Agriculture, Ecosystems and Environment 134 (2009) 168–177
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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
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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
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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
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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.
Acknowledgements
The authors thank Dr. Cui Hongxia for her technical expertise
in chlorophyll ﬂuorescence measurements. Two anonymous
reviewers are also gratefully acknowledged for their valuable
suggestions on an early version of the manuscript. D.K. Biswas is
grateful to Miss He Xiao Li and Dr. Chen Quansheng for their
help during the study period. This study was co-funded by the
Innovative Group Grant on Response of North China’s Grassland
to Global Change of Natural Science Foundation of China (No.
30821062), Beijing Natural Science Foundation (No. 8062017),
National Basic Research Program of China (2007CB106804), and
the Key Project of the Chinese Academy of Sciences (KZCX2XB2-01).
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