Journal of Experimental Botany, Vol. 52, No. 365, pp.

23752380, December 2001

Comparison of two methods used to analyse lipid

peroxidation from Vaccinium myrtillus (L.) during
snow removal, reacclimation and cold acclimation
Erja Taulavuori1,3, Eeva-Kaisa Hellstrom1, Kari Taulavuori1,2 and Kari Laine1
1
2

Department of Biology, University of Oulu, PO Box 3000, FIN-90014 Oulu, Finland

Muhos Research Station, Finnish Forest Research Institute, Kirkkosaarentie 7, FIN-91500 Muhos, Finland

Received 24 April 2001; Accepted 7 August 2001

Abstract
Malondialdehyde (MDA) concentration is a widely
used method to analyse lipid peroxidation in biological material. In plant tissues, however, certain
compounds (anthocyanins, carbohydrates) may
interfere with measurements which may lead to an
overestimation of the MDA levels. Two methods
were compared for analysing lipid peroxidation,
either uncorrected or corrected for interfering compounds. The comparison was performed in three
separate experiments with respect to cold treatments (snow removal in winter, reacclimation in
summer and cold acclimation in autumn) in bilberry
(Vaccinium myrtillus L.). During winter and autumn
the methods seem to measure different compounds,
but during active growth in the summer the difference
between the methods was less. This is obviously due
to carbohydrates which act as cryoprotectants and
increase in concentration during cold acclimation
as well as due to the anthocyanins. It is thus suggested that the validity of the uncorrected method
to measure MDA and thereby lipid peroxidation is
best in plant tissue which is in an active growth state.
Key words: Lipid peroxidation, stress, Vaccinium myrtillus,
cold acclimation.

Introduction
The major lipid classes of plant tissues are sterols, sterylglucosides, glucocerebrocides, and phospholipids, and
the proportions of free sterols and phospholipids increase
3

during cold acclimation (Steponkus, 1990). Qualitative

changes in the levels of fatty acids also occur with cold
acclimation, leading to unsaturation of the fatty acids
and subsequent protection of plant membranes against
freezing stress.
Lipid peroxidation is a widely used stress indicator of
plant membranes. The method described by Heath and
Packer is the basic protocol used or adapted in numerous studies dealing with lipid peroxidation (Heath and
Packer, 1968). This method is a very convenient assay
which is both quick and easy to perform. It is based on
malondialdehyde (MDA) production during the oxidation of polyunsaturated fatty acids. The reaction between
MDA and thiobarbituric acid (TBA) yields a reddish
colour, which peaks at 532 nm. The problem with the
method is that certain other compounds (anthocyanins
and carbohydrates) interfere with measurements at this
wavelength. Attempts to correct the interference include
omission of sucrose from the bathing solution (Lee et al.,
2000, and references therein) or the addition of PVP to
eliminate polyphenols (Blokhina et al., 1999). The method
was improved by subtracting the absorbance at 532 nm
of a solution containing the plant extract incubated
without TBA from an identical solution containing TBA
(Hodges et al., 1999).
Many recently published papers (Boo and Jung, 1999;
Di Toppi et al., 1999; Guidi et al., 1999; Chen et al.,
2000; Laukkanen et al., 2000; Somleva et al., 2000;
Mohammadi and Karr, 2001) still use the original method
(Heath and Packer, 1968; Dhindsa et al., 1981) without
correction for the interfering compounds. Overestimation
of lipid peroxidation is the obvious result in the absence
of correction. This evokes the question of how to
interpret the results: should the observed response be

To whom correspondence should be addressed. E-mail: erja.taulavuori@oulu.fi

Society for Experimental Biology 2001

2376

Taulavuori et al.

attributed to the level of stress or the acclimation against

it? For example, carbohydrate concentrations increase
during the cold hardening period in the autumn to
provide cryoprotection for a perennial plant. This may
increase the absorption at 532 nm and result in an overestimation of lipid peroxidation, which should actually
be decreased by acclimation.
The aim of this work is to compare the procedure
without (Heath and Packer, 1968) or with (Hodges et al.,
1999; Hodges and Forney, 2000) correction for interfering compounds in assessing lipid peroxidation. A special
effort has been made to relate lipid peroxidation to the
ecophysiology of cold hardiness, since membrane properties are essential for plants to maintain their vital
functions in a cold environment (Steponkus, 1990). The
work was carried out on bilberry (Vaccinium myrtillus L.),
which undergoes seasonal changes between active growth
and dormant stages. In addition, bilberry belongs to the
phanerophytes, i.e. its strategy in overwintering is to
avoid extreme cold under a well-insulating snow pack.
The above two methods used to analyse lipid peroxidation were compared on three occasions with respect to
cold: (1) snow removal during late winter, (2) artificial
reacclimation at the beginning of growth, and (3) natural
stem cold acclimation in the autumn.

Materials and methods

Experimental designs
The investigation consisted of three experiments, which were
performed in the Department of Biology and at the Botanical
Gardens of the Oulu University (northern Finland, 658 N) in
2000. The first experiment (Experiment I) was established in
March in order to manipulate bilberry (Vaccinium myrtillus L.)
by artificial removal of the snow cover. The snow pack was
removed from four (n 4) sample plots 1 m2 in size in a natural
bilberry stand growing in a spruce (Picea abies) forest in the
vicinity of the Botanical Gardens on 14 March 2000. Snow
removal was followed by assembling a plastic cover 0.5 m above
the bilberry shoots to prevent the formation of new snow cover.
The plastic cover was attached to a wooden frame with open
sides. Four control plots of the same size were marked with four
piles. The samples from the control plots were carefully dug
from beneath the snow, keeping the snowless area as small as
possible. This area was carefully recovered with powdered snow
immediately after sampling. The sampling was carried out on 14,
21 and 28 March. The outdoor minimum temperatures during
the experiment are shown in Fig. 1.
The second experiment (Experiment II) was performed in
growth chambers (Weiss Bio 1000) at the beginning of the
growing season (June). Turf patches containing mainly bilberry
vegetation were excavated and inserted into plastic boxes of
either 0.2 3 0.3 3 0.4 m or 0.2 3 0.4 3 0.6 m on 13 June. The next
day, the boxes were placed in climate chambers: one box of
each size were placed into each test unit (chamber) bringing the
total patch area to 0.36 m2. The patches contained an equal
quality and quantity of bilberry plants. In addition, excised
stems in a water container were placed in growth chambers
(1 containeruchamber). Two temperature treatments, q2 8C and

Fig. 1. Minimum temperatures during Experiment I: dashed line

denotes the control (i.e. below snow pack at the height of bilberry
plants) and the solid line ambient temperature (snow removal).
The experimental day 0 (x-axis) is the beginning of the experiment
(14 March).

q18 8C, were used to establish cold and warm conditions,

respectively. The cold treatment, i.e. reacclimation, was started
from q18 8C, and the temperature was decreased down to
q2 8C within 1 d in order to avoid a sudden temperature shock.
Two chambers (n 2) were used for both temperature treatments. Day length mimicked the outdoor photoperiod (approximately 22 h). During the light period, the maximum light
intensity was maintained using all the lamps (14 Lumilux and
3 Flora lamps). During the night break, however, 3 Flora lamps
were kept on, since total darkness does not occur at the 658 N
latitude at this time of year. The daytime irradiance peaked at
440, 550 and 615 nm (light intensity )1.5 W m2). RH was
70% in the q18 8C treatment, but could not be regulated in the
cold treatment (q2 8C). The sampling days were 14, 15, 19, and
22 June. The patches were kept well watered during the whole
period.
The third experiment (Experiment III) involved cold acclimation in both the field conditions and in the climate chambers
between 16 August and 14 September. The climate chamber
system including the plant boxes was similar to that described
for Experiment II. The temperature treatments were q2 8C and
q18 8C in the chambers, and ambient (Fig. 2) for outdoors.
The outdoor photoperiod was also programmed in chambers to
simulate outdoor conditions from 16 h 30 min to 13 h 20 min.
The plants were taken from the same forest site as in Experiment
II. The minimum ambient outdoor temperature is shown in
Fig. 2. The sampling days were 16 and 23 August and 1 and
14 September.

Sampling procedure
In each treatment, the samples were collected and placed
immediately into a styrofoam box filled with ice. They were
weighed and quickly frozen in liquid nitrogen (LN) followed by
storage in a freezer (70 8C).

Lipid peroxidation

Lipid peroxidation was analysed by both the uncorrected (U)

(Heath and Packer, 1968) and the corrected (C) methods
(Hodges et al., 1999). Both the U and the C results were

Comparison of MDA methods

2377

Fig. 2. Outdoor minimum temperature during Experiment III. The

experimental day 0 (x-axis) is the beginning of the experiment
(16 August).

obtained from the same extract with a procedure compatible

with the protocol described previously (Dhindsa et al., 1981),
except for minor modifications during homogenization. A 0.4 g
sample was homogenized in LN with a mortar and pestle. The
homogenized tissue powder was suspended in 5 ml of 0.1%
TCA on ice, and the residue of the suspension was rinsed
into a centrifuge tube with an extra 1 ml of TCA. Otherwise,
the analyses continued according to Dhindsa et al., and
they included the following steps: centrifugation, TCAuTBA
addition, a heatucool cycle and a second centrifugation
(Dhindsa et al., 1981). After that, the supernatant was divided
into two fractions, U and C. Absorbance of fraction U was read
at 532 nm subsequent to subtraction of non-specific absorption
at 600 nm. The malondialdehyde (MDA) concentration was
calculated using its extinction coefficient 155 mM1 cm1
(Heath and Packer, 1968). In the case of fraction C, absorbance
was also read at 440 nm in addition to 532 and 600 nm, and each
sample had a reference without TBA. The MDA equivalents
were calculated as described previously (Hodges et al., 1999).

Fig. 3. MDA concentration (mean"SE, n 4) in bilberry stems

measured with the uncorrected (A) and the corrected method (B) in
Experiment I. White bars denote the controls (bilberry plants covered
with snow) and grey bars denote the snow removal treatment.

Statistical analysis
The results obtained with the methods U and C were compared
with the paired sample T-test. Linear regressions between the
methods were also calculated. The data were arranged in a
hierarchic order for analysis of regression and T-tests, proceeding from general to more categorical levels: first the whole data,
and then all leaves or stems. The procedure was continued until
the split of the data, in order to find out any effects possibly
due to seasonality (Experiment), morphology (stem versus leaf )
and treatment (cold versus warm).

Results
The results are presented in detail in Figs 38. The data
show that the results obtained by the uncorrected (U) and
corrected (C) methods differ significantly (P-0.001) from
each other (Table 1). The findings concern the whole data
and the split categories, although the significance was
lower with bilberry stems in turf patches and excised
stems in a water container in Experiment II (P-0.05 and

Fig. 4. MDA concentration (mean"SE, n 2) in bilberry stems from

turf patches measured with the uncorrected (A) and the corrected
method (B) in Experiment II. White bars denote the warm (q18 8C)
treatment and grey bars the cold (q2 8C) treatment.

2378

Taulavuori et al.

Fig. 5. MDA concentration (mean"SE, n 2) in excised bilberry stems

kept in water containers measured with the uncorrected (A) or the
corrected method (B) in Experiment II. Legends as in Fig. 2.

Fig. 7. MDA concentration (mean"SE, n 2) in bilberry leaves of

excised stems kept in water containers measured with the uncorrected
(A) or the corrected method (B) in Experiment II. Legends as in Fig. 2.

Fig. 6. MDA concentration (mean"SE, n 2) in bilberry leaves from

turf patches measured with the uncorrected (A) or the corrected method
(B) in Experiment II. Legends as in Fig. 2.

Fig. 8. MDA concentration (mean"SE, n 2) in bilberry stems from

turf patches measured with the uncorrected (A) or the corrected method
(B) in Experiment III. White bars denote the warm (q18 8C) treatment,
grey bars the cold (q2 8C) treatment and black bars outdoor controls.

Comparison of MDA methods

Table 1. Comparison between the two methods: results from
regression analysis (R2) and paired sample T-test ( P-)
Data

R2

P-

All
All stems
All leaves (note: only Experiment II)
Stems
Experiment I
Experiment II
Experiment III
Experiment II
All
Bilberries in turf patches: All
Bilberries in turf patches: Stems
Bilberries in turf patches: Leaves
Excised bilberries in water container: All
Excised bilberries in water container: Stems
Excised bilberries in water container: Leaves
All: Cold treatment
Warm treatment

0.037
0.031
0.302

0.001
0.001
0.001

131
100
31

0.001
0.560
0.017

0.001
0.001
0.001

24
28
48

0.398
0.681
0.791
0.649
0.002
0.001
0.000
0.430
0.381

0.001
0.001
0.05
0.001
0.001
0.01
0.001
0.001
0.001

59
30
14
16
29
14
15
30
29

P-0.01, respectively). It is thus easy to conclude that

the two methods for lipid peroxidation do not necessarily
measure the same compound.
The regression analysis, however, yielded an interesting discovery, although the T-test showed the significant deviation between the methods. The R2 (0.56) in
Experiment II differs greatly from those in Experiments I
and III (0.00 and 0.02, respectively). HigherR2 will be
found when analysing Experiment II more carefully: the
leaves of the bilberries in turf patches show R2 0.65 and
the stems of the same plants even R2 0.79. The data
presented in Figs 4 and 6 also indicate that the changes
in MDA concentration assessed by the two methods are
similar.
The above findings suggest the possibility that the
status of lipid peroxidation, as analysed by the methods
U and C, depends on the states between active growth
and dormancy. Irrespective of the similar changes in
experiment II (Figs 4, 6), the level of MDA measured by
both methods is also closest in Experiment II (i.e. in
summer). Method C gave MDA concentrations around
or below 20 mmol g1 FW in all three experiments.
The method U, however, showed a level of 60 and
7080 mmol g1 FW MDA in late winter (Experiment I)
and autumn (Experiment III), respectively.

Discussion
In agreement with some other studies (Blokhina et al.,
1999; Hodges et al., 1999), the results suggest that lipid
peroxidation should be corrected against interfering
compounds. The uncorrected and corrected methods
obviously measure different compounds particularly
during snow removal in late winter (Experiment I) and
cold acclimation in autumn (Experiment III). However,

2379

during active growth in summer, both quantitative and

qualitative results analysed with both methods seem to
be close to each other. It is probable that the determination of other compounds (e.g. conjugated dienes
and trienes) in parallel with MDA might improve
the interpretation of the results for lipid peroxidation
(Blokhina et al., 1999).
Bilberry (Vaccinium myrtillus) stems exhibit seasonal
changes in carbohydrate concentrations. High glucose,
fructose and sucrose levels are attributed to cold hardiness or acclimation to winter, and the concentrations are
lower during active growth in the summer (Stewart and
gren, 1996;
Bannister, 1973; Pakonen et al., 1991; O
Taulavuori et al., 1997). The physiological background
is the starch hydrolysis taking place in autumn which
provides simple sugars for the cryoprotectant reserve. The
carbon assimilated during the summer is consumed in
growth processes, and the excess carbon is stored as
starch. The anthocyanins tend to accumulate in autumn
and become visible in the context of autumn coloration
of some trees. Actually, the accumulation of carbohydrates favours the formation of anthocyanins because
of their glycoside origin (Kramer and Kozlowski, 1979).
It is thus understandable that the difference in the
summer results (Experiment II) obtained both with
the uncorrected and the corrected methods is negligible
compared to the situation in late winter and autumn
(Experiments I and III).
To sum up, it can be assumed that the uncorrected
method measures both carbohydrates and MDA, while
the corrected method applies mainly to MDA. Therefore,
the corrected method should reflect lipid peroxidation,
and thereby the level of stress, better than the uncorrected
one. With this assumption, these results with respect to
cold can be interpreted as presented below.
Snow removal (Experiment I) did not cause stress
in agreement with other observations (Taulavuori et al.,
1997), according to whom there was no difference in
frost hardiness between bilberries with and without snow
cover in March. Indeed the elevated levels of MDA
measured by the uncorrected method may reflect the
competence against cold due to carbohydrates translocated from the below-ground rhizome. In Experiment
II, the continued warm treatment (q18 8C) may have
been the actual stressor compared to the reacclimation
treatment (q2 8C). It should be especially emphasized
that cold is an inaccurate term: q15 8C acts as a cold
stressor for maize (Kingston-Smith and Foyer, 2000),
while perennial species in boreal forests tolerate even
freezing temperatures during the growing season (Sakai
and Larcher, 1987). Cold acclimation (Experiment III)
in autumn begins as a consequence of the shortening
photoperiod, and high levels of carbohydrates could thus
be expected for plants in all treatments in accordance with
the interpretation of uncorrected results.

2380

Taulavuori et al.

Acknowledgement
This work was funded by the Academy of Finland.

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