Influence Of Aluminum On The Growth And Organic Acid Exudation In Alfalfa Cultivars Grown In Nutrient Solution

Journal of Plant Nutrition, 32: 618–628, 2009 Copyright © Taylor & Francis Group, LLC ISSN: 0190-4167 print / 1532-4087 online DOI: 10.1080/01904160802715430

Downloaded By: [Dutch Library Consortium (UKB) - Dekker Titles only] At: 14:26 17 March 2009

Influence of Aluminum on the Growth and Organic Acid Exudation in Alfalfa Cultivars Grown in Nutrient Solution Howard Langer,1 Mara Cea,2 Gustavo Curaqueo,1 and Fernando Borie1 1

Departamento de Ciencias Qu´ımicas, Universidad de La Frontera, Temuco, Chile 2 Instituto de Agroindustria, Universidad de La Frontera, Temuco, Chile

ABSTRACT A study was conducted to evaluate the effects of aluminum (Al) in nutrient solutions on the dry weight (DW) yield, Al and phosphorus (P) contents, and organic acid exudation in alfalfa (Medicago sativa L.). Four alfalfa cultivars (‘Robust’, ‘Sceptre’, ‘Aquarius’, and ‘California-55’) were grown in nutrient solution at pH 4.5 and 6.0, with (50 and 100 µM) and without Al. The results revealed that Al caused a significant reduction in DW, especially in pH 4.5 treatment. Organic acid exudation was affected by pH and Al treatments. Citrate and succinate exudation increased with the high Al treatment at pH 4.5. However, no relationship between pH and carboxylate exudation was observed at pH 6.0. Accumulation of P and Al in roots suggests the existence of an exclusion mechanism for Al in alfalfa. Selection of cultivars with enhanced organic exudation capacity in response to Al might be useful for alfalfa production in moderately acidic soils. Keywords: aluminum, alfalfa, citrate, succinate, dry weight yield, nutrient solution

INTRODUCTION Aluminum (Al) toxicity is considered to be a major environmental stress that limits crop and forage production in acid soils (Mora et al., 1999). Aluminum inhibits root cell elongation and division at the cellular level. The primary symptom of Al toxicity in plants is a reduction in root growth, which reduces water and nutrient uptake. Aluminum seems to be the most important species

Received 6 September 2007; accepted 27 February 2008. Address correspondence to Howard Langer, Departamento de Ciencias Qu´ımicas, Universidad de La Frontera, Casilla 54-D, Temuco, Chile. E-mail: [email protected] 618

Downloaded By: [Dutch Library Consortium (UKB) - Dekker Titles only] At: 14:26 17 March 2009

Differential Response to Aluminum in Alfalfa

619

of rhizotoxic Al, although some authors indicate that other monomeric species, such as AlOH2+ and Al(OH) 2 + , are even more toxic to soybean than Al3+ (Alva et al., 1986). Plants have evolved two major Al mechanisms: Al tolerance, known as internal tolerance and Al exclusion from the root apex (Matsumoto, 2000; Barcel´o and Poschenrieder, 2002; Kochian et al., 2004; Kochian et al., 2005). In this category, the exudation of Al-inducible organic acids has been reported to confer Al tolerance in several crop and pasture species. Both the type and amount of organic acids released in response to Al vary widely among plant species and even cultivars within species (Zheng et al., 1998). For example, Dong et al. (2004) observed that soybeans exposed to Al stimulated the release of citrate, whereas a phosphorus (P) deficiency increased the exudation of malate and oxalate instead. Several studies have reported the exudation of citrate in response to Al in some legumes species such as soybean (Yang et al., 2000; Silva et al., 2001) and bean (Miyasaka et al., 1991). On the other hand, the exudation of succinate has been reported in response to P deficiency in alfalfa plants (Lipton et al., 1987) or as a result of the overexpression of malate dehidrogenase enzyme, resulting in an increase in the exudation of citrate, oxalate, malate, succinate and acetate (Tesfaye et al., 2001). In Chile, much of the alfalfa is grown on acidic Andisols (pH < 5.5) displaying high levels of extractable Al. The inherent acidity combined with the use of ammonia fertilizers contributes to increase the levels of Al in the soil solution and thus, the Al phytotoxicity. The use of alfalfa cultivars with an increased ability to exudate organic acids may improve the alfalfa yields in moderately acidic soils. To our knowledge, this is the first attempt to elucidate the role of organic acid exudation in response to Al rhizotoxicity in alfalfa. The major aim of this study was to determine the differential response in organic acid exudation by four alfalfa (Medicago sativa L.) cultivars exposed to different Al levels and pH values in nutrient solution experiments.

MATERIALS AND METHODS Growth Conditions and Solutions Seeds of four alfalfa cultivars (‘Robust’, ‘Sceptre’, ‘Aquarius ‘and ‘California55’) were selected for shape, color, and size. Seeds were surface sterilized with ethanol (70% v/v) and sodium hypoclorite (4%), and germinated on wet filter paper in the dark for 4 d. Eight seedlings were transplanted to polypropylene pots containing 1 L of aireated Taylor and Foy nutrient solution (Taylor and Foy, 1985) with the following composition in macro- and micronutrients (mM): ammonium nitrate (NH 4 NO 3 ), 120; sodium chloride (NaCl), 150; magnesium

Downloaded By: [Dutch Library Consortium (UKB) - Dekker Titles only] At: 14:26 17 March 2009

620

H. Langer et al.

chloride (MgCl 2 6H 2 O), 162; monopotassium phosphate (KH 2 PO 4 ), 120; ammonium chloride (NH 4 Cl), 66; sodium nitrate (NaNO 3 ), 252; sodium sulfate (Na 2 SO 4 10H 2 O), 72; potassium nitrate (KNO 3 ), 330; calcium nitrate [Ca(NO 3 ) 2 4H 2 O], 762; boric acid (H 3 BO 3 ), 3.96; zinc sulfate (ZnSO 4 7H 2 O), 0.36; manganese sulfate (MnSO 4 H 2 O), 1.44; ammonium molybdate [(NH 4 ) 6 Mo 7 O 24 4H 2 O], 0.06; iron (Fe)-ethylenediaminetetraacetic acid (EDTA), 10.74; copper sulfate (CuSO 4 5H 2 O), 0.12. Nutrient solutions were renewed every 5d. Three Al levels (0, 50, and 100 µM) added in the form of aluminum chloride (AlCl 3 ) and two levels of pH (4.5 and 6.0) were tested. The pH was kept constant by daily addition of 0.1 M hydrochloric acid (HCl) or sodium hydroxide (NaOH) as required. The experiment was conducted in a controlled-environment growth chamber for 31 d at 22◦ C with a 50 to 60% relative humidity and a 16 h light photoperiod.

Collection and Analysis of Root Exudates Collection of exudates was performed according to Rosas et al. (2007). After fifteen days of growth, roots of eight intact plants that had been treated with 0 to 100 µM Al were submerged for 1 h in aerated deionized water (50 mL) and the resulting solution containing exudates was stored at −20◦ C. A short elution time was selected in order to reduce possible degradation of organic acids by microorganisms (Jones et al., 1996). In order to quantify the concentration of organic acids (citrate and succinate), root exudates were lyophilized and the residue re-dissolved in 5 mL of deionized water, filtered (0.45 µm) and used for HPLC analysis. Separation was achieved on a 250 × 4 mm reverse phase column (Merck, LiChrospher 100 RP-18, 5-µm particle size). Sample solutions (20 µL) were injected into the column, and a 200 mM ortho-phosphoric acid (pH 2.1) solution was used for isocratic elution at a flow rate of 1 mL min−1 with UV detection at 210 nm. Preliminary studies with standard organic acids indicated that recovery of the organic anions was about 98%. Aluminum speciation and the formation of Al-complexes in the nutrient solutions was evaluated using the modified GEOCHEM v.2.0. computer program (Parker et al., 1987). The total amount of organic acid exuded in 1 L of nutrient solution was considered for chemical speciation (8 plants per treatment).

Dry Weight and Chemical Analysis of Plants After 45 d, the plants were harvested, separated into roots and shoots, oven-dried (60◦ C for 48 h) and weighed. Plant samples were ashed at 500◦ C for 8 h and then analysed for Al and phosphorus (P) content following digestion with 2 M HCl. Aluminum was measured by using atomic absorption

Differential Response to Aluminum in Alfalfa

621

spectrophotometry (AAS) and P was measured colorimetrically according to the vanadophosphomolybdate method of Sadzawka et al. (2004).

Downloaded By: [Dutch Library Consortium (UKB) - Dekker Titles only] At: 14:26 17 March 2009

Statistics The experimental design was a randomized complete block with three replications. The results were analyzed by ANOVA and were compared using the Tukey Test (P ≤ 0.05).

RESULTS AND DISCUSSION Effects of Al and pH on the Growth of Alfalfa Cultivars Aluminum reduced the shoot and root DW of alfalfa growing in nutrient solutions, especially at pH 4.5 (Figure 1). The plant roots exposed to higher Al levels were brown and poorly developed, with the major effect of Al exposure observed in shoot DW. This clearly indicates that Al affected nutrient uptake, and reduced the plant growth. However, the magnitude of growth reduction depended of the alfalfa cultivar. When the plants were exposed to 50 µM Al,

Figure 1. Effects of Al on shoot and root dry weights (g dry weight pot−1 ) in four alfalfa cultivars in nutrient solutions at two pH levels. Mean of three replicates. Bars in columns indicates standar error (SE).

622

H. Langer et al.

Table 1 Relative growth reduction (%), related to the control without Al, in plants exposed to Al in nutrient solutions at pH 4.5 and 6.0 pH 4.5

Downloaded By: [Dutch Library Consortium (UKB) - Dekker Titles only] At: 14:26 17 March 2009

Cultivars California 55 Robust Sceptre Aquarius

pH 6.0

50 µM Al

100 µM Al

50 µM Al

100 µM Al

21 55 38 44

52 61 63 55

67 24 17 20

50 29 46 0

the shoot DW was reduced 21 and 55% for California 55 and ‘Robust’, respectively, in relation to the control treatments without Al. At higher Al levels (100 µM) the reduction increased to 52 and 63% for ‘California 55’ and ‘Sceptre’, respectively. Differential response in shoot DW in absence of Al likely reflects different yield potentials among alfalfa cultivars (Table 1). Similar variations in cultivar response to Al have been previously reported in some crop species (Foy, 1988). The use of alfalfa cultivars that show a high degree of Al tolerance under nutrient solutions may increase forage production under field conditions. Although Al3+ is the predominant aqueous species at low pH as shown in Table 2, other Al monomeric species such AlOH2+ , Al(OH) 2 + , and Al(OH) 4 − and the polymer Al 13 polymer present at higher pH values have been implicated in the reduction of plant yields (Kinraide, 1991). It is interesting to mention that nutrient levels in solution remained adequate to ensure the plant growth, so they were not strongly affected by the addition of Al to the nutrient solution (data not shown), which concentrates the possible toxicity of Al-OH forms at pH 6.0, more than a deficiency in some other nutrient. Levels of Al and P in the shoots of the alfalfa cultivars growing at different pH levels are shown in Table 3. Levels of P in shoots were not affected by Al treatments at both pH levels. However, an increase in Al concentrations in the shoot tissues for ‘California 55’ and ‘Robust’ was observed. On the other hand, an increase in root concentration of Al and P was observed in the plants exposed to Al. Higher levels of Al in roots were achieved by plants growing at pH 4.5 with Al (100 µM), varying from 6.35 to 7.84 g Al kg−1 DW in ‘Aquarius’ and ‘California 55’ cultivars, respectively. Levels of P in roots were from 0.28 to 0.38% in ‘Aquarius’ and ‘Sceptre’ cultivars, respectively. Our results are in agreement with those reported by Zheng et al. (2005), who studied the accumulation of Al with P in two cultivars of Fygopyrum esculentum expressing different degrees of Al tolerance, showing high concentrations of P and Al in the roots of the tolerant cultivar. These results suggest that the precipitation of Al with P in root tissues is one mechanism of reducing Al toxicity. This hypothesis is also supported by the fact that accumulation of P

Differential Response to Aluminum in Alfalfa

623

Table 2 Distribution of Al species in nutrient solutions at different levels of Al and pH as response to organic acid exudation from alfalfa roots as calculated by GEOCHEM pH 4.5 Al treatment (µM) Downloaded By: [Dutch Library Consortium (UKB) - Dekker Titles only] At: 14:26 17 March 2009

Complexes

50

100

pH 6.0 Al treatment (µM) 50

100

California 55 +3

Al Al-PO 4 −3 Al-citrate Al-OHa

17.5 30.4 43.1 7.0

8.8 15.2 8.5 66.6 Robust

— 0.03 7.8 92.23

— 0.02 19.7 80.32

Al+3 Al-PO 4 −3 Al-citrate Al-OHa

13.1 23.8 58.3 3.2

8.8 15.2 4.3 70.8 Sceptre

— 0.03 16.4 83.53

— 0.02 1.63 98.32

Al+3 Al-PO 4 −3 Al-citrate AlOHa

17.5 30.3 22.1 28.0

8.8 15.2 20.4 54.7 Aquarius

— 0.03 18.9 81.03

— 0.02 11.8 88.22

Al+3 Al-PO 4 −3 Al-citrate Al-OHa

17.5 30.3 24.5 25.5

8.8 15.2 9.4 65.7

— 0.03 19.9 80.13

— 0.02 11.0 89.02

a

Al complexed in solution and precipitated.

in roots increases in plants exposed to higher levels of Al. Precipitation of Al with P (as phosphates) in roots has been described as an important mechanism that confers Al tolerance in plants (Gaume et al., 2001). Those authors report increased precipitation of Al and a high capacity to utilize P, due to a mechanism for the active transport of Al-P complexes from the cell wall to the vacuole (V´asquez et al., 1999). The Al-dependent efflux of organic acids into the rhizosphere has been widely described as an important mechanism in plant species to minimize the toxic effects of Al in acid soils (Ma et al., 2001; Ryan et al., 2001; Barcel´o and Poschenrieder, 2002; Kochian et al., 2004). Our results indicate that the exudation of citrate increased significantly in response to Al, the magnitude of which was dependent on the alfalfa cultivar (Figure 2). Citrate exudation was higher in cultivars ‘California 55’ and ‘Robust’, especially under Al stress

624

H. Langer et al.

Table 3 Effects of pH and aluminum on the phosphorus (%) and aluminum (g kg−1 ) contents in shoots and roots in four alfalfa cultivars in nutrient solutions pH 4.5

pH 6.0

Downloaded By: [Dutch Library Consortium (UKB) - Dekker Titles only] At: 14:26 17 March 2009

Shoot Al (µM)

P) (%)

Al (g kg−1 )

Root P Al (%) (g kg−1 )

Shoot P (%)

Root

Al (g kg−1 )

P (%)

Al (g kg−1 )

California 55 0 50 100

0.14 abA 0.05 abA 0.23 0.15 abA 0.09 aA 0.32 0.14 abA 0.10 aA 0.32

0.23 0.13 abA 0.03 cdB 6.08 0.13 abA 0.06 abB 7.84 0.14 abA 0.07 aB Robust

0.29 0.36 0.30

0.08 3.70 3.04

0 50 100

0.17 abA 0.03 cA 0.29 0.15 abA 0.03 cA 0.31 0.18 aA 0.05 abA 0.30

0.30 0.16 abA 0.02 dA 6.83 0.16 abA 0.06 abcA 7.03 0.15 abA 0.07 aA Sceptre

0.35 0.40 0.35

0.04 3.66 3.10

0 50 100

0.13 abA 0.04 abA 0.27 0.17 abA 0.05 abA 0.32 0.15 abA 0.09 aA 0.38

0.27 0.11 abA 0.03 dA 7.08 0.17 aA 0.03 cdB 7.20 0.15 abA 0.03 cdB Aquarius

0.27 0.31 0.29

0.02 2.48 2.21

0 50 100

0.12 bA 0.04 abA 0.23 0.14 abA 0.06 bA 0.26 0.14 abA 0.06 bA 0.28

0.28 5.46 6.35

0.32 0.34 0.27

0.05 2.96 2.32

0.10 bA 0.02 dA 0.13 abA 0.03 dB 0.13 abA 0.04 bcdA

Within columns, means followed by the same lower case letter were not significantly different (P ≤ 0.05) according to ANOVA and Tukey’s test. Mean values within rows for each variable with the same upper case letter were not significantly different (P ≤ 0.05) according to Student’s t-test. Values of Al and P in shoots are means of three replicates, except for a single value in roots.

conditions. Previous studies performed in soybean have shown the high-Al binding capacity of citrate (Ginting et al., 1998) and the existence of varietal differences in the amount of citrate exuded from roots in response to Al exposure (Silva et al., 2001). Although plants are certainly able to concentrate succinate, however, the chemical speciation results performed by GEOCHEM indicate that succinate is not capable of forming strong complexes with Al that could potentially reduce its toxicity (Figure 3). In addition, previous have confirmed that succinate is a weak ligand with a low capacity to detoxify Al (Hue et al., 1986). Nevertheless, the exudation of organic acids including succinate from alfalfa roots has been

Downloaded By: [Dutch Library Consortium (UKB) - Dekker Titles only] At: 14:26 17 March 2009

Differential Response to Aluminum in Alfalfa

625

Figure 2. Effect of Al on the exudation of citrate (µmol h−1 g−1 root dry weight) in four alfalfa cultivars under nutrient solutions at pH 4.5. Mean of three replicates. Columns followed by different letters indicate significant differences (P ≤ 0.05) according to Tukey test. Bars in columns indicates standar error (SE).

reported by Lipton et al. (1987) as a response to limited P availability in nutrient solutions.

Figure 3. Effect of Al on the exudation of succinate (µmol h−1 g−1 root dry weight) in four alfalfa cultivars under nutrient solutions at pH 4.5. Mean of three replicates. Columns followed by different letters indicate significant differences (P ≤ 0.05) according to Tukey test. Bars in columns indicates standar error (SE).

Downloaded By: [Dutch Library Consortium (UKB) - Dekker Titles only] At: 14:26 17 March 2009

626

H. Langer et al.

Both the quality and the quantity of the organic acid exuded by plant roots are critical to reducing Al toxicity. Some estimations suggest that an equimolar concentration of citrate is required to avoid the root elongation in maize, which is considered a useful criteria for estimating Al toxicity (Zheng et al., 1998). This means that the concentration of citrate required in the nutrient solution in order to overcome Al toxicity might in the range of 50 or 100 µM. Aqueous chemical speciation of Al is largely dependent on pH. Estimates of Al speciation using GEOCHEM predicted levels of Al3+ between 8.8 and 17.5 µM at pH 4.5. However, calculations indicate that Al3+ was not present in nutrient solutions at pH 6.0 because it was mainly present in the form of Al-OH complexes. This finding is consistent with the results from similar evaluations performed by Nair and Prenzel (1978) who found that at neutral to high pH, complex formation of Al with phosphate and organic acids might reduce Al activity. In the current system, speciation estimates indicate that much of the Al in solution was complexed with citrate, varying from 22 to 58%, especially at pH 4.5. High Al concentrations in nutrient solutions resulted in poor growth of alfalfa plants. However, chemical speciation analysis showed that nutrient levels in solutions were not affected by adding Al (data not shown). Despite the fact that Al3+ was the most abundant species at pH 4.5, other Al species such as AlOH2+ , Al(OH) 2 + , or Al(OH) 4 − and polymeric Al 13 likely control Al toxicity at pH 6.0 (Kinraide, 1991). In conclusion, the present study suggests that citrate exudation by alfalfa cultivars exposed to Al is an important mechanism for reducing the rhizotoxicity of Al. However, differences in DW yield that were not fully explained by citrate exudation suggest that multiple mechanisms are important in controlling Al tolerance and increasing P acquisition in alfalfa plants growing on acid soils.

ACKNOWLEDGMENTS This work was supported by Fundaci´on Andes (Chile) C-13755-28 and DIUFRO Grant number 160602.

REFERENCES Alva, A. K., D. G. Edwards, C. J. Asher, and F. P. C. Blamey. 1986. Effects of phosphorus/aluminum molar ratio and calcium concentration on plant response to aluminum toxicity. Soil Science Society of America Journal 50: 133–137. Barcel´o, J., and C. Poschenrieder. 2002. Fast root growth responses, root exudates, and internal detoxification as clues to the mechanisms of aluminium

Downloaded By: [Dutch Library Consortium (UKB) - Dekker Titles only] At: 14:26 17 March 2009

Differential Response to Aluminum in Alfalfa

627

toxicity and resistance: a review. Environmental and Experimental Botany 48: 75–92. Dong, D., X. Peng, and X. Yan. 2004. Organic acid exudation induced by phosphorus deficiency and/or aluminium toxicity in two contrasting soybean genotypes. Physiologia Plantarum 122: 190–199. Foy, C. D. 1988. Plant adaptation to acid, aluminum-toxic soils. Communications in Soil Science and Plant Analysis 19: 959–987. Gaume, A., F. M¨achler, and E. Frossard. 2001. Aluminum resistance in two cultivars of Zea mays L.: Root exudation of organic acids and influence of phosphorous nutrition. Plant and Soil 234: 73–81. Ginting, S., B. B. Johnson, and S. Wilkens. 1998. Alleviation of aluminium phytotoxicity on soybean growth by organic anions in nutrient solutions. Australian Journal of Plant Physiology 25: 901–908. Hue, N. V., G. R. Craddock, and F. Adams. 1986. Effect of organic acids on aluminum toxicity in subsoils. Soil Science Society of America Journal 50: 28–34. Jones, D. L., A. M. Prabowo, and L. Kochian. 1996. Kinetics of malate transport and decomposition in acid soils and isolated bacterial populations: The effect of microorganisms on root exudation of malate under Al stress. Plant and Soil 182: 239–247. Kinraide, T. B. 1991. Identity of the rhizotoxic aluminum species. Plant and Soil 134: 167–178. Kochian, L. V., O. A. Hoekenga, and M. A. Pineros. 2004. How do crop plants tolerate acid soils?—Mechanisms of aluminum tolerance and phosphorous efficiency. Annual Review of Plant Biology 55: 459–493. Kochian, L. V., M. A. Pineros, and O. A. Hoekenga. 2005. The physiology, genetics and molecular biology of plant aluminum resistance and toxicity. Plant and Soil 274: 175–195. Lipton, D. S., R. W. Blanchar, and D. G. Blevins. 1987. Citrate, malate and succinate concentration in exudates from P-sufficient and P-stressed Medicago sativa L. seedlings. Plant Physiology 85: 315–317. Ma, J. F., P. R. Ryan, and E. Delhaize. 2001. Aluminum tolerance in plants and the complexing role of organic acids. Trends in Plant Science 6: 273–278. Matsumoto, H. 2000. Cell biology of aluminum toxicity and tolerance in higher plants. International Review of Cytology 200: 1–46. Miyasaka, S. C., J. G. Buta, R. K. Howell, and C. D. Foy. 1991. Mechanism of aluminum tolerance in snapbeans. Root exudation of citric acid. Plant Physiology 96: 737–743. Mora, M. L., B. Schnettler, and R. Demanet. 1999. Effect of liming and gypsum on soil chemistry, yield, and mineral composition of ryegrass grown in an acidic Andisol. Communications in Soil Science and Plant Analysis 30: 1251–1266. Nair, V. D., and J. Prenzel. 1978. Calculations of equilibrium concentration of mono- and polynuclear hydroxyaluminium species at different pH

Downloaded By: [Dutch Library Consortium (UKB) - Dekker Titles only] At: 14:26 17 March 2009

628

H. Langer et al.

hydrogen-ion concentration and total aluminum concentrations in soils. Journal of Plant Nutrition and Soil Science 141: 741–751. Parker, D. R., L. W. Zelazny, and T. B. Kinraide. 1987. Improvements to the program GEOCHEM. Soil Science Society of America Journal 52: 438–444. Rosas, A., Z. Rengel, and M. L. Mora. 2007. Manganese supply and pH influence growth, carboxylate exudation and peroxidase activity on ryegrass and white clover. Journal of Plant Nutrition 30: 253–270. Ryan, P. R., E. Delhaize, and D. L. Jones. 2001. Function and mechanism of organic anion exudation from plant roots. Annual Reviews in Plant Physiology and Plant Molecular Biology 52: 527–560. Sadzawka, A., R. Grez, M. A. Carrasco, and M. L. Mora. 2004. M´etodos de An´alisis de Tejidos Vegetales. Santiago, Chile: Comisi´on de Normalizaci´on y Acreditaci´on Sociedad Chilena de la Ciencia del Suelo. Silva, I. R., T. J. Smyth, D. Raper, T. E. Carter, and T. W. Rufty. 2001. Differential aluminum tolerance in soybean: An evaluation of the role of organic acids. Physiologia Plantarum 112: 200–210. Taylor, G. J., and C. D. Foy. 1985. Mechanisms of aluminum tolerance in Triticum aestivum L. (Wheat). II Differential pH induced by spring cultivars in nutrient solutions. American Journal of Botany 72: 702–706. Tesfaye, M., S. J. Temple, D. L. Allan, C. P. Vance, and D. A. Samac. 2001. Overexpression of malate dehydrogenase in transgenic alfalfa enhances organic acid synthesis and confers tolerance to aluminum. Plant Physiology 127: 1836–1844. V´asquez, M., C. Poschenrieder, I. Corrales, and J. Barcel´o. 1999. Changes in apoplastic aluminum during the initial growth response to aluminum by roots of tolerant maize variety. Plant Physiology 119: 435–444. Yang, Z. M., M. Sivaguru, W. J. Horst, and H. Matsumoto. 2000. Aluminium tolerance is achieved by exudation of citric acid from roots of soybean (Glycine max). Physiologia Plantarum 110: 72–77. Zheng, S. J., J. F. Ma, and H. Matsumoto. 1998. Continuous secretion of organic acids is related to aluminium resistance during relatively longterm exposure to aluminium stress. Physiologia Plantarum 103: 209–214. Zheng, S. J., J. L. Yang, Y. F. He, X. H. Yu, L. Zhang, J. F. You, R. F. Shen, and H. Matsumoto. 2005. Immobilization of aluminum with phosphorus in roots is associated with high aluminum resistance in buckwheat. Plant Physiology 138: 297–303.