Journal of Plant Nutrition, 28: 1117–1132, 2005 Copyright © Taylor & Francis Inc. ISSN: 0190-4167 print / 1532-4087 online DOI: 10.1081/PLN-200062795
Influence of Sulfate Concentration in Mineral Solution on Ryegrass Grown at Different pH and Aluminum Levels M. L. Mora,1 R. Demanet,2 E. Vistoso,3 and F. Gallardo1 1
Departamento de Ciencias Qu´ımicas, Facultad de Ingenier´ıa, Ciencias y Administraci´on, 2 Departamento de Producci´on Agropecuaria, Facultad de Ciencias Agropecuarias y Forestales, 3 Programa de Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera, Temuco, Chile
ABSTRACT The objective of this study was to clarify the effects of sulfate and phosphate at different sulfate concentrations, pH levels, and aluminum (Al) concentrations on aluminum phytotoxicity in ryegrass grown in mineral solution. The results demonstrated that, in general, both a high sulfate concentration and phosphate in the mineral solution had a positive effect on the detoxification of Al. Increased sulfate concentrations did not have a clear effect on the sulfur (S) or phosphorus (P) content within the shoot or root, because their uptake is regulated by Al uptake in the ryegrass, but a very good relationship was found between the P root content and Al root content (r = 0.944∗∗ ; p ≤ 0.01). The P and S root content and the aluminum:phosphorus molar ratio in roots were negatively correlated to shoot dry weight (r = −0.747∗∗ , −0.619∗∗ and −0.816∗∗ , respectively; p ≤ 0.01), suggesting that Al tolerance is related to P nutrition. Relative root dry weight (RTI) measured at 90 d was a more sensitive indicator of Al toxicity than relative root or shoot length (RRL, RSL). This study showed that the most important Al detoxifying mechanisms in the ryegrass were apparently physiological Al-PO4 precipitation inside the root and chemical AlSO+ 4 complex formation in the nutritive solution. Keywords: ryegrass sulfur content, ryegrass phosphorus content, aluminum toxicity, Lolium multiflorum, nutritive solution, sulfate
Received 22 March 2002; accepted 7 February 2005. Address correspondence to M. L. Mora, Departamento de Ciencias Qu´ımicas, Facultad de Ingenier´ıa, Ciencias y Administraci´on, Universidad de La Frontera, Casilla 54-D, Temuco, Chile. E-mail:
[email protected] 1117
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INTRODUCTION Toxicity of the cation Al+3 is considered to be a major environmental stress that limits world food production in acid soils (Foy, 1983). In Chile, aluminum (Al) toxicity significantly reduces crop productivity in acid soils (Mora et al., 1999), and especially affects harvests of the ryegrass Lolium multiflorum. The most pronounced symptom of affected ryegrass is stunted growth (Gallardo et al., 1999). Free Al+3 ions interfere with cell division (Morimura et al., 1978) and nuclear activity (Matsumoto et al., 1977), thus inhibiting root growth and impairing nutrient uptake (Kinraide et al., 1994; Delhaize and Ryan, 1995). The concentration of Al in soil or hydroponic solutions does not by itself determine Al phytotoxicity (Adams and Moore, 1983). Instead, toxicity appears to be determined by the availability of certain monomeric species of Al (Blamey et al., 1983). Al ion species change their forms with the pH; Al activity can be decreased by Al polymerization as pH increases (Baes and Mesmer, 1976; Nair and Prenzel, 1978) or by chelating with phosphates and organic acids. Under moderately acidic conditions (pH 4.5 to 7.0) the predominant forms are hydroxy-Al polymer ions (Bersillon et al., 1980; Nair and Prenzel, 1978). While the detrimental effects of low pH on root growth due to H+ injury are well documented by Johnson and Wilkinson (1992) and Calba et al. (1999), the effects on Al chemical compositions and behaviors have not been fully clarified, and there has been little investigation into the influence of the different Al ions on plant growth. Sulfate is able to reduce the Al+3 activity by inducing AlSO+ 4 formation (Pavan et al., 1982), but the use of CaSO4 as an Al ameliorant does not always guarantee a marked increase in crop yield under acidic condition (Kinraide et al., 1985). Several plant species have developed strategies to avoid or tolerate Al toxicity. Proposed Al resistance mechanisms can be classified as internal tolerance and exclusion (Kochian, 1995; Taylor, 1991). The main difference between these mechanisms is the site of Al detoxification: the symplasm (internal tolerance) or apoplasm (exclusion). Internal tolerance mechanisms immobilize, compartmentalize, or detoxify Al in the symplasm. In contrast, exclusion mechanisms prevent toxic Al from entering the symplasm where sensitive intracellular sites are located (Taylor, 1991); a proposed exclusion mechanism is root excretion of chelating organic substances that form stable complexes with Al+3 ions in the soil solution, which are less phytotoxic than free Al+3 ions (Hue et al., 1986). The objective of the present study was to determine the effect of sulfate concentrations on sulfate-A1 and phosphate-Al relationships on Al detoxification in ryegrass grown in mineral solution at different Al and pH levels.
Influence of Sulfate Concentration in Solution
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MATERIALS AND METHODS Growth Conditions and Solutions Seeds from Al semi-tolerant ryegrass Lolium multiflorum cv. ‘Montblanc’ were sterilized in 0.5% NaClO for 20 min, washed in deionized water, and germinated on wet filter paper in the dark for 7 d before being transferred to pots. In each pot were placed eight plants and 900 mL of Taylor & Foy nutrient solution (Taylor and Foy, 1985) in deionized water with the following composition of + 2+ macronutrients (mM): N-NO− 1.27; Mg2+ 0.27; K+ 3 3.71; N-NH4 0.31; Ca 2− 2− 0.75; S-SO4 0.12; P-HPO4 0.10; and micronutrients (uM): Fe-EDTA 17.9; B 6.6; Mn2+ 2.4; Zn2+ 0.6; Cu2+ 0.2; and Mo 0.1. Nutrient solutions were renewed every 5 d. Four Al levels (0, 100, 200 and 300 uM) in the form of AlCl3 , three sulfate levels (100, 200 and 300 uM) in the form of CaSO4 , and three pH levels (4.0, 5.0 and 5.5) were supplied. The pH was kept constant daily with diluted NaOH or HCl. The phosphate concentration was fixed in all pots at 100 uM. The experiment was conducted in a controlled-environment growth chamber for 90 d at 25 ± 2◦ C and 50%–60% relative humidity, with a 16 h photoperiod at a photon flux density of 500–600 uM s−1 m−2 . After 90 d of the experiment, root and shoot lengths and dry weights were measured; values obtained for plants grown without Al were considered to be 100%. Aluminium tolerance indices were expressed as percentages of relative dry weight (% RTI), relative root length (%RRL), and relative shoot length (%RSL). Plant samples were dried by separating shoots and roots and placing them in a 70◦ C forced-air oven for 48 h. After being weighed, plant samples were ashed at 500◦ C for 8 h and then measured for Al and phosphorus (P) content by digestion with 2 M hydrocloric acid. Aluminium was measured by atomic absorption and P colorimetrically by the vanadophosphomolybdate method. For measurement of the sulphur (S) content, the plant material was ashed at 500◦ C with magnesium nitrate, digested with 2 M hydrochloric acid, and measured turbidimetrically. All methodology is described in Sadzawka et al. (2000); the results were expressed as g kg−1 dry weight. A modified GEOCHEM computer program (version 2.0) was used to evaluate chemical Al species in solution (Sposito and Mattigod, 1980) in each treatment. The experimental design was a completely randomized block with three replicates. Analysis of variance (ANOVA) and the Tukey multiple range test were calculated at each pH using the SPSS computer program. A probability level of less than 0.05 was considered to be statistically significant.
RESULTS AND DISCUSSION In plants grown without Al in the nutritive solution (ryegrass seeds initially contained 0.23 g/kg Al), when the sulfate concentration was maintained at 100 uM,
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an increase in pH caused an increase in shoot dry weight (Table 1). Similarly, when pH was held constant at 4.0, an increase in the sulfate concentration from 200 to 300 uM increased shoot dry weight. However, when pH was set at 5.5, shoot dry weight decreased, probably as a consequence of nutrient imbalance produced by the presence of sulfate anion. When Al was added to the mineral solution, it showed a significant effect, stronger than that of pH, on both shoot and root dry weight (Tables 1 and 2). A very sensitive relationship between Al root content and root dry weight was observed (r = −0.759∗∗ ; p ≤ 0.01) (Figure 1), as well as one between Al root content and shoot dry weight (r = −0.742∗∗ ; p ≤ 0.01) (Figure 2, Table 3), in agreement with results reported by Fageria et al. (1989). At pH 4.0, P root Al content was lowest in plants grown with no Al (Table 2) and showed no significant difference at increasing sulfate concentrations in the solution. This result probably occurred because P was being used mainly as a nutrient. The S root content, on the other hand, increased significantly (P < 0.05) with increasing sulfate concentrations. When Al was added to the solution, the opposite happened. As the sulfate concentration was increased, S root content decreased due to sulfate complexes formed with the Al in solution. This can be seen in Table 4, which shows that in going from 100 to 300 uM sulfate at pH 4.0 and 100 uM Al, the percentage of AlSO+ 4 in solution increased from 4.56% to 12.72%, and Al+3 decreased from 60.17% to 54.82%; the same trend was observed with 200 µM Al. At the same time, Al root content dropped significantly, from 4.98 to 3.02 g kg−1 at 100 µM Al and from 9.19 to 5.82 g kg−1 at 200 µM Al (Table 2). Furthermore, in these conditions Al shoot content was always below 2.0 g kg−1 (Table 1), indicating that formation of the complex AlSO+ 4 in solution presented Al uptake by the plant and confirming the non-phytotoxicity of AlSO+ 4 previously reported by Cameron et al. (1986). The mechanism through which this charged species is made less toxic is not clear, but Alva et al. (1991) have indicated that sulfate complexed in the outer sphere of Al could be excluded through a steric shielding effect by plant roots. This important exclusion mechanism is regulated by the sulfate concentration in solution; the relationship between S and Al root content presents a good correlation (Table 2). At pH 5.0 and 5.5, on the other hand, AlSO+ 4 complexes were negligible (Table 4). Even though less Al accumulated in roots at pH 5.0 and 5.5 than at pH 4.0 (Table 2), levels were still high and independent of pH. This result may be because Al uptake is regulated by an acid hydrolysis mechanism (Skyllberg et al., 2001). By this process, the root Al absorption compensates for the proton release. Table 3 shows that the presence of free Al+3 drops drastically at pH levels greater than 4.0. Therefore, it is possible to hypothesize that proton release from root cation exchange or root organic acid exudates dissolves Al(OH)3 present in the rhizosphere, producing Al+3 in the solution available for root uptake.
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1.62 bc 2.00 ab 2.21 a
1.48 c 1.38 cd 1.31 cd
1.04 de 0.85 e 0.92 e
Sulfate (µM)
100 200 300
100 200 300
100 200 300
1.72 a 1.72 a 1.85 a
1.14 c 0.85 d 1.38 b
0.87 d 0.56 e 0.95 cd
Al shoot content
3.20 ab 3.49 a 2.99 abc
2.79 bc 2.72 bc 2.58 cd
1.90 e 2.04 e 2.09 de
P shoot content
1.64 a 1.33 bc 1.59 a
1.22 c 1.54 ab 1.31 bc
1.19 c 1.32 bc 1.61 a
S shoot content
2.36 b 2.16 b 2.70 ab 2.46 b 2.48 b 2.70 ab 3.19 a 2.45 b 2.30 b
0 µM Al 1.04 b 0.89 c 0.77 d
100 µM Al 1.71 ab 0.91 c 1.14 cd 0.72 d 1.77 ab 1.03 b 200 µM Al 1.03 d 1.04 b 1.11 cd 1.45 a 1.19 cd 1.06 b
2.11 a 1.62 bc 1.90 ab
P shoot content
Al shoot content
Shoot dry weight
5.0
1.87 a 1.64 bc 1.95 a
1.32 c 1.61 bc 1.79 ab
1.87 a 1.60 bc 1.55 c
S shoot content
1.22 b 1.47 b 1.19 b
1.29 b 1.13 b 1.26 b
2.37 a 1.97 a 1.51 b
Shoot dry weight
0.67 cd 0.92 b 1.14 a
0.57 d 0.67 cd 0.83 b
0.55 d 0.64 cd 0.76 bc
Al shoot content
2.84 abc 2.21 d 2.68 c
2.80 abc 2.70 bc 2.54 cd
2.14 d 3.09 ab 3.20 a
P shoot content
5.5
1.73 ab 1.67 abc 1.56 bc
1.55 bc 1.83 a 1.47 c
1.13 d 1.55 bc 1.70 abc
S shoot content
Values are means. Values with the same letter within a column are not significantly different (P < 0.05) according to Tukey’s multiple comparison test; each pH is analyzed independently.
Shoot dry weight
4.0
pH
Table 1 Influence of sulfate in nutritive solution on shoot dry weight (g pot−1 ) and aluminum, phosphorus, and sulfur content (g kg−1 ) in ryegrass at varying Al concentrations and pH levels
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0.95 a 0.92 b 0.92 b
0.91 b 0.74 bc 0.89 bc
0.41 d 0.49 cd 0.61 cd
Sulfate (µM)
100 200 300
100 200 300
100 200 300
9.19 b 9.91 a 5.82 c
4.98 d 3.99 e 3.02 f
1.47 g 1.51 g 1.40 g
Al root content
4.51 a 4.81 a 3.33 b
3.03 bc 2.72 cd 2.57 de
2.13 ef 2.25 ef 2.18 ef
P root content
1.52 abc 1.72 a 1.36 bcd
1.13 de 1.21 d 0.85 e
1.07 de 1.22 cd 1.62 ab
S root content
P root content
1.89 e 2.55 c 2.30 d 2.36 cd 3.23 a 2.16 d 3.26 a 2.30 d 2.82 b
Al root content
0 µM Al 1.21 a 1.44 d 1.07 ab 1.37 de 0.89 ab 1.07 e 100 µM Al 0.96 ab 2.25 c 0.90 ab 2.30 c 0.85 abc 2.29 c 200 µM Al 0.51 c 6.31 a 0.77 bc 5.20 b 0.71 bc 4.98 b
Root dry weight
5.0
1.60 a 1.11 bc 1.65 a
0.84 c 1.20 b 1.06 bc
0.83 c 1.20 b 1.12 bc
S root content
0.66 b 0.60 b 0.51 b
0.71 ab 0.73 ab 0.70 ab
0.98 a 0.96 a 0.79 ab
Root dry weight
5.77 a 5.80 a 5.95 a
3.75 c 3.70 c 3.51 c
1.91 d 0.95 e 1.36 de
Al root content
3.19 ab 2.97 bc 3.42 a
2.58 cd 2.89 bc 2.61 cd
2.05 e 2.30 de 1.93 e
P root content
5.5
1.15 abc 1.24 abc 1.44 a
0.91 c 1.30 ab 0.92 c
0.93 c 1.02 bc 1.08 abc
S root content
Values are means. Values with the same letter within a column are not significantly different (P < 0.05) according to Tukey’s multiple comparison test; each pH is analyzed independently.
Root dry weight
4.0
pH
Table 2 Influence of sulfate in nutritive solution on root dry weight (g pot−1 ) and aluminum, phosphorus, and sulfur content (g kg−1 ) in ryegrass at varying Al concentrations and pH levels
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Figure 1. Relationship between ryegrass root Al content (g kg−1 ) and root dry weight (g pot−1 ).
Nevertheless, some change in plant sensitivity to Al may be accounted for by pH-dependent responses to Al. Raising the pH from 4.0 to 5.0 relieved Al toxicity because free Al3+ species decreased to below 1%, and at pH 5.5 Al3+ no longer existed (Table 4). At the same time, Al(OH)3 content increased to more than 97% and 99% at pH 5.0 and 5.5, respectively. Relative root length (RRL) was used as an Al tolerance parameter for the ryegrass (Table 5), which showed greater overall Al tolerance than previous results from a short-term study of the same species (Gallardo et al., 1999). The RRL values in this study ranged between 84%–95% at 100 µM Al and 65%–89% at 200 µM Al. Relative root weight (RTI) was more sensitive to Al toxicity than RRL or RSL, making it the best indicator of these parameters; this is consistent with findings for ryegrass by Rengel and Robinson
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Figure 2. Relationship between ryegrass root Al content (g kg−1 ) and shoot dry weight (g pot−1 ).
(1989). Furthermore, root dry weight was a good predictor of shoot dry weight. Different sulfate concentrations in mineral solution favored differential Al tolerance in the ryegrass; sulfate in solution showed a positive effect on RRL only at concentrations up to 200 uM. As observed by Mora et al. (1999) in a study of acidified Chilean Andisols using gypsum, increased levels of sulfate caused considerable increases in RTI values, another indication that AlSO+ 4 complex formation in solution is an effective Al detoxifying mechanism. Sulfur and P root content were negatively correlated with shoot dry weight, (r = −0.619∗∗ and −0.747∗∗ , respectively; p ≤ 0.01) (Figures 3 and 4, Table 3), and the aluminum:phosphorus molar ratio in roots was negatively correlated with shoot dry weight, (r = −0.816∗∗ ; p ≤ 0.01) (Figure 5, Table 3),
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−0.742∗∗ −0.759∗∗
0.617∗∗
−0.747∗∗ −0.653∗∗ 0.944∗∗
P root content −0.619∗∗ −0.519∗∗ 0.683∗∗ 0.607∗∗
S root content −0.511∗∗ −0.442∗∗ 0.705∗∗ 0.695∗∗ 0.425∗∗
Al shoot content −0.429∗∗ −0.487∗∗ 0.530∗∗ 0.490∗∗ ns 0.419∗∗
P shoot content −0.265∗ ns ns ns 0.269∗ ns ns
S shoot content
−0.816∗∗ −0.568∗∗ 0.750∗∗ 0.770∗∗ 0.563∗∗ 0.533∗∗ 0.259∗ ns
Al:P root molar ratio
Correlations were calculated using the JMP computer program Version 5, where ∗ is significant at
