ISSN 1021-4437, Russian Journal of Plant Physiology, 2009, Vol. 56, No. 2, pp. 168–174. © Pleiades Publishing, Ltd., 2009. Original Russian Text © M.S. Sin’kevich, A.N. Deryabin, T.I. Trunova, 2009, published in Fiziologiya Rastenii, 2009, Vol. 56, No. 2, pp. 186–192.
RESEARCH PAPERS
Characteristics of Oxidative Stress in Potato Plants with Modified Carbohydrate Metabolism M. S. Sin’kevich, A. N. Deryabin, and T. I. Trunova Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35, Moscow, 127276 Russia; fax: 7 (495) 977-8018; e-mail:
[email protected] Received February 18, 2008
Abstract—Effects of sugars on the development of hypothermia-induced oxidative stress were studied in leaves of two potato genotypes (Solanum tuberosum L., cv. Désirée): with normal carbohydrate metabolism and a genotype with increased sugar content modified by insertion of yeast-derived invertase gene. It was found – that generation of O 2 proceeds more actively in transformed plants than in control plants. On the contrary H2O2 concentration and the catalese and peroxidase activities were lower. At the same time, the activities of superoxide dismutase were similar in plants of both genotypes. A short-term incubation of plants at –7°C confirmed that a higher freezing tolerance of transformed plants was due to low-molecular-weight components of antioxidant protection system rather than to enzymatic component. Literature data and experimental results suggest that the protective effect of sugars is caused by their ability to scavenge ROS nonspecifically under stress conditions Key words: Solanum tuberosum - oxidative stress - superoxide dismutase - reactive oxygen species - carbohydrates - transformed potato DOI: 10.1134/S1021443709020046
INTRODUCTION Accelerated generation of so-called reactive oxygen species (ROS) in plants is one of the earliest consequences of stress, low-temperature stress in particular [1]. Accumulation of highly reactive compounds, leading to a cascade of free-radical reactions called oxidative stress, is among the primary factors accounting for protein and DNA damage and for the increase in membrane permeability [2]. The system of protection against oxidative stress is known to comprise two components: the antioxidant enzymes and low-molecular-weight antioxidants. According to conventional view, based mainly on studies of thermophilic plants [3], the key role in plant protection against ROS belongs to antioxidant enzymes (superoxide dismutase (SOD), catalase, peroxydase, etc.). It was shown that the balance between oxidized and reduced compounds at the earliest stages of oxidative stress is shifted towards oxidation, while the enzymatic defense system is activated when a high level of oxidized substances is reached. Furthermore, an important condition of plant survival under stress conditions, specifically under hypothermia, is the presence of such multifunctional compounds as sugars. Therefore, accumulation of soluble Abbreviations: NBT—nitro blue tetrazolium; OD—optical density (units); ROS—reactive oxygen species; SOD—superoxide dismutase.
carbohydrates is considered an adaptive and preliminary process by many authors [4]. Chilling- and frosttolerant plants are known to efficiently synthesize and mobilize the carbohydrate reserves preaccumulated during the adaptation [5]. Moreover, the addition of exogenous sugars considerably increases plant tolerance [6]. Some authors even believe that the inferior cold tolerance of tubers in some potato cultivars is largely due to the restricted mobilization of starch reserves [7]. Recently, research on metabolism and nonspecific antioxidant properties of low-molecular-weight compounds accumulated during stress has gained new significance. For example, the capacity of scavenging ROS radicals was reported for polyamines [8], steroids [9], polyols, and phenols [10]. In the context of coldstress studies, the antioxidant properties of carbohydrates, revealed in chemical model experiments, are the matter of particular interest [11–14]. In contrast to oxidative stress in cold-sensitive plants, the features of oxidative stress and antioxidant defense system during cold treatment of cold-tolerant plants, including potato, are insufficiently characterized. The role of soluble sugars in hypothermiainduced oxidative stress is almost completely ignored. This is partly due to multiple functions of carbohydrates in living cells. In this respect, potato plants with modified carbohydrate metabolism [15] could be a
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promising model system in studies of oxidative stress in cold-tolerant plants. Earlier we demonstrated that expression in potato plants of the inserted yeast invertase gene (apoplastic localization of the enzyme) decreased sugar efflux from leaf cells and, consequently, led to accumulation of carbohydrates in photosynthesizing tissues, thus increasing cold tolerance of transformed plants as compared to nontransformed control plants [6]. Furthermore, the transformed plants were also more tolerant to oxidative stress developed during hypothermia. Several hypotheses were put forward to explain this phenomenon; one likely explanation is that the antioxidant defense system is more effective in transformed plants [16]. Therefore, it is interesting to study possible involvement of soluble carbohydrates as low-molecularweight scavengers of ROS in neutralization of oxidative stress. MATERIALS AND METHODS Plant material. Potato plants (Solanum tuberosum L., cv. Désirée) were transformed with the yeast invertase gene (inv) under control of the B33 class 1 patatin promoter, carrying also the leader peptide sequence of proteinase II inhibitor for providing the apoplastic enzyme localization (abbreviated below as the Ç33-inv plants). Nontransformed potato plants cv. Désirée were used as control plants. Plants were chosen from the collection of clones obtained during collaboration between the Max-Plank Institute of Plant Molecular Physiology (Golm, Germany) and the Chailakhyan Laboratory of Growth and Development, Timiryazev Institute of Plant Physiology (Russian Academy of Sciences). Expression of the inserted genes in the transformed plants was confirmed by means of polymerase chain reaction and by direct assay for activity of GUS reporter gene [6, 17]. In our work expression of the inserted gene constructions was earlier confirmed by elevated activity of all invertase forms in leaves of Ç33-inv plants and by elevated content of sugars [16]. Plants were propagated through in vitro micrografting and grown in the phytotron of Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, at 22°ë and 16-h photoperiod (illuminance of 4 klx) for 5 weeks on agar-based Murashige and Skoog nutrient medium [18], which contained 2% of sucrose and the following vitamins (in mg/l): 0.5 thiamine, 60.0 myoinositol, and 0.5 pyridoxine. In some experiments we analyzed the role of soluble sugars by varying sucrose concentration in the medium, because this treatment was shown to modify the intracellular carbohydrate content in leaves [16]. We used leaves without petioles, sampled from middle parts of three to five plants at the age of 5 weeks. Oxidative stress was induced by transferring test tubes with plants (without cotton-gauze plugs) into a freezing chamber with temperature of –7°ë for various RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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time intervals (30–60 min). Some plants were returned to optimal growth conditions (22°ë) to study reparative effects and the degree of damage (assays were made one day after cooling); other plants were used to measure various parameters of hypothermia-induced oxidative stress. Measurement of SOD activity. Activity of SOD cytosolic fraction (Cu,Zn-SOD) [2, 19], was measured by the method described in [20] with minor modifications. The method is based on generation of superoxide anions in the reaction of riboflavin photooxidation, which is enhanced by an indicator trap, nitro blue tetrazolium (NBT). In this reaction NBT is reduced to formazan, which has deep blue-violet color. In the presence of SOD, generation of formazan in the reaction system is slowed down. Optical density (OD) was measured against the basic buffer with SF-46 (LOMO, Russia) or Spekol-11 (Carl Zeiss, Germany) spectrophotometers at 560 nm, using cuvettes with 0.5-cm optical path length. The suppression of formazan production by 50% was taken as a unit of activity. Activity of SOD was expressed in activity units per 1 g fr wt of leaves. Hydrogen peroxide (H2O2) content was measured by color reaction with titanium chloride (TiCl4) by the method described in [19] with minor modifications. Absorbance of test solutions was measured at λ = 415 nm against 2 N H2SO4. The H2O2 content was calculated by means of a standard calibration curve for hydrogen peroxide solutions in acetone (concentration range 0.1–1 mM). Catalase activity was measured from the rate of H2O2 degradation according to the method described in [19]. Absorbance was measured with an SF-46 spectrophotometer at λ = 240 nm with 30-s intervals. Catalase activity was calculated from the formula: (∆D × n)/(ε × l × m), where ∆D is absorbance change over 1 min interval, expressed in OD units; n is dilution coefficient; ε is molar extinction coefficient of H2O2 (39.4 × 105 mol–1cm–1); m is fresh weight of the sample, g; and l is the cuvette width (1 cm). Activity of catalase was expressed in mmol of degraded H2O2/ (g fr wt min). Activity of guaiacol peroxidase was measured by the method described in [19] with minor modifications. The method is based on oxidation of aromatic compound (guaiacol) to a colored substance (tetraguaiacol). Absorbance was measured at λ = 470 nm against phosphate buffer (pH 5.5) at 30-s intervals. Activity of guaiacol peroxidase was calculated from the equation: (∆D × Vc × n × m)/(ε × Vs), where ∆D is the absorbance change over 1 min interval expressed in OD units; Vc is cuvette volume, ml; n is dilution coefficient; m is sample fresh weight, g; ε is a molar extinction coefficient (5.6 × 105 mol–1cm–1); and Vs is a sample volume, ml. Activity of guaiacol peroxidase was expressed in µmol guaiacol/(g fr wt min). Rate of ROS radical generation, predominantly – superoxide ( O 2 ) was measured by the method [21] with minor modifications, based on color reaction of No. 2
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RESULTS Our experiments provided data on generation and accumulation of reactive oxygen species (superoxide anion and hydrogen peroxide) and on key enzyme activities of the antioxidant defense system (SOD, catalase, and guaiacol peroxidase) in the control and Ç33-inv plants subjected to cold treatment. First, we compared hydrogen peroxide content and activities of enzymes responsible for its degradation (catalase and guaiacol peroxidase). Figure 1a shows that constitutive (at 22°ë) concentrations of hydrogen peroxide differed in leaves of the control and Ç33-inv plants: the concentration was approximately 1 mM in the control plants and considerably lower (0.6 mM) in the transformed plants. In the response to cold treatment, hydrogen peroxide content in plants of both genotypes increased to about 1.25 mM. The relative gain in ç2é2 content for Ç33-inv plants after cooling was 60% compared to that for noncooled plants, whereas the cold-induced increment was only 20% in control plants. After returning plants to optimal growth conditions, ç2é2 concentration in the control plants continued to grow, reaching 1.55 mM, while in B33-inv plants it decreased more than twice; i.e., it became even lower than before hypothermia. The background level of catalase activity (at 22°ë) in the transformed plants was almost twice lower than in control plants (Fig. 1b). Similar proportion was also observed for guaiacol peroxidase (Fig. 1c). Activities of catalase and guaiacol peroxidase changed similarly in plants of both studied genotypes: they increased immediately after freezing and recovered to the initial values after the plants were transferred to optimal conditions. Thus, these activities corresponded to intracellular concentrations of hydrogen peroxide. Our measurements revealed that the initial rates of superoxide anion formation (Fig. 2a) were considerably higher in leaves of transformed plants than in control plants (2.5 and 1 unit/min, respectively). However,
H2O2 concentration, mM
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adrenalin conversion to adrenochrome. Absorbance of samples was measured at λ = 480 nm with SF-46 or Specol 11 spectrophotometers, using cuvette with 1-cm – optical path length. The rate of O 2 generation was calculated from the equation: ∆D/t, where ∆D is the absorbance difference between the homogenate with adrenalin and the homogenate with water; and t is incubation period, min. Specificity of the reaction was confirmed by SOD addition (100 units). Sugar content was measured by the method [22], described in detail elsewhere [6]. Statistical data processing. Data were processed using t-test program and OriginPro 7.5 package with built in statistical functions (we used Students t-test, ê = 0.05). Figures show mean values of representative experiments made in three replicates and their standard errors.
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Fig. 1. (a) Hydrogen peroxide content, (b) catalase activity, and (c) peroxidase activity in leaves of the (1) control and (2) B33-inv plants under optimal conditions immediately after freezing, and in a day after returning plants to optimal conditions.
the rate of superoxide generation in transgenic plants exposed to cold treatment changed inconsiderably and remained on the same level one day after cooling. On the contrary, the rate of superoxide formation under hypothermic conditions in leaf tissues of the control plants increased to 2.5 unit/min; i.e., it became 2.5 times higher than the initial level. It is noteworthy that, one day after transferring cold-treated plants to optimal conditions (22°ë), the rate of superoxide generation continued to grow and reached 3.5 unit/min. According to our data, constitutive levels of the SOD activity in plants of both genotypes were almost equal, about 40 unit/g fr wt. We expected that SOD activity would follow the dynamics of the substrate (superoxide anion) formation (Fig. 2a). On the other hand, deactivation of this enzyme during stress was
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Fig. 2. (a) Rate of superoxide anion generation and (b) SOD activity in leaves of (1) control and (2) B33-inv plants under optimal conditions, immediately after hypothermia, and one day after returning the plants to optimal conditions.
Fig. 3. Activities of (a) SOD and (b) catalase in leaves of the (1) control (2) and B33-inv plants grown at 22°C on media with various concentrations of sucrose.
also reported [23]. Our experiments revealed that SOD activity in cold-treated control plants decreased by a factor of two (Fig. 2b) to 19 unit/g fr wt. One day after transferring the control plants to optimal growth conditions, SOD activity remained at a low level. At the same time, Ç33-inv plants retained the initial level of SOD activity both during and after cooling. In our work we also used another procedure for modulating the content of intracellular sugars. Growing plants on media with different sucrose concentrations allowed us to vary accumulation of intracellular carbohydrates. We obtained plants with autotrophic (grown without sucrose in the nutrient medium), heterotrophic (sucrose content 4–6%), and mixed (sucrose content 2, 4 and 6%) types of carbohydrate metabolism. Under optimal growth conditions (22°ë), SOD activity declined with increasing sucrose content in the growth medium both for control and Ç33-inv plants (Fig. 3a); i.e., the plants exhibited inverse dependence of SOD activity on sucrose concentration in the growth medium. It is important to note that SOD activity in leaves of transformed plants was lower than that in control plants. This observation suggests a lower production of hydrogen peroxide in the dismutation reaction under optimal conditions and, correspondingly, lower
activity of catalase in B33-inv plants. In contrast to our expectations, catalase activity in potato leaves increased with elevation of sucrose concentration in the growth medium, this increase being higher in the transformants than in normal plants (Fig. 3b). Since the aim of this work was to find possible role of sugars as low-molecular-weight antioxidants, it was necessary to record the processes of their free-radical oxidation under hypothermia. This task was complicated by a wide diversity of possible reaction products observed in model systems (aldehydes, ketones, lactones, and stable ë3-compounds), which are capable of generating éç· radicals [11–14]. However, the aforementioned oxidation products do not participate in classic indicator reactions for sugars, in the reaction of resorcin in particular. In this case it is possible to reveal oxidation by measuring the content of soluble carbohydrates. For this purpose plants were subjected to a shortterm (30 or 60 min) freezing at –7°ë, and then the content of soluble carbohydrates in leaves was assayed (Fig. 4). Our experiments showed that sugar content at 22°ë in the transformed plants was 30% higher than in control plants (13 vs. 10 mmol/g fr wt). There were no changes in the sugar content after a half-hour freezing. However, after one hour freezing we found a consider-
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13 Sugar content, mmol/g fr wt
12
1
11 10 9 1 0
22°C
–7°C 30 min
–7°C 60 min
Fig. 4. Sugar content in the control (1) and B33-inv (2) plants subjected to short-term (30 and 60 min) cooling at –7°C.
able decrease (by 20% of initial values) in total sugar content in the cooled plants of both genotypes. DISCUSSION Previous data on accumulation of malonic dialdehyde (MDA) and electrolyte leakage showed that the cold-induced membrane damage was less pronounced in Ç33-inv potato plants enriched with sugars than in nontransformed control plants [16, 24]. Based on these data, a hypothesis was put forward that the observed differences were caused by lower rates of ROS generation and accumulation. In order to verify this hypothesis, we analyzed the peroxide content (Fig. 1a) and the rate of superoxide anion generation (Fig. 2a) in plants of both genotypes. We observed significant increase in the rate of superoxide anion generation in control plants during freezing and after transferring the plants back to optimal conditions. In B33-inv plants, this process did not almost change (Fig. 2). At the same time, the H2O2 content after freezing were similar in plants of both genotypes. After returning plants to optimal conditions, H2O2 concentration in control plants increased significantly, while in B33-inv plants, it decreased to the level of untreated plants (Fig. 1). The data obtained indicate the more pronounced development of oxidative stress in control plants. These results agree well with our previous report on elevation membrane permeability in control plants [24]. Presumably, the threshold of oxidative stress was exceeded and there was no recovery of this parameter. Obviously, the transformed plants were subjected to stronger oxidative attack by superoxide anion already under normal growth conditions (Fig. 2a), even though the level of oxidative processes, manifested in hydrogen peroxide content (Fig. 1a), was considerably lower in the transformants than in control plants, in consistency with earlier data on dynamics of MDA accumulation [24]. We suggested that low intensity of oxidative
processes at higher rate of ROS formation is related to more efficient antioxidant defense system in B33-inv plants. The efficiency of antioxidant defense system was estimated from data on activities of key enzymes: catalase, guaiacol peroxidase (Figs. 1, 3), and SOD (Figs. 2b, 3a). Our data on rates of superoxide anion formation (Fig. 2a) might have suggested that SOD activity should be higher in B33-inv plants than in control plants. In contrast to our expectations, at 22°C activities of all antioxidant enzymes in B33-inv plants were lower than in control plants (Figs. 1–3). It should be reminded that short-term freezing increased the activities of catalase (Fig. 1) and guaiacol peroxidase. Since peroxide concentration in leaves of the studied genotypes increased (Fig. 1a) without additional activation of SOD (Fig. 2b) , and because the antioxidant system comprises two components, the existence of alternative pathway of peroxide formation (without involvement of SOD) can be inferred. Lowmolecular-weight antioxidants might compensate for low SOD activity and be integrated into a defense line, which is alternative to the enzyme defense system. It was reported [14] that mannitol, mannose, glucose, fructose, and sucrose strongly reduce toxicity of model éç·-producing system. These sugars and polyols apparently play the role of radical scavengers and might be involved in the antioxidant defense system as nonspecific low-molecular-weight antioxidants. Some research groups supported the notion that nonspecific antioxidant properties are related to a variety of free-radical oxidation pathways: attacks on individual (predominantly terminal) carbon atoms, formation of cyclic lactones, and production of unstable peroxy intermediates degrading to stable and easily metabolizabe ë3-fragments [11–13]. Based on the data reported in literature [11–14], a general view on possible reactions of ROS radicals with soluble carbohydrates could be elaborated, which is shown schematically in Fig. 5 with an example of glucose. It is noteworthy that all compounds listed above as reaction products do not belong to carbohydrates and are not detected by conventional indicator reactions for sugars. This opens a possibility to monitor carbohydrate degradation, which could be unexpectedly extensive in the case of radical reactions. Indeed, Fig. 4 shows the decrease in total content of soluble carbohydrates by 20–25% in plants of both genotypes. Such a considerable change in concentration of sugars could not be explained only by their involvement in metabolism and by activation of alternative respiratory pathway [25]. The present study indicates that a comparatively high freezing resistance of potato plants is related to enrichment of their tissues with sugars. In cold-sensitive plants the protection against oxidative stress is related to high activity of antioxidative enzymes. At variance with this, the background SOD activity is sta-
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CHO
HOCH C
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HCOH
H OH
HCOH
OH
O C O
H H OH OH
Lactones
CHO Stable aldehydes, ketones and acids
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Fig. 5. Schematic drawing of possible radical reactions between ROS and soluble carbohydrates (with glucose as an example). Oxidized carbon atoms are indicated by arrows.
bilized in potato as a typical cold-tolerant species. Under these conditions, low-molecular-weight ROS scavengers, to which soluble carbohydrates presumably belong, play more significant role in the protection against oxidative stress. ACKNOWLEDGMENTS The work was supported by the Russian Foundation for Basic Research, project no. 07-04-00601. REFERENCES 1. Kuzniak, E., Transgenic Plants: An Insight into Oxidative Stress Tolerance Mechanisms, Acta Physiol. Plant., 2002, vol. 24, pp. 97–113. 2. Scandalios, J.G., Oxygen Stress and Superoxide Dismutases, Plant Physiol., 1993, vol. 101, pp. 7–12. 3. Lukatkin, A.S., Initiation and Development of Chilling Injury in Leaves of Chilling-Sensitive Plants, Russ. J. Plant Physiol., 2005, vol. 52, pp. 542–546. 4. Chen, P.M., Burke, M.J., and Li, P.H., The Frost Hardiness of Several Solanum Species in Relation to the Freezing of Water, Melting Point Depression and Tissue Water Content, Bot. Gaz. (Chicago), 1976, vol. 137, pp. 313–317. 5. Trunova, T.I., Rastenie i nizkotemperaturnyi stress. 64-e Timiryazevskoe chtenie (Plant and Low-Temperature Stress, the 64th Timiryazev Lecture), Moscow: Nauka, 2007. 6. Deryabin, A.N., Trunova, T.I., Dubinina, I.M., Burakhanova, E.A., Sabel’nikova, E.P., Krylova, E.M., and Romanov, G.A., Chilling Tolerance of Potato Plants Transformed with a Yeast-Derived Invertase Gene under the Control of the B33 Patatin Promoter, Russ. J. Plant Physiol., 2003, vol. 50, pp. 449–454. RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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