acids. A larger proportion of root-derived C is allocated to organic acids in NO3−-fed plants, whereas with NH4+ nutrition more root-derived C is allocated to aspartate, asparagine, glutamate and glutamine (Cramer et al., 1993).
In short-term (6–12 h) uptake experiments, increased root-zone DIC promoted NO3− uptake compared with ambient root-zone DIC, whereas NH4+ uptake was decreased or unchanged (Cramer et al., 1996). Although increased NO3− uptake could be caused by increased availability of organic acid for root assimilation of NO3−, NH4+ assimilation also requires C skeletons from the TCA cycle for amino acid synthesis (Schweizer & Erismann, 1985). This and the fact that NO3− uptake by nitrate reductase (NR)-deficient mutants of barley was increased by elevated root-zone DIC indicates that there may be a direct stimulatory effect of DIC on NO3− uptake (Cramer et al., 1996). However, the question arises as to whether the influence of DIC on N uptake is sustained for longer periods of time (days).
The biological demand for CO2 or HCO3− in nongreen tissues frequently exceeds the uncatalysed equilibrium between CO2 and HCO3− (Raven & Newman, 1994). Carbonic anhydrase (CA) catalyses the reversible hydration of CO2 (Rengel, 1995). Carbonic anhydrase activity has found in both the stroma of chloroplasts (87% of total cellular activity) and the cytosol (13%) of Solanum tuberosum leaves (Rumeau et al., 1996). It is also known to occur in root nodules at high activities where it is responsible for equilibrating CO2 and HCO3−, possibly supplying PEPc with HCO3− for anaplerotic replenishment of TCA cycle intermediates or for facilitating release of CO2 from the nodules which have low gas permeability (Atkins et al., 2001). Non-nodular root tissue CA activities are lower than those of nodules. However, CA activity in Zea mays root tips was found to be 200 times higher than that of PEPc in vivo (Chang & Roberts, 1992), indicating that its activity was high enough to play a role. However, the possible variation in CA activity in roots exposed to varying CO2 concentrations is unknown.
The disparity between in vitro and in situ root PEPc activities may result from regulation in vivo (Cramer et al., 1999). Regulation is through protein phosphorylation which results in a decrease in the sensitivity of PEPc to allosteric inhibitors such as malate (Jiao & Chollet, 1991) and an increase in catalytic activity. Koga & Ikeda (1997) found that root PEPc activity was 2- to 2.5-fold higher with NH4+ nutrition than with NO3− nutrition and reached higher values in NH4+-fed wheat, barley and tomato plants compared with NO3−-fed plants. Concentration of PEPc protein, measured by Western blot analysis, was greater in the roots of plants supplied with NH4+ (Koga & Ikeda, 1997) and methionine sulfoximine suppressed the increased PEPc activity elicited by NH4+ nutrition (Koga & Ikeda, 2000). It was concluded that NH4+ assimilation is required for induction of de novo synthesis of PEPc by NH4+ nutrition. Since root-zone DIC concentrations influence NO3− and NH4+ uptake, it is possible that it would influence the production of PEPc protein or the regulation of PEPc activity.
Elevated root-zone DIC was found to stimulate NR activity in vitro and in situ in barley plants (Cramer et al., 1996). Nitrate reductase is affected by several factors including NO3− availability, pH (Kaiser & Brendle-Behnisch, 1995), light/dark, inhibitor proteins (Glaab & Kaiser, 1995) and photosynthesis (Kaiser & Brendle-Behnisch, 1991). It is active in the dephosphorylated form and partly inactivated in the phosphorylated form, although, complete inactivation requires binding of an inhibitor protein (Glaab & Kaiser, 1995). It is activated by cytosolic acidification through dephosphorylation and is inactivated by phosphorylation in response to cytosolic alkalization (Kaiser & Brendle-Behnisch, 1995). Since dissolved CO2 is a weak acid, exposure to CO2 may change the cytosolic pH, with consequences for the regulation of plant metabolism (Bown, 1985).
Although the influence of soil CO2 concentrations on soil chemistry is well known, the influence of high concentrations of soil CO2 on plant metabolism is not widely recognized. There are several reports of the influence of root-zone CO2 concentration on N acquisition/assimilation and root C fixation (Cramer, 2002). These reports are largely based on short-term experiments (1–8 h). In this investigation we extended this previous work to longer periods of time (up to 15 d). The influence of root-zone CO2 concentration on the allocation and partitioning of root-assimilated C were explored by means of a root-supplied 14CO2 pulse-chase experiment in which the changes were followed over a 24-h chase-period. The activities of key enzymes in N assimilation (NR, PEPc and CA) were also measured to determine whether the activities and regulation of these could account for changes in N and C acquisition and assimilation.
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- Materials and Methods
The increase in RGR of NO3−-fed plants caused by elevated root-zone CO2 (Fig. 1) could be due to changes in one or more of several processes including photosynthesis, respiration, NO3− uptake or partitioning of C and N (Cramer & Richards, 1999). However, there was no evidence for a sustained increase in NO3− uptake with elevated root-zone CO2 concentration (Fig. 2), although more rapid initial uptake may have contributed to greater shoot total N and biomass accumulation. The higher RGR of NO3−-compared with NH4+-fed plants (Fig. 1) was not associated with more rapid sustained uptake of N by the plants supplied with NO3− (Fig. 2), but rather may have been due to the toxicity of accumulated NH4+. Root-based synthesis of amino acids from nutrient NH4+ uptake competes with root growth resulting in increased shoot : root ratios and wilting (Cramer & Lewis, 1993). Furthermore, NH4+ can competitively exclude other cations, result in intense ATP demand for NH4+ efflux and cause rhizosphere acidification (Britto & Kronzucker, 2002). The fact that increased root-zone CO2 concentration did not increase the growth of plants supplied with NH4+ nutrition indicates that anaplerotic C alone could not ameliorate the negative effects of NH4+. Anaplerotic C fixation may not influence NH4+-induced growth depression since both anaplerotic C fixation and growth depend on shoot-derived C.
Nitrate uptake was previously found to be strongly stimulated by elevated root-zone CO2 in NR-deficient mutants of barley (Cramer et al., 1996) and in tomato (van der Merwe & Cramer, 2000) over 6–8 h uptake periods. A similar increase was observed in this study when plants were preincubated for 12 h in solutions containing low NO3− concentrations (0.2 mm) prior to the uptake experiment (Fig. 2). However, root-zone CO2 concentration had no influence on the uptake of NO3− over the longer term (15 d, Fig. 2). In previous work, the plants were either grown on NH4+ or deprived of N for c. 12 h prior to determination of NO3− uptake rates. Thus, the stimulation of NO3− uptake by elevated root-zone CO2 concentration requires depletion of NO3− within the tissue. Since stimulation of NO3− uptake has been found to be independent of NRA (Cramer et al., 1996), it has been proposed to involve an electrochemical modification facilitating NO3− uptake (M. D. Cramer & A. J. Miller, unpubl. data). Thus, stimulation of NO3− uptake by elevated root-zone CO2 could be a transient effect on the uptake kinetics, which is damped by subsequent feedback control. Inhibition of NH4+ uptake in roots aerated with 5000 p.p.m. root-zone CO2 (Fig. 2) could be due depletion of C skeletons for glutamate synthesis (van der Westhuizen & Cramer, 1998). However, increased synthesis of amino acids (Fig. 5) could also downregulate NH4+ uptake, as reported previously (Causin & Barneix, 1993; Feng et al., 1994; Glass et al., 1997).
Shoot total N concentrations of NH4+-fed plants grown with 380 p.p.m. root-zone CO2 were higher than those of NO3−- fed plants, possibly owing to a greater uptake of NH4+ and translocation to the shoot or to slower growth with NH4+-than with NO3−-nutrition (Fig. 3). The higher total N concentrations for shoots and higher total N shoot : root ratios found for NO3−-fed plants grown with 5000 p.p.m. root-zone CO2 compared with 380 p.p.m. root-zone CO2 (Fig. 3) might result from greater initial uptake, followed by translocation of reduced N to the shoot. This increased transfer to the shoot could be facilitated by the availability of anaplerotic C for amino acid synthesis. Although the proportion of 14C localized in the soluble basic fraction was not strongly influenced by the concentration of root-zone CO2 (Fig. 5), there was greater total incorporation of inorganic C (Fig. 4) and thus a greater synthesis of amino compounds.
In situ NRA was greater in plants supplied with elevated root-zone CO2. Since this was measured on plants preincubated with low concentration of NO3− (0.2 mm) for 12 h, it does not reflect the steady-state condition. In vitro NRA in leaves was decreased by elevated CO2 (Table 1) and this may represent downregulation of NRA in response to increased root assimilation of NO3− and the accompanying synthesis of amino acids (Figs 4 and 5). The increased assimilation within the root might have limited the flux of NO3− to the shoot and thus limited NRA there. Although the extent of phosphorylation seemed to have little influence on the NRA (particularly in the leaves), the removal of the inhibitor protein did significantly increase the rates of NRA. However, there were only minor differences in the phosphorylation and inhibition status of NRA between the root-zone CO2 treatments. This indicates that NRA probably responded to induction of NR expression by NO3−, rather than to post-translational regulation, and that changes in NRA activity induced by varying root-zone CO2 concentrations (Cramer et al., 1996) probably depend on NO3− uptake.
Phosphoenolpyruvate carboxylation may serve as a source of anaplerotic C during NH4+ assimilation to compensate for the loss of TCA cycle intermediates to amino acid synthesis or as a source of C for organic acid synthesis in NO3−-fed plants to maintain ionic balance in the xylem sap (Schweizer & Erismann, 1985; Cramer et al., 1993; Vuorinen & Kaiser, 1997). The reduction and assimilation of NO3− in the shoots of tomato plants could account for the higher total PEPc activity in leaves of NO3−-fed plants compared with NH4+-fed plants grown with 5000 p.p.m. root-zone CO2 (Table 1). Similar results were reported by Schweizer & Erismann (1985). Root-zone CO2 did not influence leaf or root PEPc activity of NO3−-fed plants. This reflects the steady-state situation in which NO3− uptake was not increased and 14C incorporation was not strongly increased. Decreased total root PEPc activity of NH4+-fed plants grown with 5000 p.p.m. root-zone CO2 compared with 0 p.p.m. root-zone CO2 was probably due to inhibition of NH4+ uptake by 5000 p.p.m. root-zone CO2 (Fig. 2), possibly as a consequence of feedback inhibition. According to Koga & Ikeda (1997), the increased anaplerotic PEPc activity in roots of wheat, barley and tomato transferred to NH4+ from NO3− nutrition was dependent on de novo protein synthesis. Interestingly, although PEPc activity was greater in the roots of plants supplied with NH4+ nutrition, PEPc from the NH4+-grown plants was more sensitive to inhibition by malate, indicating that the enzyme was less phosphorylated (Jiao & Chollet, 1991). Since NH4+ nutrition decreases 14C associated with organic acids (Fig. 5) and probably depletes organic acids, especially at low root-zone CO2 concentrations, it may be that PEPc in vivo was highly active in the relative absence of malate. This high root PEPc activity was also associated with greater incorporation of 14C (Fig. 4).
It is well known that CA occurs in root nodules at high activities where it is responsible for equilibrating CO2 and HCO3− (Atkins et al., 2001). Nodular CA activities range between 334 and 1863 µmol g−1 f. wt nodule s−1 (Atkins et al., 2001). This is much higher than the CA rates reported in the present investigation for root tissue (c. 10 µmol g−1 f. wt s−1). The uncatalysed conversion of CO2 to HCO3− probably occurs at rates sufficient to support the activities of root PEPc (Raven & Newman, 1994). However, inhibitors of CA activity supplied to tomato roots were previously found to inhibit respiratory O2 consumption and DI14C incorporation by tomato roots (van der Westhuizen & Cramer, 1998). Furthermore, the in vitro rates of CA activity found in this study were far higher than the in vitro rates of PEPc activity. This concurs with the in vivo results of Chang & Roberts (1992), indicating that CA could facilitate the in vivo equilibrium between HCO3− and CO2, if required.
In C3 leaves most of the CA activity is in the chloroplast stroma (Atkins et al., 1972; Fett & Coleman, 1994; Rumeau et al., 1996) and this enzyme may facilitate inorganic C diffusion into and across the chloroplast for photosynthesis by accelerating the dehydration of HCO3− to CO2 (Badger & Price, 1994; Majeau & Coleman, 1994). Elevated atmospheric CO2 concentrations have been found to result in inhibition of Rubisco and CA in Pisum sativum (Majeau & Coleman, 1996). Increased root-zone CO2 concentration results in a decrease in photosynthetic carboxylation rates (Cramer & Richards, 1999). This was suggested to result from the transport of organic C to the shoot and the decarboxylation of this in the shoot which could increase intercellular CO2 concentrations (Arteca & Poovaiah, 1982) and result in the downregulation of Rubisco (Cramer & Richards, 1999). Hibberd & Quick (2002) found evidence for a C4-like reassimilation of decarboxylation-derived CO2 in the stems of C3 plants. Possibly the decrease in leaf CA activity of NO3−-fed plants grown with 5000 p.p.m. root-zone CO2 compared with 0 p.p.m. root-zone CO2 could be due to increased availability of CO2 in the shoot. This could particularly be the case with plants supplied with NO3−, which initially accumulated a large proportion of label in the acidic fraction (organic acids) in the root tissue (Fig. 5). These organic acids when translocated to the shoot may provide a ready source of CO2 by being metabolized in the TCA cycle to liberate CO2 which is then refixed through photosynthesis. It is noteworthy that in plants supplied with NO3−, a large proportion of the soluble fraction in the leaves was associated with the neutral fraction (carbohydrates), possibly derived from refixation of CO2 derived from organic acid decarboxylation. In NH4+ plants, which are likely to transfer a greater proportion of C through the xylem as amino acids, there is perhaps less potential for such a refixation mechanism to be important.
The products of root PEPc fixation were retained to some extent in the plant tissue or exuded into the root solution. Regardless of N source, plants grown with 0 p.p.m. root-zone CO2 concentration retained (tissue + exudates) c. 66% of incorporated 14C after 24 h. By contrast, with 5000 p.p.m. root-zone CO2, NO3−- and NH4+-fed plants retained c. 86% and 77% of their incorporated 14C after 24 h, respectively. Thus, plants supplied with higher concentrations of CO2 to the root-zone lost less of the 14C supplied through respiration. This could be a result of the suppression of root CO2 loss by increased incorporation of DIC at elevated root-zone CO2 concentrations, as has been reported previously (van der Westhuizen & Cramer, 1998). High concentrations of CO2 are also known to repress the rate of respiration in roots (Nobel & Palta, 1989; Palta & Nobel, 1989) and harvested vegetables (Wills et al., 1979; Herner, 1987).
Ammonium nutrition resulted in greater DI14C incorporation than did NO3− nutrition. This was probably due to the more rapid uptake and assimilation of NH4+ than of NO3− (Murphy & Lewis, 1987), and the associated requirement for C skeletons for amino acid synthesis (Cramer et al., 1993). The initially high proportion of label associated with the basic fraction in the leaves of plants supplied with NH4+ nutrition resulted from rapid transfer of labelled amino acids from the roots to the shoots. The subsequent decrease in the amino acid fractions in the leaves was probably caused by diversion to insoluble components, metabolism of the amino acids, respiratory losses or cycling of the amino compounds back from the shoot to the root. Extensive cycling of N between the shoot and root has been documented previously (Lambers et al., 1982).
The diversion of incorporated 14C into organic acids in NO3−-fed plants and subsequent export to the shoots (Fig. 6) was consistent with previous results (Cramer et al., 1993; Cramer & Richards, 1999). Organic acids translocated from the roots to the shoots could be decarboxylated in the shoots and the CO2 reassimilated to form carbohydrates (Cramer & Richards, 1999), accounting for the greater proportion of the neutral fraction in the shoots of plants supplied with NO3− (Fig. 5). The decrease in the proportion of label associated with the acidic fraction in the roots over time could also reflect exudation from the root (Fig. 7), increased synthesis of amino acids, and subsequent allocation of amino acids and carbohydrates to structural material, as indicated by increasing allocation to the insoluble fraction over time (Fig. 4).
Greater net exudation with elevated root-zone CO2 (Fig. 6) was probably due to greater PEPc CO2 refixation, which, especially when combined with NH4+ nutrition, could result in an increased amino acid synthesis and subsequent exudation of these compounds (Cramer & van der Westhuizen, 2000). The reason for the exudation of amino acids is not clear, although it could simply be a consequence of the accumulation of amino compounds in the root or a regulatory mechanism to reduce tissue amino acid levels. A small proportion of the label exuded was associated with the organic acid fraction. The fact that the organic acids accounted for a relatively small proportion of the exuded label probably reflects the fact that the plants were grown with adequate P nutrition (2 mm). Phloem-derived sugars translocated through the root apoplast may ‘leak’ into the external solution (Jones & Darrah, 1993; Marschner, 1995), although this notion has been challenged by work on Leptochloa fusca, which may sustain diazotrophic bacteria with sugar exudates (Mahmood et al., 2002).