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Journal of Experimental Botany, Vol. 52, No. 365, pp. 2375-2380, December 1, 2001
© 2001 Oxford University Press

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 Hellström1, Kari Taulavuori1,2 and Kari Laine1

1 Department of Biology, University of Oulu, PO Box 3000, FIN-90014 Oulu, Finland
2 Muhos Research Station, Finnish Forest Research Institute, Kirkkosaarentie 7, FIN-91500 Muhos, Finland

Received 24 April 2001; Accepted 7 August 2001


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
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
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The major lipid classes of plant tissues are sterols, sterylglucosides, glucocerebrocides, and phospholipids, and the proportions of free sterols and phospholipids increase during cold acclimation (Steponkus, 1990Go). 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, 1968Go). 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., 2000Go, and references therein) or the addition of PVP to eliminate polyphenols (Blokhina et al., 1999Go). 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., 1999Go).

Many recently published papers (Boo and Jung, 1999Go; Di Toppi et al., 1999Go; Guidi et al., 1999Go; Chen et al., 2000Go; Laukkanen et al., 2000Go; Somleva et al., 2000Go; Mohammadi and Karr, 2001Go) still use the original method (Heath and Packer, 1968Go; Dhindsa et al., 1981Go) 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 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, 1968Go) or with (Hodges et al., 1999Go; Hodges and Forney, 2000Go) 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, 1990Go). 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
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
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, 65° 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. 1Go.



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  		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).

 
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 0.2x0.3x0.4 m or 0.2x0.4x0.6 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 container/chamber). Two temperature treatments, +2 °C and +18 °C, were used to establish cold and warm conditions, respectively. The cold treatment, i.e. reacclimation, was started from +18 °C, and the temperature was decreased down to +2 °C 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 65° N latitude at this time of year. The daytime irradiance peaked at 440, 550 and 615 nm (light intensity >1.5 W m-2). RH was 70% in the +18 °C treatment, but could not be regulated in the cold treatment (+2 °C). 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 +2 °C and +18 °C in the chambers, and ambient (Fig. 2Go) 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. 2Go. The sampling days were 16 and 23 August and 1 and 14 September.



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  		Fig. 2. Outdoor minimum temperature during Experiment III. The experimental day 0 (x-axis) is the beginning of the experiment (16 August).

 

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 °C).

Lipid peroxidation
Lipid peroxidation was analysed by both the uncorrected (U) (Heath and Packer, 1968Go) and the corrected (C) methods (Hodges et al., 1999Go). Both the U and the C results were obtained from the same extract with a procedure compatible with the protocol described previously (Dhindsa et al., 1981Go), 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, TCA/TBA addition, a heat/cool cycle and a second centrifugation (Dhindsa et al., 1981Go). 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 mM-1 cm-1 (Heath and Packer, 1968Go). 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., 1999Go).

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
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
The results are presented in detail in Figs 3Go–GoGoGoGo8. The data show that the results obtained by the uncorrected (U) and corrected (C) methods differ significantly (P<0.001) from each other (Table 1Go). 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 P<0.01, respectively). It is thus easy to conclude that the two methods for lipid peroxidation do not necessarily measure the same compound.



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  		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.

 


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  		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 (+18 °C) treatment and grey bars the cold (+2 °C) treatment.

 


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  		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. 2Go.

 


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  		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. 2Go.

 


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  		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. 2Go.

 


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  		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 (+18 °C) treatment, grey bars the cold (+2 °C) treatment and black bars outdoor controls.

 

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

 
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 4Go and 6Go 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 4Go, 6Go), 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 µmol g-1 FW in all three experiments. The method U, however, showed a level of 60 and 70–80 µmol g-1 FW MDA in late winter (Experiment I) and autumn (Experiment III), respectively.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In agreement with some other studies (Blokhina et al., 1999Go; Hodges et al., 1999Go), 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, 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., 1999Go).

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 Bannister, 1973Go; Pakonen et al., 1991Go; Ögren, 1996Go; Taulavuori et al., 1997Go). 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, 1979Go). 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., 1997Go), 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 (+18 °C) may have been the actual stressor compared to the reacclimation treatment (+2 °C). It should be especially emphasized that cold is an inaccurate term: +15 °C acts as a cold stressor for maize (Kingston-Smith and Foyer, 2000Go), while perennial species in boreal forests tolerate even freezing temperatures during the growing season (Sakai and Larcher, 1987Go). 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.


   Acknowledgments
 
This work was funded by the Academy of Finland.


   Notes
 
3 To whom correspondence should be addressed. E-mail: erja.taulavuori{at}oulu.fi Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Blokhina OB, Fagerstedt KV, Chirkova TV. 1999. Relationships between lipid peroxidation and anoxia tolerance in a range of species during post-anoxic reaeration. Physiologia Plantarum 105, 625–632.

Boo YC, Jung J. 1999. Water deficit-induced oxidative stress and antioxidative defences in rice plants. Journal of Plant Physiology 155, 255–261.

Chen WP, Li PH, Chen THH. 2000. Glycinebetaine increases chilling tolerance and reduces chilling-induced lipid peroxidation in Zea mays L. Plant, Cell and Environment 23, 609–618.

Dhindsa RS, Plumb-Dhindsa P, Thorpe TA. 1981. Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. Journal of Experimental Botany 32, 93–101.[Abstract/Free Full Text]

Di Toppi LS, Lambardi M, Pecchioni N, Pazzagli L, Durante M, Gabbrielli R. 1999. Effects of cadmium stress on hairy roots of Daucus carota. Journal of Plant Physiology 154, 385–391.[Web of Science]

Guidi L, Bongi G, Ciompi S, Soldatini GF. 1999. In Vicia faba leaves photoinhibition from ozone fumigation in light precedes a decrease in quantum yield of functional PSII centres. Journal of Plant Physiology 154, 167–172.[Web of Science]

Heath RL, Packer L. 1968. Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Archives in Biochemistry and Biophysics 125, 189–198.[Web of Science][Medline]

Hodges DM, DeLong JM, Forney CF, Prange RK. 1999. Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207, 604–611.[Web of Science]

Hodges DM, Forney CF. 2000. The effects of ethylene, depressed oxygen and elevated carbon dioxide on antioxidant profiles of senescing spinach leaves. Journal of Experimental Botany 51, 645–655.[Abstract/Free Full Text]

Kingston-Smith AH, Foyer CH. 2000. Bundle sheath proteins are more sensitive to oxidative damage than those of the mesophyll in maize leaves exposed to paraquat or low temperatures. Journal of Experimental Botany 51, 123–130.[Abstract/Free Full Text]

Kramer PJ, Kozlowski TT. 1979. Physiology of woody plants. London: Academic Press Inc. Ltd, 274–281.

Laukkanen H, Rautiainen L, Taulavuori E, Hohtola A. 2000. Changes in cellular structures and enzymatic activities during browning of Scots pine callus derived from mature buds. Tree Physiology 20, 467–475.[Abstract]

Lee HJ, Lee SB, Chung JS, Han SU, Han O, Guh JO, Jeon JS, An G, Back K. 2000. Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase gene are resistant to diphenyl ether herbicide oxyfluorfen. Plant and Cell Physiology 41, 743–749.

Mohammadi M, Karr AL. 2001. Membrane lipid peroxidation, nitrogen fixation and leghemoglobin content in soybean root nodules. Journal of Plant Physiology 158, 9–19.[Web of Science]

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Pakonen T, Saari E, Laine K, Havas P, Lähdesmäki P. 1991. How do seasonal changes in carbohydrate concentrations in tissues of the bilberry (Vaccinium myrtillus L.) reflect carbon resource allocation patterns? Acta Oecologia 12, 249–259.

Sakai A, Larcher W. 1987. Frost survival of plants. Responses and adaptation to freezing stress. In: Billings WD, Golley F, Lange OL, Olson JS, Remmert H, eds. Ecological studies, Vol. 62. Berlin: Springer-Verlag.

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Steponkus PL. 1990. Cold acclimation and freezing injury from a perspective of the plasma membrane. In: Katterman F, ed. Environmental injury to plants. San Diego, California: Academic Press, 1–16.

Stewart CR, Bannister P. 1973. Seasonal changes in carbohydrate content of three Vaccinium sp. with particular reference to V. uliginosum and its distribution in the British Isles. Flora 162, 134–155.[Web of Science]

Taulavuori K, Laine K, Taulavuori E, Pakonen T, Saari E. 1997. Accelerated dehardening in bilberry (Vaccinium myrtillus L.) induced by a small elevation in air temperature. Environmental Pollution 98, 91–95.[Medline]


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