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Excess cation uptake, and extrusion of protons and organic acid anions by Lupinus albus under phosphorus deficiency
Plant Science 160 (2001) 1191– 1198 www.elsevier.com/locate/plantsci
Excess cation uptake, and extrusion of protons and organic acid anions by Lupinus albus under phosphorus deficiency L. Sas a,b, Z. Rengel a,*, C. Tang a a
Soil Science and Plant Nutrition, The Uni6ersity of Western Australia, 35 Stirling Hwy, Crawley WA6009, Australia b Research Institute of Pomology and Floriculture, Skierniewice, Poland Received 26 July 2000; received in revised form 2 February 2001; accepted 6 February 2001
Abstract In symbiotically-grown legumes, rhizosphere acidification may be caused by a high cation/anion uptake ratio and the excretion of organic acids, the relative importance of the two processes depending on the phosphorus nutritional status of the plants. The present study examined the effect of P deficiency on extrusions of H+ and organic acid anions (OA−) in relation to uptake of excess cations in N2-fixing white lupin (cv. Kiev Mutant). Plants were grown for 49 days in nutrient solutions treated with 1, 5 or 25 mmol P m − 3 Na2HPO4 in a phytotron room. The increased formation of cluster roots occurred prior to a decrease in plant growth in response to P deficiency. The number of cluster roots was negatively correlated with tissue P concentrations below 2.0 g kg − 1 in shoots and 3 g kg − 1 in roots. Cluster roots generally had higher concentrations of Mg, Ca, N, Cu, Fe, and Mn but lower concentrations of K than non-cluster roots. Extrusion of protons and OA− (90% citrate and 10% malate) from roots was highly dependent on P supply. The amounts of H+ extruded per unit root biomass decreased with time during the experiment. On the equimolar basis, H+ extrusion by P-deficient plants (grown at 1 and 5 mmol P m − 3) were, on average, 2 – 3-fold greater than OA− exudation. The excess cation content in plants was generally the highest at 1 mmol P m − 3 and decreased with increasing P supply. The ratio of H+ release to excess cation uptake increased with decreasing P supply. The results suggest that increased exudation of OA− due to P deficiency is associated with H+ extrusion but contributes only a part of total acidification. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Citrate exudation; Cluster roots; Excess cations; H+ extrusion; Malate exudation; P deficiency; White lupin
1. Introduction White lupin (Lupinus albus L.) has relatively thick, coarse, lateral roots, does not form extensive mycorrhizal associations [1,2] and develops cluster roots densely packed with root hairs. The formation of cluster roots can be enhanced in response to P deficiency [2–6] and to a lesser extent to N deficiency and the N form [7,8], as well as Mn and Fe deficiency [2,3,9]. The formation of cluster roots is regulated by plant endogenous P status of the plant [3,10 –13] and phytohormones [14]. The proportion of cluster roots of the total
* Corresponding author. E-mail address: [email protected] (Z. Rengel).
root biomass generally increases with age of juvenile plants and duration of P starvation, and may reach over 60% [12]. However, the critical concentration of P in plants that is required for cluster root formation has not yet been investigated. The release of large amounts of organic acid anions (OA−) from cluster roots of P-deficient white lupin is an efficient strategy for chemical mobilisation of sparingly-soluble P sources [5]. Gerke et al. [15] found that the concentration of soluble phosphate in the rhizosphere of cluster roots of white lupin grown in two oxisols was twice that in the bulk soil despite depletion via P uptake by the plant; such finding was attributed to citrate exudation by cluster roots. The ability of cluster roots of white lupin to exude large quantities of OA− has been reported
0168-9452/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 1 ) 0 0 3 7 3 - 9
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frequently [5,11,12,16,17]. There are significant variations in the rate of organic acid anion exudation between cluster and non-cluster roots as well as along the axis of cluster roots [5]. Keerthisinghe et al. [12] demonstrated that only the youngest segment (1–3 cm) of the cluster root has significantly higher citrate efflux than the rest of the cluster root or the whole of noncluster roots. In the study by Neumann et al. [5], the rate of citric acid excretion from mature cluster roots was approximately 20-fold higher than from non-cluster roots. The mechanism of citrate exudation from cluster roots has been studied previously [12]. Since P deficiency in white lupin [5,18] and citrate excretion from cluster roots coincide with rhizosphere acidification [3,10,18], it has been suggested that citrate anions may be excreted via an anion channel with a concomitant release of protons to maintain charge balance [4,5,18]. However, the relationship between citrate exudation and proton release has not been elucidated. In this paper, we report the effect of phosphate supply on extrusion of H+ and OA− from roots of N2-fixing white lupin grown in a hydroponic culture system. Special emphasis was put on the relationship between H+ release, OA− exudation, and uptake of excess cations by plants. We also established the relationship between P supply, tissue P concentration and formation of cluster roots.
2. Methods
2.1. Plant culti6ation The experiment was conducted in a phytotron room with natural light and temperature of 20/ 12°C (day/night). Seeds of white lupin (Lupinus albus L. cv. Kiev Mutant) were germinated on a stainless-steel mesh in a 15-l plastic container containing aerated solution of 1 mM CaCl2 and 5 mM H3BO3. The plants were inoculated with Bradyrhizobium sp. (Lupinus) WU 425 during the germination stage and the first week after transplanting. After germination stage, 21 5-day-old seedlings were transferred into 5.5-l plastic pots containing nutrient solution to facilitate frequent and exact determination of H+ and organic acid
anions. The nutrient solution had the following composition (mmol P m − 3): 600 K2SO4, 200 MgSO4, 600 CaCl2, 10 FeEDTA, 0.2 CoSO4, 0.03 Na2MoO4, 5 H3BO3, 0.75 ZnSO4, 1 MnSO4 and 0.2 CuSO4. To avoid complications in the relationship between the release of acidifying agents and the excess cation uptake, the plants were reliant only on N2 fixation. Solution pH was adjusted daily to pH 6.0 using 0.1 M KOH. The volume of KOH used was recorded for later calculations of proton extrusion. Nutrient solutions were aerated continuously and were renewed every second day.
2.2. P treatments Upon transplanting, the treatments of 1, 5 or 25 mmol P m − 3 as Na2HPO4 were imposed. The concentration of P in nutrient solutions was determined using the spectrophotometric malachite/ green-molybdate method [19], and adjusted daily to the starting levels. Each treatment contained four replicates. Additional four pots containing nutrient solutions of each treatment without plants were included as the control.
2.3. Collection of root exudates Root exudates were collected weekly from the 2nd to 7th week of treatment. Roots remained intact and undamaged during collection of root exudates. Before each collection, the roots were rinsed three times with deionised water and blotted on the filter paper. The root system was then immersed for 2 h in 250 ml of aerated nutrient solution of the same composition as the solution for growing plants. The pH was monitored continuously and maintained between 5.5 and 6.0 during the collection of root exudates. Each time root exudates were collected, recovery experiments were carried out by additions of known concentrations of citrate to the root exudates. The recovery experiments indicated that 13–23% of citric acid released from the roots of white lupin plants could have been broken down and/or was taken up by the roots during the collection period of 2 h. After 2 h, the decomposition rate of citrate was higher. Therefore, we used the collection time of 2 h to minimise OA− biodegradation.
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2.4. Plant har6ests After each root exudate collection, three plants from each pot were harvested and the number of cluster roots was counted. Cluster roots were defined as those portions of secondary lateral roots bearing bottle brush-like clusters with a density of ten or more rootlets per cm [11]. Roots and shoots were separated and dried at 70°C. Shoot and root weights were recorded. The dried roots and shoots were used for determination of mineral composition.
2.5. Measurements 2.5.1. Root morphology At the final harvest (day 49), total length and diameter of roots, and the number of root tips (including cluster rootlets) were determined separately for cluster and non-cluster roots. These measurements were carried out using a Hewlett Packard’s scanner (HP ScanJet 6100C), controlled by WinRhizo software (Regent Instruments Inc., Quebec, Canada) [20]. Root surface area and volume were calculated from root length and diameter by computerised analysis. 2.5.2. Organic acids The 10-ml samples of exudate nutrient solutions were filtered through sterile Millex-GS Millipore 0.22-mm filters and directly analysed for organic acids using HPLC. Separation was conducted on a reversed phase HPLC column (Alltima C18 5 Micron, length 250 mm, i.d. 4.6 mm). Aliquots of 20 –40 ml were injected (Autosampler, Waters, USA). Detection of organic acid anions was carried out at 215 nm using a multiple wavelength spectrophotometer (UV–VIS, Waters/Millipore, USA). The solution of 0.125 M (NH4)2HPO4, adjusted to pH 2.5 with H3PO4, was used for isocratic elution with a flow rate of 1 ml min − 1 at 28°C. Standards were prepared for the following organic acid anions: citrate, L-malate, oxalate, tartarate, lactate, malonate, maleate, formate and succinate. Calibration curves were constructed for the quantification of the identified compounds present in the samples. Identification of organic acids was performed by comparing retention times and absorption spectra with those of known standards. Data acquisition and integration of chromatograms were performed using the Millennium software (1997, Waters, USA).
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2.5.3. Mineral composition in plant Concentrations of P, Ca, Mg, K, S, Na, N, Cl, Zn, Fe, Mn and Cu were determined in whole roots and shoots harvested between days 7 and 42, and in shoots, cluster roots, and non-cluster roots at the final harvest. Total nitrogen was analysed using a LECO 1000 CHN analyser. Other elements were determined using a Philips PW1400 XRF spectrometer [21]. Twelve ASPAC (Australian Soil and Plant Analysis Council) plant reference materials were used as standards, with cellulose as the blank. The calibration was verified with ASPAC white lupin reference material. The concentration of excess cations [cmol (+ ) kg − 1] was calculated from individual elements as the sum of charge concentration of K+, Ca2 + , Mg2 + 2− and Na+ minus the sum of H2PO− and 4 , SO4 − Cl . 2.5.4. H+ excretion The amount of H+ excreted by roots was calculated according to the formula, H=OHP +OHT, where OHP was the amount of OH− added to adjust the pH during plant growth, and OHT was the amount of OH− used for titrating final solutions (after growing plants) to the pH (6.0) of the no-plant control solution. Because solutions were continuously aerated, the contribution of root respiration to solution acidification was negligible. Total H+ production expressed in mmol h − 1 per plant was the average of the H+ release during 1-week period. Specific H+ (expressed in mmol g − 1 dry root weight h − 1) was calculated from the total amount of H+ extruded during the week divided by the increment in plant biomass obtained during the same period. 3. Results
3.1. Plant growth and cluster root formation The effect of low P supply on shoot dry weight was evident from day 35 of treatment and became more pronounced with time. Dry shoot weights at 1 and 5 mmol P m − 3 were 66–79 and 82– 87%, respectively, of those at 25 mmol P m − 3. The effect of P on root dry weight followed the same pattern as shoots, though less pronounced. The number of nodules, leaf number and flowering time were not significantly affected by P treatment (data not shown).
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area and volume (data not shown). However, the reverse relationship was observed in the plants grown with 25 mmol P m − 3.
3.2. Element concentrations in plant
Fig. 1. Concentrations of P in shoots (A) and roots (cluster plus non-cluster) of Lupinus albus grown at 1, 5 or 25 mmol P m − 3 for up to 49 days. Vertical bars represent means 9 S.E. (n=3), where larger than the symbol.
Plants initiated cluster roots at all P levels between days 10 and 17 of treatment. From day 21, the influence of P supply on cluster root formation was evident. The number of cluster roots was higher at 1 and 5 mmol P m − 3 than at 25 mmol P m − 3, and increased with time, the increase being greater at 1 than at 5 mmol P m − 3 (data not shown). At day 49, cluster roots of the plants grown with 1 and 5 mmol P m − 3 had significantly higher total length (cluster to non-cluster root ratio of 2.0), and a larger number of root tips (cluster to non-cluster root ratios of 2.4–3.4) in comparison with non-cluster roots. The same relationship was obtained for root weight, surface
Increasing P supply clearly increased P concentration in shoots and roots (Fig. 1). Phosphorus concentration decreased with time in plants grown at all P treatments; except that P concentration in roots at 25 mmol P m − 3 increased from day 21. Irrespective of growth stage and P treatment, the number of cluster roots correlated negatively with P concentration below 2 g kg − 1 in shoots and 3 g kg − 1 in roots (Fig. 2). By day 49, P concentrations were similar in cluster and non-cluster roots at all P supplies (data not shown). However, cluster roots had significantly higher Mg, Ca, N, Fe and Mn concentrations but lower K concentration than non-cluster roots (Fig. 3).
3.3. Root exudation In most cases, only citrate and malate were detected in root exudates. Trace amounts of succinate were detected on two occasions. There were no other OA− found in the measured samples. Citrate was the dominant OA− comprising 90% of total production regardless of P supply. Average production of malate was about 10% of total. The amounts of OA− released per unit root biomass were highest at 1 mmol P m − 3 P and lowest at 25 mmol P m − 3 from day 27 (Fig. 4). During days 41–48, the release of citrate and malate at the lowest P supply was about 3- and 14-fold than that obtained at 5 and 25 mmol P
Fig. 2. Relationship between concentrations of P in shoots and roots and number of cluster roots of Lupinus albus grown at various P levels. The lines are fitted with the Mitcherlich model. Individual points represent means of three replicates.
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Fig. 5. Specific extrusions of protons and organic acid anions (citrate plus malate) per 1000 root tips, by Lupinus albus at day 48 of treatment with 1, 5 or 25 mmol P m − 3. Bars represent means 9 S.E. (n= 4). Fig. 3. Concentrations of K, Mg, Ca, N, S, Cu, Fe and Mn in cluster and non-cluster roots of Lupinus albus measured after 49 days of treatment with 1, 5 or 25 mmol P m − 3. Vertical bars represent means 9S.E. (n=3).
m − 3, respectively. The amounts of OA− exuded generally increased with time (Fig. 4). The amount of OA− extruded per 1000 root tips measured at day 48 was four times higher at 1 mmol P m − 3 than at 5 mmol P m − 3, which, in turn, was four times higher than at 25 mmol P m − 3 (Fig. 5). From day 27, proton extrusion from roots was highly dependent on P supply, being 7-fold higher
at 1 mmol P m − 3 than at 25 mmol P m − 3 at days 41 and 48 (Fig. 4). The amount of H+ produced per unit root biomass decreased with time (Fig. 4). The amount of protons extruded per 1000 root tips at 1 mmol P m − 3 measured at day 48 was doubled as P supply increased to 5 mmol P m − 3, and was 25 times higher than at 25 mmol P m − 3 (Fig. 5). The amounts of both OA− and H+ released per plant were higher at 1 and 5 mmol P m − 3 than at 25 mmol P m − 3 at day 34, and increased with time. However, the amounts of H+ extruded by
Fig. 4. Changes in specific exudation of organic acid anions (citrate plus malate) and protons by Lupinus albus grown at 1, 5 or 25 mmol P m − 3 in nutrient solution during the experimental period. Vertical bars represent means 9 S.E. (n= 4).
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3.4. Excess cation uptake
Fig. 6. Concentrations of excess cations in shoots and roots of Lupinus albus grown at 1, 5 or 25 mmol P m − 3 during 49 days. Vertical bars represent means 9 S.E. (n=3), where larger than the symbol.
The plants grown at 1 mmol P m − 3 generally had higher concentrations of Ca and Cl and lower concentrations of K, S, Zn, Fe and Cu in roots compared with those at higher P supply. Phosphorus supply did not clearly affect concentrations of Mg, Mn and N in roots nor concentrations of K, Ca, Mg, Cl, Mn, Zn, Fe, Cu and N in shoots (data not shown). Decreasing P supply generally increased concentrations of excess cations in shoots from day 35 and in roots from day 14. The concentrations also increased with plant age up to day 28 for shoots and day 21 for roots, and then remained unchanged in roots but decreased in shoots (Fig. 6). Total uptake of excess cations was correlated with total acid production in all P treatments but the slope of the regression decreased from 0.64 at 25 to 0.17 at 1 mmol P m − 3 (Fig. 7). In other words, the ratio of H+ release to excess cation uptake decreased with increased P supply.
4. Discussion
P-deficient plants were, on average, 2– 3-fold greater than OA− exudation on an equal molar basis between days 27 and 41. Under P deficiency, the amounts of OA− exuded per plant correlated with the amounts of H+ released with a slope of the regression of 0.76 (Fig. 7).
Our data clearly proved that a continuous low P supply increased cluster root formation by white lupin plants. The increased number of cluster roots by P deficiency occurred prior to the effect on shoot growth (day 21 vs. day 35), suggesting that cluster root formation under P deficiency is not mediated by shoot growth. Similarly,
Fig. 7. The relationships between exudation of organic acid anions and H+ release (A), and between uptake of excess cations and H+ extrusion (B) by Lupinus albus grown at 1, 5 or 25 mmol P m − 3. Lines are fitted with the linear model. In (A), data at 25 mmol P m − 3 were excluded from analysis. In (B), the slopes (b) of the regression lines are indicated. Each point represents means of four replications.
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Keerthisinghe et al. [12] found that the formation of cluster roots was not related to plant dry matter yields or shoot-to-root dry matter ratios. Further, this study found that number of cluster roots was correlated inversely with P concentrations in plants, which was perceived before growth was limited. From this finding, it may be concluded that the formation of cluster roots is mediated by internal P status in plants, in accordance with other studies [3,22]. Cluster roots play an important role in the acquisition of not only P but also of other mineral nutrients [9]. The present study provided evidence that cluster roots contained higher concentrations of Mg, Ca, N, Fe and Mn, indicating higher efficiency in absorption of these ions by cluster roots than non-cluster roots. This might be caused by enhanced uptake rate of these elements by cluster roots, higher metabolic activity of these root structures [5,13], their larger surface area [9], or presumably decreased translocation of specific ions into the shoot. Potassium concentration in roots was significantly lower at 1 mmol P m − 3 than at 25 mmol P m − 3 (Fig. 6), although the K concentration in shoots was not affected by P supply (data not shown). This decreased K concentration in roots under P deficiency mainly resulted from the higher proportion of cluster roots at low P supply and the lower K concentration in cluster than noncluster roots. The results indicate that either a higher proportion of absorbed K was effluxed from the cluster roots than non-cluster roots or that K was not taken up to the same extent by the two root types. Since cluster roots of white lupin are the main site for release of H+ and organic anions under P deficiency [5,12], it is speculated that the release of organic anions is accompanied with K+ efflux (co-transport), which may also be the case in Banksia species (unpublished data). This deserves further investigation. Extrusions of both protons and OA− were highly dependent on P supply and on the stage of plant growth, but the release of H+ per unit root biomass displayed different patterns from the OA− (Fig. 4). While the increased exudation of OA− with time had resulted from the increased severity of P deficiency, the decreased release of H+ per unit roots over the experimental period might be due to a decrease in excess cation uptake. This suggests that different mechanisms are involved in proton release and OA− exudation.
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The amount of H+ extruded was always higher than the sum of OA− exuded, regardless of whether they were expressed per unit of root tips or root dry weight (Figs. 4 and 5), surface area or length (data not shown). Thus, the total acid (H+) production appeared to consist of the H+ release (and K+ efflux) associated with OA− exudation [4,5,18] and the H+ release resulting from excess cation uptake [23,24]. Under P deficiency in this study, about half of total acid (H+) extrusion during the experimental period was not accounted for from the uptake of excess cations, and was probably attributed to OA− extrusion. Plant roots extrude net H+ when cation uptake exceeds anion uptake and extrude net OH− when anion uptake exceeds cation uptake. Several studies have shown that plants can acidify soils directly by releasing protons from the roots when an excess cation over anion uptake occurs [25–28]. Legumes, reliant on N2 fixation, generally take up more cations than anions and thus extrude into the rhizosphere proportionally more H+ than OH− to balance the charges [24]. The amount of H+ extruded by legumes reliant solely on N2 fixation can be calculated from the mineral composition of the plant (non-N excess cations). Thus, Tang et al. [24] established a relationship between H+ extrusion and excess cation uptake in legume species grown in nutrient solution. In the present study, the plants were reliant on N2 fixation as the only N source. Therefore, H+ extrusion should be related to excess cation uptake. Indeed, there was a good relationship between root acid exudation and excess cation content in the plants at given P levels. However, the regression lines were all below the 1:1 line and the slope values of the lines decreased with decreasing supply of P. In other words, the ratio of H+ extrusion to excess cation uptake increased when P supply was decreased. This increased ratio correlated well with the increased OA− exudation. These results support the alternative that the exudation of OA− under P deficiency is partly accompanied by H+ efflux. Furthermore, in previous studies on root exudates under P deficiency, plants had been supplied with nitrate. White lupin plants supplied with nitrate released similar quantities of citrate [5,12], as the N2-fixing plants in our experiment. Proton release could be expected to be lower in the case of NO− 3 -fed plants as compared with N2-fixing plants. However, there is no conclusive evidence in the literature on it, and further study is warranted.
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Acknowledgements We thank Dr Z.L. Chen for valuable advice on organic acid measurements.
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Excess cation uptake, and extrusion of protons and organic acid anions by Lupinus albus under phosphorus deficiency L. Sas a,b, Z. Rengel a,*, C. Tang a a
Soil Science and Plant Nutrition, The Uni6ersity of Western Australia, 35 Stirling Hwy, Crawley WA6009, Australia b Research Institute of Pomology and Floriculture, Skierniewice, Poland Received 26 July 2000; received in revised form 2 February 2001; accepted 6 February 2001
Abstract In symbiotically-grown legumes, rhizosphere acidification may be caused by a high cation/anion uptake ratio and the excretion of organic acids, the relative importance of the two processes depending on the phosphorus nutritional status of the plants. The present study examined the effect of P deficiency on extrusions of H+ and organic acid anions (OA−) in relation to uptake of excess cations in N2-fixing white lupin (cv. Kiev Mutant). Plants were grown for 49 days in nutrient solutions treated with 1, 5 or 25 mmol P m − 3 Na2HPO4 in a phytotron room. The increased formation of cluster roots occurred prior to a decrease in plant growth in response to P deficiency. The number of cluster roots was negatively correlated with tissue P concentrations below 2.0 g kg − 1 in shoots and 3 g kg − 1 in roots. Cluster roots generally had higher concentrations of Mg, Ca, N, Cu, Fe, and Mn but lower concentrations of K than non-cluster roots. Extrusion of protons and OA− (90% citrate and 10% malate) from roots was highly dependent on P supply. The amounts of H+ extruded per unit root biomass decreased with time during the experiment. On the equimolar basis, H+ extrusion by P-deficient plants (grown at 1 and 5 mmol P m − 3) were, on average, 2 – 3-fold greater than OA− exudation. The excess cation content in plants was generally the highest at 1 mmol P m − 3 and decreased with increasing P supply. The ratio of H+ release to excess cation uptake increased with decreasing P supply. The results suggest that increased exudation of OA− due to P deficiency is associated with H+ extrusion but contributes only a part of total acidification. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Citrate exudation; Cluster roots; Excess cations; H+ extrusion; Malate exudation; P deficiency; White lupin
1. Introduction White lupin (Lupinus albus L.) has relatively thick, coarse, lateral roots, does not form extensive mycorrhizal associations [1,2] and develops cluster roots densely packed with root hairs. The formation of cluster roots can be enhanced in response to P deficiency [2–6] and to a lesser extent to N deficiency and the N form [7,8], as well as Mn and Fe deficiency [2,3,9]. The formation of cluster roots is regulated by plant endogenous P status of the plant [3,10 –13] and phytohormones [14]. The proportion of cluster roots of the total
* Corresponding author. E-mail address: [email protected] (Z. Rengel).
root biomass generally increases with age of juvenile plants and duration of P starvation, and may reach over 60% [12]. However, the critical concentration of P in plants that is required for cluster root formation has not yet been investigated. The release of large amounts of organic acid anions (OA−) from cluster roots of P-deficient white lupin is an efficient strategy for chemical mobilisation of sparingly-soluble P sources [5]. Gerke et al. [15] found that the concentration of soluble phosphate in the rhizosphere of cluster roots of white lupin grown in two oxisols was twice that in the bulk soil despite depletion via P uptake by the plant; such finding was attributed to citrate exudation by cluster roots. The ability of cluster roots of white lupin to exude large quantities of OA− has been reported
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frequently [5,11,12,16,17]. There are significant variations in the rate of organic acid anion exudation between cluster and non-cluster roots as well as along the axis of cluster roots [5]. Keerthisinghe et al. [12] demonstrated that only the youngest segment (1–3 cm) of the cluster root has significantly higher citrate efflux than the rest of the cluster root or the whole of noncluster roots. In the study by Neumann et al. [5], the rate of citric acid excretion from mature cluster roots was approximately 20-fold higher than from non-cluster roots. The mechanism of citrate exudation from cluster roots has been studied previously [12]. Since P deficiency in white lupin [5,18] and citrate excretion from cluster roots coincide with rhizosphere acidification [3,10,18], it has been suggested that citrate anions may be excreted via an anion channel with a concomitant release of protons to maintain charge balance [4,5,18]. However, the relationship between citrate exudation and proton release has not been elucidated. In this paper, we report the effect of phosphate supply on extrusion of H+ and OA− from roots of N2-fixing white lupin grown in a hydroponic culture system. Special emphasis was put on the relationship between H+ release, OA− exudation, and uptake of excess cations by plants. We also established the relationship between P supply, tissue P concentration and formation of cluster roots.
2. Methods
2.1. Plant culti6ation The experiment was conducted in a phytotron room with natural light and temperature of 20/ 12°C (day/night). Seeds of white lupin (Lupinus albus L. cv. Kiev Mutant) were germinated on a stainless-steel mesh in a 15-l plastic container containing aerated solution of 1 mM CaCl2 and 5 mM H3BO3. The plants were inoculated with Bradyrhizobium sp. (Lupinus) WU 425 during the germination stage and the first week after transplanting. After germination stage, 21 5-day-old seedlings were transferred into 5.5-l plastic pots containing nutrient solution to facilitate frequent and exact determination of H+ and organic acid
anions. The nutrient solution had the following composition (mmol P m − 3): 600 K2SO4, 200 MgSO4, 600 CaCl2, 10 FeEDTA, 0.2 CoSO4, 0.03 Na2MoO4, 5 H3BO3, 0.75 ZnSO4, 1 MnSO4 and 0.2 CuSO4. To avoid complications in the relationship between the release of acidifying agents and the excess cation uptake, the plants were reliant only on N2 fixation. Solution pH was adjusted daily to pH 6.0 using 0.1 M KOH. The volume of KOH used was recorded for later calculations of proton extrusion. Nutrient solutions were aerated continuously and were renewed every second day.
2.2. P treatments Upon transplanting, the treatments of 1, 5 or 25 mmol P m − 3 as Na2HPO4 were imposed. The concentration of P in nutrient solutions was determined using the spectrophotometric malachite/ green-molybdate method [19], and adjusted daily to the starting levels. Each treatment contained four replicates. Additional four pots containing nutrient solutions of each treatment without plants were included as the control.
2.3. Collection of root exudates Root exudates were collected weekly from the 2nd to 7th week of treatment. Roots remained intact and undamaged during collection of root exudates. Before each collection, the roots were rinsed three times with deionised water and blotted on the filter paper. The root system was then immersed for 2 h in 250 ml of aerated nutrient solution of the same composition as the solution for growing plants. The pH was monitored continuously and maintained between 5.5 and 6.0 during the collection of root exudates. Each time root exudates were collected, recovery experiments were carried out by additions of known concentrations of citrate to the root exudates. The recovery experiments indicated that 13–23% of citric acid released from the roots of white lupin plants could have been broken down and/or was taken up by the roots during the collection period of 2 h. After 2 h, the decomposition rate of citrate was higher. Therefore, we used the collection time of 2 h to minimise OA− biodegradation.
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2.4. Plant har6ests After each root exudate collection, three plants from each pot were harvested and the number of cluster roots was counted. Cluster roots were defined as those portions of secondary lateral roots bearing bottle brush-like clusters with a density of ten or more rootlets per cm [11]. Roots and shoots were separated and dried at 70°C. Shoot and root weights were recorded. The dried roots and shoots were used for determination of mineral composition.
2.5. Measurements 2.5.1. Root morphology At the final harvest (day 49), total length and diameter of roots, and the number of root tips (including cluster rootlets) were determined separately for cluster and non-cluster roots. These measurements were carried out using a Hewlett Packard’s scanner (HP ScanJet 6100C), controlled by WinRhizo software (Regent Instruments Inc., Quebec, Canada) [20]. Root surface area and volume were calculated from root length and diameter by computerised analysis. 2.5.2. Organic acids The 10-ml samples of exudate nutrient solutions were filtered through sterile Millex-GS Millipore 0.22-mm filters and directly analysed for organic acids using HPLC. Separation was conducted on a reversed phase HPLC column (Alltima C18 5 Micron, length 250 mm, i.d. 4.6 mm). Aliquots of 20 –40 ml were injected (Autosampler, Waters, USA). Detection of organic acid anions was carried out at 215 nm using a multiple wavelength spectrophotometer (UV–VIS, Waters/Millipore, USA). The solution of 0.125 M (NH4)2HPO4, adjusted to pH 2.5 with H3PO4, was used for isocratic elution with a flow rate of 1 ml min − 1 at 28°C. Standards were prepared for the following organic acid anions: citrate, L-malate, oxalate, tartarate, lactate, malonate, maleate, formate and succinate. Calibration curves were constructed for the quantification of the identified compounds present in the samples. Identification of organic acids was performed by comparing retention times and absorption spectra with those of known standards. Data acquisition and integration of chromatograms were performed using the Millennium software (1997, Waters, USA).
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2.5.3. Mineral composition in plant Concentrations of P, Ca, Mg, K, S, Na, N, Cl, Zn, Fe, Mn and Cu were determined in whole roots and shoots harvested between days 7 and 42, and in shoots, cluster roots, and non-cluster roots at the final harvest. Total nitrogen was analysed using a LECO 1000 CHN analyser. Other elements were determined using a Philips PW1400 XRF spectrometer [21]. Twelve ASPAC (Australian Soil and Plant Analysis Council) plant reference materials were used as standards, with cellulose as the blank. The calibration was verified with ASPAC white lupin reference material. The concentration of excess cations [cmol (+ ) kg − 1] was calculated from individual elements as the sum of charge concentration of K+, Ca2 + , Mg2 + 2− and Na+ minus the sum of H2PO− and 4 , SO4 − Cl . 2.5.4. H+ excretion The amount of H+ excreted by roots was calculated according to the formula, H=OHP +OHT, where OHP was the amount of OH− added to adjust the pH during plant growth, and OHT was the amount of OH− used for titrating final solutions (after growing plants) to the pH (6.0) of the no-plant control solution. Because solutions were continuously aerated, the contribution of root respiration to solution acidification was negligible. Total H+ production expressed in mmol h − 1 per plant was the average of the H+ release during 1-week period. Specific H+ (expressed in mmol g − 1 dry root weight h − 1) was calculated from the total amount of H+ extruded during the week divided by the increment in plant biomass obtained during the same period. 3. Results
3.1. Plant growth and cluster root formation The effect of low P supply on shoot dry weight was evident from day 35 of treatment and became more pronounced with time. Dry shoot weights at 1 and 5 mmol P m − 3 were 66–79 and 82– 87%, respectively, of those at 25 mmol P m − 3. The effect of P on root dry weight followed the same pattern as shoots, though less pronounced. The number of nodules, leaf number and flowering time were not significantly affected by P treatment (data not shown).
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area and volume (data not shown). However, the reverse relationship was observed in the plants grown with 25 mmol P m − 3.
3.2. Element concentrations in plant
Fig. 1. Concentrations of P in shoots (A) and roots (cluster plus non-cluster) of Lupinus albus grown at 1, 5 or 25 mmol P m − 3 for up to 49 days. Vertical bars represent means 9 S.E. (n=3), where larger than the symbol.
Plants initiated cluster roots at all P levels between days 10 and 17 of treatment. From day 21, the influence of P supply on cluster root formation was evident. The number of cluster roots was higher at 1 and 5 mmol P m − 3 than at 25 mmol P m − 3, and increased with time, the increase being greater at 1 than at 5 mmol P m − 3 (data not shown). At day 49, cluster roots of the plants grown with 1 and 5 mmol P m − 3 had significantly higher total length (cluster to non-cluster root ratio of 2.0), and a larger number of root tips (cluster to non-cluster root ratios of 2.4–3.4) in comparison with non-cluster roots. The same relationship was obtained for root weight, surface
Increasing P supply clearly increased P concentration in shoots and roots (Fig. 1). Phosphorus concentration decreased with time in plants grown at all P treatments; except that P concentration in roots at 25 mmol P m − 3 increased from day 21. Irrespective of growth stage and P treatment, the number of cluster roots correlated negatively with P concentration below 2 g kg − 1 in shoots and 3 g kg − 1 in roots (Fig. 2). By day 49, P concentrations were similar in cluster and non-cluster roots at all P supplies (data not shown). However, cluster roots had significantly higher Mg, Ca, N, Fe and Mn concentrations but lower K concentration than non-cluster roots (Fig. 3).
3.3. Root exudation In most cases, only citrate and malate were detected in root exudates. Trace amounts of succinate were detected on two occasions. There were no other OA− found in the measured samples. Citrate was the dominant OA− comprising 90% of total production regardless of P supply. Average production of malate was about 10% of total. The amounts of OA− released per unit root biomass were highest at 1 mmol P m − 3 P and lowest at 25 mmol P m − 3 from day 27 (Fig. 4). During days 41–48, the release of citrate and malate at the lowest P supply was about 3- and 14-fold than that obtained at 5 and 25 mmol P
Fig. 2. Relationship between concentrations of P in shoots and roots and number of cluster roots of Lupinus albus grown at various P levels. The lines are fitted with the Mitcherlich model. Individual points represent means of three replicates.
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Fig. 5. Specific extrusions of protons and organic acid anions (citrate plus malate) per 1000 root tips, by Lupinus albus at day 48 of treatment with 1, 5 or 25 mmol P m − 3. Bars represent means 9 S.E. (n= 4). Fig. 3. Concentrations of K, Mg, Ca, N, S, Cu, Fe and Mn in cluster and non-cluster roots of Lupinus albus measured after 49 days of treatment with 1, 5 or 25 mmol P m − 3. Vertical bars represent means 9S.E. (n=3).
m − 3, respectively. The amounts of OA− exuded generally increased with time (Fig. 4). The amount of OA− extruded per 1000 root tips measured at day 48 was four times higher at 1 mmol P m − 3 than at 5 mmol P m − 3, which, in turn, was four times higher than at 25 mmol P m − 3 (Fig. 5). From day 27, proton extrusion from roots was highly dependent on P supply, being 7-fold higher
at 1 mmol P m − 3 than at 25 mmol P m − 3 at days 41 and 48 (Fig. 4). The amount of H+ produced per unit root biomass decreased with time (Fig. 4). The amount of protons extruded per 1000 root tips at 1 mmol P m − 3 measured at day 48 was doubled as P supply increased to 5 mmol P m − 3, and was 25 times higher than at 25 mmol P m − 3 (Fig. 5). The amounts of both OA− and H+ released per plant were higher at 1 and 5 mmol P m − 3 than at 25 mmol P m − 3 at day 34, and increased with time. However, the amounts of H+ extruded by
Fig. 4. Changes in specific exudation of organic acid anions (citrate plus malate) and protons by Lupinus albus grown at 1, 5 or 25 mmol P m − 3 in nutrient solution during the experimental period. Vertical bars represent means 9 S.E. (n= 4).
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3.4. Excess cation uptake
Fig. 6. Concentrations of excess cations in shoots and roots of Lupinus albus grown at 1, 5 or 25 mmol P m − 3 during 49 days. Vertical bars represent means 9 S.E. (n=3), where larger than the symbol.
The plants grown at 1 mmol P m − 3 generally had higher concentrations of Ca and Cl and lower concentrations of K, S, Zn, Fe and Cu in roots compared with those at higher P supply. Phosphorus supply did not clearly affect concentrations of Mg, Mn and N in roots nor concentrations of K, Ca, Mg, Cl, Mn, Zn, Fe, Cu and N in shoots (data not shown). Decreasing P supply generally increased concentrations of excess cations in shoots from day 35 and in roots from day 14. The concentrations also increased with plant age up to day 28 for shoots and day 21 for roots, and then remained unchanged in roots but decreased in shoots (Fig. 6). Total uptake of excess cations was correlated with total acid production in all P treatments but the slope of the regression decreased from 0.64 at 25 to 0.17 at 1 mmol P m − 3 (Fig. 7). In other words, the ratio of H+ release to excess cation uptake decreased with increased P supply.
4. Discussion
P-deficient plants were, on average, 2– 3-fold greater than OA− exudation on an equal molar basis between days 27 and 41. Under P deficiency, the amounts of OA− exuded per plant correlated with the amounts of H+ released with a slope of the regression of 0.76 (Fig. 7).
Our data clearly proved that a continuous low P supply increased cluster root formation by white lupin plants. The increased number of cluster roots by P deficiency occurred prior to the effect on shoot growth (day 21 vs. day 35), suggesting that cluster root formation under P deficiency is not mediated by shoot growth. Similarly,
Fig. 7. The relationships between exudation of organic acid anions and H+ release (A), and between uptake of excess cations and H+ extrusion (B) by Lupinus albus grown at 1, 5 or 25 mmol P m − 3. Lines are fitted with the linear model. In (A), data at 25 mmol P m − 3 were excluded from analysis. In (B), the slopes (b) of the regression lines are indicated. Each point represents means of four replications.
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Keerthisinghe et al. [12] found that the formation of cluster roots was not related to plant dry matter yields or shoot-to-root dry matter ratios. Further, this study found that number of cluster roots was correlated inversely with P concentrations in plants, which was perceived before growth was limited. From this finding, it may be concluded that the formation of cluster roots is mediated by internal P status in plants, in accordance with other studies [3,22]. Cluster roots play an important role in the acquisition of not only P but also of other mineral nutrients [9]. The present study provided evidence that cluster roots contained higher concentrations of Mg, Ca, N, Fe and Mn, indicating higher efficiency in absorption of these ions by cluster roots than non-cluster roots. This might be caused by enhanced uptake rate of these elements by cluster roots, higher metabolic activity of these root structures [5,13], their larger surface area [9], or presumably decreased translocation of specific ions into the shoot. Potassium concentration in roots was significantly lower at 1 mmol P m − 3 than at 25 mmol P m − 3 (Fig. 6), although the K concentration in shoots was not affected by P supply (data not shown). This decreased K concentration in roots under P deficiency mainly resulted from the higher proportion of cluster roots at low P supply and the lower K concentration in cluster than noncluster roots. The results indicate that either a higher proportion of absorbed K was effluxed from the cluster roots than non-cluster roots or that K was not taken up to the same extent by the two root types. Since cluster roots of white lupin are the main site for release of H+ and organic anions under P deficiency [5,12], it is speculated that the release of organic anions is accompanied with K+ efflux (co-transport), which may also be the case in Banksia species (unpublished data). This deserves further investigation. Extrusions of both protons and OA− were highly dependent on P supply and on the stage of plant growth, but the release of H+ per unit root biomass displayed different patterns from the OA− (Fig. 4). While the increased exudation of OA− with time had resulted from the increased severity of P deficiency, the decreased release of H+ per unit roots over the experimental period might be due to a decrease in excess cation uptake. This suggests that different mechanisms are involved in proton release and OA− exudation.
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The amount of H+ extruded was always higher than the sum of OA− exuded, regardless of whether they were expressed per unit of root tips or root dry weight (Figs. 4 and 5), surface area or length (data not shown). Thus, the total acid (H+) production appeared to consist of the H+ release (and K+ efflux) associated with OA− exudation [4,5,18] and the H+ release resulting from excess cation uptake [23,24]. Under P deficiency in this study, about half of total acid (H+) extrusion during the experimental period was not accounted for from the uptake of excess cations, and was probably attributed to OA− extrusion. Plant roots extrude net H+ when cation uptake exceeds anion uptake and extrude net OH− when anion uptake exceeds cation uptake. Several studies have shown that plants can acidify soils directly by releasing protons from the roots when an excess cation over anion uptake occurs [25–28]. Legumes, reliant on N2 fixation, generally take up more cations than anions and thus extrude into the rhizosphere proportionally more H+ than OH− to balance the charges [24]. The amount of H+ extruded by legumes reliant solely on N2 fixation can be calculated from the mineral composition of the plant (non-N excess cations). Thus, Tang et al. [24] established a relationship between H+ extrusion and excess cation uptake in legume species grown in nutrient solution. In the present study, the plants were reliant on N2 fixation as the only N source. Therefore, H+ extrusion should be related to excess cation uptake. Indeed, there was a good relationship between root acid exudation and excess cation content in the plants at given P levels. However, the regression lines were all below the 1:1 line and the slope values of the lines decreased with decreasing supply of P. In other words, the ratio of H+ extrusion to excess cation uptake increased when P supply was decreased. This increased ratio correlated well with the increased OA− exudation. These results support the alternative that the exudation of OA− under P deficiency is partly accompanied by H+ efflux. Furthermore, in previous studies on root exudates under P deficiency, plants had been supplied with nitrate. White lupin plants supplied with nitrate released similar quantities of citrate [5,12], as the N2-fixing plants in our experiment. Proton release could be expected to be lower in the case of NO− 3 -fed plants as compared with N2-fixing plants. However, there is no conclusive evidence in the literature on it, and further study is warranted.
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Acknowledgements We thank Dr Z.L. Chen for valuable advice on organic acid measurements.
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