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a Biotechnology Research Institute, Chonnam National Univ., Gwangju 500-757, Korea
b Faculty of Applied Plant Science, Chonnam National Univ., Gwangju 500-757, Korea
c Department of Crop, Soil, and Environmental Sciences, Univ. of Arkansas, 1366 W. Altheimer Drive, Fayetteville, AR, USA 72704
d Department of Genetic Engineering, Chonnam National Univ., Gwangju 500-757, Korea
* Corresponding author (yikuk{at}chonnam.ac.kr).
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: AOS, active oxygen species • APX, ascorbate peroxidase • CAT, catalase • Chl, chlorophyll • GR, glutathione reductase • GSH, reduced glutathione • GSSG, oxidized glutathione • PAR, photosynthetically active radiation • PS II, photosystem II • RWC, relative water content • SOD, superoxide dismutase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Various mechanisms have been suggested to account for chilling injury or tolerance in plants (Basra, 2001). There is increasing evidence that chilling causes elevated levels of active oxygen species (AOS), which contribute significantly to chilling damage (Omran, 1980; Prasad et al., 1994; Wise and Naylor, 1987b). AOS such as superoxide 
, hydrogen peroxide (H2O2), hydroxyl radicals (OH·), and singlet oxygen (1O2), are present in plants at various levels as a result of normal aerobic metabolism. Plants have evolved antioxidant systems to protect cellular membranes and organelles from damaging effects of AOS (Foyer et al., 1991). Antioxidant enzymes, such as superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), and various peroxidases such as guaiacol peroxidase (POX, EC 1.11.17) and ascorbate peroxidase (APX, EC 1.11.1.11) can react with, and neutralize, the activity of AOS (Foyer et al., 1991; Lee and Lee, 2000; Oidaira et al., 2000; Omran, 1980; Prasad, 1996, 1997; Scandalios, 1993). In conjunction with these enzymes, antioxidant compounds such as ascorbate, glutathione, ß-carotene, and 
-tocopherol also play important roles in the removal of toxic oxygen compounds (Hodges et al., 1996; Wise and Naylor, 1987a).
Cold acclimation increases tolerance to AOS in cereals and correlates with an increase in antioxidant enzymes (Anderson et al., 1995; Scebba et al., 1998, 1999). In chilling-sensitive plants, the ability to defend against oxidative damage is inhibited by the reduction in expression of antioxidants such as ascorbate, glutathione, and -tocopherol (Wise and Naylor, 1987a), CAT (Fadzillah et al., 1996; Omran, 1980), and SOD (Michalski and Kaniuga, 1982). Chilling tolerance improved when GSH, peroxidase, and CAT levels were enhanced (Upadhyaya et al., 1989). Thus, it is important to determine the activity of various antioxidants during acclimation and chilling to assess their contribution to chilling tolerance.
The objectives of this research are to compare the physiological responses of cold-acclimated and nonacclimated rice seedlings to chilling and their capability to recover from chilling injury. We also aim to determine whether AOS-scavenging enzymes play a role in rice tolerance to chilling stress.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Evaluation of Chilling Injury
Chilling injury on leaves was evaluated by changes in relative water content (RWC) and amount of chlorophyll. Relative water content was calculated using the formula (1 - dry weight of leaf/fresh weight of leaf) x 100. Chlorophyll was extracted and assayed according to the procedure of Hiscox and Israelstam (1979). Leaves of rice seedlings (0.1 g) from each harvest were soaked in 10 mL dimethyl sulfoxide for 48 h in darkness at room temperature. Total chlorophyll content in extracts was determined spectrophotometrically. Chlorophyll content (mg/g dry weight) was calculated by means of the following formula: (20.2 x A645 + 8.02 x A663) x dilution factor (Hiscox and Israelstam, 1979).
Lipid Peroxidation
Lipid peroxidation was estimated by the level of malondialdehyde (MDA) production by a slight modification of the thiobarbituric acid (TBA) method described by Buege and Aust (1978). Rice leaves (0.1 g) were harvested and homogenized with a mortar and pestle in 5 mL of 0.5% (v/v) TBA solution in 20% (v/v) trichloroacetic acid. The homogenate was centrifuged at 20 000 x g for 15 min and the supernatant was heated in a boiling water bath for 25 min and allowed to cool in an ice bath. The supernatant was centrifuged at 20000 x g for 15 min, and the resulting supernatant was used for spectrophotometric determination of MDA. Absorbance at 532 nm was recorded and corrected for nonspecific absorbance at 600 nm. MDA concentrations were calculated by means of an extinction coefficient of 156 mM-1 cm-1 and the following formula: MDA (µmol/g fresh wt.) = [(A532 - A600)/156] x 103 x dilution factor (Zhanyuan and Bramlage, 1992).
Chl a Fluorescence and Quantum Yield Measurements
In vivo Chl a fluorescence was measured at room temperature with a pulse amplitude modulation fluorometer (PAM-2000, Walz, Effeltrich, Germany). Before measuring fluorescence, leaves were adapted in darkness for 5 min to minimize fluorescence quenching associated with thylakoid membrane energization (Krause et al., 1982). Minimal fluorescence yield, F0, was obtained on excitation of the leaves with a weak measuring beam of 0.12 µmol m-2 s-1 from a pulsed light-emitting diode. Maximal fluorescence yield, Fm, was determined after exposure to a saturating pulse of white light to close all reaction centers. The ratio of variable to maximum fluorescence (Fv/Fm) derived from the measurement was used as a measure of the maximum photochemical efficiency of photosystem II (PS II). The quantum yield of electron transport through PS II 
was calculated according to Genty et al. (1989).
Protein Extraction
Frozen leaves or roots (0.5 g) were pulverized in liquid N2 using a mortar and pestle and then resuspended in 3 mL of 100 mM potassium phosphate buffer (pH 7.5) containing 2 mM ethylenediaminetetraacetic acid (EDTA), 1% (w/v) polyvinylpyrrolidone (PVP-40), and 1 mM phenylmethylsulfonyl fluoride (PMSF). For APX assay, the extraction buffer also contained 5 mM ascorbate. The suspension was centrifuged at 15000 x g for 20 min at 4°C. The supernatant was directly used for assay of CAT, APX, and glutathione reductase (GR, EC 1.6.5.4). For the SOD assay, the supernatant was passed through a Sephadex G-25 M minicolumn (PD-10; Pharmacia, Uppsala, Sweden) with 100 mM potassium phosphate elution buffer (pH 7.5) at 4°C to remove low molecular weight inhibitors. Protein concentration was determined by the method of Bradford (1976) with bovine serum albumin as standard.
Enzyme Assays
SOD activity was determined by the procedure of Spichalla and Desborogh (1990). The reaction mixture contained 50 mM Na2CO3/NaHCO3 buffer (pH 10.2), 0.1 mM EDTA, 0.015 mM ferricytochrome C, and 0.05 mM xanthine. The reaction was initiated by addition of sufficient xanthine oxidase to produce a basal rate of ferricytochrome C reduction corresponding to an increase in absorbance at 550 nm of 0.025 units/min (V1). After V1 was established, the protein extract was added and the resulting velocity (V2) was calculated. One unit of SOD was defined as the amount of enzyme that inhibited the rate of ferricytochrome C reduction by 50% (V1/V2 = 2) in a 1-mL assay volume. CAT activity was assayed by the method of Mishra et al. (1993). The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 11 mM H2O2, and the crude enzyme extract. The reaction was initiated by addition of H2O2 to the mixture and enzyme activity was determined by monitoring the decline in absorbance at 240 nm (
= 36 M cm-1) because of H2O2 consumption. APX activity was determined by monitoring the decline in absorbance at 290 nm as ascorbate ( = 2.8 mM cm-1) was oxidized, by the method of Chen and Asada (1989). The reaction mixture contained 100 mM potassium phosphate buffer (pH 7.5), 0.5 mM ascorbate, and 0.2 mM H2O2. GR activity was determined by monitoring the decline in absorbance at 340 nm as NADPH ( = 6.2 mM cm-1) was oxidized (Rao et al., 1996). The reaction mixture contained 100 mM potassium phosphate buffer (pH 7.8), 2 mM EDTA, 0.2 mM NADPH, and 0.5 mM oxidized glutathione (GSSG). The reaction was initiated by addition of GSSG.
Native PAGE and Visualization
Isoforms of CAT, SOD, APX, and GR were separated on nondenaturating polyacrylamide gels by the procedure of Laemmli (1970) with modifications. Equal amounts of protein extracts were mixed with bromophenol blue and glycerol to a final concentration of 12.5% (v/v) and loaded on 7% T and 3% C (CAT) or 10% T and 3% C (SOD, APX, and GR) polyacrylamide gels. Gel electrophoresis was done at 4°C for 3 h with a constant current of 30 mA. For APX, however, 2 mM ascorbate was added to the electrode buffer and the gel was prerun for 30 min before the sample was loaded (Mittler and Zilinskas, 1993).
For SOD, the gel was stained according to the method of Rao et al. (1996). The gels were incubated for 25 min in a solution containing 2.5 mM nitroblue tetrazolium in darkness, followed by incubation in 50 mM potassium phosphate buffer (pH 7.8) containing 28 µM riboflavin and 28 mM tetramethyl ethylene diamine (TEMED) in darkness for 20 min. The gels were then exposed to dim light for 25 min at room temperature. In some experiments, the gels were incubated in 50 mM potassium phosphate buffer (pH 7.8) containing 3 mM KCN or 5 mM H2O2 for 30 min before staining for SOD to visualize KCN- and H2O2–sensitive isoforms (Britton et al., 1978). To visualize CAT profile, gels were stained by the procedure of Anderson et al. (1995). The gels were soaked in 3.27 mM H2O2 for 25 min, rinsed twice in distilled water, and stained in a freshly prepared solution containing 1% (w/v) potassium ferricyanide and 1% (w/v) ferric chloride. Isoforms of APX were visualized by incubating the gels for 30 min in 50 mM potassium phosphate buffer (pH 7.0) containing 2 mM ascorbate (Rao et al., 1996). The gels were then incubated in the same buffer containing 4 mM ascorbate and 2 mM H2O2 for 20 min, and then soaked in 50 mM potassium phosphate buffer (pH 7.8) containing 28 mM TEMED and 2.45 mM nitroblue tetrazolium for 15 min with gentle agitation (Rao et al., 1996). GR was detected by incubating the gels in 50 mL of 0.25 M Tris-HCl buffer (pH 7.5) containing 10 mg of 3-(4,5-dimethylthiazol-2-4)-2,5-diphenyl tetrazolium bromide, 10 mg of 2,6-dichlorophenolindophenol, 3.4 mM GSSG, and 0.5 mM NADPH in darkness for 1 h (Rao et al., 1996). The staining reaction was stopped by adding 7.5% (v/v) glacial acetic acid to the staining buffer.
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RESULTS |
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The chlorophyll content of cold-acclimated and nonacclimated rice leaves was also the same during the acclimation period (Fig. 1C). Chlorophyll content of cold-acclimated and nonacclimated plants was equally reduced by exposure to chilling temperature. Reduction in chlorophyll content continued even when plants were returned to 25°C for recovery. On the third day of recovery, chlorophyll content of cold-acclimated leaves leveled off, but that of nonacclimated leaves continued to decline. This indicates that cold-acclimated plants may recover while nonacclimated ones will not.
Lipid Peroxidation
As indicated by the level of MDA production, lipid peroxidation was barely noticeable during the acclimation period and the background level was the same between cold-acclimated and nonacclimated leaves (Fig. 1D). In nonacclimated leaves, however, lipid peroxidation occurred during chilling and increased with chilling duration. The level of lipid peroxidation continued to increase 2 d into the recovery period before starting to decline. On the other hand, lipid peroxidation was negligible in cold-acclimated leaves.
Chl a Fluorescence and Quantum Yield
Chl a fluorescence (Fv/Fm) in cold-acclimated leaves was the same as in nonacclimated leaves during the acclimation period (Fig. 2A). However, subsequent chilling rapidly reduced Chl a fluorescence in nonacclimated leaves, whereas Chl a fluorescence in cold-acclimated leaves was generally unaffected by chilling. Chl a fluorescence in nonacclimated leaves fell 0 during the first 4 d of recovery before rising close to 0.2 on the fifth day of recovery.
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Antioxidative Enzymes in Leaves
The baseline levels of antioxidative enzyme activities were generally the same between cold-acclimated and nonacclimated leaves except for APX activity during the acclimation period (Fig. 3C). No differences were found in SOD activity between cold-acclimated and nonacclimated leaves during chilling and recovery period except for 2 d after recovery (Fig. 3A). CAT activity in cold-acclimated and nonacclimated leaves was similarly affected by chilling temperature (Fig. 3B). However, CAT activity in cold-acclimated leaves showed significant recovery whereas that of nonacclimated leaves did not. Significant changes in APX activity was observed between cold-acclimated and nonacclimated leaves 2 and 3 d after acclimation (Fig. 3C). APX activity in cold-acclimated leaves was higher than in nonacclimated leaves toward the later stages of acclimation, 2 d after chilling, and all throughout the recovery period. There was generally no difference in GR activity between cold-acclimated and nonacclimated leaves during acclimation, chilling, and recovery (Fig. 3D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The earliest symptom of chilling stress is damage to the photosynthetic apparatus as evidenced by reduced CO2 fixation and altered chlorophyll a fluorescence patterns (Walker et al., 1991). This means that the most immediate effect of chilling is injury from photooxidation and reduced photosynthetic efficiency. The negative effect of cold treatment on photosynthetic capacity and chlorophyll content may be due to changes in lipid and protein components of the thylakoid membrane (Mostowska, 1997). For instance, exposing pea (Pisum sativum L.) to a chilling temperature (5°C) caused ultrastructural damage to the inner membrane of chloroplasts (Wise and Naylor, 1987b). Because of chloroplast damage, cold stress will result in loss of chlorophyll, and peroxidation of lipids will result in damage to cell membranes causing leakage of cell contents and loss of water (Koscielnak, 1993; Wilson, 1976). Therefore, visual symptoms of chilling damage include a waterlogged appearance, yellowing, wilting, inhibition of plant growth, and acceleration of senescence (Salveit and Morris, 1990).
Rice plants exposed to chilling temperature exhibited all the aforementioned symptoms; however, cold-acclimated plants showed higher tolerance to chilling stress than nonacclimated leaves. Cold-acclimated plants generally still showed similar level of injury as nonacclimated plants, but cold-acclimated plants showed the capability to recover from chilling injury. This was indicated by a positive slope of leaf fresh weight during recovery; the minimal impact of chilling on RWC, minimal lipid peroxidation, and high photosynthetic efficiency of cold-acclimated leaves; and the full recovery of carbon fixation capability of cold-acclimated plants after chilling. Although photosynthesis rates were not directly measured in this experiment, flourescence and quantum yield data are direct indicators of photosynthetic activity. When chlorophyll fluorescence and quantum yield were reduced, we concluded that photosynthesis activity of the plant was also reduced. Cold-acclimated leaves also recovered faster than nonacclimated leaves. These results suggest that cold-acclimated plants reduce oxidative damage during chilling; therefore, allowing the plant to recover from injury. A similar phenomenon was demonstrated in other cold-sensitive species, such as maize, tomato, and Arabidopsis thaliana (L.) Heynh. (Gilmour et al., 1988; Leipner et al., 1997; Venema et al., 2000).
Under optimal environmental conditions, light reaction and electron transport in photosynthesis leads to minimal production of highly reactive oxygen species, which consequently, causes some photooxidative damage to chloroplasts, carotenoids, and proteins. It is known that AOS trigger a series of deleterious processes, such as lipid peroxidation, degradation of proteins, and DNA damage in the cell (Fridovich, 1978; Halliwell and Gutteridge, 1986; Salveit and Morris, 1990; Scandalios, 1993). Plants have developed a system of enzymatic and nonenzymatic defense against oxygen radicals (Hideg, 1997). Plants also mitigate photooxidative damage through natural protectants. Under normal conditions, the antioxidant system is usually sufficient to prevent damage (Hideg, 1997). However, stressful environmental conditions impair the plant's natural capacity to dissipate excess light energy and scavenge oxygen radicals. One way of coping with stress is increasing the level of stress protectants. It was reported years ago that cold tolerance could be induced (Weiser, 1970). Decades later, many cold-inducible proteins and enzymes have been identified (Guy, 1990; Thomashow, 1990). Among these cold-inducible enzymes are the antioxidants, SOD, CAT, APX, and GR.
Lipid peroxidation, as a measure of cellular injury from chilling temperature, increased by over two-fold in nonacclimated leaves compared to cold-acclimated ones during chilling and recovery (Fig. 1D). This indicates that chilling conditions caused damage to cell membranes, which resulted in leakage of cell contents and significant water loss from leaves of nonacclimated rice plants. In maize, lipid peroxidation also increased by about two-fold in nonacclimated seedlings, compared to control or cold-acclimated seedlings, during and after chilling (Prasad, 1996). Reduced lipid peroxidation in cold-acclimated maize seedlings was due to an increased antioxidant defense system that scavenged AOS during or after chilling stress (Prasad, 1996).
High SOD activity has been associated with stress tolerance in plants because it neutralizes the reactivity of O-2, which is overproduced under stress (Bowler et al., 1992). SOD activity in cold-acclimated spinach (Spinacia oleracea L.) and wheat plants was higher compared to nonacclimated plants during exposure to low temperature (Scebba et al., 1999; Schöner and Krause, 1990). The same pattern was observed in rice plants, but only in roots (Fig. 4A) as SOD activity in leaves during chilling was not affected by cold acclimation (Fig. 3A). This is similar to what was observed in maize wherein there was no chilling response in SOD activity in mesocotyls of dark-grown seedlings (Prasad et al., 1994). It appears that in rice, induction of SOD activity in response to chilling is localized in roots. This is important for chilling tolerance because in order for rice plants to grow at low temperatures, root growth has to occur. Roots need protection from oxidative damage from cold stress. We detected two isozymes of SOD in rice leaves, and neither one was affected by cold acclimation (Fig. 5A). In spinach, new SOD isozymes were induced by cold acclimation (Bowler et al., 1992).
Two CAT isoforms were observed in rice plants (Fig. 5B). Since equal amounts of protein extracts were electrophoresed in a nondenaturing gel, the band intensity equates to enzyme activity. The activity of CAT isoform 2 in nonacclimated leaves was not present during chilling and did not recur during recovery, whereas that of cold-acclimated leaves did (Fig. 5B). In our system, higher CAT activity in both leaves and roots and high activity of CAT isozymes in cold-acclimated leaves suggests a more efficient scavenging of H2O2, which would result in better protection against this toxic molecule during chilling and recovery. In comparison to our observation, activity of CAT in Arabidopsis was either not affected by chilling temperature (O'Kane et al., 1996) or surpassed that of the control plants (Anderson et al., 1995). Other researchers have reported reduced activity of CAT in plants exposed to low temperature and bright light due to photoinactivation of CAT (Feierabend et al., 1992).
The important role of APX in relation to increased oxidative tolerance has been reported in many plants (Feierabend et al., 1992; Gupta et al., 1993; Lee and Lee, 2000; O'Kane et al., 1996). In this study, significant change in APX activity in leaves and roots was observed during acclimation and subsequent chilling and recovery between cold-acclimated and nonacclimated leaves (Fig. 3C, 4C). That is, APX activity was higher in cold-acclimated leaves and roots than in nonacclimated leaves and roots during acclimation and subsequent chilling and recovery. There were four APX isozymes and their activities were higher in cold-acclimated leaves than in nonacclimated leaves during recovery (Fig. 5C). Unlike rice, total APX activity in mesocotyls of dark-grown maize seedling was unaffected by cold acclimation or chilling (Prasad et al., 1994).
It also has been shown that GR is important in protecting many plants from oxidative stress (Aono et al., 1993; Foyer et al., 1991; Guy and Carter, 1984). No differences were observed in activity of GR isozymes between cold-acclimated and nonacclimated leaves at any time during the duration of the study (Fig. 5D). This reflected the fact that the total GR activity was not different between cold-acclimated and nonacclimated leaves (Fig. 3D). However, cold acclimation increased the GR activity in roots compared to that of nonacclimated plants when exposed to chilling temperature.
The aforementioned observations indicate that modes of chilling protection, at least in rice, are tissue specific. It appears that APX and CAT are the antioxidants well suited for protecting rice leaves from chilling injury, whereas SOD and GR are more suitable root protectants. The mechanism of this tissue specificity warrants further investigation. Other researchers reported that GR activity was increased slightly by cold acclimation and examination of GR isozyme profiles revealed three isozymes that were greatly affected by cold acclimation in maize seedlings (Anderson et al., 1995). Exposure to low temperature has also resulted in altered GR isozyme profiles in pea (Edwards et al., 1994) and spinach (Guy and Carter, 1984) that are correlated with cold tolerance.
We showed that protection from chilling injury could be induced in chilling-sensitive rice plants by cold acclimation, and this was partly due to induction of antioxidative enzyme activities including CAT and APX of leaves, and SOD, CAT, APX, and GR of roots. Chilling also invoked expression of different isozymes in cold-acclimated rice than those expressed in nonacclimated plants. These help prevent the accumulation of AOS during chilling stress. However, it should be considered that the mechanism of protection from chilling stress is complex and may involve molecules other than antioxidative enzymes (Badiani et al., 1997; Leipner et al., 1997) or ultrastructural modifications at the cellular level.
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ACKNOWLEDGMENTS |
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Received for publication May 27, 2003.
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REFERENCES |
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pH-dependent quenching. Biochim. Biophys. Acta 723:169–175.-tocopherol content of stored potato tubers. Plant Physiol. 94:1214–1218.Related articles in Crop Science:
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of photosystem in cold-acclimated and nonacclimated rice leaves during acclimation, chilling, and recovery. The plants were exposed to 15°C (cold acclimated) or 25°C (nonacclimated) for 3 d (acclimation), chilled for 3 d at 5°C (chilling), and allowed to recover for 5 d at 25°C (recovery). Values are the mean ± SE of three replicates. In some cases, the error bar is obscured by the symbol.
