- Top of page
- Materials and Methods
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.
Al exists in the soil mostly as nonphytotoxic complexes such as aluminosillicates and precipitates, but solubilization of Al is enhanced at low pH. Amongst the earliest symptoms of Al toxicity is the rapid inhibition of root growth (Delhaize & Ryan, 1995; Llugany et al., 1995). Al toxicity involves both extra- and intracellular mechanisms. Extra-cellular binding of Al to cation exchange sites in the cell wall and displacement of other cations (Godbold & Jentschke, 1998) can lead to deficiencies of cations such as Mg2+, Ca2+, Zn2+, and Mn2+ and may block Ca2+ channels at the plasma membrane (Jones et al., 1998). Intra-cellularly Al complexes cytosolic P (Crawford et al., 1998), inhibits the biosynthesis and secretion of Fe-phytosiderophores in wheat (Chang et al., 1998), damages the plasma membrane (Wagatsuma et al., 1995; Jones & Kochian, 1997), damages components of the symplasm (Kochian, 1995; Bennet, 1995) and inhibits respiration (de Lima & Copeland, 1994).
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.
- Top of page
- Materials and Methods
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.