effect of elevated carbon dioxide on nitrogen assimilation and mobilization in wheat and rye...
TRANSCRIPT
ORIGINAL ARTICLE
Effect of elevated carbon dioxide on nitrogen assimilationand mobilization in wheat and rye genotypesof different ploidy levels
Ngursangzuala Sailo • Rachana Verma •
Renu Pandey • Vanita Jain
Received: 30 November 2012 / Accepted: 18 October 2013 / Published online: 31 December 2013
� Indian Society for Plant Physiology 2013
Abstract Two wheat genotypes differing in ploidy level
viz. PBW 343 (hexaploid) and PDW 274 (tetraploid), and
rye genotype WSP 540-2 (diploid) were grown under ele-
vated CO2 (EC: 550 ± 50 ppm) and ambient CO2
(AC: 380 ppm) to study the changes in nitrogen assimila-
tory pathway enzymes. Elevated CO2 increased nitrate
reductase (NR) activity in flag leaves of the three geno-
types at milk stage, and activity was highest in rye (dip-
loid). At dough stage, hexaploid and tetraploid genotypes
showed higher NR activity in plants grown under EC.
Elevated CO2 resulted in higher expression of NR gene
(NIA1). Activity of glutamine synthetase (GS) in flag
leaves was higher in all the three genotypes under EC at
milk stage but the activity declined at dough stage. The
expression of GS1 increased in flag leaves of plants grown
under EC at both milk and dough stages, while the
expression of GS2 declined during the reproductive stages,
especially in ears of EC grown plants. Nitrate content
decreased in leaf tissues of all the three genotypes by 90
DAS in EC grown plants. This indicated enhanced nitrate
assimilation in leaves by NR under EC at reproductive
stage. However, lower GS2 expression and lower GS
activity during late reproductive phase (dough stage)
indicated inhibition of consequent steps.
Keywords Elevated carbon dioxide � Nitrate reductase �Glutamine synthetase � Nitrate content �Wheat genotypes
Introduction
During the last 110 years the atmospheric CO2 levels has
recorded an increase of about 30 % viz., from 295 (lmol mol-1)
in 1900 to 386 lmol mol-1 in 2010. By 2100 these levels
could reach between 490 to 1,260 lmol mol-1 (Carter et al.
2007). Elevated CO2 increases plant growth, especially in C3
plants, usually termed as the ‘‘carbon fertilization effect’’.
While elevated CO2 increase the C-assimilation, the growth
is often limited by the deficiency of important major and
minor mineral nutrients in the soil. Nitrogen content, rubisco
and other soluble proteins in plants grown for long periods of
time under rising CO2 declines on leaf area basis regardless
of nitrogen supply. Total nitrogen in the shoot did not
decrease under elevated CO2 compared to ambient CO2, but
the fraction of this nitrogen located in flag and penultimate
leaves was lower under elevated CO2. Decrease in rubisco:
chlorophyll ratios has been reported due to loss of leaf ru-
bisco with CO2 enrichment. However, the process of accli-
mation occurs as fewer enzymes are required to maintain
photosynthetic rates (Ainsworth and Rogers 2007).
Increased accumulation of photoassimilates in plants (Gif-
ford et al. 2000; Kant et al. 2012) as a result of elevated CO2
result in a dilution of plant N (Kirschbaum 2011). A number
of studies have demonstrated that elevated atmospheric CO2
decreased leaf N concentration. Decreased N concentration
can also be due to lower specific leaf area under elevated CO2
(Uprety et al. 2002; Moynul Haque et al. 2006).
N. Sailo � R. Verma � R. Pandey � V. Jain (&)
Division of Plant Physiology, Indian Agricultural Research
Institute, New Delhi 110012, India
e-mail: [email protected]
Present Address:
N. Sailo
National Research Center for Orchids,
Pakyong 737 106, Sikkim, India
Present Address:
V. Jain
Krishi Anusandhan Bhawan – II, Pusa Campus,
New Delhi 110012, India
123
Ind J Plant Physiol. (October–December 2013) 18(4):333–338
DOI 10.1007/s40502-013-0049-4
Although there have been many studies on the interac-
tion between elevated CO2 and the nitrogen supply, little is
known about the biochemical and molecular mechanisms
that modify nitrogen uptake, assimilation and utilization,
and the ensuing consequences for photosynthesis and
growth. The information on the changes in the expression
and activity of N assimilatory enzymes is necessary to meet
the challenges imposed by the elevated CO2 in the present
climate change scenario.
CO2 and nitrate compete for the reducing power gen-
erated during the photosynthetic process, later, therefore,
regulate nitrogen assimilation. It seems possible that the
metabolic signals produced during CO2 fixation could
regulate NR activity, although it is still unknown whether
these signals act directly on the NR enzyme itself or if they
affect regulatory proteins (Aguera et al. 1999). Therefore,
factors affecting the CO2 assimilation will also affect
nitrogen assimilation. NR is a key enzyme in the nitrogen
assimilation process, which is subjected to regulation both
at enzyme activity level and at de novo protein synthesis
and degradation level (Campbell 1999). However, little is
known about the role of elevated CO2 on NR gene
expression and enzyme activity (Geiger et al. 1998). The
ammonium formed in the plant by nitrate reduction is
incorporated into the organic molecules to form amino acid
glutamine by the glutamine synthetase (GS). GS exists in
multiple isoforms that are either cytosolic (GS1) or plas-
tidic (GS2) (Betti et al. 2012). The cytosolic GS1 is the
main enzymes for assimilating ammonium from different
sources, N2 fixation and recycling (Bernard and Habash
2009), whereas, the plastidic GS2 assimilate ammonium
produced from nitrate assimilation and photorespiration
(Cren and Hirel 1999). The aim of this work was to
investigate the effects of elevated CO2 on the activity and
gene expression of enzymes involved in nitrate assimila-
tion in wheat and rye genotypes.
Materials and methods
Two wheat genotypes, PBW-343 (Triticum aestivum,
2n = 42, AABBDD), PDW-274 (Triticum durum, 2n = 28,
AABB) and a rye genotype WSP 540-2 (Secale cereale,
2n = 14 RR) were grown under elevated CO2 (EC:
550 ± 50 ppm) in FACE facility and under ambient CO2
(AC: 380 ppm) in the field of Division of Plant Physiology,
IARI, New Delhi. Fertilizers were applied @ 150, 60 and
40 kg ha-1 of nitrogen, phosphorus and potash in form of
urea, single super phosphate and muriate of potash, respec-
tively. Nitrogen was supplied in three equal splits, where first
dose was applied as basal, second at crown root initiation and
third at the time of anthesis. Recommended agricultural
practices were followed.
Biochemical parameters
In vivo nitrate reductase activity was estimated using the
method of Klepper et al. (1971) as modified by Nair and Abrol
(1973). The nitrite produced in the reaction was estimated by
the method of Evans and Nason (1953). Absorbance was
measured using a double beam UV–Vis Spectrophotometer
(UV57045S) at 540 nm. The calibration curve was prepared
using standard sodium nitrite solution. The enzyme activity
was expressed as lmol nitrite formed g-1 dry wt h-1.
Glutamine synthetase (GS) activity was assayed fol-
lowing the method of Mohanty and Fletcher (1980).
The GS activity was calculated from the standard curve of
c-glutamyl hydroxymate as the amount of ferric c-glutamyl
hydroxymate formed and expressed as lmol c-glutamyl hy-
droxymate formed mg-1 protein h-1. Nitrate in plant tis-
sues was estimated based on hydrazine sulphate reduction
method as described by Downes (1978).
For gene expression studies of NIA2, GS1 and GS2 in
the flag leaves, pedicel and ear from milk and dough stages
were taken. RT-PCR was done using 3 sets of primers, viz.
for nitrate reductase (NIA2) and glutamine synthetase (GS1
and GS2).
Name Primer Expected length
of the product (bp)
NIA2 Forward: 50-CGCGCGAGAAGGTCC
CATGT-30400
Reverse: 50-TCCGTCTCGTCCTCCG
GCTG-30
GS1 Forward: 50-GGTTGCTCGCTACCT
TCTTG-30500
Reverse: 50-CTTCCACAGGATGGTG
GTCT-30
GS2 Forward: 50-TGGCTGGCCTGTTG
GAGGGT-30180
Reverse: 50-GTGCCCCGACGGAACC
ACAG-30
The PCR reaction conditions were as follows: The tubes
were incubated at 50 �C temperature, and subsequently run
for 25 cycles at 94 �C for 30 s, 58 �C for 20 s and 72 �C
for 30 s.
Results
The NR activity estimated in flag leaves at milk stage signif-
icantly increased in EC grown plants, irrespective of genotypes
(Fig. 1A). Rye (diploid genotype) recorded the highest NR
activity (2.2 lmol NO2- formed g-1 fr. wt h-1) in the flag
334 Ind J Plant Physiol. (October–December 2013) 18(4):333–338
123
leaves among all the genotypes grown under EC. An increase
of 56, 23 and 31 % was observed in flag leaves of EC grown
plants of diploid, tetraploid and hexaploid genotypes, respec-
tively. Only diploid genotype showed increase in NR activity
in ears and pedicels of EC grown plants, while the activity
decreased in tetraploid and hexaploid genotypes. Under AC,
diploid genotype showed highest NR (1.41 lmol NO2-
formed g-1 fr. wt h-1) activity in flag leaves, whereas, in ears
and pedicels, greater activity was recorded in tetraploid
genotype (1.26 lmol NO2- formed g-1 fr. wt h-1). Flag
leaves showed higher NR activity as compared to ears and
pedicels both under AC and EC in all the three genotypes.
At dough stage, the NR activity declined in the flag
leaves of diploid genotype under EC as compared to AC
grown plants (Fig. 1B), while the activity increased in
hexaploid and tetraploid genotypes grown under EC as
compared to plants grown under AC. In ears and pedicels,
the NR activity declined under EC grown plants of all the
three genotypes. The mean NR activity however, was
higher at dough stage as compared to milk stage in all the
three genotypes.
At milk stage, GS activity increased in flag leaves of plants
grown in EC in all the genotypes (Fig. 2A). An increase of 25,
41 and 27 % was observed in diploid, tetraploid and hexaploid
genotypes, respectively. GS activity increased in ears and
pedicles in diploid and tetraploid genotypes, whereas, hexa-
ploid genotype showed decline in activity under EC grown
plants as compared to AC grown plants. The overall activity of
GS in ears and pedicels under AC was very low in diploid
genotype (9.8 lmol mg-1 protein h-1), whereas hexaploid
and tetraploid genotypes recorded comparatively higher
activity viz. 72 and 37 lmol mg-1 protein h-1, respectively.
At dough stage, the GS activity decreased in flag leaves
and ears ? pedicels of the EC grown plants, except in the
0
1
2
3
4
5
6
7
8
FL E+P FL E+P FL E+P
AC
EC
A
B
V1 V2 V3
FL E+P FL E+P FL E+P
V1 V2 V3
Nit
rate
red
ucta
seac
tivi
ty(µ
mol
NO
2–fo
rmed
g-1
dry
wt.
h-1)
0
1
2
3
4
5
6
7
8
Nit
rate
red
ucta
seac
tivi
ty(µ
mol
NO
2–fo
rmed
g-1
dry
wt.
h-1)
Fig. 1 Changes in nitrate reductase (NR) activity in various tissues of
two wheat and a rye genotype at milk (A) and dough (B) stages in
response to ambient (AC) and elevated CO2 (EC). V1 WSP 540-2, V2
PDW-274, V3 PBW-343, FL flag leaf, E ? P ear ? pedicel
Fig. 2 Changes in glutamine synthetase (NR) activity in various
tissues of two wheat and a rye genotype at milk (A) and dough
(B) stages in response to ambient (AC) and elevated CO2 (EC). V1 WSP
540-2, V2 PDW-274, V3 PBW-343, FL flag leaf, E ? P ear ? pedicel
Ind J Plant Physiol. (October–December 2013) 18(4):333–338 335
123
ears ? pedicles of diploid genotype, wherein no significant
change was observed (Fig. 2B). Flag leaves of tetraploid
and hexaploid genotypes showed 31 and 13 % decline,
respectively under EC as compared to AC grown plants.
The expression of NR gene NIA1 was almost similar in
different tissues of the EC grown diploid plants. Increase in
expression of NR gene was observed in flag leaves of
tetraploid and hexaploid genotypes (Fig. 3). The expres-
sion of two GS isoenzymes was studied at two different
stages—milk and dough stage. At milk stage, the expres-
sion of GS1 and GS2 in flag leaves increased in EC grown
hexaploid, followed by tetraploid genotype (Fig. 4). At
dough stage, expression of GS1 increased in flag leaves and
ears of diploid and tetraploid genotypes. Slight increase in
expression of GS2 was observed in leaves and pedicles of
EC grown plants of diploid and tetraploid genotypes and no
increase was observed in hexaploid genotypes. The GS2
expression was not detectable in the ears of the hexaploid
genotype grown under EC (Fig. 5).
Nitrate content at 60 DAS, did not differ significantly in
leaves of EC and AC grown plants, irrespective of
Fig. 3 Changes in expression of nitrate reductase (NR) at milk stage
in various tissues of two wheat and a rye genotype in response to
ambient (AC) and elevated CO2 (EC). V1 WSP 540-2, V2 PDW-274,
V3 PBW-343. Lane 0 molecular ladder, 1 flag leaf AC, 2 flag leaf EC,
3 pedicel AC, 4 pedicel EC, 5 ear AC, 6 ear EC
500bp
180bp
500bp
180bp
400bp
1 2 3 4 5 60GS1
GS2
GS1
GS2
GS1
GS2
1kbp
100bp
1kbp
100bp
1kbp
100bp
500bp
180bpV1
V2
V3
Actin 1kbp
Fig. 4 Changes in expression of glutamine synthetase (GS) at milk
stage in various tissues of two wheat and a rye genotype in response
to ambient (AC) and elevated CO2 (EC). V1 WSP 540-2, V2 PDW-274,
V3 PBW-343. Lane 0 molecular ladder, 1 flag leaf AC, 2 flag leaf EC,
3 pedicel AC, 4 pedicel EC, 5 ear AC, 6 ear EC
V1
V2
V3
Actin
1 2 3 4 5 60
1kbp
100bp
1kbp
100bp
1kbp
100bp
500bp
180bp
500bp
180bp
500bp
180bp
GS1
GS2
GS1
GS2
GS1
GS2
1kbp 400bp
Fig. 5 Changes in expression of glutamine synthetase (GS) at dough
stage in various tissues of two wheat and a rye genotype in response
to ambient (AC) and elevated CO2 (EC). V1 WSP 540-2, V2 PDW-274,
V3 PBW-343. Lane 0 molecular ladder, 1 flag leaf AC, 2 flag leaf EC,
3 pedicel AC, 4 pedicel EC, 5 ear AC, 6 ear EC
0
1000
2000
3000
4000
5000
6000
7000
8000
ACEC
60 DAS 90 DAS
60 DAS 90 DAS
A
Nit
rate
con
tent
(nm
olni
trat
e g
-1dr
y w
t.)
0
1000
2000
3000
4000
5000
6000
7000
8000
ACEC
B
Nit
rate
con
tent
(nm
olni
trat
e g-1
dry
wt.
)
V1 V2 V3 V1 V2 V3
V1 V2 V3 V1 V2 V3
Fig. 6 Changes in nitrate content of leaves (A) and stem (B) in
response to ambient (AC) and elevated CO2 (EC) at 60 DAS and 90
DAS. V1 WSP 540-2, V2 PDW-274, V3 PBW-343. DAS days after
sowing
336 Ind J Plant Physiol. (October–December 2013) 18(4):333–338
123
genotypes. At 90 DAS, the plants of all the three genotypes
under EC had lower leaf NO3- content as compared to AC
grown plants. Decrease of 19, 47 and 9 % was observed in
diploid, tetraploid and hexaploid genotypes, respectively
under EC, (Fig. 6A). The diploid genotype accumulated
highest nitrate content in leaves of the plants grown under
AC. Stem NO3- content at 60 DAS also did not differ
significantly in EC and AC grown plants. However, at 90
DAS stems of hexaploid and diploid genotypes recorded 60
and 30 % increase, respectively in NO3- content under EC
as compared to AC grown plants (Fig. 6B), while 22 %
decrease was observed in tetraploid genotype under EC.
Discussion
Effects of elevated CO2 was examined on the activity of
enzymes of nitrate assimilation pathway in two wheat and
one rye genotype. Elevated CO2 in general caused increase
in NR activity in all the genotypes. Increase in NR activity
under EC has been recorded in tobacco (Geiger et al.
1998), cucumber (Larios et al. 2001), barley (Robredoa
et al. 2011) and in seagrass (Alexandre et al. 2012). Flag
leaves of EC grown plants had higher activity and
expression in all the three genotypes. Parallel increases in
the rate of nitrate uptake and assimilation vis a vis
increased rate of photosynthesis under elevated CO2, and
nitrate limiting condition have been reported by Lekshmy
et al. (2009). Carbohydrate regulates the uptake and
reduction of nitrate, stimulate the expression and the post-
translational activation of NR (Huber et al. 1996; Hanish
ten Cate and Bretelar 1998). Thus, the potential for NO3-
reduction in response to elevated CO2 was most probably
enhanced by the supply of reductant and C-skeletons
derived from photosynthesis.
The EC grown plants had lower leaf NO3- content in
leaves as compared to AC grown plants, irrespective of
genotypes, at the later reproductive stages. However, stems
accumulated more nitrate under EC as compared to AC.
The reduction in leaf nitrate content under EC could be due
to higher NR activity or blockage of nitrate movement as
suggested by Bloom et al. (2002). Reduction in leaf nitrate
content under elevated CO2 has also been reported by
Geiger et al. (1998). The rate of nitrate assimilation in
leaves exceeded the rate of nitrate uptake by a factor of
two, leading to nitrate depletion in leaves of plants grown
under EC (Matt et al. 2001; Lekshmy et al. 2009).
Glutamine synthetase plays a major role in ammonia
assimilation to form the amino acid glutamine. Biochemical
studies have shown presence of two distinct isoenzymes of
GS, GS2 in the chloroplast and GS1 in cytosol of numerous
plant species (Betti et al. 2012). The GS1 and GS2 serve
distinct roles. GS1 is normally found in the phloem, and is
particularly important for assimilating ammonium from
different sources, for both primary nitrogen fixation and
recycling (Bernard and Habash 2009), whereas, GS2, present
abundantly in chloroplast of mesophyll cell functions to
assimilate ammonium produced from nitrate reduction and
photorespiration (Cren and Hirel 1999). Higher photosyn-
thesis under elevated CO2 leads to more carbon skeletons and
ATP necessary for ammonium assimilation leading to
increase in GS activity (Robredoa et al. 2011). Consequently,
nitrogen metabolism as a whole would be more efficient
under elevated CO2 than in plants grown under ambient CO2
conditions (Natali et al. 2009). Highest GS activity was
observed in flag leaves of tetraploid genotype, which showed
29 % increase over AC grown plants at milk stage. Diploid
and tetraploid genotypes also showed greater activity in ears
and pedicles, whereas decline in activity was noticed in
hexaploid genotype under EC. GS1, which is involved in
recycling of N towards grains at maturity, has higher
expression under EC, indicating positive role of assimilate
supply in mobilization of N towards grains during matura-
tion. Larios et al. (2004) reported increased CO2 fixation at
elevated CO2, and increased carbon supply would stimulate
nitrate utilization by enhancing the expression and activity of
NR and chloroplastic GS2, thus maintaining an adequate C:N
ratio in the plant. Aguera et al. (2006) reported that elevated
CO2 levels increased the activity of GS in cucumber. Pos-
sible reason for decrease in GS2 expression could be the high
CO2 resulting in lower photorespiration and hence, lower
availability of substrate (NH4?), reduced availability of
reductant for NO3- to NO2
- conversion possibly due to
higher level of photosynthesis. High CO2 grown plant have
been shown to have high HCO3- levels, which probably
inhibited the movement of NO2- to chloroplast, leading to
inhibition of NO3- reduction (Bloom et al. 2002). Reduction
in grain N under EC could be due to loss of expression and
activity of GS2 during reproductive stage. The study indi-
cated that initial nitrate assimilation is enhanced in response
to rising CO2, but its further assimilation is inhibited, more
likely due to inhibition of ammonia assimilation in
chloroplasts.
Acknowledgments This research work was supported by Institute
project [IARI: PPH: (09): 01(3)]. Financial support to first author as
Senior Research Fellowship from Indian Agricultural Research
Institute, New Delhi is acknowledged.
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