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• The potential amelioration of Al (Al) toxicity by elevated dissolved inorganic carbon (DIC = CO2 + HCO3−) in the root medium was investigated in both NH4+- and NO3−-fed Lycopersicon esculentum (tomato) plants.
• Hydroponically grown L. esculentum seedlings were intermittently supplied with 50 µM AlCl3 and the nutrient solutions aerated with either 360 or 5000 ppm CO2. Relative growth rate (RGR), nitrogen uptake, root respiration and root incorporation of DI14C and subsequent partitioning were measured.
• Al reduced the RGR of plants grown with 360 ppm root-zone CO2. At elevated root-zone CO2, Al had no significant effect on the RGR of NO3−-fed plants whereas the RGR of the NH4+-fed plants was increased by 21%. Al decreased the respiratory quotient (Rq) by 15% at 360 ppm CO2, but had no influence at 2000 ppm CO2. Exudation of organic 14C, especially of [14C]-organic acids derived from root incorporation of DI14C, was increased by Al.
• It is concluded that elevated DIC partially ameliorated Al toxicity by providing anaplerotic carbon for organic acid synthesis.
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Carbon dioxide readily accumulates in soils to between 0.2% (v/v) and 0.5%, but can accumulate to 20% under special circumstances (Norstadt & Porter, 1984). The dissolved inorganic carbon (DIC = CO2 + HCO3−) in the soil is generated by respiration of plant roots and of other biota as well as by chemical processes in the soil. The concentration of inorganic carbon within soil air spaces depends on the vegetation type and activity (Sotomayor & Rice, 1999) and the movement of inorganic carbon through the root zone due to diffusion and advection. The DIC concentration in root tissue depends on the DIC gradient that exists between the root and the soil, with high soil DIC concentrations maintaining high tissue DIC concentrations. DIC in the root tissue is incorporated into organic acids through the activities of carbonic anhydrase and phosphoenolpyruvate carboxylase (PEPc). OAA produced from PEP carboxylation is converted to other organic acids or used as carbon skeletons for amino acid synthesis. Findings that root zone CO2 influences root respiration (Van der Westhuisen & Cramer, 1998) and nitrogen metabolism (Cramer et al., 1996) should be of concern to plant physiologists since the use of hydroponics equilibrated with ambient atmospheric CO2 concentrations for the culture plants represents an artificial situation. This is particularly true of aspects of physiology and metabolism, such as tolerance of Al, which are linked to respiratory and organic acid metabolism.
Tolerance to Al is a complex poly gene trait (Pellet et al., 1996) comprising several physiological mechanisms operating both in the cytoplasm and the rhizosphere. Mechanisms conferring Al tolerance have been suggested to include control of rhizosphere pH (Calba & Jaillard, 1997; Degenhardt et al., 1998) and exclusion of Al from the cytoplasm by complexing with substances such as mucilage (Ryan et al., 1993), organic acids (see below) and phosphate (Pellet et al., 1996). Several reports indicate that Al does occur in a soluble form in the symplasm, although, the soluble concentration is only c. 10−10 M (Ma et al., 1998). However, even such low concentrations are potentially phytotoxic. Therefore, Al accumulating plants must have internal mechanisms of detoxifying Al through the formation of complexes with organic acids (Ma et al., 1998) or P (Crawford et al., 1998) or of partitioning the Al away from sensitive areas of the cell (Vitorello & Haug, 1996).
The ability of organic acids to chelate and render Al nonphytotoxic is well established and it has been found that Al tolerant plants use organic acids, especially malate and citrate, to detoxify Al both internally and externally (Delhaize & Ryan, 1995; Ryan et al., 1995; Jorge & Arruda, 1997; Ginting et al., 1998; Ma et al., 1998; Zheng et al., 1998a; Zheng et al., 1998b). The use of organic acids to chelate Al is likely to impact on the tricarboxylic acid (TCA) cycle and consequently the anaplerotic provision of carbon to the TCA cycle through the activity of PEPc. Perturbation of carbon flow through the TCA cycle due to the use of organic acids may result in inhibition of respiration. de Lima & Copeland (1994) found that Al inhibited the respiratory activity of excised root tips and isolated mitochondria of wheat roots. Al initially inhibited the cytochrome pathway of electron transport, but after prolonged exposure of seedlings to Al there was also inhibition of alternative pathway activity. Inhibition of respiration was also associated with uncoupling of phosphorylation which may explain why Copeland & de Lima (1992) found that alcohol dehydrogenase activity was stimulated by Al. Cumming et al. (1992) found that the Al stimulated respiration of ‘Al-tolerant’ and inhibited that of ‘Al-sensitive’Phaseolus vulgaris cultivars indicating that the response of respiration to Al is genotype-specific.
Nitrate nutrition results in alkalization of the root zone while NH4+ nutrition results in acidification (Kosegarten et al., 1999), the latter possibly exacerbating Al toxicity. The reported effects of Al on NO3− uptake are variable, but Al-induced a strong decrease in NO3− uptake by maize roots and stimulated or did not influence NH4+ uptake (Calba & Jaillard, 1997). These authors concluded that inhibition of NO3− uptake by Al unmasked H+ excretion without influencing plasma membrane H+-ATPase activity. Nitrate uptake in Al-tolerant maize cultivars was not inhibited as much as in the sensitive cultivars and thus a smaller acidification of the rhizosphere occurred. Elevated concentrations of DIC have been shown to promote NO3− uptake (Cramer et al., 1996) and thus may partially protect Al-sensitive plants from Al induced acidification of the rhizosphere.
The aim of this study was to investigate whether DIC concentrations in the concentration range found in soils could partially ameliorate the influence of Al on growth of plants supplied with either NO3− or NH4+ nutrition. Changes in growth, N uptake, respiration, DI14C incorporation and exudation of organic 14C were measured in response to Al toxicity. Since plants exude organic acids to enable Al resistance, it was hypothesized that elevated DIC could partially alleviate Al toxicity since more carbon would be available through PEPc activity for organic acid synthesis enabling greater Al tolerance.
Materials and Methods
Seeds of tomato (Lycopersicon esculentum (L.) Mill. cv. F144) were pregerminated in aerated water for 24 h before planting into a 1 : 1 mixture of vermiculite and compost. Seedlings (3-wk-old) were transferred into aerated hydroponics where the hypocotyls were wrapped in foam rubber and inserted through plastic lids. Each tank contained eight plants supplied with 20 l Long Ashton nutrient medium (Hewitt, 1966) modified to contain 2 mM NaNO3 or 2 mM NH4Cl as the N source and 0.09 mM Fe EDTA as the iron source (pH 4.5). The solutions were replaced once a week for 3 wk. The pH of the solution was monitored daily and was found to be relatively stable but was corrected if it deviated by more than 0.1 pH units. Plants were grown in a glasshouse at the University of Stellenbosch between July and October. In the glasshouse midday PPFR was c. 700 µmol m−2 s−1 (over the waveband 400–700 nm), day/night temperature was 28/12°C and day/night rh 33/87% (average daily maximum and minimum). Nutrient solutions were strongly aerated with ambient air (c. 360 ppm CO2) or with air containing elevated DIC (c. 5000 ppm CO2). These CO2 concentrations were chosen to represent the aeration of nutrient solutions with both normal atmosphere and the CO2 concentration likely to be experienced in agricultural soils. Previous work (Cramer et al., 1996) has demonstrated the sensitivity of other physiological processes to these root zone CO2 concentrations. Carbon dioxide was supplied at elevated levels by enriching ambient air with industrial grade CO2. The CO2 concentrations were monitored continuously using an ADC Mk3 (Analytical Development Corporation, Hoddeston, England, UK) infrared gas analyser (IRGA). To prevent leakage of CO2 from the root zone and the consequent enrichment of atmosphere around the shoots, the lids of the hydroponic tanks receiving elevated CO2 were sealed with closed-cell foam rubber around the rim, clamped onto the tanks and the air space between the surface of the nutrient solution and the lid maintained under partial vacuum to ensure that the net airflow was inwards.
Growth analyses and biomass determination
For treatment with Al, the plants grown with Long Ashton nutrient solution were transferred to a solution of containing only 2 mM CaSO4 (pH 4.5) or 2 mM CaSO4 combined with 50 µM AlCl3 (pH 4.5) for 6 day-light h every second day. Using the chemical speciation program Minteqa2 v3.10 (Allison et al., 1991), it was estimated that 55% of the supplied Al was available as Al3+ at 20°C in this solution. To allow RGR to be calculated for each individual plant (n = 8), fresh weight was determined immediately before transfer into hydroponic solutions and every second day thereafter at a fixed time of day. Overall RGR was calculated using the natural logarithms of the fresh weight of each plant. Plants were removed from the containers, the roots gently blotted dry and weighed within 2 min of removal. A trial with and without exposure of plants to Al showed that this procedure did not significantly influence plant growth compared to plants left undisturbed over the experimental period (data not shown).
Plants were harvested 12–18 d after transplanting, depending on the experiment. Roots were blotted dry and the plants were separated into leaf, stem and root components before weighing. The leaf areas were measured using a leaf area meter (Li-Cor, Lincoln, NE, USA). Root lengths of the plants were measured using a Hewlett Packard Scanjet 6200C scanner and the image analysed with Rootedge 2.2c (Ewing & Kaspar, 1998). The root area was calculated from the root length multiplied by the root width. The plant components were dried at 80°C for 48 h and re-weighed.
Measurements of root O2 and CO2 flux were performed as described by Van der Westhuisen & Cramer (1998). Plants were acclimatized (12 h) in respiration cuvettes supplemented with Long Ashton nutrient solution (2 mM NO3−) and aerated with 360 ppm CO2. After 12 h the plants were supplied with 10 mM Mes which has limited buffering capacity at this pH (pH 4.5), 0.5 mM NaNO3 and 2 mM CaSO4 with or without 50 µM Al. Air containing either 360 or 2000 ppm CO2 was supplied through precision needle valves to each plant root at c. 1 ml s−1. Industrial grade CO2 was introduced into a cylinder of compressed air to obtain a supply of elevated CO2 (c. 2000 ppm CO2). At the flow rates used here the ΔCO2 concentrations were of the order of 100 ppm ensuring that CO2 flux could be measured against relatively high background CO2 concentrations. Use of CO2 concentrations greater than 2000 ppm was avoided for respiration measurements because this reduced the sensitivity of the measurements to unacceptable levels. Oxygen depletion was measured over 10–20 min during which time CO2 accumulation was small. After measurements, the plants were harvested, divided into root and shoot components, dried at 80°C for 48 h, weighed and the respiration rate expressed on the basis of root dry mass.
Nitrogen uptake and DI14C labelling
Seedlings grown as described previously with either NO3− or NH4+ nutrition and without exposure to Al were transferred to 300 ml containers to allow determination of uptake by depletion from the nutrient solution (n = 5). The seedlings were pretreated overnight in a Long Ashton nutrient solution with 1 mM NaNO3 or NH4Cl and aerated with 360 or 5000 ppm CO2. The seedlings were transferred to 300 ml solutions containing 2 mM CaSO4, 10 mM Mes (pH 4.5), 1 mM NaNO3 or 1 mM NH4Cl and either 0 µM or 50 µM AlCl3 for the uptake measurements. Sub-samples (1 ml) were taken at regular intervals over a period of 10 h. The pH of the nutrient solutions was measured, the NO3− concentration determined according to Cataldo et al. (1975) and the NH4+ concentration by the indophenol blue reaction (Solorzano, 1969). After 10 h, 42 nmol NaHCO3 containing 0.093 MBq NaH14CO3 was added to the nutrient solutions and aeration discontinued. Solutions were aerated for 15 s every 15 min over a period of 60 min. The plants were removed from the nutrient solutions, the roots rinsed in two volumes of 2 mM CaSO4, blotted dry, and the plants divided into shoot and root components. The components were weighed, quenched in liquid N2 and stored at −18°C. Sub-samples of the nutrient solutions (500 µl) were acidified with 500 µl of 0.3 M HCl, shaken for 12 h on a rotary shaker and then counted in a Beckman Instruments LS 5000 TD liquid scintillation counter (California, USA) with 2 m Ready-gel (Beckman) scintillation fluid. A 10 ml sample of the nutrient solution was evaporated under an air-stream and made up to 1 ml. This was separated into amino acid, organic acid and neutral fractions using ion exchange resins prepared according toAtkins & Canvin (1971). Samples were passed through 1 ml Dowex 50 W-X8 and Dowex 1 W-X8 (Sigma, St. Louis, MO, USA) columns in 1 m disposable syringes followed by 25 m 50% (v/v) ethanol. The eluate was collected as the neutral fraction. The amino acid fraction was then eluted from the Dowex 50 W column with 10 ml 2 M HCl, while the organic acid fraction was eluted from the Dowex 1 W column with 10 ml 6 M formic acid. These fractions were evaporated and made up to 1 ml. A 0.5-ml aliquot of the neutral fraction was acidified with 50 µl 0.3 M HCl and shaken for 12 h in a fume-hood. The neutral fraction and 0.5 ml of the amino and organic acid fractions were combined with 2 ml Ready-gel scintillation fluid and quantified in a liquid scintillation counter.
The plant components were homogenized with a VirTis 6303 mill (VirTis Company, Gardiner, NY, USA) in 50 ml 80% (v/v) cold ethanol and stored at −18°C for 48 h and then extracted for 60 min at 45°C. The samples were filtered through Whatman 1 filter paper and the filtrate made to volume (75 ml). The insoluble material left on the filter paper was dried at 80°C for 24 h and then a subsample was mixed with 500 µl 0.3 M HCl and shaken for 12 h. Soluene-350 (Packard, Ill, USA) was then added (2 ml) and shaken for a further 12 h and then counted by liquid scintillation counting after the samples had been mixed with 4 ml of scintillation fluid. Sub-samples (500 µl) of the soluble fraction were acidified with 50 µl 0.3 M HCl and allowed to stand in a fume-hood for 24 h before the addition of 2 ml scintillation fluid and quantification by liquid scintillation counting. The remainder of the soluble fraction was then evaporated almost to dryness under an airstream and then made up to 30 ml with water and separated into amino acid, organic acid and neutral fractions using Dowex 50 W-X8 and Dowex 1 W-X8 ion exchange resins as described above. Aliquots (1 ml) of the fractions were combined with 2 ml of scintillation fluid and quantified by liquid scintillation counting.
Results were subjected to ANOVA to determine the significance of differences between the responses to the treatments using Statgraphics 7.0 (1993). Homogeneity of variances was determined using Bartlett's test and where variances were not homogenous the results were log-transformed for statistical analysis. Where percentage data were used these were arcsine transformed (Zar, 1984) before statistical analysis. ANOVA was followed by Fisher's projected LSD tests (P < 0.05) to determine the differences between the individual treatments.
The dry weight of plants supplied with both NO3− and NH4+ nutrition combined with 360 ppm root zone CO2 was reduced in plants intermittently treated with Al (Table 1). The reduction in dry weight accumulation caused by Al was less severe with 5000 ppm root zone CO2 combined with NO3− nutrition (34%) than in plants supplied with NO3− and 360 ppm root zone CO2 (53%). In the plants supplied with NH4+ nutrition there was no change in total biomass accumulation in response to Al in plants supplied with elevated DIC, but the inhibition of growth by Al at 360 ppm was more severe (73%) than that in plants supplied with NO3−. Only small changes were observed in response to Al in the distribution biomass (weight ratios) within the plants (Table 1). However, there was a significantly higher root weight ratio for all plants treated with Al except for the plants supplied with NO3− and 5000 ppm CO2.
Table 1. Biomass characteristics of NO3−- and NH4+-fed plants treated intermittently with 0 µM and 50 µM AlCl3 combined with either 360 or 5000 ppm CO2
360 ppm CO2
5000 ppm CO2
360 ppm CO2
5000 ppm CO2
0 µM Al
50 µM Al
0 µM Al
50 µM Al
0 µM Al
50 µM Al
0 µM Al
50 µM Al
Weight ratios express the fraction of plant dry mass comprised by the leaf, stem and root components. Specific leaf and root areas are the ratios of area to dry weight. Tomato plants were grown in hydroponics and transferred to Al solutions for 6 h every second day. Letters indicating whether the treatments had a significant effect follow the mean ± SE. Different letters indicate significant differences within each row at P < 0.05 as determined by ANOVA with post hoc LSD (N = 8).
Dry weight (g)
RGR (mg g−1 d−1)
Leaf area (m2)
Root area (m2)
Specific leaf area (m2 kg−1)
Specific root area (m2 kg−1)
The average RGR for the entire growth period of both the NO3−- and the NH4+-fed plants supplied with 360 ppm root zone CO2 was decreased by Al (Table 1). However, at 5000 ppm root zone CO2 the influence of Al on the RGR was not significant in the NO3−-fed plants while the NH4+-fed plants exhibited a 21% increase in the RGR. Although the differences in RGR between the plants treated with and without Al were larger initially than subsequently, these differences were maintained throughout the growing period (Fig. 1). The irregularity of the growth curves may be ascribed to the fact that the plants were grown in a glasshouse in which light conditions could not be controlled.
Leaf area was closely correlated with leaf and total plant dry weight and thus, although Al caused some significant changes in specific leaf area (leaf area/leaf weight), these changes were relatively small (Table 1). Elevated root zone DIC concentration led to a decrease in root area of the NO3−-fed plants in comparison with those grown with 360 ppm CO2, but had no significant effect on the root area of the NH4+-fed plants (Table 1). The addition of Al to the nutrient solution of plants supplied with 360 ppm CO2 combined with either NO3−- or NH4+-nutrition led to a three- or twofold decrease in the root area, respectively. Elevated root zone DIC concentration did not protect root area development against Al-induced decreases in the NO3−-fed plants. However, at 5000 ppm root zone CO2 Al had no significant effect on root area in the NH4+-fed plants. The specific root area (root area/root weight), a measure of the tissue density and the fineness of the roots, was smaller in NO3−-fed plants supplied with Al than in control plants at both root zone DIC concentrations (Table 1). With NH4+ nutrition the specific root area was increased by Al at 360 ppm CO2 and decreased by Al at 5000 ppm CO2.
The rate of NH4+ uptake was c. twofold greater than the rate of NO3− uptake (Fig. 2). The rate of NO3− uptake was not influenced by either root zone DIC concentration or by exposure to Al. Uptake of NH4+ in the absence of Al was 44% less in plants supplied with 5000 ppm CO2 than in plants supplied with 360 ppm CO2. Inclusion of Al did not influence NH4+ uptake at 360 ppm CO2, but NH4+ uptake was 61% increased by exposure to Al at 5000 ppm CO2. With NO3− nutrition the pH of the nutrient solution increased whereas with NH4+ nutrition the pH decreased over the duration of the uptake experiment. The change in pH (ΔpH g−1 root f. wt s−1) of the nutrient solution was correlated with the uptake of NO3− and NH4+ with smaller changes in pH at lower uptake rates. The presence of Al did not have a significant influence on the changes in pH of the nutrient solution.
Aeration with 2000 ppm CO2 increased the rate of root O2 consumption, substantially decreased CO2 release and decreased the RQ's in comparison to plants aerated with 360 ppm CO2 (Fig. 3). Al induced greater increases in O2 consumption in plants supplied with 360 ppm root zone CO2 than in those supplied with 2000 ppm root zone CO2. The increase in O2 consumption in plants supplied with 360 ppm CO2 was associated with a smaller increase in CO2 release rate, resulting in a substantial decrease in the RQ after 12 h. At 2000 ppm CO2 the CO2 release rate was small and did not change upon treatment with Al.
14C uptake and incorporation
Incorporation of DI14C into acid-stable organic products (soluble and insoluble) was significantly greater in plants supplied with 5000 ppm than in those supplied with 360 ppm CO2 (Fig. 4). There was also much greater incorporation in plants supplied with NH4+ than in those supplied with NO3−. However, Al did not influence incorporation into acid stable products. Exposure to Al resulted in a decrease in the shoot : root ratio of 14C in plants supplied with NO3− nutrition, but this was not modified by the root zone DIC concentration (Fig. 4). The proportion of 14C in the amino acid fraction was twofold larger in plants supplied with NH4+ in comparison to plants supplied with NO3− (Fig. 5). This was associated with a decrease in the proportion of 14C labelled organic acids, and to a lesser extent carbohydrates, in plants supplied with NH4+. Despite the much greater total incorporation in plants supplied with 5000 ppm CO2, there were few significant differences in 14C allocation as a result of changes in root-zone DIC concentrations. Exposure to Al caused a small decrease in the allocation of 14C to amino acid synthesis and a corresponding increase in allocation to organic acids in plants supplied with NO3− and 5000 ppm CO2.
Exudation of acid-stable 14C was significantly greater in plants supplied with NH4+ nutrition than in plants supplied with NO3− nutrition (Fig. 6). With elevated root zone DIC in plants supplied with either N form, there was significantly greater exudation of 14C, especially when combined with exposure to Al. Only a small proportion of the label exuded from roots was in the form of carbohydrates and exposure to Al had no effect on the size of this fraction (Fig. 7). A larger proportion of the exudate was in the form of amino acids from roots of plants supplied with NH4+ than in plants supplied with NO3− nutrition and Al reduced the proportion of labelled amino acids exuded in plants supplied with NH4+ nutrition (Fig. 7). There was no change in the proportion of 14C exuded as organic acid with Al in plants supplied with NO3−. However, in plants supplied with NH4+ nutrition the proportion of 14C-labelled exudates comprised of organic acids was higher in plants supplied with Al (Fig. 7). Taking into account the increased total exudation of 14CO2 derived carbon (Fig. 6), the exposure to Al caused a threefold increase in exudation of organic acids in plants supplied with both NO3− and NH4+ nutrition at elevated DIC (data not shown).
Although the influence of the Al is mainly through root apices (Ryan et al., 1995), Al influenced growth as a whole. The response of the sensitive apices of the root cannot be deduced from this study. However, the aim was to determine how root zone DIC concentrations modify Al-toxicity and what implications this may have for intact plants grown in media containing artificially low DIC concentrations, such as conventional hydroponic solutions. The concentrations of CO2 supplied to the hydroponic solutions in this investigation were selected to represent the normal hydroponic system (360 ppm, ‘ambient’ CO2) and to approximate the situation in agricultural soils (5000 ppm, ‘elevated’ CO2). The incorporation of HCO3− by PEPc requires provision of glycolytically derived PEP as a carbon acceptor; thus it is likely that the responses of isolated root apices to Al (e.g. organic acid exudation) are different to those of intact roots that have a supply of shoot-derived carbohydrates.
The negative effect of Al on growth was sustained over the growth period, although the inhibition became smaller over time indicating that the plants adapted to the Al challenge (Fig. 1). The decrease in root area due to Al was much greater than the corresponding decreases in RGR and thus the changes in root area were not simply due to a decrease in growth. Conversely, Al-induced decreases in root area may have contributed to reduced growth rates. The larger negative influence of Al on the RGR's of NH4+- than of NO3−-fed plants at 360 ppm CO2 (Table 1) could be as a result of the fact that in tomato NO3− assimilation is mainly foliar, while nutrient NH4+ assimilation is largely root-based and thus depends on energy and carbon derived from root respiration (Cramer & Lips, 1995). Since Al mainly influences the root apex, the NH4+-fed plants could have been more susceptible to Al than the NO3−-fed plants because of the need for energy and carbon for NH4+ assimilation as well as for mechanisms to protect against Al toxicity. This hypothesis is supported by the observation that growth of plants exposed to Al was increased by elevated root zone DIC by 62% and 13% in plants supplied with NH4+ and NO3−, respectively (Table 1). The reduction in RGR in plants supplied with NH4+ nutrition combined with elevated root zone DIC relative to those with ambient root zone DIC may be attributed to excessive allocation of carbon to amino acid synthesis (Fig. 5). When combined with Al, elevated CO2 stimulated growth of plants supplied with NH4+ nutrition. It is speculated that diversion of organic acid to chelation of Al reduced the excessive accumulation of amino acids.
Elevated DIC prevented Al-induced growth reduction in both NO3−- and NH4+-fed plants (Table 1). Positive effects of root zone DIC on plant growth have been reported previously (Vapaavuori & Pelkonen, 1985; Bialczyk et al., 1994), although Cramer & Lips (1995) and Cramer & Richards (1999) found that the positive effect of enriched DIC on biomass production of tomato seedlings was dependant on the presence of stress. The influence of elevated DIC on plant growth may be the consequence of increased root incorporation of DIC, influence of DIC on NO3− uptake, decreases in root respiratory CO2 flux or changes in foliar gas exchange (Cramer & Richards, 1999). As demonstrated by 14C incorporation into acid-stable ethanol soluble compounds (Fig. 4), roots assimilated exogenous DIC, but the major impact of exogenous DIC may be to reduce the gradient for respiratory DIC release from the root tissue thereby increasing root tissue DIC concentrations. As reported previously (Cramer et al., 1993), the rate of DI14C incorporation by PEPc in roots was higher in plants supplied with NH4+ than in those supplied with NO3− nutrition and the incorporated 14C was preferentially allocated to amino acid synthesis (Fig. 4). DIC incorporated by plant roots, including re-fixed respiratory CO2, was also used for synthesis of amino acids, organic acids and carbohydrates (Fig. 5) and for exudation (Fig. 6). DIC-derived organic acids may ameliorate the negative effect of Al and thus improve growth of plants exposed to Al. The influence of root DIC uptake and assimilation on plant growth may be greater than the contribution of DIC to the carbon budget of the plant because of changes in the carbon partitioning between plant tissue and carbohydrates, organic acids and amino acids (Vuorinen et al., 1992). Altered root zone DIC concentration did not, however, strongly influence the distribution of biomass as indicated by leaf, stem and root weight ratios or strongly modify the response of these characters to Al.
DIC has previously been reported to increase NO3− uptake but either decrease (Cramer et al., 1999) or not influence NH4+ uptake (Cramer et al., 1996). The lack of stimulation of NO3− uptake in this study (Fig. 2) was probably due to the fact that, unlike in previous experiments (Cramer et al., 1996), the plants were grown with continuous NO3− nutrition and the pH was low. Antunes & Nunes (1997) found a rapid (1–2 min) reduction in net NO3− uptake induced by Al in Triticale seedlings and suggested that there was a toxic effect of Al on the active transport mechanism for NO3−. However, in this investigation there was no significant effect of Al on NO3− uptake. Al has been found to stimulate or not influence NH4+-uptake (Antunes & Nunes, 1997; Calba & Jaillard, 1997). In this investigation NH4+ uptake was unchanged by exposure to Al at 360 ppm root zone CO2 and was stimulated by Al at 5000 ppm root zone CO2 (Fig. 2). This stimulation of NH4+ uptake by Al at elevated root zone DIC concentrations may be related to partial relief of feedback inhibition of NH4+ uptake by amino acids (Glass et al., 1999) due to diversion of organic acids to exudation rather than amino acid synthesis by Al.
The incorporation of DIC into organic acids through root PEPc activity could account for the decreased CO2 release rate with elevated root zone DIC concentrations, as found previously in tomato (Van der Westhuizen & Cramer, 1998). The rate of O2 consumption was increased by the addition of Al at 360 ppm CO2 while the rate of CO2 release stayed constant (3 h) or decreased (12 h) and the RQ decreased (Fig. 3). de Lima & Copeland (1994) found a decrease in respiratory O2 consumption of excised wheat root tips that was largely due to decreased cytochrome pathway activity. The discrepancy between the data of the present study and that of de Lima & Copeland (1994) may be due to the use of intact roots in this study vs excised root tips. However, genotypic differences may also be important since Cumming et al. (1992) reported inhibition of respiration of intact roots by Al in ‘Al-sensitive’ and stimulation in ‘Al-tolerant’ wheat.
The decreased RQ in the presence of Al (Fig. 3) may indicate that CO2 was re-assimilated more rapidly for organic acid synthesis resulting in rapid flux of carbon through components of the TCA cycle. This could also explain the increased O2 consumption, since more NADH would be produced through increased flux of carbon through the TCA cycle for synthesis of organic acids such as malate and citrate, which have been shown to be exuded to meet the challenge of Al (Ownby & Popham, 1989; Zheng et al., 1998a). The RQ for the complete glycolytic and TCA cycle oxidation of hexose to CO2 and H2O is 1, irrespective whether pyruvate or malate derived from PEP carboxylation enters the TCA cycle (Lambers, 1997). However, if malate synthesized from carboxylation of PEP is excreted or used by plants to complex Al, then the following equation applies: Glucose + OH− + 1.5O2 + 3ADP + 3Pi → 3H2O + 3ATP + Malate + 2CO2, resulting in an RQ of 0.67 for this partial component of respiratory metabolism. Furthermore, if citrate derived from anaplerotic provision of organic acids to the TCA cycle is excreted or complexed with Al then the following equation applies: Glucose + OH−+ 1.5O2 + 2ADP + 2Pi → 3H2O + 2ATP + Citrate, resulting in an RQ of 0 for this component of respiratory metabolism. Thus the synthesis and utilization of malate or citrate to complex Al would be expected to result in a reduction in RQ. At elevated DIC concentrations the increased incorporation of HCO3− into organic acids resulted in low RQ's and exposure to Al did not appear to result in a further reduction in the RQ.
Organic acids have previously been shown to play an important role in the tolerance of plants to Al either internally or in the root zone (Delhaize & Ryan, 1995; Ginting et al., 1998; Ma et al., 1998; Zheng et al., 1998a; Zheng et al., 1998b). There were, however, only small Al-induced changes in the allocation of 14C to soluble products in NO3−-fed plants and no Al-induced changes were observed in plants supplied with NH4+ (Fig. 5). Exudation of acid-stable organic 14C was shown previously to be increased by elevated root zone DIC concentrations (Cramer & Van der Westhuizen, 2000). This result was confirmed in the present investigation and it was further found that Al significantly increased exudation of organic 14C, particularly of [14C]organic acids by roots supplied with elevated DIC (Fig. 6). The lower shoot : root ratios for the distribution of 14C in NO3−-fed plants supplied with Al than in control plants (Fig. 4) are probably due to utilization of [14C]-organic aids for chelation of Al. Thus production of organic acids for tolerance to Al is partially dependent on the incorporation of DIC by PEPc activity that supplies anaplerotic carbon to maintain the TCA cycle activity and synthesis of organic acids for excretion.
It was concluded that the effects of Al on plant growth can be partially ameliorated by elevated DIC. The effect of the DIC is dependent on the form N and is possibly the result of DIC providing carbon for synthesis of organic acids to alleviate Al toxicity. Artificially low root zone DIC concentrations in many hydroponic culture systems may thus limit the usefulness of hydroponic experiments for analysis of Al tolerance.
Funding from the National Research Foundation is acknowledged. We thank A. Viktor, C. Ward and M. February for technical assistance.