Indian Journal of Biochemistry & Biophysics
Vol. 50, April 2013, pp 139-149
Individual and interactive effects of elevated carbon dioxide and ozone on tropical
wheat (Triticum aestivum L.) cultivars with special emphasis on ROS generation
and activation of antioxidant defence system
Amit Kumar Mishra, Richa Rai and S B Agrawal*
Laboratory of Air Pollution and Global Climate Change, Department of Botany,
Banaras Hindu University, Varanasi 221005, India
Received 02 September 2012; revised 18 March 2013
The effects of elevated CO2 and O3, singly and in combination were investigated on various physiological, biochemical
and yield parameters of two locally grown wheat (Triticum aestivum L.) cultivars (HUW-37 and K-9107) in open top
chambers (OTCs). Elevated CO2 stimulated photosynthetic rate (Ps) and Fv/Fm ratio and reduced the stomatal conductance
(gs). Reactive oxygen species, lipid peroxidation, anti-oxidative enzymes, ascorbic acid and total phenolics were higher,
whereas Ps, gs, Fv/Fm, protein and photosynthetic pigments were reduced in elevated O3 exposure, as compared to their
controls. Under elevated CO2 + O3, elevated levels of CO2 modified the plant performance against O3 in both the cultivars.
Elevated CO2 caused significant increase in economic yield. Exposure to elevated O3 caused significant reduction in yield
and the effect was cultivar-specific. The study concluded that elevated CO2 ameliorated the negative impact of elevated O3
and cultivar HUW-37 was more sensitive to elevated O3 than K-9107.
Keywords: Open top chambers, Reactive oxygen species, Antioxidants, Yield, Triticum aestivum L., Elevated CO2 and O3
In past few years, anthropogenic activities have led to a
steady increase in atmospheric carbon dioxide (CO2)
and tropospheric ozone (O3) concentrations, two
important green house gases involved in global climate
change. Atmospheric concentrations of CO2 is
increasing as a result of combustion of fossil fuels,
deforestation and change in land use pattern and is
projected to continue rising to 730-1020 ppm by the
year 21001. Increasing fossil fuel consumption is
predicted to raise the production of nitrogen oxides,
while increased temperature and hydrocarbon
concentrations are expected to cause further increase in
tropospheric O3. The current annual average O3 ranges
from 25 to 34 ppb across the globe1. Tropospheric O3
concentrations are predicted to increase 20% by 20501.
Mean O3 concentrations of 45.3 and 47.3 ppb has been
_____________
*Corresponding author:
Phone: +91-9415309682; Fax: +91-542-2368174
E-mail: [email protected]
Abbreviations: AA, ascorbic acid; APX, ascorbate peroxidase;
CAT, catalase; chl, chlorophyll; Cv, cultivar; DAG, days after
germination; EC, elevated CO2; EO, elevated O3; ECO, elevated
CO2 + O3; GR, glutathione reductase; gs, stomatal conductance;
LPO, lipid peroxidation; NFCs, non-filtered chambers; OPs, open
plots; OTCs, open top chambers; PAL, phenyl alanine ammonia
lyase; PAR, photosynthetically active radiation; Ps, photosynthetic
rate; ROS, reactive oxygen species; SOD, superoxide dismutase.
reported during the year 2008 and 2009, respectively in
Varanasi2.
Both CO2 and O3 have direct effects on various
physiological aspects of plants. Elevated atmospheric
CO2 stimulates the photosynthesis in various crop
plants3. Elevated CO2 reduces stomatal conductance in
C3 and C4 plant species. Since CO2 is a substrate in
photosynthetic reaction, elevated levels of CO2
promote photosynthesis and yield of many C3-crop
plants4.
In contrast, O3 is well-known to induce foliar
injury in plants3, reduces net photosynthesis3,
decrease stomatal conductance5 and contents of
chlorophyll (chl) and carotenoids6. Ozone at higher
concentrations negatively affects crops by altering
various physiological and biochemical processes7,
which are translated in form of yield loses2. It enters
inside the leaf apoplast via the stomatal pores, where
it is rapidly converted into different reactive oxygen
species (ROS) that signal a diverse metabolic
response8. Elevated O3 also causes an up-regulation of
antioxidant defence system in plants9.
As both the gases, CO2 and O3 are rising with an
alarming rate, several experiments have been
performed in recent years to examine their impact of
elevated on crop plants. Studies on soybean10, wheat11
INDIAN J. BIOCHEM. BIOPHYS., VOL. 50, APRIL 2013
140
and peanut12 have clearly established that elevated CO2
partially or fully compensate the negative effects of O3.
Elevated CO2 causes reduction in stomatal
conductance, thereby ameliorates O3-induced oxidative
damage by suppressing its entry through stomata13.
Wheat (Triticum aestivum L.) is an O3 sensitive
crop. Keeping these facts in mind, in the present
investigation, we have investigated the level of
amelioration provided by elevated levels of CO2
against O3 in two wheat cultivars HUW-37 (dwarf
variety) and K-9107 (tall variety) in open top
chambers (OTCs) having near-natural field
conditions, and the extent of ROS production and
induction of antioxidant defence system under various
treatments of CO2 and O3.
Materials and Methods
Experimental area
The field experiments were conducted at the
Botanical garden of the Banaras Hindu University,
Varanasi, Uttar Pradesh (25°81’ N and 83°1’ E about
76 m above mean sea level) located in the eastern
Gangetic plains of India during winter between the
months of December 2010 to March 2011. During
experimental period, different meteorological
parameters, such as maximum and minimum
temperature, relative humidity, total rainfall and
sunshine hours were recorded periodically (Table 1).
Raising of plants
Two cultivars of wheat (Triticum aestivum L.) —
HUW-37 (dwarf variety) and K-9107 (tall variety)
were obtained from the Department of Genetics and
Plant Breeding, Banaras Hindu University, Varanasi
and C. S. Azad University of Agriculture and
Technology, Kanpur. The cultivars were tolerant to
Karnal bunt and resistant to rust and blight having life
spans of 125 days. The field was prepared using
standard agronomic practices. Seeds were hand sown
inside each open top chamber. Recommended dose of
fertilizers (120, 80 and 40 kg ha-1) N, P and K as urea,
superphosphate and muriate of potash, respectively
was added during the preparation of field. Half-dose
of N and full doses of P and K were supplied as basal
dressings. Another half-dose of N was given after 30
days of germination as top dressing. After 15 days of
germination, plants were thinned to one plant every
15 cm. Regular watering was done in each chamber to
maintain similar water regime. Manual weeding was
performed three to four-times during the entire course
of experiment.
Elevated CO2 and O3 fumigation
Elevated CO2 and O3 fumigation was given in
cylindrical OTCs having 1.5 m diameter and 1.8 m
height. The experimental set-up consisting of twenty
four OTCs (3 OTCs in replicate for each treatment per
cultivar) were established at the experimental site by
following the design of Bell and Ashmore14.
Treatment consisted of: (i) Non-filtered chambers
(NFCs) receiving ambient CO2 and ambient O3 as
control, (ii) NFCs with elevated CO2 (EC), (iii) NFCs
with elevated O3 (EO), and (iv) NFCs with elevated
CO2 + O3 (ECO). Plants were given elevated CO2 (700
ppm) and elevated O3 (ambient + 10 ppb) from seed
germination to the maturity from 11.00 to 15.00 h.
Elevated O3 was supplied by O3 generators that
contained UV lamps causing oxygen break down and
consequent O3 formation and elevated CO2 treatment
with CO2 cylinders having regulated gas flow. Open
plots (OPs) were also kept to assess any chamber
effect.
Air quality monitoring
Eight hourly CO2 and O3 monitoring was done in
OTCs and OPs at the experimental site using
automatic CO2 (Model LI-820, LI-COR Inc., USA)
and O3 (Model APOA 370, HORIBA Ltd., Kyoto,
Japan) analyzers throughout the growth period from
9.00 to 17.00 h. Air samples were drawn through
Teflon tube (0.35 cm in diameter) at canopy height of
plants in the chambers.
Table 1—Meteorological data of the experimental site during the study period (December 2010-March 2011)
Month/year
December 2010
January 2011
February 2011
March 2011
Rainfall
(mm)
0.0
3.2
4.6
0.3
Mean
temperature (°C)
Max.
25.4
21.4
27.1
33.9
Min.
9.2
7.0
11.3
16.2
Relative humidity
(%)
Max.
88.0
88.5
84.5
63.0
Min.
66.3
64.1
51.7
33.9
Sunshine
(h)
6.9
6.4
7.8
8.7
MISHRA et al: EFFECTS OF ELEVATED CARBON DIOXIDE AND OZONE ON WHEAT
Plant sampling and analysis
Photosynthetic pigments and physiological parameters
Plant samplings were done at 60 days after
germination (DAG). Three random samples were taken
for measurement of pigment concentration, leaf
photosynthetic rate (Ps), stomatal conductance (gs) and
chl fluorescence parameters from each treatment. The
measurements were done on third fully expanded leaf
from top of randomly selected plants. Photosynthetic
pigments, such as total chl and carotenoids were
extracted by homogenizing 100 mg of fresh leaf in
10 ml of 80% acetone, followed by centrifugation at
8000 rpm for 15 min. Optical densities (ODs) of the
filtered supernatant were taken at 480 and 510 nm
wavelengths for carotenoids and 645 and 663 nm for
chl by double beam UV-VIS Spectrophotometer
(Model 2203, Systronics, India). The concentration of
total chl and carotenoids were determined by the
formulae given by Maclachlan & Zalik15 and Duxbury
& Yentsch16, respectively.
Physiological measurements, such as Ps and gs were
recorded with the help of LICOR Photosynthetic
system (Model 6200, LICOR, Lincoln, NE, USA). The
system was calibrated using a known CO2 source
(509 ppm). The measurements were made on the day
having clear sky between 8.00 to 10.00 h on three
randomly selected plants of each treatment. During
measurements, photosynthetically active radiation
(PAR) ranged between 1100 and 1200 µ mol m-2s-1.
Measurement of chl fluorescence (Fv, Fm and Fv/Fm)
was done at ambient temperature in field conditions
with Plant Efficiency Analyzer (PEA, MK2, 9414,
Hansatech Instruments Ltd., England) on the same leaf
(where photosynthesis was measured) between 10.00
to 11.00 h. The leaves were dark adapted for 30 min
using leaf clips on the adaxial side. The leaf surface
was irradiated with a red light and fluorescence signal
was collected at excitation irradiance set at 3000 µ mol
m-2s-1 from the same surface.
Biochemical characteristics
For biochemical analysis, three plants per chamber
were taken randomly for each treatment. Total
phenolics were estimated in acetone extracts, followed
by addition of Folin-ciocalteau reagent17. Ascorbic acid
content was determined by using 2, 6-dichlorophenolindophenol (DCPIP) dye reduction method18. Lipid
peroxidation in terms of malondialdehyde (MDA)19,
ROS, such as H2O2 content20 and superoxide radical
production rate21, solute leakage22, phenyl alanine
ammonia lyase (PAL) activity23 were estimated.
141
For enzyme extraction, leaves (0.1 g) were
homogenized in 10 ml of pre-chilled 0.1 M phosphate
buffer (pH 7.0) under ice-cold conditions. The
homogenate was filtered through four layers of cheese
cloth and centrifuged at 15,000 rpm for 30 min. The
supernatant was stored at 4°C until used for assaying
the enzyme activities. An aliquot (0.5 ml) of the
supernatant was used for protein estimation by using
bovine serum albumin (BSA) as a standard24. All
steps of the enzyme extraction including
centrifugation were done at 4°C, and the assays were
performed at 25°C using a double beam UV-VIS
spectrophotometer (Model 2203, Systronics, India).
Anti-oxidative enzymes, such as peroxidase25,
catalase (CAT)26, superoxide dismutase (SOD)27,
ascorbate peroxidase (APX)28 and glutathione
reductase (GR)29 activities were assayed at 60 DAG.
Yield
Harvesting was done at 125 DAG in all the
treatments and final yield (weight of grains) in terms
of qn ha-1 was assessed. Ten replicates were sampled
from OTCs in triplicate for each treatment.
Statistical analysis
Data of pigments, photosynthesis, ROS and
antioxidants were subjected to multi-variate ANOVA
to examine the individual and combined effects of
cultivar, CO2 and O3. Duncan’s multiple range tests
were performed as post-hoc for various measurements
after subjecting to one-way ANOVA test. All the
statistical tests were performed using SPSS software
(SPSS Inc., version 16.0).
Results
Air quality monitoring
During growth period of wheat, mean
concentrations of CO2 ranged from 385.4 to 386.8
ppm and O3 ranged from 46.7 to 54.3 ppb during
December 2010 to March 2011. CO2 in elevated CO2
(EC) and elevated CO2 + O3 (ECO) chambers ranged
from 543.4 to 549.8 ppm and O3 in EO and ECO
varied from 52.3 to 60.2 ppb during December 2010
to March 2011, respectively. No significant variations
were recorded during monitoring of CO2 and O3 in
OPs and NFCs.
Pigments and physiological parameters
At 60 DAG, increments in total chl were recorded
to be 7.6 and 11.2% in HUW-37 and K-9107,
respectively under EC. Significant reduction was
INDIAN J. BIOCHEM. BIOPHYS., VOL. 50, APRIL 2013
142
observed under EO by 29 and 17.9% in HUW-37 and
K-9107, compared to NFCs. In ECO treatment, total
chl increased significantly by 4 and 5.6% in HUW-37
and K-9107, respectively at 60 DAG (Table 2).
Carotenoid content decreased significantly by 12.7,
27 and 21.6% in HUW-37 and 9.6, 24.7 and 5.7% in
K-9107, respectively under EC, EO and ECO
treatments (Table 2). Results of multi-variate
ANOVA test showed that variations in total chl and
carotenoids were significant due to Cv, CO2, O3 and
their interactions (Table 3).
EC exposure significantly increased the Ps by 29.7
and 30.8% in HUW-37 and K-9107, respectively.
Exposure to EO caused significant reduction in Ps by
13.5 and 10.7% in HUW-37 and K-9107, respectively
at 60 DAG, compared to NFCs (Table 2), while
significant increase in Ps was observed in ECO at 60
DAG (8.4 and 16.4% in HUW-37 and K-9107) (Table
2). Ps showed significant variations due to all
individual factors and their interactions, except CO2 ×
O3, Cv × O3 and Cv × CO2 × O3 (Table 3). Stomatal
conductance (gs) significantly decreased in HUW-37
(by 35.4, 39.4 and 50.6%) and K-9107 (by 18.9, 21.9
and 49.5%) in EC, EO and ECO, respectively at 60
DAG, as compared to NFCs (Table 2). Significant
variation in gs was recorded due to all individual
factors and their interactions except Cv × CO2 and
Cv × O3 (Table 3).
Table 2—Photosynthetic pigments and selected physiological parameters of wheat plants in non-filtered chambers (NFCs), elevated CO2
(EC), elevated O3 (EO) and elevated CO2 + O3 (ECO) at 60 DAG
[Values are mean ± SE]
Cultivars
HUW-37
K-9107
Treatment
NFCs
EC
EO
ECO
Total chl
0.48 ± 0.0024c
0.52 ± 0.0044a
0.34 ± 0.0020d
0.50 ± 0.0027b
Carotenoids
0.14 ± 0.0020a
0.13 ± 0.0023b
0.10 ± 0.0039d
0.11 ± 0.0061c
Ps
19.0 ± 0.45c
24.7 ± 1.11a
16.5 ± 0.75d
20.7 ± 0.10b
gs
5.9 ± 0.37a
3.8 ± 0.28b
3.6 ± 0.25b
2.9 ± 0.03c
Fv/Fm
0.814 ± 0.0009b
0.829 ± 0.0017a
0.791 ± 0.0012c
0.819 ± 0.0018b
NFCs
EC
EO
ECO
0.46 ± 0.0021c
0.51 ± 0.0032a
0.34 ± 0.0019d
0.48 ± 0.0032b
0.13 ± 0.0020a
0.12 ± 0.0042b
0.10 ± 0.0059d
0.11 ± 0.0018c
18.3 ± 0.07c
24.0 ± 0.25a
16.3 ± 0.59d
21.3 ± 0.68b
4.6 ± 0.06a
3.7 ± 0.03b
3.6 ± 0.03b
2.3 ± 0.06c
0.808 ± 0.0040c
0.836 ± 0.0026a
0.797 ± 0.0033d
0.817 ± 0.0068b
Different letters within a group of column indicate significant differences among treatments at p<0.05 according to Duncan’s test.
Table 3—F ratios and significance levels obtained from multi-variate ANOVA test performed for selected parameters of wheat plants
treated with CO2 and O3, singly and in combination
Parameters
AA
Protein
LPO
Phenol
SOD
POD
APX
CAT
GR
PAL
Ps
gs
Fv/Fm
Total chl
Carotenoids
H2O2 content
O2.Solute leakage
Cv
CO2
***
72.9
3676***
46.4***
11.6**
188.2***
3069***
179.5***
0.001ns
3331***
138.8***
11.9**
43.0***
1399***
220.4***
20.0***
19490***
72.1***
195.3***
O3
***
922.3
551.0***
1926***
2.4ns
5806***
16060***
331.2***
745.0***
521.6***
3.7ns
86.1***
86.0***
691.8***
1441***
10.7**
1364***
6895***
147.7***
CO2 × O3
***
2504
831.8***
5098***
248.8***
17460***
31210***
1089***
2218***
2117***
201.6***
38.0***
162.6***
551.8***
1880***
455.3***
3001***
13320***
505.8***
**
9.5
3267***
107.9***
143.7***
78.5***
193.4***
9.0**
66.0***
41.1***
61.5***
0.344ns
12.8**
193.6***
819.9***
108.1***
21.3***
9.8**
6.2*
Cv, cultivar; Level of significance * p<0.05, ** p<0.01, *** p<0.001, ns: not significant
Cv × CO2
**
10.4
1.3ns
8.9**
1.2ns
32.3***
767.3***
0.36ns
0.04ns
68.6***
3.4ns
0.07ns
3.3ns
234.0***
2.5ns
5.8*
147.0***
11.9**
13.5**
Cv × O3
Cv × CO2 × O3
ns
0.41
0.04ns
59.7***
4.6*
47.0***
1298***
29.1***
1.8ns
223.9***
10.3**
1.5ns
2.1ns
219.0***
18.5***
6.4*
205.5***
102.8***
4.3*
37.2***
3.2ns
122.2***
1.3ns
35.9***
389.4***
0.36ns
0.58ns
97.2***
1.5ns
0.003ns
9.6**
279.4***
31.7***
2.2ns
119.7***
173.3***
19.3***
MISHRA et al: EFFECTS OF ELEVATED CARBON DIOXIDE AND OZONE ON WHEAT
Photochemical efficiency (Fv/Fm) increased
significantly in HUW-37 and K-9107, respectively
(1.7 and 3.4%) at 60 DAG in EC. Significant
reduction in Fv/Fm ratio was observed in EO in
HUW-37 and K-9107 at 60 DAG (2.8 and 1.3%)
respectively, as compared to NFCs. In ECO, variation
observed was significant only in K-9107 at 60 DAG
(Table 2). Results of multi-variate ANOVA test
showed that variation in Fv/Fm was significant due to
Cv, CO2, O3 and their interactions (Table 3).
Biochemical characteristics
H2O2 content showed significant reduction of 16
and 41.6% in HUW-37 and K-9107 in EC, as
compared to NFCs. Significant increase in H2O2
content was noticed to be 34 and 24.5% in EO and 8.8
and 8.1% in ECO in HUW-37 and K-9107,
respectively at 60 DAG as compared to NFCs
(Fig. 1). Similarly, reduction in superoxide radical
(O2.-) observed was 23 and 33% in HUW-37 and K9107 in EC, as compared to NFCs. Significant
increments by 44.7 and 29.7% in EO and 14.4 and 6%
were recorded in ECO in HUW-37 and K-9107,
respectively at 60 DAG (Fig. 1). Results of multi-
143
variate ANOVA analysis revealed that variations in
H2O2, O2.- and solute leakage were significant due to
Cv, CO2, O3 and their interactions (Table 3).
Lipid peroxidation (LPO) decreased in case of EC
by 13 and 32.6% in HUW-37 and K-9107,
respectively (Fig. 1) and increased significantly by
66.6 and 23.6% in EO and 27 and 8.6% in ECO in
HUW-37 and K-9107, respectively at 60 DAG, as
compared to NFCs (Fig. 1). In HUW-37, solute
leakage significantly decreased by 6.6 and by 22.5 in
K-9107 in EC, as compared to NFCs. Significant
increments recorded was 20 and 18.5% in EO and 13
and 12.4% in ECO in HUW-37 and K-9107,
respectively at 60 DAG (Fig. 1). LPO and solute
leakage showed significant variations due to all
individual factors and their interactions (Table 3).
Ascorbic acid (AA) reduced by 9.2 and 15% in
HUW-37 and K-9107, respectively at 60 DAG in EC,
as compared to NFCs, but AA content increased
significantly by 25.7 and 18.7% in EO and 13.2 and
2% in ECO in HUW-37 and K-9107, respectively at
60 DAG, as compared to NFCs (Fig. 2). Results of
multi-variate ANOVA analysis showed that AA
Fig. 1—Lipid peroxidation, ROS and solute leakage of wheat plants at 60 DAG [Different letters on bars indicate significant differences
among treatments at p<0.05 according to Duncan’s test]
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INDIAN J. BIOCHEM. BIOPHYS., VOL. 50, APRIL 2013
varied significantly due to Cv, CO2, O3 and their
interactions, except Cv × O3 (Table 3).
SOD activity reduced by 12 and 14% in HUW-37
and K-9107, respectively in EC (Fig. 2). The activity
increased significantly under stress conditions and
showed increments of 16 and 20% in EO in HUW-37
and K-9107, respectively, as compared to NFCs. At
60 DAG, SOD activity also increased significantly by
5% in K-9107 in ECO (Fig. 2).
APX activity decreased by 54 and 44% in HUW-37
and K-9107, respectively at 60 DAG in EC, as
compared to NFCs. APX activity significantly
increased by 53 and 64% in EO and by 23 and 16% in
ECO in HUW-37 and K-9107, respectively at
60 DAG, as compared to NFCs (Fig. 2).
GR activity reduced in EC by 38.8 and 33.3% in
HUW-37 and K-9107, respectively at 60 DAG as
compared to NFCs. Significant increments were
recorded by 86 and 89% in EO and 54 and 44.6% in
ECO in HUW-37 and K-9107, respectively at
60 DAG, as compared to NFCs (Fig. 2). POD and
CAT also followed the same trend.
Fig. 2—Ascorbic acid content and anti-oxidative enzymes in wheat plants at 60 DAG [Bars showing different letters indicate significant
differences among treatments according to Duncan’s test at p<0.05]
MISHRA et al: EFFECTS OF ELEVATED CARBON DIOXIDE AND OZONE ON WHEAT
145
Fig. 4—Weight of grains (qn ha-1) of wheat plants grown under
NFCs, EC, EO and ECO [Bars showing different letters indicate
significant differences among treatments according to Duncan’s
test at p<0.05]
to all individual factors and their interactions, except
CO2, Cv × CO2 and Cv × CO2 × O3 (Table 3).
Protein content reduced by 7.3 and 5.7% in EC and
19 and 17.7 % in EO in HUW-37 and K-9107,
respectively at 60 DAG (Fig. 3). The variations in
protein content due to ECO were insignificant in both
the cultivars. Results of multi-variate ANOVA analysis
revealed that variations in protein was significant due
to Cv, CO2, O3 and their interactions, except Cv × CO2,
Cv × O3 and Cv × CO2 × O3 (Table 3).
Yield
Weight of grains (qn ha-1) significantly increased
by 46.2 and 54.6% in EC and 34.8 and 37.5% in
ECO. However, under EO, reduction of 39.2 and
12.4% in HUW-37 and K-9107 respectively was
observed, as compared to NFCs (Fig. 4).
Fig. 3—Protein, PAL activity and total phenolics in wheat plants at
60 DAG [Different letters on bars indicate significant differences
among treatments at p<0.05 according to Duncan’s test]
Results of multi-variate ANOVA analysis showed
that SOD, POD, APX, CAT and GR activities varied
significantly due to Cv, CO2, O3 and their
interactions, except Cv × CO2 and Cv × CO2 × O3 for
APX and CAT activities (Table 3).
In HUW-37, PAL activity increased by 23.9, 58.8
and 34.3 and by 26.7, 59.7 and 39.2% in K-9107,
respectively at 60 DAG in EC, EO and ECO, as
compared to NFCs (Fig. 3). Total phenolics increased
significantly by 17.6, 62.5 and 11.7% in HUW-37 and
24.6, 88.6 and 49.3% in K-9107, respectively at
60 DAG, as compared to NFCs (Fig. 3). PAL activity
and total phenolics showed significant variations due
Discussion
The results of the present study clearly showed
negative effects of elevated O3 at various
physiological and biochemical levels in both the
cultivars of wheat. Elevated CO2 alone and also in
combination ameliorated the negative effects of
ambient and elevated O3.
Ozone enters the leaf through stomata, reacts with
the moisture present in the sub-stomatal space and
produces ROS (O2.-, H2O2, etc.) that destroy the
structure and function of the biological membranes,
leading to electrolyte leakage8. ROS also react with
unsaturated
fatty
acids
forming
hydroxy
hydroperoxides (HHP) causing membrane disruption
due to LPO. Under elevated CO2, significant decrease
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INDIAN J. BIOCHEM. BIOPHYS., VOL. 50, APRIL 2013
in LPO revealed less generation of ROS (Fig. 5) and
thus less electrolyte leakage. MDA concentration
increased maximally in HUW-37 due to elevated O3,
suggesting its higher sensitivity. Under ECO, due to
higher concentrations of CO2 the extent of LPO was
less as compared to EO, suggesting an ameliorative
effect of CO2 against O3. K-9107 was more
responsive under ECO treatment, showing less LPO
than HUW-37. This trend was further supported by
levels of H2O2, superoxide radical (O2.-) and solute
leakage obtained in two test cultivars. The
concentration of ROS (H2O2 and O2.-) and trend of
solute leakage showed similar response as LPO,
suggesting more damage to plant cells in HUW-37
than K-9107. The results of the present experiment
clearly suggested that O3-induced oxidative damage
was drastically reduced in plants grown under
elevated levels of CO2.
In the present study, total chl increased under
elevated CO2 (EC) and elevated CO2 + O3 (ECO),
suggesting its higher production or induction. Increase
was higher in K-9107 than HUW-37. In contrast, total
chl reduced under elevated O3 (EO) exposure.
HUW-37 showed higher reduction in total chl under
elevated O3 treatment, as compared to K-9107.
Increase in chl pigments has been reported under CO2
treatment30. It is also reported that O3 induces the
formation of reactive oxygen intermediates (Fig. 5),
which destroy chl pigments by damaging the
membrane permeability of chloroplasts31 due to
higher LPO. In the present experiment, concentrations
of carotenoids reduced under all the treatments,
maximally under EO exposure. Less reduction of
carotenoids under combination of elevated CO2 and
O3 might have provided protection to chl molecules to
combat the ROS. Higher reduction in total chl and
carotenoids in HUW-37 under EO exposure suggested
greater damage of photosynthetic machinery due to
O3. The essential function of carotenoids is to protect
the photosynthetic system from the photo-oxidative
damage and their reduction at elevated O3 levels
might have increased the photo-oxidative destruction
of chl molecules. Under elevated O3 exposure,
reduction in photosynthetic pigments has been
well-documented in wheat32.
Photosynthetic rate (Ps) significantly increased
under EC and ECO treatments and reduced under EO
exposure in both the cultivars. Ps was higher in the
Fig. 5—Diagrammatic representation of effects of elevated CO2 and/or O3 in wheat plants
MISHRA et al: EFFECTS OF ELEVATED CARBON DIOXIDE AND OZONE ON WHEAT
K-9107 than HUW-37 under elevated CO2 alone and
in combined treatment of CO2 and O3 due to higher
assimilation of photosynthates. Pearcy and
Bjorkman33 reported stimulation of carboxylation
activity of Rubisco by decreasing the oxygenase
activity under CO2 enrichment, thus averting carbon
from the photo-respiratory pathway to the carbon
reduction cycle. C3 plants primarily show increase in
Ps under elevated CO2, as CO2 is often limiting for
photosynthesis under ambient conditions4. In ECO
treatment, elevated levels of CO2 might have
maintained higher levels of Rubisco content even
under high O3 concentrations hence showed higher
magnitude of photosynthesis. Under elevated O3
exposure, HUW-37 showed higher reduction in Ps
than K-9107, probably due to more reduction in the
content vis-a-vis the activity of Rubisco. Reduction in
net photosynthesis of wheat flag leaves exposed to
elevated O3 is also reported34.
Stomatal conductance (gs) decreased in all the
treatments, maximally in ECO with HUW-37
showing higher reduction than K-9107. Our results
were in agreement with earlier study3 showing
decrease in gs in rice leaves under elevated CO2 and
elevated O3, which reduced further in their combined
exposure. Under combined treatment, the differential
response of CO2 and O3 possibly led to more
reduction of gs compared to CO2 alone. Elevated CO2
induced stomatal closure through increased levels of
internal CO2 (Ci), thereby checking the O3 entry
inside the leaf mesophyll and thus reducing O3 injury.
Photochemical efficiency (Fv/Fm) was higher in EC,
but reduced under EO. Lowering of Fv (variable
fluorescence) represents thylakoid damage, whereas a
decrease in Fm (maximum fluorescence) indicates the
inhibition of PSII activity5. K-9107 showed higher
Fv/Fm than HUW-37 under EC exposure, suggesting
no damage on PSII structure. No significant
increment in Fv/Fm is reported in rice plants grown
under 700 ppm of CO235. Fv/Fm decreased under EO
exposure in both the cultivars. HUW-37 showed
higher reduction in Fv/Fm than K-9107, due to
detrimental effects of O3 on PSII. Ozone damages
PSII structure and blocks the photosynthetic electron
transport between PSII and PSI, leading to reduction
in Fv/Fm ratio5, hence higher reduction in Ps was
observed.
Ascorbic acid (AA) is a major cellular antioxidant
found in apoplastic region of the leaves and acts as a
first line of defence under oxidative stress. In the
147
present study, it showed variable response against all
the treatments in both the test cultivars. Under EC,
AA was reduced in K-9107, however, in EO, AA
increased and showed higher levels in HUW-37 than
K-9107, suggesting more utilization of AA in
apoplastic region of K-9107 to combat the adverse
effect of O3-induced ROS. AA acts as a reducing
substrate for detoxification of H2O2 in the ascorbate–
glutathione cycle. An earlier study has reported higher
levels of AA in the leaf apoplast of O3 tolerant snap
bean (Phaseolus vulgaris L.) genotypes compared to
sensitive lines36.
Plants have several enzymatic antioxidants in order
to defend themselves against oxidative stress. In the
present study, anti-oxidative enzymes, such as SOD,
POD, CAT, APX and GR significantly reduced under
elevated CO2, suggesting less generation of oxidative
stress under EC, as compared to ambient CO2. This
might be due to the fact that EC checked the entry of
ambient O3 by reducing the gs, thereby decreasing the
ROS formation. In the present experiment, the SOD
activity was higher in K-9107 in EO and ECO at 60
DAG, suggesting formation of superoxide radicals
under O3 stress. Under the combined treatment,
although the SOD activity increased, but the extent of
its activity was lower as compared to elevated O3
provided alone. Exponential increase in SOD activity
is reported in rice seedlings with increasing O3
concentrations (40, 80 and 120 ppb for 6 h for 9 days)
due to higher expression of Mn SOD isozyme genes37.
CAT and POD are found inside the cell for
detoxification of H2O2. POD usually occurs in multiple
molecular forms (isozymes) and requires H2O2 as an
essential substrate. In EO exposure, increase in POD
and CAT activities suggested higher accumulation of
H2O2 inside leaves due to elevated O3. In the present
study, both enzymes increased simultaneously under
EO and ECO to utilize excess of ROS produced, due to
higher concentrations of O3. An increase of POD
activity in 20 varieties of wheat with increments
ranging from 9 to 82% at 85 ppb O3 concentration (7 h
day−1 for 21 days) has been reported38. APX is a key
enzyme in the ascorbate-glutathione cycle and plays a
vital role in plant defence against oxidative stress by
catalysing the conversion of H2O2 to water. GR plays
an essential role in cell defence against reactive oxygen
metabolites by sustaining the reduced status of
glutathione and ascorbate pools which in turn maintain
cellular redox state under stress39. Under EO exposure,
both APX and GR activities were higher in K-9107,
148
INDIAN J. BIOCHEM. BIOPHYS., VOL. 50, APRIL 2013
clearly suggesting higher induction of anti-oxidative
defence system in K-9107 under O3 stress.
In the present study, protein content in leaves
decreased in all treatments, however, HUW-37 showed
significant reduction than K-9107 under EC and EO
treatments. CO2-induced reduction in protein has been
reported in wheat11. No significant impact of ECO on
leaf protein content suggested that EO induced
oxidative damage on protein was significantly
mitigated in plants grown under CO2 + O3 conditions.
Phenolic compounds are produced in response to
environmental stresses, including elevated CO2 and
their production is dependent on carbohydrate
availability40. Secondary compounds are formed when
relatively less carbon is allocated to growth.
Cv K-9107 showed higher accumulation of total
phenolics under various treatments than HUW-37,
suggesting more carbon assimilation than required for
growth. The higher PAL activity reflected higher
synthesis of secondary metabolites in all the treatments
in order to combat the oxidative stress generated by O3.
The test cultivars showed significant cultivarspecific response as observed in final yield (weight of
grains). Under elevated CO2 alone and in combined
treatment, increment in yield was higher in K-9107,
indicating higher allocation of photosynthates towards
developing ear ultimately affecting grain weight. In
ECO treatment, extent of amelioration due to CO2
against the damaging effect of O3 was higher in
K-9107 (less sensitive to O3) than HUW-37. Under
EO, reduction in yield was higher in HUW-37 than K9107. The reduction in the yield might be due to the
decreased Ps and less efficiency of cultivar to induce
sufficient antioxidants to combat the negative impact of
elevated O3. Reduction by 25 and 37% in weight of
seeds plant of Sonalika and HUW-510 varieties of
wheat, respectively has been reported at elevated level
of ozone2. In the present experiment, the mitigating
effect of elevated CO2 against O3 injury involved
suppression of stomatal conductance with higher
availability of substrates for detoxification and repair
processes.
Conclusions
The
present
study
demonstrated
that
photosynthesis, ROS formation and antioxidants
showed variable response under elevated CO2, O3 and
also in combination in both the wheat cultivars.
Results clearly showed that CO2 enrichment could
prevent O3-induced oxidative damage in tropical
wheat cultivars, but the effect was cultivar-specific.
On the basis of yield data, it could be concluded that
cultivar K-9107 (tall variety) was more responsive
under elevated CO2 alone and HUW-37 (dwarf
variety) was more O3 sensitive. The findings of
present experiment could be used for the cultivation
of K-9107 in the areas experiencing higher
concentrations of CO2 and/or O3.
Acknowledgements
Authors are grateful to Head, Department of Botany,
Banaras Hindu University, Varanasi for providing
necessary laboratory and field facilities. We are also
thankful to Prof. Ram Dhari, Department of Genetics
and Plant Breeding, Institute of Agricultural Sciences,
Banaras Hindu University, Varanasi and Head,
Department of Genetics and Plant breeding, C. S. Azad
University of Agriculture and Technology, Kanpur for
providing wheat seeds. Financial assistance in the form
of a research project (CST/D-1179) funded by Council
of Science and Technology (CST), Lucknow, Uttar
Pradesh is gratefully acknowledged.
References
1 IPCC Climate Change (2007) Contribution of Working
Group I to the Fourth Assessment Report of the
Intergovernmental Panel on Climate Change. In: The
Physical Science Basis, pp 996, Cambridge University Press,
Cambridge, UK
2 Sarkar A & Agrawal S B (2010) Environ Exp Bot 69, 328-37
3 Imai K & Kobori K (2008) Photosynthetica 46, 387-394
4 Agrawal M & Deepak S S (2003) Environ Pollut 121,
189-197
5 Rai R & Agrawal M (2008) Sci Total Environ 407, 679-691
6 Inada H, Yamaguchi M, Satoh R, Hoshino D, Nagasawa A,
Negishi Y, Sasaki H, Nouchi I, Kobayashi K & Izuta T (2008)
J Agric Meteorol 64, 243-255
7 Betzelberger A M, Gillespie K M, McGrath J M, Koester R P,
Nelson R L & Ainsworth E A (2010) Plant Cell Environ 33,
1569-81
8 Kangasjarvi J, Jaspers P & Kollist H (2005) Plant Cell
Environ 28, 1021-1036
9 Olbrich M, Gerstner E, Welzl G, Winkler J B & Ernst D
(2009) Plant Soil 323, 61-74
10 Gillespie K M, Rogers A & Ainsworth E A (2011) J Exp Bot
62, 2667-2678
11 Rao M V, Hale B & Ormrod D P (1995) Plant Physiol, 109,
421-432
12 Booker F L, Burkey K O, Pursley W A & Heagle A S (2007)
Crop Sci 47, 1475-1487
13 Booker F L & Fiscus E L (2005) J Exp Bot 56, 2139-2151
14 Bell J N B & Ashmore M R (1986) Design and construction of
open top chambers and method of filtration (equipments and
costs). In: Proceedings of the Second European Open Top
Chambers Workshop, Freiburg, CEC, Brussels
15 Machlachlan S & Zalik S (1963) Can J Bot 41, 1053-1062
MISHRA et al: EFFECTS OF ELEVATED CARBON DIOXIDE AND OZONE ON WHEAT
16 Duxbury A C and Yentsch C S (1956) J Mar Res 15, 91-101
17 Bray H C & Thorpe W (1954) Methods Biochem Anal 1, 27–52
18 Keller T & Schwager H S (1977) Eur J Forest Pathol 7,
338-350
19 Heath R L & Packer L (1968) Arch Biochem Biophys 125,
189-198
20 Alexieva V, Sergiev I, Mapelli S & Karanov E (2001) Plant
Cell Environ 24, 1337–1344
21 Elstner E F & Heupel A (1976) Anal Biochem 70, 616-620
22 Dijak M & Ormrod D P (1982) Environ Exp Bot 22, 395-402
23 Rao Subba P V & Tower G H N (1970)
L-phenylalanineammonia-lyase (Ustilago hordei). In: Methods
in Enzymology (Colowick S P, Kaplan, N O eds.), pp 581,
Academic Press, New York
24 Lowry O H, Rosenbourgh N J, Farr A L & Randall R J (1951)
J Biol Chem 193, 265-275
25 Britton C & Mehley A (1955) Assay of catalase and
peroxidases. In: Methods in Enzymology Colowick S P &
Kaplan N O, eds), pp 764-775, Academic Press, New York
26 Aebi H (1984) Methods Enzymol 105, 121-126
27 Beauchamp C & Fridovich I (1971) Annals Biochem 44,
276-287
28 Nakano Y & Asada K (1981) Plant Cell Physiol 22, 867–880
29 Schaedle M & Bassham J A (1977) Plant Physiol 59,
1011-1012
149
30 Habash D Z, Paul M J, Parry M A J, Keys A J & Lawlor W
(1995) Planta 197, 482-489
31 Saitanis C J, Riga-Karandinos A N & Karandinos M G (2001)
Chemosphere 42, 945-953
32 Sarkar A, Randeep R, Agrawal S B, Shibato J, Ogawa Y,
Yoshida Y, Agrawal G K & Agrawal M (2010) J Proteome
Res 9, 4565-4584
33 Pearcy R W & Bjorkman O (1983) Physiological effects. In :
CO2 and Plants: The Response to Rising Levels of Atmospheric
Carbon Dioxide (Lemon E R, ed), pp 65-105, West-view Press,
USA
34 Pleijel H, Eriksen A B, Danielsson H, Bondesson N & Selldon G
(2006) Environ Exp Bot 56, 63-71
35 Ziska L H & Teramura A H (1992) Plant Physiol 99, 473-481
36 Burkey K O, Eason G & Fiscus E L (2003) Physiol Plantarum
117, 51-57
37 Feng Z Z, Kobayashi K & Ainsworth E A (2008) Global
Change Biol 14, 2696-2708
38 Biswas D K, Xu H, Li Y G, Sun J Z, Wang X Z, Han X G &
Jiang G M (2008) Global Change Biol 14, 46-59
39 Hossain M A & Fujita M (2010) Physiol Mol Bio Plants 49,
249-279
40 Penuelas J A, Estiarte M, Kimball B A, Idso S B, Pinter Jr P J,
Wall G W, Grcia R L, Hansaker D J, LaMorte R L & Hendrix
D L (1996) J Exp Bot 47, 1463-1467