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Environmental and Experimental Botany 77 (2012) 108– 116

Contents lists available at SciVerse ScienceDirect

Environmental and Experimental Botany

journa l h o me pa g e: www.elsev ier .com/ locate /envexpbot

roteomic analysis of rice response involved in reduction of grain yield underlevated ozone stress

iroko Sawadaa,1, Setsuko Komatsub, Yohei Nanjob, Nisar Ahmad Khana, Yoshihisa Kohnoa,∗

Environmental Science Research Laboratory, Central Research Institute of Electric Power Industry, 1646 Abiko, Abiko-shi, Chiba 270-1194, JapanNational Institute of Crop Science, 2-1-18, Kannondai, Tsukuba, Ibaraki 305-8518, Japan

r t i c l e i n f o

rticle history:eceived 3 November 2011ccepted 12 November 2011

eywords:lag leafzoneroteomicsice

a b s t r a c t

Ozone threat to crop production is increasing, particularly in Asian countries. Although exposure to ozonesignificantly reduces grain yields in sensitive rice cultivars, it remains unclear what factors cause differ-ent sensitivities to ozone and consequent grain yield reduction. To clarify the ozone-induced metabolicchanges affecting rice yields, proteomic analyses were conducted of flag leaves in three rice cultivars(Kirara 397, Koshihikari, Takanari) showing various levels of yield and injury response under elevatedozone conditions. Ozone reduced grain yields in Kirara 397 and Takanari, but not in Koshihikari. Inozone treated Kirara 397, Koshihikari, and Takanari, respectively, 31, 18, and 11 proteins were differ-ently expressed. Ozone exposure decreased proteins associated with photosynthesis and glycolysis in

ield Kirara 397, but increased them in Koshihikari. Especially, Kirara 397 showed a remarkable decrease inproteins related to photosynthetic electron transport, suggesting that the elevated ozone suppressed thephotosynthetic apparatus in Kirara 397, thereby suppressing yields. However, proteins involved in photo-synthesis and other energy metabolism showed no remarkable decrease in Takanari. These results suggestthat ozone-induced yield reduction in Takanari results from mechanisms other than those inhibiting themetabolic pathway in photosynthesis.

© 2011 Elsevier B.V. All rights reserved.

. Introduction

Ozone is a major phytotoxic air pollutant in the troposphere.ecently, ozone concentration has increased rapidly in Asianeveloping countries. For example, during 1982–2003, the ozoneoncentrations in Beijing, China increased from a daily maximum ofbout 80 �g m−3 (about 40 nl l−1, ppb) to about 250 �g m−3 (about25 ppb) (Shao et al., 2006). Additionally, it was estimated that themission of anthropogenic nitrogen oxides (an ozone precursoras) in Asia under a no-further-control scenario can be expectedo increase by 350% between 1990 and 2020 (Aunan et al., 2000).

The elevated ozone concentration reduces growth and yieldsf crop plants, including rice, which is the most important foodrop in Asia (Kobayashi et al., 1995; Yonekura et al., 2005). Pangt al. (2009) showed varietal differences in rice yield responses

o elevated ozone: a hybrid Indica cultivar and a conventionalaponica cultivar showed 20.6% and 6.3% reduced grain yields,espectively, under ambient ×1.5 ozone concentration. Sawada and

∗ Corresponding author. Tel.: +81 4 7182 1181; fax: +81 4 7183 5061.E-mail address: kohno@criepi.denken.or.jp (Y. Kohno).

1 Present address: Center for Environmental Biology and Ecosystem, Nationalnstitute for Environmental Studies, Tsukuba, 305-8506, Japan.

098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2011.11.009

Kohno (2009) reported that effects of ozone exposure on grainyield depended strongly on differences among rice cultivar: mostJaponica cultivars were almost unaffected by ozone exposure. Themechanism causing varietal differences of ozone-induced yieldreduction should be clarified to alleviate ozone threats to globalrice production.

Although the mechanism of an acute, high-level ozone-inducedleaf injury has been well studied, it remains unclear what fac-tors cause grain yield reductions under long-term, relativelylow ozone stress. In general, chronic ozone exposure causes areduction in photosynthesis and growth, and induces prematurecell senescence. Leaf senescence engenders increased oxidativestress in chloroplasts, and ribulose-1,5-bisphosphate carboxy-lase/oxygenase (RuBisCO) degradation, which reduces yields (Pellet al., 1997; Schraudner et al., 1997). However, our previous reportdescribed that the ozone sensitivity in rice cultivars evaluated interms of visible injury (chlorotic or necrotic lesions) did not coin-cide with that evaluated by the grain yield reduction (Sawadaand Kohno, 2009). Moreover, accelerated leaf senescence by ele-vated ozone caused very little reduction of rice yields (Sawada

and Kohno, 2010). These results suggest that rice plants possessa mechanism controlling grain yield under ozone stress indepen-dent of leaf injury or senescence development. Yield componentsin rice cultivars exposed to ozone revealed that the filled grain

Experimental Botany 77 (2012) 108– 116 109

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Fig. 1. Daily ozone exposure patterns based on the monitoring data from the Center

H. Sawada et al. / Environmental and

ercentage (excluding sterile and unfilled grains) and 1000-graineight representing grain size were markedly decreased, rather

han the panicle number and total number of grains in the culti-ars showing yield reduction (unpublished data). The filled grainercentage and 1000-grain weight are known to be affected byhe potential photosynthetic activity and longevity of green tis-ues during ripening (Yoshida, 1981). The filled grain percentage islso affected by pollen viability. Sarkar and Agrawal (2010) showedhat yield reduction in wheat was affected differentially by losses ofollen and floret viabilities in each cultivar under elevated ozone,nd suggested that the reduced pollen viability was caused not onlyy direct damage to the pollen, but also by inhibiting the pollenevelopment by insufficient accumulation of carbohydrate. There-ore, we hypothesized that ozone stress disturbs the accumulationf carbohydrates by reduced photosynthesis and enhancement ofnergy consumption, and assimilate translocation to panicles dur-ng the grain-filling period.

Proteomic studies of ozone response have been conducted forice (Agrawal et al., 2002; Cho et al., 2008; Feng et al., 2008), beannd maize (Torres et al., 2007), poplar (Bohler et al., 2007), andoybean (Ahsan et al., 2010). These studies have used the leavesf young seedlings, but no proteomic study has been conducted ofzone response in leaves of mature plants or flag leaves, whichre closely related to yield. Additionally, varietal differences ofzone-responsive proteins in cultivars with different sensitivitieso ozone have not been investigated. Comprehensive analysis ofzone stressed plants and identification of altered metabolic activ-ties in ozone-tolerant and ozone-sensitive cultivars will elucidatehe mechanism of ozone-induced yield reduction.

In this study, to clarify the ozone-induced metabolic changesffecting the grain yield of rice, we conducted proteomic analy-es of flag leaves in rice cultivars with differing yield and injuryesponses under elevated ozone conditions during the grain-fillingeriod. Rice cultivars Kirara 397, Koshihikari, and Takanari weresed for this study, since we observed Kirara 397 and Takanarihowed significant yield reductions in response to elevated ozone,ut Koshihikari did not. Moreover, while seedlings of Kirara 397 andoshihikari showed greater leaf injury symptoms, Takanari showed

ess leaf injury under ozone stress (Sawada and Kohno, 2009). Ourypothesis was that yield reduction would be caused by differentechanism between Kirara 397 and Takanari exposed to ozone.

. Material and methods

.1. Plant materials and ozone treatment

Seeds of rice (Oryza sativa L.) cultivars Kirara 397, Koshihikarimodern Japonica cultivars), and Takanari (a hybrid Indica cul-ivar) were sown in seedling boxes and were grown for threeeeks in a glasshouse under ambient air. The seedlings were

ransplanted into pots (0.05 m2 surface area and 0.015 m3) in thelasshouse type open-top chambers (OTCs; 13.0 m2 of growthpace and 2.4 m in height) on 11 May for Kirara 397 and Koshi-ikari, and 25 May for Takanari, 2009. Each cultivar was grown

n five different pots with four plants each. All were arrayed with block design in an OTC. Fertilizer was supplied at a rate of–P2O5–K2O = 150–150–150 kg ha−1. The OTC fumigation systemas been described previously (Frei et al., 2011). The air stream

nto the OTC was passed through charcoal filters and subsequentlyupplemented with ozone at the intended treatment levels. Ozoneas generated by an electrical discharge ozone generator (Oz-24-

A, Ebara Corp., Tokyo, Japan) using oxygen-enriched dry air as the

ource gas. Ozone was treated through the mass flow controllerrom transplanting to harvest. Daily mean ozone concentrationsor 12 h were 6 (charcoal-filtered air, CF) and 73 (twice ambient

for Environmental Science in Saitama, Japan. The ozone exposure pattern of charcoalfiltered air (CF) and twice ambient ozone concentration (O3 ×2) corresponded todaily daytime 12 h mean ozone concentrations of 6 and 73 ppb, respectively.

concentration, O3 ×2) ppb with a regular diurnal pattern (Fig. 1).The designated ozone concentration was derived from the monitor-ing data at the Center for Environmental Science in Saitama duringthe 2000–2007 rice cropping seasons. The average air temperatureand relative humidity in the OTCs from May to October were 21.1 ◦Cand 77.1%, respectively. The two or three flag leaves in one pot ofeach cultivar were collected from three different pots one weekafter heading. Durations of ozone fumigation from transplantingto sampling were 63, 91, and 86 days for Kirara 397, Koshihikari,and Takanari, respectively. They were frozen in liquid nitrogen andthen stored at −80 ◦C until protein extraction.

2.2. Measurement of chlorophyll contents

Relative chlorophyll contents per unit leaf area were determinedin flag leaves treated with CF and O3 ×2 at one week after headingusing a SPAD-502 (Konica Minolta, Osaka, Japan). The SPAD read-ing was taken on one flag leaf per pot. Three SPAD readings weretaken around the midpoint of each leaf blade on one side of themidrib. Calibrations showed that relative SPAD values depend onchlorophyll concentrations (mg g−1 FW) in rice leaves. As observedpreviously in birch, wheat and potato (Uddling et al., 2007), the datain rice fitted well to exponential regression of chlorophyll concen-trations (y) on SPAD values (x) (y = 0.814e0.0345x, r2 = 0.80), while therelationship was slightly weaker than those for birch and wheat.

2.3. Measurement of the yield and yield component

Rice cultivars were harvested on 5 September (Kirara 397), 2October (Koshihikari), and 14 October (Takanari), 2009. The har-vest of each cultivar was conducted when about 80% of the grainsturned yellow. All yield data were determined by measurements of20 individual plants for each treatment. After harvesting, the plantheight and straw weight were determined. Grains were separatedfrom panicles and categorized into two groups using an automaticseed-sorting machine (FV-459A, Fujiwara Seisakusho KK, Tokyo,Japan): filled grain and unfilled grain. The quantities of total grainsand filled grains, filled grain percentage were determined. Filledgrains were unhusked and then weighed to determine the grainyield. The 1000-grain weight was calculated from the number offilled grains and the unhusked rice weight per plant.

2.4. Extraction of soluble protein

Total soluble proteins were extracted from the flag leaves. A por-tion (0.3 g fresh weight) of leaves was ground into a fine powder

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n liquid nitrogen using a chilled mortar and pestle, and trans-erred into 10 ml of acetone containing 10% (w/v) trichloroaceticcid (TCA) and 0.07% (v/v) 2-mercaptoethanol. The mixture wasonicated for 5 min, placed at −20 ◦C for 1 h and then centrifugedt 9000 × g for 20 min at 4 ◦C. The pellet was suspended with 0.5 mlf acetone containing 0.07% 2-mercaptoethanol. The solution waslaced at −20 ◦C for 1 h and then centrifuged at 20,000 × g for

min at 4 ◦C. The pellet was resuspended twice with 1.5 ml of ace-one containing 0.07% 2-mercaptoethanol and centrifuged again asescribed above. The resultant precipitate was dried by evaporationnd resuspended with 0.5 ml of lysis buffer containing 7 M urea, 2 Mhiourea, 5% CHAPS, 0.2 mM tributylphosphine. The protein solu-ion was vortexed for 1 h and centrifuged twice at 20,000 × g atoom temperature for 20 min. The supernatant was subjected tolectrophoresis.

.5. Two-dimensional polyacrylamide gel electrophoresis andmage acquisition

The solubilized proteins were quantified according to the Brad-ord method (Bradford, 1976), and applied to two-dimensionalolyacrylamide gel electrophoresis (2-DE). Total soluble protein400 �g) was mixed with 1% (v/v) Bio-Lyte ampholytes (pH 3–10),nd loaded on to an 11 cm pH 3–10 nonlinear gradient immobilizedH gradient (IPG) strip (ReadyStripTM; Bio-Rad Laboratories Inc.,ercules, CA, USA). The IPG strips were rehydrated at 50 V for 14 h,nd isoelectric focusing (IEF) was conducted at 250 V for 15 min,000 V for 1 h with a linear ramp, and finally 8000 V at 35,000 V hith a rapid ramp using a PROTEAN IEF Cell (Bio-Rad Laborato-

ies Inc.). After IEF, the IPG strips were equilibrated for 15 min inquilibration buffer (375 mM Tris–HCl, pH 8.8, 6 M urea, 20% (v/v)lycerol, and 2% (w/v) SDS) containing 2% (w/v) dithiothreitol (DTT),ith subsequent 15 min immersion in equilibration buffer contain-

ng 2.5% (w/v) iodoacetamide. Then SDS-PAGE was conducted using5% polyacrylamide gel with 5% stacking gel at 35 mA for about 2 h,r until the dye line reached the end of the gel (O’Farrell, 1975). Therotein spots were detected using Coomassie brilliant blue (CBB)taining.

Gel patterns obtained from three individual replicates werecanned using a calibrated densitometer (GS-800; Bio-Rad Labo-atories Inc.). Spots were detected and quantified using softwarePDQuest ver. 8.0.1; Bio-Rad Laboratories Inc.), based on their rel-tive volume. The amount of a protein spot was expressed as theolume of the spot, which was defined as the sum of the inten-ities of all pixels that make up the spot. To compare accuratelyubtle differences in sample loading, gel staining, and destain-ng, the volume of each spot was normalized as a percentage ofhe total volume of all the spots present in the gel. After auto-

ated detection and matching, manual editing was conducted.ignificantly altered spots were identified according to the t-testesults (P < 0.05). Proteins showing at least 1.5-fold differences inxpression values compared with the control were considered asifferentially expressed proteins and were subjected to analysis.

.6. Protein identification

Protein spots were excised from 2-DE gels stained using CBB,nd digested using a DigestPro 96 automated protein digestionystem (Intavis AG; Köln, Germany). Spots placed in 96-welllates were incubated in 50% ACN, followed by washing in 50 mMH4HCO3 for 15 min. Proteins were reduced with 10 mM DTT in0 mM NH4HCO3 for 20 min and alkylated with 40 mM iodoac-

tamide in 50 mM NH4HCO3 for 15 min, then digested with 1 pMrypsin (TPCK treated TypeXIII, Sigma–Aldrich, St. Louis, MO, USA)t 37 ◦C. The resulting peptides were concentrated and desaltedsing NuTip C-18 pipette tips (Glygen Corp., Columbia, MD, USA)

rimental Botany 77 (2012) 108– 116

and analyzed using mass spectrometry (nanoLC–MS/MS) for pro-tein identification.

Tryptic digested spots were injected using an auto samplerinto an Ultimate 3000 nanoLC (Dionex Corp., Germering, Germany)coupled to a nanospray LTQ XL Orbitrap MS (Thermo Fisher Sci-entific Inc., San Jose, CA, USA). Peptides (1 �l) were loaded in 0.1%formic acid onto a 300 �m id × 5 mm C18 PepMap trap column ata 25 �l min−1 flow rate. The peptides were eluted and separatedfrom the trap column using 0.1% formic acid in ACN on a 75 �mid × 12 cm C18 needle column (NTCC-360/75-3, Nikkyo Technos Co.Ltd., Tokyo, Japan) at a flow rate of 0.2 �l min−1. The separatedsample was sprayed with a spray voltage of 1.8 kV. The MS wasoperated in positive ion mode using software (Xcalibur ver. 1.4,Thermo Fisher Scientific Inc.). Data acquisition was set to cover ascan range of m/z 100–2000, followed by three MS/MS scans inexclusion dynamic mode. The running time of the program for spotdetection was set to 60 min.

Tandem mass spectrum DTA (text format) files were con-verted to MASCOT generic format (MGF) files using software(BioWorks ver. 3.3.1, Thermo Fisher Scientific Inc.). They were usedto search for matches in the National Center for BiotechnologyInformation database (NCBI nr) using a search engine (MASCOTver. 2.2.04, Matrix Science Ltd., London, UK). O. sativa (rice) waschosen as the taxonomy. Search parameters used fixed cysteinecarbamidomethylation and variable methionine oxidation as mod-ifications, peptide mass tolerance 10 ppm, MS/MS fragment ions0.2 Da, and one missed cleavage, with trypsin specified as the prote-olytic enzyme. Positive precursor peptide charge states of 1, 2, and3 were specified. To assign a positive match with a known protein,the following criteria were used: coverage of the mature protein bythe matching peptides must reach a minimum of 20%; at least sixindependent peptides should match; and the ion score obtainedfrom analysis with MASCOT software indicates the probabilityof a true positive identification. It must be at least 50. For pep-tides matching multiple members of a protein family, the proteinmatch was selected based on the highest score member of match-ing peptides. Positive matches were BLAST searched against theNCBI protein database (http://www.ncbi.nlm.nih.gov) for updatedannotation and identification of homologous proteins. For someidentified proteins, their sub-cellular information was predictedusing TargetP server (http://www.cbs.dtu.dk/services/TargetP/)(Emanuelsson et al., 2000).

2.7. Statistics

Software (SPSS ver. 11.0J; SPSS Japan Inc.) was used for statisti-cal analyses. To assess the statistical significance of the treatmentdifference, t-tests (with P set at 0.05) were used. Statistical analyseswere made of individual plant data for the yield and yield compo-nents, and chlorophyll content, but of the leaf sample taken fromtwo plants per pot for proteomic analysis. Two independent OTCswere used for each treatment to confirm the reproducibility of theresults.

3. Results and discussion

3.1. Biomass productivity, grain yield, and yield components

Kirara 397 exposed to ozone had significantly less plant heightand lower straw weight (P < 0.05), but those characteristics wereunaffected in Koshihikari and Takanari (Table 1). Numbers of pan-

icles showed no significant difference in any cultivars. Kirara 397and Takanari showed lower filled grain percentage and 1000-grainweight associated with ozone exposure. Grain yields were low-ered to 58% and 81% in Kirara 397 and Takanari, respectively, under

H. Sawada et al. / Environmental and Experimental Botany 77 (2012) 108– 116 111

Table 1Harvest parameters of three rice cultivars grown in charcoal filtered air (CF) and twice ambient ozone concentrations (O3).

Cultivars Plantheight (cm)

Straw weight(g/plant)

Panicle number(n/plant)

Total number ofgrain (n/plant)

Filled grainpercentage (%)

1000-grainweight (g)

Grain yield a

(g/plant)Relative yield (%)

Kirara 397CF 79.4 28.5 9.8 435 91.1 23.0 9.1 100O3 69.3*** 20.5*** 9.1 ns 333*** 73.7*** 20.9*** 5.3*** 58

KoshihikariCF 107.1 38.6 7.1 489 96.7 22.0 10.4 100O3 109.3 ns 36.4 ns 7.2 ns 519 ns 96.6 ns 21.6 ns 10.7 ns 103

TakanariCF 90.8 26.5 4.9 670 93.4 20.5 12.7 100O3 91.2 ns 22.2 ns 4.7 ns 583 ns 89.0** 19.8* 10.3* 81

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s: not significant. Asterisks denote significant differences between CF and O3 treata Grain yield was determined as rough rice grain weight in this report.

levated ozone conditions. The grain yield of Koshihikari was unaf-ected by ozone stress. The much lower grain yield in Kirara 397as partly attributable to decreased total number of grains. In Taka-ari, the total number of grains was 19% lower in ozone exposedlants compared with CF plants, although the difference was notignificant. The yield response of three cultivars under ozone stresss in agreement with description in previous reports (Sawada andohno, 2009).

.2. Effect of ozone exposure on chlorophyll content in flag leaves

Fig. 2 shows SPAD values of flag leaves in rice plants treatedith CF and O3 ×2 at one week after heading. The SPAD values

n flag leaves of Kirara 397 were significantly lower with ozonexposure (P < 0.05). In contrast, those in Koshihikari and Taka-ari were unaffected by elevated ozone. Total leaf chlorophyllontent is an indicator of the degradation of the photosyntheticpparatus (Matile, 2000). This result suggests that photosyntheticpparatus in flag leaves responded differently to ozone betweenirara 397 and Takanari, even though both cultivars showed lowerrain yields because of the ozone exposure. Ozone-tolerant beanultivar had a greater ability to dissipate excess energy via regu-ated and unregulated nonphotochemical quenching, and greaterotential capacities to reduce reactive oxygen species (ROS) than

zone-sensitive cultivar, resulting in less damage in photosyntheticpparatus under ozone stress (Guidi et al., 2010). Koshihikari andakanari might avoid damage of the photosynthetic apparatus byissipating energy and by removing ROS.

ig. 2. SPAD value of flag leaf in rice plants exposed to charcoal filtered air (CF)nd twice ambient ozone concentration (O3 ×2) at one week after heading. Aster-sk indicates a significant difference between CF and O3 treatment (P < 0.05). Barsepresent ± SE (n = 10).

(Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001).

3.3. Expression of ozone-responsive proteins in flag leaves

To identify ozone-responsive proteins during ripening periodsin three rice cultivars showing different ozone sensitivity, totalsoluble proteins were extracted from flag leaves exposed to CFand O3 ×2 from transplanting to one week after heading. Afterseparation on 2-DE and display by CBB-staining, 440, 480, and330 protein spots were detected reproducibly over a combined pIranges of 4.5–8.5 and Mr of 15–80 kDa in Kirara 397, Koshihikariand Takanari, respectively. Protein spots detected in the Indica cul-tivar Takanari leaves were less than those in the Japonica cultivarsKirara and Koshihikari leaves. The reason for this could be thatthe number of protein spots separated in 2-DE differed betweenJaponica and Indica cultivars, as suggested previously by Saruyamaand Shinbashi (1992). Number of spots separated by 2-DE mightbe different between Indica and Japonica cultivars. Quantitativeimage analyses of three replicates revealed that a total of 31 pro-tein spots showed significant expression changes with more than1.5-fold difference between the control and the ozone exposureleaves in Kirara 397 (P < 0.05; Figs. 3 and 4). Koshihikari and Taka-nari, respectively, showed significant differences in expression in18 and 11 protein spots (P < 0.05; Supplemental Fig. 1 and Fig. 4).

Among the 31 protein spots differentially expressed in Kirara397, 14 spots were up-regulated; 17 spots were down-regulatedin response to ozone stress (Fig. 4). Koshihikari leaves showed 12up-regulated and 6 down-regulated protein spots (Fig. 4). Taka-nari leaves showed 5 up-regulated and 6 down-regulated proteinspots (Fig. 4). Only one protein spot common to all three cultivarsresponded significantly to ozone stress (Ki-13). Four protein spotscommon to Kirara 397 and Koshihikari (Ki-1, 6, 11 and 18), and onespot common to Kirara 397 and Takanari (Ki-31) responded sig-nificantly to ozone stress. The protein spots were identified usingnanoLC–MS/MS. Spots Ki-17, 21, 26, Ko-1, 8, 10, Ta-4, 5, and 9 werenot identified in the NCBI database. Identified proteins varied fromsingle to multiple proteins within a spot, ranked by the ion score(Supplemental Table 1). One spot contains several proteins whenthe molecular weight and pI of proteins identified from the samespot were not distinctly different. Therefore, we inferred that allproteins identified in these spots probably contribute to the ozone-induced changes. These ozone-responsive proteins were classifiedinto several distinct categories in reference to the functional cate-gories of Zhang et al. (2010) with minor modifications and whetherthey were up-regulated or down-regulated (Fig. 5).

3.4. Energy metabolism

Proteins involved in photosynthesis were most down-regulatedin flag leaves of Kirara 397 and up-regulated in Koshihikari exposedto ozone. The RuBisCO large subunit (LSU), small subunit (SSU)and its fragments showed no significant change in any cultivar

112 H. Sawada et al. / Environmental and Experimental Botany 77 (2012) 108– 116

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ig. 3. Representative 2-DE gels of flag leaf proteins in Kirara 397 exposed to elevumbered spots differentially expressed in CF and O3 ×2 treatments.

n this experiment using mature and ripening plants, althoughroteomic analysis of young seedlings exposed to ozone in pre-ious reports showed down-regulation of RuBisCO LSU and SSUFeng et al., 2008; Ahsan et al., 2010). RuBisCO activase (spotsi-15, Ko-3 and 5) was down-regulated in Kirara 397 and up-egulated in Koshihikari. Moreover, the other proteins related to thealvin cycle (Ki-24, 28, Ko-3 and 7) were down-regulated in Kirara97, and up-regulated in Koshihikari. Feng et al. (2008) reportedhat some photosynthesis-related proteins increased with mod-rate ozone (40, 80 ppb) in Koshihikari leaves. The induction ofroteins related to the Calvin cycle probably compensate for theeduction of photosynthetic activity caused by a slight decreasen Koshihikari biomass (Table 1). Hypothetical protein OsI-20474Ta-3), which showed high homology to chloroplast phosphoglyc-rate kinase (O. sativa, 100% identity), was up-regulated with ozonexposure in Takanari. Carbonic anhydrase (Ki-22, Ko-6), whichs induced to supply CO2 to RuBisCO, was up-regulated in both

irara397 and Koshihikari. Proteins involved in light reaction, suchs photosystem II oxygen-evolving complex protein 1 (Ki-14, 15,0, and 25) were remarkably lower in Kirara 397 under elevated

ig. 4. Relative intensities of significantly changed spots with more than 1.5-fold changeoncentration (O3 ×2) exposed flag leaves (P < 0.05). Bars represent ± SE (n = 3). Spots are

zone. Proteins were extracted from samples harvested at one week after heading.

ozone conditions. Only one spot (Ko-13) involved in the lightreaction was down-regulated in Koshihikari. In Takanari, how-ever, photosystem II oxygen-evolving complex protein 1 (Ta-7)was up-regulated by ozone exposure. Reportedly, oxygen-evolvingprotein decreases in rice and soybean seedlings with acute ozoneexposure (120–200 ppb, 3–9 days) (Agrawal et al., 2002; Fenget al., 2008; Ahsan et al., 2010). The ATP synthase CF1 beta sub-unit (Ki-13) was down-regulated in Kirara 397 and Takanari, andup-regulated in Koshihikari exposed to ozone. Decreased ATP syn-thase reduces ATP production through photophosphorylation andthereby affects the Calvin cycle in photosynthesis (Agrawal et al.,2002). In mature leaves that are already subjected to early events ofsenescence, photosynthetic electron transport activity was muchlower than in young leaves in pumpkin fumigated with ozone(Ciompi et al., 1997). These results demonstrated that disruptionof electron transport proteins occurred rather than RuBisCO degra-dation in mature leaves in Kirara 397. Consequently, the proteins

related to photosynthetic apparatus were remarkably less abun-dant in Kirara 397, but they were more abundant or unaffected inKoshihikari and Takanari under elevated ozone conditions. These

in their expression between the charcoal filtered air (CF) and twice ambient ozone depicted in Fig. 3 and Supplemental Fig. 1.

H. Sawada et al. / Environmental and Expe

Fig. 5. Functional categories of proteins differentially expressed under ozone expo-sure. The black and white bars, respectively, show ozone-induced down- andup-regulated proteins. Photosynthesis includes pathways of pentose phosphate,electron transport, and photosynthesis. Energy (others) includes glycolysis, TCApathway and respiration. Metabolism (others) includes the metabolism pathwaysot

ro

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included thioredoxin peroxidase, l-ascorbate peroxidase, catalase

f nucleotides, and sugars and polysaccharides. Disease/defense (others) includeshe proteins related to stress responses.

esults were consistent with the response of chlorophyll content tozone exposure (Fig. 2).

Other energy metabolism proteins involved in the cytosolic gly-olysis and TCA cycle were affected remarkably by ozone in Kirara97 (Ki-13, 15, 19, 22, and 28), although two protein spots or noneere affected significantly in Koshihikari (Ki-13 and Ko-12) and

akanari, respectively. Four of these energy metabolism proteins inirara 397 were down-regulated with ozone exposure. In contrast,p-regulation of enolase 1 (Ki-13) and aldolase C-1 (Ko-12) in gly-olytic pathway was shown in Koshihikari leaves exposed to ozone.hang et al. (2010) reported that the enzymes involved in glycol-sis in the cytosol were up-regulated during flag leaf senescence.n senescing leaves, glycolysis and TCA-cycle genes were activatedossibly to produce energy for the degradation and transport ofrotein (Kontunen-Soppela et al., 2010). The energy produced bylycolytic enzymes in Koshihikari might be required for faster

ranslocation of metabolites from flag leaves in faster senescencerocess under ozone stress. Moreover, Bohler et al. (2007) havehown that the enzymes linked to glucose catabolism increased

rimental Botany 77 (2012) 108– 116 113

to produce more energy and reducing power for detoxificationand repair of oxidative damages in response to ozone stress inpoplar leaves. These results suggest that the up-regulation of glu-cose catabolism, which is necessary for faster senescence process,detoxification and repair of damages by ozone stress, was impededin flag leaves of Kirara 397, which showed a high sensitivity toozone.

3.5. Amino acid metabolism and photorespiration

Ozone exposure decreased expressions of proteins associatedwith amino acid metabolism in Kirara 397 (cysteine synthase; Ki-15, aspartate aminotransferase; Ki-28, glycine cleavage system Tprotein; Ki-29 and 30, serine-glyoxylate aminotransferase; Ki-30)and Takanari (NADH-dependent hydroxypyruvate reductase; Ta-6), although these proteins were increased in Koshihikari (glycinecleavage system protein; Ko-11 and 12). Especially, glycine and ser-ine metabolic pathway was inhibited by elevated ozone. In contrast,proteolysis-related proteins were induced in Kirara 397 (Ki-11 and16) and Koshihikari (Ki-11 and Ko-6) exposed to ozone. Resultssuggest that elevated ozone induced protein degradation with leafsenescence, but inhibited amino acid metabolism, thereby disturb-ing nitrogen mobilization.

Additionally, we identified many enzymes involved in photores-piration, such as glycine cleavage system protein (glycine decar-boxylase), serine-glyoxylate aminotransferase, NADH-dependenthydroxypyruvate reductase, catalase, and glycolate oxidase. Mostof these enzymes were down-regulated in Kirara 397, althoughcatalase, an antioxidative protein, was only up-regulated. Ingeneral, the rate of photosynthesis is stimulated by reducing pho-torespiration, because competition between oxygen and carbondioxide reduces the efficiency of RuBisCO. However, decrease inphotorespiration in soybean exposed to ozone coincided withozone-induced decline in net photosynthesis, which was attributedto the general metabolic injury caused by chronic ozone expo-sure (Booker et al., 1997). The photorespiratory pathway isimportant to protect plants from photoinhibition under high-intensity light (Kozaki and Takeba, 1996). Although the enzymesin photorespiratory pathway might only decrease along with theinhibition of photosynthesis, results suggest that photorespira-tion is largely suppressed in response to ozone in ozone-sensitiveKirara 397.

3.6. Multi-level defense mechanisms

Ozone entering the apoplast through stomata is decomposedrapidly to ROS. The ROS initiate a self-perpetuating peroxidationof lipids, damage nucleic acids and proteins (Rao and Davis, 2001).When ROS formation from ozone exceeds the apoplastic antioxida-tive capacity, the ROS induces an active production of further ROS.This active oxidative burst is similar to the one observed in thehypersensitive response (HR) and lead to programmed cell death.These direct and indirect ROS signals elicit downstream processesand induce expression of defense genes, and accumulation of ethy-lene, salicylic acid, jasmonic acid. These plant hormones regulateROS production and spreading of cell death, which ultimately leadsto the formation of visible ozone lesions (Kangasjarvi et al., 2005).Therefore, antioxidant defense mechanisms have been presumedas the most important to protect plant tissues against the toxiceffects of ozone and modulate ROS signals. Many proteins asso-ciated with antioxidant system changed with ozone exposure inKirara 397. Up-regulated protein spots (Ki-1, 3, 11, 22, and 27)

and OSJNBa0041A02.10 (a homolog of l-ascorbate peroxidase inSolanum lycopersicum with 73% identity). Down-regulated proteinspots (Ki-9, 12, 14, 29, and 30) included 2-cys peroxiredoxin BAS1,

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14 H. Sawada et al. / Environmental and

ehydroascorbate reductase and peroxidase. Koshihikari leavesnduced some of the same protein spots as Kirara 397 (Ki-1 and 11)n response to ozone. Identified proteins included two thioredoxineroxidases, OSJNBa0041A02.10, 2-cys peroxiredoxin BAS1, dehy-roascorbate reductase were predicted to localize in chloroplastccording to TargetP, a program for prediction the subcellular loca-ion based on the protein’s N-terminal amino acid sequence (valuesf cp [probability of chloroplast localization] 0.965, 0.869, 0.986,nd 0.898, respectively). In spite of marked up-regulation of somehloroplastic antioxidant proteins, remarkably less abundant pro-eins of the photosynthetic apparatus were observed in Kirara 397,hich suggests that Kirara 397 increased antioxidative proteins

n response to greater amount of ROS produced during ozone-nduced chloroplastic oxidative burst, but the greater amount ofroteins were insufficient to detoxify the enhanced ROS. Rao andavis (1999) showed that ozone-sensitive tobacco Cvi-0 developed

evere necrotic lesions, although it showed increased transcriptevels of antioxidant enzymes such as superoxide dismutase,lutathione reductase, and glutathione peroxidase. ChloroplasticOS production leads to the down-regulation of many nuclearenes encoding chloroplast proteins (Mahalingam et al., 2005).n contrast, Takanari leaves did not induce any antioxidant pro-ein, but showed decreased expression of thioredoxin peroxidaseTa-1).

Other disease/defense-related proteins were unaffected inakanari. Disease/defense-related proteins such as probenazole-nducible protein PBZ1 and pathogenesis-related (PR) proteinR-10a were up-regulated in both Kirara 397 (Ki-1, 3, 4, 5, 6, and) and Koshihikari (Ki-1 and 6). Ozone induced PR-proteins werelso up-regulated in response to pathogen infection during HR andystemic acquired resistance (SAR), suggesting that signal trans-uction pathways overlapped under different stresses (Sharmand Davis, 1997). The PR-proteins were induced by acute andhronic ozone exposure in proteomics research using rice seedlingsAgrawal et al., 2002; Cho et al., 2008; Feng et al., 2008). In fact, ROSre important regulators of defense-related gene expression androgrammed cell death during HR (Low and Merida, 1996). Theseesults suggest that Takanari leaves did not have active defenseechanisms responding to ozone-induced ROS or generated much

ess ROS under ozone stress, perhaps these results associate withhe fact that the formation of necrotic lesions was remarkably lowern Takanari leaves than in either Kirara 397 or Koshihikari (Sawadand Kohno, 2009).

Additionally, several folding-related proteins such as heat shockroteins (HSP) induced by pathogen and heat shock and involved inrocessing of secreted proteins, were found to be induced in Kirara97 (Ki-2, 7, and 16), but not in Takanari. In Arabidopsis, HSPs haveeen shown to be induced by high light-induced oxidative stress,nd it may be an adaptive response to limit oxidation-mediatedisulfide bridge-induced protein aggregation (Rossel et al., 2002).ohler et al. (2007) have suggested that over-expressed HSPs dur-

ng ozone stress were responsible for correct protein folding whenroteins lost their structural integrity. The induction of HSPs sug-ests that Kirara 397 suffered direct damage to the proteins duringzone stress. Only one spot (Ki-13) showed significant change inhree cultivars; it included 60 kDa chaperonin (a folding-relatedrotein), chloroplastic ATP synthase, and enolase. The expressionhange of this protein spot was coincident with ozone sensitivity ofach cultivar, as indicated by grain yield reduction. Results suggesthat these proteins are involved in yield reduction under elevatedzone.

.7. Other functions

Other identified proteins were involved in primary metabolism,rotein synthesis, signal transduction and transport. The primary

rimental Botany 77 (2012) 108– 116

metabolism-related proteins (Ki-22 and Ko-2) were up-regulatedin Kirara 397 and Koshihikari, respectively. Glyoxalase I (Ki-18)and hypothetical protein OsJ 06256 (Ko-4), which showed highhomology of putative glyoxalase I (O. sativa, 88% identity), weredown-regulated in both Kirara 397 and Koshihikari. Eukaryoticelongation factor-1 alpha (EF-1A; Ki-31) and chloroplast transla-tional elongation factor Tu (Ko-5) associated with protein synthesiswas up-regulated in both Kirara 397 and Takanari, and in Koshi-hikari, although eukaryotic translation initiation factor 3 subunit5 (Ki-12) was down-regulated in Kirara 397. Up-regulation of EF-1A has been suggested to play an important role in execution ofcell death in response to oxidative stress in animal cells (Lambertiet al., 2004). One of two proteins associated with signal transduc-tion in Kirara 397 (Ki-12 and 23) was found to increase, another wasfound to decrease. A signal transduction protein decreased (Ko-6)in Koshihikari. Two protein spots involved in transport (Ki-16 andTa-8) were increased each in Kirara 397 and Takanari, althoughone (Ko-4) was reduced in Koshihikari. Actually, OSJNB0060E08.11(Ko-4) showed high homology to chloroplastic lipocalin (Zea mays,92% identity). Porin-like protein (Ta-8), which functions as voltage-dependent anion channel (VDAC) protein, detected in Takanari,which is known as a major outer mitochondrial membrane protein,mediates the transport of metabolites. In fact, VDAC is reportedlyinvolved in programmed cell death (Godbole et al., 2003). Leavesof Takanari, however, showed no symptoms such as cell deathin seedlings and mature plants under elevated ozone conditions(Sawada and Kohno, 2009, 2010). Further study is necessary todetermine how ozone-induced change of anion channel is involvedin yield reduction of Takanari.

3.8. Comparison of the effects of ozone stress on proteins in threedifferent rice cultivars

Fig. 6 shows metabolic pathways responding to ozone, whichare likely to be involved in carbohydrate assimilation and energyproduction affecting grain yield reduction.

Although Kirara 397 increased antioxidative proteins inresponse to ozone-derived ROS, the antioxidative capacity wasinsufficient to prevent ROS induced damage. Enhanced ROS forma-tion might decompose or inhibit the repair of proteins involvedin photosynthetic electron transport in Kirara 397. Subsequentdecrease in light reaction led to the down-regulation of enzymesinvolved in the Calvin cycle. Energy production via glycolysis andTCA cycle, which is needed for faster translocation of nitrogen fromsenescing leaves under ozone stress, was down-regulated in Kirara397. Moreover, elevated ozone inhibited amino acid metabolism inspite of induced protein degradation (Fig. 5). Consequently, grainyield reduction in Kirara 397 was suggested to be caused by reducedproduction of carbohydrate and inhibition of metabolites translo-cation. The up-regulation of other defense-related proteins, such asPR-protein and HSP, could not prevent ozone-induced yield reduc-tion in Kirara 397 (Fig. 5).

Koshihikari had shown leaf injury at seedling stage (Sawadaand Kohno, 2009), but mature plant showed the acclimation toozone stress (Fig. 2). Protein involved in the light reaction hardlydecreased in flag leaves of Koshihikari, probably due to regulation ofROS and dissipation of excess energy, such as by activated photores-piration pathway. In addition, proteins related to the Calvin cyclewere up-regulated in Koshihikari exposed to ozone. Furthermore,Koshihikari could utilize the increased energy for translocationof metabolites from flag leaves by the activation of glycolyticenzymes. Therefore, the grain yield of Koshihikari exposed to ele-

vated ozone could be maintained at the same level as yield of CFplants.

In Takanari, neither the antioxidant system nor defense-relatedproteins responded under elevated ozone (Fig. 5). Changes in

H. Sawada et al. / Environmental and Experimental Botany 77 (2012) 108– 116 115

Fig. 6. Possible pathways for carbon metabolism of rice flag leaves in response to ozone stress. Arrows after the cultivar names indicate ozone-induced regulations of proteinsidentified in this study. Red arrows indicate increased expression; blue arrows indicate decreased expression. The number of arrows indicates the number of protein identifiedas each enzyme or involved in each process. Abbreviations for proteins: CAT, catalase; FBA, fructose-bisphosphate aldolase; GADPH, glyceraldehyde-3-phosphate dehydro-genase; GCV, glycine cleavage system protein; GOX, glycolate oxidase; HPR, hydroxypyruvate reductase; MDH, malate dehydrogenase; PDH, pyruvate dehydrogenase; PGK,p ase; SA Pi, phoT

paTdaobs

4

elwstrtdadpynwritucvo

hosphoglycerate kinase; RuBisCO, ribulose-1,5-bisphosphate carboxylase/oxygenbbreviations for metabolites: F-6-P, fructose-6-phosphate; Gly, glycine; glycolate-HF, tetrahydrofolic acid; Ser, serine.

roteins related to photosynthesis did not show any coherent trend,nd chlorophyll content was unaffected by elevated ozone (Fig. 2).hese results suggested that yield reduction was not caused byirect ozone damage to photosynthetic apparatus in Takanari. Sincective defense mechanisms were not induced as a response tozone-derived ROS, the entry of ozone into the leaf might haveeen suppressed by stomatal closure, resulting in reduced photo-ynthesis.

. Conclusion

Proteomic analysis of different rice cultivars showed vari-tal differences in protein responses to elevated ozone in flageaves. Proteins associated with photosynthetic electron transport

ere particularly down-regulated in Kirara 397 leaves. This resultuggests that ROS derived from ozone largely disrupted the pho-osynthetic apparatus, decreased carbohydrate assimilation, andeduced grain yields in Kirara 397. In contrast, ozone stress par-icularly induced proteins in the Calvin cycle of Koshihikari, whichid not reduce the yield, suggesting that sustained photosyntheticctivity is important for yield production under elevated ozone con-itions. Takanari showed no noteworthy changes in the metabolicathway resulting from ozone exposure. As opposed to Kirara 397,ield reduction in Takanari was probably not associated with sig-ificant suppression of photosynthesis-related proteins. Althoughe were unable to identify the mechanism of ozone-induced yield

eduction in Takanari, this study suggests that yield productionn rice is also controlled by factors other than RuBisCO degrada-ion, which is generally considered as a cause of yield reduction

nder elevated ozone condition. Further research of ozone-inducedhanges in protein expressions that is closely related with yieldariation might provide novel findings related to the mechanismf yield reduction.

BP, sedoheptulose-1,7-bisphosphatase; SGAT, serine-glyoxylate aminotransferase.sphoglycolate; 3-PGA, 3-phosphoglycerate; Ru-1,5-BP, ribulose-1,5-bisphosphate;

Acknowledgements

This work was supported by the Environmental Research andTechnology Development Fund (A0806) of the Ministry of the Envi-ronment, Japan.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.envexpbot.2011.11.009.

References

Agrawal, G.K., Rakwal, R., Yonekura, M., Kubo, A., Saji, H., 2002. Proteome analysis ofdifferentially displayed proteins as a tool for investigating ozone stress in rice(Oryza sativa L.) seedlings. Proteomics 2, 947–959.

Ahsan, N., Nanjo, Y., Sawada, H., Kohno, Y., Komatsu, S., 2010. Ozone stress-inducedproteomic changes in leaf total soluble and chloroplast proteins of soybeanreveal that carbon allocation is involved in adaptation in the early develop-mental stage. Proteomics 10, 2605–2619.

Aunan, K., Berntsen, T.K., Seip, H.M., 2000. Surface ozone in China and its possibleimpact on agricultural crop yields. Ambio 29, 294–301.

Bohler, S., Bagard, M., Oufir, M., Planchon, S., Hoffmann, L., Jolivet, Y., Hausman, J.F.,Dizengremel, P., Renaut, J., 2007. A DIGE analysis of developing poplar leavessubjected to ozone reveals major changes in carbon metabolism. Proteomics 7,1584–1599.

Booker, F.L., Reid, C.D., Brunschon-Harti, S., Fiscus, E.L., Miller, J.E., 1997. Photo-synthesis and photorespiration in soybean [Glycine max (L.) Merr.] chronicallyexposed to elevated carbon dioxide and ozone. J. Exp. Bot. 48, 1843–1852.

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of micro-gram quantities of protein utilizing the principle of protein-dye binding. Anal.Biochem. 72, 248–254.

Cho, K., Shibato, J., Agrawal, G.K., Jung, Y.-H., Kubo, A., Jwa, N.-S., Tamogami, S., Satoh,K., Kikuchi, S., Higashi, T., Kimura, S., Saji, H., Tanaka, Y., Iwahashi, H., Masuo,Y., Rakwal, R., 2008. Integrated transcriptomics, proteomics, and metabolomics

analyses to survey ozone responses in the leaves of rice seedling. J. ProteomeRes. 7, 2980–2998.

Ciompi, S., Castagna, A., Ranieri, A., Nali, C., Lorenzini, G., Soldatini, G.F., 1997. CO2

assimilation, xanthophyll cycle pigments and PSII efficiency in pumpkin plantsas affected by ozone fumigation. Physiol. Planta 101, 881–889.

1 Expe

E

F

F

G

G

K

K

K

K

L

L

M

M

O

P

16 H. Sawada et al. / Environmental and

manuelsson, O., Nielsen, H., Brunak, S., von Heijne, e.G., 2000. Predicting subcellularlocalization of proteins based on their N-terminal amino acid sequence. J. Mol.Biol. 300, 1005–1016.

eng, Y., Komatsu, S., Furukawa, T., Koshiba, T., Kohno, Y., 2008. Proteome analysisof proteins responsive to ambient and elevated ozone in rice seedlings. Agric.Ecosyst. Environ. 125, 255–265.

rei, M., Kohno, Y., Wissuwa, M., Makkar, H.P.S., Becker, K., 2011. Negative effectsof tropospheric ozone on the feed value of rice straw are mitigated by an ozonetolerance QTL. Global Change Biol. 17, 2319–2329.

odbole, A., Varghese, J., Sarin, A., Mathew, M.K., 2003. VDAC is a conserved elementof death pathways in plant and animal systems. Biochim. Biophys. Acta 1642,87–96.

uidi, L., Degl‘Innocenti, E., Giordano, C., Biricolti, S., Tattini, M., 2010. Ozone tol-erance in Phaseolus vulgaris depends on more than one mechanism. Environ.Pollut. 158, 3164–3171.

angasjarvi, J., Jaspers, P., Kollist, H., 2005. Signalling and cell death in ozone-exposedplants. Plant Cell Environ. 28, 1021–1036.

obayashi, K., Okada, M., Nouchi, I., 1995. Effects of ozone on dry matter partitioningand yield of Japanese cultivars of rice (Oryza sativa L.). Agric. Ecosyst. Environ.53, 109–122.

ontunen-Soppela, S., Parviainen, J., Ruhanen, H., Brosch, M., Keinanen, M., Thakur,R.C., Kolehmainen, M., Kangasjarvi, J., Oksanen, E., Karnosky, D.F., Vapaavuori,E., 2010. Gene expression responses of paper birch (Betula papyrifera) to ele-vated CO2 and O3 during leaf maturation and senescence. Environ. Pollut. 158,959–968.

ozaki, A., Takeba, G., 1996. Photorespiration protects C3 plants from photooxida-tion. Nature 384, 557–560.

amberti, A., Caraglia, M., Longo, O., Marra, M., Abbruzzese, A., Arcari, P., 2004.The translation elongation factor 1A in tumorigenesis, signal transduction andapoptosis: review article. Amino Acids 26, 443–448.

ow, P.S., Merida, J.R., 1996. The oxidative burst in plant defense: function and signaltransduction. Physiol. Planta 96, 533–542.

ahalingam, R., Shah, N., Scrymgeour, A., Fedoroff, N., 2005. Temporal evolution ofthe Arabidopsis oxidative stress response. Plant Mol. Biol. 57, 709–730.

atile, P., 2000. Biochemistry of Indian summer: physiology of autumnal leaf col-oration. Exp. Gerontol. 35, 145–158.

’Farrell, P.H., 1975. High resolution two-dimensional electrophoresis of proteins.J. Biol. Chem. 250, 4007–4021.

ang, J., Kobayashi, K., Zhu, J., 2009. Yield and photosynthetic characteristics of flagleaves in Chinese rice (Oryza sativa L.) varieties subjected to free-air release ofozone. Agric. Ecosyst. Environ. 132, 203–211.

rimental Botany 77 (2012) 108– 116

Pell, E.J., Schlagnhaufer, C.D., Arteca, R.N., 1997. Ozone-induced oxidative stress:mechanisms of action and reaction. Physiol. Planta 100, 264–273.

Rao, M.V., Davis, K.R., 1999. Ozone-induced cell death occurs via two distinct mech-anisms in Arabidopsis: the role of salicylic acid. Plant J. 17, 603–614.

Rao, M.V., Davis, K.R., 2001. The physiology of ozone induced cell death. Planta 213,682–690.

Rossel, J.B., Wilson, I.W., Pogson, B.J., 2002. Global changes in gene expression inresponse to high light in Arabidopsis. Plant Physiol. 130, 1109–1120.

Sarkar, A., Agrawal, S.B., 2010. Elevated ozone and two modern wheat cultivars: Anassessment of dose dependent sensitivity with respect to growth, reproductiveand yield parameters. Environ. Exp. Bot. 69, 328–337.

Saruyama, H., Shinbashi, N., 1992. Identification of specific proteins from seedembryos by two-dimensional gel electrophoresis for the discriminationbetween indica and japonica rice. Theor. Appl. Genet. 84, 947–951.

Sawada, H., Kohno, Y., 2009. Differential ozone sensitivity of rice cultivars as indi-cated by visible injury and grain yield. Plant Biol. 11 (Suppl. 1), 70–75.

Sawada, H., Kohno, Y., 2010. Effect of elevated ozone on leaf senescence and grainyield of rice cultivars. J. Jpn. Soc. Atmos. Environ. 45, 283–288 (in Japanese).

Schraudner, M., Langebartels, C., Sandermann, H., 1997. Changes in the biochemi-cal status of plant cells induced by the environmental pollutant ozone. Physiol.Planta 100, 274–280.

Shao, M., Tang, X., Zhang, Y., Li, W., 2006. City clusters in China: air and surface waterpollution. Front. Ecol. Environ. 4, 353–361.

Sharma, Y.K., Davis, K.R., 1997. The effects of ozone on antioxidant responses inplants. Free Radic. Biol. Med. 23, 480–488.

Torres, N.L., Cho, K., Shibato, J., Hirano, M., Kubo, A., Masuo, Y., Iwahashi, H., Jwa, N.S.,Agrawal, G.K., Rakwal, R., 2007. Gel-based proteomics reveals potential novelprotein markers of ozone stress in leaves of cultivated bean and maize speciesof Panama. Electrophoresis 28, 4369–4381.

Uddling, J., Gelang-Alfredsson, J., Piikki, K., Pleijel, H., 2007. Evaluating the relation-ship between leaf chlorophyll concentration and SPAD-502 chlorophyll meterreadings. Photosynth. Res. 91, 37–46.

Yonekura, T., Shimada, T., Miwa, M., Arzate, A., Ogawa, K., 2005. Impacts of tropo-spheric ozone on growth and yield of rice (Oryza sativa L.). J. Agric. Meteor. 60,1045–1048.

Yoshida, S., 1981. Fundamentals of Rice Crop Science. International Rice Research

Institute, Los Banos.

Zhang, A., Lu, Q., Yin, Y., Ding, S., Wen, X., Lu, C., 2010. Comparative proteomicanalysis provides new insights into the regulation of carbon metabolism dur-ing leaf senescence of rice grown under field conditions. J. Plant Physiol.16, 7.