Agriculture, Ecosystems and Environment 134 (2009) 168–177 Contents lists available at ScienceDirect Agriculture, Ecosystems and Environment journal homepage: www.elsevier.com/locate/agee Impacts of methods and sites of plant breeding on ozone sensitivity in winter wheat cultivars D.K. Biswas a,1,2, H. Xu a,2, J.C. Yang b, Y.G. Li a,*, S.B. Chen a, C.D. Jiang a, W.D. Li a, K.P. Ma a, S.K. Adhikary c, X.Z. Wang d, G.M. Jiang e,a,* a State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, The Chinese Academy of Sciences, 20 Nanxincun, 100093 Beijing, PR China Beijing Museum of Natural History, 126 Tianqiao ST, 100050 Beijing, PR China Agrotechnology Discipline, Khulna University, Khulna, Bangladesh d Department of Biology, Indiana University-Purdue University Indianapolis, 723 W. Michigan State, IN 46202, USA e State Key Laboratory of Crop Biology, Shandong Agricultural University, No. 61, Daizong Avenue, 271018 Tai’an, PR China b c A R T I C L E I N F O A B S T R A C T Article history: Received 5 April 2009 Received in revised form 11 June 2009 Accepted 16 June 2009 Available online 10 July 2009 Development and use of ozone (O3)-resistant crop cultivars are key measures to avoid agricultural yield reduction in a high O3 environment. However, little is known about the impacts of breeding methods and breeding sites on the development of O3 tolerance in winter wheat cultivars. To explore such impacts, 20 Chinese winter wheat cultivars bred using four breeding methods (viz. introduction, reselection, conventional breeding and hybridization) at four breeding sites having different levels of O3 exposures, were exposed to charcoal-filtered (CF) air or high O3 (82 ppb, 7 h d1) for 21 days. O3 tolerance of cultivars was assessed by the relative levels of visible injury, growth, gas exchange, dark respiration, antioxidative activities and oxidative modification of proteins and cellular membranes. We found that conventional breeding and hybridization demonstrated higher potential capacity for O3 tolerance as indicated by a higher level of ascorbate and peroxidase activity in cultivars exposed to CF air. Despite the highest potential capacity for O3 tolerance, hybridization displayed the lowest O3 tolerance as represented by antioxidative activities, oxidative stress, photosynthesis and growth. The causes of higher O3 sensitivity in hybrid cultivars included lower O3 exclusion by stomatal closure, higher reduction in antioxidative activities, higher O3-induced modification of proteins and cellular membranes, lower level of repair of O3-induced cellular damage and higher loss of assimilation rate as well as growth in O3 relative to control plants. Cultivars bred at breeding sites experiencing higher ambient O3 exposures demonstrated higher potential capacity for O3 tolerance. The observed O3 tolerance in cultivars bred by different breeding methods was uncorrelated to ambient O3 levels in breeding sites as well as to the potential O3 tolerance capacity as observed in CF air. Results from our experiment, therefore, clearly indicated that potential O3 sensitivity would have little use in predicting actual O3 tolerance of winter wheat cultivars. Our findings also suggested that sensitivity to O3 in winter wheat cultivars was related to breeding methods, but not to O3 concentrations in breeding sites. ß 2009 Elsevier B.V. All rights reserved. Keywords: Breeding sites Triticum aestivum O3 tolerance Plant breeding Winter wheat 1. Introduction Ozone (O3) tolerance is a heritable trait (Damicone and Manning, 1987; Barnes et al., 1999; Whitfield et al., 1997; Fiscus * Corresponding authors at: State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, The Chinese Academy of Sciences, 20 Nanxincun, 100093 Beijing, PR China. Tel.: +86 1062836506; fax: +86 1082595380. E-mail addresses: [email protected] (Y.G. Li), [email protected] (G.M. Jiang). 1 Present address: Department of Plant Science (ZEPS), University College Cork, Cork, Ireland. 2 Equal contribution. 0167-8809/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.agee.2009.06.009 et al., 2005) and cultivar variation in O3 sensitivity exists in wheat (Barnes et al., 1990; Velissariou et al., 1992; Pleijel et al., 2006; Biswas et al., 2008a). It has been documented that higher O3 sensitivity of modern wheat (Triticum aestivum L.) (AABBDD) is attributed to higher O3 sensitivity of Aegilops tauschii (DD), but not to Triticum turgidum ssp. durum (AABB) during speciation (Biswas et al., 2008b). These indicate the magnitude of genetic control of O3 tolerance in wheat and its exploitation through plant breeding (Biswas et al., 2008b). On the other hand, genotypic variation in O3 exclusion and detoxification (Polle et al., 1993; Fiscus et al., 2005; Biswas et al., 2008a) largely depends on growing environment as well as the degree of adaptation of an individual to a pollutant (Roose et al., 1982; Bassin et al., 2004). It also depends on crop D.K. Biswas et al. / Agriculture, Ecosystems and Environment 134 (2009) 168–177 breeding systems, gene flow, generation time and timing of selection during the life cycle of the individuals (Pitelka, 1988; Whitfield et al., 1997; Barnes et al., 1999). Although genetic variation in O3 sensitivity exits in wheat cultivars (Barnes et al., 1990; Biswas et al., 2008a) as well as in the donor species of the A, B and D genomes of modern wheat (Biswas et al., 2008b), up to now, there has been no study exploring the impacts of breeding methods and sites on the modification of O3 sensitivity in wheat. Chinese wheat breeders have succeeded greatly in improving wheat yield over years through a variety of breeding methods including introduction, reselection, conventional breeding and hybridization (Jiang et al., 2003). Over the past several decades, on the other hand, continual degradation of air quality of Asian countries, especially rising O3 levels in China, and their possible impacts on crop yield have been well documented (Chameides et al., 1999; Street and Waldhoff, 2000; Aunan et al., 2000; Wang and Mauzerall, 2004; Ashmore, 2005; Wang et al., 2007; Fuhrer, 2009). Moreover, it has been warned that O3 concentrations in many Chinese provinces exceed potentially damaging levels (>120 ppb) for many hours during the summer months (Wang et al., 2007). However, it is well known that O3 has significant negative effects on wheat yield (Fuhrer et al., 1989, 1992; Heagle et al., 2000; McKee and Long, 2001) and also a fact that Chinese wheat varieties have been bred and tested at experimental stations located mostly around metropolitan areas, where O3 concentrations are normally higher (Zheng et al., 1998; Chameides et al., 1999; Street and Waldhoff, 2000; Wang and Mauzerall, 2004). As a result, plant breeding methods having different selection procedures as well as high ambient O3 levels in breeding sites might interact with the development of O3 tolerance in wheat cultivars. Although no wheat breeding program has been directed to develop O3-resistant cultivars, natural as well as artificial selection might result in O3-tolerant crop cultivars in a breeding site having higher O3 levels (Barnes et al., 1999). Because plant breeders usually select vigorous and high-yielding individuals regardless of cause during field trials of crop improvement. In an earlier experiment (Barnes et al., 1990), O3 resistance of ten spring wheat cultivars was found to be uncorrelated with the O3 level of the breeding site. However, this has not yet been verified using cultivars released in different breeding sites having different levels of ambient O3 exposures. In addition, although O3 resistance is a heritable trait and its segregation largely depends on breeding strategy, no study has been made to assess O3 tolerance in crop cultivars bred by different breeding methods. Crop sensitivity to O3 is typically assessed by the decline in growth and/or the appearance of O3 injury. It has been verified that O3 sensitivity in wheat cultivars as determined by growth and chlorophyll fluorescence at their vegetative stage is proportional to cultivar sensitivity to O3 in terms of yield (Pleijel et al., 2006). Chlorophyll fluorescence and gas exchange represent useful and non-destructive tools for in vivo stress detection and are widely used to examine the effects of O3 on photosynthesis (Guidi et al., 1997). On the other hand, levels of leaf chlorophyll, antioxidative activities and oxidative modifications of proteins and lipid provide more insights into the mechanisms of O3 tolerance (Calatayud et al., 2003; Biswas et al., 2008a). Similarly, dark respiration quantifies the magnitude of repair of O3-induced cellular damage in plants (Amthor, 1988; Biswas et al., 2008a). Despite O3 tolerance of crop plants largely depends on breeding systems as well as degree of adaptation of an individual to a polluted environment (Damicone and Manning, 1987; Barnes et al., 1999), it is unknown if the breeding methods or breeding sites will affect the magnitude of O3 tolerance of winter wheat cultivars. It has been recently reported that hybrid rice cultivars are more susceptible to O3 in terms of yield than inbred ones, but 169 the details of physiological or biochemical mechanisms have not been studied (Pang et al., 2009; Shi et al., 2009). It is also unknown if breeding methods and ambient O3 levels in breeding sites will affect the potential capacity for O3 tolerance in crop cultivars. To clarify such uncertainties, we utilized 20 Chinese winter wheat cultivars bred using different breeding methods at different breeding sites. O3 tolerance was assessed by the levels of visible injury, growth, physiological and leaf biochemical parameters. We hypothesized that plant breeding methods and ambient O3 levels in breeding sites would interact with growth, physiological and biochemical responses of cultivars of winter wheat exposed to elevated O3 or CF air. Results from our experiment might be valuable for breeding wheat cultivars that will tolerate increased levels of O3 pollution in the future. 2. Materials and methods 2.1. Plant culture and O3 fumigation Twenty cultivars of winter wheat (Triticum aestivum L.) were obtained from the Institute of Genetics and Developmental Biology, the Chinese Academy of Sciences. All the cultivars used in this study were selected on the basis of breeding methods (viz. introduction, reselection conventional breeding and hybridization) and breeding sites (viz. Beijing, Hebei, Shandong and Shanxi provinces in China) (Table 1). Generally, winter wheat breeding in China has been directed to achieve several major targets including increasing seed size, shortening plant height, improving resistance to lodging, cold, drought, diseases and insect pests (Jiang et al., 2003). Detailed characteristics of the selected winter wheat cultivars have also been presented in a previous report (Biswas et al., 2008a). Ambient O3 concentrations (SUM06) in the selected breeding sites in 1990 were as follows: Shandong (40– 50 ppmh) > Shanxi (24–34 ppmh) > Hebei (17–27 ppmh) > BeijBeijing (7–17 ppmh) (Wang and Mauzerall, 2004) (Table 2). Similar trends of O3 concentrations in these breeding sites were also observed in elsewhere (Street and Waldhoff, 2000; Wang et al., 2007). In 2005, three seeds were sown in each of 48 plastic pots (6 cm in diameter, 9 cm in height) per cultivar in a temperature controlled double glazed greenhouse at the Institute of Botany of the Chinese Academy of Sciences, Beijing, China. Pots were filled with field clay loam soil containing organic C, total N, total P and Table 1 List of winter wheat cultivars bred following different breeding methods at different breeding sites in China. Cultivars Breeding method Breeding site Yanda 1817 Zaoyangmai Huabei 187 Beijing 6 Beijing 10 Nongda 139 Yannong 15 Hannong 2 Fengkang 2 Beinong 2 Nongda 015 Xinong 2611 Beijing 837 Jingdong 8 Nongda 3214 Gaocheng 8910 Jinmai 60 Hantan 6172 Xiaoyan 22 Heng 4338 Introduction Introduction Hybridization Resection Resection Hybridization Hybridization Hybridization Hybridization Hybridization Conventional breeding Hybridization Conventional breeding Conventional breeding Hybridization Hybridization Hybridization Hybridization Hybridization Hybridization Beijing Beijing Beijing Beijing Beijing Beijing Shandong Hebei Beijing Beijing Beijing Shanxi Beijing Beijing Beijing Hebei Shanxi Hebei Beijing Hebei 170 D.K. Biswas et al. / Agriculture, Ecosystems and Environment 134 (2009) 168–177 Table 2 Ambient O3 exposure in the four breeding sites based on 7 h (9:00–15:59) mean (M7) and cumulative O3 index (SUM06) averaged from 90 days data of different growing seasons in 1990 (Wang and Mauzerall, 2004). Name of site M7 (ppb) SUM06 (ppmh) Beijing Hebei Shandong Shanxi 43–49 48–53 55–60 53–58 7–17 17–27 40–50 24–34 total K at the rate of 1.24%, 0.045%, 296 mg kg1 and 14.7 g kg1, respectively. No chemical fertilizer was applied. Seedlings were thinned to one per pot on seven days after planting (DAP). On 11 DAP, 240 pots were moved to four open top chambers (OTC, 1.2 m in diameter, 1.6 m in height) placed in the same greenhouse. The OTCs were ventilated continuously (24 h d1) with air through activated charcoal filters attached to fan box. Seedlings were allowed to grow till 17 DAP to adapt to chamber environments before O3 exposure. During this adaptation period, all plants received charcoal-filtered air (<5 ppb O3). The gas dispensing system of the OTCs was constructed following the methodology described by Uprety (1998). The chambers were illuminated by natural daylight supplemented by fluorescence light providing a photosynthetic photon flux density (PPFD) of approximately 230 mmol m2 s1 at plant canopy height, yielding a 14 h photoperiod. The maximum PPFD in the chambers was approximately 650 mmol m2 s1. The temperature in the OTCs varied from 17 8C (night) to 30 8C (day) and the relative humidity (RH) was 67–86% during the experiment runs. Plants were kept wellwatered throughout the experiment and the hard soil crust formed after irrigation was broken to ensure better aeration in the soil. On 18 DAP, O3 was added to the charcoal-filtered air stream entering two of the chambers to maintain a concentration of 82 5 ppb, 7 h d1 (10:00–17:00 h) for 21 days. The other two chambers were set up the same way but without O3 addition. The treatments (+O3, elevated O3; CF, charcoal-filtered control) were assigned randomly to the chambers and replicated twice. O3 was generated by electrical discharge using ambient oxygen (Balaguer et al., 1995) with an O3 generator (JQ-6A, Telijie Co., Beijing) and bubbled through distilled water before entering the two high O3 chambers. Water traps were used to remove harmful compounds other than O3 (Balaguer et al., 1995). The flow of O3-enriched air to the OTCs was regulated by manual mass flow controllers. O3 concentrations in the OTCs were continuously monitored at approximately 10 cm above the plant canopy using an analyzer (APOA-360, Horiba, Ltd, Japan), which was cross-calibrated once before starting O3 treatment with another O3 monitor (ML 9810B, Eco-Tech, Canada). In order to minimize chamber effects and environmental heterogeneity, the associated treatments were rotated between the chambers with the plants being randomized within the chambers at every other day throughout the experiment. 2.2. Visible symptoms of O3 damage Visible symptoms were assessed on the 3rd youngest leaf of the main stem as well as whole plant after 21 days of O3 exposure on 39 DAP, when the plants developed a total of 5–7 expanded leaves. The percentage of mottled or necrotic area on the leaves was assessed for five plants per cultivar sampled from control and O3 fumigated chambers. 2.3. Determination of leaf biochemical parameters After exposure to O3 for 21 days, the youngest fully expanded leaves on the main stems that exhibited no visible symptoms of O3 damage were harvested from plants grown in both control and O3fumigated chambers. Leaf samples were frozen in liquid nitrogen immediately after excision and transferred to an ultra-freezer at 80 8C until the time of assay. Three samples per cultivar from each chamber (n = 6) were analyzed for biochemical parameters. Concentrations were calculated on fresh weight (f. wt.) basis. Chemicals used in analysis were obtained from Sigma Chemicals Co., St. Louis, MO, USA. Details of the methodology of biochemical analysis for chlorophyll (Chls), ascorbate (AsA), soluble protein, peroxidase activity (POD) and malondialdehyde (MDA) content were reported in an earlier publication (Biswas et al., 2008a). 2.4. Gas exchange and chlorophyll fluorescence measurements Three plants per cultivar were sampled from each chamber per treatment on the 19th day of O3 fumigation. Gas exchange of the most recently fully expanded leaf (i.e., after emergence of ligules) on the main stem was measured with a portable Gas Exchange Fluorescence System (GFS-3000, Heinz Walz, Germany). The leaves used for photosynthesis measurements were also used for dark respiration measurements. Detailed methodology for gas exchange and dark respiration was reported earlier in elsewhere (Biswas et al., 2008a). On the 20th day of O3 fumigation, after 40 min dark adaptation, modulated chlorophyll fluorescence measurements were made in the middle of intact youngest fully expanded leaves that appeared no visible symptoms of O3 damage using a PAM 2000 (Heinz, Walz, Germany). The minimum fluorescence (F0) was determined with modulated light which was sufficiently low (<1 mmol m2 s1), so as not to induce any significant variable fluorescence. The maximum fluorescence (Fm) was determined using a 0.8 s saturating pulse at 4950 mmol m2 s1. After dark-adapted fluorescence measurements, the leaf was continuously illuminated with actinic light at the intensity of 200 mmol m2 s1. The steady state fluorescence (Fs) was reached within 3 min. Then a saturating pulse was imposed to determine the maximum fluorescence in the light-adapted state (F 0m ). The minimum fluorescence in the light-adapted state (F 00 ) was determined during a brief interruption of actinic illumination in the presence of far-red illumination. After recording the fluorescence key parameters in both dark and light-adapted state, we calculated: (1) variable fluorescence, Fv = Fm F0; (2) maximum photochemical efficiency in the dark-adapted state, Fv/Fm (Krause and Weis, 1991); (3) quantum yield of PSII, FPSII ¼ ððF 0m F s Þ=F 0m Þ; (4) photochemical quenching coefficient, qP ¼ ðF 0m F s Þ=ðF 0m F 00 Þ; (5) non-photochemical quenching coefficient, qN ¼ 1 ðF 0m F 00 Þ=ðF m F 0 Þ (van Kooten and Snel, 1990) and (6) electron transport rate, ETR = Yield PAR 0.5 0.84 (Meyer et al., 1997). 2.5. Dry matter accumulation and partitioning Plants were sampled for biomass accumulation and partitioning after 21 days of O3 exposure. Four plants per cultivar (n = 8) were collected from each chamber. Plants were separated into shoot and root before being dried to constant weight in an oven at 72 8C. 2.6. Statistical analysis The experiment consisted of two randomized blocks of two treatments with 12 plants per replicate. Statistical analyses of data were performed using analysis of variance (ANOVA) in the General Linear Model procedure of SPSS (Ver. 13, SPSS, Chicago, IL, USA). The main effects of breeding methods and breeding sites in O3 and CF air were analyzed using one-way ANOVA on the measured variables. Differences between treatment were considered significant if P < 0.05. D.K. Biswas et al. / Agriculture, Ecosystems and Environment 134 (2009) 168–177 Table 3 Impacts of breeding methods and sites on the development of O3 symptoms on the 3rd youngest leaf of main stem of winter wheat cultivars. Treatment Visible symptoms (%) (a) Effects of breeding methods Introduction Reselection Con. breeding Hybridization P value 51 7 38 7 60 6 58 3 0.119 (b) Effects of breeding sites Beijing Hebei Shandong Shanxi P value 50 3 58 5 69 10 55 7 0.309 3. Results 3.1. Visible O3 injury Scoring visible symptoms after 21 days of exposure to O3 revealed that breeding methods had marginally significant effects on the development of visible symptoms in winter wheat cultivars ranging from 26 to 76% on the 3rd youngest leaf of the main stem (Table 3). Cultivars bred with reselection displayed the lowest visible O3 injury, while those bred with conventional breeding and hybridization appeared the highest. However, breeding sites had no effect on the development of visible O3 symptoms in winter wheat cultivars. Premature senescence of the leaf 1 and 2 was noted in all O3-exposed cultivars irrespective of breeding methods and sites. 3.2. Leaf biochemical properties The breeding method had a considerable effect on the levels of chlorophyll, AsA, soluble protein and MDA in cultivars exposed to CF air (Fig. 1). Cultivars bred by hybridization possessed the highest Chls content and those bred by reselection showed the lowest. Significantly higher AsA and soluble protein contents were observed in cultivars bred with conventional breeding and hybridization than in those bred with introduction and reselection. Conventional breeding and reselection showed higher MDA than introduction and hybridization. In O3-treated cultivars, the breeding method strongly influenced the levels of Chls, POD, soluble protein and MDA. Significantly higher levels of Chls, soluble protein and MDA accompanied by lower levels of POD activity were observed in cultivars bred with conventional breeding and hybridization than in those bred with introduction and resection. Breeding sites had significant effects on the levels of AsA, POD activity and MDA in CF control cultivars. A higher AsA level was noted in the cultivar bred in Shandong than in those bred in the other sites. Cultivars from Shandong and Shanxi displayed higher POD activity than those from Beijing and Hebei. The highest MDA level was found in cultivars bred in Hebei, while those bred in Shandong and Shanxi showed the lowest. In O3-exposed cultivars, the breeding site had a significant effect on the levels of AsA, POD activity, soluble proteins and MDA. Significantly higher levels of AsA and POD activity were noted in cultivars bred in Beijing, Shandong and Shanxi than in those bred in Hebei. The highest and lowest levels of soluble protein were observed in cultivars from Shandong and Hebei, respectively. Cultivars from Hebei displayed the highest levels of MDA, while those from Shandong and Shanxi showed the lowest. 171 3.3. Gas exchange and chlorophyll fluorescence Breeding methods had significant effects on gas exchange and fluorescence properties in CF-control cultivars (Figs. 2 and 3). Cultivars bred with conventional breeding and hybridization had higher Asat, ITE and Fv/Fm than those bred with introduction and reselection. Cultivars bred with introduction showed the highest Ci. The highest and lowest gs and KPSII were noted in cultivars bred with hybridization and introduction, respectively. Cultivars bred using the method introduction showed the lowest qP and ETR accompanied with the highest qN. Breeding methods showed significant effects on the impacts of elevated O3 on gas exchange and chlorophyll fluorescence. Cultivars bred with conventional breeding and hybridization had higher Asat, gs and ETR than those bred with introduction and reselection. The highest and lowest Ci were noted in cultivars bred with conventional breeding and reselection, respectively. The highest Fv/Fm was observed in cultivars bred with hybridization, while those bred with introduction were found to be the lowest. The cultivars bred with reselection showed the highest qP among breeding methods. Cultivars bred using introduction and reselection had higher Rd than those bred using conventional breeding and hybridization in CF air or high O3. CF air-exposed cultivars bred in Shanxi and Shandong had higher Asat and ITE than those bred in Beijing and Hebei. The highest and lowest Ci were observed in cultivars bred in Shandong and Hebei, respectively. O3-treated cultivars bred in Shandong and Shanxi showed higher Asat than those bred in Beijing and Hebei. Cultivars bred in Hebei demonstrated the highest gs, while that bred in Shandong showed the lowest. The highest and the lowest ITE were observed in the cultivars bred in Shandong and Beijing, respectively. Significantly higher Fv/Fm was observed in cultivars from Hebei and Shanxi than in those from Beijing and Shandong at CF air. The highest KPSII, qP and ETR accompanied with the lowest qN were noted in the cultivar bred in Shandong. The O3-treated cultivars bred from Shanxi had the highest Fv/Fm, while that bred from Shandong displayed the lowest. The highest KPSII, qP and ETR accompanied with the lowest qN were observed in the cultivar bred in Shandong. Cultivars bred in Beijing, Shandong and Shanxi had higher Rd than those bred in Hebei in CF air or elevated O3. 3.4. Biomass accumulation and partitioning In CF air, cultivars bred using methods reselection, conventional breeding and hybridization had statistically similar and higher shoot, root and total mass than those bred using the method introduction (Fig. 4). In elevated O3, cultivars bred with reselection demonstrated the highest shoot, root and total mass, while those bred with hybridization showed the lowest. There was no significant difference in biomass accumulation and partitioning in cultivars bred in different breeding sites at CF air. Breeding sites had a significant effect on the impacts of elevated O3 on biomass accumulation and partitioning in wheat cultivars. Cultivars from Beijing had considerably higher shoot mass than those from Hebei, Shandong and Shanxi under high O3. Significantly higher root mass was noted in cultivars bred in Shandong and Beijing than in those bred in Hebei and Shanxi. Cultivars from Beijing and Shandong had higher total mass than those from Hebei and Shanxi. 4. Discussion 4.1. Visible symptoms of O3 damage in winter wheat as affected by breeding methods and sites Winter wheat exhibited a large genotypic variation (from 26 to 76%) in the development of visible O3 injury on the 3rd youngest 172 D.K. Biswas et al. / Agriculture, Ecosystems and Environment 134 (2009) 168–177 Fig. 1. Effects of breeding methods and sites on leaf biochemical properties of cultivars of winter wheat exposed to CF air or elevated O3. Control plants (unfilled bar) received charcoal-filtered air (CF, <5 ppb O3) and O3-treated plants (filled bar) were exposed to 82 5 ppb O3. Percent change (line) indicates changes () in O3-exposed relative to control plants, (+O3–CF)/CF. n = 6 for each mean (1 S.E.M.). Asterisks denote significant difference between O3-treated and control plants. (*) 0.05, (**) 0.01, (***) 0.001. leaf of the main stem, indicating that all the cultivars used in this study were sensitive to O3. Our results are consistent with the earlier report on spring wheat cultivars exposed to 90 ppb O3 for 21 days (Barnes et al., 1990). Cultivars bred by hybridization and conventional breeding appeared as the most sensitive to O3, whereas those bred by reselection behaved as the most resistant to O3 in terms of visible injury. Our results suggest that cultivars were bred by hybridization mostly for their higher yields, but not for their resistance of visible O3 injury. On the other hand, breeding sites had no effect on the development of O3 injury in wheat cultivars at elevated O3. This indicates that the most O3 sensitive wheat lines in terms of visible injury could be discarded unintentionally by plant breeders during field trials or the actual O3 flux might not be high enough to develop visible injury in winter wheat cultivars in a breeding site with higher O3 concentrations. 4.2. Impacts of breeding methods on the potential and observed sensitivity to O3 in winter wheat cultivars Breeding methods exhibited a considerable variation in the magnitude of potential sensitivity to O3 in winter wheat cultivars exposed to CF air. Cultivars bred using conventional breeding and D.K. Biswas et al. / Agriculture, Ecosystems and Environment 134 (2009) 168–177 173 Fig. 2. Effects of breeding methods and sites on gas exchange and dark respiration of winter wheat cultivars exposed to CF air or elevated O3. Control plants (unfilled bar) received charcoal-filtered air (CF, <5 ppb O3) and O3-treated plants (filled bar) were exposed to 82 5 ppb O3. Percent change (line) indicates changes () in O3-exposed relative to control plants, (+O3–CF)/CF. n = 6–8 for each mean (1 S.E.M.). Asterisks denote significant difference between control and O3-treated plants. (*) 0.05, (**) 0.01, (***) 0.001. hybridization could be more tolerant to O3 as indicated by higher levels of AsA than those bred using introduction and reselection. Similarly, higher leaf soluble proteins in cultivars bred by conventional breeding and hybridization reflected higher levels of Rubisco commonly associated with higher photosynthetic activity than in those bred by introduction and reselection. This was further confirmed by higher Asat and Fv/Fm in cultivars bred by hybridization and conventional breeding. However, the highest gs and Chls content in hybrid cultivars can have led to higher O3 sensitivity as higher gas exchange and Chls content have been found to be associated with higher O3 sensitivity in wheat (Pleijel et al., 2006). On the other hand, cultivars bred using reselection and introduction may be more resistant to O3 as documented by lower gas exchange characteristic and higher levels of Rd associated with repair of O3-induced cellular damage (Biswas et al., 2008a). However, the cultivars bred by conventional breeding and hybridization demonstrated higher decease in AsA and lower increase in POD activity in high O3 relative to CF air than those bred by introduction and reselection. As a result, both hybrid and conventional bred cultivars had higher O3-induced oxidative stress 174 D.K. Biswas et al. / Agriculture, Ecosystems and Environment 134 (2009) 168–177 Fig. 3. Effects of breeding methods and sites on chlorophyll fluorescence of cultivars of winter wheat exposed to CF air or elevated O3. Control plants (unfilled bar) received charcoal-filtered air (CF, <5 ppb O3) and O3-treated plants (filled bar) were exposed to 82 5 ppb O3. Percent change (line) indicates changes () in O3-exposed relative to control plants, (+O3–CF)/CF. n = 6 for each mean (1 S.E.M.). Asterisks denote significant difference between control and O3-treated plants. (*) 0.05, (**) 0.01, (***) 0.001. as evidenced by higher relative decrease and increase in Chls and MDA, respectively. Loss of chlorophyll and associated enzymes has been proposed as consequence of structural and chemical changes by O3-induced reactive oxygen species (ROS) and therefore might be a mechanistic basis for reduced photosynthesis commonly associated with O3 exposure (Reichenauer et al., 1998; Long and Naidu, 2002; Anderson et al., 2003). Consequently, lower relative increase in MDA accompanied with lower relative decease in soluble proteins in cultivars bred by reselection and introduction suggested that O3-induced modification of proteins and cellular membrane was lower in these cultivars than in those bred by conventional breeding and hybridization. This can be further demonstrated by higher relative decreases in Asat or ITE accompanied with higher relative increase in Ci in cultivars bred using hybridization and conventional breeding than in those bred using introduction and reselection. This might be conceivable as cultivars bred by conventional breeding and hybridization had higher O3 flux and hence higher O3-induced impairment of mesophyll activity (Farage et al., 1991; Long and Naidu, 2002; Biswas et al., 2008a) or photoinhibition as indicated by higher relative decrease in Fv/Fm, KPSII, qP and ETR accompanied with higher relative increase in qN. Moreover, repair of O3-induced D.K. Biswas et al. / Agriculture, Ecosystems and Environment 134 (2009) 168–177 175 Fig. 4. Effects of breeding methods and sites on biomass accumulation and partitioning of winter wheat cultivars exposed to CF air or elevated O3. Control plants (unfilled bar) received charcoal-filtered air (CF, <5 ppb O3) and O3-treated plants (filled bar) were exposed to 82 5 ppb O3. Percent change (line) indicates changes () in O3-exposed relative to control plants, (+O3–CF)/CF. n = 8 for each mean (1 S.E.M.). Asterisks denote significant difference between control and O3-treated plants. (*) 0.05, (**) 0.01, (***) 0.001. cellular damage as determined by Rd was lower in the cultivars bred by conventional breeding and hybridization than in those bred by introduction and reselection. Although conventional breeding and hybridization had statistically similar effects on almost all biochemical and physiological parameters, hybrid cultivars appeared as the most sensitive to O3 in terms of growth. For instance, the highest relative decrease in shoot, root and total mass was noted in the cultivars bred by hybridization followed by conventional breeding and those bred using introduction and reselection showed the lowest. Our results are well consistent with the previous studies (Pang et al., 2009; Shi et al., 2009), which demonstrated that hybrid rice cultivars were more susceptible to O3 in terms of growth and yield compared to inbred ones. 4.3. Impacts of breeding sites on the potential and observed O3 tolerance in cultivars of winter wheat Breeding sites demonstrated significant effects on leaf biochemical properties of winter wheat cultivars exposed to CF air. Higher levels of antioxidative activities in cultivars bred in Shandong and Shanxi as indicated by higher levels of AsA and POD activity might be explained by the higher levels of ambient O3 exposure in these sites. Because generally plants are expected to adapt to detoxify the oxidants at a certain level as they have always been exposed to oxidative stress of the type induced by O3 (shortlived, oxidative radicals) (Paludan-Muller et al., 1999). This can be further explained by the lower levels of MDA in cultivars bred in Shandong and Shanxi. However, the cultivars bred in Hebei and Shanxi exhibited higher relative decrease in Chls, AsA, soluble proteins accompanied with a lower relative increase in POD activity than those bred in Shandong and Beijing. This suggested that cultivars bred in Beijing and Shandong had higher O3 tolerance as these cultivars maintained higher antioxidative activities at elevated O3. Available reports, however, indicated that ambient O3 levels in Beijing and Shandong were the lowest and the highest, respectively over the past several decades (Street and Waldhoff, 2000; Wang and Mauzerall, 2004; Wang et al., 2007). This indicates that O3-induced antioxidative activities in winter wheat cultivars are uncorrelated to ambient O3 levels in their breeding sites. On the other hand, cultivars bred in Beijing and Hebei showed higher relative increase 176 D.K. Biswas et al. / Agriculture, Ecosystems and Environment 134 (2009) 168–177 in MDA, suggesting higher O3-induced membrane lipid peroxidation in these cultivars than those bred in Shanxi and Shandong. These results were in accordance with the O3 concentrations in the breeding sites suggesting that O3-caused membrane lipid peroxidation as determined by MDA might be strongly regulated by an oxidant such as O3. However, the physiological response of cultivars found to be more strongly linked to O3 environment of the breeding sites. It has been well established that plant response to O3 is correlated with the flux of O3 into the leaf through stomatal conductance (Musselman and Massmann, 1999; Pleijel et al., 2007). The highest and lowest O3 flux or O3-induced loss of mesophyll activity as documented by gs in O3 or increase in Ci were observed in cultivars bred in Hebei and Shandong, respectively. This suggests that cultivars bred in a breeding site with higher O3 levels demonstrated higher O3 exclusion by partial stomatal closure than those bred in a breeding site with lower O3 levels. Our results also indicated that O3-induced impairment of mesophyll cells in winter wheat cultivars was mainly regulated by O3 flux, which determined the magnitude of oxidative stress as well as loss in Asat (Biswas et al., 2008a). Consequently, the highest and the lowest relative decrease in Asat or ITE were observed in cultivars bred in Hebei and Shandong, respectively. This can also be partly explained by an increase in Rd as the cultivars bred in Shandong and Shanxi experiencing higher O3 exposures had higher degree of repair of O3-induced cellular damage as documented by higher relative increase in Rd. However, the cultivars bred in Beijing and Shandong demonstrated statistically similar and higher O3 tolerance as indicated by a lower relative decrease in shoot, root and total mass than those bred in Shanxi and Hebei. The results obtained here, therefore, do not provide sufficient evidence to elucidate that O3 tolerance in terms of growth is related to higher O3 concentrations in the breeding sites since ambient O3 levels in Shandong and Shanxi are higher than in Beijing and Hebei (Street and Waldhoff, 2000; Wang and Mauzerall, 2004; Wang et al., 2007). It might be conceivable by the fact that high O3 concentrations are normally associated with hot sunny weather, which can lead to a greater incidence of water stress, and hence, a reduced stomatal conductance (Davison and Barnes, 1998). Consequently, the limited O3 flux might be not enough to decrease fitness to the extent that it produces a significant selection pressure for O3 tolerance (Davison and Barnes, 1998; Zheng et al., 1998). In addition, it should also be noted that higher O3 tolerance in wheat cultivar bred in Shandong based on a single cultivar, and therefore there is a need for further studies to make a definite conclusion on the cultivar response to O3 in this breeding site. 5. Conclusions Plant breeding methods demonstrated a significant variation in the potential and observed O3 sensitivity in winter wheat cultivars in terms of biochemical and physiological effects and growth. Hybrid cultivars with higher potential capacity for O3 tolerance appeared as the most sensitive to O3. Our study therefore indicated that recent yield improvement in wheat mostly by hybrid cultivars would be negatively affected by higher O3 environment in the future. This can be validated as higher yield or harvest index (HI) of hybrid cultivars will certainly reduce investments in other organs or functions, which can progress the magnitude of O3 sensitivity determined at vegetative stage. In addition, higher O3 flux and higher O3-induced oxidative stress along with lower levels of repair of O3-induced cellular damage will enhance reduction in photosynthesis as well as yield in hybrid cultivars, as grain yield of wheat mostly depends on current photosynthesis of flag leaf. Nevertheless, the observed O3 tolerance in winter wheat cultivars was uncorrelated with ambient O3 exposures in the breeding sites. This discrepancy might be related to the magnitude of O3 flux, which can be regulated by a range of environmental factors. Our findings, therefore, suggest that higher O3 concentrations in the breeding sites have played a little role in developing O3 tolerance in winter wheat cultivars through natural selection. It was also concluded that a plant breeding program targeting an improvement in the physiological and biochemical basis of O3 tolerance in crop cultivars might be useful to combat agricultural yield reduction by high O3 in the future. 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