PHYSIOLOGIA PLANTARUM 83: 463-^168, Copenhagen 1991

Effect of aluminium on lipid peroxidation, siiperoxide dismutase,

catalase, and peroxidase activities in root tips of soybean (Glycine
max)
Ismail Cakmak and Walter J. Horst

Cakmak, 1. and Horst, W. J. 1991. Effect of aluminium on lipid peroxidation,

superoxide dismutase, catalase,, and perosidase activities io root tips of soybean
{Glycine max). - Physiol. Plant 83: 463-468.

Inhibition of root elongation and modification of membrane properties are sensitive

responses of plants to aluminium. The present paper reports on the effect of Al on
lipid peroxidation and activities of enzymes related to production of activated oxygen
species. Soybean seedlings {Gtycine max L. cv. Sito) were precultured in solution
culture for 3-5 days and then treated for 1-72 h with Al (AlCi3) concentrations
ranging from 10 to 75 \>.M at a constant pH of 4.1. In response to Al suppiy, lipid
peroxidation in the root tips (< 2 cm) was enhanced only after longer durations of
treatment. Aluminium-dependent increase in lipid peroxidation was intensified by
Fe'"^ (FeSOj). A close relationship existed between lipid peroxidation and inhibition
of root-elongation rate induced by Al and/or Fe toxicity and/or Ca deficiency. Besides
enhancement of iipid peroxidation in the crude extracts of root tips due to Ai, the
activities of superoxide dismutase (EC 1.15.1.1) and peroxidase (EC 1.11.1.7) in-
creased, whereas catalase (EC 1.11.1.6) activity decreased. This indicates a greater
generation of oxygen free radicals and related tissue damage. The results suggest that
iipid peroxidation is part of the overail expression of Al toxicity in roots and that
enhanced lipid peroxidation by oxygen free radicals is a consequence of primary
effects of Al on membrane structure.
Key words - Aluminium toxicity, cataiase, Glycine max, lipid peroxidation, mem-
brane damage, peroxidase, root elongation, soybean, superoxide dismutase.

/. Cakmak, Depi of Soil Science and Planl Nutrition, Univ. of Cukurova, Adana,
Turkey and W. J. Horsi. {corresponding author), Institut fiir Pflanzenernahrung,
Univ. Hannover, Herrenhauser Str. 2, D-3000 Hannover 21, Germany.

charge of protoplasts and Al tolerance of plant species

Introduction (Wagatsuma and Akiba 1989) also indicate a distinct
Inhibition of root elongatioti is a sensitive response of effect of A! on membrane integrity.
soybean to aluminium (Horst and Klotz 1990). The Increasing evidence exists that membrane injury by
primary target of Al at the cellular level might be the various stress factors is related to an increased produc-
plasma metnbrane. This view is supported by results tion of highly toxic oxygen free radicals. In Fe- and
showing alterations of plasma irtembrane properties by Zn-deficient plant roots (Cakmak et al, 1987, Cakmak
interference of Al with membrane lipids (Vierstra and and Marschner 1988a,b), aod in plant tissues injured
Haug 1978, Deleers et al. 1985, Hunter and Etherton mechanically (Salin and Bridge 1981) or by virus in-
1989) or membrane proteins (McDonald et al, 1987, fection (Doke and Ohashi 1988), the induction of an
Caldwell 1989), The modification of the permeability of enzyme system producing superoxide radicals (Oj") has
the plasma membrane to electrolytes (Horst et al. 1991, been demonstrated. This enzyme system is mostly lo-
Cakmak and Horst 1991) and nonelectrolytes by Al cated in plasma membranes and cell walls and uses
(Zhao et al. 1987) and the relationship between surface NAD(P)H as a reductant for O,, The existence of an

Received 16 May, 1991; revised 22 July, 1991

Physiol. PJanl. 83. lWl 463

 i producing peroxidase in cell walls (Gross et uid nitrogen. Root-tip material (300 mg) was homoge-
al. 1976) is now well established. An increase in perox- nized in 3 ml 0.1% TCA solution. The homogenate was
idase activity is a common response to various oxidative centrifuged at 15 000 g for 10 min and 0.5 ml of the
stress factors (Gaspar et al. 1985). Enhanced produc- supernatant obtained was added to 1.5 ml 0.5% TBA in
tion of oxygen free radicals are responsible for stress- 20% TCA, The mixture was incubated at 90°C in a
dependent peroxidation of membrane lipids (Elstner shaking water bath for 20 min, and the reaction stopped
1987). Superoxide dismutase (SOD) and catalase are by placing the reaction tubes in an ice-water bath. Then,
important defence systems of plants against oxygen free the samples were centrifuged at 10 000 g for 5 min, and
radicals. Therefore, increased activities of these en- the absorbance of the supernatant was read at 532 nm.
zymes may be considered as circumstantial evidence for The value for non-specific absorption at 600 nm was
enhanced production of such radicals (Fridovich 1986, subtracted. The amount of MDA-TBA complex (red
Eistner et al, 1988). pigment) was calculated from the extinction coefficient
In the literature, very little information is available on 155 mM"' cm"'.
the relationships between Al toxicity, oxygen free rad- For the measurement of enzyme activities, root tips
icals and related membrane damage. From animal phys- (< 2 cm), usually 500 mg fresh weight, were homoge-
iology research it is known that Al in the presence of Fe nized in 10 ml HEPES-KOH buffer (pH 7.8) containing
or HiOi may enhance lipid peroxidatioo in phospholipid 0.1 mM EDTA. The homogenate was centrifuged at
liposomes isolated from rats (Gutteridge et al. 1985, 15000 g for 15 min. All operations were performed at
Quinlan et al. 1988), Furthermore, it has been reported, 4°C. In the supernatant, superoxide dismutase (SOD)
that modifications of membrane structure (e.g. by Al, was assayed by a photochemical method as described by
see above), may stimulate oxygen radical production Giannopolitis and Ries (1977). Reaction mixtures (3
and facilitate lipid peroxidation (Demopoulos 1973, ml) consisted of 50 mM HEPES-KOH (pH 7.8), 0.1
Vladimirov et al. 1980). The present paper reports on mJW EDTA, 50 mM Na,CO, (pH 10.2), 12 mM L-meth-
studies of the effect of Al on lipid peroxidation and ionine, 75 [xM NBT, appropriately diluted enzyme ex-
activities of enzymes related to oxygen free radical pro- tract (0 to 300 nl) and 1 |xM riboflavin. One unit SOD
duction. The objective of the study was to contribute to activity was defined as the amount of enzyme required
a better understanding of short-term effects of Al on to result in a 50% inhibition of the rate of NBT reduc-
membrane integrity. tion measured at 560 nm. Activities of catalase and
peroxidase were measured according to Chance aod
Abbreviations -- MDA, malondialdehyde; NBT, ;j-nitro blue Maehly (1955). The reaction mixture for catalase con-
tetrazolium chloride; SOD, superoxide dismutase; TBA, thio- sisted of 25 mM potassium phosphate buffer (pH 6,8),
barbituric acid. 10 mM HyO, and diluted enzyme extract in a total
volume of 1 ml. The decomposition of HIOT was fol-
lowed by the decline in absorbance at 240 nm. For
Materials and methods peroxidase, the reaction mixture consisted of 25 mM
potassium phosphate buffer (pH 6.8), 10 mM H^O,,
Soybean {Glycine max L, cv. Sito) plants were growo 0.05% guaiacol and diluted enzyme extract. The ox-
under controlled climatic conditions at 28/25°C day/ idation of guaiacol was followed at 470 nm. Protein was
night temperatures, light/dark regimes of 16/8 h, light measured by the method of Bradford (1976).
intensity at table height of 280 [imol (Sylvania
VHO cool white, 215 W lamps) and 70% relative hu- Aluminium in root tips was determined by atomic
midity. Seeds were germinated in quartz sand moist- absorption spectrometry after drying at 65°C and dry
ened with 10 mM CaSOj. After 3 ^ days, seedlings were ashing at 450°C overnight.
transferred to 22-1 plastic vessels.
The composition of the nutrient solution was as fol-
lows (iiM): 750 KNO3, 325 MgCNO,),, 10 KH2PO4,
1000 CaSOj, 8 H3BO,, 0,2 MnSOj, o'.2 ZnSO^, 0,2 Tab. 1. Primary root elongation, Al concentration and lipid
CUSO4 and 0,2 (NH4)6M07O24. Concentrations of Al peroxidation in root tips (< 2 cm) as dependent on the dura-
tion of Al supply (40 [iM], At harvest soybean plants (cv. Sito)
(AlClj) and Fe (FeSO4) in the nutrient solution and were 7 days old. Mean ± SD, n = 3; ND = not determined.
treatment duration differed with each experiment as
indicated in the legends to the figures and tables. The Duration Increase in root A! Lipid
pH of the nutrient solution was adjusted to 4.1 and kept of AI length (cm) concentration peroxidation
constant with 0.01 M HCl or KOH by automatic litra- supply (h) (mgg"' DW) (nmol MDA
-Al +A1 g-' FW)
tion.
For the measurement of lipid peroxidation in root 0 ND 14.011,6
tips, the TBA test which determines MDA as an end 1 0.25+0.05 O.22±0.05 0.6+0.0 14.9+0.6
product of lipid peroxidation (Heath and Packer 1968) 6 2,3±0.3 1,7+0.2 1.410.2 16.0+0.6
was used. The root tips (< 2 cm) were isolated in 24 7.310.6 4.110.4 2.0±0.1 17.610,7
48 19.811.9 4.511.0 ND 20.4±2.1
distilled water (pH 4,1) and immediately frozen in liq-

464 Physiol. Plant. 83. IW!

Tab. 2. Effect of Al supply on primary root elongation and lipid Tab. 4. Effect of Ca. Al, and Fe supply on primary root
peroxidatioB in root tips (< 2 cm) of soybean plants (cv. Sito). elongation and hpid peroxidation in root tips (< 2 cm) of
The duration of Al treatments was 32 h following preculture soybean plants (cv. Sito). Duration of treatments was 24 h
for 3 days without Al. Mean ± SD, n = 3. following preculture for 3 days at 1 000 fiM Ca without Al and
Fe. Mean + SD, n = 3.
Al supply Root elongation Lipid peroxidation
M) (cm |32 h]"') (nmol MDA g^' FW) Supply
- elongation peroxiaaiion
Ca Fe Al (nmol MDA
0 9.81 i.O 10.510.4 (cm |24h]-')
10 10.410.4 11.4+1.1 g-' FW)
20 8.910.9 11.710.7
40 5.8+0.6 14.211.3 1000 0 0 7.11015 8.411.6
250 0 0 5.511.0 10.211.0
100 0 0 3.610.9 11.611.3
1000 0 40 3.810.4 11.310.3
Results 1000 40 0 4.4±0.9 1L8+0.0
250 40 0 3.210.6 15.4+1.1
Root growth of the A!-sensitive soybean cultivar used in
100 40 0 2.7+0.7 16.812.9
these experiments was severely depressed by 40 [iM Al 1000 40 40 3.3+0.5 16.4+1.1
(Tab. 1): Only 1 h after Al was supplied root elongation
was slightly reduced and by 48 h had been almost com-
pletely inhibited. Aluminium concentrations in the root ment with high Al supply (Tab. 5) indicating an in-
tips increased with the duration of Al treatment. After 6 creased level of the superoxide radical. This was con-
h Al supply, a slight increase in lipid peroxidation was firmed in other experiments including those conducted
evident and increased further up to 48 h. Lipid perox- using lower Al concentrations (Tab. 6). and those with
idation in root tips was rather insensitive to lower Al prolonged Al treatment (Tab. 7). In contrast to SOD,
supplies which did not lead lo severe depression of root activity of catalase tended to decrease with increasing
elongation (Tab. 2), but was very sensitive to high Al supply after 24 h (Tab. 6) and clearly decreased after
FeSO., supply, which inhibited root elongation to a de- 72 h of Ai treatment (Tab. 7). Peroxidase activity was
gree comparable to that obtained with a high Al supply
(Tab. 3). In the presence of Al the promotion of lipid
peroxidation by Fe was even more distinct. Not only Fe
and Al toxicity but also Ca deficiency inhibited root
growth and enhanced lipid peroxidation (Tab. 4). De-
pression of root elongation and enhancement of iipid
peroxidation were most severe at low Ca or high Al
supply in the presence of Fe. Regardless of the factors
involved, the relationship between root elongation rate -20-
and lipid peroxidation can be described by a highly
<
significant regression (Fig. 1). Q

Lipid peroxidation mediated by activated oxygen spe-

cies should be accompanied by changes in activities of
enzymes involved in oxygen metabolism. Activity of
15
SOD was increased in root tips after short-term treat-
Tab. 3. Effects of Al and Fe supply (FeSO^) on primary root
elongation and lipid peroxidation in root tips (< 2 cm) of
soybean plants (cv. Sito). Duration of Al and Fe treatments
was 24 h following precuiture for 3 days without Al and Fe.
Mean ± SD, n = 3. 10
Al supply Fe supply Root Lipid peroxidation
(|iM) ' 1((tM) elongation (nmol MDA
(cm[24h]-') g'' FW)

0 0 7.810.6 8.911.1
0 10 7.7+0.4 9.3+0.6
4.0+0.7 12.7+0.9
0
0
40
80 3.1+0.6 18.710.5
c 0 2 4 6 8
Root elongation [cm (24 h)'"']
40 0 3.7+0.9 12.1+0.3
40 10 3.2±0.6 I6.7±0.7 Fig. 1. Relationship between primary root elongation and lipid
40 40 3.010.4 18.3+0.4 peroxidation in root tips (< 2 cm) of soybean plants (cv. Sito).
40 80 1.810.5 23.6±0.3 Variation was due to Al, Fe and Ca supply. Data compiled
from Tabs 3 and 4.

Physiol PIml; 8.1. 1991 465

Tab. 5. Effect of Al treatment (75 iiM) on activity of superox- estimation of lipid peroxidation by this method is well
ide dismutase (SOD) in root tips (< 2 ctn) of soybeati plants correlated to other tnethods such as HPLC, fluorescent
(cv. Sito). Plants were precultured for 3 days without AL, Mean analysis, electron spin resonance spectroscopy, and UV
± SD, n =3.
absorbtion of conjugated dienes (Gutteridge 1987,
Al treatment SOD activity Pompella et al. 1987, Janero and Burghardt 1989, Yang
duration (h) et al. 1991).
(Ug-'FW) (U mg ' protein)
Using both TBA assay and fluorescent analysis, Gut-
teridge et al. (1985) and Quinlan et al. (1988) showed
0 102±21 18.7±1.2 with bovine phospholipids and rat liver microsomes that
1 109+ 2 19.6±1.5
6 127± 8 20.1±1.0 Al alone exerted only a slight effect on lipid perox-
24 140± 9 23.711.1 idatioo. However, together with Fe, Al was highly ef-
fective in stimulating lipid peroxidation. Similarly, lipid
peroxidation in soybean root tips measured with the
drastically enhanced even at lower Al supplies and shor- TBA assay was enhanced in response to Al supply only
ter Al treatment duration (Tabs 6 and 7). after longer treatment duration (Tabs 1 and 2) and,
particularly, in the presence of Fe (Tab. 3). In accord-
ance with Gutteridge et al. (1985) and Gutteridge
Discussion (1987) we suggest that Al potentiates or facilitates lipid
Although the thiobarbituric acid (TBA) assay is the peroxidation by disorganizing membrane structure.
most extensively used test for the measurement of lipid In the literature there are several lines of evidence
peroxidation in cell menibranes and isolated lipids (Gi- indicating that modification of membrane structure can
rotti et al. 1985, Halliwell and Gutteridge 1990) limita- activate production of oxygen free radicals and induce
tions of the test have been documented (Gutteridge and lipid peroxidation, especially via dislocated hemopro-
Halliwell 1990, Halliwell and Gutteridge 1990). The teins and nonheme iron compounds (Demopoulos 1973,
lack of specificity of the TBA assay has been especially Vladimirov et al. 1980). It has repeatedly been reported
discussed for animal and humati materials but, to our that several Fe compounds have a high potential to
knowledge, not for plant roots. In such materials sev- promote free radical production and thus peroxidation
eral compounds other than malondialdehyde (MDA) of membrane lipids (Elstner 1987, Gutteridge 1987).
may react with TBA to produce absorption at 532 nm. Interactions of Al with membrane lipids and proteins
On the other hand there are many reports showing that and consequent impairment of membrane structural

Tab. 6. Effect of Al supply (24 h) on root elongation, and activities of superoxide dismutase (SOD), catalase and peroxidase in
root tips (< 2 cm) of soybean plants (cv. Sito) precultured for 6 days without Al.

Al supply Root SOD Catalase Peroxidase

elongation
(cm [24 h]-') ( U g - ' FW) (U mg-' (U g-' FW) (U mg ' (U g-' FW) (U mg-'
protein) protein) protein

0 6.3 222 19.9 5.4 0.49 154 14

20 5.2 247 20.6 4.4 0.37 270 22
40 3.8 277 26.6 3.9 0.38 286 28
80 2.6 277 27.7 4.8 0.48 328 33
LSD 0.05 1.0 32 2.9 0.4 0.05 26 5

Tab. 7. Effect of Al supply (3 days) on root elongation and activities of superoxide dismutase (SOD), catalase and peroxidase in
root tips (<2 cm) of soybean plants (cv. Sito) precuitured for 5 days without Al.

Al supply Root SOD Catalase Peroxidase

elongation
(cm [72 h]-') (U g-' FW) (U mg-' (U g-" FW) (U mg-' (U g-' FW) (U mg-'
protein) protein) protein)

0 19.7 239 42 5.9 1.02 103 17.7

10 18.4 263 41 5.5 0.87 150 23.9
20 13.9 322 38 4.4 0.53 234 26.4
40 401 . 47 3.8 0.45 327 38.2
3.6
LSD 0.05 3.9 86 7 1.6 0.17 98 9.8

466 Physiol. Plant. 83. 1991

properties are also well documented (see Introduction), ion, peroxidase activity showed an increase with dura-
Increases in lipid peroxidation as a result of Ca defi- tion and level of Al treatment, whereas catalase activity
ciency (Tab. 4) may be interpreted along the same lines decreased (Tabs 6 and 7). This indicates that in Al
(Gutteridge 1976), However, a more direct effect of treated roots, HjOj is mostly consumed in oxidation
Ca'* on lipid peroxidation cannot be excluded, Babiz- processes, such as lipid peroxidation, rather than detox-
hayev (1988) explained enhanced lipid peroxidatioo in ified. Similar patterns of peroxidase and catalase activ-
liposomes at low Ca^* concentrations (1-10 (iM) by ities have been found in plant tissues under toxicity
release of Fe"* from exchange sites and thus enhance- caused by oxygen (Foster and Hess 1980) and Fe (Hen-
ment of hydroxyl radical production via the metal-cata- dry and Brocklebank 1985),
lyzed Haber-Weiss reaction, at the same time as inhib- Our results suggest that lipid peroxidation is part of
ition of lipid peroxidation by higher Ca-^ coocentrations the overall expression of Al toxicity in roots. It appears
(100-1000 fiM) may be caused by complexation and that enhanced lipid peroxidation is the consequence of
thus incacfivation of the superoxide anion radicals. primary effects of Al on membrane structure rather
The observed lipid peroxidation was less sensitive to than the reason for the modification of membrane prop-
Al than the inhibition of root elongation. Therefore, the erties by AI, However, the method used to estimate
close relationship between root elongation rate and lipid peroxidation and enzyme activities in the crude
lipid peroxidation, independent of the factor respon- extracts of whole root tips is not very sensitive, because
sible for growth inhibition (Fig, 1), suggests that lipid initial effects of Al are mainly restricted to the outer
peroxidation is the consequence rather than the primary cortex cells as indicated by the localization of Al (Kin-
cause of Al injury to plant roots. raide 1988) and Al-induced callose accumulation (Wis-
Especially after prolonged Al treatment, root tips semeier et al, 1987),
became brown which may indicate oxidation of phenolic
compounds to cytotoxic quinones (Elstoer 1987), The Acknowledgements - We thank the Deutsche Forschungsge-
cytotoxicity of quinones is believed to be mediated by meinschaft for financial support, H. Marschner, Institute for
Plant Nutrition, Univ. of Hohenheim for stimulating discus-
their one-electron reduction to semiquinone radicals, sions and Mr P. D. Seward, Institute for Plant Nutrition and
which autooxidize to form Oj- and other reiated toxic Environmental Researeh, Diiimen, for correeting the English
O, species (Kappus 1986, Blstner 1987), This phenom- text.
enon is responsible for drug toxicity in human cells
(Kappus 1986) and involved in plants in parasite de-
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