Revista Brasileira de FisiologiaVegetal, 11(3):137-143, 1999.
ALUMINUM EFFECTS ON LIPID PEROXIDATION AND
ON THE ACTIVITIES OF ENZYMES OF OXIDATIVE
METABOLISM IN SORGHUM1
Paulo Henrique Pereira Peixoto2*, José Cambraia3, Renato Sant’Anna3, Paulo
Roberto Mosquim4 and Maurílio Alves Moreira5
Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa, MG, 36571000, Brasil.
ABSTRACT - Seedlings of two sorghum (Sorghum bicolor (L.) Moench) cultivars with differential
tolerance to Al were exposed to 0 and 185 µM of Al, in a pH 4.0 nutrient solution, for 10 days and, then,
lipid peroxidation and the activity of enzymes of the oxidative metabolism were determined. Lipid
peroxidation increased in the root system of the two cultivars, especially in the sensitive one. In the
presence of Al, superoxide dismutase and cinnamyl alcohol dehydrogenase activities increased only in
the Al-tolerant cultivar. In this cultivar the largest reduction in phenylalanine ammonia lyase activity also
occurred. So, this cultivar probably accumulated less amounts of reactive oxygen species and of toxic
phenolic compounds and, consequently, showed smaller lipid peroxidation. On the other hand, largest
increases in peroxidase and polyphenoloxidase activities were observed in the Al-sensitive cultivar in the
presence of Al. In this cultivar a greater accumulation of peroxides and phenolic compounds probably
occurred resulting in more lipid peroxidation. The changes in ascorbate peroxidase and catalase activities
in both cultivars suggest a reduced contribution of these enzymes in the mechanism of peroxide
decomposition. The results indicate that the Al-tolerant cultivar produces smaller concentrations of reactive
oxygen species and/or it possesses more efficient enzymatic mechanisms of removal and/or neutralization
of these radicals than the Al-sensitive cultivar.
Additional index terms: Al toxicity, free oxygen radicals, peroxides, Sorghum bicolor.
EFEITOS DO ALUMÍNIO SOBRE A PEROXIDAÇÃO DE LIPÍDIOS E A ATIVIDADE
DE ENZIMAS DO METABOLISMO OXIDATIVO EM SORGO
RESUMO – Plântulas de dois cultivares de sorgo (Sorghum bicolor (L.) Moench) com tolerância diferencial ao Al foram expostas a 0 e 185 µM de Al, em solução nutritiva, pH 4,0, durante 10 dias e, então,
determinou-se peroxidação dos lipídios e a atividade de enzimas do metabolismo oxidativo. A peroxidação
de lipídios aumentou no sistema radicular dos dois cultivares, com maior intensidade no sensível. Os
aumentos nas atividades das dismutases de superóxidos e das desidrogenases dos álcoois cinamílicos,
observados apenas no cultivar tolerante, e a maior redução na atividade das amônia liases da fenilalanina,
resultaram, provavelmente, em menores acúmulos de espécies reativas de oxigênio e de compostos fenólicos
tóxicos e, consequentemente, menor peroxidação de lipídios no sistema radicular deste cultivar, na presença do Al. Por outro lado, os maiores aumentos nas atividades das peroxidases e das oxidases de
polifenóis, observados no sistema radicular do cultivar sensível na presença do Al, sugerem ter ocorrido,
neste caso, maiores acúmulos de peróxidos e de compostos fenólicos, resultando em maior peroxidação
de lipídios neste cultivar. As modificações nas atividades das catalases e das peroxidases do ascorbato
sugerem uma contribuição reduzida dessas enzimas nos mecanismos de decomposição de peróxidos em
ambos os cultivares. Os resultados sugerem que cultivar tolerante produz menores concentrações de
espécies reativas de oxigênio e/ou possui mecanismos enzimáticos de remoção e/ou de eliminação desses
radicais livres mais eficientes do que o cultivar sensível.
Termos adicionais para indexação: peróxidos, radicais livres, Sorghum bicolor, toxicidade por Al.
1
Received 05/05/1999 and accepted 18/08/1999.
Pesquisa financiada parcialmente pelo CNPq.
2* Prof. Adjunto, D.S., Depto. Botânica, ICB, UFJF, Juiz de Fora,
MG, 36036-330; Bolsista da CAPES; Autor para correspondência: FAX: (032) 229-3216; E-mail: phpp@icb.ufjf.br.
3 Prof. Titular, Ph.D., Departamento de Biologia Geral - UFV,
Viçosa, MG; Pesquisador do CNPq.
4 Prof. Titular, D.S., Departamento de Biologia Vegetal - UFV,
Viçosa, MG.
5 Prof. Titular, Ph.D., Departamento de Bioquímica e Biologia
Molecular - UFV, Viçosa, MG.
137
Peixoto et al.
138
INTRODUCTION
Aluminum (Al) is one of the major factors limiting
growth and productivity of important crops in many acid
soils throughout the world. Considerable research has
been directed to elucidating the mechanisms of Al toxicity
and plant tolerance in recent years, however, these processes are still poorly understood (Delhaize & Ryan,
1995; Kochian, 1995). One of the most important Al
effects seems to be on the structure and function of the
plasma membrane. Aluminum strongly binds to
phospholipids, alters the total and relative abundance of
phospholipids and other membrane lipids and changes
the degree of fatty acid unsaturation (Zhang et al., 1996;
1997). There is also evidence that Al toxicity can cause
excessive generation of reactive oxygen species (ROS)
and an increase in peroxidation and/or breakdown of
membrane lipids (Gutteridge et al., 1985; Cakmak &
Horst, 1991; Ono et al., 1995). As a consequence, there
is a significant modification in membrane permeability,
ion transport and in the activity of a number of membranebound enzymes, especially H+-ATPase (Cooke & Burden,
1990).
The reactive oxygen species, especially O2.- and H2O2,
are commonly present in plant cells as a result of normal aerobic metabolism. Plants, however, usually
possess mechanisms of cellular protection to keep these
oxygen species below damaging levels, which include
the action of several enzymes and/or the presence of
some antioxidant substances (Scandalios, 1993). When
plants are stressed (by high levels of Al for instance),
however, the production and/or accumulation of reactive
oxygen species can exceed the capacity of the
scavenging systems, resulting in an increase in lipid
peroxidation and, consequently, damages to plasma
membrane structure and function (Gutteridge et al., 1985;
Cakmak & Horst, 1991; Ono et al., 1995; Richards et al.,
1998).
The objective of this work was to evaluate the influence
of Al on lipid peroxidation and the role of several enzymes
of the oxidative metabolism in the production and/or
degradation of reactive oxygen species, peroxides and
other oxidative substances in two sorghum cultivars with
differential tolerance to Al.
MATERIALS AND METHODS
Two sorghum cultivars (Sorghum bicolor (L.) Moench)
obtained from the Centro Nacional de Pesquisas do Milho e do Sorgo (CNPMS/Embrapa), Sete Lagoas, MG,
one Al-tolerant (BR006R), and the other Al-sensitive
(BR007A) were studied.
The seeds were surface sterilized with 0.5% sodium
hypochlorite for 20 min, thoroughly rinsed with deionized
water and then germinated in rolls of neutral pH
“germtest” paper. After seven days, the seedlings were
selected and transferred to polyethylene pots with 1.6 L
of Clark’s nutrient solution, pH 4.0 (Clark, 1975), in the
absence or presence of Al (185 µM of Al, as
Al2(SO4)3.18 H2O salt). All experiments were conducted
in a growth room with controlled temperature (25±3ºC),
under photosynthetically active radiation of 230 µmoles
m-2s-1, and a photoperiod of 16 hours. Volume and pH
corrections of the nutrient solution were carried out daily
and continuous aeration was supplied. Ten days after Al
treatment, the seedlings were harvested, washed in
deionized water, divided in shoot and root system and
the fresh material used to evaluate lipid peroxidation,
production of volatile aldehydes and enzyme assays.
The experiments followed a 2 x 2 factorial
arrangement: two sorghum cultivars, two Al levels, with
three replicates. The results were subjected to an analysis
of variance (ANOVA) and the means were statistically
compared by Tukey’s test at 5%.
Lipid peroxidation
Samples of fresh material of 0.2 g were homogenized
in 4 mL of 1% (w/v) trichloroacetic acid (TCA) solution.
The homogenate was filtered through four layers of
cheesecloth and then centrifuged at 12,000 g for 15 min.
One mL aliquots of the supernatants were added to 3
mL of 0.5% (w/v) thiobarbituric acid (TBA) in 20% (w/v)
TCA and the tubes were incubated in a shaking waterbath at 95 0C for 2 h. The reaction was stopped by placing
the reaction tubes into an ice bath. The tubes were
subsequently centrifuged at 9,000 g for 10 min, the
absorbance of the supernatant was measured at 532 and
660nm (Cakmak & Horst, 1991) and the concentration of
the malondialdehyde (MDA)-TBA complex produced was
calculated using the molar extinction coefficient of 155
mM-1 cm-1.
Production of volatile aldehydes
Volatile aldehydes were extracted from 0.2 g of the
root system and of the shoot fresh tissues in 5 mL of 2%
(v/v) ethanol at 25 0C for 2 h, under constant stirring.
One mL aliquot was transferred to a test tube containing
1 mL of 0.1% (m/v) 3-methyl-2-benzothiazolinone
hydrazone (MBTH) and then 2.5 mL of 0.23% (m/v)
FeCl3.6H2O was added. After 5 min of incubation, 6.0 mL
of acetone was added and the absorbance read at 635
nm (Reis et al., 1989). The amount of volatile aldehyde
produced was estimated using a formaldehyde calibration
curve.
Enzyme assays
Two hundred mg of the root system and the shoot
fresh tissues were homogenized in 10 mL of 0.1 M
potassium phosphate buffer, pH 6.8, containing 0.1 mM
EDTA. After filtration through four layers of cheesecloth,
the homogenate was centrifuged at 12,000 g for 20 min
and the supernatant was used as source of crude enzyme.
All steps to obtain enzyme preparation were carried out
at 4ºC.
The activity of peroxidases (POX, EC 1.11.1.7) was
determined by adding 100 µL of the crude enzyme
preparation, diluted in the proportion of 1:25 (v/v), to 4.9
mL of a solution containing 25 mM potassium phosphate
buffer, pH 6.8, 20 mM pyrogallol and 20 mM H2O2. After
incubation of the solution at 250C for 1 min, the reaction
was stopped by adding 0.5 mL of 5% (v/v) H2SO4 and
the absorbance was read at 420 nm (Kar & Mishra, 1976).
The enzyme activity was calculated using the molar
extinction coefficient of 2.47 mM-1 cm-1.
R. Bras. Fisiol. Veg., 11(3):137-143, 1999.
Aluminum effects on lipid peroxidation . . .
The activity of polyphenol oxidases (PPO, EC
1.10.3.2, EC 1.10.3.1 and EC 1.14.18.1) was determined
using the same methodology described before (for POX)
(Kar & Mishra, 1976) but omitting H2O2 from the reaction
mixture. The enzyme activity was calculated as described
before for POX.
The activity of catalases (CAT, EC 1.11.1.6) was
determined by adding 100 µL of crude enzyme
preparation to 2.9 mL of a solution containing 12.5 mM
H2O2 and 50 mM potassium phosphate buffer, pH 7.0,
and by measuring the absorbance decrease at 240 nm,
at 300C (Havir & McHale, 1987). The enzyme activity was
calculated using the molar extinction coefficient of 36 M1
cm-1.
The activity of superoxide dismutases (SOD, EC
1.15.1.1) was determined by adding 50 µL the crude
enzyme preparation to a solution containing 13 mM
metionine, 75 µM p-nitro blue tetrazolium chloride (NBT),
100 nM EDTA and 2 µM riboflavin in a 50 mM sodium
phosphate buffer, pH 7.8 (Del Longo et al., 1993). The
reaction took place in a chamber under illumination of a
15W fluorescent lamp kept inside a box covered with
aluminum foil at 25ºC. The reaction was started turning
the fluorescent lamp on and stopped 5 min later turning
it off (Giannopolitis & Ries, 1977). The blue formazane
produced by NBT photoreduction was measured by the
increase in absorbance at 560 nm. The blank mixture
had the same composition but it was kept in the dark.
One SOD unit was defined as the amount of enzyme
required to inhibit 50% of the NBT photoreduction.
The activity of lipoxygenases (LOX, EC 1.13.11.12)
was determined by adding 33 µL of the crude enzyme
preparation (with no EDTA in the extraction medium) to
a reaction mixture consisting of 4 µL of 10 mM sodium
linoleate in 0.36% (v/v) Tween-20 and 2.0 mL of 50 mM
sodium phosphate buffer, pH 6.0 (Axelrod et al., 1981).
The absorbance increase at 234 nm was recorded during
1.5 min, at 250C, and the enzyme activity was calculated
using the molar extinction coefficient of 25,000 M-1 cm-1.
The crude enzyme preparation for ascorbate
peroxidases (APX, EC 1.11.1.11) was obtained in similar way, but ascorbate was added to the extraction medium
to obtain the final concentration of 1 mM. The activity of
APX was determined by adding 100 µL of this crude
enzyme preparation to 2.9 mL of a reaction medium
containing 0.8 mM ascorbate and 1.0 mM H2O2 in 50
mM potassium phosphate buffer, pH 6.0. The absorbance
decrease was measured at 290 nm, at 250C (Koshiba,
1993). The enzyme activity was calculated using the molar
extinction coefficient of 2.8 mM-1 cm-1.
The crude enzyme preparation for phenylalanine
ammonia-lyases (PAL, EC 4.3.1.5) was obtained by
homogenizing 0.15 g of fresh tissue in 15 mL of an
extraction medium containing 20 mM β-mercaptoethanol,
0.1 M sodium borate buffer, pH 8.8, and 5% (m/v)
insoluble polyvinylpyrrolidone (PVP). After filtration
through four layers of cheesecloth, the homogenate was
centrifuged at 12,000 g for 20 min. The enzyme activity
was determined by adding 1 mL of the crude enzyme
preparation to a reaction medium containing 1 mL of 0.2
M sodium borate buffer, pH 8.8, and 1 mL of 0.1 M L-
139
phenylalanine. After incubation for 1 h at 300C, the
reaction was stopped by adding 0.1 mL of 6 N HCl and
the absorbance was determined at 290 nm (Cahill &
McComb, 1992). The enzyme activity was calculated
using the molar extinction coefficient of 104 mM-1 cm-1.
The crude enzyme preparation for cinnamyl alcohol
dehydrogenases (CAD, EC 1.1.1.195) was obtained by
homogenizing 0.2 g of fresh tissues in 3 mL of an
extraction medium containing 40 mM β-mercaptoethanol
and 100 mM potassium phosphate buffer, pH 7.3. The
enzyme activity was determined by adding 200 µL this
crude enzyme preparation to 2.8 mL of a reaction
medium containing 100 µM sinapyl alcohol, 100 µM
NADP+ and 100 µM Tris-HCl buffer, pH 9.3 (Mitchell et
al., 1994). The increase of absorbance was measured
at 340 nm, at 300C and the enzyme activity was calculated
using the molar extinction coefficient 6.22 mM-1 cm-1.
RESULTS AND DISCUSSION
In the absence of Al, lipid peroxidation, measured by
the MDA-TBA complex concentration in plant tissues,
was statistically identical in both parts of the plants of
the two cultivars (Figure 1, A). In the presence of Al, a
considerable increase on lipid peroxidation was observed
in the root system with a greater intensity in the Alsensitive cultivar (200%) when compared to the Al-tolerant
one (126%). Under these conditions, the Al-sensitive cultivar showed a lipid peroxidation about 71% greater than
the Al-tolerant cultivar. In the shoot, Al did not have a
significant effect on lipid peroxidation in either cultivar
and there was no difference between them.
The results found in this experiment were similar to
those obtained by Cakmak & Horst (1991) who also
demonstrated lipid peroxidation in whole soybean roots
after long-term Al treatments. According to these authors,
Al alone is not capable of promoting lipid peroxidation if
applied during short periods of time and at low
concentration. The Al concentration and the exposure
time used in this experiment were superior to those used
by the previously mentioned authors. On the other hand,
Ono et al. (1995) argue that oxidative alterations in
membrane lipids could be the result of a combined action
of Al and Fe, usually quite soluble in acid environments.
According to Gutteridge et al. (1985), Al potentiates and/
or facilitates lipid peroxidation when it binds to the
membrane phospholipids. Although some authors
consider the lipid peroxidation more consequence than
the primary cause of the Al toxic effects, there is evidence
showing that Al promotes the oxidation of phenolic
compounds which result in the production of O2.- and other
oxygen toxic species that would potentiate lipid
peroxidation (Cakmak & Horst, 1991).
Since the MDA-TBA concentration found in the Alsensitive cultivar in the presence of Al was superior to
that observed in the Al-tolerant cultivar and the toxic
effects depend on their capacity and/or their efficiency to
eliminate these active oxygen species, the results found
here do not leave any doubt about the superiority of the
Al-tolerant cultivar under stressing conditions. In the shoot,
neither cultivar show significant changes in the lipid
peroxidation intensity, probably because the Al
R. Bras. Fisiol. Veg., 11(3):137-143, 1999.
140
Peixoto et al.
70
120
aA
A
60
aA
B
aA
100
aA
50
aA
aA
aB
40
aA
aB
80
60
aA
aB
aB
30
bA
µ
20
40
bA
bA
20
10
0
bB
0
Root syst em
Shoot
Root syst em
Shoot
FIGURE 1 – Aluminum effects on MDA-TBA complex (A) and volatile aldehyde (B) concentrations in
tissues of root system and shoot of two sorghum cultivars. Al-sensitive, 0 µM of Al; Al-sensitive, 185
µM of Al; n Al-tolerant, 0 µM of Al; n Al-tolerant, 185 µM of Al. Means with the same small letters (in
each cultivar, in absence and presence of aluminum) and by the same capital letters (between cultivars,
in the absence and presence of aluminum) for each part of the plant do not statistically differ by the
Tukey test at 5%.
concentration was lower in this part of the plant (Gonçalves et al., 1996) and also because the root system
seems to be more sensitive to the Al toxic effects
(Cakmak & Horst, 1991).
In the absence of Al, the volatile aldehyde
concentration was greater in the Al-tolerant cultivar both
in the root system and in the shoot (Figure 1, B). In the
presence of Al, the volatile aldehyde concentration in
the root system increased 6 and 2.4-fold in the Al-sensitive
and Al-tolerant cultivars respectively. The volatile aldehyde
concentration in the root tissues, which in the absence of
Al was 93% greater in the Al-tolerant cultivar, became
29% greater in the Al-sensitive cultivar in the presence
of this cation. In the shoot, Al had no significant influence
on the production of volatile aldehydes in either cultivar,
although the Al-tolerant cultivar showed an average of
47% more volatile aldehydes than the Al-sensitive one.
Therefore, in this experiment, the most intense effect of
Al on volatile aldehyde concentration was observed in
the root system, especially in the Al-sensitive cultivar.
Aldehydes are very harmful to the cellular metabolism,
inhibiting enzymes, reducing the mitosis rate and inhibiting
tubulin synthesis, required for the achromatic spindle
formation (Wilson Jr. & McDonald Jr., 1986). The results
obtained, which are quite similar to those indicated by
the MDA-TBA test (Figure 1, A), strengthen the idea that
Al in the nutrient solution or substances produced during
Al treatment can cause greater peroxidative damages in
the Al-sensitive cultivar than in the Al-tolerant one.
In the absence of Al, POX activity in the root system
was greater in the Al-sensitive cultivar but in the shoot
there was no statistical difference between them (Figure
2, A). In the presence of Al, POX activity increased in
the root system of both cultivars with a greater intensity
in the Al-sensitive cultivar. In the shoot, an increase in
the POX activity was also observed but with less intensity
than in the root system and the cultivars did not differ
statistically. Increase in POX activity in the presence of
Al was also observed in soybean roots, especially after
a long-term stress and under high Al concentrations
(Cakmak & Horst, 1991). Apparently, Al activates a latent
POX in tissues and/or induces the appearance of isoforms
by an increase in the production of H2O2, phenolic
compounds and/or hydroperoxides, important substrates
for these enzymes (Siegel, 1993; Esaki et al., 1996). The
activity of this enzyme depends not only on species, plant
development stage and other factors (Siegel, 1993) but
apparently also on the degree of Al toxicity, which is
generally higher in the root system, as it has been verified
in this experiment.
In the absence of Al, APX activity in the root system
was greater in the Al-tolerant cultivar but, in the shoot, it
was greater in the Al-sensitive cultivar (Figure 2, B). In the
presence of Al, a 34% increase in the enzyme activity was
observed in the root system of the Al-sensitive cultivar but
a 52% decrease in the Al-tolerant cultivar. Under these
circumstances, the Al-sensitive cultivar exhibited an APX
activity 54% greater than the Al-tolerant cultivar. In the
shoot, the APX activity was reduced by Al treatment about
31% and 46% in the Al-sensitive and Al-tolerant cultivars,
respectively. These results suggest that the Al-enhanced
H2O2 production and/or accumulation in the root system
of the Al-sensitive cultivar may have contributed to increase
lipid peroxidation in membranes (Peixoto, 1998) and/or
tissues of this cultivar (Figure 1, A and B). The largest APX
activity observed in the presence of Al, especially in the
root system of the Al-sensitive cultivar, strengthens the
possibility that a greater accumulation of H2O2 in the tissues
of this cultivar may be occurring. However, since the H2O2
concentration has not actually been measured, the
ascorbate oxidation by phenolic compounds and, or the
action of other non-specific enzymes cannot be discarded
since the enzyme preparation has not been submitted to
any kind of purification.
R. Bras. Fisiol. Veg., 11(3):137-143, 1999.
141
Aluminum effects on lipid peroxidation . . .
30
1,4
1,2
aA
A
25
aA
B
aA
aA
bB
bA
1
20
aB
bA
0,8
bB
aA
0,6
aA
15
aA
aB
bB
bA
10
bB
0,4
5
µ
0,2
0
0
Root syst em
Shoot
0,7
0,6
Root syst em
Shoot
600
aA
D
C
aA
500
aA
aA aA
aB
0,5
400
0,4
aA
aA aB
aA
300
aA
bB
0,3
bA
bA
bA
0,2
200
bB
100
0,1
0
0
Root syst em
Shoot
Root syst em
Shoot
6
0,8
aA
E
F
5
aA
0,6
aA aB
bA
aA
aA
aA
4
aA
3
0,4
aB
2
0,2
aA
bA
bA
(
1
0
Root syst em
bB
aB
0
Root syst em
Shoot
Shoot
120
10
aA
G
100
8
H
aA
aA
80
bA
6
bA
aB
bA
bB
aA
aA
60
bB
aA
aA
4
40
2
aA aA
20
(µ
aB aB
0
0
Root syst em
Shoot
Root syst em
Shoot
FIGURE 2 - Aluminum effects on the activities of oxidative enzymes in tissues of root system and shoot
of two sorghum cultivars. Al-sensitive, 0 µM of Al; Al-sensitive, 185 µM of Al; n Al-tolerant, 0 µM of Al;
n Al-tolerant, 185 µM of Al. Means with the same small letters (in each cultivar, in absence and
presence of aluminum) and by the same capital letters (between cultivars, in the absence and presence
of aluminum) for each part of the plant do not statistically differ by the Tukey test at 5%.
R. Bras. Fisiol. Veg., 11(3):137-143, 1999.
142
Peixoto et al.
In the absence of Al, no difference between cultivars
was observed in the root system but in the shoot CAT
activity was greater in the Al-tolerant cultivar (Figure 2,
C). After Al treatment the enzyme activity decreased in
both parts of the plant of both cultivars. In the shoot, the
Al-tolerant cultivar showed an enzyme activity 22% higher
than the Al-sensitive cultivar. Similar results have been
obtained by Richards et al. (1998) who has also observed
a reduction in specific mRNA for these catalases in Altreated plants. Increase in POX activity but reduction in
CAT activity in the presence of Al indicate a higher
consumption of H2O2 by oxidative processes rather than
its removal by the action of enzymes of the detoxifying
mechanism (Cakmak & Horst, 1991). The reduction in
CAT activity observed in both parts of the plant of both
cultivars, however, suggests a limited involvement of these
enzymes in the H2O2 elimination mechanisms in response
to Al toxicity.
In the absence of Al, SOD activity in the root system
was greater in the Al-sensitive cultivar but there was no
difference between cultivars in the shoot (Figure 2, D).
In the presence of Al, however, the enzyme activity
increased only in the Al-tolerant cultivar (43%), and
became 12% higher than in the Al-sensitive cultivar. In
the shoot, the enzyme activity did not change in the
presence of Al and there was no difference between the
cultivars. The SOD activity was comparatively much
greater than that of other enzymes. This seems to be
necessary since O2.- and other reactive oxygen species
are highly toxic to metabolism. So, plants usually maintain
a higher SOD basal activity, which is important for cellular
protection (Scandalios, 1993). The greater activity of SOD
in the root system of the Al-tolerant cultivar in the
presence of Al would probably result in an increase in
H2O2 concentration demanding a larger participation of
the enzymes of the H2O2 elimination system such as the
membrane-bound POX, as suggested by Ezaki et al.
(1996). Since the SOD activity in the root system of the
Al-sensitive cultivar was not significantly changed by Al
in the nutrient solution, the observed increase in APX in
this par t of the plant (Figure 2, B) would be a
consequence of ascorbate oxidation by phenolic
compounds present in the nonpurified enzyme extracts.
The PPO activity was not statistically changed in
either part of the control plants of both cultivars (Figure
2, E). In the presence of Al, a 31% increase in enzyme
activity was observed but only in the root system of the
Al-sensitive cultivar. This increase suggests that
production of some precursor of reactive oxygen species
biosynthesis may be occurring. This possibility seems to
be quite plausible since Al interferes with boron
(Lukaszewski & Blevins, 1996) and zinc (Gonçalves et
al., 1996) absorption, causing deviation in the pentose
phosphate cycle that results in phenol production. This is
usually followed by an increase in PPO activity
(Marschner, 1995).
In the absence of Al, the LOX activity was much
greater in the root system and the Al-sensitive cultivar
exhibited higher enzyme activity than the Al-tolerant
cultivar in both parts of the plants (Figure 2, F). In the
presence of Al, reductions of 85% and 77% were
observed in the root system but increases of 120% and
150% were observed in the shoot of the Al-sensitive and
Al-tolerant cultivars, respectively. Since lipid peroxidation
increased in the roots of both cultivars in the presence of
Al (Figure 1, A and B), an increase in the LOX activity
was expected since the catabolic product of the action of
these enzymes on polyunsaturated fatty acids are usually
malonic aldehyde and other aldehydes (Gutteridge et al.,
1985). However, it is important to point out that the
enzyme activity assay was carried out only after ten days
of Al treatment and probably is not exhibiting the average
effect during the stress period. On the other hand, since
Al concentration in the shoot is usually much lower than
in the root system (Gonçalves et al., 1996), Al interference
on the free radical elimination mechanisms and
consequently on phenol accumulation was much lower,
resulting in an increase in LOX activity.
In the absence of Al, the Al-tolerant cultivar showed
higher PAL activity than the Al-sensitive cultivar in both
parts of the plant (Figure 2, G). In the presence of Al,
the enzyme activity decreased 20% and 30% in the root
system of the Al-sensitive and Al-tolerant cultivars,
respectively. Under these conditions the Al-tolerant cultivar still showed 35% higher enzyme activity than the
Al-sensitive cultivar. In the shoot, the enzyme activity did
not change in the presence of Al but the Al-tolerant cultivar showed a significantly higher activity. The reduction
of the PAL activity observed in the root system in the
presence of Al in both cultivars may be the result of an
inhibitory effect of accumulated phenolic compounds
(Sato et al., 1982; Cahill & McComb, 1992) or of some
kind of control mechanism operating in the transcription
of this enzyme (Campbell & Sederoff, 1996). The greater
reduction in the enzyme activity in the Al-tolerant cultivar
may also have contributed to lower the intensity of lipid
peroxidation of membranes (Peixoto, 1998) and tissues
in this cultivar (Figure 1, A and B).
In the absence of Al, there was no statistical difference
in CAD activity between cultivars for both plant parts
(Figure 2, H). In the presence of Al, a 17% reduction
was observed in the CAD activity in the root system of
the Al-sensitive cultivar, but a 38% increase in the Altolerant cultivar. In the shoot, the enzyme activity was
not significantly changed by the presence of Al and there
was no statistical difference between cultivars. The
reduction in CAD activity in the root system of the Alsensitive cultivar caused by Al, though it may not be
exactly reflecting the average activity with time of Al
treatment, clearly indicates that Al may directly affect
CAD or at least have an indirect effect by inducing Zn
deficiency as suggested by Sarni et al. (1984). On the
other hand, the increase observed in the Al-tolerant cultivar suggests that more lignification may be occurring in
this cultivar. The Al-tolerant cultivar probably is using this
strategy to reduce phenolic compounds and, or H2O2
accumulation, which are highly toxic to the cellular
metabolism (Cakmak & Horst, 1991; Siegel, 1993).
Monitoring changes in CAD and PAL activities will
certainly allow a better understanding of the importance
of these enzymes to lignification and to tolerance of plants
to Al.
R. Bras. Fisiol. Veg., 11(3):137-143, 1999.
Aluminum effects on lipid peroxidation . . .
The increase in CAD and SOD activities and also the
greater reduction in PAL activity observed in the presence
of Al probably resulted in lower O2.- and toxic phenols
accumulation, reducing lipid peroxidation in the root
system of the Al-tolerant cultivar. On the other hand, the
greater increase in POX activity and the increase in PPO
activity observed in the root system of the Al-sensitive
cultivar in the presence of Al suggest that a larger H2O2
and toxic phenos accumulation may have occurred in this
cultivar, resulting in an increased oxidative damage to its
membranes (Peixoto, 1998) and tissues (Figure 1, A and
B). Additionally, this would explain the larger reductions
in CAD and LOX activities in the plants of this cultivar
since such substances are potential inhibitors of these
enzymes. The reductions observed in CAT activity in the
root system of plants of both cultivars and in APX activity
in the root system of the Al-tolerant cultivar in the Al
presence suggest a reduced contribution of these
enzymes in the peroxide decomposition mechanisms,
strengthening the hypothesis that this toxic substance
may be consumed especially in the peroxidative reactions
under Al stress conditions.
AKNOWLEDGEMENTS
The authors acknowledge the financial support and
fellowships of the Conselho Nacional de Pesquisa e
Desenvolvimento Científico (CNPq), Brasília, DF, Brasil.
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