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Re-aeration – induced oxidative stress and antioxidative defenses in hypoxically pretreated lupine roots
J. Plant Physiol. 161. 415 – 422 (2004) http://www.elsevier-deutschland.de/jplhp
Re-aeration – induced oxidative stress and antioxidative defenses in hypoxically pretreated lupine roots Małgorzata Garnczarska1 *, Waldemar Bednarski2, Iwona Morkunas3 1
Department of Plant Physiology, Faculty of Biology, A. Mickiewicz University, Al. Niepodległo´sci 14, 61-713 Poznan, ´ Poland
2
Institute of Molecular Physics, Polish Academy of Science, Smoluchowskiego 17, 60-179 Poznan, ´ Poland
3
Department of Plant Physiology, Agricultural University, Wołynska ´ 35, 60-637 Poznan, ´ Poland
Received February 6, 2003 · Accepted June 1, 2003
Summary The level of free radicals and activities of antioxidative enzymes were examined in roots of lupine seedlings (Lupinus luteus L.) that were deprived of oxygen by subjecting them to root hypoxia for 48 and 72 h and then re-aerated for up to 24 h. Using electron paramagnetic resonance (EPR), we found that the exposure of previously hypoxically grown roots to air caused the increase in free radicals level, irrespective of duration of hypoxic pretreatment. Immediately after re-aeration the level of free radicals was two times higher than in aerated control. The EPR signal with the g-values at the maximum absorption of 2.0057 and 2.0040 implied that the paramagnetic radicals are derived from a quinone. Directly after re-aeration of hypoxically pretreated roots, the activity of superoxide dismutase (SOD, EC 1.15.1.1) increased to its highest value, followed by a decline below the initial level, whereas activities of catalase (CAT, EC 1.11.1.6) and peroxidase (POX, EC 1.11.1.7) were diminished or only slightly influenced during reaeration. The electrophoretic patterns of the soluble extracts show 4 isozymes of SOD, 4 isozymes of POX and 1 isozyme of CAT. The level of H2O2 was enhanced or lowered by re-aeration, depending on the previous duration of hypoxia. At the onset of re-aeration products of lipid peroxidation were present at a three-fourth of the levels found in aerobic control. Their levels increased after prolonged exposure to air but remained lower than those in aerobic control even after 24 h of re-aeration. Re-admission of oxygen resulted in about 20 % rise in oxygen uptake by root axes segments immediately after transfer of roots from hypoxia and the high uptake rates were observed over whole re-aeration period. Oxygen consumption by root tips was significantly reduced just after transfer from hypoxic conditions as compared to aerated control but after 24 h of re-aeration even approached the control level. The results are discussed in relation to the ability of lupine roots to cope with oxidative stress caused by re-aeration following hypoxic pretreatment. Key words: catalase – electron paramagnetic resonance – hydrogen peroxide – hypoxia – lipid peroxidation – lupine – peroxidase – re-aeration – superoxide dismutase Abbreviations: CAT = catalase. – EPR = electron paramagnetic resonance. – MDA = malondialdehyde. – POX = peroxidase. – SOD = superoxide dismutase. – TBARS = thiobarbituric acid reactive substances * E-mail corresponding author: [email protected] 0176-1617/04/161/04-415 $ 30.00/0
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Małgorzata Garnczarska, Waldemar Bednarski, Iwona Morkunas
Introduction Many plant species sustain injury or even die when the soil becomes excessively wet through transient flooding. O2 deprivation is the primary stress experienced by plants during flooding, so plants must cope with hypoxic or even anoxic conditions. The response to oxygen deficiency in several species is associated with a number of biochemical, morphological, and anatomical changes in both the root and the shoot (reviewed by Ricard et al. 1994, Vartapetian and Jackson 1997). Plants cannot survive the prolonged oxygen defecit, however, the ability of plant tissues to survive anoxic stress can be increased by hypoxic pretreatment. The phenomenon of acclimation to anaerobiosis has been reported for wheat (Waters et al. 1991), maize (Saglio et al. 1988), rice (Ellis and Setter 1999) and lupine (Garnczarska and Ratajczak 2003). Injury and death of roots under oxygen deficiency have been attributed to the accumulation of toxic end products of anaerobic metabolism, to lowering of energy charge, or to lack of substrates for respiration (Drew 1997). Although it is clear that most of the injuries are incurred during oxygen deficiency, the restoration of normal O2 conditions is also damaging to plant tissue. Under these conditions, damage is mainly brought about by a burst of reactive oxygen species formed when oxygen becomes available again as electron acceptor for electron-saturated redox chain (Biemelt et al. 2000). Conditions favouring the formation of reactive oxygen species, such as low energy charge, high levels of reducing equivalents, and saturated electron transport chains, usually prevail in plant tissues when oxygen supply is restricted (Van Toai and Bolls 1991). The main sites of reactive oxygen species production in the plant cell are the organelles with highly oxidizing metabolic activities or with sustained electron flows: chloroplasts, mitochondria and peroxisomes (Dat et al. 2000, Corpas et al. 2001). In non-photosynthetic tissues, mitochondria are the main source of reactive oxygen species (Puntarulo et al. 1991). Components of the electron transport chain may reduce oxygen, thus leading to the formation of superoxide radicals. Superoxide is converted to hydrogen peroxide by disproportionation reaction. The hydroxyl radical is then formed from superoxide anion and H2O2 via the metal-catalyzed Haber-Weiss reaction. Under normal growing conditions, reactive oxygen species are maintained at a non-damaging level by low molecular mass antioxidants (ascorbate, glutathione, tocopherols) and enzymes (superoxide dismutase, catalase, peroxidase), which either scavenge or utilize them (Elstner and Osswald 1994). However, these defence mechanisms can be overwhelmed during environmental stress, resulting in an excess production of reactive oxygen species (Prasad et al. 1994). Despite considerable progress on some aspects of tolerance and acclimation to low oxygen regimes, there are divergent opinions as to the cause of death when plant tissue is deprived of oxygen. Determining whether injury takes place
directly under oxygen deficiency or on re-exposure to air could underlie the problem of whether or not post-hypoxic (post-anoxic) injury contributes to loss of plant survivability. As we described previously lupine seedlings have mechanisms for sensing and adapting to O2 deficiency in roots and in shoots. Hypoxia led to a reduction of root elongation and, as expected, the activities of alcohol and lactate dehydrogenases increased during hypoxia (Garnczarska 2002). Hypoxic acclimation to anoxia in lupine roots seems to be associated with avoidance of lactate accumulation and cytoplasmic acidosis because of lowered lactate dehydrogenase activity, combined with increased alcohol dehydrogenase activity and therefore increased potential for ethanolic fermentation to continue glycolysis and permit seedlings to survive (Garnczarska and Ratajczak 2003). However, hypoxia (anoxia) tolerance, as judged by the ability to return to an aerobic environment, may involve also other metabolic strategies for dealing with oxygen deficiency and subsequent re-aeration. Reoxygenation damage, known as «reperfusion injury», is well documented in human and animal systems (Halliwell and Gutteridge 1985), but also occurs after re-aeration in plants which are deprived of oxygen due to flooding (Crawford 1992). Post-hypoxic (post-anoxic) injury after transfer from hypoxia (anoxia) to normoxia has been reported with Iris germanica rhizomes (Hunter et al. 1983), rice seedlings (Ushimaru et al. 1992), soybean seedlings (Van Toai and Bolles 1991) and wheat seedlings (Albrecht and Wiedenroth 1994, Biemelt et al. 1998). The aim of the present study was to examine whether posthypoxia induces an oxidative stress in lupine roots. Therefore, the concentration of free radicals and there activities, and isoenzyme patterns of antioxidative enzymes were determined in hypoxically grown and subsequently re-aerated roots as well as in roots of continuously aerated control seedlings. In addition, the concentrations of hydrogen peroxide and malondialdehyde were measured and oxygen consumption by root tips and root axes was estimated. Furthermore, roots ability to survive hypoxia and subsequent re-aeration was studied.
Materials and Methods Plant material Seeds of yellow lupine (Lupinus luteus L. cv. Juno) were allowed to imbibe on moistened filter paper at 25 ˚C in the dark. After 60 h, 70 germinated seeds were transferred for 12 h to growth vessels containing 2.5 L of aerated 1/5 strength Knop nutrient solution (acclimation to growth conditions). The roots were in contact with the solution while the shoots were in the gas phase. All experiments were carried out in the dark at 25 ˚C.
Gas treatments After growing under aerated conditions for 24 h and 0 h from the initiation of the experiments, seedlings were subjected to hypoxia by flush-
Oxidative stress in lupine under post-hypoxia ing nutrient solution with N2 gas (99.99 % N2) for 48 h and 72 h, respectively. At the start of the hypoxic pretreatment seedlings were 3 and 4 d after imbibition, respectively. Hypoxically pretreated seedlings were then re-aerated for 30 min, 2 h, 4 h and for some estimation for 24 h. The nutrient solution of control plants was continuously flushed with air for 72 h. Following the gassing treatments, roots were rinsed carefully in water, dried with filter paper and used either immediately or frozen in liquid nitrogen. The contact with air during this preparation lasted about 2 min, so roots harvested immediately after hypoxic pretreatment were labelled as re-aerated for 2 min. Oxygen concentration in the vessels was monitored with an oxygen meter (Dissolved Oxygen Meter 9300, Jenway). Dissolved oxygen concentrations were 20 % O2 v/v for aerobic control and no detectable oxygen concentration in solution flushed with pure nitrogen.
Viability determination Root tips were considered to be viable if they resumed elongation during 72 h recovery period (re-oxygenation). Elongation was followed by measuring root length at the end of recovery period. A reference mark was applied 10 mm behind the root tip at the end of re-aeration period following hypoxic pretreatment.
Electron paramagnetic resonance (EPR) The samples of 6 roots frozen in liquid nitrogen were lyophylized in a Jouan LP3 freeze dryer and introduced into EPR-type quartz tubes (4 mm diameter). The concentration of free radicals was measured applying EPR spectrometer recording signals at X-band (microwave frequency – 9.4 GHz). EPR spectra were recorded as the first derivative of microwave absorption. Computer numerical double integration of the first derivative curve (Ayscough 1967) enabled us to obtain the total spin intensity for each sample. Since samples were of different weight, the areas under the EPR absorption curve per 1 mg of each sample were calculated. Because relative intensity of the lines was influenced by the quality factor of the resonator, an error of the results generated the above way would approach twelve percent. To avoid this problem the line intensities of the samples were compared to the intensity of the lines of the standard (Al2O3: Cr3 + ) that was kept permanently in resonance cavity. This way an uncertainty generated by the quality factor of the resonator could be eliminated and real relative intensities of EPR signals might be measured.
Protein extraction About 1.0 g of liquid nitrogen powered samples was homogenised with three volumes of cold 50 mmol/L potassium phosphate buffer (pH 7.0) containing 1 mmol/L EDTA and 5 % (w/v) polyvinylopyrrolidone (PVP). The slurry was centrifuged at 20,000 g for 20 min at 4 ˚C and the supernatants were used for enzyme assays. The protein concentration in the samples was estimated according to Bradford (1976).
Enzyme analyses Superoxide dismutase activity assay was based on the method of Beauchamp and Fridovich (1971) as described by Donahue et al. (1997). Catalase activity was assayed according to the method of Aebi (1974). Total peroxidase activity was measured according to Na-
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kano and Asada (1981). The reliability of the extraction and assay procedures was checked by recovery of activity of a commercially available enzymes added to the extraction buffer prior to tissue extraction. The recovery of enzyme activities was 95 – 97%.
Electrophoretic procedure Samples of crude roots extracts were electrophoresed in 10 % (SOD, POX) or 8 % (CAT) polyacrylamide slab gels at pH 8.9 under nondenaturing conditions according to Davis (1964). SOD activity was detected on gels using the photochemical procedure of Beauchamp and Fridovich (1971). The different types of SOD were differentiated by performing the activity stains in gels previously incubated in 3 mmol/L KCN or 5 mmol/L H2O2 as described in Asada et al. (1975). Isoenzymes of CAT were visualised on gels by the method of Woodbury et al. (1971). The diaminobenzidine reaction was used for staining isoenzymes of POX by the method of Ros Barcelo (1987).
Determination of hydrogen peroxide The measurement was performed by recording the oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone (MBTH) with its formaldehyde azine in the presence of horseradish peroxidase according to Capaldi and Taylor (1983).
Assay of 2-thiobarbituric acid – reactive material The level of lipid peroxidation in roots was measured as malondialdehyde (MDA) content determined by reaction with thiobarbituric acid (TBA) according to the method of Dhindsa and Matowe (1981) as reported in Ren et al. (1999) without addition of 3,5-diizobutyl-4-hydroxytoluene (BHT) as antioxidant.
Measurement of root respiration Root tips and root axes segments (approximately 10 mm long) were, immediately after excision, transferred into an oxygen electrode incubation chamber containing 3 mL of fully aerated stirred nutrient solution. The decrease of the oxygen concentration was polarographically measured by a Clark type digital model 10 electrode (Rank Brothers, Cambride, UK). After the measurements, the root segments were blotted and weighed to allow expression of the rate of respiration per gram fresh weight.
Results Electron paramagnetic spectra of lupine roots hypoxically pretreated for 48 and 72 h and then transferred to aerobic conditions for 2 min, 30 min, 2 h and 4 h were measured at room temperature using X-band EPR spectrometer. The EPR spectra with g-values of 2.0057 and 2.0040 for several samples are shown in Figure 1. A relative concentration of free radicals was compared in control vs. hypoxically pretreated and subsequently re-aerated roots. The exposure of previously hypoxically pretreated lupine roots to increased oxy-
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Małgorzata Garnczarska, Waldemar Bednarski, Iwona Morkunas
Figure 1. Electron paramagnetic spectra of free radicals in lupine roots subjected to hypoxia for 48 h (48 H) followed by re-aeration for 2 min, 30 min, 2 h and 4 h ( + 2 min air, + 30 min air, + 2 h air, + 4 h air) compared to aerated control roots.
Figure 3. Activities of SOD (A), CAT (B) and POX (C) in lupine roots subjected to hypoxia for 48 h (48 H, light grey bars) and 72 h (72 H, dark grey bars) followed by re-aeration for 2 min, 30 min, 2 h and 4 h ( + 2 min, + 30 min, + 2 h, + 4 h) compared with continuously aerated control roots (white bars). Means ± SD of four replicates.
Figure 2. Concentration of free radicals in lupine roots subjected to hypoxia for 48 h (48 H, light grey bars) and 72 h (72 H, dark grey bars) followed by re-aeration for 2 min, 30 min, 2 h and 4 h ( + 2 min, + 30 min, + 2 h, + 4 h) compared with continuously aerated control roots. The values for post-hypoxically treated roots are expressed as percentages of that for aerated control. The concentration of free radicals in aerated control was 18.2 Arb. U · mg –1 DW. The data is typical of data obtained in three replicate experiments.
gen availability caused a twofold increase in concentration of free radicals, irrespective of duration of hypoxic pretreatment. (Fig. 2). For longer re-aeration periods the amount of free radicals decreased but was still above the control level. Immediately after hypoxically pretreated roots were returned to air, the activity of SOD increased to its highest value, followed by a slow decline below the initial level (Fig. 3 A). Four different SOD isozymes were detected in PAGE, each of them being Cu, Zn-SOD (Fig. 4 A). In fact, both H2O2 and KCN inhibited the enzyme leading to the disappearance of the bands (data not shown). The enhanced activity of CAT was observed over the 2 h re-aeration period of
Oxidative stress in lupine under post-hypoxia
Figure 4. Isoenzyme patterns of SOD, CAT and POX in lupine roots subjected to hypoxia for 48 h (48 H) and 72 h (72 H) followed by reaeration for 2 min, 30 min, 2 h and 4 h ( + 2 min, + 30 min, + 2 h, + 4 h) compared with continuously aerated control roots. The different isoforms are numbered from cathode to anode. For SOD 100 µg proteins were loaded per each well, for CAT 25 µg and for POX 75 µg.
Table 1. Malondialdehyde (MDA) and H2O2 concentrations in lupine roots hypoxically pretreated for 48 h (48 H) and 72 h (72 H) and subsequently re-aerated for 2 min, 4 h and 24 h ( + 2 min air, + 4 h air, + 24 h air) compared to aerated control roots. Means ± SD of four replicates. Growth conditions
H2O2 concentration (nmol · g – 1 FW)
MDA concentration (nmol · g – 1 FW)
Control 48 H + 2 min air 48 H + 4 h air 48 H + 24 h air 72 H + 2 min air 72 H + 4 h air 72 H + 24 h air
11.75 ( ± 0.91) 12.94 ( ± 0.82) 13.42 ( ± 1.14) 13.16 ( ± 1.09) 10.31 ( ± 0.98) 9.16 ( ± 0.73) 9.14 ( ± 0.67)
90.1 ( ± 6.2) 68.4 ( ± 4.4) – 82.8 ( ± 5.8) 66.6 ( ± 3.6) – 81.8 ( ± 5.4)
roots grown under hypoxia for 48 h, whereas the activity of this enzyme tended to decrease during re-aeration of roots subjected to hypoxic conditions for 72 h and after 4 h of reaeration was clearly below the control level (Fig. 3 B). Gel stained for CAT activity revealed one isoform (Fig. 4 B). POX
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activity slightly increased after the start of re-aeration and declined after 4 h of exposure to air (Fig. 3 C). Four POX isoforms were present in lupine roots (Fig. 4 C). Superoxide dismutase, catalase and peroxidase zymogram analyses showed that synthesis of new isoforms was not induced by re-aeration. The amount of hydrogen peroxide slightly increased after re-aeration of roots hypoxically pretreated for 48 h and was fairly constant over the post-hypoxic period (Table 1). In seedlings hypoxically pretreated for 72 h the concentration of hydrogen peroxide gradually decreased during post-hypoxia. In order to estimate the extent of peroxidative damage to lipids in hypoxically pretreated lupine roots after exposure to air, the level of TBARS, an index of lipid peroxidation, was measured and expressed as MDA concentration. Immediately after re-exposure to air lupine roots contained 75 % of the MDA found in aerobic control (Table 1). After 24 h of re-aeration this value increased to about 90 % of the control level. Polarygraphic studies performed on 10 mm lupine root tips revealed that the respiration immediately after 48 and 72 h of hypoxic pretreatment was reduced to 38 % and 57%, respectively, as compared to control root tips (Table 2). After 24 h of re-exposure to oxygen following hypoxic pretreatment for 72 h the respiration of root tips was significantly increased to 738.2 nmole O2 · min –1 · g –1 FW. Oxygen consumption in control root tips was 636.1nmole O2 · min –1 · g –1 FW. In contrast, in root tips subjected to hypoxia for 48 h and then re-aerated for 24 h respiration was reduced to 44 % as compared to control. Re-admission of oxygen resulted in a 20 % rise of oxygen uptake (compared with the control) by root axes segments excised from roots previously grown under hypoxia for 48 and 72 h (Table 2). After 4 h of re-aeration oxygen consumption by root axes segments exceeded the control by 45 % and 31 %, respectively. Because of the marked differences in oxygen consumption following re-aeration of lupine root tips hypoxically pretreated for 48 and 72, it was of interest to determine whether there were differences in their ability to survive hypoxia and re-
Table 2. Oxygen consumption by 10 mm root tips and root axes of lupine roots hypoxically pretreated for 48 h (48 H) and 72 h (72 H) and subsequently re-aerated for 2 min, 4 h and 24 h ( + 2 min air, + 4 h air, + 24 h air) compared to aerated control. Means ± SD of five replicates. Growth conditions
Control 48 H + 2 min air 48 H + 4 h air 48 H + 24 h air 72 H + 2 min air 72 H + 4 h air 72 H + 24 h air
Oxygen consumption (nmol O2 · min – 1 · g – 1FW) Root tips
Root axes
636 ( ± 47) 240 ( ± 29) 333 ( ± 34) 278 ( ± 30) 365 ( ± 42) 632 ( ± 49) 738 ( ± 55)
109 ( ± 12) 133 ( ± 18) 158 ( ± 23) 130 ( ± 21) 131 ( ± 19) 143 ( ± 24) 147 ( ± 26)
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Discussion
Figure 5. Elongation (A) and viability (B) of lupine roots subjected to hypoxia for 48 h (48 H, light grey bars) and 72 h (72 H, dark grey bars) followed by re-aeration for 2 min, 30 min, 2 h and 4 h ( + 2 min, + 30 min, + 2 h + 4 h) compared with continuously aerated control roots (white bars). A. elongation per root during 72 h of recovery; B. percent viability. The experiment was repeated twice with 12 plants each. Error bars indicate SD.
aeration. We investigated the effects of hypoxic pretreatment for 48 and 72 h followed by re-aeration for 4 and 24 h on root elongation and survival of roots tips. Root survival was based on growth during 72 h recovery period. Lupine roots hypoxically pretreated for 72 h showed a rapid root extension during recovery period (Fig. 5 A), but following 24 h of re-aeration root elongation was slowed down to the control level. The roots of seedlings hypoxically pretreated for 48 h and subsequently re-aerated were more susceptible to treatment, with a significant decrease in survival after 24 h of re-aeration (Fig. 5 B). In about 30 % of these roots the deformation of 2 – 3 mm root tips occurred just after hypoxic pretreatment (root tips were water soaked and translucent) and continued further during reaeration. The root tips of seedlings hypoxically pretreated for 72 h and then re-aerated were more tolerant to the stress. Root survival after recovery period remained at 100 %.
The experimental protocol used in this study utilized lupine seedlings with their roots exposed to oxygen deprived conditions and their shoots remaining in ambient air. This would simulate conditions similar to those encountered by lupine plants in the field. The roots were grown in solution flushed with pure nitrogen (no detectable oxygen concentration). However, the internal O2 movement from shoots to roots would create hypoxic conditions in root tissues. The direct detection of stable free radicals in lupine roots by EPR spectroscopy revealed that re-oxygenation after a period of oxygen deprivation intensifies the synthesis of free radicals. Immediately after hypoxically pretreated roots were returned to air the concentration of free radicals increased to its highest value (204 and 187% of free radicals found in control tissue) and then decreased after recovery in air for 30 min, 2 h and 4 h but was clearly above the control level (Fig. 2). The radical spectra consisted of two only slightly split lines visible as a broadening of the EPR signal at high magnetic field (Fig. 1). EPR analyses conducted on desiccated moss revealed an accumulation of stable free radicals with two g-values of 2.0054 and 2.0023 (Atherton et al. 1993). It was found that these two g-value were consistent with quinone radicals. Atherton et al. (1993) suggested that the reason of the radicals might be quinones involved in electron transport chains. Cakmak and Marschner (1988) observed zinc-dependent changes of semiquinone radical signals measured by elecron spin resonance spectrocopy in cotton roots. It has been shown that superoxide anion is generated at the level of the three enzymatic complexes interacting with ubiquinone (coenzyme Q) and that this anion is the only reactive oxygen species generated suggesting a monoelectronic donor likely the ubisemiquinone, a quinone radical intermediate of the redox Q-cycle (Du et al. 1998). Purvis (1997) pointed out that plant mitochondria are capable of generating large quantities of superoxide radical when the level of Q reduction is high. The higher SOD activity in lupine roots immediately after transfer from hypoxic conditions (Fig. 3) may be due to increased formation of superoxide anion as the enhanced activity of this enzyme is an indicator of increased levels of superoxide anion in plant cells. According to Monk et al. (1987) an increase in SOD activity already apparent under anoxia is of advantage for plants which face enhanced superoxide formation during post-anoxia. The strong induction of SOD activity under anoxia found in the flooding-tolerant Iris pseudacorus was discussed as an evolutionary advantage of plants being thus prepared for re-aeration. In soybean short-term anoxia favoured subsequent production of superoxide radicals. Longer anoxic treatment lead to higher SOD activities and therefore to a more efficient protection (Van Toai and Bolles 1971). Neither hypoxia nor subsequent re-aeration caused significant changes in SOD activity in wheat roots (Biemelt et al. 2000). However, anoxia led to the appearance of new isoforms and the increase in SOD activity. By contrast, the activ-
Oxidative stress in lupine under post-hypoxia ities of six antioxidative enzymes including SOD were reduced in submerged rice seedlings, but approached control levels following the restoration of aerobic conditions (Ushimaru et al. 1992). The level of hydrogen peroxide was enhanced or lowered in lupine roots, depending on previous duration of hypoxia (Table 1). It is worth recalling that hydrogen peroxide actually measured by a given method always reflects the balance between H2O2-generating and H2O2-consuming processes. Thus low H2O2 level after re-oxygenation following hypoxic treatment for 72 h can be due either to a low intrinsic production rate or to efficient scavenging by detoxifying enzymes such as CAT and POX. Concomitant with decrease in the amount of hydrogen peroxide, the activity of CAT was diminished, whereas the activity of POX remained nearly unchanged (Fig. 3). However, the decrease in amount of hydrogen peroxide was correlated with diminished activity of SOD. The amount of hydrogen peroxide slightly increased after reaeration of roots hypoxically pretreated for 48 h, indicating enhanced formation of reactive oxygen, although CAT and POX showed a transient increase in activity during first two hours of post-hypoxia. MDA remained at levels lower than those found in aerobic control during the 24 h that followed the transfer of hypoxically grown lupine roots to air (Table 1), indicating that lupine roots are provided with an efficient mechanism for protection against lipid peroxidation under these conditions. Increase in SOD activity should contribute to protection of lipids against peroxidation upon the return of roots to air, although this protection in the posthypoxic period may depend also on a different mechanism. Rhizomes of an anoxia-sensitive species of Iris, in which no changes in SOD activity can be detected during anoxic treatment, are subject to considerable peroxidation of lipids after returning from anaerobic to aerobic conditions, while no such damage is observed with rhizomes of an anoxia tolerant species within the same genus, in which a marked increase in the activity of SOD can be detected during anoxic treatment (Hunter et al. 1983). By contrast, rice seedlings did not show such an increase in the activity of SOD during the hypoxic period but were equipped with an efficient system for protection against any lipid peroxidation caused by rapid increase in oxygen tension that occurred upon their exposure to air (Ushimaru et al. 1994). Lupine root tips lost most of their respiration capacity immediately after hypoxic pretreatment. In contrast to this, root axes segments showed a marked increase in oxygen uptake over a whole 24 h re-aeration period (Table 2). An increase in oxygen uptake following re-aeration was accompanied by a rapid root extension (Fig. 5 A). Albrecht and Wiedenroth (1994) showed an increase in oxygen uptake following reaeration as a response to high energy demands caused by re-activated metabolism. An explanation of differences in oxygen consumption between lupine root tips and axes may be that more mature root zones, in which cells are fully vacuolated, are much more resistant to oxygen deprivation than
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the metabolically highly active tip zones. Even aerenchymatous roots are limited in their ability to conduct O2 over long distances to the apical zone. In cereal root tips, lower energy metabolism (Drew et al. 1985) and production of ethanol, alanine, and lactate suggest that anoxia can develop (Gibbs et al. 1995). Pavelic et al. (2000) showed that potato cells gradually lost most of their respiration capacity after a 12 to 24 h of anoxic treatment followed by a 24-h post-anoxic period. The post-anoxic recovery of cell respiration depended on the duration of the anoxic pretreatment and was correlated with cell viability. Surprisingly, our results showed that all lupine roots hypoxically pretreated for 72 h and subsequently re-aerated were viable and continued to elongate during recovery period but roots hypoxically pretreated for 48 h were less tolerant to hypoxia and subsequent re-aeration (Fig. 5 B). Before transfer to hypoxia for 48 h lupine seedlings were grown under aerated conditions for 24 h from the initiation of the experiment and at the start of the hypoxic pretreatment their root caps were coated by mucigel (data not shown). In contrast, roots hypoxically pretreated for 72 h were not grown in aerated nutrient solution from the initiation of the experiment, and at the onset of hypoxia their root caps did not produce as much mucigel. The greater susceptibility of roots subjected to hypoxic treatment for 48 h may be a consequence of the inhibition of oxygen diffusion by mucigel produced by cells of the root cap. Division and elongation might cease in cells of root tips because of development of anoxic zones. The elongation of lupine roots was retarded immediately after re-aeration and ceased in 75 % of roots re-aerated for 24 h and this cessation was likely to be due to the death of 2 – 3 mm root tips. The evidence obtained in this study strongly suggests that re-aeration following hypoxia imposes an oxidative stress in lupine roots. The effects of oxidative stress were indicated by an increase in the level of free radical and the induction of SOD activity immediately after re-oxygenation of hypoxically pretreated roots. However, the capacity of lupine roots to keep balance between the formation and detoxification of activated oxygen species seems to be sufficient to counteract harmful effects induced by re-aeration since hydrogen peroxide content and MDA concentration decreased even after longer hypoxic pretreatment. The differences in response to stress between root tips and root axes might be a consequence of higher resistance of root axes consisted of mature, fully-differentiated cells to oxygen deprivation and subsequent re-oxygenation. We conclude that the re-oxygenation stress per se was not the key factor for cell death of root tips. The experiment clearly demonstrates a coincidence of greater susceptibility to hypoxia followed by re-aeration with development of anoxic zones in root tips. Despite clear evidence that hypoxic pretreatmant was the critical step in plant response, we cannot exclude the possibility that re-aeration could have contributed to the retardation of root elongation.
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Ellis MH, Setter TL (1999) Hypoxia induces anoxia tolerance in completely submerged rice seedlings. J Plant Physiol 154: 219 – 230 Elstner EF, Osswald W (1994) Mechanisms of oxygen activation during plant stress. Proceedings of the Royal Society of Edinburgh 102 B: 131–154 Garnczarska M (2002) Hypoxic induction of alcohol and lactate dehydrogenases in lupine seedlings. Acta Physiol Plant 24: 265 – 272 Garnczarska M, Ratajczak L (2003) Hypoxia induces anoxia tolerance in roots and shoots of lupine seedlings. Acta Physiol Plant 25: 47– 53 Gibbs J, de Bruxelle G, Armstrong W, Greenway H (1995) Evidence for anoxic zones in 2 – 3 mm tips of aerenchymatous maize roots under low O2 supply. Aust J Plant Physiol 22: 723–730 Halliwell B, Gutteridge JMC (1985) Free Radicals in Biology and Medicine. Clarendon Press, Oxford Hunter MI, Hetherington AM, Crawford RMM (1983) Lipid peroxidation – a factor in anoxia tolerance in Iris species? Phytochemistry 22: 1145–1147 Monk LS, Fagerstedt KV, Crawford RMM (1987) Superoxide dismutase as an anaerobic polypeptide. A key factor in recovery from oxygen deprivation in Iris pseudacorus? Plant Physiol 85: 1016–1020 Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22: 867– 880 Pavelic D, Arpagaus S, Rawyler A, Brändle R (2000) Impact of postanoxia stress on membrane lipids of anoxia-pretreated potato cells. A re-appraisal. Plant Physiol 124: 1285–1292 Prasad TK, Anderson MD, Stewart CR (1994) Acclimation, hydrogen peroxide and abscisic acid protect mitochondria against irreversible chilling injury in maize seedlings. Plant Physiol 105: 619 – 627 Puntarulo S, Galleano M, Sanchez RA, Boveris A (1991) Superoxide anion and hydrogen peroxide metabolism in soybean embryonic axes during germination. Biochim Biophys Acta 1074: 277– 283 Purvis AC (1997) Role of the alternative oxidase in limiting superoxide production by plant mitochondria. Physiol Plant 100: 165–170 Ren H-X, Wang Z-L, Chen X, Zhu Y-L (1999) Antioxidative responses to different altitudes in Plantago major. Environ Exp Bot 42: 51– 59 Ricard B, Couee I, Raymond P, Saglio PH, Saint-Ges V, Pradet A (1994) Plant metabolism under hypoxia and anoxia. Plant Physiol Biochem 32: 1–10 Ros Barcelo A (1987) Quantification of lupin peroxidase isoenzymes by densitometry. Ann Biol 14: 33 – 38 Saglio PH, Drew MC, Pradet A (1988) Metabolic acclimation to anoxia induced by low (2 – 4 kPa partial pressure) oxygen pretreatment (hypoxia) in root tips of Zea mays. Plant Physiol 86: 61– 66 Ushimaru T, Shibasaka M, Tsuji H (1992) Development of the O2 – -detoxification system during adaptation to air of submerged rice seedlings. Plant Cell Physiol 33: 1065–1071 Ushimaru T, Shibasaka M, Tsuji H (1994) Resistance to oxidative injury in submerged rice seedlings after exposure to air. Plant Cell Physiol 35: 211– 218 Van Toai T, Bolles CS (1991) Postanoxic injury in soybean (Glycine max) seedlings. Plant Physiol 97: 588 – 592 Vartapetian BB, Jackson MB (1997) Plant adaptation to anaerobic stress. Ann Bot 79: 3 – 20 Waters I, Morrell S, Greenway H, Colmer TD (1991) Effects of anoxia on wheat seedlings. II. Influence of O2 supply prior to anoxia on tolerance to anoxia, alcoholic fermentation, and sugar levels. J Exp Bot 42: 1437–1447 Woodbury W, Spencer AK, Stahmann MA (1971) An improved procedure using ferricyanide for detecting catalase isozymes. Anal Biochem 44: 301– 305
Re-aeration – induced oxidative stress and antioxidative defenses in hypoxically pretreated lupine roots Małgorzata Garnczarska1 *, Waldemar Bednarski2, Iwona Morkunas3 1
Department of Plant Physiology, Faculty of Biology, A. Mickiewicz University, Al. Niepodległo´sci 14, 61-713 Poznan, ´ Poland
2
Institute of Molecular Physics, Polish Academy of Science, Smoluchowskiego 17, 60-179 Poznan, ´ Poland
3
Department of Plant Physiology, Agricultural University, Wołynska ´ 35, 60-637 Poznan, ´ Poland
Received February 6, 2003 · Accepted June 1, 2003
Summary The level of free radicals and activities of antioxidative enzymes were examined in roots of lupine seedlings (Lupinus luteus L.) that were deprived of oxygen by subjecting them to root hypoxia for 48 and 72 h and then re-aerated for up to 24 h. Using electron paramagnetic resonance (EPR), we found that the exposure of previously hypoxically grown roots to air caused the increase in free radicals level, irrespective of duration of hypoxic pretreatment. Immediately after re-aeration the level of free radicals was two times higher than in aerated control. The EPR signal with the g-values at the maximum absorption of 2.0057 and 2.0040 implied that the paramagnetic radicals are derived from a quinone. Directly after re-aeration of hypoxically pretreated roots, the activity of superoxide dismutase (SOD, EC 1.15.1.1) increased to its highest value, followed by a decline below the initial level, whereas activities of catalase (CAT, EC 1.11.1.6) and peroxidase (POX, EC 1.11.1.7) were diminished or only slightly influenced during reaeration. The electrophoretic patterns of the soluble extracts show 4 isozymes of SOD, 4 isozymes of POX and 1 isozyme of CAT. The level of H2O2 was enhanced or lowered by re-aeration, depending on the previous duration of hypoxia. At the onset of re-aeration products of lipid peroxidation were present at a three-fourth of the levels found in aerobic control. Their levels increased after prolonged exposure to air but remained lower than those in aerobic control even after 24 h of re-aeration. Re-admission of oxygen resulted in about 20 % rise in oxygen uptake by root axes segments immediately after transfer of roots from hypoxia and the high uptake rates were observed over whole re-aeration period. Oxygen consumption by root tips was significantly reduced just after transfer from hypoxic conditions as compared to aerated control but after 24 h of re-aeration even approached the control level. The results are discussed in relation to the ability of lupine roots to cope with oxidative stress caused by re-aeration following hypoxic pretreatment. Key words: catalase – electron paramagnetic resonance – hydrogen peroxide – hypoxia – lipid peroxidation – lupine – peroxidase – re-aeration – superoxide dismutase Abbreviations: CAT = catalase. – EPR = electron paramagnetic resonance. – MDA = malondialdehyde. – POX = peroxidase. – SOD = superoxide dismutase. – TBARS = thiobarbituric acid reactive substances * E-mail corresponding author: [email protected] 0176-1617/04/161/04-415 $ 30.00/0
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Małgorzata Garnczarska, Waldemar Bednarski, Iwona Morkunas
Introduction Many plant species sustain injury or even die when the soil becomes excessively wet through transient flooding. O2 deprivation is the primary stress experienced by plants during flooding, so plants must cope with hypoxic or even anoxic conditions. The response to oxygen deficiency in several species is associated with a number of biochemical, morphological, and anatomical changes in both the root and the shoot (reviewed by Ricard et al. 1994, Vartapetian and Jackson 1997). Plants cannot survive the prolonged oxygen defecit, however, the ability of plant tissues to survive anoxic stress can be increased by hypoxic pretreatment. The phenomenon of acclimation to anaerobiosis has been reported for wheat (Waters et al. 1991), maize (Saglio et al. 1988), rice (Ellis and Setter 1999) and lupine (Garnczarska and Ratajczak 2003). Injury and death of roots under oxygen deficiency have been attributed to the accumulation of toxic end products of anaerobic metabolism, to lowering of energy charge, or to lack of substrates for respiration (Drew 1997). Although it is clear that most of the injuries are incurred during oxygen deficiency, the restoration of normal O2 conditions is also damaging to plant tissue. Under these conditions, damage is mainly brought about by a burst of reactive oxygen species formed when oxygen becomes available again as electron acceptor for electron-saturated redox chain (Biemelt et al. 2000). Conditions favouring the formation of reactive oxygen species, such as low energy charge, high levels of reducing equivalents, and saturated electron transport chains, usually prevail in plant tissues when oxygen supply is restricted (Van Toai and Bolls 1991). The main sites of reactive oxygen species production in the plant cell are the organelles with highly oxidizing metabolic activities or with sustained electron flows: chloroplasts, mitochondria and peroxisomes (Dat et al. 2000, Corpas et al. 2001). In non-photosynthetic tissues, mitochondria are the main source of reactive oxygen species (Puntarulo et al. 1991). Components of the electron transport chain may reduce oxygen, thus leading to the formation of superoxide radicals. Superoxide is converted to hydrogen peroxide by disproportionation reaction. The hydroxyl radical is then formed from superoxide anion and H2O2 via the metal-catalyzed Haber-Weiss reaction. Under normal growing conditions, reactive oxygen species are maintained at a non-damaging level by low molecular mass antioxidants (ascorbate, glutathione, tocopherols) and enzymes (superoxide dismutase, catalase, peroxidase), which either scavenge or utilize them (Elstner and Osswald 1994). However, these defence mechanisms can be overwhelmed during environmental stress, resulting in an excess production of reactive oxygen species (Prasad et al. 1994). Despite considerable progress on some aspects of tolerance and acclimation to low oxygen regimes, there are divergent opinions as to the cause of death when plant tissue is deprived of oxygen. Determining whether injury takes place
directly under oxygen deficiency or on re-exposure to air could underlie the problem of whether or not post-hypoxic (post-anoxic) injury contributes to loss of plant survivability. As we described previously lupine seedlings have mechanisms for sensing and adapting to O2 deficiency in roots and in shoots. Hypoxia led to a reduction of root elongation and, as expected, the activities of alcohol and lactate dehydrogenases increased during hypoxia (Garnczarska 2002). Hypoxic acclimation to anoxia in lupine roots seems to be associated with avoidance of lactate accumulation and cytoplasmic acidosis because of lowered lactate dehydrogenase activity, combined with increased alcohol dehydrogenase activity and therefore increased potential for ethanolic fermentation to continue glycolysis and permit seedlings to survive (Garnczarska and Ratajczak 2003). However, hypoxia (anoxia) tolerance, as judged by the ability to return to an aerobic environment, may involve also other metabolic strategies for dealing with oxygen deficiency and subsequent re-aeration. Reoxygenation damage, known as «reperfusion injury», is well documented in human and animal systems (Halliwell and Gutteridge 1985), but also occurs after re-aeration in plants which are deprived of oxygen due to flooding (Crawford 1992). Post-hypoxic (post-anoxic) injury after transfer from hypoxia (anoxia) to normoxia has been reported with Iris germanica rhizomes (Hunter et al. 1983), rice seedlings (Ushimaru et al. 1992), soybean seedlings (Van Toai and Bolles 1991) and wheat seedlings (Albrecht and Wiedenroth 1994, Biemelt et al. 1998). The aim of the present study was to examine whether posthypoxia induces an oxidative stress in lupine roots. Therefore, the concentration of free radicals and there activities, and isoenzyme patterns of antioxidative enzymes were determined in hypoxically grown and subsequently re-aerated roots as well as in roots of continuously aerated control seedlings. In addition, the concentrations of hydrogen peroxide and malondialdehyde were measured and oxygen consumption by root tips and root axes was estimated. Furthermore, roots ability to survive hypoxia and subsequent re-aeration was studied.
Materials and Methods Plant material Seeds of yellow lupine (Lupinus luteus L. cv. Juno) were allowed to imbibe on moistened filter paper at 25 ˚C in the dark. After 60 h, 70 germinated seeds were transferred for 12 h to growth vessels containing 2.5 L of aerated 1/5 strength Knop nutrient solution (acclimation to growth conditions). The roots were in contact with the solution while the shoots were in the gas phase. All experiments were carried out in the dark at 25 ˚C.
Gas treatments After growing under aerated conditions for 24 h and 0 h from the initiation of the experiments, seedlings were subjected to hypoxia by flush-
Oxidative stress in lupine under post-hypoxia ing nutrient solution with N2 gas (99.99 % N2) for 48 h and 72 h, respectively. At the start of the hypoxic pretreatment seedlings were 3 and 4 d after imbibition, respectively. Hypoxically pretreated seedlings were then re-aerated for 30 min, 2 h, 4 h and for some estimation for 24 h. The nutrient solution of control plants was continuously flushed with air for 72 h. Following the gassing treatments, roots were rinsed carefully in water, dried with filter paper and used either immediately or frozen in liquid nitrogen. The contact with air during this preparation lasted about 2 min, so roots harvested immediately after hypoxic pretreatment were labelled as re-aerated for 2 min. Oxygen concentration in the vessels was monitored with an oxygen meter (Dissolved Oxygen Meter 9300, Jenway). Dissolved oxygen concentrations were 20 % O2 v/v for aerobic control and no detectable oxygen concentration in solution flushed with pure nitrogen.
Viability determination Root tips were considered to be viable if they resumed elongation during 72 h recovery period (re-oxygenation). Elongation was followed by measuring root length at the end of recovery period. A reference mark was applied 10 mm behind the root tip at the end of re-aeration period following hypoxic pretreatment.
Electron paramagnetic resonance (EPR) The samples of 6 roots frozen in liquid nitrogen were lyophylized in a Jouan LP3 freeze dryer and introduced into EPR-type quartz tubes (4 mm diameter). The concentration of free radicals was measured applying EPR spectrometer recording signals at X-band (microwave frequency – 9.4 GHz). EPR spectra were recorded as the first derivative of microwave absorption. Computer numerical double integration of the first derivative curve (Ayscough 1967) enabled us to obtain the total spin intensity for each sample. Since samples were of different weight, the areas under the EPR absorption curve per 1 mg of each sample were calculated. Because relative intensity of the lines was influenced by the quality factor of the resonator, an error of the results generated the above way would approach twelve percent. To avoid this problem the line intensities of the samples were compared to the intensity of the lines of the standard (Al2O3: Cr3 + ) that was kept permanently in resonance cavity. This way an uncertainty generated by the quality factor of the resonator could be eliminated and real relative intensities of EPR signals might be measured.
Protein extraction About 1.0 g of liquid nitrogen powered samples was homogenised with three volumes of cold 50 mmol/L potassium phosphate buffer (pH 7.0) containing 1 mmol/L EDTA and 5 % (w/v) polyvinylopyrrolidone (PVP). The slurry was centrifuged at 20,000 g for 20 min at 4 ˚C and the supernatants were used for enzyme assays. The protein concentration in the samples was estimated according to Bradford (1976).
Enzyme analyses Superoxide dismutase activity assay was based on the method of Beauchamp and Fridovich (1971) as described by Donahue et al. (1997). Catalase activity was assayed according to the method of Aebi (1974). Total peroxidase activity was measured according to Na-
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kano and Asada (1981). The reliability of the extraction and assay procedures was checked by recovery of activity of a commercially available enzymes added to the extraction buffer prior to tissue extraction. The recovery of enzyme activities was 95 – 97%.
Electrophoretic procedure Samples of crude roots extracts were electrophoresed in 10 % (SOD, POX) or 8 % (CAT) polyacrylamide slab gels at pH 8.9 under nondenaturing conditions according to Davis (1964). SOD activity was detected on gels using the photochemical procedure of Beauchamp and Fridovich (1971). The different types of SOD were differentiated by performing the activity stains in gels previously incubated in 3 mmol/L KCN or 5 mmol/L H2O2 as described in Asada et al. (1975). Isoenzymes of CAT were visualised on gels by the method of Woodbury et al. (1971). The diaminobenzidine reaction was used for staining isoenzymes of POX by the method of Ros Barcelo (1987).
Determination of hydrogen peroxide The measurement was performed by recording the oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone (MBTH) with its formaldehyde azine in the presence of horseradish peroxidase according to Capaldi and Taylor (1983).
Assay of 2-thiobarbituric acid – reactive material The level of lipid peroxidation in roots was measured as malondialdehyde (MDA) content determined by reaction with thiobarbituric acid (TBA) according to the method of Dhindsa and Matowe (1981) as reported in Ren et al. (1999) without addition of 3,5-diizobutyl-4-hydroxytoluene (BHT) as antioxidant.
Measurement of root respiration Root tips and root axes segments (approximately 10 mm long) were, immediately after excision, transferred into an oxygen electrode incubation chamber containing 3 mL of fully aerated stirred nutrient solution. The decrease of the oxygen concentration was polarographically measured by a Clark type digital model 10 electrode (Rank Brothers, Cambride, UK). After the measurements, the root segments were blotted and weighed to allow expression of the rate of respiration per gram fresh weight.
Results Electron paramagnetic spectra of lupine roots hypoxically pretreated for 48 and 72 h and then transferred to aerobic conditions for 2 min, 30 min, 2 h and 4 h were measured at room temperature using X-band EPR spectrometer. The EPR spectra with g-values of 2.0057 and 2.0040 for several samples are shown in Figure 1. A relative concentration of free radicals was compared in control vs. hypoxically pretreated and subsequently re-aerated roots. The exposure of previously hypoxically pretreated lupine roots to increased oxy-
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Małgorzata Garnczarska, Waldemar Bednarski, Iwona Morkunas
Figure 1. Electron paramagnetic spectra of free radicals in lupine roots subjected to hypoxia for 48 h (48 H) followed by re-aeration for 2 min, 30 min, 2 h and 4 h ( + 2 min air, + 30 min air, + 2 h air, + 4 h air) compared to aerated control roots.
Figure 3. Activities of SOD (A), CAT (B) and POX (C) in lupine roots subjected to hypoxia for 48 h (48 H, light grey bars) and 72 h (72 H, dark grey bars) followed by re-aeration for 2 min, 30 min, 2 h and 4 h ( + 2 min, + 30 min, + 2 h, + 4 h) compared with continuously aerated control roots (white bars). Means ± SD of four replicates.
Figure 2. Concentration of free radicals in lupine roots subjected to hypoxia for 48 h (48 H, light grey bars) and 72 h (72 H, dark grey bars) followed by re-aeration for 2 min, 30 min, 2 h and 4 h ( + 2 min, + 30 min, + 2 h, + 4 h) compared with continuously aerated control roots. The values for post-hypoxically treated roots are expressed as percentages of that for aerated control. The concentration of free radicals in aerated control was 18.2 Arb. U · mg –1 DW. The data is typical of data obtained in three replicate experiments.
gen availability caused a twofold increase in concentration of free radicals, irrespective of duration of hypoxic pretreatment. (Fig. 2). For longer re-aeration periods the amount of free radicals decreased but was still above the control level. Immediately after hypoxically pretreated roots were returned to air, the activity of SOD increased to its highest value, followed by a slow decline below the initial level (Fig. 3 A). Four different SOD isozymes were detected in PAGE, each of them being Cu, Zn-SOD (Fig. 4 A). In fact, both H2O2 and KCN inhibited the enzyme leading to the disappearance of the bands (data not shown). The enhanced activity of CAT was observed over the 2 h re-aeration period of
Oxidative stress in lupine under post-hypoxia
Figure 4. Isoenzyme patterns of SOD, CAT and POX in lupine roots subjected to hypoxia for 48 h (48 H) and 72 h (72 H) followed by reaeration for 2 min, 30 min, 2 h and 4 h ( + 2 min, + 30 min, + 2 h, + 4 h) compared with continuously aerated control roots. The different isoforms are numbered from cathode to anode. For SOD 100 µg proteins were loaded per each well, for CAT 25 µg and for POX 75 µg.
Table 1. Malondialdehyde (MDA) and H2O2 concentrations in lupine roots hypoxically pretreated for 48 h (48 H) and 72 h (72 H) and subsequently re-aerated for 2 min, 4 h and 24 h ( + 2 min air, + 4 h air, + 24 h air) compared to aerated control roots. Means ± SD of four replicates. Growth conditions
H2O2 concentration (nmol · g – 1 FW)
MDA concentration (nmol · g – 1 FW)
Control 48 H + 2 min air 48 H + 4 h air 48 H + 24 h air 72 H + 2 min air 72 H + 4 h air 72 H + 24 h air
11.75 ( ± 0.91) 12.94 ( ± 0.82) 13.42 ( ± 1.14) 13.16 ( ± 1.09) 10.31 ( ± 0.98) 9.16 ( ± 0.73) 9.14 ( ± 0.67)
90.1 ( ± 6.2) 68.4 ( ± 4.4) – 82.8 ( ± 5.8) 66.6 ( ± 3.6) – 81.8 ( ± 5.4)
roots grown under hypoxia for 48 h, whereas the activity of this enzyme tended to decrease during re-aeration of roots subjected to hypoxic conditions for 72 h and after 4 h of reaeration was clearly below the control level (Fig. 3 B). Gel stained for CAT activity revealed one isoform (Fig. 4 B). POX
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activity slightly increased after the start of re-aeration and declined after 4 h of exposure to air (Fig. 3 C). Four POX isoforms were present in lupine roots (Fig. 4 C). Superoxide dismutase, catalase and peroxidase zymogram analyses showed that synthesis of new isoforms was not induced by re-aeration. The amount of hydrogen peroxide slightly increased after re-aeration of roots hypoxically pretreated for 48 h and was fairly constant over the post-hypoxic period (Table 1). In seedlings hypoxically pretreated for 72 h the concentration of hydrogen peroxide gradually decreased during post-hypoxia. In order to estimate the extent of peroxidative damage to lipids in hypoxically pretreated lupine roots after exposure to air, the level of TBARS, an index of lipid peroxidation, was measured and expressed as MDA concentration. Immediately after re-exposure to air lupine roots contained 75 % of the MDA found in aerobic control (Table 1). After 24 h of re-aeration this value increased to about 90 % of the control level. Polarygraphic studies performed on 10 mm lupine root tips revealed that the respiration immediately after 48 and 72 h of hypoxic pretreatment was reduced to 38 % and 57%, respectively, as compared to control root tips (Table 2). After 24 h of re-exposure to oxygen following hypoxic pretreatment for 72 h the respiration of root tips was significantly increased to 738.2 nmole O2 · min –1 · g –1 FW. Oxygen consumption in control root tips was 636.1nmole O2 · min –1 · g –1 FW. In contrast, in root tips subjected to hypoxia for 48 h and then re-aerated for 24 h respiration was reduced to 44 % as compared to control. Re-admission of oxygen resulted in a 20 % rise of oxygen uptake (compared with the control) by root axes segments excised from roots previously grown under hypoxia for 48 and 72 h (Table 2). After 4 h of re-aeration oxygen consumption by root axes segments exceeded the control by 45 % and 31 %, respectively. Because of the marked differences in oxygen consumption following re-aeration of lupine root tips hypoxically pretreated for 48 and 72, it was of interest to determine whether there were differences in their ability to survive hypoxia and re-
Table 2. Oxygen consumption by 10 mm root tips and root axes of lupine roots hypoxically pretreated for 48 h (48 H) and 72 h (72 H) and subsequently re-aerated for 2 min, 4 h and 24 h ( + 2 min air, + 4 h air, + 24 h air) compared to aerated control. Means ± SD of five replicates. Growth conditions
Control 48 H + 2 min air 48 H + 4 h air 48 H + 24 h air 72 H + 2 min air 72 H + 4 h air 72 H + 24 h air
Oxygen consumption (nmol O2 · min – 1 · g – 1FW) Root tips
Root axes
636 ( ± 47) 240 ( ± 29) 333 ( ± 34) 278 ( ± 30) 365 ( ± 42) 632 ( ± 49) 738 ( ± 55)
109 ( ± 12) 133 ( ± 18) 158 ( ± 23) 130 ( ± 21) 131 ( ± 19) 143 ( ± 24) 147 ( ± 26)
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Discussion
Figure 5. Elongation (A) and viability (B) of lupine roots subjected to hypoxia for 48 h (48 H, light grey bars) and 72 h (72 H, dark grey bars) followed by re-aeration for 2 min, 30 min, 2 h and 4 h ( + 2 min, + 30 min, + 2 h + 4 h) compared with continuously aerated control roots (white bars). A. elongation per root during 72 h of recovery; B. percent viability. The experiment was repeated twice with 12 plants each. Error bars indicate SD.
aeration. We investigated the effects of hypoxic pretreatment for 48 and 72 h followed by re-aeration for 4 and 24 h on root elongation and survival of roots tips. Root survival was based on growth during 72 h recovery period. Lupine roots hypoxically pretreated for 72 h showed a rapid root extension during recovery period (Fig. 5 A), but following 24 h of re-aeration root elongation was slowed down to the control level. The roots of seedlings hypoxically pretreated for 48 h and subsequently re-aerated were more susceptible to treatment, with a significant decrease in survival after 24 h of re-aeration (Fig. 5 B). In about 30 % of these roots the deformation of 2 – 3 mm root tips occurred just after hypoxic pretreatment (root tips were water soaked and translucent) and continued further during reaeration. The root tips of seedlings hypoxically pretreated for 72 h and then re-aerated were more tolerant to the stress. Root survival after recovery period remained at 100 %.
The experimental protocol used in this study utilized lupine seedlings with their roots exposed to oxygen deprived conditions and their shoots remaining in ambient air. This would simulate conditions similar to those encountered by lupine plants in the field. The roots were grown in solution flushed with pure nitrogen (no detectable oxygen concentration). However, the internal O2 movement from shoots to roots would create hypoxic conditions in root tissues. The direct detection of stable free radicals in lupine roots by EPR spectroscopy revealed that re-oxygenation after a period of oxygen deprivation intensifies the synthesis of free radicals. Immediately after hypoxically pretreated roots were returned to air the concentration of free radicals increased to its highest value (204 and 187% of free radicals found in control tissue) and then decreased after recovery in air for 30 min, 2 h and 4 h but was clearly above the control level (Fig. 2). The radical spectra consisted of two only slightly split lines visible as a broadening of the EPR signal at high magnetic field (Fig. 1). EPR analyses conducted on desiccated moss revealed an accumulation of stable free radicals with two g-values of 2.0054 and 2.0023 (Atherton et al. 1993). It was found that these two g-value were consistent with quinone radicals. Atherton et al. (1993) suggested that the reason of the radicals might be quinones involved in electron transport chains. Cakmak and Marschner (1988) observed zinc-dependent changes of semiquinone radical signals measured by elecron spin resonance spectrocopy in cotton roots. It has been shown that superoxide anion is generated at the level of the three enzymatic complexes interacting with ubiquinone (coenzyme Q) and that this anion is the only reactive oxygen species generated suggesting a monoelectronic donor likely the ubisemiquinone, a quinone radical intermediate of the redox Q-cycle (Du et al. 1998). Purvis (1997) pointed out that plant mitochondria are capable of generating large quantities of superoxide radical when the level of Q reduction is high. The higher SOD activity in lupine roots immediately after transfer from hypoxic conditions (Fig. 3) may be due to increased formation of superoxide anion as the enhanced activity of this enzyme is an indicator of increased levels of superoxide anion in plant cells. According to Monk et al. (1987) an increase in SOD activity already apparent under anoxia is of advantage for plants which face enhanced superoxide formation during post-anoxia. The strong induction of SOD activity under anoxia found in the flooding-tolerant Iris pseudacorus was discussed as an evolutionary advantage of plants being thus prepared for re-aeration. In soybean short-term anoxia favoured subsequent production of superoxide radicals. Longer anoxic treatment lead to higher SOD activities and therefore to a more efficient protection (Van Toai and Bolles 1971). Neither hypoxia nor subsequent re-aeration caused significant changes in SOD activity in wheat roots (Biemelt et al. 2000). However, anoxia led to the appearance of new isoforms and the increase in SOD activity. By contrast, the activ-
Oxidative stress in lupine under post-hypoxia ities of six antioxidative enzymes including SOD were reduced in submerged rice seedlings, but approached control levels following the restoration of aerobic conditions (Ushimaru et al. 1992). The level of hydrogen peroxide was enhanced or lowered in lupine roots, depending on previous duration of hypoxia (Table 1). It is worth recalling that hydrogen peroxide actually measured by a given method always reflects the balance between H2O2-generating and H2O2-consuming processes. Thus low H2O2 level after re-oxygenation following hypoxic treatment for 72 h can be due either to a low intrinsic production rate or to efficient scavenging by detoxifying enzymes such as CAT and POX. Concomitant with decrease in the amount of hydrogen peroxide, the activity of CAT was diminished, whereas the activity of POX remained nearly unchanged (Fig. 3). However, the decrease in amount of hydrogen peroxide was correlated with diminished activity of SOD. The amount of hydrogen peroxide slightly increased after reaeration of roots hypoxically pretreated for 48 h, indicating enhanced formation of reactive oxygen, although CAT and POX showed a transient increase in activity during first two hours of post-hypoxia. MDA remained at levels lower than those found in aerobic control during the 24 h that followed the transfer of hypoxically grown lupine roots to air (Table 1), indicating that lupine roots are provided with an efficient mechanism for protection against lipid peroxidation under these conditions. Increase in SOD activity should contribute to protection of lipids against peroxidation upon the return of roots to air, although this protection in the posthypoxic period may depend also on a different mechanism. Rhizomes of an anoxia-sensitive species of Iris, in which no changes in SOD activity can be detected during anoxic treatment, are subject to considerable peroxidation of lipids after returning from anaerobic to aerobic conditions, while no such damage is observed with rhizomes of an anoxia tolerant species within the same genus, in which a marked increase in the activity of SOD can be detected during anoxic treatment (Hunter et al. 1983). By contrast, rice seedlings did not show such an increase in the activity of SOD during the hypoxic period but were equipped with an efficient system for protection against any lipid peroxidation caused by rapid increase in oxygen tension that occurred upon their exposure to air (Ushimaru et al. 1994). Lupine root tips lost most of their respiration capacity immediately after hypoxic pretreatment. In contrast to this, root axes segments showed a marked increase in oxygen uptake over a whole 24 h re-aeration period (Table 2). An increase in oxygen uptake following re-aeration was accompanied by a rapid root extension (Fig. 5 A). Albrecht and Wiedenroth (1994) showed an increase in oxygen uptake following reaeration as a response to high energy demands caused by re-activated metabolism. An explanation of differences in oxygen consumption between lupine root tips and axes may be that more mature root zones, in which cells are fully vacuolated, are much more resistant to oxygen deprivation than
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the metabolically highly active tip zones. Even aerenchymatous roots are limited in their ability to conduct O2 over long distances to the apical zone. In cereal root tips, lower energy metabolism (Drew et al. 1985) and production of ethanol, alanine, and lactate suggest that anoxia can develop (Gibbs et al. 1995). Pavelic et al. (2000) showed that potato cells gradually lost most of their respiration capacity after a 12 to 24 h of anoxic treatment followed by a 24-h post-anoxic period. The post-anoxic recovery of cell respiration depended on the duration of the anoxic pretreatment and was correlated with cell viability. Surprisingly, our results showed that all lupine roots hypoxically pretreated for 72 h and subsequently re-aerated were viable and continued to elongate during recovery period but roots hypoxically pretreated for 48 h were less tolerant to hypoxia and subsequent re-aeration (Fig. 5 B). Before transfer to hypoxia for 48 h lupine seedlings were grown under aerated conditions for 24 h from the initiation of the experiment and at the start of the hypoxic pretreatment their root caps were coated by mucigel (data not shown). In contrast, roots hypoxically pretreated for 72 h were not grown in aerated nutrient solution from the initiation of the experiment, and at the onset of hypoxia their root caps did not produce as much mucigel. The greater susceptibility of roots subjected to hypoxic treatment for 48 h may be a consequence of the inhibition of oxygen diffusion by mucigel produced by cells of the root cap. Division and elongation might cease in cells of root tips because of development of anoxic zones. The elongation of lupine roots was retarded immediately after re-aeration and ceased in 75 % of roots re-aerated for 24 h and this cessation was likely to be due to the death of 2 – 3 mm root tips. The evidence obtained in this study strongly suggests that re-aeration following hypoxia imposes an oxidative stress in lupine roots. The effects of oxidative stress were indicated by an increase in the level of free radical and the induction of SOD activity immediately after re-oxygenation of hypoxically pretreated roots. However, the capacity of lupine roots to keep balance between the formation and detoxification of activated oxygen species seems to be sufficient to counteract harmful effects induced by re-aeration since hydrogen peroxide content and MDA concentration decreased even after longer hypoxic pretreatment. The differences in response to stress between root tips and root axes might be a consequence of higher resistance of root axes consisted of mature, fully-differentiated cells to oxygen deprivation and subsequent re-oxygenation. We conclude that the re-oxygenation stress per se was not the key factor for cell death of root tips. The experiment clearly demonstrates a coincidence of greater susceptibility to hypoxia followed by re-aeration with development of anoxic zones in root tips. Despite clear evidence that hypoxic pretreatmant was the critical step in plant response, we cannot exclude the possibility that re-aeration could have contributed to the retardation of root elongation.
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Małgorzata Garnczarska, Waldemar Bednarski, Iwona Morkunas
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