Aluminum-stress response in oat genotypes with monogenic tolerance

Environmental and Experimental Botany 74 (2011) 114–121

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Aluminum-stress response in oat genotypes with monogenic tolerance Graciela Castilhos a , Júlia Gomes Farias b , Adriano de Bernardi Schneider a , Paulo Henrique de Oliveira a , Fernando Teixeira Nicoloso b , Maria Rosa Chitolina Schetinger c , Carla Andréa Delatorre a,∗ a Programa de Pós-graduac¸ão em Fitotecnia, Departamento de Plantas de Lavoura, Faculdade de Agronomia, Universidade Federal do Rio Grande do Sul, 91501-970 Porto Alegre, RS, Brazil b Programa de Pós-Graduac¸ão em Agrobiologia, Departamento de Biologia, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil c Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil

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Article history: Received 22 June 2010 Received in revised form 4 May 2011 Accepted 5 May 2011 Keywords: Avena sativa Oxidative stress Aluminum localization Regrowth

a b s t r a c t Aluminum (Al) toxicity is one of the limiting factors in agricultural productivity on acid soils. The oat genotypes UFRGS17 and UPF91Al100-1-4 have been developed through different breeding programs and are classified as Al-tolerant. Genetic analysis indicated that the Al tolerance in each genotype is conferred by one gene. Pyramiding Al tolerance genes may increase the tolerance level. The objectives of this study were to determine whether the Al-tolerance observed in both genotypes is conferred by different genes and to investigate the tolerance mechanisms. The aluminum tolerance of the F2 population derived from the UFRGS17 × UPF91Al100-1-4 cross was estimated by using the regrowth of the primary root, and the tolerance was compared to the parental frequency distribution. The tolerance mechanism was investigated in seedlings after Al exposure for seven days. Al accumulation in the root apex was visualized, and the activity of antioxidant enzymes, the content of non-protein thiol groups and the concentrations of ascorbic acid, superoxide and H2 O2 in the root apex, the differentiated root region and the shoots were measured. Al tolerance in UFRGS17 and UPF91Al100-1-4 is conferred by the same gene. A diverse pattern of Al accumulation was obtained for the genotypes, suggesting that the external detoxification is not the main mechanism of tolerance. The tolerant genotypes were able to manage the oxidative stress caused by Al on the other hand the sensitive genotype showed higher oxidative stress and lipid oxidation. The relative importance of each antioxidant component could not be established because different responses were observed between the Al-tolerant genotypes. It is possible that the Al-tolerance gene codifies an upstream component, such as a transcription factor, which once induced by Al, ends up activating, among other pathways, the antioxidative system. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Aluminum (Al) toxicity is an important limitation to yield in acid soils or fields with subsoils that have a pH below 5 (Von Uexküll and Mutert, 1995). The most distinct symptom of Al toxicity is the inhibition of root growth, which limits the capacity of the plant to explore the soil, affecting the uptake of water and nutrients (Kochian, 1995; Sivaguru et al., 2000). The main mechanism causing root growth inhibition in response to Al is unknown. Tolerance to Al is observed in some plant species and varies among genotypes (Delhaize and Ryan, 1995; Huan-Xin et al., 2009; Lisitsyn, 2000; Liu et al., 2007; Nava et al., 2006). External and internal mechanisms may be used to cope with Al toxi-

∗ Corresponding author at: UFRGS, Faculdade de Agronomia, Departamento de Plantas de Lavoura, PO Box 15100, 91501-970, Porto Alegre, RS, Brazil. Tel.: +55 51 33086005; fax: +55 51 33086576. E-mail address: [email protected] (C.A. Delatorre). 0098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2011.05.007

city (Huang et al., 2009; Kochian, 1995; Kochian et al., 2004; Liu et al., 2007). In cereals, the Al tolerance has been suggested to depend on organic acid secretion, reducing the entrance of Al into the root (Ligaba et al., 2009; Liu et al., 2007, 2009; Matsumoto, 2005; Ryan and Delhaize, 2010; Sasaki et al., 2004). However, other mechanisms may also be used for tolerance, as observed in maize (Giannakoula et al., 2010) and rice (Huang et al., 2009). The existence of different mechanisms might explain differences in the Al tolerance levels, both within and between species. Pyramiding mechanisms may allow for the development of more tolerant crops, but their development requires detailed information about mechanisms and interactions. Oats are considered one of the most Al tolerant small grains; yet research on the Al tolerance systems in oats is scarce. Genetic studies indicate that oat Al tolerance is controlled by one or two dominant genes (Nava et al., 2006; Oliveira et al., 2005; SanchezChacon et al., 2000; Wagner et al., 2001), but there is no data about the tolerance mechanism.

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Different methodologies have been used to classify genotypes as Al-tolerant. For oats, the most efficient methodology uses the regrowth rate of the primary root after exposure to Al (Sanchez-Chacon et al., 2000). The oat genotypes UFRGS17 and UPF91Al100-4-1 have been classified as Al-tolerant (Nava et al., 2006; Oliveira et al., 2005; Wagner et al., 2001). Based on the analysis of F6 inbred lines from UFRGS17 × UFRGS930598 (Al-sensitive) and UPF91Al100-4-1 × UFRGS930598, it was established that the tolerance of both Al-tolerant genotypes was controlled by only one gene found in each genotype (Nava et al., 2006; Oliveira et al., 2005). A previous study had shown limited Al access to the internal tissues of UFRGS17 roots if compared to UFRGS930598, suggesting that external detoxification by organic acid secretion might be the main tolerance mechanism (Limberger et al., unpublished). Several reports have shown increases in the reactive oxygen species (ROS) content due to Al, leading to oxidative stress (Boscolo et al., 2003; Cakmak and Horst, 1991; Ezaki et al., 2000; Jones et al., 2006; Ribeiro et al., 2010; Tabaldi et al., 2009; Tamás et al., 2006; Yamamoto et al., 2003; Yin et al., 2010b). Despite being a non-transition metal, Al has a pro-oxidant activity via the formation of the aluminum superoxide semi reduced radical ion (Exley, 2004). The ability to reduce the oxidative stress after Al exposure may be an important component of Al tolerance (Giannakoula et al., 2010; Jones et al., 2006; Tamás et al., 2006; Yin et al., 2010a). There are indicators that suggest that the tolerance of UFRGS17 to Al may be related to oxidative stress (Pereira et al., 2011). The identification of different mechanisms in Al-tolerant oat genotypes would open the possibility of pyramiding and increasing Al tolerance. To test the hypothesis that the monogenic Al tolerance of both UFRGS17 and UPF91Al100-4-1 is due to different genes, the aluminum tolerance of the F2 population derived from the UFRGS17 × UPF91Al100-1-4 cross was estimated by using the regrowth of the primary root, and the tolerance was compared to the parental frequency distribution; and to test the hypothesis that there were two different tolerance mechanisms, one based on reduction of oxidative stress and another on external detoxification, the activity of antioxidant enzymes, the content of non-protein thiol groups and the concentrations of ascorbic acid, superoxide and H2 O2 in the root apex, the differentiated root region and the shoots were measured, as well as the Al accumulation in the root apex was visualized. 2. Materials and methods 2.1. Plant material The aluminum tolerant oat genotypes UFRGS17 and (COR2/CTZ3/PENDEK/ME1563/76-29-76-23/75-28/CI833) UPF91Al100-1-4 (8014/301/CRcps/SRcpx/JHG-8) were obtained from the UFRGS Oat Breeding Program and the UPF Oat Breeding Program, RS, Brazil. These genotypes were used in the mechanism studies. The aluminum-sensitive line UFRGS930598 (UFRGS15/UFRGS881920) was used as a negative control. The F2 population from the UFRGS17 × UPF91Al100-1-4 cross and the parental genotypes were used in the genetic study. 2.2. Genetics of Al tolerance Seeds from the parental genotypes and from the six F2 populations from the UFGRS17 × UPF91Al100-1-4 cross were hulled, disinfected with sodium hypochlorite at 2% for 3 min and rinsed three times in sterile distilled water. About 100 individuals were evaluated for each F2 population. For each parental genotype and for UFRGS930598 at least 100 seeds were analyzed. The rate of root regrowth was evaluated using the methodology described by

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Nava (2005). Sterilized seeds were transferred onto germination paper and kept at a constant temperature (19 ± 2 ◦ C). Seedlings with 1 cm-long roots were arranged on plastic pot lids adapted with plastic screens, these were then placed over 5 l plastic pots kept at 22 ◦ C ± 2 ◦ C and in constant light. The hydroponic solution was aerated and the pH was adjusted to 4.5. Two hydroponic solutions were used. Pre-germinated seeds were first grown for 48 h in a solution free of aluminum; they were then transferred into a solution with aluminum for another 48 h and finally transferred back to a the aluminum-free solution for an additional 72 h. The root regrowth of the primary root of each seedling was measured starting from the point of root thickening (caused by Al). The aluminum-free solution referred to as the complete nutrient solution contained 4 mM Ca(NO3 )2 ·4H2 O, 2 mM MgSO4 ·7H2 O, 4 mM KNO3 , 0.435 mM (NH4 )2 SO4 , 0.5 mM KH2 PO4 , 2 ␮M MnSO4 ·1H2 O, 0.3 ␮M CuSO4 ·5H2 O, 0.8 ␮M ZnSO4 ·7H2 O, 30 ␮M NaCl, 0,1 ␮M Na2 MoO4 ·2H2 O, 10 ␮M H3 BO3 . The iron source was a high performance iron chelate, comprised of 6% of chelate iron and 92% of an orthoisomer, with 0.9 mM of iron in the solution. The aluminumsolution was at a concentration of one tenth of the complete solution. To avoid complexing with aluminum, the KH2 PO4 was replaced by KCl. The aluminum source was aluminum sulfate (Al2 (SO4 )3 ·18H2 O) at the final concentration of 740 ␮M, which has shown better constancy in distinguishing Al tolerance between oat genotypes (Nava et al., 2006; Sanchez-Chacon et al., 2000; Wagner et al., 2001). The experiment was repeated twice. 2.3. Mechanism studies 2.3.1. Growth conditions Seeds from UFGRS 17, UPF91Al100-1-4 and UFRGS930598 (Alsensitive) were treated as described in Section 2.2. Seedlings with approximately 2 cm-long roots were arranged on plastic pot lids adapted with plastic screens, these were placed over 5 l plastic pots filled with an aerated nutrient solution. The nutrient solution was changed every 48 h and was equal in composition to the complete solution described in Section 2.2, except for the replacement of KH2 PO4 with KCl. The aluminum stress was induced by adding 740 ␮M AlCl3 ·6H2 O. The Al source was altered because the toxic effect is more pronounced with AlCl3 than with Al2 (SO4 )3 – this result may be a consequence of their differential solubility. Phosphate was omitted from the solution to avoid interaction with Al ions, which would reduce the Al activity (initial Al3+ activity was 6.46 × 10−6 evaluated by Minteq). The pH of the solution was kept adjusted to 4.5. The plants were grown in a growth chamber at 22 ± 2 ◦ C and with constant light for seven days. Each biological replicate contained 250 seedlings. 2.3.2. Enzyme extraction and quantification The shoot, root and the root tip from the seedlings were separated, and the individual tissues were homogenized in 100 mM phosphate buffer (pH 7.5), 1 mM EDTA, 3 mM dl-dithiothreitol and 5% (m/v) insoluble PVP. The homogenate was centrifuge at 10,000 × g for 30 min at 4 ◦ C and the resultant supernatant used for the enzyme assay. Protein concentration was determined following Bradford (1976) using bovine serum albumin as the standard. 2.3.3. Enzyme activities Catalase (CAT) activity was assayed following the methodology described by Azevedo et al. (1998) with small modifications. The degradation of H2 O2 at 25 ◦ C was measured by the absorbance at 240 nm every 10 s for 1 min in a solution containing 1 mL of 100 mM phosphate buffer (pH 7.5), 2.5 ␮L of 30% H2 O2 (v/v) and 25 ␮L plant extract. Activity was expressed as ␮mol min−1 mg−1 protein. Glutathione reductase (GR) and ascorbate peroxidase (APX) activities were measured according to Azevedo et al. (1998) and Nakano and

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Asada (1981), respectively. Superoxide dismutase (SOD) activity was evaluated in a native 10% polyacrylamide gel assay. Samples containing 30 ng of protein were added to the gel and 30 mA were applied for 3 h at 4 ◦ C. The activity bands were revealed following the method described by Beauchamp and Fridovic (1971) and modified by Azevedo et al. (1998). 2.3.4. Estimation of the lipid peroxides and hydrogen peroxide content, as well as the histological staining of superoxide and hydrogen peroxide The method of El-Moshaty et al. (1993) was used to determine the lipid peroxide content and to evaluate membrane integrity. The lipid peroxides were expressed as nmol malondialdehyde (MDA) mg−1 protein. Hydrogen peroxide content was assayed using the method described by Loreto and Velikova (2001). Histological staining of the superoxide in 2 cm-long root segments was conducted as described by Wang et al. (2007). The method of Thordal-Chistensen et al. (1997) was used for the histological staining of hydrogen peroxide in 2 cm-long root segments. 2.3.5. Ascorbic acid (AsA) and non-protein thiol group (NPSH) quantification Oat tissue was homogenized in a solution containing 50 mM Tris–HCl (pH 7.5), after centrifugation at 3000 rpm for 10 min, 10% TCA at a proportion 1:1 (v/v) was added to the supernatant. A new centrifugation was carried out to remove the protein. Ascorbic acid was quantified using the procedure described by Jacques-Silva et al. (2001). The non-protein thiol concentration was assayed spectrophotometrically. An aliquot of the sample (400 ␮L) was added to 550 ␮L of 1 M phosphate buffer (pH 7.4) and 50 ␮L of 10 mM 5-5-dithio-bis 2-nitrobenzoic acid. A cysteine standard curve was used to calculate the amount of thiol groups in the samples. 2.3.6. Al-staining Roots were washed in distilled water for 15 min and stained with 0.2% hematoxylin and 0.02% KIO3 for other 15 min at room temperature. The roots were fixed using an ethanol and xylol series. Segments measuring 1 cm and taken from the root tip were embedded in paraffin using standard histological procedures. Transversal cuts (10 ␮m) were obtained using a microtome and were observed using an optical microscope.

Fig. 1. Primary root regrowth distribution of the F2 population from UPF91Al1001-4 × UFRGS17 cross and parental genotypes.

sensitive control UFRGS930598. Data from the biochemical analysis were submitted to an analysis of variance and a correlation analysis. Treatment means were compared using Duncan’s range test at a 5% error probability. 3. Results Initially seven oat genotypes were screened by Al tolerance using the regrowth method. UFRGS17 and UPF91Al100-1-4 presented higher tolerance and were chosen for this study, in contrast UFRGS930598 showed the highest sensitivity and was chosen as the negative control (Table S1). 3.1. Al-tolerance in the UFRGS17 × UPF91Al100-1-4 F2 populations The parental genotypes showed statistically similar means for the main root regrowth after exposure to Al (UFRGS17 = 12.5 ± 2.9, UPF91Al100-1-4 = 16.9 ± 5.1), and the regrowth differed from the Al-sensitive genotype UFRGS930598 (5.9 ± 1.8). Great amplitude in the primary root regrowth was observed for both parent genotypes; UFRGS17 regrowth varied from 5.1 to 27.5 mm, UPF91Al100-4-1 varied from 4.9 to 30.3 mm and the Al-sensitive UFRGS930598 from 3.1 to 11.6 mm. The frequency distribution of the F2 populations was similar to those obtained for the parental genotypes, indicating that the Al tolerance in the parental genotypes is controlled by the same gene (Fig. 1).

2.4. Statistical analysis 3.2. Al localization The experiments followed a completely randomized design. The frequency distribution of the data from the regrowth analysis was used to estimate the number of genes involved in the tolerance. The threshold between aluminum sensitive and tolerant genotypes was based on the root regrowth frequency distribution shown by the

Al accumulated in different tissues within the Al-tolerant genotypes. In the UFRGS17 roots, Al was observed mainly in the epidermal cells (Fig. 2B), however in UPF91Al100-1-4 Al was observed also in the cortex and in the vascular tissue (Fig. 2C). The

Fig. 2. Aluminum localization by hematoxylin staining in transversal cuts (10 ␮m) of root apex from Avena sativa genotypes after seven days of exposure to aluminum (740 ␮M). (A) UFRGS930598, (B) UFRGS17 and (C) UPF91Al100-1-4 (optical magnitude 200×).

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Al-accumulation pattern for the UPF91Al100-1-4 roots was very similar to the one observed for the UFRGS930598 roots, which is an Al-sensitive genotype (Fig. 2). 3.3. Antioxidative metabolism The Al-sensitive oat genotype UFRGS930598 was also used for comparison in the antioxidative metabolism analysis. The responses of the antioxidative metabolism to Al were affected by the organ and the genotype. The triple interaction (Al x genotype x organ) was significant (˛ = 0.01) for all of the traits evaluated, except for the H2 O2 content, which showed an interaction between genotypes and organs. Negative correlation was observed between NPSH (r = −0.80, ˛ = 0.01), AsA (r = −0.74, ˛ = 0.01), H2 O2 (r = −0.30, ˛ = 0.5), CAT (r = −0.73, ˛ = 0.01) and lipid peroxide (MDA). The AsA (r = 0.56, ˛ = 0.5) and NPSH (r = 0.76, ˛ = 0.01) content were positively correlated to the CAT activity. 3.3.1. Effects of Al on the enzymatic antioxidative metabolism Three SOD isoforms were observed, and these isoforms were named SOD1, SOD2 and SOD3 based on their gel position. SOD1 and SOD2 showed higher activity in the roots, whereas SOD3 was more active in the shoots (Fig. 1). The Al-tolerant genotypes had contrasting responses in the differentiated root zone, the UFRGS17 genotype had increased the SOD activity, mostly in with respect to SOD1; conversely, UPF91Al1001-4 showed reduction in SOD activity. No Al effect was observed in the Al sensitive genotype at this root region. However, the presence of Al in the root tip reduced the SOD activity in the sensitive genotype and increased it in the tolerant ones; in this case SOD2 was the main isoform affected in UFRGS17. SOD3 activity was elevated in the shoots and was similar between the genotypes and independent of Al presence. However, the SOD1 and 2 activities were increased by Al in UFRGS17 and UFRGS930598 shoots (Fig. 3).

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In the absence of Al, APX activity was much higher in the UFRGS17 roots and was more than 4 times the activity observed in UPF91Al100-1-4 roots. However, after seven days of Al exposure the activity was reduced to values similar to the UPF91Al100-1-4 roots. No Al-effect was observed in UPF91Al100-1-4 in any organ (Fig. 4A). The CAT activity was also higher in the UFRGS17 and to a lesser extent in UPF91Al100-1-4, however, this effect was seen only in the differentiated root region and in the absence of Al. After Al exposure the activities in the differentiated root region were reduced by 90 and 85% respectively. Al did not affect the CAT activity in the roots of the Al sensitive oat genotype. Interestingly, the CAT activity was kept high in the shoots independent of Al presence; the exception was the Al-sensitive genotype in which the activity was reduced by half by Al (Fig. 4B). In addition, UFRGS17 roots showed increased GR activity in response to Al, not only in the differentiated region (approximately two times), but also in the root tip (four times). No effect of Al was observed in UPF91Al100-1-4 roots (Fig. 4C).

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Fig. 3. Effect of aluminum (740 ␮M) over seven days on SOD isoform activity in the root tip, differentiated root region and shoot of Avena sativa genotypes UFRGS17 (1), UFRGS930598 (2) and UPF91Al100-1-4 (3).

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Fig. 4. Antioxidant metabolism on the root tip, the differentiated root region and the shoot of Avena sativa genotypes UFRGS17, UPF 91Al100-1-4 (Al-tolerant) and UFRGS930598 (Al-sensitive) after seven days of exposure to aluminum (740 ␮M). Ascorbate peroxidase activity (A), catalase activity (B), glutathione reductase activity (C), non-protein thiol group content (D) and ascorbic acid content (E). Means and SD are shown. Capital letters compare genotypes inside the dose and organ, and small letters compare Al doses inside genotype and organ by Duncan’s test at ˛ = 0.05.

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Fig. 5. Hydrogen peroxide (A) and lipid peroxide (B) accumulation on root tip, differentiated root region and shoots of Avena sativa genotypes UFRGS17, UPF91Al100-1-4 (Al-tolerant) and UFRGS930598 (Al-sensitive) after seven days of exposure to aluminum (740 ␮M). Means and SD are shown. Capital letters compare genotypes inside the dose and organ, and small letters compare Al doses inside genotype and organ by Duncan’s test at ˛ = 0.05.

the UFRGS17 shoots and reduced them in UPF91Al100-1-4 and in the Al sensitive UFRGS930598. Small differences were observed in the roots with the presence of Al in the tolerant genotypes, which showed higher NPSH levels than the sensitive ones in the differentiated root region. UFRGS17 also had higher NPSH content in the root tip (Fig. 4D). Another important antioxidant, AsA, was affected by Al presence in the roots. The AsA content increased in both of the Al-tolerant oat genotypes, but AsA content was reduced in the Al-sensitive genotype. UFRGS17 presented higher amounts of AsA under Al conditions. No difference was observed in the AsA content in the shoots between treatments or genotypes (Fig. 4E). 3.3.3. Effect of Al on hydrogen peroxide, superoxide and lipid peroxide levels It was not possible to visualize the significant differences in the H2 O2 levels in the roots among genotypes using histological staining. H2 O2 was present in the epidermal cells independent of Al presence, and a slight increase in H2 O2 was observed after seven days of Al exposure (Supplemental 1). Neither of the spectrophotometric measurements was able to differentiate between the Al-tolerant oat genotypes in the presence of Al. The Al-sensitive genotype, UFRGS930598, presented a significant increase in H2 O2 content in all organs evaluated after seven days of exposure to 740 ␮M Al. Conversely, both Al-tolerant genotypes showed no change in the level of H2 O2 in the root (Fig. 5A). The amount of superoxide staining in the roots was considerably different between the Al-tolerant oat genotypes after seven days of Al exposure (Supplemental 2). The UPF91Al100-1-4 roots showed much less superoxide accumulation; when there was accumulation it was mainly in the vascular tissue. In UFRGS17, the accumulation of superoxide was more extensive, but still less than in the Al-sensitive oat genotype (Supplemental 2). Lipid peroxide was evaluated using the MDA content. Little effect was seen on lipid peroxidation due to the Al in the roots in Altolerant oat genotypes (Fig. 5B). In the roots submitted to Al stress, the levels of MDA were smaller for UFRGS17 than for UPF91Al1001-4. No difference was observed between these two genotypes in the shoots in the presence of Al. UFRGS930598 had increased the MDA levels in both roots and shoots in the presence of Al (Fig. 5B). 4. Discussion Al presence is harmful to plants, and the first symptom of Al-induced damage is the inhibition of root growth. Different strategies have been developed to evaluate Al tolerance. In oats, the most efficient method is the analysis of root regrowth after exposure to Al (Sanchez-Chacon et al., 2000). This methodology correlates well with plant aluminum tolerance per se (Nava et al., 2006). In this work, this methodology was used to verify if the Al

tolerance of UFRGS17 and UPF91Al100-1-4 were controlled by the same gene. The Al-tolerance was confirmed for both parental genotypes. The Al-sensitivity was again observed in UFRGS930598, the negative control. However, the genotypes showed great amplitude of regrowth. Similar amplitudes had been observed previously (Nava et al., 2006; Oliveira et al., 2005). Variations from residual heterozygosis at the Al tolerance locus are not expected for UFRGS17, an oat variety developed in 1996. Additionally, the seeds used in this study were derived from individual panicles. Nava et al. (2006) suggest that the amplitude may be derived from the incomplete expression of the dominant allele, or from the intrinsic variation of the methodology. Other factors that are not genetic may be involved in the Al tolerance expression. Lisitsyn (2000) suggests the strong dependence of Al tolerance on the conditions during seed development. The effect of environmental conditions, such as temperature, on oat Al tolerance has also been observed (Limberger, 2006). If the tolerance mechanism requires changes in the plant metabolism, it is expected that other environmental and internal signals modulate its expression. Based on the frequency distribution for the F2 populations and the parental genotypes, the hypothesis of two different genes cannot be accepted. If different genes were controlling the Al-tolerance in each parental genotype an expressive number of Al-sensitive F2 individuals would be expected. The amplitude of the F2 population was smaller than the parental amplitude (Fig. 1), thus both parental genotypes contain the same major gene controlling Al tolerance. The tolerance mechanism and the function of the only major gene controlling Al tolerance in these two oat genotypes are not known. The most common tolerance mechanism in cereals is related to organic acid secretion and to the external Al detoxification (Fontecha et al., 2007; Liu et al., 2009; Liu et al., 2007; Magalhães et al., 2007; Matsumoto, 2005). Field studies using recombinant inbred lines from the UFRGS17 × UFRGS930598 cross indicate that the Al tolerance mechanism of UFRGS17 is due not to a constitutive trait, but to an inducible one (Nava et al., 2006). Previous studies indicated that UFRGS17 was able to limit the entrance of Al in the root tissues (Limberger et al., unpublished), and this action was confirmed here. However, UPF91Al100-1-4 was not efficient in limiting Al entrance since Al was observed in the cortex and in the vascular tissue in the root tip of this Al-tolerant genotype (Fig. 2). Based on the discrepancy in Al accumulation between the genotypes, the hypothesis that the gene conferring Al tolerance acts by external detoxification could not be confirmed. Al accumulation occurs mainly in the cell apoplast where Al slowly crosses the cell membrane and increases in concentration to toxic levels (Vazquez et al., 1999). Al can give rise to the production of reactive oxygen species (ROS) in plant cells (Cakmak and Horst, 1991; Meriga et al., 2004; Mohan Murali Achary et al., 2008; Yamamoto et al., 2003). Maize, tobacco, soybeans and potato

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tolerant genotypes reduce the production of ROS through Al stress, leading to reduced oxidative stress (Darkó et al., 2004; Du et al., 2010; Giannakoula et al., 2010; Tabaldi et al., 2009; Yin et al., 2010a). The possibility that the Al-tolerance in both genotypes could be related to an efficient antioxidative system was evaluated. The data obtained in this study corroborate the relation between Al toxicity and ROS production. The Al-sensitive oat genotype UFRGS930598 showed higher H2 O2 and superoxide levels, as well as higher lipid peroxide in the roots in response to Al; whereas the Al-tolerant oat genotypes showed smaller values for both traits, suggesting the ability to cope with the Al-induced ROS production after seven days of exposure (Supplemental 1, 2 and Fig. 5). Reduced levels of lipid peroxide and ROS indicate that the antioxidative system was effective in both Al-tolerant oat genotypes. To cope with the oxidative stress, plants have evolved several enzymatic and non-enzymatic mechanisms including peroxidases, superoxide dismutases, catalases, glutathione reductase and other low-molecular mass antioxidants such as ascorbic acid, glutathione and carotenoids (Miller et al., 2008; Mittler et al., 2004; Noctor et al., 2002). Thus, the activities of enzymes involved in this process were evaluated aiming to verify if any of them could play a main role in Al tolerance. Negative correlation between lipid peroxide and NPSH and AsA content and CAT activity was found. Three SOD isoforms were identified in the oat tissues; of these three isoforms, only the activity of SOD3 was not affected by Al (Fig. 3). An increase in SOD activity was observed for both Al-tolerant oat genotypes UFRGS17 and UPF91Al100-1-4. In maize and soybean, Al tolerant genotypes also showed increased SOD activity upon Al exposure (Du et al., 2010; Giannakoula et al., 2010). Despite the increase in SOD activity, the Al-tolerant genotypes showed different responses for each SOD isoform, and the accumulation of superoxide was absolutely different among tolerant genotypes (Supplemental 2), indicating that the SODs tested are unlikely candidates for the major Al-tolerance gene. SOD acts on superoxide anion producing hydrogen peroxide, which requires action of other enzymes to remove it. APX is involved in the reduction of hydrogen peroxide to water using ascorbic acid as the electron donor (Mohan Murali Achary et al., 2008). The increase in APX activity is in general related to elevated levels of AsA and a reduced H2 O2 content (Dipierro et al., 2005). APX activity was constant in UPF91Al100-1-4, despite the presence of Al, and was reduced in UFRGS17. APX activity in the absence of Al in the UFRGS17 roots was four times higher than for UPF91Al100-1-4. This amount might be sufficient for ROS detoxification in the first hours of Al exposure. Even after seven days of Al exposure, the levels of APX in the root tip were higher in the Al-tolerant genotypes than in the sensitive one (Fig. 4A). The alleviation of the stress condition at the root tip is crucial to maintaining cell division and elongation. Furthermore, it is possible that other enzymes also contribute to the detoxification at different times over the course of the Al exposure. An increase in CAT activity is considered an important defense mechanism against H2 O2 , especially in the peroxisome of leaves (Darkó et al., 2004). In tea (Ghanati et al., 2005), wheat (Darkó et al., 2004) and potato (Tabaldi et al., 2009), increases in CAT activity were observed at specific times of Al exposure in tolerant genotypes. Al did not cause increases in CAT activity in the oat genotypes (Fig. 4B). CAT activity was higher in the shoot, independent of the Al condition, suggesting that in oats this antioxidant enzyme is more important in minimizing the ROS produced by photosynthesis. A reduction in the CAT activity was observed only in the differentiated root region for the tolerant oat genotypes and in the shoot region for the sensitive one (Fig. 4B). The effect on the shoot suggests that either Al is translocated to leaves, or that photosynthesis is affected by root homeostasis in the sensitive oat genotype. A reduction in activity may be due to the blockage of essential functional groups

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such as –SH in the enzymes, or might be due to the displacement of essential metal ions by Al (Schützendübel and Polle, 2005). As opposed to other antioxidant enzymes, CAT does not require reducer power to transform H2 O2 into H2 O and O2 , therefore resulting in less affinity for H2 O2 (Mohan Murali Achary et al., 2008). In tolerant maize roots, the reduction in CAT activity was also observed in the presence of Al, suggesting that other peroxidases were responsible for the H2 O2 degradation (Boscolo et al., 2003). In barley the cell wall peroxidases were found to play an important role in H2 O2 detoxification during Al stress (Tamás et al., 2004). Again, the initial CAT activity was much higher in the differentiated root region of UFRGS17, suggesting that the pre-existing levels of CAT might be helpful at the onset of the Al stress. A positive correlation was found between CAT activity and the NPSH levels (r = 0.76) and AsA levels (r = 0.56). The low APX and CAT activity observed could be related to the Al exposure time. The highest H2 O2 levels after Al exposure have been observed at different times in different plants; for barley they were observed in the first 24 h (Tamás et al., 2004) and for pumpkin in the first 48 h (Dipierro et al., 2005). ROS affects gene regulation by activating MAPK cascades that end up inducing antioxidant enzymes, thus keeping a balance between signaling and damage (Desmond et al., 2008; Foyer and Noctor, 2009; Jaspers and Kangasjärvi, 2010). If the oxidative burst occurs in the first hours of Al exposure, after seven days the low enzyme activity may indicate that the burst was controlled and other countermeasures were taken. This hypothesis is reinforced by the reduced accumulation of H2 O2 and lipid peroxide in the Al-tolerant oat genotypes. GR was the only enzyme related to H2 O2 detoxification that shows higher activity in the UFRGS17 roots after seven days of Al exposure than in the absence of Al, and this enzyme may have an important antioxidant role at least under conditions of extended Al exposure (Fig. 4C). Interestingly, APX, CAT and GR activities in the root tips of UPF91Al100-4-1 were similar, independently of Al presence. Similarly, no difference was observed in the levels of the non-enzymatic components of the antioxidative metabolism. Non-protein thiol group (NPSH) content also did not differ; only the AsA level was higher at the roots after Al exposure. Evaluation at an early time point might help to understand the oxidative behavior of this Altolerant oat genotype. AsA levels seem to be associated with dehydroascorbate reductase (DHAR) activity, which regenerates ascorbate (Yin et al., 2010a); this enzyme activity was not assayed. AsA is suggested as a major antioxidant involved in Al-stress mitigation in tobacco (Devi et al., 2003) and rice (Guo et al., 2005). In the Al-tolerant oat genotypes, the AsA levels increased in roots in the presence of Al. This increment may confer protection, by detoxifying especially H2 O2 (Noctor and Foyer, 1998), hence reducing lipid peroxidation and DNA damage (Yin et al., 2010a). The lipids of cell membrane are formed to some extent by unsaturated fatty acids, and their double bonds are excellent targets for ROS attack (Nordberg and Arnér, 2001); in addition, lipid peroxide derived aldehydes may amplify the Al symptoms (Yin et al., 2010b). Lipid peroxidation was assessed via the MDA content. The levels of MDA were not increased at the root tip by Al exposure in the Al-tolerant oat genotypes. Indeed, in the presence of Al, MDA levels were smaller in the Al-tolerant oat genotypes in all organs if compared to the Al-sensitive oat UFRGS930598. Lipids are the first cellular target for oxidative stress, leading to cell damage and reduced growth (Domínguez et al., 2009; Tabaldi et al., 2009). However, oxidative stress is not the only cause of reduced root growth due to Al. In maize, MDA levels did not increase, but a reduction in the root growth was observed, suggesting that other process were involved (Boscolo et al., 2003).

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Taken together, the data indicated that the two Al-tolerant oat genotypes were able to manage the oxidative stress efficiently by minimizing the accumulation of lipid peroxides and thus reducing membrane damage. This antioxidative response can be considered an important mechanism in oat Al tolerance because ROS and lipid peroxide accumulation was observed in the Al-sensitive oat. The relative significance of each antioxidant element is still not clear, and a diverse response was observed among the Al-tolerant genotypes. No changes in the antioxidant enzymes were observed in the root of UPF91Al100-1-4, except for the SOD, whereas in UFRGS17, the SOD and GR could be considered the principal players, although the higher activity of CAT and APX in the absence of Al in this genotype might also have an effect in the beginning of the Al stress. Therefore, the major gene conferring Al tolerance to both UFRGS17 and UPF91Al100-1-4 does not codify an enzyme directly involved in the antioxidant metabolism. Other enzymes or substances not evaluated here play a role in the antioxidant response and may be affected by the major Al-tolerance gene. Nitric oxide, for example, has been correlated with reduction of oxidative stress caused by Al (Wang and Yang, 2005). It is possible that the Al-tolerance gene codifies an upstream component of the tolerance, such as a transcription factor, which once induced by Al, affects cascades activating, among other pathways, the antioxidative system. The differences observed between the genotypes might be explained by different alleles in the genes located downstream in the response pathway, as a consequence of their diverse genetic background. Recently, two transcription factors involved in Al signal transduction pathways have been identified, STOP1, a zinc-finger protein from A. thaliana (Iuchi et al., 2007), which shares 41% identity with ART1 from rice (Yamaji et al., 2009). The stop1 mutant displays more than 100 genes down regulated under Al, including AtALMT1, ALS3/STAR2, STOP2, MATE and other genes related to ion homeostasis (Iuchi et al., 2007; Sawaki et al., 2009). ART1 regulates 31 genes including STAR1, STAR2, and MATE, genes required for Al tolerance in rice. However these transcription factors were not enough to explain the differences in Al tolerance observed among genotypes neither in Arabidopsis nor in rice. Analysis of the antioxidative metabolism in inbred lines from crosses between the Al-tolerant oat genotypes and a sensitive one might elucidate the importance of this mechanism to Al-tolerance in oats. Acknowledgments The authors would like to thank the CAPES Foundation and FAPERGS Foundation (Brazil) for the scholarships provided for the first and fourth authors, respectively. Special thanks are due to Fábio Berndt and Jonatan Anton for technical support in the experiments. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.envexpbot.2011.05.007. References Azevedo, R.A., Alas, R.M., Smith, R.J., Lea, P.J., 1998. Response of antioxidant enzymes to transfer from elevated carbon dioxide to air and ozone fumigation, in leaves and roots of wild-type and a catalase-deficient mutant of barley. Physiologia Plantarum 104, 280–292. Beauchamp, C., Fridovic, I., 1971. Superoxide dismutase-improved assays and an assay applicable to acrylamide gels. Analytical Biochemistry 44, 276–279. Boscolo, P.R., Menossi, M., Jorge, R.A., 2003. Aluminum-induced oxidative stress in maize. Phytochemistry 62, 181–189.

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