REGULAR PAPER

Changes in C–N metabolism under elevated CO2 and temperaturein Indian mustard (Brassica juncea L.): an adaptation strategyunder climate change scenario

Chandra Shekhar Seth • Virendra Misra

Received: 19 March 2014 / Accepted: 26 July 2014 / Published online: 23 September 2014

� The Botanical Society of Japan and Springer Japan 2014

Abstract The present study was performed to investigate

the possible role of carbon (C) and nitrogen (N) metabolism

in adaptation of Indian mustard (Brassica juncea L.) grow-

ing under ambient (370 ± 15 ppm) and elevated CO2

(700 ± 15 ppm), and jointly in elevated CO2 and tempera-

ture (30/22 �C for day/night). The key enzymes responsible

for C–N metabolism were studied in different samples of

Brassica juncea L. collected from ambient (AMB), elevated

(ELE) and ELExT growth conditions. Total percent amount

of C and N in leaves were particularly estimated to establish

a clear understanding of aforesaid metabolism in plant

adaptation. Furthermore, key morphological and physio-

logical parameters such as plant height, leaf area index, dry

biomass, net photosynthetic rate, stomatal conductance,

transpiration, total protein and chlorophyll contents were

also studied in relation to C/N metabolism. The results

indicated that the C-metabolizing enzymes, such as (ribu-

lose-1,5-bisphosphate carboxylase/oxygenase, phospho-

enolpyruvate carboxylase, malate dehydrogenase, NAD-

malic enzyme, NADP-malic enzyme and citrate synthase)

and the N-metabolizing enzymes, such as (aspartate amino

transferase, glutamine synthetase, nitrate reductase and

nitrite reductase) showed significantly (P \ 0.05) higher

activities along with the aforesaid physiological and bio-

chemical parameters in order of ELE [ ELExT [ AMB

growth conditions. This is also evident by significant

(P \ 0.05) increase in percent contents of C and N in leaves

as per said order. These findings suggested that improved

performance of C–N metabolism could be a possible

approach for CO2 assimilation and adaptation in Brassica

juncea L. against elevated CO2 and temperature prevailing

in climate change scenarios.

Keywords Aspartate aminotransferase � Brassica juncea

L � Climate change � Carbon di oxide � Nitrate reductase �Rubisco carboxylase and/or oxygenase

Introduction

Global climate change is a ground reality and a continuous

process that warrants immediate attention. Two main

implications of climate change are the rise in atmospheric

CO2 concentration and increase in global temperature. A

detailed knowledge of plant responses are needed against

the projected concentration of CO2 and temperature, which

could be helpful to understand how plants are currently

responding and how they could adapt under climate change

scenario (Stitt and Krapp 1999). Plants display enormous

adaptive capability to survive under adverse climatic con-

ditions which could involve changes at anatomical, mor-

phological, physiological, and biochemical levels. These

changes enable plants to combat harsh climatic conditions

and maintain a reasonably efficient metabolic activity to

compensate the harmful effects of temperature and ele-

vated CO2 conditions (Hogy et al. 2010; Morison and

Lawlor 1999). Among the various metabolic pathways for

adaptation, C–N metabolism is unique under high CO2 and

temperature environment because CO2 fixed via photo-

synthesis should be channelizing in the nitrogen assimila-

tion pathway to maintain homeostasis in plants (Boomiraj

et al. 2010; Geiger et al. 1999). Relatively, larger studies

C. S. Seth (&)

Department of Botany, University of Delhi, Delhi 110007, India

e-mail: seth_bhu@yahoo.co.in

V. Misra

CSIR-Indian Institute of Toxicology Research,

Lucknow 226001, India

123

J Plant Res (2014) 127:793–802

DOI 10.1007/s10265-014-0664-9

are focused on physiological aspects of photosynthesis and

transpiration and not much has been reported on combined

role of C and N metabolic enzymes in plant adaptation

despite the fact that these enzymes play an important role

in CO2 harvesting and N assimilation with concomitant

synthesis of several compounds such as ribulose 1,5-bis-

phosphate (RuBP), phosphoenolpyruvate (PEP), oxaloac-

etate (OAA), 2-oxoglutarate, malate, citrate, aspartate

(Asp), asparagine, glutamate (Glu) and glutamine (Aubry

et al. 2011; Carmen Antolın et al. 2010). It is evident that

C–N metabolism work together and involve complex

interactions between photosynthesis, photorespiration,

respiration and various aspects of nitrogen metabolism,

particularly glutamine synthase–glutamine: 2-oxoglutarate

aminotransferase (GS–GOGAT) cycle (Lancien et al.

2000; Lawlor 2002). In brief, nitrate reductase (NR) is the

first enzyme in N metabolism, helping in reduction of

nitrate (NO3-) to nitrite (NO2

-), which is further reduced

to ammonia/ammonium (NH3/NH4?) by nitrite reductase

(NiR). This ammonia/ammonium is assimilated via GS–

GOGAT cycle in plant cells (Nunes-Nesi et al. 2010; Wang

et al. 2013). While ribulose-1,5-bisphosphate carboxylase/

oxygenase (Rubisco) is the primary C-assimilating enzyme

in plants, phosphoenolpyruvate carboxylase (PEPCase)

catalyzing the reaction of CO2 and PEP to the four carbon

compound OAA carries out various functions other than

CO2 fixation. Namely, 2-oxoglutarate derived from OAA

by the tricarboxylic acid (TCA) cycle, works as carbon

skeletons that allow ammonium assimilation (Aubry et al.

2011). OAA also follows two pathways: it may be used for

Asp production by aspartate amino transferase (AspAT) or

produce malate by malate dehydrogenase (MDH). This

malate is further converted to 2-oxoglutarate and Glu with

the help of the TCA cycle and GOGAT enzymes, respec-

tively. In this way, Asp and Glu work as substrates for

ammonium metabolism, and by getting a continuous sup-

ply of ammonium from soil or through photorespiration/

amino acid catabolism, nitrogen assimilation is going on in

plant cells with GS–GOGAT cycle. Activity of AspAT is

essential as it facilitates the conversion of Asp and

2-oxoglutarate to OAA and Glu, and vice versa. A sche-

matic presentation of the aforesaid pathway with focused

enzymes and related metabolites is given in Fig. 1.

Rapeseed-mustard (Brassica spp.) is a major group of

oilseed crop with India being the second largest cultivator

after China. Although there has been a significant increase

in oilseed production since the 1960s, demand for future

oilseeds production is likely to go up due to the increase in

population and their earning. Mustard is sensitive to cli-

matic variables, particularly changes in carbon dioxide and

temperature, therefore climate change could have signifi-

cant effects on its production (Boomiraj et al. 2010). There

are limited studies performed to assess the impact of

elevated CO2 and temperature on oilseed crops compared to

cereals. There are some reports on synergistic action of

tropospheric ozone and carbon dioxide on yield and nutri-

tional quality of Indian mustard (Brassica juncea (L.)

Czern.) (Singh et al. 2013); response to elevated CO2 of

Indian mustard and sunflower growing on copper contam-

inated soil (Tang et al. 2003); and effect of elevated CO2

and moisture stress on the carbon and nitrogen contents in

Brassica juncea (Uprety and Rabha 1999). In light of this,

an attempt has been made to explore the adaptive mecha-

nisms of this plant for growth under elevated CO2 and

temperature by considering C–N metabolism and gaseous

exchange. Activities of C-metabolizing enzymes viz. Ru-

bisco, PEPCase, malate dehydrogenase (MDH), NAD-

malic enzyme (NAD-ME), NADP-malic enzyme (NADP-

ME), and citrate synthase (CS) along with the activities of

N-metabolizing enzymes like AspAT, GS, NR and NiR

were determined to gain insight on the possible role of this

metabolism. Moreover, total percentage of C and N content

in leaves were particularly estimated to correlate the

activity of C–N metabolic enzymes and establish a clear

understanding for combined role of these enzymes in C–N

metabolism for CO2 assimilation and finally adaptation

under elevated CO2 and temperature conditions.

Materials and methods

Plant material and growth conditions

The seeds were germinated and grown (ca. 6 weeks) in

ambient, elevated CO2, and elevated CO2 and temperature

Fig. 1 Schematic presentation about the role of PEPCase in

inorganic N assimilation with the help of malate dehydrogenase

(MDH), tri carboxylic acid (TCA) cycle enzymes, AspAT and GS–

GOGAT cycle (glutamine synthetase–glutamine: 2-oxoglutarate

aminotransferase)

794 J Plant Res (2014) 127:793–802

123

conditions until the early vegetative stage (6th leaf stage). In

brief, plants were grown in 5-L pots in a 1:1:1 mixture of top-

soil, sand, and perlite and placed in growth chambers (LGC-

6301, Lab Tech India, Pvt. Ltd) equipped with light, tem-

perature, and CO2 control. Plants were grown in three sets as

ambient (370 ± 15 ppm), elevated CO2 (700 ± 15 ppm)

and jointly in elevated CO2 and temperature conditions (30/

22 �C for day/night, respectively) with a day length of 14 h

and a light level of 1,000 lmol m-2 s-1 PAR (photosyn-

thetic active radiation). Temperature in control set i.e.

ambient (AMB) and elevated CO2 (ELE) experimental set

was kept 5 �C below (25/17 �C day/night, respectively) to

the treated set i.e. ELExT which was almost similar to the

temperature of growing season of this plant. Plants were

rotated at least once per week to avoid position effects in the

chambers and about 20–25 uniformly sized plants from each

set were selected within each chamber for experimental use.

All the pots were equally irrigated with water and fertilized

regularly with a nitrogen source (20 mM potassium nitrate,

sigma brand) at a regular interval of time (every other week)

and left for 3 months for acclimatization in the aforesaid

chambers. After the acclimatization of plants, experiments

were carried out and one large sampling was done in

between 9.00 and 11.00 AM in liquid N2 for assaying of

different biochemical parameters. Harvested samples were

brought into the laboratory and kept in a deep freezer at -

80 �C for further use. Briefly, youngest leaves were har-

vested for AMB, ELE and ELExT conditions and used for

various analyses. For one set such as ambient, the second

nodal leaves of around ten plants were harvested and pooled

in one group (biological replicate-1). The experiments were

repeated in the same manner for biological replicate-2 and

biological replicate-3. Likewise sampling was done for the

other two sets (ELE and ELExT) and used for the entire

biochemical assay described in this study.

Measurement of physiological response in AMB, ELE

and ELExT conditions

Some of the important morphological and physiological

parameters relating to photosynthetic responses of plants

were observed. These observations included plant height,

leaf area index (LAI), dry biomass (DM), net photosyn-

thetic rate (PN), stomatal conductance (gs), transpiration

rate (E), and total protein and chlorophyll content growing

in AMB, ELE and ELExT growth conditions. These phys-

iological parameters were recorded in three different plants

representing three replications of each experimental set.

Prior to sampling of leaves for C–N metabolic enzymes,

total chlorophyll and protein assay; PN, gs and E were

observed on the same plant using a portable open gas

exchange system at the same CO2 levels and temperature at

which the plants were growing and a fix photosynthetic

photon flux density (PPFD) of 1,000 lmol m-2 s-1 in

between 9.00 and 11.00 AM. For DM estimation entire

plants were harvested, washed properly to remove adherent

soil, and blotted on tissue and filter paper. They were first

open air dried for 10–15 days and then kept in an oven

(60 ± 5 �C) for 72 h and estimated dry weight on balance

until weight loss became constant. Measurements of above

parameters were taken on three randomly selected plants

per treatment and finally the mean was taken for all the

measured values. Approximately 500 mg fresh leaves were

homogenized in 1.0 ml of 0.2 M phosphate buffer (pH 7.0)

and centrifuged at 5,000 rpm for 10 min. A 0.5-ml portion

of the supernatant was mixed with 1.0 ml of 10 % (w/v)

trichloroacetic acid for total soluble protein precipitation.

The protein pellet was washed with acetone to remove

pigments and dissolved in 1.0 ml of 4 % (w/v) NaOH.

Finally protein content was estimated spectrophotometri-

cally (590 nm) by following the method of Bradford (1976),

using bovine serum albumin as a standard. Total chloro-

phyll contents were extracted by following the method of

Hiscox and Israeclstam (1979). Chlorophyll was extracted

by homogenizing 100 mg of fresh leaves in 5 ml of 80 %

chilled acetone solution and after centrifugation for 10 min

at 7,500 rpm, optical density of supernatant was measured

at 480, 510, 645 and 663 nm using UV–visible spectro-

photometer (SPECORD 200, Analytikjena). Chlorophyll

contents were determined using the formula given by Arnon

(1949) and expressed as mg g fresh weight-1 (g FW-1).

Carbon and nitrogen analysis in plant samples

Leaves of approximately similar morphology and phenol-

ogy of each experimental set were collected in paper bags

and dried in an oven (60 ± 5 �C) for a week. Dried plant

matter was crushed into very fine powder and analyzed for

percent amount of C and N using a CHNS analyzer (Ele-

mentar, vario micro). In brief, about 10–15 mg of each

plant sample was mixed with tungsten oxide, (3–4 mg) as a

catalyst, and packed in a very thin tin board. Similar pro-

cedure was repeated for all the plant samples. The packed

sample boats were placed in the sample holder of the

instrument for analysis. Sulfanilamide was used as a stan-

dard, which has a fixed percent amount of C (41.8 %), N

(16.25 %), H (4.65 %) and S (18.62 %).

Rubisco (EC 4.1.1.39) activity

Rubisco activity was determined by the spectrophotometric

method of Lilley and Walker (1974), modified by Gerard

and Driscoll (1996). Frozen leave tissues (100 mg) were

homogenized in chilled mortar with 1 ml of a CO2-free

extraction buffer (0.1 M Tris–HCl buffer containing 2 mM

EDTA, 10 mM MgCl2, 20 % glycerol, 10 %

J Plant Res (2014) 127:793–802 795

123

polyvinylpolypyrrolidone (PVPP), 1 % v/v Triton X-100,

50 mM dithiothreitol (DTT), 100 mM Na ascorbate and

10 mM NaHCO3, at pH 7.6). The homogenate was cen-

trifuged at 16,0009g for 2 min at 4 �C and the supernatant

was used for Rubisco activity. Total Rubisco activity was

determined after enzyme activation by pre-incubation of

more than 10 min at 0 �C in presence of 10 mM NaHCO3

and 10 mM MgCl2, at pH 7.2. The assay mixture contained

50 mM HEPES, 10 mM NaHCO3, 20 mM MgCl2, 0.2 mM

NADH, 5 mM ATP, 5 mM phosphocreatine, 5 units (U) of

creatine phosphokinase (CPK), and 5 units of glycer-

aldehyde-3-phosphate dehydrogenase/3-phosphoglycerate

kinase, at pH 7.8. Rubisco activity was calculated by rate

of decrease in absorbance (A340) to a rate of NADH oxi-

dation. Oxygenase activity was monitored by measuring

the O2 uptake with an oxygen electrode at 25 �C. In this

procedure, all the solutions were saturated with O2, using a

CO2-free O2 gas. The activated enzyme was assayed in the

medium containing 100 mM Bicine buffer (pH 8.2),

0.2 mM EDTA, 0.5 mM RuBP and 20 mM MgCl2 (Pierce

et al. 1982).

PEPCase (EC 4.1.1.31), NADP-ME (EC 1.1.1.40)

and NAD-ME (EC 1.1.1.39) activity

Extraction of PEPCase, NADP-ME and NAD-ME were

performed by grinding 100 mg of frozen leaf samples in a

extraction buffer containing 50 mM Tris–HCl buffer (pH

7.5), 1.0 mM MgCl2, 5.0 mM DTT, 1.0 mM phenylmeth-

ylsulfonyl fluoride, 2 % (w/v) PVPP, 10 % (v/v) glycerol

and 0.1 % (v/v) Triton X-100. The extract was centrifuged

at 12,000g for 10 min at 4 �C and supernatant was used for

determining enzyme activity. PEPCase was determined by

coupling its activity to malate dehydrogenase-catalysed

NADH oxidation in 1.5 ml final volume of a standard

buffer containing 100 mM Tris–HCl (pH 8.0), 5 mM

MgCl2, 2.5 mM PEP, 0.2 mM NADH, 10 mM NaHCO3,

and 15 lg ml-1 MDH. Assays were initiated by adding

aliquots of the protein extracts determined by the Bradford

procedure using BSA as a standard and NADH oxidation

was determined at 340 nm by spectrophotometer at 25 �C

(Ashton et al. 1990). NADP-ME was assayed following

NADP? reduction at 340 nm (e = 6,200); however, NAD-

ME was assayed in the direction of malate decarboxylation

by measuring NADH formation at 340 nm (e = 6,220)

detailed by Ashton et al. (1990).

CS (EC 4.1.3.7) and MDH (EC 1.1.1.37) activity

Citrate synthase was extracted in 250 mM of Tris–HCl

buffer (pH 8.0) and 0.5 M sucrose, and assayed as

described by Bogin and Wallace (1969). However, malate

dehydrogenase was extracted in 0.2 M potassium

phosphate buffer (pH 7.4) and assayed by monitoring the

oxidation of NADH described by Davies (1969).

AspAT (EC 2.6.1.1) activity

AspAT was extracted by grinding approximately 100 mg

of leaves in 1 ml of extraction buffer composed of 200 mM

Tris–HCl buffer (pH 7.5), 2 mM EDTA and 20 % (v/v)

glycerol (Ireland and Joy 1990). The assay was initiated

with a reaction mixture composed of 50 mM of HEPES-

KOH buffer (pH 7.5), 25 mM of 2-oxoglutarate, 5 U of

MDH and 100 ll of crude enzyme extract and the reaction

was initiated by adding 0.2 mM of NADH. The kinetic

study was performed by measuring the gradual decline in

the optical density of the enzyme reaction at 340 nm for

10 min.

GS (EC 6.3.1.2) activity

Glutamine synthetase was extracted by grinding about

200 mg of leaves in 1 ml of extraction buffer composed of

50 mM Tris–HCl buffer (pH 7.8), 1 mM EDTA, 10 mM

MgSO4, 5 mM monosodium Glu, 10 % (v/v) glycerol and

2 % (w/v) PVPP. The reaction was initiated by taking

50 mM monosodium Glu, 5 mM of hydroxylamine

hydrochloride, 50 mM of MgSO4, 20 mM of ATP and

200 ll of crude enzyme extract in 2.0 ml of microcentifuge

tubes. Above chemical mixtures were left for 30 min at

room temperature and terminated by adding 700 ll mixture

of 0.67 M ferric chloride and 0.37 M HCl. Optical density

of the brown colored compound was taken at 540 nm and

enzyme activity was calculated by following the standard

curve of c-glutamylhydroxamate (Lea et al. 1990).

NR (EC 1.6.6.1) activity

Nitrate reductase activity was measured by following the

method of Harley (1993). Approximately 100 mg of leaves

were crushed in 1 ml of chilled extraction buffer composed

of 500 mM MOPS-KOH buffer (pH 7.5), 1 mM EDTA,

0.5 mg ml-1 DTT and 0.1 % TritonX-100 and centrifuged

at 8,000 rpm for 10 min at 4 �C. Enzyme activity was

determined by using a reaction mixture composed of

25 mM potassium phosphate buffer (pH 7.5), 10 mM

KNO3 and 0.25 mM NADH and 100 ll of crude enzyme

extract and distilled water to make a final volume of 500 ll

and left for 30 min at room temperature. Thereafter, the

reaction was stopped by adding 25 ll of zinc acetate

(0.1 M). The treated reaction mixtures were centrifuged at

8,000 rpm for 10 min and a suitable aliquot of supernatant

fraction (500 ll) was removed for nitrite estimation. This

isolated supernatant was mixed with 500 ll of coloring

reagent made up with 1.0 ml of 1 % (w/v) sulfanilamide in

796 J Plant Res (2014) 127:793–802

123

1.5 N HCI and 1.0 ml of 0.02 % (w/v) N-(1-naphthyl)

ethylenediamine dihydrochloride (NED) solution prepared

in distilled water. After standing for 10 min, optical density

was determined at 540 nm (SPECORD 200, Analytikjena).

The amount of nitrite released was read from a standard

curve of nitrite and the activity is defined as lmol NO2-

produced h-1 g FW-1.

NiR (EC 1.7.7.1) activity

Nitrite reductase was extracted by grinding 100 mg of leaf

tissue in 1 ml of 50 mM potassium phosphate buffer (pH

8.8) containing 1 mM EDTA, 25 mM cystein and 3 % (w/

v) BSA (Ramirez et al. 1966). The assay mixture consisted

of 700 ll of 100 mM potassium phosphate buffer (pH 7.5),

50 ll of 5 mM KNO2, 50 ll of enzyme extract and 50 ll

of methyl viologen (2 mg/ml) and final volume was made

to 900 ll by adding 50 ll distilled water. Enzyme assay

was started by adding 100 ll of sodium dithionite

(25 mg ml-1 in 290 mM NaHCO3 solution) and incubated

for 30 min at 30 �C. At the end of the incubation period,

50 ll of the assay mixture was added to 950 ll of water

and vortexed immediately to oxidize the dithionite. The

amount of nitrite used up by NiR was estimated by adding

freshly prepared 500 ll of sulfanilamide [1 % (w/v) in

1.5 N HCl] and 500 ll of 0.02 % (w/v) NED solution. The

solution was incubated at 30 �C for 10 min and the

absorbance was read at 540 nm. The amount of nitrite used

up by nitrite reductase was estimated from a standard curve

of nitrite and expressed as lmol NO2- used h-1 g FW-1.

Statistical analysis

All the experiments were performed in triplicates using three

biological replicates and values are presented as

mean ± standard deviation (SD). The level of significant

differences between the results of AMB and ELE/ELExT

were calculated through multiple comparison test by Dunnet

method at a critical difference of P \ 0.05 and P \ 0.005.

Results and discussion

Physiological response and percent contents of CHNS

in leaves collected from AMB/ELE/ELExT growth

conditions

To gain a clear understanding of plant responses against

elevated CO2 and temperature conditions, physiological

parameters like photosynthesis, transpiration, stomatal

conductance, dry biomass and photosynthetic pigments

were estimated. Total percent contents of carbon and

nitrogen in leaves were particularly observed as these are

the first insight into the efficiency of photosynthesis and

finally C–N metabolism. Percentage of C (37.80, 42.65,

38.87 %) were found for AMB, ELE and ELExT, respec-

tively; and N (2.90, 3.95, 3.10 %) were found for AMB,

ELE and ELExT, respectively (Table 1). In contrast to

earlier reports (Aubry et al. 2011; Nunes-Nesi et al. 2010;

Wang et al. 2013), the present findings showed a reverse

pattern of higher nitrogen content in ELE as compared to

AMB which could be due to the regular supply of nitrogen

fertilizer (20 mM KNO3) in growth conditions helping in

higher activity of C and N metabolic enzymes under ele-

vated CO2 and temperature compared to ambient. In

addition, since the dry samples contain little water, respi-

ratory loss for CO2 would affect elemental contents per

gram dry weight. Considering these observations it is

important to determine the characteristic responses of

enzymes associated with C–N metabolism. Results

obtained for gaseous exchange as photosynthesis, transpi-

ration, stomatal conductance along with the dry biomass

and photosynthetic pigments are presented in Table 2. The

trend of photosynthetic pigments (chlrophyll a, chlrophyll

b, and total chlrophyll), Pn, gs and E under AMB, ELE and

ELExT conditions clearly reflect the adaptation strategy of

plants under elevated CO2 and temperature. Overall, the

responses for all the above said parameters were found

maximum in ELE followed by ELExT and AMB, except gs

and E which were recorded in reverse order: AMB [ E-

LE [ ELExT. These results indicate that elevated CO2 and

temperature triggered a clear increase in all the physio-

logical parameters, particularly photosynthetic efficiency

of plants for adaptation. It is known that adaptation can be

achieved through certain metabolic pathways and under

elevated CO2 and temperature scenario the key pathway

could be C–N metabolism. The response of this metabo-

lism can be achieved primarily by inducing the enzymes

activity participating to complete this pathway.

Response of C-metabolic enzymes in Indian mustard

under AMB, ELE and ELExT conditions

There are quite a few experiments in which Indian mustard

was observed for change in C-metabolism (photosynthesis

Table 1 Total percent contents of CHNS (per gram DW) in Indian

mustard leaves samples collected from AMB, ELE and ELExT

growth conditions

Growthconditions

% N % C % H % S

AMB 2.90 ± 0.23 37.80 ± 1.10 1.46 ± 0.12 0.37 ± 0.03

ELE 3.95 ± 0.35* 42.65 ± 1.45* 1.95 ± 0.17* 0.42 ± 0.05

ELExT 3.10 ± 0.26 38.87 ± 1.16* 1.58 ± 0.14 0.39 ± 0.04

All the values are mean of triplicates ± SD. Significant differences *P \ 0.05between the results of AMB, ELE and ELExT

J Plant Res (2014) 127:793–802 797

123

and respiration) under AMB, ELE and ELExT conditions.

CO2 has a direct impact on carboxylation capacity of Ru-

bisco and thereby the rate of photosynthesis. In this con-

text, comprehensive assays about the activities of different

enzymes related to C-metabolism were performed in plant

samples collected from respective growth conditions.

Results obtained for C metabolic enzymes viz. Rubisco,

PEPCase, MDH, NAD-ME, NADP-ME and CS are shown

in Fig. 2a–f. Rubisco is the primary carbon assimilating

enzyme and final activity was found to be higher by 108

and 39 % in ELE and ELExT, respectively, compared to

those growing in AMB (Fig. 2a). Likewise RuBP oxy-

genase activity was found to be higher by 75 and 25 % in

ELE and ELExT, respectively (Fig. 2b). The findings

revealed that the plant is adapted to high CO2 and tem-

perature via the balanced activity of Rubisco in terms of

carboxylation and oxygenation. Balanced activity of Ru-

bisco carboxylase and oxygenase is a useful strategy in

plant adaptation under elevated CO2 and temperature as it

induces photorespiration which results in lower suscepti-

bility to photo inhibition or photo damage, and the

advantage arises largely due to increased electron trans-

port, increased capacity to assimilate, or capacity to dis-

sipate energy through various mechanisms (Streb et al.

1998). Rate of photorespiration which was decreased

under elevated CO2 and increased under ELTxT as

compared to AMB is also confirmed by the gas exchange

analysis (Table 2). The present observation is in confor-

mity with earlier reports where altitude related increase in

activity or content of Rubisco was reported in Selinum

vaginatum (Pandey et al. 1984), barley and wheat (Kumar

et al. 2006) and Aconogonum weyrichii (Sakata et al.

2006) for adaptation. PEPCase is the another important

C-metabolizing enzyme which was found to be signifi-

cantly (P \ 0.05) higher by 85 and 39 % in ELE and

ELExT, respectively, as compared to those growing in

AMB (Fig. 2c). Enhanced activity of PEPCase suggests

an altered carbon metabolism for optimal photosynthetic

performance, as this could play an important role for

adaptation by capturing environmental or photo respired

CO2. Increased PEPCase activity was reported earlier in

Glycine soja with increase in altitude of 500 to 3,650 m

(Pyankov et al. 2001), and in succulents such as Umbili-

cus rupestris, Kalanchoe blossfeldiana and Mesembryan-

themum crystallinum, in response to high light regimes

(Brulfert et al. 1993; Reyss and Prioul 1975; Shimono

et al. 2010), suggesting its role in adaptation of C3 plants

under different stress scenarios. In brief, enhanced activity

of PEPCase was an important observation because it is

involved in multifaceted physiological processes and

works as a bridging factor for C–N-metabolism. The

possible fate of OAA, a product of PEPCase catalyzed

reaction could be towards malate/citrate/Asp synthesis.

The conversion of OAA towards malate is taking place

with the combined action of enzymes viz. NAD-ME,

NADP-ME and MDH while towards citrate and/or Asp

occurs through the enzymes, CS and AspAT, respectively

(Britto and Kronzucker 2005; Yang et al. 2010). Present

results revealed a slight increase in the activity of NAD-

ME, MDH and CS under ELE and ELExT conditions as

compared to AMB. In this context, MDH activity was

found to be enhanced by 7 and 3 % for ELE and ELExT,

respectively (Fig. 2d), and NAD-ME activity was found to

be 40 and 20 % for ELE and ELExT, respectively,

compared to those of AMB (Fig. 2e). Activity of NADP-

ME could not be detected in any of the plant samples.

Table 2 Different physiological responses of Indian mustard under AMB, ELE and ELExT growth conditions

Parameters Plant responses

AMB ELE ELExT

Plant height (cm) 26.2 ± 1.07 35.5 ± 1.14** 29.4 ± 1.45*

LAI 0.90 ± 0.06 1.23 ± 0.15* 1.09 ± 0.08

Total dry biomass (roots ? shoots) 24.985 ± 0.31 33.09 ± 0.43** 27.40 ± 0.35*

PN 20.89 ± 0.23 27.32 ± 0.72* 21.43 ± 0.67

gs 350.58 ± 15.27 306.56 ± 13.21* 285.47 ± 11.18**

E 9.51 ± 0.55 7.13 ± 0.49* 6.17 ± 0.45**

Total soluble protein content 43.13 ± 1.6 51.45 ± 1.9* 46.62 ± 1.8

Chlorophyll a 1.12 ± 0.03 1.34 ± 0.04* 1.21 ± 0.03*

Chlorophyll b 0.85 ± 0.01 0.94 ± 0.02* 0.90 ± 0.03*

Total chlorophyll content 1.97 ± 0.05 2.28 ± 0.03* 2.10 ± 0.04*

Plant height (cm), Leaf area index (LAI), Total dry biomass (DM) [g 100 g FW-l], net photosynthetic rate (PN) (lmol CO2 m-2 s-1), stomatal

conductance (gs), transpiration rate (E) [mmol H2O m-2 s-1], total protein (mg g FW-1) and chlorophyll (mg g FW-1) content. All the values

are mean of triplicates ± SD. Significant differences *P \ 0.05 and **P \ 0.005 between the results of AMB, ELE and ELExT

798 J Plant Res (2014) 127:793–802

123

Similarly, CS activity was found to be enhanced by 27

and 11 % for ELE and ELExT, respectively, compared to

those of AMB (Fig. 2f). These indicated that the routing

of OAA towards malate and/or citrate is not a preferred

pathway. Present observations are also in conformity to

earlier reports of slightly enhanced activity of NAD-ME,

MDH and CS in barley and wheat growing under envi-

ronmental stress of high altitudes (Kumar et al. 2006).

These findings along with the earlier reports suggest

responsiveness of Rubisco, PEPCase for C-metabolism

and its adaptive role together with N-metabolism in Indian

mustard under elevated CO2 and temperature, prevailing

in climate change scenarios.

Response of N-metabolic enzymes in Indian mustard

under AMB, ELE and ELExT conditions

Responses of N-metabolic enzymes were studied under the

regular supply of nitrogen fertilizer during the experi-

mental growth of plants. AspAT, GS, NR and NiR activi-

ties were particularly estimated for analyzing N

metabolism in Indian mustard (Fig. 3a–d). In contrast to

slightly enhanced activities of MDH, NAD-ME and CS

obtained for C-metabolism, Indian mustard showed sig-

nificantly (P \ 0.05) higher activity of AspAT and GS,

suggesting a preferred pathway for routing of OAA

towards Asp, rather than to malate and/or citrate. In this

Fig. 2 Activities of different

enzymes related to carbon

metabolism in leaf samples of

Indian mustard growing under

AMB, ELE and ELExT growth

conditions. a–f Depicts the

activities of Rubisco

carboxylase, Rubisco

oxygenase, PEPCase, MDH,

NAD-ME and CS, respectively

and the values are presented as

mean ± SD. Significant

differences *P \ 0.05 and

**P \ 0.005 between the

results of AMB, ELE and

ELExT

J Plant Res (2014) 127:793–802 799

123

context, AspAT activity was found to be significantly

(P \ 0.05) higher by 53 and 30 % (Fig. 3a) while GS

activity was defined to be 57 and 33 % (Fig. 3b) in ELE

and ELExT, respectively, compared to the activity found in

AMB. The present results are in conformity to earlier work

where a preferred shift towards Asp synthesis was recorded

in barley and wheat at high altitudes (Kumar et al. 2006). It

is important to maintain a balance between C and N fixa-

tion and assimilation in plant cells. It is evident that CO2

coming from atmosphere and/or generated in photorespi-

ration could be fixed by PEPCase and the outcome inter-

mediate OAA is routed to Asp and Glu synthesis by AspAT

and GOGAT, respectively (Fig. 1). The source of NH3 for

GS catalyzed reactions could be provided either externally

from soil or by high photo-respiratory activity as indicated

by the observed increase in oxygenase activity of Rubisco

(Fig. 2b). This is also reported that higher photo-respira-

tory activity could provide ammonia in Homogyne alpina,

Ranunculus glacialis and Soldanella alpina under stress

condition of high altitudes (Streb et al. 1998). Besides this,

the externally supplied nitrogen as nitrate could be assim-

ilated by enhanced activity of NR and NiR. Present study

results revealed NR and NiR activity in order of

ELE [ ELExT [ AMB. Figure 3c showed a significant

(P \ 0.05) increase in NR activity with a minimum in

AMB (5.5 lmol NO2- produced h-1 g FW-1) and maxi-

mum in ELE (8.5 lmol NO2- produced h-1 g FW-1),

however NiR activity was found to be (30 lmol NO2-

remained h-1 g FW-1) and (48 lmol NO2- remained

h-1 g FW-1), respectively (Fig. 3d). Nitrite reductase

activity was found to be higher than NR because the NiR

gene could be expressed stronger than NR and could be

induced by both NO3- and/or NO2

-, however NR activity

is induced only by NO3- (Aslam and Huffaker 1989; Wang

et al. 2007). It is very important to mention here that the

present experiment can be extended under limited supply

of nitrogen to explore the clear performance of N-metab-

olism as the activity of these enzymes is nearly dependent

on the availability of nitrogen. The data found under EL-

TxT condition is discussed more elaborately than ELE and

AMB because in all results plants responded in a common

pattern of ELE [ ELExT [ AMB which indicates tem-

perature has antagonistic interaction with elevated CO2 and

decreasing the positive impact of elevated CO2. This could

be due to the fact that temperature above the optimum level

always has negative impacts on plants metabolism. More-

over, the present study is more focused towards adaptation

strategy achieved by C–N metabolism in Indian mustard

jointly under elevated CO2 and temperature scenarios.

Overall, Indian mustard, a primary oil producing and

economically important plant will likely experience high

radiation load, CO2, temperature, limited soil nitrogen and

so on during the extreme pressure of climate change which

would resulted in significant decrease in productivity.

Fig. 3 Activities of different

enzymes related to nitrogen

metabolism in leaf samples of

Indian mustard growing under

AMB, ELE and ELExT growth

conditions. a–d Showing the

activities of AspAT, GS, NR

and NiR, respectively. Values

are presented as mean ± SD.

Significant differences

*P \ 0.05 and **P \ 0.005

between the results of AMB,

ELE and ELExT

800 J Plant Res (2014) 127:793–802

123

Present findings have implications in C-sequestering

pathways reported earlier in barley and wheat at high

altitudes that also conserves N (Kumar et al. 2006). This

earlier pathway proposed only on the basis of metabolic

fluxes from activities measured from extracts, is also sup-

ported on the basis of physiological responses observed at

the level of LAI, DM, PN, gs, E, total protein and chloro-

phyll contents under elevated CO2 and temperature con-

dition in the present experiment (Table 2). Besides this, the

work has implications in generating transgenic Indian

mustard efficiently conserving C and N which can mitigate

the requirement of nitrogen fertilizer, particularly, in cli-

mate change scenarios. Therefore, enhanced activities of

Rubisco carboxylase and/or oxygenase, PEPCase, AspAT,

GS, NR and NiR in Indian mustard under elevated CO2 and

temperature suggests a combined role of above said

enzymes in C–N metabolism, CO2 and nitrogen assimila-

tion and its importance in adaptation under climate change

scenarios.

Conclusions

The present study emphasizes the possible role of C and N

metabolism in Indian mustard adaptation to growing in

elevated CO2 and temperature conditions. An important

observation was that plants responded to high CO2 and

temperature environments through changes in C–N

metabolism and gaseous exchange. Enhanced activities of

Rubisco carboxylase and/or oxygenase, PEPCase, MDH,

NAD-ME, CS coupled with activities of AspAT, GS, NR

and NiR suggests the possible role of this metabolism

whereby plants could efficiently conserve C and N for

adaptation in high CO2 and temperature environment. It was

also observed that C–N metabolism could play a crucial role

in climate change scenarios by trapping efficient amount of

atmospheric CO2 through Rubisco carboxylase and/or

oxygenase, PEPCase and their further assimilation in

nitrogenous compounds via GS, AspAT, NR and NiR pro-

vided the availability of nitrogen. This would be a realistic

approach in plant adaptation under climate change scenar-

ios, which is primarily due to the rise in atmospheric CO2

and temperature. In light of this, further research targeting

to enhanced performance of N metabolism under limited

supply of nitrogen, expected under climate change, in

Indian mustard along with economically important plants,

which are at risk of extinction, along with particularly rare

and endangered plants, under the extreme pressure of cli-

mate change, is an urgent requirement.

Acknowledgments The first author expresses his deep gratitude to

the Department of Botany, University of Delhi, Delhi-110007, India

for providing the research and development grant to continue the

research activity. Prof. R.R. Singh, Department of Botany, University

of Lucknow-226001, India is gratefully acknowledged for his valu-

able suggestions and support in the present experiment.

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