J Agro Crop Sci (2014) ISSN 0931-2250

OXIDATIVE STRESS

Cultivar Specific Response of CO2 Fertilization on Two

Tropical Mung Bean (Vigna radiata L.) Cultivars: ROS
Generation, Antioxidant Status, Physiology, Growth, Yield
and Seed Quality
A. K. Mishra & S. B. Agrawal
Laboratory of Air Pollution and Global Climate Change, Department of Botany, Banaras Hindu University Varanasi, Uttar Pradesh, India

Keywords Abstract
antioxidants; CO2; growth; mung bean;
reactive oxygen species; yield Increasing atmospheric carbon dioxide concentration (CO2) is an important
component of global climate change that will have a significant impact on the
Correspondence productivity of crop plants. In recent years, growth and yield of agricultural crop
Prof. S. B. Agrawal plants have been shown to increase with elevated CO2 (EC) and have enticed
Laboratory of Air Pollution and Global
considerable interest due to variation in the response of crop plants. In this study,
Climate Change, Department of Botany,
comparative response of two mung bean cultivars (HUM-2 and HUM-6) was
Banaras Hindu University, Varanasi-221005,
India evaluated against EC at different growth stages under near-natural conditions for
Tel.: +91 9415309682 two consecutive years. The plants were grown in ambient as well as EC
Fax: +91 542 2368174 (700 ppm) in specially designed open-top chambers. Under elevated CO2,
Email: sbagrawal56@gmail.com marked down-regulation of reactive oxygen species (ROS) levels, membrane dis-
ruption and activities of superoxide dismutase and catalase were noticed in both
Accepted February 27, 2014
the cultivars, but the extent of reduction was more in HUM-6. As compared to
ambient CO2, EC increased total chlorophyll, photosynthetic rate, growth and
doi:10.1111/jac.12057
yield parameters. Cultivar-specific response was noticed as HUM-6 showed
higher increase in yield attributes than HUM-2. Under CO2 treatment, soluble
protein and reducing sugars decreased while total soluble sugars and starch
showed an opposite trend. Principal component analysis showed that both the
cultivars responded more or less similarly to EC in their respective groupings of
physiological and growth parameters, but the magnitude of ROS and antioxida-
tive enzymes was variable. The experimental findings depict that both the culti-
vars of mung bean showed contrasting response against EC and paved the way
for selecting the suitable cultivar having higher productivity in a future high-CO2
environment.

carbon assimilation of plants, the rise in CO2 levels would

Introduction
Increasing the crop productivity to meet growing human
food demand is a challenging issue in view of global climate
change. Atmospheric carbon dioxide (CO2) levels have lin-
early increased from approximately 280 parts per million
(ppm) during pre-industrial times to the current level more
than 390 ppm (Mishra et al. 2013a). In past few years,
anthropogenic activities led to a rapid increase in global
CO2 concentration. As per future projections, the concen-
trations of CO2 will reach to 700 ppm by the year 2100
(IPCC 2007). As CO2 acts as an essential substrate for
have a significant impact on crop production and global
food security. Future global food security will depend on
primary physiological processes of crop plants and will be
affected by combined effects of climate change factors
including rise of CO2 concentrations (Tausz-Posch et al.
2013). The primary effects of rising CO2 on plants have
been well documented and include higher rates of photo-
synthesis (Garcia et al. 1998). Variability in crop responses
to the elevated CO2 (EC) made the agricultural productiv-
ity and food security vulnerable to the climate change
(Uprety et al. 2009). In view of the irreversible rise of

© 2014 Blackwell Verlag GmbH, 200 (2014) 273–289 273

Mishra and Agrawal
atmospheric CO2 concentration, there is an urgent need for
developing crop varieties with the potential of increased
utilization of CO2 to feed the growing population (Kant
et al. 2012).
Under EC, for a given electron transport rate, the forma-
tion of reactive oxygen species (ROS) may alleviate due to
more consumption of electrons during carbon fixation,
which in turn may restrict electron channelling into ROS-
producing pathways, such as photorespiration (Asada
1999). This may reduce the need for the activation of an-
tioxidative defence system inside the plant cells (Badiani
et al. 1998, di Toppi et al. 2002). Previous studies address-
ing oxidative stress and antioxidative defence responses of
crop plants against EC have reported reduced photo-oxida-
tive stress (Aranjuelo et al. 2008) as well as significantly
lower concentrations of ascorbic acid (Aranjuelo et al.
2008, Gillespie et al. 2011) and glutathione (Robinson and
Sicher 2004, Aranjuelo et al. 2008) as compared to ambient
CO2. However, contrasting results have been reported by
Jia et al. (2010), di Toppi et al. (2002) and Badiani et al.
(1998) with some findings that EC can generate the oxida-
tive stress (Qiu et al. 2008).
Several studies have shown the CO2 fertilization effect on
crop growth and yield. An increase of 30 % in plant growth
and yield has been reported when CO2 concentration has
been doubled from 330 to 660 ppm (Kimball and Idso
1983). Seneweera and Conroy (2005) have reported expan-
sion of leaf blade in wheat at EC (700 ppm). Positive effects
of EC on photosynthesis, water use efficiency (WUE) and
growth of C3 plants, such as wheat, rice, soybean, pigeon
pea and cotton (Kimball et al. 2002, Ziska et al. 2004, Ains-
worth and Long 2005, Saha et al. 2011), and pulse crops,
such as black gram (Vanaja et al. 2007), mung bean (Das
et al. 2002) and green gram (Srivastava et al. 2001), have
been well documented. It is predicted that yield of most
agricultural crops will increase under EC with productivity
increases in the range of 15–41 % for C3 crops and 5–10 %
for C4 crops (IPCC 2007).
In free-air CO2 enrichment (FACE) experiments with
clover (Trifolium repens) and soybean (Glycine max),
increase in photosynthetic carbon uptake and carbohydrate
content in leaves along with higher biomass has been
reported at EC (Ainsworth et al. 2003, Rogers et al. 2004).
Vanaja et al. (2006) reported enhancement in phenotypical
parameters (total dry weight, stem dry weight, root dry
weight, leaf dry weight, shoot length, root length and leaf
area) at 600 ppm CO2 in two important rain-fed food
crops, viz. sorghum (Sorghum bicolor L. Moench.) and
black gram (Vigna mungo L. Happer), and two oil seed
crops, viz. sunflower (Helianthus annuus L.) and ground-
nut (Arachis hypogaea L.), using open-top chamber
approach. Photosynthetic acclimation and productivity of
mung bean cultivars (Pusa Baisakhi, PS-16 and P-105)
274
have been reported under EC concentration
(650
50 ppm) using open-top chambers (OTCs; Shar-
ma-Natu et al. 2004).
Mung bean (Vigna radiata L.) is a rich source of protein
and a major leguminous C3 crop plant of the Indian sub-
continent. Studies attempting to characterize the effect of
CO2 enrichment on ROS generation, antioxidant activity,
physiology, growth, yield and seed quality of mung bean
from the Indo-Gangetic plains of India are not available, so
this study has been conducted with the objectives (i) to
study the effect of EC on induction of ROS and activation
of antioxidative defence system in two mung bean cultivars;
(ii) to analyse the cultivar-specific response of EC on pho-
tosynthetic pigments, physiology and growth of cultivars at
different development stages; (iii) to evaluate the effects of
EC on various yield attributes; and (iv) to assess variations
in seed quality, if any, under CO2 enrichment. The experi-
ment was performed to test the hypothesis that whether
CO2 fertilization simply affects the productivity or it also
alters the quality of produce.
Materials and Methods
Experimental site
The field experiment was performed during summer season
between the months of April and June 2011 and then
repeated from April to June 2012 at the experimental field
of Botanical garden of the Banaras Hindu University, Vara-
nasi, Uttar Pradesh (25°810 N and 83°10 E about 76 m above
mean sea level), located in the eastern Gangetic plains of
India. At the experimental site, soil was alluvial, pale brown
in colour and sandy loam in texture (sand 45 %, silt 28 %
and clay 27 %) having slightly alkaline pH (7.2–7.4).
During the experimental period, meteorological parame-
ters such as maximum and minimum temperature, relative
humidity, total rainfall and number of sunshine hours were
collected from an observatory of Indian Meteorological
Division (IMD), situated at Banaras Hindu University,
Varanasi, India.
Selection of cultivars
A preliminary experiment was conducted on six popular
cultivars of mung bean, viz. HUM-1, HUM-2, HUM-6,
HUM-12, HUM-23 and HUM-24, to find out the most and
least responsive cultivars against EC. The cultivars were
exposed to EC (700 ppm) in specially designed OTCs. Plant
heights and total biomass were recorded at 20 days after
germination (DAG) and observed that HUM-2 was least
responsive and HUM-6 showed highest response against EC
(data not shown). On the basis of the observed results,
HUM-2 (Malviya Jagriti) and HUM-6 (Malviya Janpriya)
© 2014 Blackwell Verlag GmbH, 200 (2014) 273–289

were selected for further study. Both the cultivars were
widely grown by local farmers in and around Varanasi.
Seeds were obtained from the Department of Genetics and
Plant Breeding, Banaras Hindu University, Varanasi. Both
the cultivars have life span of 60–65 days from seed germi-
nation to maturity. HUM-2 and HUM-6 were released in
the year 2000 and 2001, respectively, having yield potentials
of 12–15 and 12–14 qn, respectively. The cultivars were
moderately resistant to Cercospora leaf spot, powdery mil-
dew disease and mung bean yellow mosaic virus.
Raising of plants
The field was prepared using standard agronomic practices.
Seeds were hand sown inside each OTC. After 7 days of
germination, plants were thinned to one plant every 15 cm.
Twenty-five plants were maintained inside each OTC. Dur-
ing the preparation of field, recommended doses of N, P
and K (20, 40 and 20 kg ha1, respectively) were applied as
urea, single super-phosphate and muriate of potash,
respectively. A half-dose of N and full doses of P and K
were provided as basal dressings. Another half-dose of N
was supplied after 10 days of germination as top dressing.
Irrigation was provided to each chamber to maintain simi-
lar water regime. Manual weeding was performed 3–4 times
over the course of the experiment.
Elevated CO2 treatment
Twelve OTCs were installed at the experimental site by fol-
lowing the design of Bell and Ashmore (1986). The OTCs
were cylindrical with 1.5 m diameter and 1.8 m height. Our
experimental design was completely randomized block hav-
ing three chambers (n = 3) for each treatment, so a total of
six chambers per cultivar. Open plots (OPs) (n = 3) were
also kept to assess any chamber effect per cultivar. The
chambers were made up of clear acrylic plastic. Each of the
OTCs was attached with non-filtered high-speed blower
having three air volume changes per minute. In this study,
chambers receiving ambient atmospheric CO2 (non-filtered
chambers (NFCs)) served as control while CO2 concentra-
tion in the other one was increased up to 700 ppm (EC). As
carbon dioxide is utilized mainly during day hours, plants
were exposed to EC (700 ppm) during all daytime hours,
7 day week1 from germination to seed maturation. The
increased concentration of CO2 was selected on the basis of
projected concentration (700 ppm) by the end of the cen-
tury (IPCC 2007). EC was dispensed with CO2 cylinders
having regulated gas flow. Microclimatic measurements
were continuously taken within the OTCs and OPs at can-
opy height. Temperature and relative humidity were,
respectively, 0.1–0.2 °C and 2–4 % higher inside the cham-
bers compared with OPs. The light intensity in the OTCs
© 2014 Blackwell Verlag GmbH, 200 (2014) 273–289
Effect of CO2 Enrichment on Two Tropical Mung Bean Cultivars
was 94–95 % of the ambient level/OPs. So, OTCs provided
the near-natural conditions such as OPs.
CO2 monitoring
Continuous daytime CO2 monitoring was performed in
OTCs and OPs at the experimental site using automatic
CO2 (Model LI-820; LI-COR Biosciences, Lincoln, NE,
USA) analyzer throughout the growth period of the crop.
Air samples were drawn through a tube at canopy height
inside the chamber/OPs.
ROS, lipid peroxidation and antioxidative enzymes
Superoxide radical (.O2) production rate was determined
by following the method described by Elstner and Heupel
(1976) with some modifications; 0.5 g of leaves was
homogenized in 3 ml phosphate buffer (65 mM, pH 7.8) at
4 °C. The reaction mixture was then centrifuged at 2000 g
for 10 min. The supernatant (1 ml) was then incubated at
25 °C in phosphate buffer (65 mM, pH 7.8) and hydrox-
ylamine hydrochloride (10 mM) for 20 min. After the
incubation, sulphanilamide (1 ml, 8.5 mM) and a-
naphthylamine (1 ml, 3 mM) were added. The reaction
mixture was kept at 25 °C for 20 min. Finally, absorbance
was recorded at 530 nm.
Hydrogen peroxide (H2O2) content was determined by
following the protocol of Alexieva et al. (2001); 0.1 g of
leaf tissue was crushed in 0.1 % trichloroacetic acid
(TCA) and homogenized at 4 °C. After centrifugation at
10 000 g for 15 min, the supernatant was kept in dark for
1 h after mixing with phosphate buffer (10 mM, pH 7.0)
and potassium iodide (1 M). The absorbance of the result-
ing solution was recorded at 390 nm.
Malondialdehyde (MDA) content, a product of lipid
peroxidation (LPO), was estimated by thiobarbituric acid
(TBA) reaction following the protocol of Heath and Packer
(1968). Solute leakage (SL) was performed by following the
method described by Dijak and Ormrod (1982).
Superoxide dismutase (SOD) activity was measured as
50 % reduction of nitroblue tetrazolium (NBT) as given by
Beauchamp and Fridovich (1971). Catalase (CAT) was
extracted by homogenizing 0.1 g of fresh leaves at 4 °C in
100 mM cold phosphate buffer (pH 7.2) containing 0.5 %
Triton-X. The activity of CAT was assessed in 100 ll phos-
phate buffer, 400 ll H2O2 (200 mM) and 100 ll enzyme
extract. A decrease in H2O2 was measured at 240 nm
according to the method of Aebi (1984).
Photosynthetic pigments and physiological parameters
Photosynthetic pigments such as total chlorophyll and car-
otenoids were extracted by homogenizing 100 mg of fresh
275
Mishra and Agrawal
leaves in 10 ml of 80 % acetone followed by centrifugation
at 4500 g. The supernatant was filtered, and optical densi-
ties were taken at 645 and 663 nm wavelengths for chloro-
phyll and 480 and 510 nm for carotenoids and by Double
Beam UV-VIS Spectrophotometer (Model 2203; Systronics,
Ahmedabad, India). The concentration of total chlorophyll
and carotenoids was estimated by the formulae given by
Maclachlan and Zalik (1963) and Duxbury and Yentsch
(1956), respectively.
Photosynthetic rate (Ps), stomatal conductance (gs),
internal CO2 concentration (Ci) and transpiration rate (E)
were determined using portable photosynthesis system
(Model LI-6200; LI-COR Inc.,). WUE was calculated as a
ratio of photosynthesis to stomatal conductance. Before the
measurements, the system was calibrated using a known
CO2 source of 509 ppm concentration. The measurements
were made on the third fully expanded mature leaf from
the top of each plant on cloud-free days between 08.00 and
10.00 h at 20, 40 and 60 DAG on three randomly selected
plants from each treatment. During the measurements,
photosynthetically active radiation (PAR) ranged between
1100 and 1200 mmol m2 s1.
Plant sampling and analysis
After fumigation, plant samples were taken from each
treatment at 20, 40 and 60 DAG for various growth and
biomass analyses. Three plants were taken randomly from
each chamber. Monoliths of 10 9 10 9 20 cm3 size con-
taining intact roots were carefully dug from each chamber.
To remove attached soil particles, thorough washing was
performed by placing the monoliths on sieves of 1 mm
mesh size under running tap water. Growth parameters
recorded were plant height, number of nodules, number of
leaves and leaf area. Leaf area was measured using a porta-
ble leaf area meter (Model LI-3000; LI-COR Inc.,). Compo-
nent plant parts (root, shoot and leaf) were separated and
oven-dried at 80 °C to obtain constant weight for biomass
determination. Dry weight of each plant part was taken.
For the determination of biomass allocation pattern,
growth indices, such as relative growth rate (RGR), net
assimilation rate (NAR), leaf area ratio (LAR) and root/
shoot ratio (RSR), were calculated on dry weight data using
modified formulae of Hunt (1982).
Yield attributes
In both the years of experiment, harvesting was performed
at 80 DAG. The number of pods plant1, weight of
pods plant1, number of seeds plant1 (NOSPP), weight
of seeds plant1, harvest index (HI) and test weight (TW;
weight of 1000 seeds) were evaluated at harvest. Ten plants
were sampled from OTCs for each cultivar and treatment.
276
Seed quality parameters
Seed samples were oven-dried, grinded and passed through
a 2.0-mm sieve. Three replicates were taken from each treat-
ment. For extracting sugars and starch, 50 mg powdered
seed sample was boiled with 5 ml 80 % ethanol (v/v) and
centrifuged. The pellets were successively washed with 80 %
ethanol four times and centrifuged after each washing.
Finally, the pellets were washed with distilled water and cen-
trifuged again. The supernatant collected after each washing
was used for estimating soluble and reducing sugars (RSs)
and pellets for extracting starch. Total soluble sugar (TSS)
and starch were estimated by following phenol/H2SO4 col-
orimetric assay (Dubois et al. 1956). RS content was deter-
mined by following the method of Somogyi-Nelson
(Herbart et al. 1971). Protein content in seeds was esti-
mated by following the method as described by Lowry et al.
(1951). Total free amino acids (TFAAs) were estimated by
following the methodology of Moore and Stein (1948).
Statistical analyses
Results of ROS, antioxidative enzymes, physiological and
growth parameters were subjected to multivariate ANOVA to
examine the effects of year (Y), age (A), cultivar (Cv) and
treatment (T). Results of yield attributes were analysed
through multivariate ANOVA using Y, Cv and T as factors.
Two-way ANOVA was used to observe the effects of Cv and T
on seed quality parameters. The significance of difference
between the treatments was calculated using paired-sample
t-test. Correlation analysis between A, T and different
parameters was assessed using Pearson’s correlation analy-
sis. Principal component analysis (PCA) was performed
using data of ROS, antioxidative enzymes, physiological
and growth parameters at 20, 40 and 60 DAG. The method
was based on the correlation matrix with the rotation
method of varimax with Kaiser’s normalization. Loading
values of more than 0.5 in both the components were con-
sidered to be significantly influenced by CO2. The PCA
allows the identification of interrelated variables.
All the statistical tests were performed using SPSS software
(version 16.0; SPSS Inc., Chicago, IL, USA). The yearwise
variations in different parameters of plants in relation to
CO2 enrichment except yield attributes were not significant
(Table 1); hence, the data used for figures in the present
paper were of 2011.
Results
Meteorological parameters
Maximum mean temperature was highest during May in
2011 and 2012 (40.7 and 40.9 °C, respectively) and lowest
© 2014 Blackwell Verlag GmbH, 200 (2014) 273–289
 Effect of CO2 Enrichment on Two Tropical Mung Bean Cultivars

Table 1 Significance levels obtained from multivariate ANOVA test performed for photosynthetic pigments, biochemical, physiological and growth
parameters of both the cultivars of Vigna radiata L. under elevated CO2 treatment for two consecutive years of experiment (2011 and 2012)

Y9 Y9 Y9 A9 Y9
Parameter Y A Cv T Y9A Y 9 Cv Y9T A 9 Cv A9T Cv 9 T A 9 Cv A9T Cv 9 T Cv 9 T A 9 Cv 9 T

TC ns *** *** *** ns ns ns *** *** *** ns ns ns ns ns

CAR ns *** *** *** ns ns ns *** *** *** ns ns ns *** ns
LPO ns *** *** *** ns ns ns *** *** ** ns ns ns *** ns
HP ns *** *** *** ns ns ns *** ** ns ns ns ns ns ns
SR ns *** *** *** ns ns ns *** *** *** ns ns ns ns ns
SL ns *** ns *** ns ns ns *** *** * ns ns ns *** ns
SOD ns *** *** *** ns ns ns *** *** *** ns ns ns *** ns
CAT ns *** ns *** ns ns ns * ns ns ns ns ns ns ns
Ps ns *** *** *** ns ns ns * *** *** ns ns ns ns ns
Ci ns *** *** *** ns ns ns *** *** *** ns ns ns *** ns
gs ns *** *** *** ns ns ns *** *** *** ns ns ns * ns
E ns ** ** *** ns ns ns *** *** ns ns ns ns ns ns
WUE ns *** * *** ns ns ns ** *** * ns ns ns ns ns
PH ns *** *** *** ns ns ns *** * *** ns ns ns ns ns
NOL ns *** ** *** ns ns ns ** *** * ns ns ns ns ns
LA ns *** ns *** ns ns ns *** *** ns ns ns ns * ns
NON ns *** * *** ns ns ns * *** ns ns ns ns ns ns
DRW ns *** *** *** ns ns ns *** *** *** ns ns ns * ns
DSW ns *** *** *** ns ns ns ** *** ns ns ns ns ns ns
DLW ns *** ** *** ns ns ns *** ** ns ns ns ns ns ns
TB ns *** *** *** ns ns ns ns *** * ns ns ns ns ns

Y, year; A, age; Cv, cultivar; T, treatment; TC, total chlorophyll; CAR, carotenoids; LPO, lipid peroxidation; HP, hydrogen peroxide content; SR, super-
oxide radical production rate; SL, solute leakage; SOD, superoxide dismutase activity; CAT, catalase activity; Ps, photosynthetic rate; Ci, internal CO2
concentration; gs, stomatal conductance; E, transpiration rate; WUE, water use efficiency; PH, plant height; NOL, number of leaves; LA, leaf area;
NON, number of nodules; DRW, dry root weight; DSW, dry shoot weight; DLW, dry leaf weight; TB, total biomass. Level of significance *P < 0.05,
**P < 0.01, ***P < 0.001, ns, not significant.

during June in 2011 (37 °C) and during April in 2012 410
2011 2012
(38.6 °C). Minimum mean temperature varied from 20.7–
405
26.6 °C during 2011 to 22.3–28.6 °C during 2012. Maxi-
Conccentration oof CO2 (pp m)

mum mean relative humidity was highest during June 400

(73.8 %) in 2011 and 56.4 % during April in 2012. Total
395
rainfall was higher during the first year (241.9 mm) than
the second year (97.4 mm) of experiment. The number of 390
sunshine hours was maximum during May in 2011 and
385
2012 (9.5 and 9.4 h, respectively) and minimum during
June in 2011 and 2012 (5.6 and 6.2 h, respectively). 380

375

CO2 monitoring 370

In the first year of experiment (2011), mean daytime ambi- April May June

ent CO2 concentration varied from 389.8 to 387.2 ppm

during April to June 2011 (Fig. 1). During second year
(2012), mean daytime ambient CO2 concentration varied
from 391.9 during April to 392.4 ppm during June (Fig. 1).
ROS, lipid peroxidation and antioxidative enzymes
Under EC treatment, ROS (.O2 and H2O2) showed signifi-
cant reductions at 20 and 40 DAG in both the cultivars as
Fig. 1 Diurnal profile of ambient CO2 (ppm) at the experimental site of
two consecutive years (2011 and 2012) of experiment.
compared to their controls. .O2 and H2O2 decreased by
11.8 and 23.5 % in HUM-2 and 24.8 % and 27.8 % in
HUM-6 at 40 DAG, respectively (Fig. 2). At 40 DAG, LPO
reduced in case of EC by 17.6 % and 22.9 % in HUM-2
and HUM-6, respectively, as compared to controls (Fig. 2).

© 2014 Blackwell Verlag GmbH, 200 (2014) 273–289 277

Mishra and Agrawal

NFCs EC
Superoxide radical production rate
0.5 0.35

0.30
(nmol min–1 g–1 fresh wt.)

0.4 ns
ns

(µmol g–1 fresh wt.)

ns
ns 0.25

H2O2 content
* **
0.3 **
0.20
** *
0.2 * 0.15
** **
0.10
0.1
0.05

0.0 0.00
ns
1.0 ns ns
* ns *
(nmol ml–1 fresh wt.)

* **

Solute leakage (%)

** 30
MDA equivalents

*
0.8 *
**
0.6 20

0.4
10
0.2

0.0 0
Superoxide dismutase activity

(mM H2O2 decomposed min–1

4 ** 0.5
ns ***
(Unit g fresh wt.)

Catalase activity
*** 0.4

g–1 fresh wt.)

** *
3
** * ns Fig. 2 Reactive oxygen species (.O2 produc-
ns 0.3
–1

2 ns
tion rate and H2O2), lipid peroxidation, solute
0.2
leakage and activities of superoxide dismutase
1 * and catalase (CAT) of HUM-2 and HUM-6 at
0.1
20, 40 and 60 days after germination at ambi-
0 0.0 ent (NFCs) and elevated CO2 (mean
S.E.)
20 40 60 20 40 60 20 40 60 20 40 60
DAG DAG DAG DAG *P < 0.05, **P < 0.01, ***P < 0.001, ns: not
HUM-2 HUM-6 HUM-2 HUM-6 significant.

SL showed similar trend as LPO in both the cultivars compared to their respective controls (Fig. 3). Under EC
(Fig. 2). Results of multivariate ANOVA showed that varia- treatment, carotenoid content decreased significantly by
tions observed in .O2 and H2O2 were significant due to A, 14.7 % in HUM-2 and 8.1 % in HUM-6 at 40 DAG as
Cv, T and their interactions except Cv 9 T in H2O2 compared to ambient CO2 (Fig. 3). Results of multivariate
(Table 1). Decrease in LPO and SL was significant due to ANOVA test showed that variations in total chlorophyll and
all individual factors and their interactions except Cv in SL carotenoids were significant due to all individual factors
(Table 1). and their interactions except A 9 Cv 9 T in total chloro-
Reductions in the activities of antioxidative enzymes phyll (Table 1).
such as SOD and CAT were observed in both the cultivars. Photosynthetic rate (Ps) increased in both the cultivars
SOD activity decreased significantly by 11.8 % and 14.7 % of mung bean growing in EC as compared to those in
in HUM-2 and HUM-6, respectively, at 40 DAG as com- ambient CO2 (Fig. 3). Ps increased significantly by 25.4 %
pared to controls (Fig. 2). At 40 DAG, CAT activity also and 29.2 % in HUM-2 and HUM-6 at 40 DAG, respec-
showed significant reductions by 64.3 % in HUM-6 only. tively. Increase in Ps was significant at all the ages of sam-
Analysis of multivariate ANOVA revealed that variations pling in Cv HUM-6 (Fig. 3). Significant reductions were
recorded in SOD and CAT activities were significant due to recorded in gs by 21 % and 18.6 % at 20 DAG and 33.9 %
all individual factors and their interactions except Cv, and 26 % at 40 DAG, respectively, in HUM-2 and HUM-6
A 9 T, Cv 9 T and A 9 Cv 9 T in CAT (Table 1). as compared to their controls (Fig. 3). Transpiration rate
(E) also showed similar trends as gs in both the cultivars
(Fig. 3). Significant increment in internal CO2 (Ci) concen-
Photosynthetic pigments and physiological parameters
tration was observed by 9 % and 14 %, respectively, in
Variations in total chlorophyll were significant at all the HUM-2 and HUM-6 at 40 DAG (Fig. 3). WUE increased
ages of sampling in both the cultivars. At 40 DAG, incre- significantly by 47.5 % and 61.5 % in HUM-2 and HUM-
ments in total chlorophyll were noticed to be 30.6 % and 6, respectively, at 40 DAG as compared to ambient CO2
38.9 % in HUM-2 and HUM-6, respectively, under EC as (Fig. 3). Results of multivariate ANOVA showed that Ps and

278 © 2014 Blackwell Verlag GmbH, 200 (2014) 273–289

 Effect of CO2 Enrichment on Two Tropical Mung Bean Cultivars

NFCs EC
0.6 0.14
**
** * 0.12
0.5 **
* ** ** **

Total chlorophyll
(mg g–1 fresh wt.)

(mg g–1 fresh wt.)

** ns
0.10
0.4 ns ns

Carotenoids
0.08
0.3
0.06
0.2
0.04
0.1 0.02

0.0 0.00
**
* 6
** *
ns

Stomatal conductance
20 *

Photosynthesis rate
(µmol CO2 m–2 s–1)

(mol m–2 s–1)

*
15 * * 4
**
ns
10 ns
2
5

0 0
ns

(µmol CO2 m–2 s–1/mol m–2 s–1)

ns ns ns * ** 0.8

Water use efficiency

** ns
Transpiration rate

30 *
ns ns
(mmol m–2 s–1)

* 0.6
20
0.4

10
0.2

0 0.0
20 40 60 20 40 60
600 * ** DAG DAG
ns * ** * HUM-2 HUM-6
Internal CO2

400
(ppm)

Fig. 3 Photosynthetic pigments and physio- 200

logical parameters of mung bean plants in
non-filtered chambers and elevated CO2 at
20, 40 and 60 days after germination 0
20 40 60 20 40 60
(mean
S.E.). *P < 0.05, **P < 0.01, DAG DAG
*** < 0.001, ns: not significant. HUM-2 HUM-6

WUE vary significantly due to all the individual factors and 40 and 60 DAG in HUM-6 as compared to ambient CO2
their interactions except A 9 Cv 9 T (Table 1). Varia- (Fig. 4). The number of leaves showed significant varia-
tions in Ci and gs were significant due to all individual fac- tions due to all individual factors and their interactions
tors and their interactions (Table 1). Reduction in E was except A 9 Cv 9 T (Table 1).
significant due to all the individual factors and their inter- Significant increment in leaf area was observed at all the
actions except Cv 9 T and A 9 Cv 9 T (Table 1). ages of sampling. At 60 DAG, leaf area increased by 20.5 %
and 22.3 % in HUM-2 and HUM-6, respectively, in EC as
compared to ambient CO2 (Fig. 4). In EC, the number of
Morphological parameters
nodules significantly increased by 43 % and 50 % in
Plant height increased continuously under EC at 20, 40 and HUM-2 and HUM-6, respectively, at 60 DAG (Fig. 4).
60 DAG as compared to ambient CO2 in both the cultivars. Results of multivariate ANOVA revealed that variations in leaf
At 60 DAG, plant height increased significantly by 19 % area and number of nodules were significant due to A, T
and 26 % in HUM-2 and HUM-6, respectively, in EC and their interactions (Table 1).
(Fig. 4). Results of multivariate ANOVA test showed that var-
iation in plant height was significant due to A, Cv, T and
Biomass accumulation and allocation
their interactions except A 9 Cv 9 T (Table 1).
In EC treatment, significant increase in the number of Increase in root biomass was significant at all the ages of
leaves was observed by 21.3 % and 24.6 %, respectively, at sampling in HUM-6 under EC treatment (Fig. 5). Root,

© 2014 Blackwell Verlag GmbH, 200 (2014) 273–289 279

Mishra and Agrawal

NFCs EC
100 35
*
* ns
* 30
80
* ** **

Number of leaves
Plant height (cm)

ns 25
ns
* ns
60 * 20

40 15

10
20
5

0 0
600 **
* 12
* * * * *
500 **

Number of nodules
10
Leaf area (cm 2)

400
8
* *
300 ns 6
ns
200 4
100 2
Fig. 4 Morphological parameters of mung
0 0
20 40 60 20 40 60 20 40 60 20 40 60 bean plants at 20, 40 and 60 days after germi-
DAG DAG DAG DAG nation (mean
S.E.). *P < 0.05, **P < 0.01,
HUM-2 HUM-6 HUM-2 HUM-6 ***P < 0.001, ns: not significant.

shoot and leaf biomass increased significantly by 36.4 %,
33.9 % and 29 %, respectively, in HUM-6 at 60 DAG as
compared to ambient CO2 (Fig. 5). In EC treatment, total
biomass increased significantly by 29.6 % and 19.8 % in
HUM-2 and by 32.6 % and 33.6 % in HUM-6, respec-
tively, at 40 and 60 DAG as compared to their respective
controls (Fig. 5). Significant variations in root, shoot and
total biomass were noticed due to all the individual factors
and their interactions except Cv 9 T in shoot biomass.
However, for leaf biomass, it varied significantly due to A,
Cv, T, A 9 Cv and A 9 T as proved by multivariate ANOVA
(Table 1). Biomass allocation was higher towards shoot
and leaf portions of EC plants as compared to root (Fig. 5).
However, in both the cultivars, major biomass was accu-
mulated in shoot portion in control as well as in EC-treated
plants.
Variations observed in RGR, NAR and LAR were insig-
nificant at all the ages of sampling (Fig. 6). Significant
increment in RSR was observed in EC treatment (17.2 %)
in HUM-6 as compared to ambient CO2 (Fig. 6).
Yield attributes
Elevated CO2 treatment positively modified the yield attri-
butes of mung bean cultivars (Table 2). The increase in
the number of pods plant1 was significant only in
HUM-6 (18.8 %) as compared to ambient CO2 (Table 2).
Weight of pods plant1 increased significantly in HUM-2
(24.2 %) and HUM-6 (33.1 %) treated with EC as com-
pared to ambient CO2 (Table 2). Significant increments
in number and weight of seeds plant1 were 34.6 % and
25.3 % in HUM-2 and 40.6 % and 33.9 % in HUM-6,
respectively, in EC. In the second year of experiment,
weight of seeds plant1 increased significantly by 34.6 %
in HUM-2 and 40 % in HUM-6 under EC treatment as
compared to ambient CO2. In both the years, under EC
treatment, variations in HI were insignificant in both the
cultivars (Table 2). Weight of 1000 seeds (TW) increased
significantly by 10.6 % and 17.7 % during 2011 and
11.6 % and 20.2 % during 2012 in HUM-2 and HUM-6,
respectively, as compared to ambient CO2. Variations in
NOSPP and TW were significant yearwise (Table 2). Mul-
tivariate ANOVA revealed that variations in yield attributes
due to Cv and T were significant except HI under EC
treatment (Table 2).
Seed quality parameters
In EC treatment, soluble protein content (SPC) in seeds
reduced significantly by 9.9 % in Cv HUM-2 as com-
pared to ambient CO2 (Fig. 7). Variations observed in
TFAAs were insignificant in both the cultivars under EC
exposure (Fig. 7). TSS increased significantly by 9.3 %
and 15.1 % in HUM-2 and HUM-6, respectively, in EC
as compared to ambient CO2 (Fig. 7). In EC, significant
decrease in RSs was noticed to be 9.4 % and 8.9 % in
HUM-2 and HUM-6, respectively. Starch content (SC)
increased significantly by 15.5 % in HUM-6 under EC
treatment. Results of two-way ANOVA showed that SPC,
TFAA and RS varied significantly due to all the individual
factors (Fig. 7). Variations noticed in TSS were significant
only due to treatment (Fig. 7). SC showed significant
changes due to all individual factors and their interactions
(Fig. 7).

280 © 2014 Blackwell Verlag GmbH, 200 (2014) 273–289

 Effect of CO2 Enrichment on Two Tropical Mung Bean Cultivars

NFCs EC
8 18
**
** * 16
**

Shoot biomass (g)

6 * ns 14

Root biomass (g)

12
10
4
* ns * 8
ns
ns 6
2 ns
4
2
0 0
**
* ns * * 30
8 *
*

Total biomass (g)

Leaf biomass (g)
*
6 20
ns
ns
4
* ns
10
Fig. 5 Effect of elevated CO2 (EC) on biomass
2
accumulation and allocation in two cultivars of
mung bean at different stages of growth in
0 0
non-filtered chambers and EC. Values are 20 40 60 20 40 60 20 40 60 20 40 60
mean
S.E. *P < 0.05, **P < 0.01, DAG DAG DAG DAG
***P < 0.001, ns: not significant. HUM-2 HUM-6 HUM-2 HUM-6

NFCs EC
0.16 35
0.14 ns ns
30
Relative growth rate

0.12 ns
ns 25

Leaf area ratio

(g g–1 day–1)

ns ns ns
0.10

(cm2 g–1)
20
0.08 ns
15
0.06
ns
ns 10
0.04
0.02 5
ns
ns
0.00 0
ns
0.014 * 0.5
Net assimilation rate

0.012 ns ns
(mg cm–2 day–1)

Root shoot ratio

ns 0.4
0.010 ns
0.008 0.3
ns ns
0.006
0.2
Fig. 6 Growth indices of HUM-2 and HUM-6 0.004
ns ns 0.1
at different stages of growth in non-filtered 0.002
ns ns
chambers and elevated CO2 (mean
S.E.). 0.000
0–20 20–40 40–60 0–20 20–40 40–60 20 40 60 20 40 60
0.0
*P < 0.05, **P < 0.01, ***P < 0.001, ns: not DAG DAG DAG DAG
significant. HUM-2 HUM-6 HUM-2 HUM-6

trend may be ascribed to more decrease in .O2 production

Discussion
rate and H2O2 levels. In general, it is thought that up/
The present study conducted in near-natural conditions down-regulation of plant antioxidant systems is controlled
revealed differential response of mung bean cultivars by the extent of oxidative stress (Polle et al. 1997). Under
exposed to EC. Data obtained in the present experiment EC, higher reductions in the activities of SOD and CAT in
showed that plants grown under EC will have lower antiox- HUM-6 may reflect its higher capability to cope with vari-
idative enzyme activity because of decrease in cellular ROS ous stress events. According to Halliwell and Gutteridge
production. Under EC, decrease in MDA equivalents, (1989), EC would result in an increased pCO2/pO2 ratio,
which reflects the membrane LPO, was noticed in both the with consequent possible reduction of ROS produced in
cultivars, but the extent of reduction was higher in Cv the plant cell and virtual elimination of photorespiration in
HUM-6 than in HUM-2. The possible explanation for this C3 plants (Bowes 1991). These effects may lead to

© 2014 Blackwell Verlag GmbH, 200 (2014) 273–289 281

Mishra and Agrawal

Table 2 Yield attributes of mung bean cultivars in non-filtered chambers (NFCs) and elevated CO2 (EC) in two consecutive years of experiment (val-
ues are mean
S.E.)

Year Cultivar Treatment NOPoPP NOSPP WOPoPP WOSPP HI TW

2011 HUM-2 NFCs 22.1
1.17 171.2
14.11 9.18
0.60 7.5
0.74 0.29
0.01 43.2
1.30
EC 25.8
1.76 ns 230.5
16.78* 11.4
0.75** 9.4
0.48* 0.30
0.02 ns
47.8
0.75*
HUM-6 NFCs 34.6
1.66 301.9
17.11 14.2
0.86 11.8
0.57 0.46
0.03 43.5
0.71
EC 41.1
1.95** 424.6
8.37*** 18.9
0.44** 15.8
0.47*** 0.51
0.01 ns
51.2
0.90***
2012 HUM-2 NFCs 21.6
0.99 169.9
13.50 8.78
0.51 7.0
0.62 0.27
0.02 41.3
1.17
EC 24.9
1.65 ns 228.8
15.23* 10.8
0.45** 8.9
0.43* 0.29
0.01 ns
46.1
0.74*
HUM-6 NFCs 33.5
1.43 298.0
17.50 13.7
0.56 11.2
0.58 0.45
0.03 42.6
0.79
EC 39.9
1.69** 417.2
6.53*** 18.0
0.52** 15.1
0.41*** 0.49
0.02 ns
51.2
0.66***
F ratios and significance levels from multivariate ANOVA
Y 0.7 ns 5.7* 2.0 ns 2.3 ns 1.6 ns 5.5*
Cv 151.5*** 248.3*** 200.8*** 183.1*** 160.5*** 11.2**
T 20.0*** 78.3*** 58.7*** 57.0*** 0.6 ns 86.1***
Y 9 Cv 0.04 ns 0.03 ns 0.05 ns 0.06 ns 1.1 ns 0.53 ns
Y9T 0.01 ns 0.009 ns 0.17 ns 0.006 ns 0.1 ns 0.18 ns
Cv 9 T 1.7 ns
9.2** 7.2** 6.3* 0.2 ns 5.7*
Y 9 Cv 9 T 0.005 ns 0.006 ns 0.02 ns 0.008 ns 0.7 ns 0.07 ns

NOPoPP, number of pods plant1; NOSPP, number of seeds plant1; WOPoPP, weight of pods plant1; WOSPP, weight of seeds plant1; HI, harvest
index; TW, test weight; Y, year; Cv, cultivar; T, treatment. Level of significance *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.

relaxation of antioxidant defence system inside the plant
cell (Badiani et al. 1993).
In our experiment, total chlorophyll increased under EC
and it was more in HUM-6 than in HUM-2, suggesting
higher induction of chlorophyll molecules from precursors
as compared to ambient CO2. An increase in chlorophyll
pigments has been reported under CO2 enrichment (Mish-
ra et al. 2013b). Significant positive correlation between
treatment and total chlorophyll has been noticed in HUM-
2 (r = 0.62, P < 0.01) and HUM-6 (r = 0.92, P < 0.001)
(Tables 3 and 4). Carotenoids are accessory pigments, and
their essential role is to protect the photosynthetic system
from the photo-oxidative damage. Concentrations of carot-
enoids reduced in both the cultivars, maximally under
HUM-2. Less reduction of carotenoids in HUM-6 might
have provided more protection to chlorophyll molecules
against photo-oxidative damage.
In the present investigation, the Ps, Ci and WUE values
in leaves of mung bean were higher, while the gs and E were
lower under EC than ambient CO2. Cv HUM-6 showed
higher rate of photosynthesis than HUM-2. In HUM-6,
higher increment in Ps was found to be associated with
more increase in internal CO2 concentration (Ci), which
might have increased the carboxylation activity of Rubisco
and the capacity for photosynthetic electron transport and
ribulose-1,5-bisphosphate regeneration, and thereby the
photosynthetic rate. Significant positive correlations were
observed between Ps and Ci in HUM-2 (r = 0.82,
P < 0.001) and HUM-6 (r = 0.92, P < 0.001) (Tables 3
and 4). Our results are in agreement with previous findings
on mung bean (Uprety et al. 1996, Sharma-Natu et al.
2004). According to Long et al. (2004), the increase in pho-
tosynthesis due to EC results from two properties of Rubi-
sco: (i) the Km of the enzyme for CO2 is close to the
current atmospheric concentration, and EC stimulates the
rate of carboxylation; and (ii) CO2 competitively inhibits
the oxygenation reaction, which produces glycolate, result-
ing in photorespiration. The second effect is particularly
important because it increases the efficiency of net CO2
uptake by decreasing the loss of CO2 from photorespiration
and diverting ATP and NADP, from the photorespiratory
metabolism to photosynthetic assimilation (Sujatha et al.
2008). Thus, the efficiency of net photosynthesis increases,
regardless of other factors that limit gross photosynthetic
rate (Long and Drake 1992).
Stomatal conductance (gs) generally decreases in
response to high CO2 (Ainsworth and Rogers 2007), but
the effect is variable and subject to environmental feedback
(Leakey et al. 2006). Decrease in gs was higher in HUM-2,
suggesting lower gaseous exchange from the outer environ-
ment, thereby less Ci compared to HUM-6. Under EC,
reduction in gs was not found associated with concurrent
decline in photosynthesis because the ratio between inter-
nal CO2 and atmospheric CO2 ((Ci/Ca), an index of the
limitation of photosynthesis) was nearly identical for plants
grown at ambient or EC (Long et al. 2004). This suggests
that Ci being high in EC-treated plants led to continued
increase in Ps even at reduced gs. Under EC treatment,
reduction in transpiration rate (E) was noticed in both the
mung bean cultivars; however, decrease was higher in
HUM-6. The result indicated less water loss in HUM-6
under CO2-enriched condition. Uprety et al. (1996) found

282 © 2014 Blackwell Verlag GmbH, 200 (2014) 273–289

 Effect of CO2 Enrichment on Two Tropical Mung Bean Cultivars

NFCs EC
20 0.5
Cv *** Cv ***
ns
T *** T ***
* Cv × T ns Cv × T ns

Total free amino acids

ns
0.4
15

Seed protein
0.3

(mg g–1)
(mg g–1)
ns
10
0.2

5
0.1

0 0.0
* Cv ns ** Cv **
T *** T *** *
50 Cv × T ns * Cv × T ns
20

Total soluble sugars

Reducing sugars
40
15

(mg g–1)

(mg g–1)
30
10
20

10 5

0 Cv ***
0
HUM-2 HUM-6
T ***
120
Cv × T ** *

100 ns
(mg g–1)
Starch

80

60
Fig. 7 Variations in seed quality parameters of
mung bean cultivars in non-filtered chambers 40

and elevated CO2. Values are mean
S.E. 20

*P < 0.05, **P < 0.01, ***P < 0.001, ns: not
0
significant. HUM-2 HUM-6

similar results in mung bean exposed to EC. At 40 DAG,
WUE was also higher in HUM-6 than in HUM-2. The pos-
sible reason for this trend may be that increase in CO2 con-
centration will decrease the stomatal conductance, thereby
reducing water flux and ultimately decreasing evapotrans-
piration (Li et al. 2010). The correlations between WUE
and E were negative and significant in HUM-2 (r = 0.92,
P < 0.001) and HUM-6 (r = 0.85, P < 0.001) (Tables 3
and 4).
Plant height increased continuously under EC at all the
ages of sampling as compared to ambient CO2 in both the
cultivars. Under CO2 enrichment, HUM-6 showed more
positive response than HUM-2, which may be due to
increased allocation of photoassimilates towards shoot
growth. A significant positive correlation between treat-
ment and plant height was noticed in HUM-6 (r = 0.51,
P < 0.05) (Table 4). The number of leaves and leaf area are
two important parameters for plant survival and growth.
Increase in number of leaves and leaf area was significant in
HUM-6, which may be attributed to greater partitioning of
assimilates. In the present experiment, increase in leaf area
also suggested that EC provided sufficient photosynthates
for leaf expansion. Pal et al. (2004) reported increase in leaf
area of a fodder crop, clover, under EC.
In EC treatment, increase in the number of nodules was
more in Cv HUM-6 than in HUM-2. Higher photosyn-
thetic efficiency along with increased photosynthetic
surface area led to increased nodulation under EC. Signifi-
cant positive correlations were observed between leaf area
and number of nodules in HUM-2 (r = 0.96, P < 0.001)
and HUM-6 (r = 0.87, P < 0.001) (Tables 3 and 4). There-
fore, in the present study, increased level of CO2 not only
increased photosynthesis but also enhanced the biological
nitrogen fixation capacity of mung bean plants. Increase in
nodulation and nitrogen fixation in legumes by increased
assimilate availability has been reported by Rao and Ghildi-
yal (1985).
In the present experiment, EC clearly imparts its positive
effects during the dry matter distribution, which was
reflected in terms of increase in dry weight of root, shoot
and leaf. Increased root growth contributes to root biomass
and root dry weight under EC regardless of species or study
conditions (Rogers et al. 1996). We observed an increase in
plant biomass under EC, and the relative increase was
higher in HUM-6 compared to HUM-2 (Fig. 5). Enhanced
photosynthesis led to increased allocation of photosynth-
ates to the growing organs (root, shoot and leaves) and
ultimately resulted in higher total biomass in HUM-6 com-
pared to HUM-2. Increment in total biomass under EC
treatment may be ascribed to higher assimilation and accu-
mulation of photosynthates due to higher number of leaves
and leaf area (Mishra et al. 2013a). Under CO2 enrichment,
increase in root biomass in grasslands (Le Cain et al. 2006)
and in weight of roots in black gram (Vanaja et al. 2007)

© 2014 Blackwell Verlag GmbH, 200 (2014) 273–289 283

 284
Mishra and Agrawal

Table 3 Correlation coefficient (r) between photosynthetic pigments, physiological, growth and biochemical parameters under CO2 treatment in HUM-2#

TC CAR Ps Ci gs E WUE PH NOL LA NON DRW DSW DLW TB LPO HP SR SL SOD CAT

ns ns ns ns ns ns ns
A 0.40 0.50* 0.09 0.35 0.56* 0.18 0.01 0.87*** 0.73*** 0.81*** 0.79*** 0.76*** 0.86*** 0.77*** 0.83*** 0.87*** 0.88*** 0.75*** 0.24 0.78*** 0.17
ns ns ns ns ns ns ns ns ns ns ns ns ns ns
T 0.62** 0.46 0.90*** 0.72*** 0.49* 0.79*** 0.89*** 0.39 0.35 0.46 0.40 0.46 0.23 0.32 0.29 0.21 0.39 0.59* 0.37 0.47 0.32
ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns
TC 0.54* 0.33 0.11 0.46 0.44 0.37 0.25 0.16 0.21 0.26 0.23 0.42 0.35 0.38 0.05 0.55* 0.53* 0.15 0.21 0.01
ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns
CAR 0.28 0.29 0.94*** 0.10 0.22 0.44 0.75*** 0.41 0.33 0.35 0.30 0.26 0.30 0.82*** 0.34 0.29 0.46 0.83*** 0.12
ns ns ns ns ns ns ns
Ps 0.82*** 0.34 0.82*** 0.98*** 0.49* 0.23 0.58* 0.56* 0.63** 0.45 0.55* 0.51* 0.12 0.33 0.52* 0.55* 0.40 0.37
ns ns ns ns ns ns
Ci 0.40 0.76*** 0.85*** 0.71*** 0.34 0.80*** 0.76*** 0.79*** 0.68** 0.73*** 0.72** 0.29 0.07 0.28 0.53* 0.58* 0.46
ns ns ns ns ns ns ns ns ns ns
gs 0.17 0.29 0.54* 0.77*** 0.53* 0.44 0.47* 0.41 0.36 0.41 0.83*** 0.37 0.28 0.36 0.88*** 0.18
ns ns ns ns ns ns ns ns ns ns ns
E 0.92*** 0.26 0.06 0.36 0.32 0.39 0.19 0.33 0.27 0.12 0.58* 0.69** 0.63** 0.22 0.39
ns ns ns ns ns ns ns ns
WUE 0.43 0.16 0.53* 0.50* 0.58* 0.39 0.51* 0.46 0.03 0.43 0.60** 0.62** 0.35 0.41
ns ns ns
PH 0.73*** 0.97*** 0.93*** 0.92*** 0.93*** 0.90*** 0.93*** 0.75*** 0.57* 0.34 0.09 0.81*** 0.35
ns ns ns
NOL 0.65** 0.56* 0.56* 0.54* 0.51* 0.54* 0.89*** 0.56* 0.38 0.41 0.86*** 0.30
ns ns ns
LA 0.96*** 0.96*** 0.94*** 0.93*** 0.95*** 0.67** 0.48* 0.25 0.23 0.79*** 0.37
ns ns ns ns
NON 0.97*** 0.94*** 0.95*** 0.96*** 0.63** 0.46 0.26 0.23 0.75*** 0.24
ns ns ns ns
DRW 0.95*** 0.95*** 0.97*** 0.61** 0.42 0.21 0.28 0.74*** 0.36
ns ns ns
DSW 0.96*** 0.99*** 0.63** 0.59** 0.42 0.18 0.69** 0.31
ns ns ns ns
DLW 0.98*** 0.56* 0.45 0.27 0.25 0.66** 0.30
ns ns ns
TB 0.61** 0.53* 0.35 0.22 0.70*** 0.32
ns
LPO 0.74*** 0.62** 0.51* 0.93*** 0.10
ns
HP 0.94*** 0.49* 0.51* 0.03
ns ns
SR 0.56* 0.36 0.16
ns ns
SL 0.26 0.35
ns
SOD 0.23

A, age; T, treatment; TC, total chlorophyll; CAR, carotenoids; Ps, photosynthetic rate; Ci, internal CO2 concentration; gs, stomatal conductance; E, transpiration rate; WUE, water use efficiency; PH,
plant height; NOL, number of leaves; LA, leaf area; NON, number of nodules; DRW, dry root weight; DSW, dry shoot weight; DLW, dry leaf weight; TB, total biomass; LPO, lipid peroxidation; HP,
hydrogen peroxide content; SR, superoxide radical production rate; SL, solute leakage; SOD, superoxide dismutase activity; CAT, catalase activity. #Data used from first-year experiment (2011). Level
of significance *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.

© 2014 Blackwell Verlag GmbH, 200 (2014) 273–289

 Table 4 Correlation coefficient (r) between photosynthetic pigments, physiological, growth and biochemical parameters under CO2 treatment in HUM-6#

TC CAR Ps Ci gs E WUE PH NOL LA NON DRW DSW DLW TB LPO HP SR SL SOD CAT

ns ns ns ns ns ns ns ns
A 0.01 0.04 0.04 0.15 0.79*** 0.45 0.19 0.81*** 0.80*** 0.89*** 0.67** 0.79*** 0.81*** 0.79*** 0.81*** 0.90*** 0.83*** 0.77*** 0.09 0.88*** 0.43
ns ns ns ns ns ns ns ns ns ns
T 0.92*** 0.14 0.90*** 0.78*** 0.40 0.66** 0.87*** 0.51* 0.44 0.39 0.55* 0.47 0.36 0.49* 0.41 0.17 0.31 0.62** 0.78*** 0.34 0.51*
ns ns ns ns ns ns ns ns
TC 0.01 0.94*** 0.93*** 0.37 0.72** 0.94*** 0.47* 0.39 0.44 0.60** 0.53* 0.43 0.54* 0.48* 0.13 0.36 0.57* 0.78*** 0.31 0.61**
ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns
CAR 0.22 0.26 0.48* 0.06 0.14 0.17 0.30 0.03 0.20 0.23 0.31 0.15 0.26 0.44 0.40 0.12 0.07 0.39 0.53*
ns ns ns ns ns ns ns
Ps 0.92*** 0.22 0.61** 0.93*** 0.41 0.30 0.39 0.59** 0.53* 0.44 0.52* 0.48* 0.01 0.49* 0.57* 0.78*** 0.17 0.68**
ns ns ns ns ns ns ns ns ns ns
Ci 0.88*** 0.26 0.16 0.29 0.50* 0.44 0.36 0.41 0.39 0.12

© 2014 Blackwell Verlag GmbH, 200 (2014) 273–289

0.11 0.59** 0.56* 0.62** 0.76*** 0.05 0.65**
ns ns ns ns
gs 0.65** 0.44 0.85*** 0.85*** 0.83*** 0.63** 0.67** 0.62** 0.73** 0.66** 0.95*** 0.69** 0.32 0.24 0.97*** 0.32
ns ns
E 0.85*** 0.67** 0.67** 0.72** 0.80*** 0.70** 0.65** 0.77*** 0.69** 0.52* 0.09 0.09 0.53* 0.64** 0.60**
ns ns ns ns
WUE 0.59** 0.52* 0.60** 0.78*** 0.69** 0.62** 0.71** 0.66** 0.24 0.27 0.39 0.74*** 0.41 0.74***
ns ns
PH 0.95*** 0.92*** 0.84*** 0.89*** 0.85*** 0.90*** 0.88*** 0.84*** 0.53* 0.29 0.23 0.91*** 0.55*
ns ns ns
NOL 0.88*** 0.77*** 0.80*** 0.77*** 0.83*** 0.80*** 0.87*** 0.58* 0.31 0.18 0.92*** 0.43
ns ns
LA 0.87*** 0.94*** 0.93*** 0.96*** 0.95*** 0.83*** 0.55* 0.44 0.28 0.89*** 0.68**
ns ns ns
NON 0.92*** 0.90*** 0.92*** 0.92*** 0.59*** 0.21 0.18 0.42 0.69*** 0.74***
ns ns ns
DRW 0.98*** 0.97*** 0.99*** 0.66*** 0.34 0.33 0.31 0.75*** 0.81***
ns ns ns
DSW 0.96*** 0.99*** 0.64*** 0.37 0.41 0.23 0.72*** 0.78***
ns ns ns
DLW 0.98*** 0.70*** 0.38 0.31 0.34 0.79*** 0.79***
ns ns ns
TB 0.66** 0.37 0.37 0.28 0.75*** 0.78***
ns ns
LPO 0.85*** 0.56* 0.01 0.98*** 0.25
ns ns
HP 0.81*** 0.35 0.75*** 0.05
ns ns
SR 0.54* 0.43 0.06
ns
SL 0.14 0.58*
ns
SOD 0.36

A, age; T, treatment; TC, total chlorophyll; CAR, carotenoids; Ps, photosynthetic rate; Ci, internal CO2 concentration; gs, stomatal conductance; E, transpiration rate; WUE, water use efficiency; PH,
plant height; NOL, number of leaves; LA, leaf area; NON, number of nodules; DRW, dry root weight; DSW, dry shoot weight; DLW, dry leaf weight; TB, total biomass; LPO, lipid peroxidation; HP,
hydrogen peroxide content; SR, superoxide radical production rate; SL, solute leakage; SOD, superoxide dismutase activity; CAT, catalase activity. #Data used from first-year experiment (2011). Level
of significance *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.
Effect of CO2 Enrichment on Two Tropical Mung Bean Cultivars

285
Mishra and Agrawal
1.5
1.0
13
17 1
14
10
20 21
10
1
15 13
Component 2
0.5 14
19
11
16
11
16 15
0.0
12
22
17 3
–0.5
20 21 22
18
12 19 18
–1.0
–1.0 –0.5 0.0 0.5
Component 1
Fig. 8 The principal component analysis (PCA) of mung bean cultivars,
HUM-2 and HUM-6. The scatter plot of both the cultivars lies on com-
ponent 1 and component 2. Closed (black circle) and open (white circle)
symbols denote different parameters of HUM-2 and HUM-6, respec-
tively, under ambient and elevated CO2. Each symbol represents the
mean values for the PC scores of the three replicates of each parameter.
For the convenience, the parameters are denoted by numbers, (1) CO2,
carbon dioxide; (2) PH, plant height; (3) NOL, number of leaves; (4) LA,
leaf area; (5) NON, number of nodules; (6) DRW, dry root weight; (7)
DSW, dry shoot weight; (8) DLW, dry leaf weight; (9) TB, total biomass;
(10) Ps, photosynthetic rate; (11) gs, stomatal conductance; (12) E, tran-
spiration rate; (13) WUE, water use efficiency; (14) Ci, internal CO2 con-
centration; (15) TC, total chlorophyll; (16) CAR, carotenoids; (17) LPO,
lipid peroxidation; (18) HP, hydrogen peroxide content; (19) SR, super-
oxide radical production rate; (20) SL, solute leakage; (21) SOD, super-
oxide dismutase activity; (22) CAT, catalase activity.
has been reported. The stimulation of C3 plant growth by
EC was closely associated with the increase in photosynthe-
sis (Long et al. 2006). The increase in growth and photo-
synthesis has been primarily affected in C3 plants as rising
CO2 reduces the oxygenase activity of Rubisco, the primary
CO2-fixing enzyme (Pearcy and Bjorkman 1983). Under
EC exposure, increase in growth parameters may be attrib-
uted from higher carbohydrate flux (Mulchi et al. 1992).
EC enhances the photosynthesis rate in the leaves of almost
all C3 species that are well supplied with nutrients (Long
and Drake 1992). Peng et al. (2004) reported that EC stim-
ulates photosynthesis rate and thus increased the biomass
production. According to Long et al. (2004), under CO2
enrichment, increased biomass is due to higher rate of pho-
tosynthesis and net assimilation capacity by inhibiting oxy-
genation and promoting carboxylation of ribulose
bisphosphate carboxylase (RuBP). Root, leaf and total bio-
mass showed significant positive correlations with Ps in
both the cultivars (Tables 3 and 4). Increase in RSR under
EC may be ascribed to greater biomass partitioning to the
roots due to higher net photosynthetic rate. This might be
an adaptation to sequester more carbon for the initiation
of more number of roots (Uprety et al. 1996).
286
The yield components were positively affected by EC in
both the mung bean cultivars. Cv HUM-6 showed better
yield as compared to HUM-2. The higher yield in HUM-6
may be attributed to more increase in number of
pods plant1, NOSPP and weight of seeds plant1. Under
high-CO2 environment, variations in the number of pods
5 6
5
84 may be the key determinant of the response of seed yield
9 2
6
7 8 (Bunce 2009). Increase in seed yield by EC may be primar-
9
7
2 ily associated with increased number of seeds (Jablonski
4
3 et al. 2002, Pleijel and Uddling 2012). The result may be
explained due to enhanced assimilate production resulting
from higher rate of photosynthesis under CO2 enrichment
1.0 1.5 (Mulholland et al. 1997). No significant effect of EC was
observed on HI, and it revealed that CO2 stimulated the
production of reproductive and vegetative parts of the
above-ground biomass to the same extent in both the test
cultivars. Weight of 1000 seeds (TW) was also higher in the
HUM-6, which may be attributed to higher weight gain
under EC environment. We speculate that the advanced
phenological development in HUM-6 likely contributed to
the increase in reproductive biomass at the time of harvest,
revealing higher seed yield under EC. The results indicated
that increased supply of photoassimilates and its higher
translocation to reproductive parts contributed to the culti-
var-specific variations in yield response under EC (De
Costa et al. 2007).
Under EC treatment, contents of soluble protein and
total free amino acids (TFAAs) decreased in seeds of both
the mung bean cultivars. The reduction in protein content
was significant only in HUM-2. Studies have shown the
reduction in soluble protein content under EC environ-
ment (Hogy et al. 2009). Under EC exposure, dilution of
protein occurs if the increase in biomass accumulation is
larger than the increase in N acquisition (Loladze 2002).
This phenomenon is referred to as growth dilution and
leads to reduced concentration of protein with increased
yield under EC. Reduction in nearly all individual and total
amino acids has been reported (Hogy et al. 2009). How-
ever, in our experiment, the variations recorded in TFAA
in seeds were insignificant in both the cultivars. Besides
this, stimulation and accumulation of non-nitrogenous
compounds, that is, TSSs and starch, has been observed
under EC. TSSs increased significantly due to CO2 in both
the cultivars. Higher increase in TSS content in Cv HUM-6
treated with EC may be attributed to greater stimulation of
photosynthesis compared to ambient CO2. Booker et al.
(2007) reported that under EC, increase in photosynthesis
along with reduction in photorespiration is responsible for
higher carbohydrate availability. SC also showed similar
response as TSS in both the cultivars. Under CO2 enrich-
ment, higher concentration of starch may be due to the
increase in the capacity of sinks to assimilate photosynth-
ates in leaves (Wang et al. 2013). The results of the present
© 2014 Blackwell Verlag GmbH, 200 (2014) 273–289

study support the hypothesis that EC not only affects the
productivity and quality of mung bean cultivars but also
increases the biological nitrogen-fixing abilities.
In our study, biochemical, physiological and growth trait
variances were extracted in two components by a PCA
approach. The presence of least sensitive, most sensitive and
moderately sensitive parameters was observed for HUM-2
and HUM-6. From the results of PCA, it was observed that
EC significantly affected physiological and growth parame-
ters of both the cultivars to the same extent (Fig. 8). In
HUM-6, higher reductions in the activities of SOD and
CAT and less ROS generation might have led to higher
translocation of photoassimilates towards reproductive
parts. This differential translocation played an important
role in deciding greater yield in HUM-6; however, in HUM-
2, assimilates might have diverted more towards antioxida-
tive defence system rather than to the reproductive parts.
In summary, the results obtained in the present study
suggested contrasting response of mung bean cultivars
against EC. Our results clearly proved that EC decreased the
formation of ROS vis-
a-vis activities of antioxidative
enzymes revealing reduced oxidative stress environment
inside the cell and contributed to the varying response of
the cultivars. Data also showed that increased level of CO2
imparts its beneficiary effect on the number of nodules,
which may enhance the biological nitrogen fixation ability
of mung bean plants. The overall effect of CO2 enrichment
was an increase in total biomass and seed yield, which may
be due to increased photoassimilation capacity of the test
cultivars. EC not only affected the yield positively but also
enhanced the growth of above-ground vegetative parts.
Thus, it is not only helpful in solving the problem of food
security but also the need of fodder for cattle and/or green
manure for agriculture. The results can be exploited by
plant breeders and biotechnologists in selecting the more
adapted genotypes that will produce more and maintain
desirable quality characteristics in a future high-CO2 world.
Acknowledgements
Authors are thankful to Head, Department of Botany and
Coordinator, Center of Advanced Study in Botany, Banaras
Hindu University, Varanasi, for providing necessary field
and laboratory facilities and to Prof. M. N. Singh, Depart-
ment of Genetics and Plant Breeding, Institute of Agricul-
tural Sciences, Banaras Hindu University, for providing
seeds of mung bean. The authors are also grateful to Coun-
cil of Science and Technology (CST), Lucknow, Uttar Pra-
desh, for providing financial assistance in the form of a
research project (CST/D-1179). Amit Kumar Mishra is
grateful to Center of Advanced Study in Botany, Banaras
Hindu University, for awarding the Junior Research
Fellowship.
© 2014 Blackwell Verlag GmbH, 200 (2014) 273–289
Effect of CO2 Enrichment on Two Tropical Mung Bean Cultivars
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