Involvement of the leaf antioxidant system in the response to soil flooding in two Trifolium genotypes differing in their tolerance to waterlogging

Plant Science 183 (2012) 43–49

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Involvement of the leaf antioxidant system in the response to soil flooding in two Trifolium genotypes differing in their tolerance to waterlogging L. Simova-Stoilova a,∗ , K. Demirevska a , A. Kingston-Smith b , U. Feller c a

Plant Stress Molecular Biology Department, Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, 1113 Sofia, Bulgaria IBERS Aberystwyth University, United Kingdom c Institute of Plant Sciences, University of Bern, Altenbergrain 21, CH-3013 Bern, Switzerland b

a r t i c l e

i n f o

Article history: Received 2 June 2011 Received in revised form 1 November 2011 Accepted 10 November 2011 Available online 17 November 2011 Keywords: White clover Red clover Oxidative stress Soil flooding Stress tolerance

a b s t r a c t A comparative study of the response to waterlogging in a tolerant (Trifolium repens L., white clover cultivar Rivendel) and susceptible (Trifolium pratense L., red clover cultivar Raya) plants was undertaken to reveal the possible link between plant performance and oxidative stress protection mechanisms in leaves. Two weeks of soil waterlogging induced visible leaf damage in the susceptible genotype. In the tolerant one, signs of stress suffering appeared a week later. Waterlogging induced hydrogen peroxide accumulation in both clover species. The content of lipid hydroperoxides markedly increased in the sensitive plants along with stress prolongation, while in the tolerant ones their initial rise was followed by return to control levels. In the leaves of both genotypes ascorbic acid content increased following treatment, accompanied by transient increase in oxidized ascorbate. Superoxide dismutase (SOD) isoforms responded differently to the treatment, CuZn SOD isoforms being inhibited; catalase activity diminished while peroxidase activity increased and a new peroxidase isoform was detected after prolonged waterlogging in both clover species. Results support more pronounced oxidative secondary stress in red clover leaves as a result of waterlogging with progressively increased oxidative membrane injury, protein loss, and peroxidase activity enhancement. White clover presented relative protein stability and earlier and more active ascorbate involvement in the antioxidative protection. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Flooding of different degree (from soil waterlogging to full submergence) and duration (days to months) is a worldwide encountered environmental stress which imposes severe constraint on plant growth and productivity [1,2]. The predicted climate change is expected to increase the frequency and severity of excessive rainfall and other extreme events, resulting in soil waterlogging [2,3]. Soil is considered to be waterlogged if the available water fraction on the surface layer is at least 20% higher than the field capacity or if there is freestanding water on the soil surface [4]. Waterlogging is a unique kind of stress with primary effect – impeded gas diffusion through water-saturated soil pores (104 slower diffusion of gases dissolved in water compared to air), leading to root hypoxia and dysfunction [5]. The prevailing anaer-

Abbreviations: ASC, ascorbate; CAT, catalase; DW, dry weight; FW, fresh weight; GPX, non-specific peroxidase; MDA, malondialdehyde; PAGE, polyacrilamide gel electrophoresis; ROS, reactive oxygen species; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances. ∗ Corresponding author. Tel.: +359 2 9792681; fax: +359 2 8729952. E-mail address: [email protected] (L. Simova-Stoilova). 0168-9452/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2011.11.006

obic microbial processes in the soil result in accumulation of toxic substances, in decrease of soil redox potential, reduction of soil nitrate, sulphate, manganese and ferric oxide [6,7]. Thus, due to decreased root hydraulic conductivity and functioning as well as altered soil composition, waterlogging stress imposes on shoots: disturbance of water and nutrient uptake, stomata closure and decline in photosynthesis, secondary toxicity stress, which increase with prolongation of stress [3,6]. As a result of waterlogging plant growth could be substantially inhibited and premature senescence could be induced [1]. The necessity to produce ATP and NADPH in roots anaerobically via glycolysis and ethanolic fermentation provokes at the whole plant level “energy crisis” and “carbohydrate crisis” with reduced capacity to replenish the exhausted sugar and starch reserves because of inhibition of photosynthesis [3]. In a relatively short time period, metabolic acclimation with induction of the production of the so-called “anaerobic stress proteins” which include enzymes of the glycolytic and fermentative pathway can help plants to tolerate root hypoxia [1,5]. Adaptation to long-term flooding stress is directly linked to drastic morphological changes in the root system, development of aerenhyma and new adventitious roots which improve internal gas exchange [8]. Prolonged waterlogging inevitably leads to secondary oxidative stress development both in roots and in shoots [1]. The prevention of reactive

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oxygen species (ROS) formation and countering of oxidative damage is a highly relevant defense mechanism both during short and long-duration waterlogging stress [3]. It is considered that the main source of oxidative stress in shoots during waterlogging stress is the well documented inhibition of the photosynthetic activity [9–11], which results in imbalance between light capture and utilization and may generate enhanced superoxide radical production by the Mehler reaction in chloroplasts [12]. Increases in the levels of ROS in plant leaves under flooding stress have been demonstrated previously [13–15]. Due to the short lifespan of ROS, their damaging effects are usually restricted to the sites of their production [16]. Accordingly antioxidant protection under hypoxia is complex and highly compartmentalized, comprising non-enzyme and enzyme components [17]. The main low-molecular antioxidant scavengers – ascorbate and glutathione, are normally present in the tissues in millimolar concentrations, chloroplasts and cytosol being particularly rich in these compounds, and in stress conditions their levels increase [18]. The enzymes of the superoxide dismutase family (SOD, EC 1.15.1.1), located both in the organelles and the cytosol, are playing a central role in the defense against ROS [16]. These work in concert with the enzymes and compounds of the ascorbate–glutathione cycle; with catalases in peroxisomes (CAT, EC 1.11.1.6) which remove H2 O2 generated in photorespiration, and with the broad specificity peroxidases (GPX, EC 1.11.1.7) located in vacuoles, cell walls and the cytosol [16]. Hydrogen peroxide diffusing to vacuoles could be reduced by vacuolar peroxidases using phenolics as primary electron donors [17]. Generation of ROS is an unavoidable consequence of electron transport and maintaining the balance between SOD, CAT and GPX activities is crucial for maintaining the steady-state level of superoxide and H2 O2 and for preventing the formation of highly cytotoxic hydroxyl radicals [16]. Any imbalance in this protective systems leads to oxidative damage to lipids and proteins. Evidence of increased level of thiobarbituric acid reactive substances (TBARS) as a result of lipid peroxidation is reported in the leaves of different plants under waterlogging stress [11,13,15,19–21]. The activity of antioxidant enzymes often increases after exposure to stress conditions which increase ROS generation but these enzymes are also susceptible to oxidative damage [1]. Given the link between ROS generation and hypoxia/anoxia it is possible that the capacity of delaying and coping with oxidative stress events could be linked with waterlogging tolerance in various plant species [19–21]. The attention of most authors in the study of flooding stress is directed mainly to metabolic changes in the root. Relatively little attention has been paid to the effects of this kind of stress on leaves despite the fact that leaves are the main source of proteins in leafy crops for human and animal consumption, and are important in ensuring grain filling. Here we have focused on red (Trifolium pratense L.) and white (Trifolium repens L.) clovers which are used as a protein source for ruminant and non-ruminant animal nutrition and also are important plant species in meadow plantations. Clovers are widely distributed in Europe’s middle latitudes, red clover being relatively sensitive to hypoxia [22,23], while white clover is moderately tolerant to flooding stress compared with other clover species [24]. Previous studies on the effect of waterlogging on clover have addressed root morphology, aerenchyma formation, nitrogen fixation and induction of some secondary metabolites [23,25,26]. Sensitivity to waterlogging differs between clover species and even between cultivars and ecotypes of the same species [8,27], but the underlying mechanisms remain poorly understood. However, a comparative study of the differential effect of acute ozone stress on white and red clover species has highlighted the importance of higher constitutive levels of some peroxidases in the antioxidative defense system of white clover as a possible reason for its better ozone stress tolerance [28].

In this work we have tested the hypothesis that differences in visible symptoms relating to poor tolerance to waterlogging could be due to constitutive differences in the functioning of the antioxidant defense system under excess water stress. The aim of the present study was to compare the main indicators of leaf oxidative damage and the content of certain oxidative stress protective metabolites and enzymes in two clover genotypes with different extent of leaf damage and stress tolerance as a result of the same waterlogging treatment. 2. Materials and methods 2.1. Plant material and stress treatment Certified seeds of T. repens L. cv Rivendel and T. pratense L. cv Raya were purchased from the “Cultivar seeds” commercial agency in Bulgaria. Seeds were presoaked in distilled water and germinated at room temperature on moist filter paper in the dark for one day. Imbibed seeds with protruding radicles were planted in pots (150 mm deep, 75 mm diameter, 20 seeds per pot) filled with Vam potting soil, Holland, with larger external dark containers. Vam potting soil composition was 20% organic material, 25% dry material, NPK (14-16-18) fertilizer 1 kg m−3 with trace elements, organic fertilizer 1 kg m−3 NPK (7-6-6) with trace elements, electrical conductivity 1.5 mS cm−1 and pH 5.5–6.5. Pots were irrigated regularly to maintain 70% of maximal soil water capacity. Growth chamber conditions were: light intensity 150 ␮mol photons m−2 s−1 , 12 h photoperiod, at 24/18 ◦ C day/night temperature. Plants were maintained under control conditions until day 21, thereafter waterlogging treatment was imposed to one half of the pots by slowly filling the external containers with tap water in upward direction until 2 cm of standing water appeared above the soil surface. Standing water above soil surface was maintained throughout the whole treatment by adding water in the external pot. The second half of the plants was kept at optimal water regime. Leaf samples from control and waterlogged plants were taken at day 14 after the beginning of the treatment (C14 and W14) and a week later at day 21 of waterlogging (C21 and W21). Analyses were performed on randomly mixed leaf samples comprising the mature fully expanded leaves from 10 to 12 plants, harvested from two different pots per species and treatment. Protein content and enzyme activities were assayed on samples quickly frozen in liquid nitrogen and stored at −70 ◦ C prior to extraction. For all other analyses fresh plant material was used. 2.2. Growth parameters, leaf pigment and protein content Root and shoot length and fresh weight (FW) were registered on 9 individual plants. For determination of dry weight (DW) per FW ratio, roots and shoots were dried at 105 ◦ C to constant weight. Total chlorophylls were extracted from 0.1 g FW mixed leaf sample with 80% acetone and estimated according to Lichtenthaler [29]. Protein quantity was determined using the method of Bradford [30] with bovine serum albumin as a standard. 2.3. Hydrogen peroxide and MDA determination Leaves (0.5 g FW) were homogenized in 5 ml of 0.1% trichloroacetic acid. At the time of grinding, 50 mg Polyclar AT was added. The homogenate was centrifuged at 10,000 × g for 30 min. Hydrogen peroxide content was assayed with the redox active indicator xylenol orange according to Wolff [31]. Values were calculated using standard curve with known amount of H2 O2 . Lipid peroxidation was estimated using the TBARS assay. The optical density was read at 440, 532 and 600 nm and malondialdehyde (MDA) content was calculated according to [32].

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2.4. Low molecular mass antioxidants Low molecular mass thiol compounds were determined as described by Edreva and Hadjiiska [33] and were assumed to represent mainly GSH. Briefly, 0.5 g of fresh leaf tissue was ground in a mortar with 5 ml of ice-cold freshly made 2.5% (w/v) 5sulphosalicylic acid. The homogenate was centrifuged at 15,000 × g for 30 min. Color reaction was developed by incubation for 30 min at room temperature of 500 ␮l of supernatant with 1.25 ml 400 mM Tris–HCl buffer pH 7.8 containing 20 mM EDTA and 50 ␮l Ellman’s reagent. Optical density at 412 nm was registered and the level of non-protein thiol groups was calculated using ε = 13,600 M−1 cm−1 for the reaction product 2-nitro-5-benzoic acid. The ascorbate pool (total and reduced) was assayed according to [34] on the basis of reduction of Fe3+ to Fe2+ by ascorbate in acid solution and complexation of Fe2+ with ␣,␣ -dipyridyl leading to a pink color. The absorbance at 525 nm was measured and ascorbate content was quantified using a standard curve. Oxidized ascorbate was estimated from the difference between total and reduced ascorbate. 2.5. Activities and izoenzyme forms of certain antioxidative enzymes Leaf material (0.5 g FW) was homogenized in ice-cold 50 mM Tris–HCl buffer pH 7.5 containing 2 mM MgCl2 , 2 mM CaCl2 , 10 mM ␤-mercaptoethanol, 2 mM phenyl-methanesulphonylfluoride, 0.005% Triton-X 100, 50 mg Polyclar AT and centrifuged at 15,000 × g for 30 min at 4 ◦ C. Activities of the antioxidative enzyme isoforms were estimated as previously described by using in-gel staining methods. One hundred micrograms of total protein was separated at 4 ◦ C on a native 7.5% PAGE (CAT and GPX) or 10% native PAGE (SOD). SOD activity was visualized and SOD types differentiated according to [35]. SOD isoforms were differentiated by pre-stain incubation for 30 min in 50 mM potassium phosphate buffer, pH 7.8 containing 5 mM H2 O2 . MnSOD and FeSOD are resistant to such a treatment, whereas Cu–Zn SODs are inhibited. CAT isoenzymes were stained following [36], and GPX isoforms were determined according to [37] using diaminobenzidine as a substrate. 2.6. Statistical analysis Results were based on at least three independent replicates and the whole experiment was repeated two times. Representative data from one experiment are shown. Data for each parameter were analyzed by multifactor ANOVA (Statgraphics plus version 2.1) at level of significance P < 0.05. Means and standard deviations are given; values marked with different letters in the tables and figures are significantly different. Zymograms were not statistically analyzed as negative stainings are not strictly quantitative. 3. Results 3.1. Growth inhibition and morphological responses to waterlogging Typical physiological and morphological response to waterlogging stress was observed in both clover species. Visible leaf stress symptoms appeared in red clover at W14 while some leaf damage in white clover was seen only a week later, at W21. Soil flooding for 2–3 weeks resulted in significant growth inhibition (Table 1) and in dramatic morphological changes in the root system with disappearance of deeply situated root parts and formation of lateral roots close to the soil surface (data not shown). Despite this unidirectional response to waterlogging concerning root system, the red clover cultivar presented more severe shoot FW reduction

Fig. 1. Control and waterlogged white and red clover plants after 21 days of stress.

which progressed with stress prolongation (58% and 77% reduction of shoot FW at 14 and 21 days of waterlogging, compared to the respective control plants), while waterlogging-induced decrease in fresh weight in the white clover cultivar seemed to “stabilize” at 65% (Table 1). Both genotypes had significant and progressive increase in DW to FW ratio in the shoots, most probably linked with disturbance in water balance. 3.2. Symptoms of premature senescence in the leaves Control plants did not show any symptoms of leaf injury throughout the experiment (Fig. 1). In red clover visible yellowing and wilting of individual leaves was noticed within 2 weeks of waterlogging (data not shown), which became more pronounced with prolongation of stress, whereas in white clover a minor leaf yellowing was observed only after 3 weeks of waterlogging (Fig. 1). In control plants leaf pigment content presented a slight tendency to increase during the period of stress treatment (Table 2), whereas in waterlogged plants the content of chl a + b diminished with stress prolongation (by 22% at W14 and 44% at W21 for white clover, by 47% at W14 and 60% at W21 for red clover, compared to the respective age controls). The same tendency was noticed in carotenoid changes (decrease by 14% at W14 and 42% at W21 for white clover, by 44% at W14 and 55% at W21 for red clover, compared to the respective age controls). Chla/b and chla + b/car ratios were not significantly changed. Leaf total soluble protein content was relatively stable in the white clover cultivar, whereas waterlogging resulted in a decrease in foliar protein in the red clover cultivar (by 36% at W14 and more at W21). Pigment and protein loss was more pronounced in the waterlogging-sensitive genotype in concert with the visible symptoms.

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Table 1 Effect of waterlogging on shoot length, fresh and dry biomass accumulation in white clover cv. Rivendel and red clover cv. Raya. Genotype

Treatment

Length (cm)

White clover (cv. Rivendel)

C 14 W 14 C 21 W 21

15.1 10.3 14.2 13.0

± ± ± ±

1.3d 2.2a 3.3c,d 2.1b,c

FW (mg) 865 299 894 433

± ± ± ±

80c 25a,b 147c,d 83b

DW to FW ratio 0.0845a 0.1084c 0.0955b 0.1301d,e

Red clover (cv. Raya)

C 14 W 14 C 21 W 21

18.5 11.2 19.4 9.9

± ± ± ±

1.1e 1.5a,b 2.0e 2.2a

1046 435 1072 245

± ± ± ±

175d,e 86b 88e 43a

0.1212d 0.1349e 0.1377e 0.1544f

Treatments: W14 – waterlogged plants following 14 days of stress, C14 – control plants of the same age as W14; W21 – waterlogged plants following 21 days of stress, C21 – control plants, same age. Means ± standard deviations are given from measurements on 9 individual plants. For each parameter, values followed by different letters are significantly different at P < 0.05.

Table 2 Effect of waterlogging on leaf pigment and total soluble protein content in the leaves of white clover cv. Rivendel and red clover cv. Raya. Genotype

Treatment

Chl a (mg.g−1 FW)

Chl b (mg.g−1 FW)

Carotenoids (mg.g−1 FW)

Protein (mg.g−1 FW)

White clover (cv Rivendel)

C 14 W 14 C 21 W 21

1.57 1.23 1.94 1.09

± ± ± ±

0.08d 0.06c 0.06f 0.07b

0.52 0.40 0.67 0.38

± ± ± ±

0.08b,c 0.04a,b 0.10d,e 0.06a,b

0.43 0.37 0.55 0.32

± ± ± ±

0.04c 0.04b,c 0.04d 0.03a,b

26.05 26.15 22.67 24.75

± ± ± ±

5.95a,b 5.45a,b 2.525a,b 4.65a,b

Red clover (cv Raya)

C 14 W 14 C 21 W 21

1.72 0.93 2.12 0.84

± ± ± ±

0.13e 0.02a 0.07g 0.07a

0.56 0.28 0.72 0.31

± ± ± ±

0.09c,d 0.03a 0.11e 0.05a

0.54 0.30 0.64 0.29

± ± ± ±

0.05d 0.02a 0.03e 0.02a

30.1 19.25 22.95 15.60

± ± ± ±

5.5b 1.55a,b 1.05a,b 1.2a

Treatments: W14 – waterlogged plants following 14 days of stress, C14 – control plants of the same age as W14; W21 – waterlogged plants following 21 days of stress, C21 – control plants, same age. Means ± standard deviations are given from 3 independent replicates. For each parameter, values followed by different letters are significantly different at P < 0.05.

3.3. Accumulation of hydrogen peroxide and oxidative damage to lipids in the leaves

conserved in both genotypes. It seems that in red clover the ASC pool was engaged in the stress response only a week later (W21).

Increases in the steady state level of the relatively stable active oxygen species H2 O2 and of membrane lipid peroxidation products is considered to reflect oxidative stress. Hydrogen peroxide and MDA levels in control (C14, C21) and waterlogged (W14, W21) plants are presented in Fig. 2. The red clover cultivar had higher constitutive H2 O2 content than the white clover cultivar and there was a tendency in increase of H2 O2 concentration in leaves with plant age in both clover types. Compared to control plants, plants flooded for 2 weeks had significantly elevated leaf H2 O2 content in both genotypes (W14) but without further accumulation after one additional week of stress treatment (W21). In both clover species, the content of TBARS was also higher after 2 weeks of waterlogging (W14) compared to the controls (C14). In white clover, however, MDA levels returned to that of controls a week later, whereas in red clover lipid peroxidation continued to increase with stress prolongation (W21).

3.5. Leaf SOD, CAT and GPX isoenzyme changes Total enzyme activity may not reflect changes in the activities of distinct enzyme isoforms, while activity staining after

3.4. Response of the nonenzymatic ROS scavenging system to waterlogging Leaf ascorbate and glutathione pools are the main components of the non enzymatic antioxidative defense system in plants which readily respond to oxidative stress. In Fig. 3 the content of lowmolecular antioxidant thiol compounds (represented mainly by glutathione) and ascorbate in the leaves of control (C14, C21) and waterlogged (W14, W21) plants is presented. Leaf low-molecular thiol compounds did not change dramatically during application of waterlogging stress conditions. Ascorbic acid content increased following treatment in both clover types, although this occurred earlier in white clover plants (W14) with transient rise of the oxidized ascorbate, which reflected active involvement of ASC in the detoxification of ROS before visible stress symptoms. Thereafter, the ratio of reduced to oxidized ascorbate was more or less

Fig. 2. Hydrogen peroxide and malondialdehyde content in the leaves of control (C14, C21) and waterlogged (W14 – for 2 weeks, W21 – for 3 weeks) clover plants. White columns – hydrogen peroxide, stripped columns – lipid peroxidation level. The columns represent means ± SD of three replicates from a representative of two independent experiments. Different letters above columns mark values significantly different at P < 0.05.

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Fig. 3. Low molecular mass thiol content, oxidized and reduced ascorbate in the leaves of control (C14, C21) and waterlogged (W14, W21) clover plants. Checked columns – thiol level, white part of columns – the part of oxidized ascorbate, grey part of columns – reduced ascorbate. The columns represent means ± SD of three replicates from a representative of two independent experiments. Different letters above columns mark values significantly different at P < 0.05.

electrophoretic separation is informative both for total activity and for changes in enzyme isoforms. In Fig. 4 are presented the results of in-gel staining for SOD, CAT and GPX activity after separation of the extract protein in non-denaturing PAGE. Data are on a leaf protein basis, which corresponds to the relative proportion of the enzyme in the total protein content. Four isoforms of SOD, one of CAT and several forms of GPX were clearly visible. Bands of SOD isoforms were established using preincubation with H2 O2 which inhibits CuZn SOD isoforms (data not shown), and compared with other published results for the respective plant species [38,39]. Based on inhibition by H2 O2 and comparison with data published by other authors, two Mn SOD or Fe SOD (SOD1 and 2) and two Cu/Zn SOD (SOD3 and 4) isoforms were revealed. Predominant forms were SOD2 and 4 in white clover, SOD2 in red clover which remained unchanged following treatment. One of the CuZn SOD forms in white clover (cytosolic as reported by [39], who obtained similar SOD pattern for white clover) and both CuZn SOD forms in red clover were inhibited as a result of waterlogging stress. Catalase activity increased in control plants with age but diminished in both clover types after 3 weeks of stress treatment. Several isoforms of peroxidase were detected in activity stained gels and some new enzyme forms with lower molecular mass appeared after prolonged waterlogging in both genotypes (GPX* for white clover, GPX** for red clover). In red clover the higher GPX activity at W21 correlated with more expressed visible stress symptoms. 4. Discussion This study was focused on the role of leaf oxidative status and major ROS defense systems as implicated in soil flooding stress, comparing two clover species. Both white and red clover responded similarly to waterlogging stress with substantial shoot growth inhibition and diminution in leaf chlorophyll and carotenoid content. Similar parallel loss of chlorophylls and carotenoids were observed Vicia faba leaves under flooding stress [40]. Pigment

Fig. 4. Isoenzyme profiles of SOD, CAT and GPX in the leaves of white and red clover control and flooded plants. Abbreviations below the images: C14, W14 – control and waterlogged plants at 14th day of treatment, C21, W21 – control and waterlogged plants at 21st day of treatment. Different isoforms are indicated at the sides. Gels are loaded with 100 ␮g protein per lane.

changes did not match exactly those typical of senescence in which carotenoids usually persist while chlorophyll pigments are progressively degraded and chl/car ratio is changed [41]. These changes could be explained rather by adjustment of the photosynthetic apparatus to the reduction in photosynthetic activity under this kind of stress, reported by several authors in different plant species [9–11]. On the other hand, carotenoids have additional stress protective role as ROS scavengers [16] and their decline could make chloroplasts more vulnerable to oxidative damage. The red clover cultivar under study suffered greater growth inhibition and presented earlier and more severe visible stress symptoms, as well as tendency to diminution in leaf protein content, while in the white clover cultivar leaf protein was almost unchanged. Protein loss in red clover was in concert with increasing malondialdehyde accumulation in this genotype after 3 weeks of waterlogging as an index of oxidative damage of cell constituents in general. A rise in the level of various ROS has been shown by some authors in the leaves of different plant species subjected to waterlogging stress [13,15]. It could be speculated that the main cause of protein decrease in the red clover genotype, besides general inhibition of biosynthetic processes under stress, could be due to possible oxidative damage to proteins, which could mediate enhanced proteolysis. Once generated, ROS act usually randomly and nonselectively. Oxidatively damaged proteins, which are particularly prone to proteolysis, could be degraded by an ATP-independent proteasomal activity [42]. Another feature reported in the leaves of flooded plants is an increase in leaf titrable acidity indicating some acidification of the cytoplasm [43]. Cytoplasmic acidosis may lead

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to protease activation and increased proteolysis, manifested as protein loss. Upregulation of cysteine protease proteins was detected in tomato leaves subjected to waterlogging stress by proteomic analysis [44]. These assumptions should be verified by further analysis of the changes in leaf protein pattern and proteolytic activity under waterlogging stress. The detected higher content of H2 O2 following 14 days of stress along with TBARS accumulation in both clover species support increased oxidative strain on leaves under root hypoxia. However the lack of further increase in H2 O2 a week later plus some diminution in activities of CAT and certain SOD isoforms indicated that the oxidative stress was rather moderate. In other species a more severe oxidative stress situation was detected in the leaves under waterlogging, manifested by elevated SOD and CAT activities as well as by progressive increase in H2 O2 accumulation [11,13,21]. A plausible explanation for the relatively moderate oxidative stress level is the ability of clover species to counteract the negative effects of soil hypoxia by changing root morphology and developing shallow adventitious roots with increased aerenchyma, thus improving internal gas exchange within the whole plant [8,26]. At present we can only speculate about subcellular location of the elevated H2 O2 content. Blokhina et al. [45] have visualized cytochemically an enzyme-driven H2 O2 formation in plasma membranes and the apoplast in four plant species during anoxic stress. The increase in the general peroxidase activity and the detection of many isoforms of this enzyme, including appearance of new ones under the applied stress in both genotypes, rather suggests that active generation of H2 O2 takes place in the apoplast to be used as a peroxidase substrate. Besides protection of plant tissues against oxidative damage, peroxidases are also known to promote cell wall lignification and protein cross-linking [46]. The same authors observed an appearance of new GPX isoform under drought stress in clover, as we found new GPX isoforms under prolonged waterlogging stress. The localization and precise function of the new enzyme form remain to be elucidated. Plant peroxidases belong to a superfamily that contains three different classes and have several isoforms with specific functions, assisting growth inhibition and raising barriers to chemical toxicity [47]. In both genotypes an active involvement of ascorbate in counteraction of oxidative stress damage was registered, especially after more prolonged waterlogging stress. An early involvement of antioxidative enzymes in stress protection, as well as a subsequent predominant role of the low-molecular antioxidant compounds accompanied by diminution in enzymatic ROS protection has been documented in different plant species [10,11,13,40]. In our study, we revealed different response of the individual SOD isoforms to flooding stress, which may indicate variation in the oxidative stress load in different cell compartments. The predominant SOD2 band (most probably Fe-containing chloroplastic isoform as reported by [39]) was relatively stable, whereas SOD3 in white and red clover (CuZn-containing isoforms most probably of cytoplasmic origin) and SOD4 in red clover were inhibited. Our results differ from the reported reduction in chloroplastic Fe SOD activity in barley plants under flooding stress [9], which could probably be explained by species peculiarities. It seems that in clover leaves the active involvement of ascorbate and GPX is the prevailing response under waterlogging stress. Along with the common trends, some differences in the leaf antioxidative protection under waterlogging stress were found when comparing the two clover genotypes. The white clover cultivar tended to diminish oxidative damage to lipids with stress prolongation and leaf protein content did not change significantly while in the red clover cultivar lipid peroxidation increased further with duration of the stress along with enhanced activity of GPX bands. This was in accordance with the more pronounced growth inhibition and increased visible symptoms of leaf injury. In

white clover, ROS protection via ascorbate appeared to have been involved earlier in the stress response than in red clover. Probably the severity of symptoms under waterlogging stress in red clover could be linked to relatively later ascorbate engagement in the stress response as compared to white clover plants. More rapid mobilization of various constituents of the antioxidative protection in the more tolerant genotypes was found in comparative studies on other plant species under waterlogging, which was linked with delay in the stress injury [19,21,48]. However, it is not excluded that other mechanisms besides oxidative stress-driven injury play also a role in the waterlogging susceptibility of red clover. 5. Conclusions In this study, the red clover leaves suffered more from oxidative stress under soil waterlogging than the leaves of the more tolerant white clover genotype, evidenced by (i) progressively increased oxidative membrane injury, (ii) protein loss, (iii) both CuZn SOD isoforms with diminished activity, (iv) greater GPX involvement in the stress response. White clover seemed to be able to partly recover from oxidative stress and minimize leaf damage in contrast to the red clover genotype, manifested by (i) return of MDA to the level of controls with stress prolongation, (ii) relative protein stability, (iii) earlier and more active ASC involvement as a ROS scavenger. Acknowledgements This study was supported by bilateral projects between BAS, IBERS Aberystwyth University, and Institute of Plant Sciences, University of Bern. The skilled technical assistance of A. Kostadinova and R. Nenkova is greatly acknowledged. References [1] R.K. Sairam, D. Kumutha, K. Ezhimathi, P.S. Deshmukh, G.C. Srivastava, Physiology and biochemistry of waterlogging tolerance in plants, Biol. Plant 52 (2008) 401–412. [2] P. Perata, W. Armstrong, L.A.C.J. Voesenek, Plants and flooding stress, New Phytol. 190 (2011) 269–273. [3] T.D. Colmer, L.A.C.J. Voesenek, Flooding tolerance: suits of plant traits in variable environments, Funct. Plant Biol. 36 (2009) 665–681. [4] P.K. Aggarwal, N. Kalra, S. Chander, H. Pathak, Infocrop: a dynamic simulation model for the assessment of crop yields losses due to pests, and environmental impact of agro-ecosystemsin tropical environments, Agric. Syst. 89 (2006) 1–25. [5] M. Irfan, Sh. Hayat, Q. Hayat, Sh. Afroz, A. Ahmad, Physiological and biochemical changes in plants under waterlogging, Protoplasma 241 (2010) 3–17. [6] C.W.P.M. Bloom, L.A.C.J. Voesenek, Flooding: the survival strategies of plants, Trends Ecol. Evol. 11 (1996) 290–295. [7] I.M. Unger, P.P. Motavalli, R-M. Muzika, Changes in soil chemical properties with flooding: a field laboratory approach, Agric. Ecosyst. Environ. 131 (2009) 105–110. [8] M.R. Gibberd, J.D. Gray, Ph.S. Cocks, T.D. Colmer, Waterlogging tolerance among a diverse range of Trifolium accessions is related to root porosity lateral root formation and aerotropic rooting, Ann. Bot. 88 (2001) 579–589. [9] R.Y. Yordanova, L.P. Popova, Photosynthetic response of barley plants to soil flooding, Photosynthetica 39 (2001) 515–520. [10] S. Ahmed, E. Nawata, M. Hosokawa, Y. Domae, T. Sakuratani, Alterations in photosynthesis and some antioxidant enzymatic activities of mungbean subjected to waterlogging, Plant Sci. 163 (2002) 117–123. [11] Z. Hossain, M.F-M. Lopez-Climent, V. Arbona, R.M. Perez-Clemente, A. GomezCadenas, Modulation of the antioxidant system in citrus under waterlogging and subsequent drainage, J. Plant Physiol. 166 (2009) 1391–1404. [12] A. Edreva, Generation and scavenging of reactive oxygen species in chloroplasts: a submolecular approach, Agric. Ecosyst. Environ. 106 (2005) 119–133. [13] B. Yan, Q. Dai, X. Liu, S. Huang, Z. Wang, Flooding induced membrane damage, lipid oxidation and activated oxygen generation in corn leaves, Plant Soil 179 (1996) 261–268. [14] R.Y. Yordanova, K.N. Christov, L.P. Popova, Antioxidative enzymes in barley plants subjected to soil flooding, Environ. Exp. Bot. 51 (2004) 93–101. [15] R. Jamei, R. Heidari, J. Khara, S. Zare, Hypoxia induced changes in the lipid peroxidation, membrane permeability, reactive oxygen species generation, and antioxidative response systems in Zea mays leaves, Turk. J. Biol. 33 (2009) 45–52.

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