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Response of embryo axes of germinating seeds of yellow lupine to Fusarium oxysporum
Plant Physiology and Biochemistry 42 (2004) 493–499 www.elsevier.com/locate/plaphy
Original article
Response of embryo axes of germinating seeds of yellow lupine to Fusarium oxysporum Iwona Morkunas a,*, Waldemar Bednarski b, Monika Kozłowska a a
Department of Plant Physiology, August Cieszkowski Agricultural University, Wołyn´ska 35, 60-637 Poznan´, Poland b Institute of Molecular Physics, Polish Academy of Science, Smoluchowskiego 17, 60-179 Poznan´, Poland Received 15 March 2004; accepted 10 May 2004 Available online 15 June 2004
Abstract Defence responses of embryo axes of Lupinus luteus L. cv. Polo were studied 48–96 h after inoculation with Fusarium oxysporum Schlecht f.sp. lupini. The infection restricted the growth of embryo axes, the lengths of infected embryo axes 72 and 96 h after inoculation were 11 and 12 mm less in the controls, respectively, while their masses c. 0.03 g less than in the controls. The concentration of H2O2 in embryo axes of inoculated germinating seeds was higher than in the control. This was probably a consequence of oxidative burst as well as H2O2 generation by the invading necrotrophic fungal pathogen. EPR-based analyses detected the presence of free radicals with g1 and g2 values of 2.0052 ± 0.0004 and 2.0031 ± 0.0005, respectively. Concentrations of the radicals 72 and 96 h after inoculation were 50% higher than in the control. The values of the spectroscopic splitting coefficients suggest that they are quinone radicals. However, inoculated embryo axes possess a number of adaptive mechanisms protecting them from oxidative damage. A twofold increase in catalase (CAT, EC 1.11.1.6) activity was evidenced in embryo axes infected with F. oxysporum Schlecht f. sp. lupini, as compared to the control 48–96 h after inoculation. Superoxide dismutase (SOD, EC 1.15.1.1) activity 96 h after inoculation was 80% higher than in the control. Furthermore, EPR-based analyses revealed a higher concentration of Mn2+ ions after 72 h for inoculated embryo axes, as compared to the control. On the other hand, no increase was detected in the concentration of thiobarbituric acid reactive substances (products of lipid peroxidation) in infected embryo axes. The protective mechanisms induced in lupine embryo axes in response to F. oxysporum Schlecht f.sp. lupini were compared with responses to infections with pathogenic fungi elicited in other plant families. © 2004 Elsevier SAS. All rights reserved. Keywords: Free radicals; Fusarium oxysporum; Germinating seeds; Hydrogen peroxide; Lupinus luteus; Lipid peroxidation; Oxidative stress
1. Introduction Adverse environmental conditions and deep sowing make germinating seeds prone to fungal infections. Susceptibility to fungal attack is particularly evident at the earliest stages of seed germination, when intensive structural and metabolic changes are involved in embryo activation [37,38]. At this early phase of plant development, environmental stimuli may
Abbreviations: ABTS, 2,2′-Azino-bis (3-ethylbenz-thiazoline)-6sulfonic acid; CAT, catalase; EPR, electron paramagnetic resonance; H2O2, hydrogen peroxide; TBARS, thiobarbituric acid reactive substances; NBT, nitro blue tetrazolium; ROS, reactive oxygen species; SOD, superoxide dismutase. * Corresponding author. E-mail address: [email protected] (I. Morkunas). © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2004.05.007
modify various biochemical pathways, interfering with the distribution of metabolites between donor (cotyledon) and acceptor (embryo axis) tissues. Perturbations in sugar distribution, indispensable for respiration, growth, and development of embryo axes, diminish their defence capacity [30,31]. Germinating lupine seeds and seedlings are frequently infected with F. oxysporum, including f.sp. lupini [11]. These pathogens cause Fusarium wilt and pre-emergent sprout root rot and post-emergent seedling rot. Phytopathologists pay a lot of attention to fungi of the genus Fusarium, since they play a major role in pathogenesis of vascular plants. However, there is little data on physiological reactions elicited by F. oxysporum during germination. It is widely believed that plants have coevolved with pathogens to develop complex mechanisms, both constitutive and inducible, to cope with the infection [1,20]. One of the short-term
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responses that take place upon the pathogen invasion is an enhanced generation of free radicals, including reactive oxygen species (ROS) [12,18,33]. ROS derive from molecular oxygen by stepwise incomplete electron uptake, finally leading to complete oxygen reduction and production of H2O [18]. ROS accumulation is closely associated with the induction of plant defence reactions against pathogens, such as the hypersensitive reaction, defence gene expression, and cell wall strengthening via cross-linking reactions of phenylpropanoids and proteins [22]. Under conditions of normal healthy growth, plants possess a number of enzymatic and non-enzymatic mechanisms of detoxification to efficiently scavenge for either the ROS themselves or their secondary reaction products [5]. Major enzymes of the defence system, which eliminate ROS directly, are catalases, superoxide dismutases [25], and ascorbate peroxidases [21]. During prolonged periods of oxidative stress, however, these mechanisms become overwhelmed, resulting in tissue damage. In this study we investigated the role of oxidative stress in early response of Lupinus luteus L. cv. Polo to infection with F. oxysporum Schlecht f.sp. lupini during germination. The levels of free radicals and hydrogen peroxide, as well as changes in activities of antioxidative enzymes (CAT and SOD) were assessed. As a secondary objective, we estimated membrane damages on the basis of the level of products of lipid peroxidation—thiobarbituric acid reactive substances (TBARS).
2. Results 2.1. Growth of yellow lupine embryo axes Changes in length and fresh weight of embryo axes of germinating lupine seeds under the influence of F. oxysporum are shown in Fig. 1. The growth of embryo axes was retarded as a consequence of inoculation; the length of infected embryo axes 72 h after inoculation was 11 mm and their mass 0.032 g less than control axes. Later on, i.e. 96 and
Fig. 1. Effects of F. oxysporum Schlecht f.sp. lupini on the length (A) and fresh weight (B) of embryo axes of germinating lupine seeds.
120 h after inoculation, we observed only a slight increase in embryo length difference, which reached 12 and 13 mm, respectively. Simultaneously, the differences in fresh weight between infected and control embryo axes decreased to 0.031 and 0.028 g, respectively. 2.2. Hydrogen peroxide H2O2 content determined in embryo axes of germinating lupine seeds was influenced by their inoculation with F. oxysporum (Fig. 2). The increase in H2O2 content was quite stable, ranging from 15% to 24%. 2.3. EPR signals of free radicals and Mn2+ ions Examples of typical wide-scan EPR spectra of the control and infected tissues are shown in Fig. 3A. In both cases the spectra consist of free radicals and Mn2+ lines. Free radicals gave signals with two g-values of 2.0052 ± 0.0004 and 2.0031 ± 0.0005, both in the control and in infected embryo axes. Their levels were in the same ratio within experimental accuracy for all studied samples. Fig. 3A presents the differences between signal intensities of control and infected embryo axes vs. cultivation time. Free radical signals decreased between 48 and 96 h in control tissues, in contrast to the infected tissues, where the intensity of free radicals remained at the same high level. Fig. 3B and C shows concentrations of free radicals and Mn2+ ions in the control and infected embryo axes vs. cultivation time, respectively. The high levels of free radicals (3.9 × 1015 g–1 DW) and Mn2+ ions were observed after 48 h for control tissues. Free radical concentrations were almost at the same level of 3.3−3.7 × 1015 g–1 DW for infected tissues. The concentration of free radicals was about 43% (72 h) and 50% (96 h) higher in infected embryo axes than in the control. We found that 72 h after inoculation, Mn2+ concentration was about 38% higher than
Fig. 2. Concentration of H2O2 (nmole H2O2 per 1 g fresh weight) in embryo axes of germinating lupine seeds infected with F. oxysporum f.sp. lupini and in the control.
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Fig. 4. Changes in the activities of CAT (A) and SOD (B) in embryo axes of germinating lupine seeds infected with F. oxysporum f. sp. lupini and in the control.
Fig. 3. Typical wide-scan EPR spectra of embryo axes of germinating lupine seeds (A) and concentrations of free radicals (B) and manganese ions (C); i, embryo axes infected with F. oxysporum f. sp. lupini; c, control. Arrows indicate the signals of free radicals and manganese sextets.
in the control (Fig. 3C). However, earlier on and later on, Mn2+ concentration in embryo axes of germinating seeds inoculated with F. oxysporum was lower than in the control. 2.4. Antioxidative enzymes Results of assays of catalase (CAT) and superoxide dismutase (SOD) activities are illustrated in Fig. 4. An increase in CAT activity was observed both in embryo axes of germinating seeds that were inoculated with F. oxysporum and in the control 48–96 h of culture (Fig. 4A). The stimulating effect of inoculation of embryo axes with F. oxysporum on CAT activity was revealed—the activity was 2–2.5 times higher in infected embryo axes than in the control 48–96 h after inoculation. In contrast to these results, SOD activity was initially slightly (by 16%) lower in embryo axes of infected seeds than in the control (Fig. 4B). Later on, an increasing trend in SOD activity was observed: the value in infected seeds increased by 33% after 72 h and by 80% after 96 h, as compared to the control. 2.5. Thiobarbituric acid reactive substances Fig. 5 shows the results of analyses of concentration of thiobarbituric acid reactive substances (TBARS) in lupine
Fig. 5. Concentration of TBARS in embryo axes of germinating lupine seeds infected with F. oxysporum f. sp. lupini and in the control.
embryo axes of germinating seeds under the influence of F. oxysporum infection. During all measurements, TBARS concentration in infected and non-infected embryos was roughly similar except that 48 h after inoculation the concentration was 13% lower in infected axes.
3. Discussion 3.1. Hydrogen peroxide production An enhancement of ROS production during the oxidative burst is one of the earliest defence reactions elicited in response to infection with pathogenic microorganisms [23,29]. That different ROS may trigger opposite effects in plants depending on their spatiotemporal distribution and subcellular concentrations [18]. H2O2—a relatively stable oxygen form, which was assayed in this paper—is a key molecule not only because it shows a direct antimicrobial activity but also
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because it is a factor mediating cell wall strengthening [4,27]. Studies performed by Lu and Higgins [26] demonstrated that H2O2 may remarkably inhibit the growth of Cladosporium fulvum (a pathogenic fungus), and that the H2O2 concentration effective in killing the fungus is considerably lower than the concentration causing plant cell death. In addition, other studies have shown that acting at a relatively low concentration, H2O2 could be a factor inducing the expression of defence-related genes, including genes coding for CAT [16,17,35]. The data presented in our study indicate that in infected embryo axes of yellow lupine the level of H2O2 is 15–24% higher than in the control (Fig. 2). This result suggests that H2O2 generation in infected embryo axes may be one of the strategies of their defence against invasion by a necrotrophic pathogen. However, it cannot be excluded that the fungus F. oxysporum generates some H2O2, too. Recent investigations show that necrotrophic pathogens, like Botrytis cinerea, use oxidative processes during their attack and invasion of plant tissues. Those investigations suggest a role for the so-called ROS as fungal pathogenicity factors, which include hydrogen peroxide, so it is often difficult to distinguish experimentally between the actual sources of the ROS [15,28]. 3.2. Electron paramagnetic resonance spectra and concentrations of free radicals EPR-based analyses carried out in this study show that in tissues infected with F. oxysporum, concentrations of free radicals with g values of 2.0052 ± 0.0005 and 2.0031 ± 0.0004 were relatively high during all measurements, reaching 3.3–3.7 × 1015 of free radicals per 1 g DW (Fig. 3A,B). Their levels 72 and 96 h after inoculation were 50% higher than in the control. The value of the spectroscopic splitting coefficient indicates that it may be the quinone radical. Accumulation of similar stable radicals, which had two g values of 2.0054 and 2.0023, and originated from a single free radical species, was observed by Atherton et al. [3] on desiccated moss. Moreover, Atherton et al. [3] suggested that the precursors of these radicals might be either quinones involved in electron transport pathways, or simple phenolic metabolites, or more complex polyphenols. Application of EPR allowed Muckenschnabel et al. [33] to demonstrate the appearance of a pronounced free radical signal with a g value of 2.0035 as a result of infection of Arabidopsis thaliana by B. cinerea. In an earlier work of the same research team the increases in intensity of the EPR signal with a g value of 4.27 (Fe III), as well as a single stable free radical signals, were evidenced to result from infection of bean leaves with B. cinerea [32]. We believe that the apparent increase in concentration of quinone radicals reported here might result from a H2O2 burst occurring at an early stage of infection with a pathogen. At later stages of infection, H2O2 becomes massively converted to hydroxyl radical, which—being a strong one-electron oxidizing agent—reacts non-specifically with other organic compounds and destroys
in this way the biological activity of macromolecular structures and cell membranes [19,39]. Also the changes in concentrations of free radicals observed in our study in control embryo axes are interesting. Unlike in infected tissues, the level of free radicals in control tissues decreased during the experiment. The highest concentration was observed 48 h after the beginning of the experiment. This indicates that intensive metabolic processes took place in the axes of germinating seeds during the rupture of the seed coat by the radicle (24−48 h after the beginning of the experiment). Similar observations were made by Schopfer et al. [36] in healthy, actively germinating radish seeds. They showed that a strong rise in ROS release is initiated in germinating seeds shortly before the radicle protrudes through the seed coat. These ROS may also originate from the seed coat (the living aleurone layer) as well as the embryo. Moreover, it was demonstrated that the production of ROS by germinating seeds is an active, developmentally controlled physiological function, presumably for protecting the emerging seedling against attack by pathogens. 3.3. Lipid peroxidation The most important observation in this paper is the detection of the large progressive increase in CAT activity with time both in the control and in infected embryo axes. We suppose that the strong activation of CAT protects cells against an excess of H2O2 and thus against considerable membrane degradation caused by oxidative stress. A small but progressive increase was recorded in the level of TBARS (products of lipid peroxidation) vs. time in the control samples, but no significant differences between control and infected tissues were found (Fig. 5). Moreover, 48 h after inoculation a 13% decrease in TBARS content was observed, in comparison with the control. No signs of an infectionrelated increase in levels of MDA and 4-hydroxy-2-nonenal (another product of lipid peroxidation) have been found in bean leaves subjected to a strong oxidation stress resulting from infection with B. cinerea [33]. This is in contrast to observations made with pathogen-inoculated tissues from other plant families [12,32]. It is also in conflict with the results of Conklin et al. [10], who showed that increased MDA levels were formed in an ascorbic-acid-deficient Arabidopsis mutant when subjected to oxidative stress (by ozone) [10]. In our study, the observed decrease in TBARS between control and infected tissues may be a consequence of the limited stability of those products and their metabolization by the fungus [33]. 3.4. Activity of antioxidative enzymes and the intensity of the Mn2+ EPR signal SOD is thought to be the key enzyme involved in H2O2 production [34] and our data are consistent with this notion, as judged by a significant increase in SOD activity detected in embryo axes of germinating lupine seeds infected with
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F. oxysporum. Namely, SOD activity in embryo axes of infected seeds was 33% higher (72 h after inoculation) and 80% higher (96 h after inoculation) than in the control. An interesting adaptation seems to be the observed presence of Mn2+ ions in germinating lupine seeds, detected by EPR simultaneously with signals of free radicals, as reflected in the sextets visible in Fig. 3A. The high level of Mn2+ ions in embryo axes 72 h after inoculation may aim at supporting the synthesis of Mn–SOD (Fig. 3C), as we observed then the highest intensity of infection, reflected in results of measurements of length and fresh weight (Fig. 1). No Mn2+ ions were present in the external environment in which the seeds germinated, so the observed result may attest to a higher transport of Mn2+ ions from cotyledons to embryo axes in infected embryos. The mechanism of this phenomenon cannot be explained without detailed investigations into ion distribution in the seedling during infection. The decrease in concentration of Mn2+ ions in embryo axes 96 h after inoculation may be determined by the physiological stage of germination, because of the beginning of the autotrophic growth stage. The effective concentration of free radicals in plant tissues is a result of a dynamic equilibrium between the rates of their production and scavenging. SOD and CAT were shown to be involved in the regulation of H2O2 levels in plant tissues [8,22,24]. In our study, we observed a marked induction of CAT in material infected with F. oxysporum, as its level was there about 2.5 higher than in the control (Fig. 4A). Thus the activation of this enzyme was disproportionately higher than H2O2 generation. The relatively low accumulation of H2O2 was probably due to the high CAT activation, also 48 h after the beginning of the experiment. However, the role of this enzyme in the plant seems to be more complex. This would confirm the observations made by Małolepsza and Urbanek [27] who detected a decrease in CAT activity when H2O2 concentration was high in tomato leaves infected with B. cinerea, so could be regarded as evidence for an overwhelming of this antioxidant enzyme. The high efficiency of the antioxidative system observed in this study, in particular with reference to catalase in embryo axes of germinating lupine seeds, is one of the most important elements of their defence against oxidative stress. It seems that the protective mechanisms induced in response to pathogen attack in embryo axes of germinating lupine seeds were effective enough not to allow considerable damage of membrane structures. Interestingly, the protective responses elicited in order to stop the development of an invading pathogen were not sufficient to enable the normal development of lupine seedlings. 4. Methods 4.1. Plant material Yellow lupine (Lupinus luteus L. cv. Polo) seeds of S-elite class were used in the experiments. The seeds were surface-
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sterilized, immersed in sterile water and left in a thermostat (25 °C). After 6 h of imbibition, the seeds were transferred onto filter paper (in Petri dishes) and immersed in a small amount of water in order to support further imbibition. After subsequent 18 h, imbibed seeds were sown in pots (45 seeds per pot) containing sterilized perlite, inoculated with a spore suspension of F. oxysporum f.sp. lupini and allowed to germinate at 23 °C at an irradiance of 130 µmole m–2 s–1 (Philips TLD 58W/84 fluorescent lamps) under a light regime of 12 h light/12 h dark. As a control, non-inoculated germinating seeds were used. Embryo axes were removed 48, 72 and 96 h after inoculation in order to perform analyses. Since our aim was to investigate if lupine seedlings are able to restrict the development of the invading pathogen, the length and fresh weight of embryo axes was analysed till 120 h after inoculation. Results of the measurements of length and fresh weight were means for 40 embryo axes. In all assays, samples of the same fresh weight were compared. 4.2. Fungi and inoculation F. oxysporum f.sp. lupini strain K-1018 (further named F. oxysporum) was obtained from the Bank of Pathogens, Institute of Plant Protection, Poznan´. The pathogen was grown on a PDA medium (agar-Difco) in light at 24 °C. The spore suspension used for inoculation was prepared from a 3 week-old culture and was applied at a concentration of 5·× 106 spores per 1 ml. 4.3. Assay of hydrogen peroxide The embryo axes (1 g of fresh weight) were ground in 100 mM potassium phosphate buffer (pH 7.0) with Polyclar AT, in a chilled mortar on ice. The homogenates were centrifuged at 15 000 × g for 20 min. The concentration of H2O2 was determined according to Bergmeyer and Bernt [7]. The reaction mixture contained: 400 µl of plant extract, 2 ml of 100 mM KH2PO4/K2HPO4 (pH 7.0) buffers, 50 µl of horseradish peroxidase (147 U ml–1), and 50 µl of ABTS solution (50 mM). The concentration of H2O2 was estimated by measuring absorbance at 415 nm against a calibration curve and was expressed as nmole H2O2 per 1 g of fresh weight. 4.4. Electron paramagnetic resonance (EPR) Samples of embryo axes (1 g of fresh weight) were frozen in liquid nitrogen and lyophilized in the freeze dryer Jouan LP3. The lyophilized material was introduced into EPR-type quartz tubes of 4 mm in diameter. Concentrations of free radicals and Mn2+ ions were measured by an EPR spectrometer (SEX-2543 RADIPOAN), which recorded signals at the X-band (microwave frequency 9.4 GHz) using a magnetic modulation frequency of 100 kHz. The spectra were recorded at room temperature with a low microwave power of the klystron (<1 mW). The linear characteristic of the EPR line intensities vs. microwave power allowed us to avoid the
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saturation and line broadening effects for all studied paramagnetic centres. The EPR signal was monitored by measuring the first derivative of microwave absorption. Computer numerical double integration of the first derivative curve Ascough [2] enabled to obtain the total spin intensity of each sample. Since the samples were of equal volume, but of different weight, the areas under the EPR curve per 1 g of sample were calculated. Because relative intensity of the lines was influenced by the quality factor of the resonator, an error in the results generated in this way would approach 12%. To avoid this problem, the line intensities of the samples were compared to the intensity of the lines of the standard that was kept permanently in the resonance cavity (Cr3+ lines in a crystal of Al2O3:Cr3+). In this way, uncertainty generated by the change in quality factor of the resonator could be eliminated and real relative intensities of EPR signals were measured. The sloping baseline evident for each spectrum (noise of the spectrometer) was subtracted before the double integration procedure. EPR lines were integrated over a field range of the order of four linewidths.
used for determining lipid peroxidation with the thiobarbituric acid reaction as described by Dhindsa and Matowe [13]. 4.7. Statistical analysis All determinations were performed in at least three replicates in six independent experiments. Statistical analysis was made based on Student’s t-test, with the level of significance ␣ = 0.05; S.D. was calculated and its range is shown in the figures.
Acknowledgements The authors would like to thank Prof. L. Ratajczak, A. Mickiewicz University, Poznan´ for fruitful discussion and Dr. Remlein-Starosta, Institute of Plant Protection, Poznan´, for cultures of F. oxysporum Schlecht f. sp. lupini. This work was supported by the Polish Committee for Scientific Research (KBN, grant no. 3P06R 052 24).
4.5. Enzymatic assays References Embryo axes in samples of 1 g each were homogenized in 5 ml of 50 mM phosphate buffer (pH 7.0), containing 0.5 M NaCl and 1% PVP at 4 °C and centrifuged at 15 000 × g for 15 min. The activity of catalase, CAT (EC 1.11.1.6), was determined by measuring H2O2 consumption [14]. The reaction mixture contained 50 mM sodium phosphate buffer (pH 7.0), 15 mM H2O2 and plant extract. The reaction was started by introducing H2O2 to the reaction mixture. CAT activity was assessed by measuring absorbance at 240 nm against a calibration curve. The activity of the enzyme was expressed as U per 1 mg of protein. The activity of superoxide dismutase, SOD (EC 1.15.1.1), was assayed by measuring its ability to inhibit the photochemical reduction of NBT by the method of Beauchamp and Fridovich [6]. The 3 ml reaction mixture contained 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 µM NBT, 0.1 mM EDTA and 30 µl of enzyme extract and 2 µM riboflavin (introduced as the last reagent to the reaction mixture). The reaction was started by switching on the light (two 15 W fluorescent lamps placed 30 cm below the test tubes) and proceeded for 15 min. Samples without the enzymatic extract in the examined tests were selected so that the absorption difference between the blank and examined tests was about 50%. The amount of the enzyme that caused the inhibition of NBT reduction by 50% was taken as a unit of SOD activity. Protein was determined according to Bradford [9], by using bovine serum albumin as a standard.
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Samples of embryo axes (500 mg) were ground in 4 ml of 5% trichloroacetic acid and centrifuged at 5000 × g for 15 min. The supernatants were mixed with 50 mg of activated charcoal and centrifuged at 15 000 × g for 30 min, and then
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[27] U. Małolepsza, H. Urbanek, The oxidants and antioxidant enzymes in tomato leaves treated with o-hydroxyethylorutin and infected with Botrytis cinerea, Eur. J. Plant Pathol. 106 (2000) 657–665. [28] A.M. Mayer, R.C. Staples, N.L. Gil-ad, Mechanisms of survival of necrotrophic fungal plant pathogens in hosts expressing the hypersensitive response, Phytochemistry 58 (2001) 33–41. [29] M.C. Mehdy, Active oxygen species in plant defense against pathogens, Plant Physiol. 105 (1994) 467–472. [30] I. Morkunas, M. Kozłowska, W. Bednarski, Generation of free radicals at seed germination stage of yellow lupin infected with Fusarium oxysporum under carbohydrate stress, J. Appl. Genet. 43A (2002) 27–34. [31] I. Morkunas, M. Garnczarska, W. Bednarski, W. Ratajczak, S. Waplak, Metabolic and ultrastructural responses of lupine embryo axes to sugar starvation, J. Plant Physiol. 160 (2003) 311–319. [32] I. Muckenschnabel, B. Williamson, B.A. Goodman, G.D. Lyon, D. Stewart, N. Deighton, Markers for oxidative stress associated with soft rots in French beans (Phaseolus vulgaris) infected by Botrytis cinerea, Planta 212 (2001) 376–381. [33] I. Muckenschnabel, B.A. Goodman, B. Williamson, G.D. Lyon, N. Deighton, Infection of leaves of Arabidopsis thaliana by Botrytis cinerea: changes in ascorbic acid, free radicals and lipid peroxidation products, J. Exp. Bot. 367 (2002) 207–214. [34] K. Ogawa, S. Kanematsu, K. Asada, Intra- and extra-cellular localization of “cytosolic” CuZn-superoxide dismutase in spinach leaf and hypocotyls, Plant Cell Physiol. 37 (1996) 790–799. [35] A.N. Polidoros, J.G. Scandalios, Role of hydrogen peroxide and different classes of antioxidants in the regulation of catalase and glutathione S-transferase gene expression in maize (Zea mays L.), Physiol. Plant. 106 (1999) 112–120. [36] P. Schopfer, C. Plachy, G. Frahry, Release of reactive oxygen intermediates (superoxide radicals, hydrogen peroxide, and hydroxyl radicals) and peroxidase in germinating radish seeds controlled by light, gibberellin, and abscisic acid, Plant Physiol. 125 (2001) 1591– 1602. [37] M.T. Smith, J. Van Staden, Infochemicals: the seed-fungus-root continuum, Environ. Exp. Bot. 35 (2) (1995) 113–123. [38] A. Srobarova, A. Pavlov, The resistance of wheat to Fusarium culmorum and its metabolites: a survey, J. Appl. Genet. 43A (2002) 193–196. [39] M.W. Sutherland, The generation of oxygen radicals during host plant responses to infections, Physiol. Mol. Plant Pathol. 39 (1991) 79–93.
Original article
Response of embryo axes of germinating seeds of yellow lupine to Fusarium oxysporum Iwona Morkunas a,*, Waldemar Bednarski b, Monika Kozłowska a a
Department of Plant Physiology, August Cieszkowski Agricultural University, Wołyn´ska 35, 60-637 Poznan´, Poland b Institute of Molecular Physics, Polish Academy of Science, Smoluchowskiego 17, 60-179 Poznan´, Poland Received 15 March 2004; accepted 10 May 2004 Available online 15 June 2004
Abstract Defence responses of embryo axes of Lupinus luteus L. cv. Polo were studied 48–96 h after inoculation with Fusarium oxysporum Schlecht f.sp. lupini. The infection restricted the growth of embryo axes, the lengths of infected embryo axes 72 and 96 h after inoculation were 11 and 12 mm less in the controls, respectively, while their masses c. 0.03 g less than in the controls. The concentration of H2O2 in embryo axes of inoculated germinating seeds was higher than in the control. This was probably a consequence of oxidative burst as well as H2O2 generation by the invading necrotrophic fungal pathogen. EPR-based analyses detected the presence of free radicals with g1 and g2 values of 2.0052 ± 0.0004 and 2.0031 ± 0.0005, respectively. Concentrations of the radicals 72 and 96 h after inoculation were 50% higher than in the control. The values of the spectroscopic splitting coefficients suggest that they are quinone radicals. However, inoculated embryo axes possess a number of adaptive mechanisms protecting them from oxidative damage. A twofold increase in catalase (CAT, EC 1.11.1.6) activity was evidenced in embryo axes infected with F. oxysporum Schlecht f. sp. lupini, as compared to the control 48–96 h after inoculation. Superoxide dismutase (SOD, EC 1.15.1.1) activity 96 h after inoculation was 80% higher than in the control. Furthermore, EPR-based analyses revealed a higher concentration of Mn2+ ions after 72 h for inoculated embryo axes, as compared to the control. On the other hand, no increase was detected in the concentration of thiobarbituric acid reactive substances (products of lipid peroxidation) in infected embryo axes. The protective mechanisms induced in lupine embryo axes in response to F. oxysporum Schlecht f.sp. lupini were compared with responses to infections with pathogenic fungi elicited in other plant families. © 2004 Elsevier SAS. All rights reserved. Keywords: Free radicals; Fusarium oxysporum; Germinating seeds; Hydrogen peroxide; Lupinus luteus; Lipid peroxidation; Oxidative stress
1. Introduction Adverse environmental conditions and deep sowing make germinating seeds prone to fungal infections. Susceptibility to fungal attack is particularly evident at the earliest stages of seed germination, when intensive structural and metabolic changes are involved in embryo activation [37,38]. At this early phase of plant development, environmental stimuli may
Abbreviations: ABTS, 2,2′-Azino-bis (3-ethylbenz-thiazoline)-6sulfonic acid; CAT, catalase; EPR, electron paramagnetic resonance; H2O2, hydrogen peroxide; TBARS, thiobarbituric acid reactive substances; NBT, nitro blue tetrazolium; ROS, reactive oxygen species; SOD, superoxide dismutase. * Corresponding author. E-mail address: [email protected] (I. Morkunas). © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2004.05.007
modify various biochemical pathways, interfering with the distribution of metabolites between donor (cotyledon) and acceptor (embryo axis) tissues. Perturbations in sugar distribution, indispensable for respiration, growth, and development of embryo axes, diminish their defence capacity [30,31]. Germinating lupine seeds and seedlings are frequently infected with F. oxysporum, including f.sp. lupini [11]. These pathogens cause Fusarium wilt and pre-emergent sprout root rot and post-emergent seedling rot. Phytopathologists pay a lot of attention to fungi of the genus Fusarium, since they play a major role in pathogenesis of vascular plants. However, there is little data on physiological reactions elicited by F. oxysporum during germination. It is widely believed that plants have coevolved with pathogens to develop complex mechanisms, both constitutive and inducible, to cope with the infection [1,20]. One of the short-term
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responses that take place upon the pathogen invasion is an enhanced generation of free radicals, including reactive oxygen species (ROS) [12,18,33]. ROS derive from molecular oxygen by stepwise incomplete electron uptake, finally leading to complete oxygen reduction and production of H2O [18]. ROS accumulation is closely associated with the induction of plant defence reactions against pathogens, such as the hypersensitive reaction, defence gene expression, and cell wall strengthening via cross-linking reactions of phenylpropanoids and proteins [22]. Under conditions of normal healthy growth, plants possess a number of enzymatic and non-enzymatic mechanisms of detoxification to efficiently scavenge for either the ROS themselves or their secondary reaction products [5]. Major enzymes of the defence system, which eliminate ROS directly, are catalases, superoxide dismutases [25], and ascorbate peroxidases [21]. During prolonged periods of oxidative stress, however, these mechanisms become overwhelmed, resulting in tissue damage. In this study we investigated the role of oxidative stress in early response of Lupinus luteus L. cv. Polo to infection with F. oxysporum Schlecht f.sp. lupini during germination. The levels of free radicals and hydrogen peroxide, as well as changes in activities of antioxidative enzymes (CAT and SOD) were assessed. As a secondary objective, we estimated membrane damages on the basis of the level of products of lipid peroxidation—thiobarbituric acid reactive substances (TBARS).
2. Results 2.1. Growth of yellow lupine embryo axes Changes in length and fresh weight of embryo axes of germinating lupine seeds under the influence of F. oxysporum are shown in Fig. 1. The growth of embryo axes was retarded as a consequence of inoculation; the length of infected embryo axes 72 h after inoculation was 11 mm and their mass 0.032 g less than control axes. Later on, i.e. 96 and
Fig. 1. Effects of F. oxysporum Schlecht f.sp. lupini on the length (A) and fresh weight (B) of embryo axes of germinating lupine seeds.
120 h after inoculation, we observed only a slight increase in embryo length difference, which reached 12 and 13 mm, respectively. Simultaneously, the differences in fresh weight between infected and control embryo axes decreased to 0.031 and 0.028 g, respectively. 2.2. Hydrogen peroxide H2O2 content determined in embryo axes of germinating lupine seeds was influenced by their inoculation with F. oxysporum (Fig. 2). The increase in H2O2 content was quite stable, ranging from 15% to 24%. 2.3. EPR signals of free radicals and Mn2+ ions Examples of typical wide-scan EPR spectra of the control and infected tissues are shown in Fig. 3A. In both cases the spectra consist of free radicals and Mn2+ lines. Free radicals gave signals with two g-values of 2.0052 ± 0.0004 and 2.0031 ± 0.0005, both in the control and in infected embryo axes. Their levels were in the same ratio within experimental accuracy for all studied samples. Fig. 3A presents the differences between signal intensities of control and infected embryo axes vs. cultivation time. Free radical signals decreased between 48 and 96 h in control tissues, in contrast to the infected tissues, where the intensity of free radicals remained at the same high level. Fig. 3B and C shows concentrations of free radicals and Mn2+ ions in the control and infected embryo axes vs. cultivation time, respectively. The high levels of free radicals (3.9 × 1015 g–1 DW) and Mn2+ ions were observed after 48 h for control tissues. Free radical concentrations were almost at the same level of 3.3−3.7 × 1015 g–1 DW for infected tissues. The concentration of free radicals was about 43% (72 h) and 50% (96 h) higher in infected embryo axes than in the control. We found that 72 h after inoculation, Mn2+ concentration was about 38% higher than
Fig. 2. Concentration of H2O2 (nmole H2O2 per 1 g fresh weight) in embryo axes of germinating lupine seeds infected with F. oxysporum f.sp. lupini and in the control.
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Fig. 4. Changes in the activities of CAT (A) and SOD (B) in embryo axes of germinating lupine seeds infected with F. oxysporum f. sp. lupini and in the control.
Fig. 3. Typical wide-scan EPR spectra of embryo axes of germinating lupine seeds (A) and concentrations of free radicals (B) and manganese ions (C); i, embryo axes infected with F. oxysporum f. sp. lupini; c, control. Arrows indicate the signals of free radicals and manganese sextets.
in the control (Fig. 3C). However, earlier on and later on, Mn2+ concentration in embryo axes of germinating seeds inoculated with F. oxysporum was lower than in the control. 2.4. Antioxidative enzymes Results of assays of catalase (CAT) and superoxide dismutase (SOD) activities are illustrated in Fig. 4. An increase in CAT activity was observed both in embryo axes of germinating seeds that were inoculated with F. oxysporum and in the control 48–96 h of culture (Fig. 4A). The stimulating effect of inoculation of embryo axes with F. oxysporum on CAT activity was revealed—the activity was 2–2.5 times higher in infected embryo axes than in the control 48–96 h after inoculation. In contrast to these results, SOD activity was initially slightly (by 16%) lower in embryo axes of infected seeds than in the control (Fig. 4B). Later on, an increasing trend in SOD activity was observed: the value in infected seeds increased by 33% after 72 h and by 80% after 96 h, as compared to the control. 2.5. Thiobarbituric acid reactive substances Fig. 5 shows the results of analyses of concentration of thiobarbituric acid reactive substances (TBARS) in lupine
Fig. 5. Concentration of TBARS in embryo axes of germinating lupine seeds infected with F. oxysporum f. sp. lupini and in the control.
embryo axes of germinating seeds under the influence of F. oxysporum infection. During all measurements, TBARS concentration in infected and non-infected embryos was roughly similar except that 48 h after inoculation the concentration was 13% lower in infected axes.
3. Discussion 3.1. Hydrogen peroxide production An enhancement of ROS production during the oxidative burst is one of the earliest defence reactions elicited in response to infection with pathogenic microorganisms [23,29]. That different ROS may trigger opposite effects in plants depending on their spatiotemporal distribution and subcellular concentrations [18]. H2O2—a relatively stable oxygen form, which was assayed in this paper—is a key molecule not only because it shows a direct antimicrobial activity but also
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because it is a factor mediating cell wall strengthening [4,27]. Studies performed by Lu and Higgins [26] demonstrated that H2O2 may remarkably inhibit the growth of Cladosporium fulvum (a pathogenic fungus), and that the H2O2 concentration effective in killing the fungus is considerably lower than the concentration causing plant cell death. In addition, other studies have shown that acting at a relatively low concentration, H2O2 could be a factor inducing the expression of defence-related genes, including genes coding for CAT [16,17,35]. The data presented in our study indicate that in infected embryo axes of yellow lupine the level of H2O2 is 15–24% higher than in the control (Fig. 2). This result suggests that H2O2 generation in infected embryo axes may be one of the strategies of their defence against invasion by a necrotrophic pathogen. However, it cannot be excluded that the fungus F. oxysporum generates some H2O2, too. Recent investigations show that necrotrophic pathogens, like Botrytis cinerea, use oxidative processes during their attack and invasion of plant tissues. Those investigations suggest a role for the so-called ROS as fungal pathogenicity factors, which include hydrogen peroxide, so it is often difficult to distinguish experimentally between the actual sources of the ROS [15,28]. 3.2. Electron paramagnetic resonance spectra and concentrations of free radicals EPR-based analyses carried out in this study show that in tissues infected with F. oxysporum, concentrations of free radicals with g values of 2.0052 ± 0.0005 and 2.0031 ± 0.0004 were relatively high during all measurements, reaching 3.3–3.7 × 1015 of free radicals per 1 g DW (Fig. 3A,B). Their levels 72 and 96 h after inoculation were 50% higher than in the control. The value of the spectroscopic splitting coefficient indicates that it may be the quinone radical. Accumulation of similar stable radicals, which had two g values of 2.0054 and 2.0023, and originated from a single free radical species, was observed by Atherton et al. [3] on desiccated moss. Moreover, Atherton et al. [3] suggested that the precursors of these radicals might be either quinones involved in electron transport pathways, or simple phenolic metabolites, or more complex polyphenols. Application of EPR allowed Muckenschnabel et al. [33] to demonstrate the appearance of a pronounced free radical signal with a g value of 2.0035 as a result of infection of Arabidopsis thaliana by B. cinerea. In an earlier work of the same research team the increases in intensity of the EPR signal with a g value of 4.27 (Fe III), as well as a single stable free radical signals, were evidenced to result from infection of bean leaves with B. cinerea [32]. We believe that the apparent increase in concentration of quinone radicals reported here might result from a H2O2 burst occurring at an early stage of infection with a pathogen. At later stages of infection, H2O2 becomes massively converted to hydroxyl radical, which—being a strong one-electron oxidizing agent—reacts non-specifically with other organic compounds and destroys
in this way the biological activity of macromolecular structures and cell membranes [19,39]. Also the changes in concentrations of free radicals observed in our study in control embryo axes are interesting. Unlike in infected tissues, the level of free radicals in control tissues decreased during the experiment. The highest concentration was observed 48 h after the beginning of the experiment. This indicates that intensive metabolic processes took place in the axes of germinating seeds during the rupture of the seed coat by the radicle (24−48 h after the beginning of the experiment). Similar observations were made by Schopfer et al. [36] in healthy, actively germinating radish seeds. They showed that a strong rise in ROS release is initiated in germinating seeds shortly before the radicle protrudes through the seed coat. These ROS may also originate from the seed coat (the living aleurone layer) as well as the embryo. Moreover, it was demonstrated that the production of ROS by germinating seeds is an active, developmentally controlled physiological function, presumably for protecting the emerging seedling against attack by pathogens. 3.3. Lipid peroxidation The most important observation in this paper is the detection of the large progressive increase in CAT activity with time both in the control and in infected embryo axes. We suppose that the strong activation of CAT protects cells against an excess of H2O2 and thus against considerable membrane degradation caused by oxidative stress. A small but progressive increase was recorded in the level of TBARS (products of lipid peroxidation) vs. time in the control samples, but no significant differences between control and infected tissues were found (Fig. 5). Moreover, 48 h after inoculation a 13% decrease in TBARS content was observed, in comparison with the control. No signs of an infectionrelated increase in levels of MDA and 4-hydroxy-2-nonenal (another product of lipid peroxidation) have been found in bean leaves subjected to a strong oxidation stress resulting from infection with B. cinerea [33]. This is in contrast to observations made with pathogen-inoculated tissues from other plant families [12,32]. It is also in conflict with the results of Conklin et al. [10], who showed that increased MDA levels were formed in an ascorbic-acid-deficient Arabidopsis mutant when subjected to oxidative stress (by ozone) [10]. In our study, the observed decrease in TBARS between control and infected tissues may be a consequence of the limited stability of those products and their metabolization by the fungus [33]. 3.4. Activity of antioxidative enzymes and the intensity of the Mn2+ EPR signal SOD is thought to be the key enzyme involved in H2O2 production [34] and our data are consistent with this notion, as judged by a significant increase in SOD activity detected in embryo axes of germinating lupine seeds infected with
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F. oxysporum. Namely, SOD activity in embryo axes of infected seeds was 33% higher (72 h after inoculation) and 80% higher (96 h after inoculation) than in the control. An interesting adaptation seems to be the observed presence of Mn2+ ions in germinating lupine seeds, detected by EPR simultaneously with signals of free radicals, as reflected in the sextets visible in Fig. 3A. The high level of Mn2+ ions in embryo axes 72 h after inoculation may aim at supporting the synthesis of Mn–SOD (Fig. 3C), as we observed then the highest intensity of infection, reflected in results of measurements of length and fresh weight (Fig. 1). No Mn2+ ions were present in the external environment in which the seeds germinated, so the observed result may attest to a higher transport of Mn2+ ions from cotyledons to embryo axes in infected embryos. The mechanism of this phenomenon cannot be explained without detailed investigations into ion distribution in the seedling during infection. The decrease in concentration of Mn2+ ions in embryo axes 96 h after inoculation may be determined by the physiological stage of germination, because of the beginning of the autotrophic growth stage. The effective concentration of free radicals in plant tissues is a result of a dynamic equilibrium between the rates of their production and scavenging. SOD and CAT were shown to be involved in the regulation of H2O2 levels in plant tissues [8,22,24]. In our study, we observed a marked induction of CAT in material infected with F. oxysporum, as its level was there about 2.5 higher than in the control (Fig. 4A). Thus the activation of this enzyme was disproportionately higher than H2O2 generation. The relatively low accumulation of H2O2 was probably due to the high CAT activation, also 48 h after the beginning of the experiment. However, the role of this enzyme in the plant seems to be more complex. This would confirm the observations made by Małolepsza and Urbanek [27] who detected a decrease in CAT activity when H2O2 concentration was high in tomato leaves infected with B. cinerea, so could be regarded as evidence for an overwhelming of this antioxidant enzyme. The high efficiency of the antioxidative system observed in this study, in particular with reference to catalase in embryo axes of germinating lupine seeds, is one of the most important elements of their defence against oxidative stress. It seems that the protective mechanisms induced in response to pathogen attack in embryo axes of germinating lupine seeds were effective enough not to allow considerable damage of membrane structures. Interestingly, the protective responses elicited in order to stop the development of an invading pathogen were not sufficient to enable the normal development of lupine seedlings. 4. Methods 4.1. Plant material Yellow lupine (Lupinus luteus L. cv. Polo) seeds of S-elite class were used in the experiments. The seeds were surface-
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sterilized, immersed in sterile water and left in a thermostat (25 °C). After 6 h of imbibition, the seeds were transferred onto filter paper (in Petri dishes) and immersed in a small amount of water in order to support further imbibition. After subsequent 18 h, imbibed seeds were sown in pots (45 seeds per pot) containing sterilized perlite, inoculated with a spore suspension of F. oxysporum f.sp. lupini and allowed to germinate at 23 °C at an irradiance of 130 µmole m–2 s–1 (Philips TLD 58W/84 fluorescent lamps) under a light regime of 12 h light/12 h dark. As a control, non-inoculated germinating seeds were used. Embryo axes were removed 48, 72 and 96 h after inoculation in order to perform analyses. Since our aim was to investigate if lupine seedlings are able to restrict the development of the invading pathogen, the length and fresh weight of embryo axes was analysed till 120 h after inoculation. Results of the measurements of length and fresh weight were means for 40 embryo axes. In all assays, samples of the same fresh weight were compared. 4.2. Fungi and inoculation F. oxysporum f.sp. lupini strain K-1018 (further named F. oxysporum) was obtained from the Bank of Pathogens, Institute of Plant Protection, Poznan´. The pathogen was grown on a PDA medium (agar-Difco) in light at 24 °C. The spore suspension used for inoculation was prepared from a 3 week-old culture and was applied at a concentration of 5·× 106 spores per 1 ml. 4.3. Assay of hydrogen peroxide The embryo axes (1 g of fresh weight) were ground in 100 mM potassium phosphate buffer (pH 7.0) with Polyclar AT, in a chilled mortar on ice. The homogenates were centrifuged at 15 000 × g for 20 min. The concentration of H2O2 was determined according to Bergmeyer and Bernt [7]. The reaction mixture contained: 400 µl of plant extract, 2 ml of 100 mM KH2PO4/K2HPO4 (pH 7.0) buffers, 50 µl of horseradish peroxidase (147 U ml–1), and 50 µl of ABTS solution (50 mM). The concentration of H2O2 was estimated by measuring absorbance at 415 nm against a calibration curve and was expressed as nmole H2O2 per 1 g of fresh weight. 4.4. Electron paramagnetic resonance (EPR) Samples of embryo axes (1 g of fresh weight) were frozen in liquid nitrogen and lyophilized in the freeze dryer Jouan LP3. The lyophilized material was introduced into EPR-type quartz tubes of 4 mm in diameter. Concentrations of free radicals and Mn2+ ions were measured by an EPR spectrometer (SEX-2543 RADIPOAN), which recorded signals at the X-band (microwave frequency 9.4 GHz) using a magnetic modulation frequency of 100 kHz. The spectra were recorded at room temperature with a low microwave power of the klystron (<1 mW). The linear characteristic of the EPR line intensities vs. microwave power allowed us to avoid the
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saturation and line broadening effects for all studied paramagnetic centres. The EPR signal was monitored by measuring the first derivative of microwave absorption. Computer numerical double integration of the first derivative curve Ascough [2] enabled to obtain the total spin intensity of each sample. Since the samples were of equal volume, but of different weight, the areas under the EPR curve per 1 g of sample were calculated. Because relative intensity of the lines was influenced by the quality factor of the resonator, an error in the results generated in this way would approach 12%. To avoid this problem, the line intensities of the samples were compared to the intensity of the lines of the standard that was kept permanently in the resonance cavity (Cr3+ lines in a crystal of Al2O3:Cr3+). In this way, uncertainty generated by the change in quality factor of the resonator could be eliminated and real relative intensities of EPR signals were measured. The sloping baseline evident for each spectrum (noise of the spectrometer) was subtracted before the double integration procedure. EPR lines were integrated over a field range of the order of four linewidths.
used for determining lipid peroxidation with the thiobarbituric acid reaction as described by Dhindsa and Matowe [13]. 4.7. Statistical analysis All determinations were performed in at least three replicates in six independent experiments. Statistical analysis was made based on Student’s t-test, with the level of significance ␣ = 0.05; S.D. was calculated and its range is shown in the figures.
Acknowledgements The authors would like to thank Prof. L. Ratajczak, A. Mickiewicz University, Poznan´ for fruitful discussion and Dr. Remlein-Starosta, Institute of Plant Protection, Poznan´, for cultures of F. oxysporum Schlecht f. sp. lupini. This work was supported by the Polish Committee for Scientific Research (KBN, grant no. 3P06R 052 24).
4.5. Enzymatic assays References Embryo axes in samples of 1 g each were homogenized in 5 ml of 50 mM phosphate buffer (pH 7.0), containing 0.5 M NaCl and 1% PVP at 4 °C and centrifuged at 15 000 × g for 15 min. The activity of catalase, CAT (EC 1.11.1.6), was determined by measuring H2O2 consumption [14]. The reaction mixture contained 50 mM sodium phosphate buffer (pH 7.0), 15 mM H2O2 and plant extract. The reaction was started by introducing H2O2 to the reaction mixture. CAT activity was assessed by measuring absorbance at 240 nm against a calibration curve. The activity of the enzyme was expressed as U per 1 mg of protein. The activity of superoxide dismutase, SOD (EC 1.15.1.1), was assayed by measuring its ability to inhibit the photochemical reduction of NBT by the method of Beauchamp and Fridovich [6]. The 3 ml reaction mixture contained 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 µM NBT, 0.1 mM EDTA and 30 µl of enzyme extract and 2 µM riboflavin (introduced as the last reagent to the reaction mixture). The reaction was started by switching on the light (two 15 W fluorescent lamps placed 30 cm below the test tubes) and proceeded for 15 min. Samples without the enzymatic extract in the examined tests were selected so that the absorption difference between the blank and examined tests was about 50%. The amount of the enzyme that caused the inhibition of NBT reduction by 50% was taken as a unit of SOD activity. Protein was determined according to Bradford [9], by using bovine serum albumin as a standard.
[1] [2] [3]
[4] [5]
[6]
[7]
[8]
[9]
[10]
[11]
4.6. Lipid peroxidation [12]
Samples of embryo axes (500 mg) were ground in 4 ml of 5% trichloroacetic acid and centrifuged at 5000 × g for 15 min. The supernatants were mixed with 50 mg of activated charcoal and centrifuged at 15 000 × g for 30 min, and then
[13]
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