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Antioxidative Enzymes Offer Protection from Chilling Damage in Rice Plants

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Antioxidative Enzymes Offer Protection from Chilling Damage in Rice Plants
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  Antioxidative Enzymes Offer Protection from Chilling Damage in Rice Plants Yong In Kuk,* Ji San Shin, Nilda R. Burgos, Tay Eak Hwang, Oksoo Han, Baik Ho Cho,Sunyo Jung, and Ja Ock Guh ABSTRACT  the spring. This results in chilling injury to rice cropsand sometimes to freezing injury due to spring frosts or Rice ( Oryza sativa  L.) is a tropical crop, but is also grown in occasional night freezing temperatures. Also, rice crops temperateregionsinlatespringtosummer.Coldtemperaturedamageis a common problem for early-planted rice in temperate countries.  planted very early in the USA (i.e., March) grow very Physiological responses to chilling, including antioxidative enzyme  slowly and will eventually mature at the same time as activity, were investigated in rice to identify mechanisms of chilling rice planted in mid-April to early May. If chilling injury tolerance. Plants were exposed to 15  C (cold-acclimated) or 25  C could be avoided and early-planted rice would develop (nonacclimated) for 3 d, under 250   mol m  2 s  1 photosynthetically within the same timeframeasrice planted in late spring, active radiation (PAR). All plants were then exposed to chilling tem- alternative cropping systems for rice would be possible perature at 5  C for 3 d and allowed to recover at 25  C for 5 d. Leaf  and the efficiency of using farm resources could be im- fresh weight, relative water content, lipid peroxidation, chlorophyll a proved. Understanding the physiological and biochemi- fluorescence, and quantum yield showed that cold-acclimated leaves cal mechanisms involved in cold tolerance would help werelessaffectedbychillingcomparedtononacclimatedleaves.Cold-acclimated leaves also recovered faster from chilling injury than non-  us improve cold tolerance in rice plants. acclimated leaves. We analyzed the isozyme profile and activity of   Various mechanisms have been suggested to account superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase for chilling injury or tolerance in plants (Basra, 2001). (APX), and glutathione reductase (GR). Significant induction of ex- There is increasing evidence that chilling causes ele- pression and activity of antioxidative enzymes CAT and APX in vated levels of active oxygen species (AOS), which con- leaves and SOD, CAT, APX, and GR in roots were observed. We tribute significantly to chilling damage (Omran, 1980; deduced that CAT and APX are most important for cold acclimation Prasad et al., 1994; Wise and Naylor, 1987b). AOS such and chilling tolerance. Increased activity of antioxidants in roots is as superoxide (O  2  ), hydrogen peroxide (H 2 O 2 ), hy- more important for cold tolerance than increased activity in shoots. droxylradicals(OH·),andsingletoxygen( 1 O 2 ),arepres- Chilling-sensitivericeplantscanbemadetolerantbycoldacclimation. ent in plants at various levels as a result of normalaerobic metabolism. Plants have evolved antioxidantsystems to protect cellular membranes and organelles F ood crops  of tropical and subtropical srcins suchfromdamagingeffectsofAOS(Foyeretal.,1991).Anti-asrice ( Oryzasativa L.), corn( Zeamays L.), tomatooxidant enzymes, such as superoxide dismutase (SOD,( Lycopersicon esculentum  Mill.), and soybean [ Glycine EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), and various max  (L.) Merr.] are now cultivated in areas where tem-peroxidases such as guaiacol peroxidase (POX, ECperatures fall well below the optimum required for their1.11.17) and ascorbate peroxidase (APX, EC 1.11.1.11)normal growth and development. Of these crops, ricecan react with, and neutralize, the activity of AOSis most susceptible to chilling temperatures (DeDatta,(Foyer et al., 1991; Lee and Lee, 2000; Oidaira et al.,1981). Indica rice is known to be more sensitive to pho-2000; Omran, 1980; Prasad, 1996, 1997; Scandalios,toinhibition at chilling temperature than Japonica rice1993). In conjunction with these enzymes, antioxidant(Hetherington et al., 1989). Many attempts have beencompounds such as ascorbate, glutathione,   -carotene,made to improve cold tolerance in plants. One of theand   -tocopherol also play important roles in the re-methods tested is cold acclimation. It is now known thatexposure of chilling-sensitive plants, such as maize and moval of toxic oxygen compounds (Hodges et al., 1996;tomato, to temperatures slightly above chilling reduces Wise and Naylor, 1987a).chilling injury (Anderson et al., 1995; Gilmour et al., Cold acclimation increases tolerance to AOS in cere-1988; Leipner et al., 1997; Prasad et al., 1995; Prasad, als and correlates with an increase in antioxidant en-1996; Scebba et al., 1999; Venema et al., 2000). Cold zymes (Anderson et al., 1995; Scebba et al., 1998, 1999).acclimationinriceplantshasnotyetbeenstudied.Inthe In chilling-sensitive plants, the ability to defend againstUSA,riceplantingisbeingpushedasearlyaspossiblein oxidativedamageisinhibitedbythereductioninexpres-sion of antioxidants such as ascorbate, glutathione, and  -tocopherol(WiseandNaylor,1987a),CAT(Fadzillah Y.I. Kuk and S. Jung, Biotechnology Research Institute, Chonnam et al., 1996; Omran, 1980), and SOD (Michalski and National Univ., Gwangju 500-757, Korea; J.S. Shin, T.E. Hwang, B.H.Cho, and J.O. Guh, Faculty of Applied Plant Science, Chonnam Na-  Kaniuga, 1982). Chilling tolerance improved when GSH, tional Univ., Gwangju 500-757, Korea; N.R. Burgos, Department of  peroxidase,andCATlevelswereenhanced(Upadhyaya Crop, Soil, and Environmental Sciences, Univ. of Arkansas, 1366 W. et al., 1989). Thus, it is important to determine the Altheimer Drive, Fayetteville, AR, USA 72704; O. Han, Departmentof Genetic Engineering, Chonnam National Univ., Gwangju 500-757,Korea.Received27May2003.*Correspondingauthor(yikuk@chonnam. Abbreviations:  AOS, active oxygen species; APX, ascorbate peroxi-ac.kr).dase; CAT, catalase; Chl, chlorophyll; GR, glutathione reductase;GSH, reduced glutathione; GSSG, oxidized glutathione; PAR, photo-Published in Crop Sci. 43:2109–2117 (2003). ©  Crop Science Society of America synthetically active radiation; PS II, photosystem II; RWC, relativewater content; SOD, superoxide dismutase.677 S. Segoe Rd., Madison, WI 53711 USA 2109  2110  CROP SCIENCE, VOL. 43, NOVEMBER–DECEMBER 2003 MDA(  mol/g freshwt.)  [(A 532  A 600 )/156]  10 3  dilution activity of various antioxidants during acclimation and factor (Zhanyuan and Bramlage, 1992). chilling to assess their contribution to chilling tolerance.The objectives of this research are to compare the Chl a Fluorescence and Quantum physiological responses of cold-acclimated and nonac- Yield Measurements climated rice seedlings to chilling and their capabilitytorecoverfromchillinginjury.Wealsoaimtodetermine  In vivo Chl a fluorescence was measured at room tempera- whether AOS-scavenging enzymes play a role in rice  ture with a pulse amplitude modulation fluorometer (PAM-2000, Walz, Effeltrich, Germany). Before measuring fluores- tolerance to chilling stress. cence, leaves were adapted in darkness for 5 min to minimizefluorescence quenching associated with thylakoid membrane MATERIALS AND METHODS energization (Krause et al., 1982). Minimal fluorescence yield,F 0 , was obtained on excitation of the leaves with a weak mea- Plant Growth and Treatment Conditions suring beam of 0.12  mol m  2 s  1 from a pulsed light-emittingSeeds of chilling-sensitive Indica rice ‘Taebaekbyeo’ werediode. Maximal fluorescence yield, F m , was determined aftersoaked in water for 4 d at 25  C, sown in commercial pottingexposuretoasaturatingpulseofwhitelighttocloseallreactionmix (peat moss, vermiculite, and zeolite), and placed in thecenters.Theratioofvariabletomaximumfluorescence(F v /F m )greenhouse at 30/25  C (day/night) temperature. At 8 d afterderived from the measurement was used as a measure of theseeding, roots of seedlings were washed with distilled watermaximum photochemical efficiency of photosystem II (PS II).and the seedlings were transferred to containers (48 by 32 byThe quantum yield of electron transport through PS II (Y   7 cm) with half-strength Hoagland’s nutrient solution. The  F/F  m ) was calculated according to Genty et al. (1989).plants were grown at 30/25  C (day/night) temperature, 70%relative humidity, with a 14-h photoperiod under fluorescent Protein Extraction white light (250   mol m  2 s  1 ) in a growth chamber. At theFrozen leaves or roots (0.5 g) were pulverized in liquid N 2 three-leaf stage, seedlings were placed at 15  C (cold-accli-using a mortar and pestle and then resuspended in 3 mL of mated) or 25  C (nonacclimated) for 3 d under a 14-h photope-100m M  potassiumphosphatebuffer(pH7.5)containing2m M  riod.Lightperiodstartedat0600h.Theacclimatedandnonac-ethylenediaminetetraaceticacid(EDTA),1%(w/v)polyvinyl-climated seedlings were then exposed to chilling at 5  C forpyrrolidone (PVP-40), and 1 m M   phenylmethylsulfonyl fluo-3 d and allowed to recover for 5 d at 25  C. For the evaluationride (PMSF). For APX assay, the extraction buffer also con-of various parameters, three plants were harvested daily fromtained 5 m M   ascorbate. The suspension was centrifuged ateach treatment, 7 h after the onset of light period, from the15 000    g  for 20 min at 4  C. The supernatant was directlystart of low temperature acclimation to the fifth day of recov-used for assay of CAT, APX, and glutathione reductase (GR,ery. Acclimation and stress treatments were also imposed 7 hEC 1.6.5.4). For the SOD assay, the supernatant was passedafter the onset of light period. Harvesting was done at thethrough a Sephadex G-25  M   minicolumn (PD-10; Pharmacia,sametimeeachdaytoavoidcomplicationsfromdiurnalfluctu-Uppsala, Sweden) with 100 m M   potassium phosphate elutionations in plant biochemical processes.buffer (pH 7.5) at 4  C to remove low molecular weight inhibi-tors. Protein concentration was determined by the method of  Evaluation of Chilling Injury Bradford (1976) with bovine serum albumin as standard.Chilling injury on leaves was evaluated by changes in rela-tivewatercontent(RWC)andamountofchlorophyll.Relative Enzyme Assays watercontentwascalculatedusingtheformula(1  dryweightSOD activity was determined by the procedure of Spichallaof leaf/fresh weight of leaf)    100. Chlorophyll was extractedandDesborogh(1990).Thereactionmixturecontained50m M  and assayed according to the procedure of Hiscox and Isra-Na 2 CO 3 /NaHCO 3  buffer (pH 10.2), 0.1 m M   EDTA, 0.015 m M  elstam (1979). Leaves of rice seedlings (0.1 g) from each har-ferricytochrome C, and 0.05 m M   xanthine. The reaction wasvest were soaked in 10 mL dimethyl sulfoxide for 48 h ininitiated by addition of sufficient xanthine oxidase to producedarkness at room temperature. Total chlorophyll content ina basal rate of ferricytochrome C reduction corresponding toextracts was determined spectrophotometrically. Chlorophyllan increase in absorbance at 550 nm of 0.025 units/min (V 1 ).content (mg/g dry weight) was calculated by means of theAfter V 1  was established, the protein extract was added andfollowing formula: (20.2    A 645    8.02    A 663 )    dilutionthe resulting velocity (V 2 ) was calculated. One unit of SODfactor (Hiscox and Israelstam, 1979).was defined as the amount of enzyme that inhibited the rateof ferricytochrome C reduction by 50% (V 1 /V 2  2) in a 1-mL Lipid Peroxidation assay volume. CAT activity was assayed by the method of Mishra et al. (1993). The reaction mixture contained 50 m M  Lipid peroxidation was estimated by the level of malondial-dehyde (MDA) production by a slight modification of the thio- potassium phosphate buffer (pH 7.0), 11 m M   H 2 O 2 , and thecrude enzyme extract. The reaction was initiated by additionbarbituric acid (TBA) method described by Buege and Aust(1978). Rice leaves (0.1 g) were harvested and homogenized of H 2 O 2  to the mixture and enzyme activity was determinedby monitoring the decline in absorbance at 240 nm ( ε  36  M  with a mortar and pestle in 5 mL of 0.5% (v/v) TBA solutionin 20% (v/v) trichloroacetic acid. The homogenate was centri- cm  1 ) because of H 2 O 2  consumption. APX activity was deter-mined by monitoring the decline in absorbance at 290 nm asfuged at 20 000   g for 15 min and the supernatant was heatedin a boiling water bath for 25 min and allowed to cool in an ascorbate ( ε    2.8 m M   cm  1 ) was oxidized, by the methodof Chen and Asada (1989). The reaction mixture containedice bath. The supernatant was centrifuged at 20 000    g  for15min,andtheresultingsupernatant wasusedforspectropho- 100 m M   potassium phosphate buffer (pH 7.5), 0.5 m M   ascor-bate, and 0.2 m M   H 2 O 2 . GR activity was determined by moni-tometric determination of MDA. Absorbance at 532 nm wasrecorded and corrected for nonspecific absorbance at 600 nm. toring the decline in absorbance at 340 nm as NADPH ( ε   6.2 m M   cm  1 ) was oxidized (Rao et al., 1996). The reactionMDA concentrations were calculated by means of an extinc-tion coefficient of 156 m M   1 cm  1 and the following formula: mixture contained 100 m M   potassium phosphate buffer (pH  KUK ET AL.: PROTECTION FROM CHILLING DAMAGE IN RICE PLANTS  2111 7.8), 2 m M   EDTA, 0.2 m M   NADPH, and 0.5 m M   oxidizedglutathione (GSSG). The reaction was initiated by additionof GSSG. Native PAGE and Visualization Isoforms of CAT, SOD, APX, and GR were separatedon nondenaturating polyacrylamide gels by the procedure of Laemmli (1970) with modifications. Equal amounts of proteinextracts were mixed with bromophenol blue and glycerol toa final concentration of 12.5% (v/v) and loaded on 7% T and3% C (CAT) or 10% T and 3% C (SOD, APX, and GR)polyacrylamide gels. Gel electrophoresis was done at 4  C for3 h with a constant current of 30 mA. For APX, however,2 m M   ascorbate was added to the electrode buffer and thegel was prerun for 30 min before the sample was loaded (Mit-tler 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 asolution containing 2.5 m M   nitroblue tetrazolium in darkness,followed by incubation in 50 m M   potassium phosphate buffer(pH 7.8) containing 28   M   riboflavin and 28 m M   tetramethylethylene diamine (TEMED) in darkness for 20 min. The gelswere then exposed to dim light for 25 min at room tempera-ture. In some experiments, the gels were incubated in 50 m M  potassium phosphate buffer (pH 7.8) containing 3 m M   KCNor 5 m M   H 2 O 2  for 30 min before staining for SOD to visualizeKCN- and H 2 O 2 –sensitive isoforms (Britton et al., 1978). Tovisualize CAT profile, gels were stained by the procedure of Anderson et al. (1995). The gels were soaked in 3.27 m M  H 2 O 2  for 25 min, rinsed twice in distilled water, and stainedin a freshly prepared solution containing 1% (w/v) potassiumferricyanide and 1% (w/v) ferric chloride. Isoforms of APXwere visualized by incubating the gels for 30 min in 50 m M  potassium phosphate buffer (pH 7.0) containing 2 m M   ascor-bate (Rao et al., 1996). The gels were then incubated in thesame buffer containing 4 m M   ascorbate and 2 m M   H 2 O 2  for20min,andthensoakedin50m M  potassiumphosphate buffer(pH 7.8) containing 28 m M   TEMED and 2.45 m M   nitrobluetetrazolium 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-dimethyl-thiazol-2-4)-2,5-diphenyl tetrazolium bromide, 10 mg of 2,6-dichlorophenolindophenol, 3.4 m M   GSSG, and 0.5 m M  NADPH in darkness for 1 h (Rao et al., 1996). The stainingreaction was stopped by adding 7.5% (v/v) glacial acetic acidto the staining buffer. RESULTSPhysiological Responses to Chilling Typical symptoms of chilling injury are wilting, yel-lowing of leaves, and inhibition of growth. Leaf freshweightsofcold-acclimatedandnonacclimated riceseed- Fig. 1. Changes in (A) leaf fresh weight, (B) relative water content, ling were similar within 3 d of acclimation (Fig. 1A).  (C) chlorophyll content, and (D) lipid peroxidation in cold-accli-mated and nonacclimated rice leaves during acclimation, chilling, Leaf fresh weight of nonacclimated plants rapidly de- and recovery. The plants were exposed to 15  C (cold acclimated) clined when exposed to chilling temperature and plants or 25  C (nonacclimated) for 3 d (acclimation), chilled for 3 d at did not recover from the chilling treatment. Leaf fresh 5  C (chilling), and allowed to recover for 5 d at 25  C (recovery). weight of cold-acclimated plants also declined signifi- Values are the mean    SE of three replicates. In some cases, the cantly 1 d after exposure to chilling temperature. Al-  error bar is obscured by the symbol. though the cold-acclimated plants did not completelyrecover from chilling injury compared with untreated There was no difference in RWC of cold-acclimatedand nonacclimated rice leaves during the acclimationcontrol plants, the cold-acclimated plants showed a gen-eral increase in leaf fresh weight compared with nonac- period (Fig. 1B). The RWC of all plants declined duringchilling, but RWC of cold-acclimated rice leaves wasclimated plants during the recovery period.  2112  CROP SCIENCE, VOL. 43, NOVEMBER–DECEMBER 2003 less affected by chilling than nonacclimated leaves. TheRWC of plants in neither treatment returned to srcinallevels observed before chilling was imposed.The chlorophyll content of cold-acclimated and non-acclimated rice leaves was also the same during theacclimation period (Fig. 1C). Chlorophyll content of cold-acclimated and nonacclimated plants was equallyreduced byexposure to chillingtemperature. Reductionin chlorophyll content continued even when plants werereturnedto25  Cforrecovery. Onthethirddayofrecov-ery, chlorophyll content of cold-acclimated leaves lev-eled off, but that of nonacclimated leaves continued todecline. This indicates that cold-acclimated plants mayrecover while nonacclimated ones will not. Lipid Peroxidation As indicated by the level of MDA production, lipidperoxidation was barely noticeable during the acclima-tion period and the background level was the samebetween cold-acclimated and nonacclimated leaves(Fig. 1D). In nonacclimated leaves, however, lipid per-oxidation occurred during chilling and increased withchilling duration. The level of lipid peroxidation contin-ued to increase 2 d into the recovery period beforestartingtodecline.Ontheotherhand,lipidperoxidationwas negligible in cold-acclimated leaves. Chl a Fluorescence and Quantum Yield Fig. 2. Changes in (A) maximum photochemical efficiency (F v  /F m ) Chl a fluorescence (F v /F m ) in cold-acclimated leaves and (B) quantum yield (  F/F  m ) of photosystem in cold-acclimated was the same as in nonacclimated leaves during the  and nonacclimated rice leaves during acclimation, chilling, andrecovery. The plants were exposed to 15  C (cold acclimated) or acclimation period (Fig. 2A). However, subsequent 25  C (nonacclimated) for 3 d (acclimation), chilled for 3 d at 5  C chilling rapidly reduced Chl a fluorescence in nonaccli- (chilling),andallowedtorecoverfor 5dat25  C(recovery).Values mated leaves, whereas Chl a fluorescence in cold-accli- are the mean    SE of three replicates. In some cases, the error mated leaves was generally unaffected by chilling. Chl  bar is obscured by the symbol. a fluorescence in nonacclimated leaves fell 0 during thefirst 4 d of recovery before rising close to 0.2 on the observed between cold-acclimated and nonacclimatedfifth day of recovery. leaves2and3dafteracclimation(Fig.3C).APXactivitySimilartoChlafluorescence,quantumyieldinnonac- in cold-acclimated leaves was higher than in nonaccli-climated leaves dropped rapidly during the chilling pe- mated leaves toward the later stages of acclimation, 2 driod and was not restored during recovery (Fig. 2B). after chilling, and all throughout the recovery period.However, quantum yield of cold-acclimated leaves was There was generally no difference in GR activity be-reduced slowly during chilling, compared to nonaccli- tween cold-acclimated and nonacclimated leaves duringmatedleaves,and was restored tothe srcinallevelafter acclimation, chilling, and recovery (Fig. 3D).3 d of recovery. Antioxidative Enzymes in RootsAntioxidative Enzymes in Leaves Activities of SOD, CAT, APX, and GR in cold-accli-mated and nonacclimated roots during acclimation andThe baseline levels of antioxidative enzyme activitieswere generally the same between cold-acclimated and subsequent chillingweregenerallythe same(Fig.4A–D).However,rootsofcold-acclimatedplantsshowedhighernonacclimated leaves except for APX activity duringthe acclimation period (Fig. 3C). No differences were activity of antioxidative enzymes during the later phaseof the recovery period compared to roots of nonaccli-found in SOD activity between cold-acclimated andnonacclimated leaves during chilling and recovery pe- mated plants. SOD activity in cold-acclimated roots washigher than in nonacclimated roots 2 and 3 d after accli-riodexceptfor2dafterrecovery(Fig.3A).CATactivityin cold-acclimated and nonacclimated leaves was simi- mation and 1 d after chilling (Fig. 4A). Although SODactivity decreased in roots of all plants during recoverylarly affected by chilling temperature (Fig. 3B). How-ever, CAT activity in cold-acclimated leaves showed regardlessof acclimation,the SOD activity in cold-accli-mated roots was higher than in nonacclimated roots 3significant recovery whereas that of nonacclimatedleaves did not. Significant changes in APX activity was and 5 d after recovery. No significant change in CAT  KUK ET AL.: PROTECTION FROM CHILLING DAMAGE IN RICE PLANTS  2113 Fig. 3. Changes in (A) SOD, (B) CAT, (C) APX, and (D) GR activi-ties in cold-acclimated and nonacclimated rice leaves during accli- Fig. 4. Changes in (A) SOD, (B) CAT, (C) APX, and (D) GR activi-mation, chilling, and recovery. The plants were exposed to 15  C tiesincold-acclimatedandnonacclimatedricerootsduringacclima-(cold acclimated) or 25  C (nonacclimated) for 3 d (acclimation), tion, chilling, and recovery. The plants were exposed to 15  C (coldchilled for 3 d at 5  C (chilling), and allowed to recover for 5 d at acclimated) or 25  C (nonacclimated) for 3 d (acclimation), chilled25  C (recovery). Values are the mean    SE of three replicates. In for 3 d at 5  C (chilling), and allowed to recover for 5 d at 25  Csome cases, the error bar is obscured by the symbol. (recovery). Values are the mean  SE of three replicates. In somecases, the error bar is obscured by the symbol. activitywas observedbetweencold-acclimated andnon-acclimated roots during acclimation and subsequent alsosimilarbetweencold-acclimatedandnonacclimatedroots during acclimation and subsequent chillingchilling (Fig. 4B). However, CAT activity in cold-accli-mated roots was much higher than in nonacclimated (Fig. 4C). However, APX activity in cold-acclimatedroots was much higher than in nonacclimated roots dur-rootsduringrecovery.SimilartoCAT,APXactivitywas
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