Scientia Horticulturae 259 (2020) 108813

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Antioxidant performance and aluminum accumulation in two genotypes of T

Solanum lycopersicum in response to low pH and aluminum availability and
under their combined stress
Lucélia Borgoa, , Flávio Henrique Rabêloa, Giselle Carvalhoa, Thiago Ramiresb,
⁎

Ana Julia Righettoc, Fernando Ângelo Piottoa, Luis Felipe Boarettoa, Ricardo Azevedoa
a
College of Agriculture Luiz de Queiroz, University of São Paulo, 13418-900, Piracicaba, Brazil
b
Department of Mathematics, Federal University of Technology, 86812-460, Apucarana, Brazil
c
Agronomic Institute of Paraná, 86047-902, Londrina, Brazil

ARTICLE INFO ABSTRACT

Keywords: The mechanisms of Aluminum (Al) tolerance are dependent on the plant species and genotypes, but the results
Enzymatic activity attributed to the toxic effect of Al can also be influenced by pH. Our aim was to study the effect of Al and pH in
Lipid peroxidation the antioxidative system of two tomato genotypes in response to Al in hydroponic system. The genotypes
Oxidative stress Moneymaker (MM) and Calabash Rouge (CR) were grown in Hoagland and Arnon (1950) nutrient solution pH
Tolerance mechanisms
6.0 until the complete establishment in hydroponic system. Then the plants were transferred to the treatments:
Tomato
minimum medium at pH 4.2, minimum medium at pH 6.5 and minimum medium pH 4.2 + 20 mmol L−1
AlCl3.6H2O. The sampling of plant material was performed in the times: zero (immediately before exposure to
the treatments), 24 and 48 h after their beginning. Aluminum accumulation in MM roots was higher than in CR
after 48 h. The highest hydrogen peroxide (H2O2) concentration was observed in the shoots at control and at low
pH without Al in the roots of CR. Malondialdehyde (MDA) concentration in CR roots did not present alteration
along the 48 h, remaining near or below the control, whereas genotype MM presented a lower MDA con-
centration after 48 h in the presence of Al in relation to CR. In the roots of the genotype CR, superoxide dis-
mutase (SOD) activity was lower at pH 4.2+Al and higher at pH 4.2. We evidenced that the antioxidative system
seems more involved in response to low pH than Al toxicity, and that genotype CR presents mechanisms of Al
tolerance possibly more related to the lower Al translocation from roots to shoots than with the protective
activity of antioxidant enzymes.

1. Introduction 2016), causing damages to the cell membranes, enzymatic alterations

and inhibition of cell division and nuclear DNA synthesis (Singh et al.,
The toxicity caused by aluminum (Al) is a limiting factor for food 2017). Another alteration in cell metabolism that can be detected some
production in soils with pH values below than 5.5, since in these con- minutes after exposure to Al is the rise in production of reactive oxygen
ditions Al is solubilized and Al3+ ions are dissolved in the soil solution, species (ROS) and free radicals, such as superoxides (O2%−), hydroxyls
primarily inhibiting the growth of the root system and reducing the (OH%-), hydrogen peroxides (H2O2) and singlet oxygen (1O2) (Liu et al.,
productivity of the crops (Aggarwal et al., 2015). Aluminum directly 2014). The imbalance between the production of these species and their
affects roots; therefore, the first damages are caused in the apoplast, degradation can lead to overload of the cellular antioxidative system,
where the cell elongation process is inhibited because of Al ligation to resulting in a process known as oxidative stress, leading to enzyme
the cell walls of the rhizodermis and of the external part of the root inactivation, protein oxidation, lipid peroxidation of the cell mem-
cortex, hardening them and preventing them from performing the re- branes, among others (Sharma and Dubey, 2007; Sujkowska-
modeling process, necessary for cell elongation (Kopittke et al., 2015). Rybkowska, 2012; Bhoomika et al., 2013; Matsumoto and Motoda,
Aluminum also affects root growth by interfering in water and nutrient 2013; Furlan et al., 2018). To counteract these ROS, plants synthesize a
uptake, such as the cations Ca2+, Mg2+, K+, NH4+ (Moustaka et al., variety of antioxidant enzymes, such as superoxide dismutase (SOD, EC

⁎
Corresponding author.
E-mail addresses: borgolucelia@hotmail.com, lborgo@usp.br (L. Borgo).

https://doi.org/10.1016/j.scienta.2019.108813
Received 3 June 2019; Received in revised form 13 August 2019; Accepted 26 August 2019
Available online 12 September 2019
0304-4238/ © 2019 Elsevier B.V. All rights reserved.
L. Borgo, et al. Scientia Horticulturae 259 (2020) 108813

1.15.1.1), catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, this sense, our first hypothesis is that the antioxidant enzymatic system
EC 1.11.1.11) (Grato et al., 2005; Matsumoto and Motoda, 2012). is more activated in conditions of high H+ concentrations and Al ex-
Nevertheless, the synthesis of antioxidant enzymes is only one of the posure in attempt to avoid damages in the cell wall, lipid peroxidation
metabolic mechanisms activated by the plants in this context, still in and protein oxidation. On the other hand, we believe that tomato
general, it constitutes an important indicator of both biotic and abiotic genotypes that show an active antioxidant enzymatic system can
stresses (Arunakumara et al., 2013; Ezaki et al., 2013; Liu et al., 2014). growth more and taken up more Al in this situation, performing our
In a more specific way to Al, the exudation of organic acids, such as second hypothesis.
malate, citrate and oxalate, constitutes a strategy that minimizes the
entry of Al in the root cells, complexing Al in the rhizosphere and re- 2. Methods
ducing its uptake, accumulation and cell damages (Ryan et al., 2009;
Horst et al., 2010; Brunner and Sperisen, 2013). Many mechanisms of 2.1. Plant material and plant growth
Al tolerance are already well elucidated; for instance, the external
mechanisms related to root external structure which avoids Al entry in The study was conducted in greenhouse at 27 ± 1 °C with a 16 h
the cells, and the internal mechanisms regarding the processes of photoperiod. The seeds of the tomato (Solanum lycopersicum L.) geno-
complexation and cell compartmentalization of Al, where it becomes types Moneymaker (MM) and Calabash Rouge (CR) were scarified in a
non-toxic when bound to certain cellular compounds (Bian et al., 2013). solution containing 50% sodium hypochlorite for 20 min followed by
Nonetheless, it is worth emphasizing that the damages caused by Al and three rinses with distilled water and subsequently sown in trays con-
the mechanisms involved in its tolerance are variable and dependent on taining vermiculite as substrate, with a daily supply of deionized water.
the species and genotypes (Smith et al., 2011; Arunakumara et al., After seedling emergence (seedlings with 8–10 cm), a supply of
2013; Furlan et al., 2018). Therefore, species and genotypes which Hoagland and Arnon (1950) nutrient solution modified for tomato
naturally present distinct levels of Al tolerance have been increasingly started, containing 5 mmol L−1 Ca(NO3)2.4H2O, 5 mmol L-1 KNO3,
studied, since the mechanisms attributed to the Al tolerance are specific 2 mmol L-1 MgSO4.7H2O, 1 mmol L-1 KH2PO4, 116.5 μmol L-1 H3BO3,
and complex (Dark et al., 2004; Giannakoula et al., 2010; Inostroza- 22.8 μmol L-1 MnCl2.4H2O, 1.9 μmol L-1 ZnSO4.7H2O, 0.8 μmol L-1 Cu-
Blancheteau et al., 2012; Sujkowska-Rybkowska, 2012; Bhoomika SO4.5H2O, 0.9 μmol L-1 H2MoO4.4H2O and 89.9 μmol L−1 Fe-EDTA, pH
et al., 2013; Cai et al., 2013; Ezaki et al., 2013; Matsumoto and Motoda, 6.0; at 15% of the ionic strength. Thirty days after the sowing, the
2013; Liu et al., 2014; Furlan et al., 2018). Thus, the studies involving seedlings were stored in a styrofoam support and transferred to a hy-
strategies to elucidate the different mechanisms for Al tolerance in droponics system composed of plastic trays covered with black bags to
different plant species and among genotypes have generated a great avoid the entry of light. Each tray received 12 l of the nutrient solution
variety of information that have been used in programs for plant of Hoagland and Arnon (1950) at pH 6.0. After four days of acclima-
breeding (Bian et al., 2013; Alcntara et al., 2015). tization, the ionic strength was gradually increased from 15 to 35%.
Another important factor strictly related to the toxicity caused by Al The aeration of the nutrient solution was performed by a compressor
is the effect from the exposure to high proton (H+) concentrations in with a system of compressed air hoses.
low pH (Ikka et al., 2007). The toxicity caused by H+ (low pH of the After the period of hydroponics system establishment (34 days after
soil solution) can cause damages to the plants in some soil types, where sowing), the seedlings were transferred to a new solution containing
there is no toxicity by Al3+, for instance in the tropical regions of 0.01 mol L−1 KCl, 0.02 mol L-1 CaCl2.2H2O and 0.0595 mol L−1 KOH
America, where more than 30% of the acidic soils do not contain toxic (minimal medium) to apply the treatments. The choice for a minimal
Al concentrations (George et al., 2012). Since Al toxicity occurs in medium was based on the literature where there are evidences that in
combination with acidity, the effects of the two types of stress are full nutrient solution containing Al, the metal may form complexes with
difficult to be independently analyzed. Hence, several cell responses nutrients, mainly with P (Zheng et al., 1998). Therefore, we used a
attributed exclusively to the effect of Al can be strongly influenced by simple solution formulated using Visual Mintec™ software, where was
the alteration of H+ activity in the rhizosphere (Balkovi et al., 2014; performed Al3+ solution activity (Tables 1 and 2).
Cristancho et al., 2014). There are divergent opinions on whether the
high H+ concentration alone, in low pH, limits plant growth or not 2.2. Experimental design
(Shavrukov and Hirai, 2015). However, other studies proposed that the
toxic effect of H+ has been underestimated (Kidd and Proctor, 2001) The treatments consisted of three different minimal medium for
and needs to be recognized as a significant limitation for the cultivation
in acidic soils (Lefebvre et al., 2009). Table 1
Given the context presented, our purpose with this study was to Composition of the minimal medium used in hydroponics during the treat-
evaluate the effect of Al and pH in the antioxidative system of two ments.
tomato genotypes (Solanum lycopersicum) in hydroponics system, in
Species Concentration (μmol L−1) Activity (μmol L−1)
attempting to finding differences in mechanisms of physiological and
biochemical response to the combination of Al3+ and H+ stress or these Al(OH)2+ 0.059407 0.047392
factors separately. Tomato plants are a preeminent model system for Al(OH)3 (aq) 1.492E-4 1.5178E-4
genetic and biochemistry studies in plants. It is also the most intensively Al(OH)4− 7.462E-7 5.9529E-7
Al+3 115.47 14.611
investigated Solanaceous species, with simple diploid genetics, a short
Al2(OH)2+4 0.010243 2.7568E-4
generation time, routine transformation technology, and availability of Al3(OH)4+5 4.6698E-5 1.6448E-7
rich genetic and genomic resources (Barone et al., 2008). It has there- AlCl+2 0.84788 0.34342
fore become a goal of modern plant breeding to screen wild genetic AlOH+2 2.9021 1.1754
Ca+2 1862.9 754.54
resources for valuable traits that could be introduced into modern
CaCl+ 137.07 109.35
varieties to improve specific traits. More specifically related to acidity, CaOH+ 1.5182E-6 1.2111E-6
and consequently Al toxicity, it is an interesting model to studies of Cl− 72321 57695
metals transport to the fruits, for example. Moreover, the genotype H+ 156.62 124.94
Moneymaker (MM) is regarded as a model for several types of studies in K+ 67959 54215
KCl (aq) 1541 1567.7
plants (Arie et al., 2007) and Calabash Rouge (CR) has been uptake less
KOH (aq) 7.4503E-6 7.5793E-6
Al when compared to other genotypes (Nogueirol et al., 2015), sug- OH− 1.0084E-4 8.0447E-5
gesting the existence of tolerance mechanisms for Al in this genotype. In

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L. Borgo, et al. Scientia Horticulturae 259 (2020) 108813

Table 2 2.5. Determination of hydrogen peroxide (H2O2) and malondialdehyde

Precipitates in the simple hydroponics solution containing 20 mmol L−1 Al. (MDA) concentrations
Ion Remaining concentration in solution (μmol L−1) Precipitate
Hydrogen peroxide quantification was performed following the
methods described by Alexieva et al. (2001). The samples were ma-
+3
Al 115.47 99.423
Ca+2 2000 0
cerated in trichloroacetic acid (TCA) 1 g L−1, in the proportion of 0.3 g
Cl− 7400 0
K+ 69500 0
of leaf tissue to 2 ml of buffer and 0.5 g of root to 2 ml of buffer, to-
gether with 0.04 g of polyvinylpolypyrrolidone (PVPP). After the
complete homogenization, the samples were transferred to tubes and
tomato growth (minimal medium pH 4.2; minimal medium pH 6.5; and centrifuged at 4 °C and 10,000 rpm for 10 min. From the supernatant,
minimal medium pH 4.2 + 20 mmol L−1 AlCl3.6H2O - real concentra- 200 μL were collected and mixed with 200 μL of 100 mmol L−1 po-
tion of 115.47 μmol L−1 Al+3) and two exposure times (24 and 48 h) in tassium phosphate buffer (pH 7.5) and 800 μl of 1 mol L−1 potassium
each minimal medium. The control consisted of plants collected im- iodide solution. The tubes remained in the dark, on ice for 1 h and at
mediately before exposure to the treatments (0 h), cultivated in room temperature for 15 min to stabilize the reaction. Then, a read in
Hoagland and Arnon (1950) nutrient solution at pH 6.0 for each gen- spectrophotometer was performed at 390 nm.
otype Moneymaker (MM) and Calabash Rouge (CR). Aluminum con- Lipid peroxidation was determined by measuring the concentration
centration was based on the study performed by Steiner et al. (2012). of reactive substances in 2-thiobarbituric acid (TBA), estimated as MDA
The experimental design was performed in random blocks, with three equivalent, following the method described by Heath and Packer
replicates by treatment and 12 plants by replicate. (1968). The samples were macerated in 1 g L−1 TCA in the proportion
The plant material (shoots and roots) collected after each exposure of 0.3 g of leaf tissue to 2 ml of buffer and 0.5 g of root tissue to 2 ml of
time were immediately frozen in liquid N and stored at −80 °C for buffer, with the addition of 0.04 g of PVPP. After complete homo-
biochemical analysis and dried in a forced ventilation stove to de- genization, the sample was centrifuged at 4 °C and 10,000 rpm for
termine dry mass production, Al concentration and Al accumulation. 10 min. The volume of 0.25 ml was collected and transferred to another
tube, together with 1 ml of solution containing 200 g L−1 TCA and 5 g L 1
−
Were used per treatment 24 plants, being 12 plants harvested 24 h
after the beginning of treatment and other 12 plants after 48 h. The TBA. The mixture was placed in water bath for 30 min at 95 °C, fol-
control consisted of 12 plants of each cultivar established in the hy- lowed by cooling on ice. Then, reads were performed in a spectro-
droponic system containing Hoagland and Arnon (1950) nutrient so- photometer at 535 and 600 nm.
lution pH 6.0, harvested just before Al and different pH values (4.2 and
6.5) exposure. In each evaluation period (0, 24 and 48 h) 6 plants were 2.6. Protein extraction and determination
used to Al concentration and accumulation measurements and dry mass
production and 6 plants to measurements of H2O2, MDA, soluble pro- The samples were homogenized in a 100 mmol L−1 potassium
tein and antioxidant enzymes activities. phosphate buffer (pH 7.5), in the proportion of 0.5 g of sample from the
For pH maintenance in the minimal medium, 2 buffers were used. leaf tissue to 3 ml of buffer and 0.5 g of sample from the root tissue to
Buffer Homopiperazine-1,4-bis (2-ethanesulfonic acid), pKa 4.32 was 2 ml of buffer, containing 1 mmol L-1 ethylene diamine tetraacetic acid
used in the concentration of 1 mmol L−1 in the treatments at pH 4.2 and (EDTA), 3 mmol L−1 dithiothreitol (DTT) and 40 g L-1 PVPP, according
buffer MES (2-(N-morpholino) ethanesulfonic acid), pKa 6.15 at to Gomes-Junior et al. (2007). The homogenized material was cen-
10 mmol L-1 in the treatment pH 6.5, which is in the optimal pH range trifuged at 4 °C and 10,000 rpm for 30 min and the supernatant was
for plant cultivation in a hydroponics system (De Kreij et al., 1999; aliquoted and stored in a freezer at −80 °C until the moment of the
Domingues et al., 2012; Sardare and Admane, 2013). The choice of analyses. The concentration of total soluble proteins was determined
buffers was based on their pKa values, appropriate for the buffering following the method described by Bradford (1976), using bovine
range used in the experiments, as well as in evidences of non-toxicity in serum albumin (BSA) as standard.
plant cells (Borgo, 2017).
2.7. Determination of the enzymatic activity in a spectrophotometer

2.3. Determination of dry mass production Superoxide dismutase (SOD) activity in spectrophotometer was
determined by the method described by Giannopolitis and Ries (1977),
Dry mass production from shoot and root system was determined in in which the activity is measured by the ability of the enzyme in in-
an analytical scale after drying the plant material in a forced ventilation hibiting the photochemical reduction of the compound tetrazolium-ni-
stove at 60 °C for 72 h. troblue chloride (NBT). The reaction solution (3 mL) was composed of
50 mmol L−1 phosphate buffer (pH 7.8), 75 μmol L-1 NBT, 3 μmol L-1
riboflavin, 13 mmol L-1 methionine, 0.1 mmol L-1 EDTA and 25 μL of
2.4. Determination of Al concentration and Al accumulation protein extract. The solution was added to glass tubes and irradiated
with white light (15 W fluorescent lamp) for 5 min. After the exposure
The plant material (shoot and roots) collected was placed in a forced period, the solution was analyzed in a spectrophotometer at 560 nm.
ventilation stove at 60 °C for 72 h, and subsequently milled in a Wiley- Catalase (CAT) activity in a spectrophotometer was determined
type mill. The plant material was then subjected to the nitric-perchloric according to the method described by Kraus et al. (1995), with mod-
digestion (Sarruge and Haag, 1974), with further determination of Al ifications of Azevedo et al. (1998). The reaction medium contained 1 ml
concentration by atomic absorption. Aluminum accumulation was ob- of 30 mmol L−1 H2O2 solution in 0.1 mol L−1 potassium phosphate
tained by the product of Al concentration by the dry mass production. buffer (pH 7.5). The reaction started with the addition of 25 μL of plant
The Al concentration and Al accumulation were not performed in the extract and the activity was determined following H2O2 decomposition
treatment pH 6.5 because obviously the solution was not supplied with at intervals of 10 s for 1 min at the absorbance of 240 nm.
Al, and any Al present in solution would not be soluble at pH 6.5, which Ascorbate peroxidase (APX) activity was determined following the
is not the case in the minimal medium pH 4.2. method described by Nakano and Asada (1981). The reaction medium
(1 mL) contained 25 μL of sample and 975 μL of 80 mmol L−1 potassium
phosphate buffer solution (pH 7.0), which contained 500 μmol L−1 as-
corbate (AsA), 100 μmol L−1 EDTA and 135 μmol L−1 H2O2. The read

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L. Borgo, et al. Scientia Horticulturae 259 (2020) 108813

Table 3
Shoot and root dry mass production (g/plant) of the genotypes Moneymaker (MM) and Calabash Rouge (CR) after 24 and 48 h in minimal medium pH 4.2, minimal
medium pH 6.5 and minimal medium pH 4.2 with the addition of 20 mmol L−1 Al. The control (time 0) refers to the plants collected immediately before exposure to
the treatments, thus in pH 6.0.
Shoot pH Time p-values
Treatment
0 24 48 0 vs 24 h 0 vs 48 h 24 vs 48 h

MM Control (6.0) 0.16 – – – – –

6.5 – 0.17 0.24 < 0.001 < 0.001 < 0.001
4.2 – 0.59 1.07 < 0.001 < 0.001 < 0.001
4.2+Al – 0.61 0.69 < 0.001 < 0.001 < 0.001
CR Control (6.0) 0.16 – – – – –
6.5 – 0.40*** 0.40*** < 0.001 < 0.001 0.388
4.2 – 0.42*** 0.94*** < 0.001 < 0.001 < 0.001
4.2+Al – 0.60 0.69 < 0.001 < 0.001 < 0.001

Root pH Time p-values

Treatment
0 24 48 0 vs 24 h 0 vs 48 h 24 vs 48 h

MM Control (6.0) 0.03 – – – – –

6.5 – 0.04 0.06 < 0.001 < 0.001 < 0.001
4.2 – 0.09 0.13 < 0.001 < 0.001 < 0.001
4.2+Al – 0.08 0.09 < 0.001 < 0.001 < 0.001
CR Control (6.0) 0.04 – – – – –
6.5 – 0.13*** 0.13*** < 0.001 < 0.001 0.001
4.2 – 0.09 0.17*** < 0.001 < 0.001 < 0.001
4.2+Al – 0.08 0.11** < 0.001 < 0.001 0.004

***1% and **5% of significance obtained in T test, when compared the averages between MM and CR at same time (column).

was performed in spectrophotometer for 1 min at 290 nm. The molar than genotype CR. However, the genotype MM exposed to pH 4.2+Al
extinction coefficient was 2.8 mM−1 cm 1. after 48 h presented higher Al accumulation in the roots than the
−

genotype CR (Table 5).

2.8. Statistical analysis

3.2. Increases in H2O2 concentration induced by pH and Al resulted in lipid

To compare the effect of the time, a longitudinal model was fitted
peroxidation and protein oxidation
for each response variable, considering the times 0, 24 and 48 h as
factor. We also considered the t-test to compare the effects of different
The genotype MM presented higher H2O2 concentrations in the
genotypes at 24 h or 48 h. The analyses have been obtained using the R
shoots and roots after 24 and 48 h in relation to the control, while the
software (Team, 2014).
genotype CR presented lower H2O2 concentrations in the shoots and
higher H2O2 concentrations in the roots (Table 6). Otherwise, H2O2
3. Results concentrations after 48 h decreased or remained stable in relation to
24 h, except in the shoots and roots of the genotype CR exposed to pH
3.1. Dry mass production is more affected by high Al concentration than 4.2, which presented higher H2O2 concentrations in this time and
high H+ concentration higher H2O2 concentrations in relation to the genotype MM.
Lipid peroxidation (expressed by MDA concentration) increased in
The dry mass production in the shoots and roots of both genotypes the shoots of the genotype MM and decreased in the shoots of the
increased after 24 and 48 h of exposure to the treatments in relation to genotype CR after 24 and 48 h of exposure to the treatments in relation
the control (Table 3). Otherwise, the dry mass production in the shoots to the control. In the roots, lipid peroxidation decreased after 24 and
of the genotype CR exposed to pH 6.5 did not increase after 48 h in 48 h in relation to control, with exception in the roots of the genotype
relation to 24 h. After 48 h, the shoot growth of both genotypes was CR grown at pH 4.2 (Table 7). There was no difference between lipid
more negatively affected when the plants were exposed to pH 6.5, peroxidation observed at 24 and 48 h in the shoots of the genotype MM
followed by pH 4.2+Al. In the roots, the results were similar to the grown at pH 4.2, and in the roots of the genotype CR grown at pH 4.2
shoots for the genotype MM, but the genotype CR presented lower dry and 4.2+Al. In general, the lipid peroxidation observed in the both
mass production at pH 4.2+Al. In general, the root dry mass produc- tissues of the genotype CR was higher than in the genotype MM
tion of the genotype CR was higher than MM, independently of the pH (Table 7).
(Table 3). In general, the soluble protein concentration in the shoots and roots
Aluminum concentration increased over the time in the shoots and of both genotypes grown at pH 4.2+Al after 24 and 48 h was higher
roots of both genotypes in relation to the control (Table 4). Obviously, than the control, except in the shoots of the genotype MM (Table 8).
the higher Al concentration in the shoots and roots of both genotypes After 48 h of exposure to the treatments, there was a reduction in the
were observed in the plants exposed to pH 4.2+Al, followed by pH 4.2. soluble protein concentration in the shoots of both genotypes in relation
The genotype MM presented higher Al concentration in the shoots and to 24 h, but this did not happen in the roots. The soluble protein con-
roots than the genotype CR when the plants were grown at pH 4.2+Al centration in the shoots of the genotype CR exposed to low pH with or
(Table 4). As observed for Al concentration (Table 4), Al accumulation without Al was higher than MM, independently of the exposure time.
increased over the time in the shoots and roots of both genotypes in The same pattern was observed in the roots of the plants grown at pH
relation to the control, and the higher Al accumulations were observed 4.2, but the opposite was observed for the plants grown at pH 4.2+Al
at pH 4.2+Al (Table 5). In general, the genotype MM presented higher after 24 h (Table 8).
Al accumulations in the shoots and lower Al accumulations in the roots

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L. Borgo, et al. Scientia Horticulturae 259 (2020) 108813

Table 4
Shoot and root aluminum (Al) concentration (mg kg−1 DW) of the genotypes Moneymaker (MM) and Calabash Rouge (CR) after 24 and 48 h in minimal medium pH
4.2, minimal medium pH 6.5 and minimal medium pH 4.2 with the addition of 20 mmol L−1 Al. The control (time 0) refers to the plants collected immediately before
exposure to the treatments, thus in pH 6.0.
Shoot pH Time p-values
Treatment
0 24 48 0 vs 24 h 0 vs 48 h 24 vs 48 h

MM Control (6.0) 9.85 – – – – –

6.5 – 0 0 < 0.001 < 0.001 –
4.2 – 11.51 12.67 < 0.001 < 0.001 < 0.001
4.2+Al – 43.19 66.84 < 0.001 < 0.001 < 0.001
CR Control (6.0) 8.28 – – – – –
6.5 – 0 0 < 0.001 < 0.001 –
4.2 – 10.18** 9.36*** < 0.001 < 0.001 0.001
4.2+Al – 20.27** 24.55*** < 0.001 < 0.001 < 0.001

Root pH Time p-values

Treatment
0 24 48 0 vs 24 h 0 vs 48 h 24 vs 48 h

MM Control (6.0) 24.31 – – – – –

6.5 – 0 0 < 0.001 < 0.001 –
4.2 – 24.69 25.21 < 0.001 < 0.001 < 0.001
4.2+Al – 441.26 1144.7 < 0.001 < 0.001 < 0.001
CR Control (6.0) 25.95 – – – – –
6.5 – 0 0 < 0.001 < 0.001 –
4.2 – 30.09*** 32.93*** < 0.001 < 0.001 < 0.001
4.2+Al – 425.60** 440.32*** < 0.001 < 0.001 < 0.001

***1% and **5% of significance obtained in T test, when compared the averages between MM and CR at same time (column).

3.3. SOD, CAT and APX activities are changed by the exposure time, exposure time (Table 9).
genotype, pH and Al presence Catalase activity measured in the shoots of both genotypes after 24
and 48 h of exposure to the treatments was lower than the control, but
In general, in the shoots both genotypes presented higher SOD ac- the same pattern was not observed in the roots (Table 10). In general,
tivity after 24 and 48 h in relation to the control (Table 9). In the CAT activity in the shoots and roots of both genotypes exposed to the
shoots, SOD activity of both genotypes was higher after 48 h in relation treatments for 48 h was similar or lower to 24 h. In general, CAT ac-
to 24 h, but the opposite was observed in the roots. In the roots of the tivity in the roots of the genotype CR was lower than in the roots of the
genotype MM, SOD activity was higher at pH 6.5 than in pH 4.2 and pH genotype MM (Table 10).
4.2+Al, but it is not observed for the genotype CR. The genotype CR In the shoots of the genotype CR there was a clear increase in APX
presented higher SOD activity in the shoots than genotype MM at pH activity after 24 and 48 h of exposure to the treatments in relation to the
6.5 and lower SOD activity at pH 4.2 and 4.2+Al, independently of the control, but this happened with the genotype MM only after 48 h

Table 5
Shoot and root aluminum (Al) accumulation (μg/plant) of the genotypes Moneymaker (MM) and Calabash Rouge (CR) after 24 and 48 h in minimal medium pH 4.2,
minimal medium pH 6.5 and minimal medium pH 4.2 with the addition of 20 mmol L−1 Al. The control (time 0) refers to the plants collected immediately before
exposure to the treatments, thus in pH 6.0.
Shoot pH Time p-values
Treatment
0 24 48 0 vs 24 h 0 vs 48 h 24 vs 48 h

MM Control (6.0) 2.31 – – – – –

6.5 – 0 0 < 0.001 < 0.001 –
4.2 – 5.74 11.59 < 0.001 < 0.001 < 0.001
4.2+Al – 24.99 42.3 < 0.001 < 0.001 < 0.001
CR Control (6.0) 2.31 – – – – –
6.5 – 0 0 < 0.001 < 0.001 –
4.2 – 4.25*** 4.50*** < 0.001 < 0.001 0.021
4.2+Al – 12.25*** 16.90*** < 0.001 < 0.001 < 0.001

Root pH Time p-values

Treatment
0 24 48 0 vs 24 h 0 vs 48 h 24 vs 48 h

MM Control (6.0) 1.12 – – – – –

6.5 – 0 0 < 0.001 < 0.001 –
4.2 – 2.52 4.07 < 0.001 < 0.001 < 0.001
4.2+Al – 51.92 111.79 < 0.001 < 0.001 < 0.001
CR Control (6.0) 1.18 – – – – –
6.5 – 0 0 < 0.001 < 0.001 –
4.2 – 3.38*** 6.27*** < 0.001 < 0.001 < 0.001
4.2+Al – 77.84*** 99.53*** < 0.001 < 0.001 < 0.001

***1% and **5% of significance obtained in T test, when compared the averages between MM and CR at same time (column).

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L. Borgo, et al. Scientia Horticulturae 259 (2020) 108813

Table 6
Hydrogen peroxide (H2O2) concentration (nmol g−1 FW) in the shoot (A) and root (B) of the genotypes Moneymaker (MM) and Calabash Rouge (CR) after 24 and
48 h in minimal medium pH 4.2, minimal medium pH 6.5 and minimal medium pH 4.2 with the addition of 20 mmol L−1 Al. The control (time 0) refers to the plants
collected immediately before exposure to the treatments, thus in pH 6.0.
Shoot pH Time p-values
Treatment
0 24 48 0 vs 24 h 0 vs 48 h 24 vs 48 h

MM Control (6.0) 762.12 – – – – –

6.5 – 1275.62 891.71 < 0.001 < 0.001 < 0.001
4.2 – 1144.69 1128.69 < 0.001 < 0.001 0.445
4.2+Al – 1249.69 1089.41 < 0.001 < 0.001 < 0.001
CR Control (6.0) 1461.35 – – – – –
6.5 – 1323.60 1011.62 < 0.001 < 0.001 0.001
4.2 – 1144.69 1336.17** < 0.001 0.009 0.006
4.2+Al – 1039.94*** 915.24*** < 0.001 < 0.001 0.006

Root pH Time p-values

Treatment
0 24 48 0 vs 24 h 0 vs 48 h 24 vs 48 h

MM Control (6.0) 125.07 – – – – –

6.5 – 214.67 218.73 < 0.001 < 0.001 0.506
4.2 – 223.33 201.48 < 0.001 < 0.001 0.015
4.2+Al – 242.03 218.73 < 0.001 < 0.001 0.024
CR Control (6.0) 147.83 – – – – –
6.5 – 209.7 214.79 < 0.001 < 0.001 0.307
4.2 – 157.72** 271.71*** 0.076 < 0.001 < 0.001
4.2+Al – 237.08 236.66** < 0.001 < 0.001 0.898

***1% and **5% of significance obtained in T test, when compared the averages between MM and CR at same time (column).

(Table 11). The genotype CR grown without Al presented higher APX production in the shoots of the genotypes MM and CR after 48 h de-
activity in the shoots after 48 h than 24 h, but the opposite was ob- monstrated that dry mass production at pH 4.2 was higher than the
served for the plants grown at pH 4.2+Al. In general, in the roots of others treatments (Table 3). This means at a first moment that Al3+ is
both genotypes, APX activity was higher after 48 h than 24 h in the more harmful to tomato plants than high H+ concentrations. Other-
plants grown at pH 4.2 and pH 4.2+Al. Ascorbate peroxidase activity wise, the dry mass production of the genotype MM was more inhibited
was similar between both genotypes, independently of the treatments at pH 6.5 than by low pH or low pH + Al (Table 3), which can be
(Table 11). explained by the Acid Growth Theory. This theory says that the extent
of growing cell walls occurs much more rapidly at acidic pH than at
neutral pH (6.5 is close to 7). The acidification of the cell wall resulting
4. Discussion from the extrusion of H+ ions through the membrane causes relaxation
and extension of cell wall via expansins that catalyzes pH-dependent
The evaluation of morphological parameters such as dry mass

Table 7
Malondialdehyde (MDA) concentration (nmol g−1 FW) in the shoot (A) and root (B) of the genotypes Moneymaker (MM) and Calabash Rouge (CR) after 24 and 48 h
in minimal medium pH 4.2, minimal medium pH 6.5 and minimal medium pH 4.2 with the addition of 20 mmol L−1 Al. The control (time 0) refers to the plants
collected immediately before exposure to the treatments, thus in pH 6.0.
Shoot pH Time p-values
Treatment
0 24 48 0 vs 24 h 0 vs 48 h 24 vs 48 h

MM Control (6.0) 0.725 – – – – –

6.5 – 0.86 1.38 < 0.001 < 0.001 < 0.001
4.2 – 0.78 0.84 0.164 0.018 0.156
4.2+Al – 1.02 1.35 < 0.001 < 0.001 < 0.001
CR Control (6.0) 1.69 – – – – –
6.5 – 1.72*** 1.41 0.048 < 0.001 0.004
4.2 – 1.47*** 1.85*** < 0.001 < 0.001 < 0.001
4.2+Al – 1.15 0.91*** < 0.001 < 0.001 0.007

Root pH Time p-values

Treatment
0 24 48 0 vs 24 h 0 vs 48 h 24 vs 48 h

MM Control (6.0) 0.685 – – – – –

6.5 – 0.45 0.16 < 0.001 < 0.001 < 0.001
4.2 – 0.51 0.18 < 0.001 < 0.001 < 0.001
4.2+Al – 0.35 0.16 < 0.001 < 0.001 0.011
CR Control (6.0) 0.553 – – – – –
6.5 – 0.52 0.14 0.212 < 0.001 < 0.001
4.2 – 0.57** 0.57*** 0.319 0.201 0.684
4.2+Al – 0.29 0.28** < 0.001 < 0.001 0.711

***1% and **5% of significance obtained in T test, when compared the averages between MM and CR at same time (column).

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L. Borgo, et al. Scientia Horticulturae 259 (2020) 108813

Table 8
Soluble protein concentration (mg mL−1) in the shoot (A) and root (B) of the genotypes Moneymaker (MM) and Calabash Rouge (CR) after 24 and 48 h in minimal
medium pH 4.2, minimal medium pH 6.5 and minimal medium pH 4.2 with the addition of 20 mmol L−1 Al. The control (time 0) refers to the plants collected
immediately before exposure to the treatments, thus in pH 6.0.
Shoot pH Time p-values
Treatment
0 24 48 0 vs 24 h 0 vs 48 h 24 vs 48 h

MM Control (6.0) 6.20 – – – – –

6.5 – 5.88 5.52 0.246 0.03 0.186
4.2 – 5.38 4.88 < 0.001 < 0.001 0.003
4.2+Al – 5.95 4.83 0.184 < 0.001 < 0.001
CR Control (6.0) 6.07 – – – – –
6.5 – 5.27 4.44*** 0.006 < 0.001 0.008
4.2 – 6.03*** 5.28 0.913 0.028 0.061
4.2+Al – 6.16 6.13** 0.652 0.738 0.894

Root pH Time p-values

Treatment
0 24 48 0 vs 24 h 0 vs 48 h 24 vs 48 h

MM Control (6.0) 1.39 – – – – –

6.5 – 1.38 1.19 0.975 0.447 0.559
4.2 – 1.71 1.96 0.086 0.011 0.221
4.2+Al – 1.81 2.32 < 0.001 < 0.001 0.015
CR Control (6.0) 1.66 – – – – –
6.5 – 1.52 1.53 0.469 0.489 0.971
4.2 – 1.6 1.58*** 0.336 0.227 0.792
4.2+Al – 2.23** 2.13 0.001 0.004 0.496

***1% and **5% of significance obtained in T test, when compared the averages between MM and CR at same time (column).

extension and stress relaxation of cell walls (Rayle and Cleland, 1992). which control its uptake (Ryan and Delhaize, 2010) and can be asso-
More specifically in roots, it is very interesting to compare results from ciated with the release of organic acids from the roots to protect
different reactions in response to low pH and its combination with growing sensible apex, as well as with the detoxification of Al3+ ions in
Al+3. Kinraide (1998), for instance, concluded that Al+3 was at least 10 the apoplast (Kinraide et al., 2005; Ryan et al., 2009; Horst et al.,
times more toxic than high H+ concentrations, based on the responses 2010). This result show us that our second hypothesis is partially
of root elongation in wheat seedlings. In our study, we also observed wrong, since the root dry mass production of the genotype CR was
that Al3+ was more toxic to the plants than high H+ concentrations higher (Table 3) and Al accumulation (Table 5) lower than genotype
(Table 3). MM. Additionally, based on these results, there are evidences that
Regarding to the lower Al concentration and Al accumulation ob- genotype MM presents less efficient mechanisms in relation to CR to
served mainly in the shoots of the genotype CR compared to MM reduce Al translocation from roots to shoots and reduce ROS production
(Tables 4 and 5), it is suggested that there are external mechanisms in the roots because of exposure to it (Table 4). Matsumoto and Motoda

Table 9
Activity of the enzyme superoxide dismutase (SOD) (μmol min.−1 mg prot.−1) in the shoot (A) and root (B) of the genotypes Moneymaker (MM) and Calabash Rouge
(CR) after 24 and 48 h in minimal medium pH 4.2, minimal medium pH 6.5 and minimal medium pH 4.2 with the addition of 20 mmol L−1 Al. The control (time 0)
refers to the plants collected immediately before exposure to the treatments, thus in pH 6.0.
Shoot pH Time p-values
Treatment
0 24 48 0 vs 24 h 0 vs 48 h 24 vs 48 h

MM Control (6.0) 6.71 – – – – –

6.5 – 7.09 7.56 < 0.001 < 0.001 < 0.001
4.2 – 7.69 8.73 < 0.001 < 0.001 < 0.001
4.2+Al – 7.01 7.56 < 0.001 < 0.001 < 0.001
CR Control (6.0) 6.82 – – – – –
6.5 – 7.91*** 9.35*** < 0.001 < 0.001 < 0.001
4.2 – 3.71*** 7.99*** < 0.001 < 0.001 < 0.001
4.2+Al – 6.81*** 6.96*** 0.696 < 0.001 0.001

Root pH Time p-values

Treatment
0 24 48 0 vs 24 h 0 vs 48 h 24 vs 48 h

MM Control (6.0) 29.32 – – – – –

6.5 – 29.41 26.01 0.046 < 0.001 < 0.001
4.2 – 24.05 20.79 < 0.001 < 0.001 < 0.001
4.2+Al – 22.65 17.59 < 0.001 < 0.001 < 0.001
CR Control (6.0) 24.59 – – – – –
6.5 – 18.44*** 26.69*** < 0.001 < 0.001 < 0.001
4.2 – 26.97*** 25.98*** < 0.001 < 0.001 < 0.001
4.2+Al – 25.98*** 19.13*** < 0.001 < 0.001 < 0.001

***1% and **5% of significance obtained in T test, when compared the averages between MM and CR at same time (column).

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L. Borgo, et al. Scientia Horticulturae 259 (2020) 108813

Table 10
Activity of the enzyme catalase (CAT) (μmol min.−1 mg prot. 1) in the shoot (A) and root (B) of the genotypes Moneymaker (MM) and Calabash Rouge (CR) after 24
−

and 48 h in minimal medium pH 4.2, minimal medium pH 6.5 and minimal medium pH 4.2 with the addition of 20 mmol L−1 Al. The control (time 0) refers to the
plants collected immediately before exposure to the treatments, thus in pH 6.0.
Shoot pH Time p-values
Treatment
0 24 48 0 vs 24 h 0 vs 48 h 24 vs 48 h

MM Control (6.0) 19.93 – – – – –

6.5 – 15.3 11.86 < 0.001 < 0.001 0.001
4.2 – 13.65 14.81 < 0.001 < 0.001 0.276
4.2+Al – 16.03 15.23 0.008 0.003 0.495
CR Control (6.0) 23.2 – – – –
6.5 – 13.76 13.97** < 0.001 < 0.001 0.784
4.2 – 14.95 11.56*** < 0.001 < 0.001 0.002
4.2+Al – 15.51 16.37 < 0.001 < 0.001 0.309

Root pH Time p-values

Treatment
0 24 48 0 vs 24 h 0 vs 48 h 24 vs 48 h

MM Control (6.0) 23.8 – – – – –

6.5 – 29.57 22.34 < 0.001 0.221 0.001
4.2 – 33.21 20.01 < 0.001 0.168 < 0.001
4.2+Al – 19.96 22.85 < 0.001 < 0.001 0.921
CR Control (6.0) 19.31 – – – – –
6.5 – 27.91 18.64 < 0.001 0.653 0.005
4.2 – 23.38*** 25.66 0.01 0.001 0.157
4.2+Al – 19.04** 16.22*** 0.625 0.001 0.006

***1% and **5% of significance obtained in T test, when compared the averages between MM and CR at same time (column).

(2013) reported that the higher ROS production in Pisum sativum plants than in root tissue in both genotypes (Table 7), with the most pro-
exposed to Al occurred in ruptures of the roots due to the low exudation nounced difference detected in relation to the treatment pH 6.5 in the
of organic acids and low efficiency of the antioxidant system. genotype CR. Similar results were shown by Martins et al. (2011),
The production of ROS, such as H2O2, can also increase the lipid where MDA concentration was significantly higher in the shoot of
peroxidation of the cell membranes and protein oxidation, but in some Plantago algarbiensis and in the roots of both Plantago algarbiensis and
plants these effects do not interfere directly in root growth and biomass Plantago almogravensis grown at pH 4.5 in comparison with higher pH
production (Yamamoto et al., 2001; Boscolo et al., 2003; Furlan et al., values, but without negative effects on the in vitro proliferation and
2018), which possibly occurred with genotype CR, which presented rooting. This indicates the occurrence of damages in the cell mem-
higher MDA concentrations than genotype MM (Table 7) and higher dry branes, which were analytically detectable before the appearance of
mass production (Table 3) at the end of the study compared to genotype differences in growth (Martins et al., 2011), as occurred with genotype
MM. The MDA concentration in response to pH 4.2 was higher in shoot CR in which dry mass production in treatment at low pH did not suffer

Table 11
Activity of the enzyme ascorbate peroxidase (APX) (μmol min.−1 mg prot. 1) in the shoot (A) and root (B) of the genotypes Moneymaker (MM) and Calabash Rouge
−

(CR) after 24 and 48 h in minimal medium pH 4.2, minimal medium pH 6.5 and minimal medium pH 4.2 with the addition of 20 mmol L−1 Al. The control (time 0)
refers to the plants collected immediately before exposure to the treatments, thus in pH 6.0.
Shoot pH Time p-values
Treatment
0 24 48 0 vs 24 h 0 vs 48 h 24 vs 48 h

MM Control (6.0) 0.43 – – – – –

6.5 – 0.38 0.44 0.023 0.455 0.034
4.2 – 0.43 0.5 0.937 0.088 0.158
4.2+Al – 0.45 0.5 0.43 0.018 0.124
CR Control (6.0) 0.33 – – – – –
6.5 – 0.41 0.44 < 0.001 < 0.001 0.004
4.2 – 0.37 0.43 0.117 < 0.001 0.011
4.2+Al – 0.41 0.38** < 0.001 < 0.001 0.034

Root pH Time p-values

Treatment
0 24 48 0 vs 24 h 0 vs 48 h 24 vs 48 h

MM Control (6.0) 1.13 – – – – –

6.5 – 1.44 1.09 < 0.001 < 0.001 < 0.001
4.2 – 1.03 1.04 0.106 0.153 0.82
4.2+Al – 0.88 1.08 < 0.001 0.326 0.006
CR Control (6.0) 1.03 – – – – –
6.5 – 1.28** 1.13 < 0.001 < 0.001 0.001
4.2 – 0.91 1.16 0.125 0.089 0.035
4.2+Al – 0.93 1 0.134 0.636 0.37

***1% and **5% of significance obtained in T test, when compared the averages between MM and CR at same time (column).

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L. Borgo, et al. Scientia Horticulturae 259 (2020) 108813

Fig. 1. Schematic overview showing the main plant responses in relation to the parameters assayed in the roots and shoot of two Solanum lycopersicum genotypes
(CR = Calabash Rouge; MM = Moneymaker) exposed to low pH (minimum medium at pH 4.2) and low pH + Al (minimum medium pH 4.2 + 20 mmol L−1
AlCl3.6H2O) for 24 and 48 h. Al = Aluminum; MDA = malondialdehyde (indicator of lipid peroxidation); H2O2 = hydrogen peroxide; APX = ascorbate peroxidase;
CAT = catalase; SOD = superoxide dismutase; Protein = soluble total protein.

negative impacts after 48 h of cultivation (Table 3). MDA (Table 7) concentrations and lower soluble protein concentrations
In general, the low pH induced higher MDA production in the two (Table 8) in plants maintained at pH 4.2 without Al in relation to the
tomato genotypes when compared to low pH + Al (Table 7). Kinraide plants grown with Al supply, mainly in shoot, also evidencing a dif-
(1993) postulated that there might be a protective mechanism, where ferent response standard for the two stressing factors and for the two
the Al+3 ions in a low concentration (real Al concentration tested in this genotypes.
study was 115.47 μmol L−1) can establish the membranes, reducing the Soluble protein concentration is an indicator of changes in cell
negative charge of their surface, thus reducing the interaction with H+ metabolism (Martins et al., 2011). In the shoots of the genotype CR, a
and, therefore, reducing exosmosis. In this sense, this result could in- higher soluble protein concentration was observed at pH 4.2 and at pH
dicate us that the antioxidant enzymatic system of both tomato geno- 4.2+Al in relation to pH 6.5 (Table 8), which did not happen with the
types assayed in this study can be less efficient to scavenge ROS induced genotype MM. It is known that this biochemical parameter responds to
only by H+ compared to H+ + Al3+ because the protective mechanism a wide variety of stresses (Singh and Tewari, 2003) and that higher
induced by Al3+ cited above. Therefore, our first hypothesis that the soluble protein concentrations have been reported in several plant
antioxidant enzymatic system is more activated in conditions of high species under adverse growth conditions (Ashraf and Harris, 2004; He
H+ concentrations and Al exposure apparently is not correct. Martins and Huang, 2007). In this sense, is possible that the genotype CR pre-
et al. (2013) evaluating two Plantago species at pH 4.0 demonstrated sents an antioxidant system more efficient than MM to avoid protein
that MDA concentration were high in the shoot only in one of the two oxidation when the plants are exposed to Al and high H+ concentra-
species and that the activity of the antioxidant enzymes was increased tions. This result show us that is very important to assay different
or not affected in the two species, suggesting that the high enzymatic genotypes in studies of this type since the intra-specific genetic varia-
activity was not enough to protect the seedlings against the oxidative tion can indicate us more precisely which protection mechanisms are
damages. involved in the tolerance to Al and high H+ concentrations.
Most of the studies in plants report that the largest part of H2O2 Regarding to the oxidative stress caused by Al, it is capable of in-
produced in plant cells has been concentrating on the photosynthetic ducing a rote of well characterized responses which includes the pro-
activity of the chloroplasts and peroxisomes as main H2O2 sources, but duction of antioxidant enzymes such as SOD, CAT, APX, among others
there are evidences that the extracellular matrix also becomes an im- (Shamsi et al., 2008; Xu et al., 2012). Nevertheless, little is known
portant H2O2 production site, especially during the responses to en- about the induction of antioxidant compounds in response to acidity. In
vironmental stresses (Ślesak et al., 2007). Indeed, the highest H2O2 the genotypes MM and CR, SOD activity was in general higher in the
concentration in the tomato genotypes assayed in this study was de- roots than shoots (Table 9), which can result in higher oxidative da-
tected in the shoots (Table 6), especially in the control treatment of the mages in the shoots because of the lower activity of this enzyme (Grato
genotype CR and at pH 6.5 after 24 h. However, after 48 h the highest et al., 2005; Bhoomika et al., 2013). However, there were no visual
H2O2 production was detected at pH 4.2, being 46% higher than the symptoms of toxicity in the shoots, which can be related to the lower
treatment pH 4.2+Al. It suggests that H2O2 is not only a signaling generation of O2%− (Gill and Tuteja, 2010; Matsumoto and Motoda,
molecule capable of triggering responses for defense to stress, but also, 2012, 2013). In general, in the roots of the genotypes MM and CR, SOD
in relatively high concentrations, it plays an important role in the pri- (Table 9), CAT (Table 10) and APX (Table 11) activities were similar or
mary metabolism (Ślesak et al., 2007), since the highest values of dry lower at pH 4.2+Al compared to pH 4.2. A reduction in SOD, CAT and
mass production were observed at pH 4.2 (Table 3). Still, H2O2 can APX activities indicate us in this case that the generation of ROS in the
regulate the expression of genes that are involved in the synthesis of roots of both genotypes grown at pH 4.2+Al was lower than pH 4.2,
enzymes of the cellular antioxidative system (Neill et al., 2002a, b; which is evidenced by the lower lipid peroxidation (Table 7) and pro-
Geisler et al., 2006). Genotype CR presented higher H2O2 (Table 6) and tein oxidation (Table 8). This result indicates us again that our first

9
L. Borgo, et al. Scientia Horticulturae 259 (2020) 108813

hypothesis is not correct. Azevedo, R.A., Alas, R.M., Smith, R.J., Lea, P.J., 1998. Response of antioxidant enzymes
Superoxide dismutase induction in response to the treatments in the to transfer from elevated carbon dioxide to air and ozone fumigation, in the leaves
and roots of wild-type and a catalase-deficient mutant of barley. Physiol. Plant. 104,
shoots occurred in a distinct way for the two genotypes: at low pH 280–292.
without Al the genotype MM presented a rapid induction (in 24 h) and Balkovič, J., Kollár, J., Šimonovič, V., Žarnovičan, H., 2014. Plant assemblages respond
the genotype CR a later induction (48 h) possibly as a mechanism for sensitively to aluminium solubility in acid soils. Community Ecol. 15, 94–103.
Barone, A., Chiusano, M.L., Ercolano, M.R., Giuliano, G., Grandillo, S., Frusciante, L.,
adaptation to stress. In addition, in the roots of the genotype CR an 2008. Structural and functional genomics of tomato. Int. J. Plant Genomics 820274-
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enzymes to aluminum toxicity in two rice (Oryza sativa L.) cultivars with marked
and CAT activities in the roots of rice plants were suppressed at low pH; presence and elevated activity of Fe-SOD and enhanced activities of Mn-SOD and
however, APX activity was significantly induced. In our study APX catalase in aluminum tolerant cultivar. J. Plant Growth Regul. 71, 235–252.
activity was also induced at low pH in the shoots of the genotype CR Bian, M., Zhou, M., Sun, D., Li, C., 2013. Molecular approaches unravel the mechanism of
acid soil tolerance in plants. Crop J. 1, 91–104.
(Table 11), while CAT activity in the roots of both genotypes was higher
Borgo, L., 2017. Evaluation of buffers toxicity in tobacco cells: homopiperazine-1, 4-bis
in the treatments without Al supply, mainly in treatment pH 4.2 (2-ethanesulfonic acid) is a suitable buffer for plant cells studies at low pH. Plant
(Table 10). Catalase activity was suppressed in the shoots of the gen- Physiol. Biochem. 115, 119–125.
otypes comparing the control with the final time 48 h, whereas it pre- Boscolo, P.R.S., Menossi, M., Jorge, R., 2003. Aluminum induced oxidative stress in
maize. Phytochemistry 62, 181–189.
sented a fast induction in the roots of the genotype MM (24 h). Thus, Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram
CAT was demonstrated to respond differently in the different tissues quantities of protein utilizing principle of protein-dye-binding. Anal. Biochem. 72,
evaluated. Data obtained by Martins et al. (2013) showed that SOD, 248–254.
Brunner, I., Sperisen, C., 2013. Aluminum exclusion and aluminum tolerance in woody
CAT, APX and guaiacol peroxidase (GPX) in Plantago algarbiensis and plants. Front. Plant Sci. 4, 172. https://doi.org/10.3389/fpls.2013.00172.
Plantago almogravensis were induced both by Al and by the acidity in the Cai, M., Wang, N., Xing, C., Wang, F., Wu, K., Du, X., 2013. Immobilization of aluminum
medium. The most significant induction of these enzymes occurring with mucilage secreted by root cap and root border cells is related to aluminum
resistance in Glycine max L. Environ. Sci. Pollut. Res. 20, 8924–8933.
when the seedlings grown at low pH compared to plants grown at pH Cristancho, R.J.A., Hanafi, M.M., Omar, S.R.S., Rafii, M.Y., 2014. Aluminium speciation
5.75. These authors concluded that both Plantago species can accumu- of amended acid tropical soil and its effects on plant root growth. J. Plant Nutr. 37,
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The fact of the genotype CR had accumulated less Al in the shoots Domingues, D.S., Takahashi, H.W., Camara, C.A., Nixdorf, S.L., 2012. Automated system
compared to genotype MM indicates the existence of mechanisms for Al developed to control pH and concentration of nutrient solution evaluated in hydro-
ponic lettuce production. Comput. Electron. Agric. 84, 53–61.
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The higher MDA concentrations in the roots and shoots of the genotype virginicus L. Environ. Exp. Bot. 93, 35–44.
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Declaration of Competing Interest Academic Press, London, pp. 409–472.
Giannakoula, A., Moustakas, M., Syros, T., Yupsanis, T., 2010. Aluminum stress induces
up-regulation of an efficient antioxidant system in the Al-tolerant maize line but not
The authors declare that they have no conflict of interests.
in the Al-sensitive line. Environ. Exp. Bot. 67, 487–494.
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Acknowledgement tative relationship with water-soluble protein on seedlings. Plant Physiol. 59,
315–318.
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