Differential responses in two varieties of winter wheat to elevated ozone concentration under fully open-air field conditions

Transcription

1 Global Change Biology (2011) 17, , doi: /j x Differential responses in two varieties of winter wheat to elevated ozone concentration under fully open-air field conditions ZHAOZHONG FENG*, JING PANG*w, KAZUHIKO KOBAYASHI*, JIANGUO ZHUz and DONALD R. ORT *Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo , Japan, wacademy of Resource and Environment, Hubei University, Wuhan , China, zstate Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Sciences, Chinese Academy of Sciences, Nanjing , China, Photosynthesis Research Unit, USDA/ARS & Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Abstract Two modern cultivars [Yangmai16 (Y16) and Yangfumai 2 (Y2)] of winter wheat (Triticum aestivum L.) with almost identical phenology were investigated to determine the impacts of elevated ozone concentration (E-O 3 ) on physiological characters related to photosynthesis under fully open-air field conditions in China. The plants were exposed from the initiation of tillering to final harvest, with E-O 3 of 127% of the ambient ozone concentration (A-O 3 ). Measurements of pigments, gas exchange rates, chlorophyll a fluorescence and lipid oxidation were made in three replicated plots throughout flag leaf development. In cultivar Y2, E-O 3 significantly accelerated leaf senescence, as indicated by increased lipid oxidation as well as faster declines in pigment amounts and photosynthetic rates. The lower photosynthetic rates were mainly due to nonstomatal factors, e.g. lower maximum carboxylation capacity, electron transport rates and light energy distribution. In cultivar Y16, by contrast, the effects of E-O 3 were observed only at the very last stage of flag leaf ageing. Since the two cultivars had almost identical phenology and very similar leaf stomatal conductance before senescence, the greater impacts of E-O 3 on cultivars Y2 than Y16 cannot be explained by differential ozone uptake. Our findings will be useful for scientists to select O 3 -tolerant wheat cultivars against the rising surface [O 3 ] in East and South Asia. Nomenclature: %D 5 the fraction of photons dissipated in the antenna %P 5 the fraction of photons utilized in PSII photochemistry %X 5 the fraction of absorbed photons by PSII neither used in photochemistry nor dissipated in the PSII A 5 photosynthetic rate AOT40 5 the cumulative O 3 exposure over a threshold of the 1 h average [O 3 ]of 40 ppb during daytime A sat 5 light-saturated photosynthesis Car 5 Carotenoid Chl 5 Chlorophyll C i 5 intercellular CO 2 concentration AQY 5 apparent quantum efficiency DAFE 5 days after full expansion of flag leaves Fv 0 /Fm 0 5 actual photochemical efficiency of PSII in the saturated light gs 5 stomatal conductance J max 5 the maximum rate of electron transport l 5 stomatal limitation to photosynthesis M7 5 the mean of the daily 7 h average [O 3 ] MDA 5 malondialdehyde O 3 5 ozone OTC 5 open-top chambers Correspondence: K. Kobayashi, tel , fax , aclasman@mail.ecc.u-tokyo.ac.jp 580 r 2010 Blackwell Publishing Ltd

2 VARIETAL DIFFERENCE IN WINTER WHEAT RESPONSES TO O PPFD 5 photosynthetic photon flux density PhiPSII 5 light-adapted apparent quantum efficiency of PSII qp 5 quenching of photochemical efficiency of PSII SUM06 5 sum of hourly average [O 3 ] 0.06 ppm during daytime Vc max 5 the maximum carboxylation efficiency Y16 5 wheat variety Yangmai 16 Y2 5 wheat variety Yangfumai 2. Keywords: chlorophyll fluorescence, gas exchange, ozone, ozone uptake, variety, winter wheat Received 3 November 2009 and accepted 15 December 2009 Introduction Ozone (O 3 ) is among the most important air pollutants in many parts of the world causing significant biological and economic damage to crop plants (Wahid et al., 1995; Fuhrer & Booker, 2003; Morgan et al., 2003; Ashmore, 2005; Ashmore et al., 2006; Felzer et al., 2007; Wang et al., 2007; Feng et al., 2008, 2009; Fuhrer, 2008; Feng & Kobayashi, 2009). It is well documented that chronic exposure to elevated O 3 concentration ([O 3 ]) causes a range of adverse effects on plants including reduced photosynthetic activity, altered carbon allocation, diminished biomass accumulation, reduced yield and accelerated senescence, with or without visible injury (Kobayashi et al., 1995; Farage & Long, 1999; Morgan et al., 2004, 2006; Ashmore, 2005; Pleijel et al., 2006; Feng et al., 2007; Biswas et al., 2008). A meta-analysis of 53 peer-reviewed chamber studies showed that elevated [O 3 ] (averaged 73 ppb) decreases leaf photosynthetic rate (A) by 20% and grain yield by 29% in wheat plants compared with those grown in carbon-filtered air (Feng et al., 2008), which suggested that decreased photosynthesis was a key factor driving yield loss in wheat exposed to elevated [O 3 ]. The meta-analysis also indicated that the negative effects of O 3 increased with the duration of leaf exposure to elevated [O 3 ], implying cumulative effects of O 3 during wheat leaf development (Feng et al., 2008). For field-grown plants, photosynthesis in saturating light (A sat ) is normally co-limited by stomatal factors controlling gas exchange along with biochemical and photochemical processes of photosynthesis. Most results indicated that stomatal limitation (l) ofa was not increased despite significant reduction of stomatal conductance (gs) in response to elevated [O 3 ] (Fiscus et al., 1997; Farage & Long, 1999; Zheng et al., 2002; Morgan et al., 2004). The central biochemical processes controlling photosynthesis are, the maximum carboxylation efficiency (Vc max ), which reflects in vivo activity of Rubisco activity (Rogers & Humphries, 2000), and the maximum rate of RuBP regeneration (J max ) (Sage, 1990). Chamber results indicated that reduced A sat from growth at elevated [O 3 ] was due primarily to a lower Vc max (Atkinson et al., 1988; Farage & Long, 1995, 1999; Reid & Fiscus, 1998; Zheng et al., 2002; Morgan et al., 2004; Fiscus et al., 2005). Chlorophyll a fluorescence measurements showed decreased photochemical efficiency and quantum yield of PSII in the light as a result of growth at elevated [O 3 ] that depended on [O 3 ] dose and crop species (Castagna et al., 2001; Calatayud et al., 2002, 2004; Zheng et al., 2002; Biswas et al., 2008). The effects of growth at elevated [O 3 ] on photosynthesis are strongly influenced by genetic background, developmental stage as well as interacting environmental factors. To date, most O 3 exposure results are based on experiments in controlled environment enclosures such as greenhouses and closed or open-top chambers (OTC). Enclosures can substantially modify the plant canopy microclimate (McLeod & Long, 1999) and thereby alter both qualitatively and quantitatively the effects of O 3 exposure (Nussbaum & Fuhrer, 2000; Piikki et al., 2008). Morgan et al. (2004, 2006) reported that O 3 -induced inhibition of photosynthesis and losses in yield of soybean under fully open-air field conditions were even greater than the losses reported in earlier chamber studies. Therefore, it is necessary to investigate the basis for the negative effects of O 3 on wheat in systems where artificial perturbations to the soil plant atmosphere continuum are minimal. Ozone-sensitive and tolerant cultivars or clones have been investigated for numerous crop species e.g. soybean (Robinson & Britz, 2000), wheat (Heagle et al., 2000; Biswas et al., 2008), and snap bean (Phaseolus vulgaris L.) (Guzy & Heath, 1993). At least in some crops, O 3 tolerance has been shown to be a heritable trait involving the antioxidant system and high apoplastic ascorbate (ASC) content (Fiscus et al., 2005). Modern wheat cultivars are reported to be more sensitive to O 3 than older accessions, which was largely attributed to higher gs in modern cultivars allowing for greater O 3 uptake (Barnes et al., 1990; Pleijel et al., 2006; Biswas et al., 2008). Since high gs is necessary for high production, from an agronomic view point, it is more important to compare among modern high gs

3 582 Z. FENG et al. varieties to identify sources of tolerance other than restricting ozone flux into leaves by low gs. In this work, we studied two modern cultivars of winter wheat with nearly identical phenology in terms of photosynthetic response to elevated [O 3 ] in a field experiment by using open-air O 3 fumigation systems built in China. Our objectives were (1) to compare the varieties in response to elevated [O 3 ]; (2) and to test if stomatal O 3 uptake contributed to the differential response between the varieties. Materials and methods Experiment site The experiment was conducted in Xiaoji town, Jiangdu county, Jiangsu province, China ( E, N). This site has been in continuous cultivation for more than 1000 years with rice wheat or rice rapeseed rotations. The soil is Shajiang Aquic Cambosols with a sandy-loamy texture. The region has a subtropical marine climate with mean annual precipitation of mm, mean annual temperature of 16 1C, a total annual sunshine duration of hours, and a frost-free period of 4230 days. Fumigation treatment Three 240 m 2 plots were treated with elevated [O 3 ] (hereinafter called E-O 3 plots) and three equal size plots were maintained at ambient [O 3 ] (hereinafter called A-O 3 plots). The target [O 3 ] for E-O 3 plots was 50% higher than the A-O 3. All the E-O 3 plots were separated from the other plots by at least 70 m to avoid cross-contamination. The experimental design was based on completely randomized plots allocated to either A- O 3 or E-O 3, and split into subplots of wheat cultivars. In the E- O 3 plots, crops were grown within 14 m in diameter octagons with a perimeter of eight 6 m ABS pipes. A mixed gas consisting of about 5% O 3 and 95% O 2 was produced by an O 3 generator (KCF-BT0.2; Jiangsu Koner Ozone Co. Ltd., Yangzhong, China). Using a mass flow controller, the O 3 /O 2 mixture was released in a stream of compressed air into the plots through ABS pipes positioned 50 cm above the canopy height. [O 3 ] at the middle point of each plot was measured every 20 s by O 3 analyzer (model 49C; Thermo Environmental Instruments, Franklin, MA, USA). Based on the wind direction and wind speed, O 3 achieved a concentration within 15% of the set point maintained 90% of the time, and within 20% of the set point for 95% of the time. Mean [O 3 ] throughout the O 3 fumigation period had the coefficient of variation of only 2.5% across 13 locations within an E-O 3 ring. Owing to the dew in the earlier morning, E-O 3 treatment was added from 9:00 hours to sunset except when raining or when the background [O 3 ] lower than 20 ppb or higher than 170 ppb. In the ambient plots, plants were grown under A-O 3 without perimeter pipes. Owing to low temperature and A-O 3 o30 ppb before tillering stage, ozone fumigation began on March 5, 2008 at the initiation of tillering stage of wheat and continued until harvest. Plant material The winter wheat varieties cv. Yangmai 16 (medium gluten cultivar, hereafter called Y16) and Yangfumai 2 (weak gluten cultivar, hereafter called Y2) were selected due to similar phenology, as shown in Table 1. Standard cultivation practices common to the region were followed in all experimental plots. The seeds of two cultivars were sown in two of five subplots (each about 11 m 2 ) which were distributed randomly in each plot of A-O 3 and E-O 3 treatments on November 15, 2007, at a density of 210 plants m 2. Healthy flag leaves fully unfolded on the same day were marked and used to make the following measurements. Gas exchange and fluorescence measurements The marked plants were excised predawn, as described by Morgan et al. (2004), placed in water and quickly taken to a laboratory where they were kept in low light (o20 mmol m 2 s 1 ) until 30 min before the measurement, at which time they were light acclimated at 400 mmol m 2 s 1. Gas exchange and fluorescence measurements were made using a LI-6400 photosynthesis system (LICOR, Lincoln, NE, USA) fitted with a leaf chamber fluorometer (LCF). Two detached flag leaves per subplot for either cultivar with three replicates were selected to measure A vs. intercellular CO 2 concentration (C i ) and A vs. photosynthetic photon flux density (PPFD) curves every 3 7 days from their initial fully expanded state until senescence was visible (80% yellow). The automatic program in the LI-6400 photosynthesis system was used to generate the response of A to C i. Net photosynthesis and chlorophyll fluorescence characteristics were determined simultaneously. Measurements were taken by changing [CO 2 ] in LCF in 11 steps (380, 300, 200, 100, 50, 400, 400, 600, 800, 1000 and 1200 mmol mol 1 ) under a constant PPFD of 1500 mmol m 2 s 1, block temperature of 25 1C and relative humility of 50% 70%. Both steady-state (Fs) and maximal (Fm 0 ) fluorescence were logged along with standard photosynthetic parameters. The time allowed for the instrument to reach steady state at each [CO 2 ] was 240 s. The instrument logged values when the stability or steady state had been reached as indicated by total coefficient of variation 3%. Vc max, J max and l were determined following the method of Farquhar & Sharkey (1982), as described previously (Long & Bernacchi, 2003). The software in the instrument provided data on the fluorescence parameters including actual photochemical efficiency of PSII in the saturated light (Fv 0 / Fm 0 ), quenching of photochemical efficiency of PSII (qp), and the quantum yield of noncyclic electron transport (PhiPSII). gs, C i and the fluorescence parameters were extracted from measurements at [CO 2 ] 380 mmol mol 1. The fraction of radiation absorbed that was dissipated in the antenna (%D) and utilized in PSII photochemistry (%P) were estimated as 1 (Fv 0 / Fm 0 ) 100 and (Fv 0 /Fm 0 ) qp 100, respectively (Demmig- Adams et al., 1996). The fraction of absorbed radiation by PSII

4 VARIETAL DIFFERENCE IN WINTER WHEAT RESPONSES TO O Table 1 Phenology of the two cultivars of winter wheat Yangmai16 (Y16) and Yangfumai2 (Y2) in the ambient (A-O 3 ) and elevated (E-O 3 )[O 3 ] plots Cultivar Treatment Sowing Elongation Flag leaf full expansion Booting Flowering Maturity Y16 A-O 3 15/11/ /03/ /04/ /04/ /04/2008 3/06/2008 E-O 3 27/03/ /04/ /04/ /04/2008 5/06/2008 Y2 A-O 3 3/04/ /04/ /04/ /04/2008 4/06/2008 E-O 3 3/04/ /04/ /04/ /04/2008 5/06/2008 neither used in photochemistry nor dissipated in the PS2 antennae (%X) was estimated as (Fv 0 /Fm 0 ) (1 qp) 100 (Demmig-Adams et al., 1996). To determine the response of A to PPFD, chamber [CO 2 ]was set at 380 mmol mol 1 and block temperature to 25 1C. PPFD was gradually reduced from 2000 to 0 mmol m 2 s 1 in nine steps (2000, 1500, 1000, 500, 250, 200, 150, 75 and 0). The initial slopes of the A-PPFD curves were fit with a linear function to estimate the maximum apparent quantum yield (AQY). The whole curves were fit to a nonrectangular hyperbola with a least square curve fitting procedure to derive A sat. Leaf pigment content After measurements of gas exchange and fluorescence, the middle part of flag leaves was punched and then extracted in 95% ethanol in the dark for 72 h at 4 1C. The extract was then assayed for chlorophyll (Chl) and carotenoid (Car) by using the specific absorption coefficients of Lichtenthaler (1987). Malondialdehyde (MDA) content Flag leaves were sampled at noon on the same day that gas exchange measurements were conducted. MDA was analyzed as a 2-thiobarbituric acid-reactive metabolite (TBA) following the method of Heath & Parker (1968). Two punches of the middle part of leaf samples (0.06 g) were homogenized in a prechilled mortar and pestle in 1 ml ice-cold 6% (w/v) trichloroacetic acid (TCA) and centrifuged at 6000 g for 10 min at 4 1C. Assay mixture containing 0.1 ml aliquot of supernatant and 0.2 ml of 0.6% (w/v) TBA was incubated in a water bath at 95 1C for 15 min and then rapidly cooled in an ice-bath. After centrifugation ( g for 10 min at 4 1C), the supernatant absorbance at 532 nm was determined. The values corresponding to nonspecific absorption (600 nm) and sugar disturbance absorption (450 nm) were subtracted. Concentration of MDA was calculated using the following equation: C MDA (mmol L 1 ) (A 532 A 600 ) 0.56 A 450 (Heath & Parker, 1968). random variable and other variables to fixed variables, using JMP software (SAS Institute, USA). Comparison of means between A-O 3 and E-O 3 on individual dates of measurement was done on Student s-t statistics for each cultivar. A difference between the means was considered significant if P Results Ozone exposure Wheat plants were exposed to ozone fumigation in the field from March 5, 2008 through to May 28, 2008, with a total of 60 days effective fumigation. No fumigation was given on 25 days during this period due to 24 rainy or cloudy days and 1 day that power was lost. The consistent phenology of Y16 and Y2 ensured that the O 3 exposure dose was same in both cultivars. During the full 85 days fumigation, the mean of the daily 7 h average [O 3 ] (M7), accumulated O 3 exposure over a threshold of 40 ppb (AOT40) and sum of hourly average [O 3 ] 60 ppb (SUM06) in the E-O 3 were 27%, 110% and 152% higher than those in A-O 3, respectively (Table 2). As shown in Fig. 1, the M7 in A-O 3 in May was much higher than that before May. During flag leaf development, M7, AOT40 and SUM06 in the E-O 3 treatment were 26.7%, 88.9% and 110% higher than those in ambient air, respectively. The mean [O 3 ] during flag leaf development was much higher than that for the overall fumigation period (Table 2), since the higher [O 3 ] occurred more frequently in the later growth stage (Fig. 1). M7 in A-O 3 averaged 52 ppb with a maximum of 110 ppb. The observed maximum ambient 1 h mean [O 3 ] was 140 ppb in the afternoon, suggesting a serious O 3 pollution in this region of China. Statistical analysis The experiment was laid out according to a split plot design with either of the [O 3 ] levels assigned to a ring, i.e. the main plot, which was split into subplots of cultivars. Datasets were based on the mean values for each subplot. The data for each dependent variable was subjected to the analysis of variance with mixed linear model, in which the ring is assigned to the Leaf senescence E-O 3 significantly accelerated the senescence of flag leaves as indicated by a significant interaction between O 3 and leaf age in Chl, Car and MDA contents (Table 3), and by progressively increasing difference between A-O 3 and E-O 3 in the contents (Fig. 2a d, g and h).

5 584 Z. FENG et al. Table 2 O 3 exposure indices in the ambient (A-O 3 ) and elevated (E-O 3 )[O 3 ] plots and the percentage of actual elevation (1 %) during the whole fumigation (85 days) and during flag leaf development (49 days), respectively Whole fumigation Flag leaves development M7 (ppb) AOT40 (ppm.h) SUM06 (ppm.h) M7 (ppb) AOT40 (ppm.h) SUM06 (ppm.h) A-O E-O % Mean [O 3 ] is calculated based on daily average concentration of 7 h period from 09:00 to 16:00 hours (M7). The accumulated exposure over a threshold of 40 ppb (AOT40) and sum of hourly average [O 3 ] X60 ppb (SUM06) for the season are calculated from the 1 h average of daytime concentrations in A-O 3 and E-O 3 plots. Fig. 1 Daily 7 h average [O 3 ] for ambient (A-O 3 ) and elevated (E-O 3 )[O 3 ] treatments from tillering stage of winter wheat to final harvest under fully open-air field conditions. When compared with A-O 3, a statistically significant difference first emerged in cultivar Y2 at 30 days after full expansion of flag leaves (DAFE) and persisted throughout the remainder of the O 3 fumigation period (Fig. 2a, c and g). The effects of O 3 elevation appeared more than 10 days later for the cultivar Y16 than Y2 (Fig. 2b, d and h). The varietal difference in the accelerated senescence was indeed significant for Chl and Car contents and marginally significant for MDA as shown by the interaction between O 3, leaf age and cultivar (Table 3). Gas exchange and photosynthetic capacity E-O 3 induced significant stomatal closure in Y2 beginning at 26 DAFE, which was not the case in Y16 (Fig. 3c and d). The marginally significant interaction between cultivar and O 3 supported the varietal difference in the stomatal response to E-O 3 (Table 3). In addition to the reduced gs, E-O 3 significantly decreased A sat and increased C i along with aging of flag leaves in Y2 (Fig. 3a and e), whereas these effects were not observed in Y16 despite the significant difference at the last two measurements (Fig. 3b and f). The significant interaction between O 3, leaf age and cultivar also indicated that the progressive increase (O 3 by leaf age interaction) in the effects of O 3 on A sat and C i differed between the cultivars (Table 3). There was no significant change in stomatal limitation (l) in either cultivar due to exposure to O 3 (Fig. 4g and h, Table 3), which is in good accordance with the decrease in A sat and gs, and the increase in C i. It can be inferred that nonstomatal factors were the major contributors to reduced photosynthesis rate of Y2 grown at E-O 3. Relative to A-O 3, photosynthetic capacity in E-O 3, as indicated by Vc max and J max values, was reduced significantly in both Y2 and Y16 at the final a few measurements (Fig. 4a d, Table 3). Reduced Vc max /J max was also found in O 3 -treated Y2

6 VARIETAL DIFFERENCE IN WINTER WHEAT RESPONSES TO O Table 3 Analysis of variance of the effects of O 3, leaf age, cultivar and their interactions on investigated variables in winter wheat cultivars Yangmai 16 (Y16) and Yangfumai 2 (Y2) O 3 Leaf age Cultivar O 3 Leaf age O 3 Cultivar Cultivar Leaf age O 3 Cultivar Leaf age Chl Car Chl a/b MDA A sat gs C i Vc max J max Vc max /J max l AQY qp PhiPSII %P %D %X P-values were calculated from mixed linear model analysis. Chl, chlorophyll; Car, Carotenoid; MDA, malondialdehyde; A sat, light-saturated photosynthesis; gs, stomatal conductance; C i, intercellular CO 2 concentration; Vc max, the maximum carboxylation efficiency; J max, the maximum rate of electron transport; l, stomatal limitation to photosynthesis; AQY, apparent quantum efficiency qp, quenching of photochemical efficiency of PSII; PhiPSII, light-adapted apparent quantum efficiency of PSII; %D, the fraction of photons dissipated in the antenna; %P, the fraction of photons utilized in PSII photochemistry; %X, the fraction of absorbed photons by PSII neither used in photochemistry nor dissipated in the PSII. plants since 33 DAFE in comparison with ambient plants (Fig. 4e), suggesting that O 3 induced a larger loss in in vivo Rubisco activity relative to capacity for RuBP regeneration. Chlorophyll a fluorescence E-O 3 caused a significantly earlier and larger decline of AQY in Y2 than Y16, as indicated by significant interaction between O 3, leaf age and cultivar (Fig. 5a and b, Table 3). E-O 3 significantly decreased qp and PhiPSII in both Y2 and Y16 at the final a few measurements (Fig. 5c f, Table 3). Significant interactions between O 3 and leaf age (Table 3) suggest that light capture capacity of flag leaves exposed to E-O 3 decreased more with leaf aging than that at A-O 3. Although there was no significant difference between A-O 3 and E-O 3 in AQY, PhiPSII or qp at flowering stage (Fig. 5 at 20 DAFE), a significantly different responses between the two cultivars was observed at late grain filling stage with a larger decrease in Y2 than in Y16 (Fig. 5, Table 3). The distribution of photons absorbed by PSII antennae was changed significantly. The %P was significantly reduced in E-O 3 for both Y2 and Y16 at late developmental stages (Fig. 6a and b, Table 3), as presaged by the decrease in PhiPSII. It follows that %D was increased significantly in O 3 treated plants at the final three measurements with a larger extent in Y2 than Y16, as indicated by the significant interaction between O 3, leaf age and cultivar (Fig. 6c and d, Table 3). With leaf aging, %X increased in the ambient plants for both cultivars (Fig. 6e and f), however, E-O 3 significantly decreased %X at the final a few measurement in either cultivar (Fig. 6e and f, Table 3). In Y2, often the responses of all investigated fluorescence parameters to E-O 3 were observed later than those of gas exchange, implying that the impairment of the dark reactions of photosynthesis occurred earlier than impairment of process of the light reactions of photosynthetic rate for sensitive cultivar exposed to E-O 3. On the other hand, Y16 showed similar time response to E-O 3 between fluorescence parameters and gas exchange. Discussion In this first ever study on impacts of ozone on wheat in fully open-air field condition, two cultivars of common phenology (Table 1) were investigated. Significant interactions between cultivar, leaf age and O 3 were found

7 586 Z. FENG et al. Fig. 2 Chlorophyll (Chl) and carotenoid (Car) content, the ratio of chlorophyll a/b (Chl a/b) and malondialdehyde content (MDA) in the flag leaves of winter wheat cultivars Yangfumai 2 (Y2) (a, c, e, g) and Yangmai 16 (Y16) (b, d, f, h) grown in ambient (A-O 3 ) and elevated (E-O 3, approximately 1.27 ambient) [O 3 ] treatments from tillering stage to final harvest under fully open-air field conditions. At each time point, statistically significant differences between O 3 treatment for each cultivar are noted with an asterisk at P Error bars represent SD of the means. (Table 3), thereby demonstrating that the responses of these two cultivars to E-O 3 were different during later developmental stages. The O 3 -induced effects of an increase in average [O 3 ] for the 85 days fumigation period from 44.4 ppb in the ambient to 56.4 ppb in the treatment plot occurred about 10 days earlier in Y2 than in Y16, showing Y2 s greater sensitivity to ozone. Different mechanisms of ozone impact on the two cultivars Chamber results indicated that increasing [O 3 ] accelerated foliar senescence in many plants (Pell et al., 1994; Finnan et al., 1998; Miller et al., 1999; Ribas et al., 2005). Senescence is regulated by processes involving chlorophyll degradation, photosynthetic decline and lipid peroxidation (Pell et al., 1994; Mulholland et al., 1997; Farage & Long, 1999; Calatayud et al., 2004; Pleijel et al., 2006). From our study, the 27% elevation of average [O 3 ] from 44.4 to 56.4 ppb caused significant decreases in both Chl and A sat and an increase in MDA in Y2, but no significant effects were observed in Y16 until the final stages of grain filling. MDA concentration, which represents the state of membrane lipid peroxidation, has been shown to be correlated with the degree of O 3 exposure to plants (Ranieri et al., 1996; Calatayud et al., 2004; Ariyaphanphitak et al., 2005; Biswas et al., 2008). Compared with ambient plants, higher MDA in O 3 -treated flag leaf appeared much earlier in Y2 than Y16. From these results, E-O 3 significantly accelerated senescence of the flag leaves more in Y2 than Y16,

8 VARIETAL DIFFERENCE IN WINTER WHEAT RESPONSES TO O Fig. 3 Light-saturated photosynthetic rate (A sat ), stomatal conductance (gs) and intercellular CO 2 concentration (C i ) in the flag leaves of winter wheat cultivars Yangfumai 2 (Y2) (a, c, e) and Yangmai 16 (Y16) (b, d, f) grown in ambient (A-O 3 ) and elevated (E-O 3, approximately 1.27 ambient) [O 3 ] treatments from tillering stage to final harvest under fully open-air field conditions. At each time point, statistically significant differences between O 3 treatment levels for each cultivar are noted with an asterisk at P Error bars represent SD of the means. which caused significant decreases in photosynthetic capacity observed in Y2 rather than in Y16. There are some uncertainties about the role of the stomata in ozone-induced reductions of photosynthetic rates. Ozone can cause opening of stomata, but, in most cases, causes closure (Saxe, 1991), which was in general considered not a direct effect of O 3 but a response to an increased C i resulting from the inhibition of carbon assimilation (Heath & Taylor, 1997). In the present experiment, l was not affected in Y2 during O 3 exposure, although gs of flag leaves was decreased by nearly 15%, suggesting that stomatal closure was not a primary contributor to the reduced A sat seen in Y2. A/C i response curves were used to determine if changes in A can be attributed to nonstomatal factors. Our results showed that O 3 induced significant reductions in Vc max and J max with the decline in Vc max being larger for Y2 (Fig. 4a, c and e). Similar findings were reported for soybean under open-air elevation of [O 3 ] (Morgan et al., 2004). Most enclosure studies on wheat indicate that chronic O 3 fumigation primarily affected Rubisco activity with little effect on the capacity for generating RuBP (Lehnherr et al., 1987; McKee et al., 1995; Farage & Long, 1999). Ozone-induced decrease in Rubisco activity and content appears to be a common feature of ozone exposure across species as it has been reported in Glycine max (Morgan et al., 2004), Pisum sativum (Farage & Long, 1995) and Plantago major (Zheng et al., 2002). The underlying mechanism for the O 3 -induced decline in quantum efficiency was investigated using chlorophyll a fluorescence. During flag leaf development in the two cultivars, E-O 3 induced a significant decrease in excitation energy reaching the PSII reaction centers, as indicated by the decrease in Fv 0 /Fm 0 (1 D%), representative of the efficiency of excitation capture by open PSII reaction centers. qp of flag leaves iny2 decreased late in the season, indicating a reduced

9 588 Z. FENG et al. Fig. 4 The maximum velocity of carboxylation efficiency (Vc max ), the maximum rate of electron transport (J max ), the ratio of Vc max /J max, stomatal limitation to photosynthesis (l) in the flag leaves of winter wheat cultivars Yangfumai 2 (Y2) (a, c, e, g) and Yangmai 16 (Y16) (b, d, f, h) grown in ambient (A-O 3 ) and elevated (E-O 3, approximately 1.27 ambient) [O 3 ] treatments from tillering stage to final harvest under fully open-air field conditions. At each time point, statistically significant differences between O 3 treatment levels for each cultivar are noted with an asterisk star at P Error bars, represent SD of the means. net rate of re-oxidation of QA which in turn resulted in a larger fraction of closed PSII centers and thus a lower PSII quantum efficiency in E-O 3. A significant decrease in qp and PhiPSII may correlate with a decrease in the proportion of available excitation energy used in the photochemistry, as indicated by lower %P in O 3 -treated plants. The results support the notion that the decrease in the quantum yield of PSII electron transport may be a mechanism to down-regulate photosynthetic electron transport so that production of ATP and NADPH is maintained in equilibrium with the decreased demand in the Calvin cycle in ozonated leaves. Similar findings have been reported for spinach, lettuce, snap bean,and wheat exposed to E-O 3 (Calatayud et al., 2002, 2004; Flowers et al., 2007). Possible causes for the different response between Y2 and Y16 to O 3 It is well known that O 3 penetrates plant leaves through open stomata and dissolves into the apoplastic fluid, suggesting that plants with large gs would allow a higher O 3 flux into the air spaces surrounding the mesophyll cells. Numerous studies indicated that crop/cultivar/clone sensitivity to O 3 was correlated with gs before O 3 exposure in wheat (Pleijel et al., 2006; Biswas et al., 2008) and bush bean (Elagoz et al., 2006). The role of gs as a determinant of ozone sensitivity was further illustrated in experiments with plants exposed simultaneously to O 3 and elevated atmospheric CO 2 or drought, which showed reduced O 3

10 VARIETAL DIFFERENCE IN WINTER WHEAT RESPONSES TO O Fig. 5 The maximum apparent quantum efficiency (AQY), quenching of photochemical efficiency of PSII (qp) and light-adapted apparent quantum yield of PSII (PhiPSII) in the flag leaves of winter wheat cultivars Yangfumai 2 (Y2) (a, c, e) and Yangmai 16 (Y16) (b, d, f) grown in ambient (A-O 3 ) and elevated (E-O 3, approximately 1.27 ambient) [O 3 ] treatments from tillering stage to final harvest under fully open-air field conditions. At each time point, statistically significant differences between O 3 treatment levels for each cultivar are noted with an asterisk at P Error bars, represent SD of the means. injury, presumably due in large part to lower gs and consequently lowered O 3 flux (McKee et al., 1995; Fiscus et al., 1997, 2005; Reid & Fiscus, 1998; Morgan et al., 2003; Feng et al., 2008, 2009). We tested if the different responses between Y2 and Y16 to E-O 3 can be related with the difference in gs between the two cultivars. In Fig. 3c, E-O 3 induced significant stomatal closure in Y2 starting at 26 DAFE, which suggests that injury by E-O 3 occurred after 23 DAFE. An ANOVA result with the gs values measured before 26 DAFE showed a slightly larger gs (0.386 mol m 2 s 1 ) in Y16 than that (0.336 mol m 2 s 1 ) in Y2 with no significant difference (P ). The higher sensitivity in Y2 cannot therefore be accounted for by the varietal difference in gs. Our results with the detached leaves are consistent with a result of gas exchange measurement with intact leaves of the same cultivars in the field. The measurement was done with a LI-6400 system under saturating PPFD at 1500 mmol m 2 s 1 and CO 2 concentration at 380 ppm. The results showed gs values (A-Y2: mol m 2 s 1 ; A-Y16: mol m 2 s 1 ) in the same range as our measurements (A-Y2: mol m 2 s 1 ; A-Y16: mol m 2 s 1 ) with no difference between the cultivars at an early stage of flag leaf life span (April 23) (J. Liang, unpublished results). Besides stomatal control, another popular notion suggests that ASC could act as the first line of defense against O 3 damage (Chameides, 1989; Conklin & Barth, 2004; Fiscus et al., 2005). In our experiments, higher apoplastic ASC was indeed found in Y16, relative to Y2 (Z. Feng et al., unpublished results). Moreover, higher antioxidant enzymes were found in O 3 -treated leaves of Y16, in comparison with Y2 (Z. Feng et al., unpublished results). All these results suggested that antioxidant system in the apoplast and the symplast in Y16 protected against E-O 3. In summary, with the same phenology of two cultivars, Y2 and Y16 were similar in physiological traits in ambient rings. However, the response to 27% increase of [O 3 ] in Y2 was about 10d earlier than that in Y16 in most investigated variables, suggesting that Y2 is more sensitive to O 3 than Y16. Other factors than gs

11 590 Z. FENG et al. Fig. 6 The fraction of photons utilized in PSII photochemistry (%P), the fraction of photons dissipated in the antenna (%D); the fraction of absorbed photons by PSII neither used in photochemistry nor dissipated in the PSII (%X) in the flag leaves of winter wheat cultivars Yangfumai 2 (Y2) (a, c, e) and Yangmai 16 (Y16) (b, d, f) grown in ambient (A-O 3 ) and elevated (E-O 3, approximately 1.27 ambient) [O 3 ] treatments from tillering stage to final harvest under fully open-air field conditions. At each time point, statistically significant differences between O 3 treatment levels for each cultivar are noted with an asterisk at P Error bars, represent SD of the means. contributed to the differential response to O 3 between the two cultivars. Our findings will be useful for scientists to select O 3 -tolerant wheat cultivars against the rising surface [O 3 ] in East and South Asia. Acknowledgements This study was supported by Eco-Frontier Fellowship (07-C062-03), the Global Environment Research Fund (C-062) of the Ministry of Environment, Japan, The International S & T Cooperation Program of China (2009DFA31110), Fellowship of Japan Society for the Promotion of Science (P09120) and the International Cooperation Key Project of Chinese Academy of Sciences (GJHZ0748). We are grateful to Prof. G. Liu and Mr. H. Y. Tang for their technical support in the free-air ozone release system. References Ariyaphanphitak W, Chidthaisong A, Sarobol E, Bashkin VN, Towprayoon S (2005) Effects of elevated ozone concentrations on Thai Jasmine rice cultivars (Oryza sativa L.). Water Air and Soil Pollution, 167, Ashmore MR (2005) Assessing the future global impacts of ozone on vegetation. Plant, Cell and Environment, 28, Ashmore MR, Toet S, Emberson L (2006) Ozone a significant threat to future world food production? New Phytologist, 170, Atkinson CJ, Robe SV, Winner WE (1988) The relationship between changes in photosynthesis and growth for radish plants fumigated with SO2 and O3. New Phytologist, 110, Barnes JD, Velissariou D, Davison AW, Holevas CD (1990) Comparative ozone sensitivity of old and modern Greek cultivars of wheat. New Phytologist, 116, Biswas DK, Xu H, Li YG, Sun JZ, Wang XZ, Han XG, Jiang GM (2008) Genotypic differences in leaf biochemical, physiological and growth responses to ozone in 20 winter wheat cultivars released over the past 60 years. Global Change Biology, 14, Calatayud A, Lglesias DJ, Talon M, Barreno E (2004) Response of spinach leaves (Spinacia oleracea L.) to ozone measured by gas exchange, chlorophyll a fluorescence, antioxidant systems, and lipid peroxidation. Photosynthetica, 42, Calatayud A, Ramirez JW, Iglesias DJ, Barreno E (2002) Effects of ozone on photosynthetic CO2 exchange, chlorophyll a fluorescence and antioxidant systems in lettuce leaves. Physiologia Plantarum, 116, Castagna A, Nali C, Ciompi S, Lorenzini G, Soldatini GF, Ranieri A (2001) Ozone exposure affects photosynthesis of pumpkin (Cucurbita pepo) plants. New Phytologist, 152, Chameides W (1989) The chemistry of ozone deposition to plant leaves: the role of ascorbic acid. Environmental Science and Technology, 23, Conklin PL, Barth C (2004) Ascorbic acid, a familiar small molecule intertwined in the response of plants to ozone, pathogens, and the onset of senescence. Plant, Cell and Environment, 27, Demmig-Adams B, Adams WW, Baker DH, Logan BA, Bowling DR, Verhoeven AS (1996) Using chlorophyll fluorescence to assess the fraction of absorbed light

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