CHAPTER-I
INTRODUCTION
Wheat (Triticum aestivum L.) is one of the main staple crops throughout the world. In
Punjab, during 2011-12 it was cultivated over an area of 35.13 lac hectares with production of
179 lac tones per hectare. Productive agriculture needs a large amount of expensive
nitrogenous fertilizers (Good et al 2004). However, overdose of nitrogen (N) application is
not only detrimental to crops but also life threatening to human population. Improving
nitrogen use efficiency (NUE) of crop plants is thus of key importance. NUE mainly depends
on how plants extract inorganic N from the soil, assimilate nitrate (NO3-) and ammonium
(NH4+) ions and recycle organic N. Efforts have been made to study the biochemical
mechanisms involved in N uptake, assimilation and remobilization in crop plants.
Identification or development of genotypes that can effectively absorb and accumulate high
concentrations of N can grow well and give high yield under low doses of N was the major
goal of our present research.
Nitrogen is an important nutrient required for crop development and is a major yield
determining factor (Schimel and Bennett 2004, Bardgett 2005). It is a component of many
biological compounds that plays a major role in crop yield capacity (Ahmad 2000, Cathcart
and Swanton 2003) and its deficiency constitutes one of the major limiting factors for cereal
production (Shah et al 2003). However, over-use of N fertilizers is economically costly to the
low-value crop producers (Ju et al 2009). Also, there is extensive concern in relation to the N
that is not used by the plant which is lost by leaching of NO3- or denitrification from the soil
and loss of ammonia to the atmosphere, all of which can have deleterious enivormental
effects and health hazards (Camargo et al 2005, Luo et al 2006, Anjana et al 2007).
Efficient use of N by wheat is needed to sustain or increase yield and quality, whilst
reducing the negative impacts of crop and fertilizer production on the environment (Hirel et al
2007, Foulkes et al 2009). NUE has been defined as grain production per unit of available N
and is separated into two components i.e. N uptake efficiency (total plant N per unit soil N)
and N utilization efficiency (grain biomass per unit of total plant N) (Moll et al 1982). NUE
in plants depends on N availability in the soil and on how plants use N throughout their life
span. Genetic variation in NUE has been reported in wheat (Khalilzadeh et al 2011).
Maximizing NUE in wheat, leading to a lower input of N is the need of the hour and can be
brought about by identifying responsive genotypes. The use of such genotypes could be
beneficial in agro-ecosystem while maintaning high yields (Sepehr et al 2009, Sorgona et al
2011).
Nitrogen use efficiency varies with plant growth and development. Wheat life cycle
can be divided into two phases (Hirel et al 2007). In the first phase, i.e. the vegetative phase,
1
young developing roots and leaves behave as sink organs for the assimilation of inorganic N
into amino acids (Hirel and Lea 2001). These amino acids are further used for the synthesis of
enzymes and proteins mainly involved in building up plant architecture and the different
components of the photosynthetic machinery. At later stage of plant development generally
starting after flowering, the remobilization of the N accumulated by these parts behave as
source of N by providing amino acids released from protein hydrolysis that are subsequently
exported to reproductive and storage organs.
Studies have shown that grain N in wheat mainly represents N accumulated in the
vegetative parts till anthesis and then translocated to kernel during the reproductive phase. N
distribution showed that 60-92% of the N accumulating in the wheat grain originates from the
translocation from vegetative tissue after anthesis (Aynehband et al 2010). The assimilation
of inorganic N into organic form has marked influence on plant productivity and crop yield.
In all higher plants, inorganic N is first reduced to ammonia prior to its incorporation into
organic form or can be used in the form of NO3- under particular conditions of soil
composition. NO3- uptake is actually a step of assimilation which further reduces NO3- to
nitrite (NO2-) followed by NH4+ before it gets incorporated into amino acids (Crawford and
Forde 2002). NH4+ assimilation in the plants generally occurs via glutamine synthetase (GS)
pathway but when in excess is assimilated via glutamate dehydrogenase (GDH) (Lam et al
2003).
Entire process of NO3- uptake and assimilation depends upon the activities of various
enzymes i.e. nitrate reductase (NR), nitrite reductase (NIR), GS, glutamate synthase
(GOGAT) and GDH. Reduction of NO3- is the first step that occurs in cytosol, catalyzed by
NR, which reduces NO3- to NO2- (Meyer and Stitt 2001). NO3- reduction takes place in both
roots and shoots but is spatially separated between the cytoplasm where the reduction of NO3takes place and plastids/chloroplasts where NO2- reduction occurs. NO2- is highly toxic if it
accumulates as such in plants, however it is further reduced to NH4+ ions (Meyer and Stitt
2001).
Ammonium ions originating from NO3- reduction and also from photorespiration or
amino acid recycling are mainly assimilated into organic form as glutamine and glutamate,
which serves as the N donors in the biosynthesis of all amino acids, nucleic acids and other N
containing compounds such as chlorophyll in the plastid/chloroplast by the so called
GS/GOGAT cycle (Oliveira et al 2002). The plastidic isoform of GS fixes NH4+ on a
glutamate molecule to form glutamine which further reacts with 2-oxoglutarate to form two
molecules of glutamate being catalysed by the plastidic ferredoxin dependent GOGAT.
Better NUE should leads to maximum yield and better quality per unit of N applied.
Conventional breeding procedures have been performed empirically over the last two decades
(Masclaux et al 2000) and these approaches have been successful in terms of yield
2
enhancement. However, no further attempt has been made on the biochemical basis for
improving NUE. In addition, to obtain a high grain protein content and good quality
parameters, most of the absorbed N need to be translocated to the grain before maturity.
However, less effort has been laid on wheat compared with other cereals such as rice (Obara
et al 2001) and maize (Gallais and Hirel 2004, Hirel et al 2005a) or model plants such as
tobacco (Tercé-Laforgue et al 2004a) and Arabidopsis (Diaz et al
2005). Also, our
knowledge on the regulation of N acquisition and assimilation has however, remained
fragmentary in limited genotypes. Moreover, modern cultivation of wheat involves large land
areas with intensive inputs of agriculture that are highly laborious in conventional breeding.
Alternate way of using detached tiller culture where cell sap entering the grain can be
effectively manipulated and is comparatively fast and convenient method to study in vivo N
metabolism. Similarly, hydroponic culture is useful for studying N metabolism in root/shoot
organs as these techniques have been successfully employed earlier for other crops also.
Understanding the role of various enzymes involved in N metabolism will help in
identifying wheat genotypes that can efficiently use N at sub optimal doses. Results obtained
will be complemented on activities of N assimilating enzymes at different growth stages in
diverse genotypes at different N doses. Such information can lead to germplasm enhancement
for better N uptake and metabolizing capacity. Therefore, in the present study 18 selected
genotypes based on their commercial relevance or distinct genetic background were chosen to
determine the NUE with the following objectives:
1)
To study early stage genotypic differences in N uptake, nitrate reductase and
glutamine synthetase activity in wheat seedlings grown hydroponically.
2)
To determine differences in efficiency of N utilization pathway in different wheat
genotypes under field conditions.
3)
To determine N use efficiency of wheat genotypes grown in field and relate it to
various biochemical parameters.
3
CHAPTER-II
REVIEW OF LITERATURE
The relevant literature available on the present study has been discussed under the
following headings:
2.1 Introduction
2.2 Nitrogen sources available to crop
2.3 Nitrate uptake
2.4 Ammonium uptake
2.5 Nitrate metabolizing enzymes
2.6 Ammonium assimilating enzymes
2.7 Nitrogen metabolites
2.8 Effect of nitrogen on yield attributes and nitrogen use efficiency (NUE)
2.9 Use of tiller culture and hydroponic culture techniques for study of nitrogen
metabolism
2.1 INTRODUCTION
Wheat (Triticum aestivum L.) is an important crop next to rice and maize. It is grown
mainly for carbohydrates in form of starch (65-75%), sugars (1-3%) and proteins (8-14%)
(Bos et al 2005). Plants have fundamental dependence on inorganic nitrogen (N) (Good et al
2004, Karungi et al 2006, Barbanti et al 2007) and 85–90 million metric tonnes of
nitrogenous fertilizers are added to the soil annually (Giller 2004). As N is often the most
limiting nutrient for crop yield in many regions of the world (Giller 2004, Karungi et al 2006,
Barbanti et al 2007) and the increase of agricultural food production worldwide over the past
four decades has been associated with a 7-fold increase in the use of N fertilizers (Nemati and
Sharifi 2012) which has raised serious concerns due to environmental pollution arising from
leaching, ammonia volatilization and dentrification (Chen et al 2004). The most typical
examples of environmental impacts are the eutrophication of freshwater (London 2005) and
marine ecosystems (Tilman et al 2002, Beman et al 2005). In addition, there can be gaseous
emission of N in form of toxic ammonia into the atmosphere (Gioacchini et al 2006).
Furthermore, farmers are facing increasing economic threat with the rising cost of fossil fuels
required for production of N fertilizers. To address both economic and ecological issues, there
is need to identify cultivars that can efficiently utilize N. This will not only reduce pollution
but will favour low fertilizer input which otherwise is quite expensive (Gouis et al 2000,
Delmer 2005).
In wheat, 60-95% of the grain N comes from the remobilization of N stored in roots
and shoots before anthesis (Habash et al 2006). A less important fraction of grain N comes
from post-flowering N uptake and translocation to the grain. After flowering both the size and
4
the N content of the grain can be significantly reduced under N-deficient conditions (Dupont
and Altenbach 2003). However, it is still not clear whether it is plant N availability (including
the N taken up after anthesis and the remobilized N originating from uptake before anthesis)
or storage protein synthesis that limits the determination of grain yield in general and grain
protein deposition in particular (Martre et al 2003). In parallel, whole-plant physiological
studies (Hirel et al 2005a, b) combined with 15N-labelling experiments preferably performed
in the field were undertaken (Gallais et al 2006). These experiments helped in the
identification of some of the key molecular and biochemical traits and nitrogen use efficiency
(NUE) components that govern the adaptation to N-depleted environments before and after
grain filling in lines or hybrids exhibiting variable capacities to take up and utilize N
(Martin et al 2005, Kichey et al 2007).
The assimilation and metabolism of inorganic N in plants is a complex process
involving a series of enzymes. For instance NUE depends on uptake, reduction, translocation,
temporary storage in vacuoles, energy availability and so on. Plants are able to accumulate
NO3- to high concentrations, the majority of which is concentrated in the vacuole by a NO 3
2/H+ exchanger (De Angeli et al 2006). Plants also have the capacity to grow successfully in a
wide range of available NO3- applications. Because of these two properties, the uptake of N
by plant roots is not a simple process, as there would appear to be four different transport
systems operating: (a) constitutive high-affinity (cHATS), (b) nitrate-inducible high-affinity
(iHATS), (c) constitutive low-affinity (cLATS) and (d) nitrate-inducible low-affinity (iLATS)
(Glass 2003, Okamoto et al 2006).
For N management at the whole-plant level, the arbitrary separation of the plant life
cycle into two phases (Masclaux et al 2000) remains simplistic, since it is well known that N
recycling can occur before flowering for the synthesis of new proteins in developing organs
(Lattanzi et al 2005). In addition, during the assimilatory phase, the NH4+ incorporated into
free amino acids is subjected to a high turnover as a result of photorespiratory activity and it
needs to be immediately reassimilated into glutamine and glutamate (Hirel and Lea 2001,
Novitskaya et al 2002).
2.2 NITROGEN SOURCES AVAILABLE TO CROP
Nitrogen is the most abundant element in the atmosphere still its conversion to usable
form by the plants is an expensive process. The N sources directly available to plants include
inorganic N compounds such as NO3- and NH4+ as well as organic compounds such as amino
acids (e.g. glycine, alanine, glutamic acid, aspartic acid) (Lea and Azevedo 2006) and small
peptides (Schimel and Bennett 2004, Bardgett 2005). The major sink for the N is ultimately
protein, which is available for human consumption in the form of grain (Landry and Delhaye
2007). The productivity of grain in the form of grain yield can be enhanced if N uptake and its
5
utilization efficiency is increased or in other words by increasing NUE.
Although generally low, soil N availability can fluctuate greatly in both space and
time due to factors such as precipitation, temperature, wind, soil type and pH. Therefore, the
preferred form in which N is taken up depends on plant adaptation to soil conditions.
Generally, plants adapted to low pH and reducing soils tend to take up NH4+ or amino acids
whereas plants adapted to higher pH and more aerobic soils prefer NO3- (Maathuis 2009).
NO3- uptake occurs at the root level and NO3- transport systems as already discussed have
been shown to coexist in plants and to act co-ordinately to take up NO3- from the soil and
distribute it within the whole plant (Tsay et al 2007). Regardless of the form in which N is
supplied viz. urea, NH4+ or NO3-, the microbial process of denitrification ensures that NO3- is
the most abundant form of N available to the plant. The availability of organic acids is critical
for the supply of carbon skeleton needed for amino acid synthesis. This necessitates the
coordinated regulation of multiple metabolic and regulatory pathways (including N and
carbon) by NO3- as a signal. The presence of multiple isoforms of many of the NO 3responsive enzymes and their differential regulation by internal metabolites and external
signals constitute sophisticated regulatory controls which are not yet fully understood.
2.3 NITRATE UPTAKE
Nitrate, the most preferred source for most plants is taken up by active transport
through the roots, distributed through the xylem and assimilated by the sequential action of
the enzymes nitrate reductase (NR) and nitrite reductase (NIR). The end-product, NH4+, is
incorporated into amino acids via the glutamine synthetase (GS) and glutamate synthase
(GOGAT) cycle (Sanchez et al 2004) (Fig. 1). In the soil, NO3– is carried towards the root by
bulk flow and is taken up by epidermal and cortical cells of the root. Within the root
symplasm, NO3– has four fates: (i) reduction to NO2– by the enzyme NR (ii) efflux back
across the plasma membrane to the apoplasm (iii) influx and storage in the vacuole or (iv)
transport to the xylem for long-distance translocation to the shoot. Following translocation to
the shoot, NO3– leave the xylem and enter the leaf apoplasm to reach mesophyll cells where it
is either reduced to NO2– or stored in the vacuole (Marquez et al 2005).
In regard to energy costs, assimilation of NO3- requires the energy equivalent of 20
mol ATP (Basra and Goyal 2002). The concentration of NO3- in the soil is variable, both
spatially and temporally (Miller et al 2007), depending on soil type, fertilizer addition,
microbial activity etc. NO3- concentration in the soil have been reported by a number of
surveys and reviews that range from very low levels of a few hundred micromolar to around
20 mM, the highest up to 70 mM. NO3– is acquired by LATS and HATS. The LATS displays
linear kinetics and its contribution to NO3- uptake becomes significant at external NO3concentrations above 1 mM. The HATS are able to take-up NO3– at low concentrations
6
(between 1 μM and 1 mM) (Miller et al 2007). Due to the abundant availability of
photosynthetic reductants, leaf mesophyll cells are the main sites of NO3- reduction.
Fig. 1 Nitrogen metabolism in plants
The efficiency of net NO3- uptake rate is under negative feedback control by NO3accumulation (Orsel et al 2002, Chen et al 2004). Therefore, when NO3- supply is higher than
the plant demand, the decrease in NO3- accumulation might be due to the decrease of NO3uptake as a result of the negative feedback regulation by accumulated NO3- which will end up
in the NO3- build up in soil and NO3- pollution (Stohr 1999). External factors such as NO3concentration, light intensity and temperature as well as internal factors such as N metabolites
(NH4+ and glutamine) regulate the rate of NO3- uptake.
2.4 AMMONIUM UPTAKE
Primary sources of NH4+ include direct NH4+ uptake from the soil and the reduction
of NO3− and atmospheric N while the secondary sources consist of amino acid catabolism
following protein degradation, photorespiratory N cycling and the production of NH4+ by
phenylalanine ammonia lyase and asparaginase. In addition to the common pathway of NO3−
assimilation for generating ammonia, the major part, up to 90% of the NH4+ fed into the
GS/GOGAT cycle (Xun et al 2007), is derived from the mitochondrial glycine decarboxylase
(GDC) reaction, which is an integral part of the photorespiratory carbon and N cycle (Wingler
et al 2000, Hirel and Lea 2001, Bauwe and Kolukisaoglu 2003).
The pathway for NH4+ assimilation in higher plants has been well-documented. NH4+
uptake also takes place in a biphasic manner, involving LATS and HATS. Toxicity symptoms
frequently occur if crop plants are grown in NH4+ in the absence of NO3– (Britto and
Kronzucker 2002). Both HATS and LATS for NH4+ uptake are present in plant roots that are
constitutive and do not seem to be significantly induced by NH4+ (Glass et al 2002).
7
2.5 NITRATE METABOLISING ENZYMES
2.5.1 Nitrate Reductase (NR)
Nitrate reductase mediates two-electron reduction of NO3– to NO2– by NAD/NADPdependent by the reaction:
NR
NO3- + 2H+ + 2e-
NO2- + H2O
Nitrate reductase is a homodimer, with two identical subunits joined and held
together by the molybdenum (Mo) cofactor and is known to be substrate inducible (Cazetta
and Villela 2004). Each monomer has molecular mass of about 100-110 KDa (Pathak et al
2008) and is a complex protein containing flavin adenine dinucleotide (FAD), heme
(cytochrome b557) and a Mo cofactor (a prosthetic group) in 1:1:1 (Marquez et al 2005). Thus,
some interactions between the effects of N sources and Mo fertilization on NR have been
reported. NR exists in multiple copies as well as multiple NADH/ NADPH isoforms with
tissue specific distribution.
Nitrate reductase has two hydrophilic hinge regions situated between the cofactor
binding regions which are susceptible to the proteolytic cleavage. Hinge 1 is between Mo
cofactor and cyt b and hinge 2 is between cyt b and FAD (Campbell 2001). The reducing
power for NO3- reduction is normally supplied by NADH. But in some plants, a biospecific
enzyme (that is able to use either NADH or NADPH as a substrate) is present and helps in
plant metabolism studies and its environment relationship (Stolz and Bazu 2002, Cazetta and
Villela 2004). NR is present in the cytosol of the mesophyll cells of the crop plants. However,
there is growing evidence that NR can also be located outside the plasma memebrane
(Morozkina and Zvyagilskaya 2007). The regulation of NR is remarkably complex and is
subjected to mechanisms controlling both its synthesis and catalytic activity. NR gene
expression is induced by NO3– and other factors such as light or sugars (Appenroth et al 2000,
Klein et al 2000) and is repreesed by glutamine or related downstream metabolites that are
formed from NO3– (Balotf et al 2012).
There is evidence suggesting that cytosolic NR is the first enzyme and limiting step in
the pathway of NO3– assimilation in most plants (Cazella and Villela 2004, Balotf et al 2012).
Leaf NR seems to be dependent on the source of N (Cazella and Villela 2004) and on a
continuous supply of NO3- through the xylem (Kawachi et al 2002). At the molecular level,
NR can be phosphorylated at a serine residue, creating a binding site for 14-3-3 proteins and
binding of 14-3-3 protein inhibitors to phosphorylated NR inactivates this enzyme completely
in the presence of divalent cations (Huber et al 2002, Huber 2007, Lambeck et al 2010). In
general, NR activity increased with the growth of the plant upto flowering and thereafter
declined under irrigated conditions (Singh et al 2003). It has been suggested that increasing
the amount of N supply has increased the NR activity in rice (Fan et al 2007) and strawberry
8
(Taghavi et al 2004). Nathawat et al (2005) reported that NR is maximum with NO3– fertilizer
and minimum with NH4+ form. In green bean (Phaseolous vulgaris) activities of NR and NIR
increases in pods and seeds at N dose of 18 and 24 mM compared to control (Sanchez et al
2004).
2.5.2 Nitrite Reductase (NIR)
Nitrite formed in the first step by NR is further reduced to NH4+ by the ferredoxindependent NIR (Meyer and Stitt 2001) by following reaction:
NIR
NO2- + 8H+ + 6e-
NH4+ + 2H2O
The ferredoxin dependent NIR is monomeric protein with molecular mass near 63
kDA (Hirasawa et al 2009). NIR contains a covalently bound 4Fe-4S cluster, one molecule of
FAD and one siroheme prosthetic group. Siroheme is a cyclic tetrapyrolle with one Fe-atom
in the center. Its structure is different from that of heme as it contains additional acetyl and
propionyl residues derived from pyrrole synthesis. The 4Fe-4S cluster, FAD and siroheme
forms an electron transport chain by which electrons are transferred from Fd to NO2-. NIR has
very high affinity for NO2-.
Nitrite reductase is present in the chloroplast and leucoplast of leaves and roots,
respectively. But the capacity for NO2- reduction in the chloroplast is much greater than that
for NO2- reduction in cytosol. Therefore, all NO2- formed by NR can be completely converted
to NH4+. Transgenic plants lack NIR activity so that NO2- accumulates in them. As a result,
plants had greatly reduced level of ammonia, glutamine and proteins in the leaf tissue. NO2- is
never allowed to accumulate in the cells because of its toxicity (Ali et al 2007). NIR utilizes
reduced Fd as electron donor which is supplied by photosystem1 as a product of
photosynthetic electron transport. To a much lesser extent, the Fd required for NO2- reduction
in a leaf can also be provided during darkness by NADPH, which is generated by the
oxidative pentose phosphate pathway that operates in chloroplast and leucoplast of leaves and
roots, respectively (Yonekura-Sakakibara et al 2000).
In plants, NIR has highest activity when grown with NO3- form and lowest with NH4+
form of N. However, in Brassica juncea, the NIR activity has maximum percent reduction
with NH4+ form and minimum with the NO3- form of N at pre-flowering (Nathawat et al
2005). The levels of NR and NIR enzyme activities in crude extracts from L japonicas plant
increased with the increase in NO3- concentration (Orea et al 2005).
2.6 AMMONIUM ASSIMILATING ENZYMES
Ammonium formed from NO2- is toxic if it accumulates in large amounts so its
further assimilation is very important. NH4+ is assimilated to amino acids by the set of
9
following enzymes:
2.6.1 Glutamine Synthetase (GS)
2.6.2 Glutamate Synthase (GOGAT)
2.6.3 Glutamate Dehydrogenase (GDH)
2.6.1 Glutamine Synthetase (GS)
Glutamine synthetase is the sole port of entry into amino acids in higher plants. It is
the central enzyme responsible for the assimilation of NH4+ ions and recycling in plants
(Gallardo et al 1999). GS catalyzes the ATP-dependent fixation of NH4+ to the δ-carboxyl
group of glutamate to form glutamine (Helling 1994).
GS
L-Glutamate + ATP +
NH4+
L-Glutamine + ADP + Pi + H+
Plants contain two types of GS, namely GS1 and GS2 located in cytosol and
chloroplast, respectively, the former being encoded by set of five genes and the latter by one
gene (Hirel et al 2007, Tabuchi et al 2007). GS1 is present mainly in non-green tissues such
as seeds, roots, flowers and nodules but also in the phloem companion cells of leaves. GS2 is
the major GS isoform being located in chloroplast of mesophyll cell (Suarez et al 2002). It is
a dodecamer protein with molecular weight of 350-400 kDa and it has very high affinity for
NH4+. The plastidic isoform of GS2 catalyzes the incorporation of NH4+ into glutamate
producing glutamine and cytosolic GS1 causes the assimilation of NH4+ coming from the soil.
Cytosolic and plastidic isoforms are regulated differently within specific cell types and organs
and in response to different developmental, metabolic and environmental conditions. This
specialization ensures a rapid reassimilation of NH4+ derived from multiple sources (Miflin
and Habash 2002).
The two major GS isoforms in cytosol/chloroplast play primarily non-overlapping
roles in plant N assimilation, with plastidic GS holding a major role in NH4+ assimilation
within photorespiratory N cycling (Forde and Lea 2007). The roles and regulation of plastidic
GS have been discussed by various authors (Zozaya-Hinchliffe et al 2005, Betti et al 2006).
Accumulation of GS product glutamine acts as one of the signals for NR inactivation. GS1
has been proposed as a key component of NUE in plants (Miflin and Habash 2002) and its
metabolic role is particularly important for N remobilization and recycling in woody plants
(Suarez et al 2002, Gallardo et al 2003). Leaf GS activity is a good indicator of the transition
of sink leaves to source leaves during leaf ageing in both C3 (Masclaux et al 2000) and C4
plants (Hirel et al 2005a,b). It has been reported that the determination of total GS activity is
more informative and facilitate the monitoring of leaf senescence and the N remobilization
process (Diaz et al 2008).
A number of studies have suggested a role for cytosolic GS in the remobilization of
10
the inorganic form of N from senescing leaves for grain filling processes. The localization of
cytosolic GS in the vascular tissues and the developmental regulation of this enzyme suggests
its important role in N remobilization. Cytosolic GS is necessary for grain filling as reported
in rice and maize using mutants and also from studies of QTL analysis (Hirel et al 2001,
Obara et al 2004, Habash et al 2007, Hirel et al 2007). Levels of N assimilatory enzymes do
not limit primary N assimilation and hence yield (Andrews et al 2004). The relationship
between kernel number and GS activity suggests that a high GS activity is required to avoid
embryo abortion just after fertilization (Gallais and Coque 2005).
In rice, maximum GS activity was found at 180 kg/ha N dose which is approximately
three times as detected in flag leaves of control plants (Gaur et al 2012). Comparative results
in rice was also reported by Zheng-xun et al (2007). GS activity increased in response to
NH4+ supplementation in radish as reported by Sood et al (2002). Similar effects with NH4+ on
GS activity have also been reported by Sugiharto and Sugiyama (1992). GS activity in leaves
of wheat that had been treated to foliar N fertilization was nine times as compared to control
plants (Skrumsager et al 2009).
2.6.2 Glutamate Synthase (GOGAT)
Glutamate synthase catalyzes the reductant dependent conversion of glutamine and 2oxoglutarate to two molecules of glutamate, one molecule can be utilized as a substrate for the
synthesis of glutamine via GS reaction and other can be used for further metabolic reactions.
GOGAT has two isoforms:
(i) Ferredoxin dependent plastidic isoform (Fd-GOGAT)
(ii) NADH dependent cytosolic isoform (NADH-GOGAT).
The plastidic isoforms of both enzymes (GS2 and Fd-GOGAT) are involved in
primary NH4+ assimilation, while their cytosolic isoforms are involved in secondary
assimilation (Miflin and Habash 2002).
Ferredoxin dependent GOGAT: GOGAT is an Fe-S flavoprotein and a large monomeic
protein with a molecular weight of 140-160 kDa.
GOGAT
L-Glutamine + 2-oxoglutarate + Fd (reduced)
2-Glutamate + Fd (oxdized)
It may represent upto 1% of the protein content of leaves and is localized in
chloroplast of leaves. NH4+ once formed is incorporated initially into the amide position of
glutamine through the plastidic isoform of GS (GS2) (Peat and Tobin 1996, Tobin and
Yamaya 2001, Martin et al 2006) following which the N is transferred to the α-amino position
of glutamate by plastidic isoform of GOGAT (Fd-GOGAT) (Hirel and Lea 2001, Forde and
Lea 2007), thus providing glutamate for NH4+ assimilation. The net outcome of the GS–
GOGAT cycle is the production of glutamate, which can then be incorporated into other
11
amino acids through the action of aminotransferases or transaminases (Forde and Lea 2007).
Activity of this enzyme increases during leaf development in the light.
NADH dependent GOGAT: This enzyme catalyzes the reaction using NADH as reducing
power.
GOGAT
L-Glutamine + 2-oxoglutarate + NADH + H+
2-Glutamate + NAD+
Generally, in green leaves and non-green tissues like seeds and roots, the activity of
the NADH-dependent enzyme is 2-25 folds lower in comparison to Fd-dependent activity
(Hayakawa et al 1999). Although Fd-GOGAT plays a critical role in the reassimilation of
NH4+ released by glycine decarboxylase during photorespiration, NADH-GOGAT assimilates
NH4+ from both primary and secondary sources during N remobilization (Lea and Miflin
2003). It is important in the utilization of N in grain filling and its activity in developing
grains is positively related to yield (Andrews et al 2004).
2.6.3 Glutamate Dehydrogenase (GDH)
Glutamate dehydrogenase is one of the few enzymes capable of releasing amino N
from amino acids to give a keto acid and ammonia that can be separately recycled to be used
in respiration and amide formation, respectively. GDH catalyzes the following reaction:
GDH
Glutamate + NADP+
NH3 + 2-oxoglutarate + NADPH + H+
Glutamate dehydrogenase is a homohexameric, mitochondrial enzyme that reversibly
catalyzes the reversible amination/deamination between 2- oxoglutarate and glutamate using
either NADH or NADPH with comparable efficacy. This enzyme plays a dual role in
companion cells, either in mitochondria when N availability is low or in the cytosol when N
concentration increases above certain threshold (Tercé-Laforgue et al 2004b). It has been
reported that GDH is 25 % more efficient than GOGAT in the synthesis of L-glutamate (Osuji
et al 2009). It is mainly responsible for glutamate catabolism under carbon and N limiting
conditions (Aubert et al 2001). The native GDH has molecular mass of about 239 KDa. In the
GDH pathway, cofactor for the glutamate to 2-oxoglutarate reaction which produces NH4+ as
a bi-product is NAD+. Its cofactor for the reverse rection, 2-oxoglutarate to glutamate is
NADP+. This reverse reaction uses NH4+ to incorporate N and 2-oxoglutarate to glutamate.
These NADH or NADPH forms of GDH are present in the mitochondria and chloroplast,
respectively.
The role of GDH in N management and recycling has recently been reviewed in a
number of whole-plant physiological studies performed on tobacco (Terce-Laforgue et al
2004a) and maize (Hirel et al 2005b). Its physiological role in N metabolism has been the
subject of controversy as it has never been clearly demonstrated that the enzyme plays a
significant role either in NH4+ assimilation or carbon recycling in plants (Dubois et al 2003,
12
Lea and Miflin 2003, Terce-Laforgue et al 2004b, Forde and Lea 2007).
Although some important changes in leaf enzyme activity were observed, both
during plant development and at the different leaf stages, no significant correlation was
obtained between GDH activity and all the other metabolic markers. This finding reiterates
the fact that GDH is probably not directly involved in the control of N management (TerceLaforgue et al 2004b). Although some evidence suggests a role for GDH in NH4+
assimilation, most studies indicate that GDH functions primarily in the deamination direction,
thereby producing NH4+ in the mitochondria (Lea and Miflin 2003). It is most likely that GDH
functions as a shunt to divert some of the carbon skeleton from N metabolism to carbon
metabolism and the tricarboxylic acid cycle (Miflin and Habash 2002, Lea and Miflin 2003).
2.7 NITROGEN METABOLITES
2.7.1 Nitrogen Content
Nitrogen is necessary for the normal growth and development and is an essential
component of enzymes, nucleic acids and regulatory proteins (Seebauer et al 2004). Further,
it is used by higher plants in various physiological processes, including absorption, vacuole
storage, xylem transport and incorporation into organic forms (Lasa et al 2002). Total N
content of plant is an indication of the plant’s capacity to accumulate N (Azeez and Adetunji
2007). Leaf proteins and in particular photosynthetic proteins of plastids are extensively
degraded during senescence, providing an enormous source of N that plants can provide to
supplement the nutrition of growing organs such as new leaves and seeds. N remobilization
has been studied in several plant species through the ‘apparent remobilization’ method which
is the determination of the amount of total N present in the different plant organs at different
times of development and through N long-term labelling, which allows the determination of
fluxes (Gallais et al 2006).
In Arabidopsis and oilseed rape, it has been shown that N can be remobilized from
senescing leaves to expanding leaves at the vegetative stage as well as from senescing leaves
to seeds at the reproductive stage (Malagoli et al 2005, Diaz et al 2008, Lemaître et al 2008).
The contribution of leaf N remobilization to rice, wheat or maize grain N content is cultivar
dependent, varying from 50 to 90% (Masclaux et al 2001). As reported by Khan et al (2009),
N concentration in wheat leaves increased by increasing N concentration either in soil or by
foliar applications. The highest N content in grain (2.25%) and straw (0.48%) was found at
150% N application followed by 100% N and 50% N (Khalque et al 2008).
2.7.2 Amino Acid Content
Inorganic N is assimilated into amino acids which play a pivotal role in plants (Lea
and Miflin 2003). The major transport form of reduced N are amino acids, which are
transported predominantly in the phloem (Tilsner et al 2005). The N in glutamate and
13
glutamine can be transferred to a wide variety of amino acids, nucleic acids (Forde and Lea
2007), ureides (Amarante et al 2006) and polyamines (Alburquerque et al 2006). N
fertilization can be used for nutritional improvement of cereals by increasing and maintaining
contents of essential amino acids and proteins (Thanapornpoonpong et al 2008).
Specific amino acids can subsequently become precursors for all N-containing
organic molecules such as proteins, chlorophyll, cytochrome/phytochrome, secondary
metabolites and nucleic acids. The product of GS enzyme, glutamine, is itself the main form
of organic N for transport in the phloem of rice (Yamaya and Oaks 2004) and in the xylem of
poplar (Couturier et al 2010). In plant cells, N assimilation is compartmentalized between the
cytosol and chloroplast, in relation to the different sources of NH4+. The content of amino
acids, an important N- containing compounds in plant biomass is significantly affected by N
nutrition (Pavlík et al 2010).
In maize, under low N conditions, the amino acid content decreased significantly and
continuously during leaf development. Slightly decreasing amino acid content under low N
supply could be interpreted as the dilution of a stable organic N pool by an increasing leaf
volume. The high N treatment led to increased amino acid content. High N supply caused an
overall increase in amino acid content and the large part of which is stored in the vacuole
(Losak et al 2010).
2.7.3 Protein Content
Nitrogen is a constituent of amino acids, which are required to synthesize proteins
and other related compounds which plays a role in almost all plant metabolic processes
(Mokhele et al 2012). The protein content depends critically on the availability of all the
amino acids in requiste proportions, which in turn depends on the balance between
assimilation of NO3- into NH4+ and the incorporation of the latter into amino acids (Ali et al
2008). The whole plant processes such as N acquisition, translocation and mobilization of
carbon and N are important in the determination of protein concentration. Decrease in N,
amino acid and protein content with ageing can be attributed to the N dilution process (Diaz et
al 2008). Protein composition of the wheat grain is influenced by genotype as well as by
environmental conditions. It indicates that although increased N supply correlated
significantly to an increase in protein, its effect on grain protein also depends on the cultivar
sown due to difference in use of available N (Abedi et al 2010).
The amount of N significantly affected the quality of grain protein. Protein content
increases significantly with higher N doses in Brassica juncea as reported by Sahoo et al
(2000). In the mustard, highest recovery of applied N in the form of protein was observed
under the influence of 30, 60 and 90 kg/ha N, respectively, compared to control (Meena and
Sumeriya 2003). As discussed earlier, the protein content of wheat increased with high N
rates (Nasseri et al 2009, Rahimizadeh et al 2010). Increased N dose also increased sugar,
14
chlorophyll and protein content in rape leaves (Saha et al 2003).
Stitt (1999) indicated that NO3- induces genes involved in different aspects of carbon
metabolism, including the synthesis of organic acids used for amino acid synthesis. These
results suggest that the highest N rate increases the amino acid synthesis in the leaves and this
stimulate the accumulation of protein. The coordination and optimal functioning of the
metabolic pathways for N and carbon assimilation in plants are critical in determining plant
growth and ultimately, biomass accumulation (Krapp et al 2005). Hussain et al (2006)
reported that genotypic variability in protein content may be affected not only by
physiological traits but also by N supply in the soil.
2.7.4 Chlorophyll Content
There was increase in chlorophyll content with increase in N application over the
control in Brassica (Sharma and Chandra 2004) and lettuce (Konstantopoulou et al 2012).
Chlorophyll content also increased with the growth of the plant upto flowering (Singh et al
2003). Genotypes having the higher chlorophyll content had higher grain yield which
indicated a positive association between chlorophyll content and grain yield (Bilagi et al
2008). According to Tranaviciene et al (2007), contents of photosynthetic pigments were
higher at higher fertilization rates in wheat. Variation in grain yield at different N levels was
related to the differences in size of photosynthetic surface and to the efficiency of sink
activity (Hokmalipour and Darbandi 2011).
It has been suggested that leaf N assimilation is energetically more efficient than root
N assimilation when photosynthesis is light-saturated (Marquez et al 2005). In wheat, the
increased dry matter is due to the increased light intercepting area, resulting in more
assimilation of photosynthates and increase in filling rate (Warriach et al 2002). Boussadia et
al (2010) in olive reported that low levels of leaf N reduced chloropyhll concentration.
The increase in chlorophyll concentration with increasing N is important for the plant
to enhance CO2 assimilation and thereby achieving a greater leaf area and yield (Leon et al
2007, Konstantopoulou et al 2012). In contrast, N deficiency results in lower chlorophyll
levels and limited leaf area (Broadley et al 2001). Decreased plant biomass production due to
N shortage was associated with reductions in both leaf area and leaf photosynthetic capacity
(Zhao et al 2005). As reported in maize, remobilized N comes from the proteolytic
degradation of leaf and stalk proteins and mainly from the photosynthetic enzyme, Ribulose1,5-bisphosphate carboxylase oxygenase (Rubisco). Therefore, when remobilization is active,
during the ageing process, photosynthesis decreases and thus also N-uptake (Triboi and
Triboi-Blondel 2002).
2.7.5 Sugar Content
Nitrogen assimilation needs carbon which is provided by carbohydrates (Taiz and
Zeiger 2002). N fertilizer increases sugar content in sweet sorghum (Almodares et al 2010).
15
The percent increase in invert sugars under high N supply was greatest at the earlier growth
stages (Robertson et al 1996). Thus, as the amount of N absorption increased, more
carbohydrates are needed. There are many studies showing that the plant can sense its reduced
N status and regulate the metabolism of inorganic N (Miflin and Habash 2002).
In wheat, it has been reported that the increase in dry matter is due to the increased
light intercepting area, resulting in more assimilation of photosynthates (Warraich et al 2002).
Almodares et al (2009) reported that N fertilizer increased soluble carbohydrates content
(sucrose) in sweet sorghum. This relationship between N fertilizer and consumption of
soluble carbohydrates in plants may be due to N assimilation (Almodares et al 2009). Few
reports indicate correlation between sugar and photosynthetic pigment. High amount of
hexoses preceded the loss of photosynthetic activity thereby leading to sucrose and starch
accumulation. In maize, the use of labeled N fertilizer shows that the greater part absorbed N
is allocated to the kernels (Ta and Weiland 1992).
2.8 EFFECT OF NITROGEN ON YIELD ATTRIBUTES AND NITROGEN USE
EFFICIENCY (NUE)
2.8.1 Yield attributes
Grain yield is the main target of crop production. It is the final result of different steps
that can be studied through the yield components. N fertilization significantly increased the
grain yield. Number of spikes per m2, grains per spike and thousand grain weight were
considered as the most important yield components of wheat, which responded differently to
N levels. Rahman et al (2011) reported that N application has tremendous effect on tiller
formation and survival of tillers. Usually the number of grains per spike is determined at
panicle primordial formation stage which strongly dependent on genetic factors rather than
management factors (Schwarte et al 2006).
The enhancement of grain yield per plant could be because under optimal N nutrition
CO2 assimilation is favourably upregulated resulting in an adequate supply of
photoassimilates to the developing meristems which maintains their growth (Shah 2008).
Thus, the capacity of grains to grow substantially increased probably because more cells with
greater enzyme capacity are produced (Lawlor 2002). Under such an enhanced sink potential,
the availability of ample nutrients can be expected to cause more grain filling and thereby
increase overall yield at harvest (Shah 2008). The N status of the plant around two weeks
before anthesis determines the number of kernels per cob (Below 1987). An application of N
at later stages of maize delayed phenological development (Amanullah et al 2009), increased
crop growth rate, leaf area per plant, plant height, kernel number and higher biomass at
anthesis as well as at maturity that resulted in high yield (Amanullah et al 2008, Hokmalipour
et al 2010).
16
With the application of high rates of N fertilizer, grain yield increased in B. juncea
(Singh et al 2002, Thanki et al 2004), corn (Hokmalipour et al 2010) and barley (Beatty et al
2010). Application of 120 kg ha−1 N fertilizer increased yield up to 40% for Amaranth and
94% for quinoa as compared to control as reported by Schulte aufʼ m Erly et al (2005). A
similar trend in yield across different N rates has been reported by Zeidan et al (2006).
Increased grain yield due to increased N application could be ascribed to increased biomass
production, improved harvest index and increased seed set (Miri et al 2012). Positive effect of
N on grain yield and yield attributes of sweet sorghum was reported by Hugar et al (2010).
Plant height and number of grains per ear increased with increasing N levels in corn
as reported by Nemati and Sharifi (2012). Increase in grains per ear at higher N levels might
be due to the lower competition for nutrients that allow plants to accumulate more biomass
with higher capacity to convert more photosynthates into sink (Zeidan et al 2006). Number of
grains per ear plays an important role in determining grain yield. Increase in N application
from 60 to 180 kg N/ha significantly increased thousand grain weight due to increased
photosynthetic activity that increases accumulation of metabolites (Hokmalipour et al 2010).
In wheat, the role of N in encouraging metabolic processes consequently their growth,
spike initiation and grain filling is responsible for the increase of spike length, number of
spikelets and grains/spike, thousand grain weight and ultimately grain yield/fed (Sawires
2000, El-Gizawy 2005). As expected, modern cultivars generally outyield older ones across a
range of fertility and N levels (Coque and Gallais 2007) and yield gain is associated with an
increase in the C/N ratio as a result of an improvement of N-uptake during the grain filling
period (Gallais and Coque 2005).
2.8.2 Nitrogen Use Efficiency (NUE)
Nitrogen use efficiency (NUE) is defined as the yield of grain per unit of available N
in the soil (including the residual N present in the soil and the fertilizer) (Moll et al 1982).
Infact, NUE is more complex and the ways of estimating NUE depends on the crop and the
harvestable product (Good et al 2004). NUE can be divided into two processes: uptake
efficiency (N uptake efficiency, NupE); the ability of the plant to remove N from the soil as
NO3- and NH4+ ions and the utilization efficiency (N utilization efficiency, NutE); the ability
to use N to produce grain yield (Moose and Below 2009, Beatty et al 2010) (Fig. 2). Total
NupE can be calculated from the N present in the above-ground parts of the crop at maturity
divided by all the N supplied by fertilizer and soil during the growing season. Total NutE is
the total dry matter i.e. yield (grain plus straw) divided by all N in the above-ground parts of
the crop at maturity.
Efficient N utilization is crucial for economic wheat production and protection of
ground and surface water (Vukovic et al 2008). Better N utilization is an integration of better
N assimilation and remobilization within the genotypes (Good et al 2004). Uptake and
17
partitioning between straw and grain are the two major components of N economy in plants.
The relative contribution of these processes to NUE varies among genetic population and
environment (Alizadeh and Ghaderi 2006). Differences in absorption, assimilation and
utilization of absorbed N have been described (Rizzi et al 1995).
In addition to the improvement of N fertilization, soil management and irrigation
practices, there is need to improve NUE in cereals by selecting new hybrids or cultivars from
the available ancient and modern germplasm collection (Alva et al 2005, Atkinson et al
2005). Consequently, the effective use of plant genetic resources will be required to meet the
challenge posed by the world's expanding demand for food to fight against hunger and the
protection of the environment (Hirel et al 2007). It is therefore imperative to identify the
limiting steps in the control of NUE i.e. N uptake and N assimilation during growth and
development. Less effort has been laid in wheat over other cereals (Hirel et al 2005a) or
model plants such as tobacco (Masclaux et al 2000, Terce–Lafrgue et al 2004a) and
Arabidopsis (Loudet et al 2003, Diaz et al 2005).
Field-based studies have shown differences in the NUE of barley genotypes by
Sinebo et al (2004), Abeledo et al (2008), Anbessa et al (2009). For maize, genotype
comparison under low and high N inputs revealed that the genotypes that were adapted to low
N supply were different from those adapted to high N supply (Gallais and Coque 2005).
Similarly in Indian mustard genotypes, NUE showed that plants with high NupE and high
physiological NutE are not only able to take up N efficiently but also utilize it efficiently.
Genotypes with high NupE accumulated higher N content than those with low NupE under
limited N conditions. High physiological NutE is essential for optimum grain yield. Some
genotypes either stop accumulating or show a net loss of shoot N between anthesis and
maturity which could be associated with superior pre-anthesis N accumulation capacity.
Therefore, genetic recombination of genotypes possessing superior activities for individual
steps in the N assimilation process can be used for obtaining genotypes having enhanced
NUE.
Paponov et al (2005) observed that at low N supply differences among maize hybrids
for NUE were largely due to variation in utilization of accumulated N but with high N they
were largely due to variation in uptake efficiency. They concluded that variation of NUE
results from differences among genotypes and levels of N supplied i.e. depends on their
interaction. However, absence of significant interaction has been reported by Spanakakis
(2000). In general, presence or absence of interactions strongly depends upon the behavior of
the genotypes (Alizadeh and Ghaderi 2006).
Enhancing NUE is especially relevant to cereal crops for which large amount of N
fertilizers are required to attain maximum yield and NUE is estimated to be far less than 50%
18
Fig. 2 Diagramatic representation of the key terms used to describe wheat nitrogen use
efficiency (NUE), NUpE – nitrogen uptake efficiency, NUtE – nitrogen utilization
efficiency
(Murphy et al 2007). Despite its importance, a clear understanding of the major mechanisms
and inheritance of NUE is lacking as NUE in plants is a complex trait and in addition to soil
N availability, it can also depend on a number of internal and external factors such as
photosynthetic carbon fixation to provide precursors required for amino acid biosynthesis or
respiration to provide energy (Lewis et al 2000). However, for most plant species, NUE
mainly depends on how plant extracts inorganic N from the soil, assimilate NO3- and NH4+
and recycle organic N (Masclaux-Daubresse et al 2010).
NUE decreased with increasing dose of N fertilizer (Khalque et al 2008, Vukovic et
al 2008, Nasseri et al 2009) which may be due to excessive N losses that decreased N
utilization efficiency. Jamaati-e-Somarin et al (2008) and Kanampiu et al (1997) reported
similar results in wheat and potato tuber. Lopez-Bellido and Lopez-Bellido (2001) indicated
that in wheat a decrease in NUE with increasing fertilizer rates is because grain yield rises
less compared to N supply in soil and fertilizer. Higher NUE leads to maximum tillers, high
number of grains per panicle and high thousand grain weight in wheat (Khalque et al 2008)
and maize (Hanan et al 2008, Nasser and El-Gizay 2009).
2.9 USE OF TILLER AND HYDROPONIC CULTURE TECHNIQUES IN STUDY OF
NITROGEN METABOLISM
Alternative way of studying the effect of different N concentrations is through the use
of liquid culture technique. In this technique, detached ears are cultured in liquid medium for
19
studying the response of genotypes to different N concentrations. It follows standard in vivo
procedure for manipulating nutrient entering the peduncle and finally to the grain.
Due to the difficulty in maintaining specific N ratios in soils, hydroponic culture
technique helps in mantaining seedlings for experimentation. Hydroponic culture is suitable
in order to investigate plant metabolism. In higher plants, NO3- assimilation can take place in
leaves as well as in roots, the contribution of each of these organs being dependent on the
species and on the external NO3- concentration (Orea et al 2005).
Amino acid concentration measured in the roots and shoots of barley grown in
hydroponic growth chamber conditions at 0.5, 4 and 8 mM NO3- showed significant
differences (Beatty et al 2010). This is evident from the results of Konstantopoulou et al
(2012) that the NO3- concentration in both the inner and outer leaves increased with
increasing N application in lettuce. Uptake rates of all nutrients increased with increasing
nutrient solution concentration in lettuce (Samarakoon et al 2006). Total N uptake and grain
N content in wheat (Bellido et al 2007) and sugarcane (Weigel et al 2010) increased with
increase in N fertilizer.
20
CHAPTER-III
MATERIALS AND METHODS
In order to fulfill the objectives, the present study was conducted on 18 wheat
(Triticum aestivum L.) genotypes viz: PBW 621, PBW 636, PBW 590, DBW 17, HD 2967,
PBW 509, BW 9178, BW 9183, BW 8989, BW 9022, PBW 343, PBW 550, GLU 1101, GLU
1356 GLU 2001, GLU 700, PH132-4836 and PH132-4840 for identifying and characterizing
nitrogen (N) efficient genotype during two successive years (2009-10 and 2010-2011). The
seeds were procured from the Department of Plant Breeding and Genetics, Punjab
Agricultural University, Ludhiana and experiments were carried out under following
headings:
3.1 Biochemical studies on field raised crop under different nitrogen levels
3.2 Laboratory studies (Liquid and hydroponic culture techniques) at different
nitrogen doses
3.1 BIOCHEMICAL STUDIES ON FIELD RAISED CROP UNDER DIFFERENT
NITROGEN LEVELS
3.1.1 Experimental Set Up in the Field
During first year (2009-10), twelve genotypes of wheat (Triticum aestivum L.) viz.
PBW 621, PBW 636, PBW 590, DBW 17, HD 2967, PBW 509, BW 9178, BW 9183, BW
8989, BW 9022, PBW 343 and PBW 550 were used for characterization of NUE. During
second year (2010-11), alongwith twelve genotypes, six addtional lines (GLU 1101, GLU
1356 GLU 2001, GLU 700, PH132-4836 and PH132-4840) relevant to this study were
included. The crop was raised under normal planting time i.e. 28th October, 2009 and 29th
October, 2010 in the experimental fields of Department of Plant Breeding and Genetics, PAU,
Ludhiana under 4 different N levels including the presently recommended N dose (RDN)
(120 Kg N/ha), suboptimal N doses [RDN-50% (60 Kg N/ha) and RDN-25% (90 Kg N/ha)]
and supra-optimal N dose [RDN+25% (150 Kg N/ha)]. The crop was grown in plots
measuring 1mx50cm in three replications. Each plot consisted of 4 rows with row to row
spacing of 9 inches and between the plots was 40 cm. Basal dose of diammonium phosphate
(2.0 Kg during 2009-10 and 2.5 Kg during 2010-11) and NPK (1.9 Kg during 2009-10 and
2.4 Kg during 2010-11) was added to the soil at the time of sowing. Urea at the rate of 60, 90,
120 and 150 kg/ha was applied, half dose at the time of sowing and half at 1st irrigation.
Parentage and important features of the studied genotypes is given below:
Genotype
Parentage
Important features
PBW 621 - KAUZ//ALTAR84/AOS/3 - Released variety for timely sown irrigated conditions
MILAN/KAUZ/4/HUITES
21
PBW 636 - KSWW1/PBW 552
- Winter wheat × Spring wheat derivative
PBW 590 - WH594/RAJ3812//W485 - Released variety for late sown irrigated conditions
DBW 17 - CMH79A95/3*CNO79//
- Released variety for timely sown irrigated conditions
RAJ3777
HD 2967 - ALD/COC//URES/3/
- Released variety for timely sown irrigated conditions
HD2160M/HD2278
PBW 509 - W1634/PBW 381
- Released variety for late sown irrigated conditions
BW 9178 - INQ 91*3/TUKURU//
DBW18
Durable rust resistant stocks
BW 9183 - INQ 91*3/TUKURU//
DBW18
BW 8989 - C 273/2*PBW534
Introgression lines with genetic input from tall
BW 9022 - C 591/3*PBW343
traditional cultivars
PBW 343 - ND/VG 9144//KAL/
BB/3/YACO’S’/4/Vec#5
- Widely sown released variety for timely sown
irrigated conditions
PBW 550 - WH594/RAJ3856//W485 - Good grain released variety for timely sown irrigated
conditions
GLU 1101 - GLUPRO/3*C518
(BWL 0992)
GLU 1356 - GLUPRO/3*PBW554
(BWL 0975)
GLU 2001- GLUPRO/3*PBW568
Lines with high grain protein content conferred
by Gpc-B1 gene originally introgressed from
Triticum dicoccoides (Tetraploid wild wheat)
(BWL 0977)
GLU 700 - GLUPRO/3*PBW568
(BWL 0985)
PH132-4836 - PH132/WL711//PBW343 Lines with high grain protein content (Mechanism
PH132-4840 - PH132/WL711//PBW343 not known)
Collection of samples: Flag leaf was excised and used for biochemical analysis. Samples
were collected at 9 am daily so as to minimize the effect of diurnal variation. The tissue was
dried in hot air oven at 110°C for 10 min followed by drying at 60°C till constant
weight. All parameters were studied at three stages of plant growth and development i.e.
tillering (30 days after sowing; DAS), anthesis (about 90-100 DAS depending upon genotype)
and post-anthesis (15 days post anthesis). Fresh tissue was used for enzyme assays and
cholorophyll content whereas stored (dried) tissue was used for N content, amino acids,
soluble proteins, sugars and starch analysis.
22
3.1.2 Extraction and Assay of Nitrate Reductase and Nitrite Reductase
3.1.2a Nitrate reductase (NR, EC 1.6.6.1) (Jaworski 1971)
Reagents:
 0.1 M Sodium Phosphate Buffer (pH 7.5): 72 ml of sodium dihydrogen phosphate
was added to 28 ml of disodium hydrogen orthophosphate.
 1% n-propanol
 0.5 M potassium nitrate
 1% (w/v) Sulfanilamide in 1 N HCL: Dissolved 1g of sulphanilamide in 100 ml of 1
N HCL.
 0.02% N-(1-napthyl) ethylene diamine dihydrochloride (NEDD) – Dissolved 20 mg
of NEDD in 100 ml of distilled water.
Extraction: 200 mg of tissue was suspended in 5 ml of 0.1 M phosphate buffer (pH 7.5), 0.05
ml of n-propanol and 0.25 g KNO3. Nitrogen gas (N2) was passed through the incubation
media to make the atmosphere free from O2. The vials were sealed and incubated in a
metabolic shaker at 30oC in dark for 90 min.
Assay: The release of nitrite (NO2-) into the medium was determined by treating 1 ml of
aliquot with 1 ml of 1% sulphanilamide in 1M HCl and 0.02% NEDD. The total volume was
made to 10 ml with distilled water. After 20 min, the absorbance was recorded at 540 nm
using Spectronic-20. The standard curve was prepared using 0-10 µg of KNO2. NR activity
was expressed as µmol NO2- formed h-1 g-1 FW.
3.1.2b Nitrite reductase (NIR, EC 1.7.7.1) (Verner and Ferari 1971)
Reagents:
 0.1 M Sodium Phosphate Buffer (pH 7.5): 72 ml of sodium dihydrogen phosphate
was added to 28 ml of disodium hydrogen orthophosphate.
 20 µg Chloramphenicol

Sodium nitrite (NaNO2)
 Dimethylsulfoxide (DMSO)
 1% (w/v) Sulfanilamide in 3 N HCL: Dissolved 1g of sulphanilamide in 100 ml of 3
N HCL.
 0.02% N-(1-napthyl) ethylene diamine dihydrochloride (NEDD) – Dissolved 20 mg
of NEDD in 100 ml of distilled water.
Extraction: 200 mg tissue was suspended in 2 ml of 0.1 M sodium phosphate buffer (pH
7.5), chloramphenicol and NaNO2. 0.1 ml aliquot was immediately removed to calculate an
accurate measurement of the initial NO2- concentration in the medium. After 40 min, again
0.1 ml aliquot of the medium was removed for NO2- determination. The amount of NO2- taken
up by the tissue was determined from the difference between the final and the initial NO 223
concentration of the medium. Dimethylsulfoxide (50% v/v) was then added to the medium
and the vials were placed on a hot plate until the medium started boiling. Under these
conditions, the NO2- of tissue rapidly leaked back into the medium. After cooling, 0.2 ml
aliquot was removed for NO2- determination. The difference between this NO2- concentration
and the initial concentration measures the amount of NO2- reduced.
Assay: Nitrite was determined colorimetrically by adding 1 ml each of 1% sulphanilamide in
3N HCl and 0.02% NEDD to 0.1 ml of aliquot. Absorbance at 540 nm was determined after
centrifugation at 2000 g for 10 min. The standard curve was prepared using 0-10 µg of
NaNO2. NIR activity was expressed as µmol NO2- released h-1 g-1 FW.
3.1.3 Extraction of Glutamine Synthetase, Glutamate Synthase and Glutamate
Dehydrogenase
Reagents:
 0.1 M Sodium Phosphate Buffer (pH 7.5): 72 ml of sodium dihydrogen phosphate
was added to 28 ml of disodium hydrogen orthophosphate.
 Cysteine (5 mM)
 Polyvinypyrollidone (300 mg)
 Ammonium sulphate (70%)
Extraction: 1g of fresh leaves were macerated in 10 ml of 0.1M sodium phosphate buffer
(pH 7.5) containing 5 mM cysteine and 300 mg polyvinypyrollidone (PVP). After
centrifugation at 10,000 g for 10 min, the proteins were precipitated using 70% ammonium
sulphate and kept overnight. After centrifugation at 15,000 g for 20 min, the pellet so obtained
was dissolved in 0.1M phosphate buffer (pH 7.5) and dialysed for 36 h. This extract was then
used for enzyme assays.
3.1.3a Assay of glutamine synthetase (GS, EC 6.3.1.2) (Kanamori and Matsumoto 1974)
Reagents:
 0.2 M Tris HCl Buffer (pH 7.5)
 0.05 M ATP (pH 7.0)
 0.5 M Sodium glutamate
 1 M MgSO4
 0.1 M NH2OH (pH 7.0)
 0.1 M Cysteine
 0.37 M FeCl3.6H2O
 0.67 M HCl
 0.2 M TCA
 γ-glutamyl hydroxamate
Assay: Reaction mixture consisted of 0.5 ml of 0.2 M Tris-HCl buffer (pH 7.5), 0.2 ml of
0.05 M ATP (pH 7.0), 0.5 ml of 0.5 M sodium glutamate, 0.1 ml of 1 M NH2OH (pH 7.0)
24
(freshly prepared), 0.1 ml of 0.1 M cysteine and appropriate enzyme solution. The final
volume was made to 3 ml with distilled water. The reaction was started by the addition of
sodium glutamate which was omitted in the blank. After incubation at 30 oC for 15 min, γglutamyl hydroxamate formed was determined by adding 1ml of ferric chloride reagent (0.37
M FeCl3:6H2O, 0.67 N HCl and 0.2 M TCA). The reaction mixture was clarified by
centrifugation and absorbance was read at 540 nm, using γ-glutamyl hydroxamate (0.4-2.0
µM) as the standard. The GS activity was expressed as µmol γ-glutamyl hydroxamate formed
min-1 g-1 FW.
3.1.3b Assay of glutamate synthase (GOGAT, EC 1.4.1.14) (Bulen 1956)
Reagents:
 0.2 M Tris HCl Buffer (pH 8.3)
 0.2 M α-Ketoglutaric acid
 1.5 M Glutamine
 4.0 mM NADH
Assay: Reaction mixture consisted of 1ml of 0.2 M Tris HCl buffer (pH 8.3), 0.2 ml of 0.2M
α-ketoglutaric acid, 0.2 ml of 1.5 M ammonium sulphate, 0.2 ml of 4.0 mM NADH and
appropriate enzyme solution. The decrease in absorbance at 340 nm was recorded after every
15 sec for 3-4 min at 30oC. The enzyme activity was expressed as µmol NADH oxidized min1
g-1 FW.
3.1.3c Assay of glutamate dehydrogenase (GDH, EC-1.4.1.2) (Bulen 1956)
Reagents:
 0.2 M Tris HCl Buffer (pH 8.3)
 0.2 M α-Ketoglutaric acid
 1.5 M Ammonium sulphate
 4.0 mM NADH
Assay: Assay of GDH activity was similar to GOGAT except ammonium sulphate was used
in place of glutamine. The enzyme activity was expressed as µmol NADH oxidized min-1 g-1
FW.
3.1.4 Extraction and Estimation of Metabolites
3.1.4a Nitrogen content (McKenzie and Wallace 1954)
Reagents:
 Conc. H2SO4
 Mixture of CuSO4.5H2O and K2SO4 in the ratio of 0.7 g:8.0 g (Catalyst mixture).
 40% NaOH
 Boric acid: Dissolved 20 g of reagent grade boric acid in about 1 L of hot distilled
water, cooled and added 50 ml of 0.1 % alcoholic solution of bromocresol green and
25
35 ml of 0.1% methyl red solution and final volume was made to 5 L.
 0.1 N HCl
Extraction and estimation: Took 1 g finely powdered sample in digestion tubes. Added
10ml of conc. H2SO4 and catalyst mixture (8g K2SO4 and 0.7g CuSO4.5H2O) and digested the
sample at 418oC for 20 min at the Kjeltec digestor until the solution becomes clear. After this,
tubes were transferred to the Kjeltec distillation unit and in the steam chamber of distillation
unit, 40 % NaOH was added to the digested sample in the tube which resulted in the
production of NH3 which then reacted with boric acid (present in the titration flask) to form
ammonium borate. This ammonium borate was titrated with 0.1 N HCl till bluish green color
changes to pink. A blank was prepared identically but without sample.
3.1.4b Amino acids and soluble proteins
Extraction (Singh et al 1978): Dried leaves and grains (0.1- 0.2 g) were extracted twice with
continuous stirring with 5 ml of 0.1 N NaOH for 30 min followed by centrifugation at 14,000
x g for 15 min. The supernatant so obtained were pooled. To 2 ml of supernatant, 2 ml chilled
20 % TCA was added and mixed thoroughly. After aging for 1 h at 4oC, the contents were
centrifuged at 14,000 x g for 15 min and the precipitates were dissolved in 0.5 N NaOH for
protein estimation. Supernatant was used for amino acid while precipitates for soluble protein
estimation.
Estimation of amino acids (Lee and Takahashi 1966)
Reagents: Prepared ninhydrin reagent by reagents A:B:C in the ratio of 5:12:2.
 Reagent A: 1 % ninhydrin in 0. 5 M citrate buffer (pH 5.5)
 Reagent B: Pure glycerol
 Reagent C: 0.5 M citrate buffer
Procedure: To 0.2 ml of the extract, added 5 ml of ninhydrin reagent. The reaction mixture
was heated in water bath for 12 min and after cooling the test tubes under running tap water
the absorbance was read at 570 nm against reagent blank. Using the standard curves of Lglycine (0.03 – 0.24 µM) run simultaneously, the conc. of amino acids were determined.
Estimation of soluble proteins (Lowry et al 1951)
Reagents:
 Reagent A: 2 % sodium carbonate in 0.1 N sodium hydroxide.
 Reagent B: 0.5 % copper sulphate in 1 % solution of sodium potassium tartarate.
 Reagent C: Reagent mixture was prepared immediately before use by mixing reagent
(i) and reagent (ii) in 50:1 ratio.
 Reagent D: Folin-Ciocalteau phenol reagent (1N).
Procedure: To 1 ml of appropriately diluted test solution, 5 ml of reagent C was added and
the contents were mixed well. The mixture was allowed to stand for 10 min at room
26
temperature. Then 0.5 ml of reagent D was added and shaken rapidly. The mixture was kept
for 30 min and intensity of blue color so developed was read at 520 nm. The concentration of
soluble proteins was calculated from the BSA standard (20-100 g) run simultaneously.
3.1.3c Chlorophyll Content (Arnon 1949)
Reagents: Ammonical acetone: Prepared by mixing 81.8 ml acetone and 0.2 ml
ammonium hydroxide and making final volume 100 ml with distilled water.
Extraction and estimation: 0.5 g fresh tissue was homogenized in 5 ml of ammonical
acetone. The sample was then centrifuged at 3000 g for 3 min. The absorbance of the
supernatant was determined at 645, 663 and 710 nm wavelength. 710 nm wavelength was
used as an isoelectric point which was deducted from other absorbance readings.
Total chlorophyll (mg/g) = (20.2 x A 645 ) – (8.02 x A 663 )
V
1000 x W
3.1.4d Sugars and Starch
Extraction: 0.5 g dried leaves and grains were homogenized with deionised water and the
extract was filtered through three layers of muslin cloth and supernatant obtained was used for
estimation of total sugars. The sugar free residue from filtration was used for starch
estimation. Sediment of the extract that filtered in sugar content dried, weighed and boiled
with deionised water. Content of soluble sugars were expressed as mg g-1 DW.
Estimation of sugars (Dubois et al 1956)
Reagents:
 Conc. sulphuric acid
 Phenol 5% (w/v redistilled)
Procedure: To 1 ml of appropriately diluted sugar extracts (within the range of
authentic sugar standards), 1 ml of 5 % phenol was added followed by addition of 5
ml of conc. sulphuric acid, poured directly in the centre of the test tube to ensure a
proper mixing of the solutions. The tubes were cooled at room temperature for 10
min and for another 20 min under running tap water. Intensity of the orange color
developed was measured at 490 nm against reagent blank. The concentration of tot al
sugars (as glucose) was calculated from the glucose standards (10 -60 g) run
simultaneously.
Estimation of starch: The starch was estimated from the sugar free extracts with phenolsulphuric acid method using standard glucose as already described for estimation of total free
sugars in this section. The concentration of starch was calculated by multiplying the value of
glucose concentration so obtained in the test extract by a factor of 0.9.
3.1.4 Yield and its Attributes
3.1.4a Grain yield
The grain yield of each plot was recorded and expressed as kg/sqm.
27
3.1.4b Biomass
It was determined by taking the weight of above ground dried plants along with the
ears. The biomass was expressed as kg/plant.
3.1.4c 1000-grain weight (g)
A random sample of 1000 grains were taken from the bulk of each plot and weighed.
3.1.4d Plant Height (cm)
Length of three randomly selected plants from each plot was measured from the base
to the tip and mean values computed.
3.1.4e Nitrogen Use Efficiency (NUE)
It was calculated by the following formula:
NUE =
3.2
LARORATORY
Grain yield
Nitrogen fertilizer application
STUDIES
(LIQUID
AND
HYDROPONIC
CULTURE
TECHNIQUES) AT DIFFERENT NITROGEN DOSES
3.2.1 Liquid Culture Technique
Two wheat (Triticum aestivum L.) cultivars namely, PBW 621 and PBW 343 were
selected for tiller culturing experiment. Ears at mid-milky stage, i.e. 12–15 days post anthesis
(DPA) were cut under water below penultimate node and cultured according to the method of
Asthir and Bhatia (2011) keeping twelve replications for each treatment. To study the
response of these cultivars to different N doses, culture media having three concentrations of
L-glutamine viz. 5 mM, 17 mM and 25 mM were used. After adjusting the pH of the culture
solution to 5.5, the medium was ultra filtered through 0.22 μM millipore membrane. Before
culturing, the flag leaf and its sheath were removed and stems were surface sterilized with 40
% ethanol followed by quick washing with distilled water. Ear-heads carrying 20 cm peduncle
length from the cut end were placed (one ear-head per tube) in culture tube containing 35 ml
cold-sterilized liquid medium. These cultured ear-heads were then transferred to water bath
maintained at 2–4 °C in the natural day light conditions. After required culturing for 5 days,
the grains were separated and used for analysis of activities of NR, NIR, GS, GOGAT, GDH
and amino acids and proteins by methods as discussed in earlier sections.
Composition of culture medium: The basic culture medium used for culturing detached ears
was of the following composition.

Major elements solution
Compunds
Concentration (g/L)
Solution
CaCl2
8.80
A
KH2PO4
24.00
B
+MgSO4.7H2O
7.57
28

Minor elements solution
H3BO3


6.20
MnSO4.4H2O
22.30
ZnSO4.4H2O
8.60
KI
0.83
Na2MoO4.2H2O
0.25
CuSO4.5H2O
0.026
CoCl2.6H2O
0.026
Iron solution
FeSO4.7H2O
5.37
Na2EDTA
7.45
Vitamin solution
Thiamine-HCl
Myo inositol
0.04
10.00
Before using, the above solutions were mixed in the following proportion
Major element solution A
50 ml
Major element solution B
5 ml
Minor element solution
Iron solution
Vitamin solution
10 ml
5 ml
10 ml
In addition, the above basic culture solution contained 117 mM sucrose as source of
N. After adjusting the pH of the culture solution to 5.0 with NaOH solution, the total
volume of the mixture was raised to one litre.
3.2.2
Hydroponic Culture Technique
Six wheat genotypes (PBW 621, PBW 636, GLU 1356, BW 8989, GLU 700 and
PBW 343) were grown hydroponically in a greenhouse in plastic trays containing 8 L
Hoagland solution. Seed from each genotype were sprouted in a controlled environment in
paper towels. After the seeds germinated, the seedlings were transferred to trays in the
greenhouse. Each tray contained one genotype at one N rate. The trays contained Hoagland
solution (Hoagland and Arnon 1950) that supplied all essential nutrients with N which was
added at the rate of 0, 2 and 6 mM in the form of potassium nitrate. The solutions were
aerated continuously through the use of an air compressor. pH of the solution was monitored
daily and maintained at 6.0-6.5 with either 1N HCl or 1N NaOH to prevent any iron
deficiency symptoms. Solution was replaced after every 3 days. Plants were harvested at 12
days after transfer to the greenhouse. The plants were separated into shoots and roots and
analyzed for various parameters (activities of NR, GS and amino acids, soluble proteins, N
29
content and N uptake) by methods discussed in earlier sections.
Table 1: Composition of Hoagland solution is as follows:
Compound
Amount (g/L)
 Ca(NO3)2.4H2O
236.1
 KNO3
101.1
 KH2PO4
136.1
 MgSO4.7H2O
246.5
 Trace elements (Prepare 1L)
H3BO3
2.8
MnCl2.4H2O
1.8
ZnSO4.7H2O
0.2
CuSO4.5H2O
0.1
NaMoO4
0.025
 Fe-EDTA (pH of KOH solution was adjusted to 5.5 using H2SO4. To this EDTA.2Na
and FeSO4.7H2O was added to make final volume of IL)
EDTA.2Na
10.4
FeSO4.7H2O
7.8
KOH
56.1
7 ml Ca(NO3)2, 5 ml KNO3, 2 ml KH2PO4, 2 ml MgSO4, 1 ml trace elements and 1 ml FeEDTA stock solutions were mixed and the final volume was made to 1 L with distilled
water.
Statistical analysis
All the values were mean of three replicates. Data obtained was subjected to split plot
design at 5% level of CD using CPCS1 software developed by department of Statistics,
Punjab Agricutural University, Ludhiana.
30
CHAPTER-IV
RESULTS AND DISCUSSION
Nitrogen (N) is one of the major essential elements for crop growth and development
and is extensively used in modern agriculture to maximize crop yields. However, its excessive
use leads to environmental problems such as acidification of soil and water resources,
eutrophication of coastal marine ecosystems and increased greenhouse gas emission (Lawlor
et al 2001). To optimize its uptake and use, genotypes with high nitrogen use efficiency
(NUE) need to be identified and characterized. Although the genotype takes up N efficiently
from the soil, still some of the unutilized N remains in form of non-protein-N. Thus,
development of such N-efficient genotypes that will not only grow well at low N levels but
will also give high yield which will reduce production cost of N and will limit N pollution
risks. Regulatory aspects of N uptake and assimilation will enable to identify marker traits
optimizing NUE in wheat. An attempt has been made in the present study to understand
various traits related to NUE viz. nitrate reductase (NR), nitrite reductase (NIR), glutamine
synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH) and
metabolites (contents of N, amino acid, protein, chlorophyll and sugar). Yield attributes
associated with NUE were also measured in the studied genotypes.
During first year (2009-10), twelve genotypes of wheat (Triticum aestivum L.)
namely PBW 621, PBW 636, PBW 590, DBW 17, HD 2967, PBW 509, BW 9178, BW 9183,
BW 8989, BW 9022, PBW 343 and PBW 550 were selected for characterization of NUE.
PBW numbers, DBW 17 and HD 2967 represent released cultivars for different sowing
conditions under north western plain zone of India while BW numbers represent advanced
breeding lines. These genotypes were tested for various physiological and biochemical
response at four different N levels including the presently recommended N dose (RDN) (120
Kg N/ha), suboptimal N doses [RDN-50% (60 Kg N/ha) and RDN-25% (90 Kg N/ha)] and
supra-optimal N dose [RDN+25% (150 Kg N/ha)] at three stages of plant growth and
development i.e. tillering (30 days after sowing; DAS), anthesis (about 90-100 DAS
depending upon genotype) and post-anthesis (15 days post anthesis). During second year of
the study, six new lines (GLU 1101, GLU 1356 GLU 2001, GLU 700, PH132-4836 and
PH132-4840) were included to the already existing set of 12 genotypes. These lines possessed
very high grain protein content. Four of these lines i.e. GLU 1101, GLU 1356 GLU 2001,
GLU 700 have Gpc-B1 gene which possess a known mechanism for accelerated translocation
of nutrients from leaves to grains resulting in high content of protein alongwith Fe and Zn.
Remaining two genotypes PH132-4836 and PH132-4840 also possessed high grain protein
content but their mechanism is unknown. These genotypes infact, served as benchmark for
comparison with other genotypes under the influence of varied N levels.
31
The results of experiments conducted during this study have been presented and
discussed under the following headings:
4.1 Biochemical studies on field raised crop under different nitrogen levels
4.2 Physiological studies on field raised crop under different nitrogen levels
4.3 Laboratory studies (liquid and hydroponic culture techniques) at different
nitrogen doses
4.1 BIOCHEMICAL STUDIES ON FIELD RAISED CROP UNDER DIFFERENT
NITROGEN LEVELS
4.1.1 Genotypic Variation With Respect to Nitrate and Ammonium Assimilating
Enzymes in Leaves
4.1.1a Nitrate reductase
Nitrate reductase is the first enzyme of N assimilatory pathway catalyzing the
conversion of nitrate (NO3-) to nitrite (NO2-) (Rosales et al 2011). It is the rate-limiting step in
N assimilation as it is a substrate inducible enzyme and operates in cytoplasm. It represents
the capacity of the plant to reduce inorganic N (Masclaux et al 2000, Fan et al 2007). The
relationship of the NR activity with the rest of the enzymes involved in NO 3− assimilation
clearly indicates its importance in determining N status of the plant (Keresi et al 2008). N
applied in the form of urea is converted into NH4+ by urease present in the soil (Witte 2011).
NH4+ is further converted to NO2- and NO3- by the two step process of nitrification involving
nitrosomonas and nitrobacter bacteria. NO3- thus formed is taken up by roots and is
assimilated in the form of amino acids and proteins (Bateman and Baggs 2005).
The activity of NR in leaves increased till anthesis and thereafter decreased with plant
development at all N doses (Fig 1). Higher activity of this enzyme till anthesis was infact
controlled by high substrate level in the form of NO3- that is translocated from roots to leaves.
During both years of the study i.e. 2009-10 and 2010-11, NR activity was significantly
affected by various N levels as well as by different genotypes and was found to be maximum
at RDN+25% as depicted in fig 1. The interaction of N levels and various genotypes showed
that some genotypes were significantly better at low N doses. Comparing N doses during year
2009-10, NR activity decreased by 40% and 14% with the application of two lower doses of
N i.e. RDN-50% and RDN-25%, respectively as compared to optimum dose (RDN). More or
less similar decrease was observed during the second year of study also.
The range of NR activity at tillering (0.97 to 2.02 μmol NO2- formed/h/g FW),
anthesis (2.05 to 4.11 μmol NO2- formed/h/g FW) and post-anthesis (1.49 to 3.40 μmol NO2formed/h/g FW) stages indicated maximum activity at anthesis stage (Table 1). Substantial
activity of NR was observed in PBW 621 at RDN-25% during tillering (2.18 μmol NO2formed/h/g FW), anthesis (4.38 μmol NO2- formed/h/g FW) and post-anthesis (3.57 μmol
32
NO2- formed/h/g FW) stages while PBW 590 revealed higher activity at RDN+25%. During
tillering stage, PBW 636 (1.85 μmol NO2- formed/h/g FW) showed higher activity of NR at
RDN-50% compared to other genotypes. Higher activity of NR in PBW 621 and PBW 636 at
low N dose probably indicates their higher uptake as well as utilization efficiency over other
genotypes. Reports in the literature indicate that translocation in the roots become efficient at
lower doses of N due to presence of various carriers or transport systems (Balotf et al 2012).
From our results it appeared that genotypic variation exists for NR activity (Tables 1 and 2)
and is possibly connected to differences in the rate of uptake and accumulation of NO3- ions in
the tissues (Abrol 1990).
Fig. 1 Effect of different doses of nitrogen (RDN-25%, RDN-50%, RDN and
RDN+25%) on nitrate reductase activity at tillering, anthesis and post anthesis
developmental stages of wheat genotypes during years 2009-10 and 2010-11
During second year (2010-11), the general trend of NR activity was more or less
similar to the first year. The range of NR was from 0.70 to 2.30 (tillering), 1.61 to 4.45
(anthesis) and 0.94 to 3.67 μmol NO2- formed/h/g FW (post-anthesis) as shown in table 2. The
superiority of PBW 621 with regard to NR activity was confirmed at RDN-25% during
tillering (2.03 μmol NO2- formed/h/g FW), anthesis (4.52 μmol NO2- formed/h/g FW) and
post-anthesis (4.19 μmol NO2- formed/h/g FW) stages while PBW 590 was efficient at higher
dose of N. There was variation for NR activity with respect to genotypes at lowermost dose of
N (RDN-50%). Among the lines with Gpc-B1 gene, GLU 1356 showed highest NR activity at
RDN-50% (3.38 μmol NO2- formed/h/g FW) during post-anthesis stage only.
During both years, low NR efficiency of widely grown cultivar PBW 343 was
observed. The reduction in this activity might be either due to reduction in enzyme level or
due to inactivation of the enzyme. Overall PBW 621 revealed higher trend in this activity at
lower dose of N. Genotypes with high NR activity might have an efficient NO3- induction
system which includes active enzyme protein, higher rates of enzyme synthesis and steady
state of NR-mRNA levels with respect to genotypes having low NR activity. High NR
33
Table 1:
Effect of different doses of nitrogen on leaf nitrate reductase activity (μmol NO2- formed/h/g FW) at three developmental stages of
wheat during year 2009-10
Nitrate reductase (μmol NO2- formed/h/g FW)
Tillering stage
N Doses
Genotypes
Anthesis stage
Post-anthesis stage
34
RDN50%
RDN25%
RDN
RDN+
25%
Mean
RDN50%
RDN25%
RDN
RDN+
25%
Mean
RDN50%
RDN25%
RDN
RDN+
25%
Mean
PBW 621
0.70
2.18
2.20
2.42
1.87
2.48
4.38
4.57
4.99
4.11
2.54
3.57
3.64
3.85
3.40
PBW 636
1.85
1.78
1.97
2.47
2.02
2.38
2.67
3.70
3.89
3.16
1.69
2.40
2.88
2.61
2.39
PBW 590
0.95
1.07
2.26
2.91
1.80
2.86
3.65
3.81
4.34
3.67
1.92
2.67
4.00
4.78
3.34
DBW 17
0.86
1.03
1.54
1.94
1.34
2.28
2.59
2.93
3.08
2.72
1.95
2.13
2.34
2.97
2.35
HD 2967
1.01
1.30
1.96
1.92
1.55
2.78
3.00
2.87
4.83
3.37
2.17
3.00
3.51
3.40
3.02
PBW 509
0.23
1.00
1.13
1.61
0.99
2.59
3.01
3.12
3.89
3.15
1.60
2.15
2.41
2.30
2.11
BW 9178
0.32
0.59
1.77
1.93
1.15
2.57
2.97
4.23
4.96
3.68
1.27
1.78
2.04
3.44
2.13
BW 9183
1.44
1.67
1.78
1.80
1.67
2.30
3.01
3.96
3.99
3.32
1.35
1.08
1.58
1.97
1.49
BW 8989
0.97
1.84
1.92
2.09
1.70
1.34
3.34
3.89
4.00
3.14
1.13
2.00
1.81
1.91
1.71
BW 9022
0.98
1.38
1.66
2.00
1.50
1.29
3.07
4.16
4.58
3.27
1.04
1.90
3.09
4.55
2.64
PBW 343
0.11
0.52
1.00
2.24
0.97
1.34
2.61
1.55
2.70
2.05
1.68
2.05
2.21
3.31
2.31
PBW 550
0.78
1.81
1.33
1.63
1.39
2.48
3.47
3.57
3.81
3.33
1.82
2.73
2.68
3.25
2.62
0.85
1.35
1.71
2.08
Mean
CD (5%)
A- 0.024, B- 0.112, AB- 0.216
A- N doses, B-Genotypes, AB-Interaction
1.50
2.23
3.15
3.53
4.09
3.25
1.68
2.29
2.68
3.19
2.46
A- 0.049, B- 0.182, AB- 0.227
34
A- 0.043, B- 0.196, AB- 0.272
Table 2: Effect of different doses of nitrogen on leaf nitrate reductase activity (μmol NO 2- formed/h/g FW) at three developmental stages of wheat
during year 2010-11
N Doses
Genotypes
PBW 621
PBW 636
PBW 590
DBW 17
HD 2967
PBW 509
35
BW 9178
BW 9183
BW 8989
BW 9022
PBW 343
PBW 550
GLU 1101
GLU 1356
GLU 2001
GLU 700
PH132-4836
PH132-4840
RDN50%
1.95
1.82
1.18
0.96
0.55
0.63
0.66
0.59
0.58
0.43
0.52
1.29
0.63
1.07
1.29
0.33
0.82
0.77
0.89
Tillering stage
RDN- RDN
RDN+
25%
25%
2.03
1.67
1.65
1.40
1.48
1.61
1.52
0.74
0.62
0.95
0.58
1.43
1.07
1.39
1.34
0.96
1.28
1.16
1.27
2.14
1.96
2.23
1.79
1.40
1.62
1.62
1.06
1.14
1.31
0.76
1.52
1.16
1.65
1.57
1.10
1.32
1.19
1.47
3.08
2.31
2.99
2.83
2.43
1.83
1.75
1.35
0.91
1.40
0.94
1.90
1.01
1.74
1.81
1.47
1.66
1.54
1.83
Mean
CD (5%)
A- 0.059, B- 0.105, AB- 0.202
A- N doses, B-Genotypes, AB-Interaction
Nitrate reductase (μmol NO2- formed/h/g FW)
Anthesis stage
RDNRDNRDN
Mean
RDN+
Mean
50%
25%
25%
2.30
1.94
2.01
1.74
1.46
1.42
1.39
0.94
0.81
1.02
0.70
1.53
0.97
1.46
1.50
0.96
1.27
1.17
1.37
3.63
4.13
2.28
2.24
3.31
2.55
2.76
1.40
1.47
1.69
1.32
2.41
2.36
3.34
2.40
1.42
2.24
1.51
2.36
4.52
4.27
3.59
3.6
3.27
2.84
3.39
1.4
2.37
2.08
1.32
2.57
2.39
3.81
3.26
2.34
2.35
2.17
2.86
4.69
4.33
4.28
4.32
2.48
3.47
3.42
2.41
2.42
2.26
1.42
3.24
2.26
4.48
3.60
3.62
2.81
3.26
3.27
A- 0.052, B- 0.103, AB- 0.214
35
4.37
4.49
4.97
4.45
3.41
3.67
3.83
3.49
2.32
3.23
2.38
4.26
4.84
4.42
3.51
3.61
3.28
4.48
3.83
4.45
4.31
3.63
3.65
3.12
3.13
3.35
2.18
2.15
2.32
1.61
3.12
2.96
4.01
3.19
2.75
2.67
2.86
3.08
RDN50%
2.35
2.36
2.39
2.35
1.33
1.39
2.45
1.37
1.38
1.40
0.31
1.44
1.43
3.38
0.56
1.40
2.14
1.33
1.71
Post-anthesis stage
RDNRDN
RDN+
25%
25%
4.19
2.35
3.46
2.5
2.49
1.57
2.46
1.57
1.35
1.37
0.49
1.41
1.49
3.40
1.52
2.47
2.36
1.43
2.10
3.64
3.32
4.31
3.32
2.35
2.58
2.52
2.31
1.54
1.41
1.39
2.32
2.46
3.42
1.55
2.48
2.45
2.48
2.55
4.36
4.41
4.52
3.5
3.33
3.45
3.43
2.34
2.36
2.49
1.58
2.35
3.34
4.42
3.37
2.60
3.42
2.48
3.21
A- 0.052, B- 0.105, AB- 0.193
Mean
3.64
3.11
3.67
2.92
2.38
2.25
2.72
1.90
1.66
1.67
0.94
1.88
2.18
3.66
1.75
2.24
2.59
1.93
2.39
genotypes maintained the smaller pool of tissue NO3- levels whereas the low NR genotypes
maintained larger vacuole pool of NO3-. (Hakeem et al 2011).
Since NO3- induces NR, the rate of NO3- uptake at the site of induction is the main
controlling factor for NR activity as reported by Hakeem et al (2011). Faster rates of NO3remobilization may be an important factor for maintaining NR activity (Fan et al 2007). NR
could play an important role in regulating the levels of NO3- and amino acids in the cells.
Therefore, NR activity can be viewed as a marker enzyme of plant ability to utilize soil N in
form of NO3- (Raimanova and Trckova 2007). Approximately, 35% of the total variation in
NUE is known to be accounted by NR activity. Increase in NR activity improves N utilization
(Pathak et al 2008) which in turn is reflected in terms of plant biomass as discussed in
subsequent section. These findings are further supported from the studies conducted in the
past on wheat wherein the applicability of NR activity could be considered as an index for
determining N availablility in the plant (Souza et al 2002). Furthermore, manipulating NR
activity may be beneficial for both plant quality and productivity and thus may open up new
perspectives toward the improvement of NUE.
4.1.1b Nitrite reductase
Nitrite is highly reactive molecule in plant cells. As soon as it is formed it gets
immediately transported from the cytosol to chloroplast of leaves and plastid of roots. NIR is
a chloroplastic enzyme and is involved in the memebrane stability of chloroplast. Chloroplast
breakdown during senescence involves NIR proteolysis. In chloroplast and plastid, NO2- is
further reduced to NH4+ by NIR (Rosales et al 2011). The response of NIR to N doses was
quite similar to NR. NIR activity also increased from tillering to anthesis and thereafter
decreased during post-anthesis stage. Activity of NIR significantly increased with increasing
dose of N (Fig 2). Our observation on NIR activity was similar to the findings as reported by
Forde (2000) that high levels of N led to an increase in the activity of N assimilating enzymes.
During both years i.e. 2009-10 and 2010-11, NIR activity was significantly affected by
various N levels and genotypes with significant interaction at all stages.
During year 2009-10, as compared to optimum dose NIR activity decreased by 34%
and 17% with the application of RDN-50% and RDN-25%, respectively. However, during
year 2010-11, decrease in NIR activity was less (23% and 13%, respectively) compared to
first year. In the year 2009-10, NIR activity ranged from 0.16 to 0.34 (tillering), 0.36 to 0.84
(anthesis) and 0.32 to 0.70 μmol NO2- released/h/g FW (post-anthesis) (Table 3). In the first
year, genotype PBW 621 was distintly superior over other genotypes at RDN-50% showing
higher activity during anthesis (0.62 μmol NO2- released/h/g FW) and post-anthesis (0.44
μmol NO2- released/h/g FW) stages. Also at RDN-25%, PBW 621 showed highest activity
during tillering (0.35 μmol NO2- released/h/g FW), anthesis (0.93 μmol NO2- released/h/g
FW) and post - anthesis (0.69 μmol NO2- released/h/g FW) stages over other genotypes. HD
36
Fig. 2
Effect of different doses of nitrogen (RDN-25%, RDN-50%, RDN and
RDN+25%) on nitrite reductase activity at tillering, anthesis and post anthesis
developmental stages of wheat genotypes during years 2009-10 and 2010-11
2967 seems to be best adapted to optimum dose (RDN) while DBW 17 revealed highest
activity at RDN+25%.
Similar to first year study, year 2010-11 also depicted similar trend in NIR activity
with respect to studied genotypes. During this year, PBW 621 showed high NIR activity at
RDN-50% (0.44and 0.62 μmol NO2- released/h/g FW) and at RDN-25% (0.49 and 0.85 μmol
NO2- released/h/g FW) during tillering and post-anthesis stages, respectively (Table 4). It was
observed that PBW 636 was efficient for NIR at optimum and higher doses. For this enzyme,
genotype PBW 343 showed least activity during both years. Genotype PBW 621 was found to
give comparable results for NIR also during both years.
Nitrate reductase and NIR activities were significantly inhibited in NH4+ treated
leaves showing a similar pattern of regulation for both (Ali et al 2007). NH4+ represses the
expression of nitrate transporters (NRT) transcripts which results in less NO3- uptake (Balotf
et al 2012). This depicts that NIR enzyme works in association with NR. Levels of NR and
NIR activities in crude extracts from L. japonicus also increased with the NO3- concentration
present in the nutrient solution (Orea et al 2005). Since most of the studies conducted in the
past involved the manipulation of NR and NIR activities, further studies are required to prove
that N uptake efficiency (NupE) and N utilization efficiency (NutE) are controlled by NR or
NIR in roots and shoots of crops grown under various N fertilization regimes.
4.1.1c Glutamine synthetase
Glutamine synthetase is a multi-functional enzyme in N metabolism. In higher plants,
the entire N in plant is channeled through the reaction catalyzed by GS (Hirel and Lea 2001).
Thus, an individual N atom pass through the GS reaction many times (Coque and Gallais
2006) following uptake from the soil during assimilation and remobilization to final storage
protein (Gallais et al 2006). Therefore, GS is one of the checkpoints in N-assimilation as the
product of GS reaction is supplied in constant amount to GOGAT. Determination of total GS
37
Table 3: Effect of different doses of nitrogen on leaf nitrite reductase activity (μmol NO 2- released /h/g FW) at three developmental stages of wheat during year
2009-10
Nitrite reductase (μmol NO2- released/h/g FW)
Tillering stage
Anthesis stage
Post-anthesis stage
N Doses RDNRDNRDN
RDNRDN- RDN RDN+
RDN- RDNRDN
RDN+
Mean
Mean
RDN+
Mean
50%
25%
50%
25%
50%
25%
25%
25%
25%
Genotypes
38
PBW 621
0.24
0.35
0.37
0.41
0.34
0.62
0.93
0.89
0.93
0.84
0.44
0.69
0.80
0.86
0.70
PBW 636
0.11
0.29
0.30
0.32
0.26
0.37
0.70
0.84
0.98
0.72
0.36
0.46
0.51
0.65
0.50
PBW 590
0.22
0.20
0.29
0.38
0.27
0.44
0.49
0.55
0.83
0.58
0.40
0.47
0.57
0.63
0.52
DBW 17
0.12
0.20
0.20
0.22
0.18
0.42
0.63
0.87
1.00
0.73
0.36
0.40
0.39
0.94
0.52
HD 2967
0.26
0.27
0.40
0.40
0.33
0.45
0.50
0.90
0.98
0.70
0.30
0.30
0.35
0.64
0.40
PBW 509
0.29
0.27
0.29
0.30
0.29
0.44
0.58
0.57
0.58
0.54
0.38
0.39
0.44
0.47
0.42
BW 9178
0.16
0.20
0.24
0.29
0.22
0.41
0.47
0.62
0.90
0.60
0.35
0.33
0.47
0.65
0.45
BW 9183
0.15
0.12
0.17
0.20
0.16
0.31
0.40
0.44
0.49
0.41
0.27
0.28
0.49
0.67
0.43
BW 8989
0.14
0.23
0.26
0.52
0.29
0.29
0.44
0.42
0.53
0.42
0.28
0.36
0.40
0.37
0.35
BW 9022
0.27
0.25
0.36
0.35
0.31
0.44
0.46
0.63
0.76
0.57
0.29
0.33
0.41
0.39
0.35
PBW 343
0.07
0.14
0.24
0.25
0.18
0.26
0.33
0.27
0.59
0.36
0.25
0.27
0.37
0.40
0.32
PBW 550
0.18
0.30
0.27
0.34
0.27
0.44
0.66
0.79
0.86
0.69
0.43
0.39
0.39
0.40
0.40
Mean
0.18
0.23
0.28
0.33
0.26
0.41
0.55
0.65
0.79
0.60
0.34
0.39
0.46
0.59
0.45
CD (5%)
A- 0.014, B- 0.023, AB- 0.054
A- N doses, B-Genotypes, AB-Interaction
A- 0.021, B- 0.044, AB- 0.081
38
A- 0.030, B- 0.057, AB- 0.092
Table 4: Effect of different doses of nitrogen on leaf nitrite reductase activity (μmol NO 2- released /h/g FW) at three developmental stages of wheat during year
2010-11
Nitrite reductase (μmol NO2- released/h/g FW)
N Doses
Genotypes
PBW 621
PBW 636
PBW 590
DBW 17
HD 2967
39
PBW 509
BW 9178
BW 9183
BW 8989
BW 9022
PBW 343
PBW 550
GLU 1101
GLU 1356
GLU 2001
GLU 700
PH132-4836
PH132-4840
RDN50%
0.44
0.28
0.23
0.38
0.27
0.24
0.13
0.14
0.14
0.14
0.14
0.35
0.13
0.36
0.23
0.15
0.39
0.25
0.24
Tillering stage
RDN- RDN
RDN+
25%
25%
0.49
0.35
0.33
0.38
0.32
0.25
0.16
0.15
0.21
0.19
0.15
0.34
0.14
0.45
0.35
0.16
0.40
0.38
0.29
0.47
0.48
0.36
0.38
0.36
0.36
0.37
0.29
0.31
0.24
0.18
0.35
0.23
0.45
0.36
0.23
0.42
0.51
0.35
0.52
0.56
0.42
0.49
0.45
0.36
0.36
0.27
0.36
0.23
0.24
0.46
0.34
0.48
0.46
0.25
0.48
0.54
0.40
Mean
CD (5%)
A- 0.012, B- 0.036, AB- 0.051
A- N doses, B-Genotypes, AB-Interaction
Mean
RDN50%
0.48
0.41
0.33
0.41
0.35
0.30
0.26
0.21
0.25
0.20
0.18
0.38
0.21
0.44
0.35
0.20
0.42
0.42
0.32
0.72
0.83
0.62
0.55
0.66
0.56
0.66
0.42
0.45
0.46
0.33
0.62
0.44
0.65
0.54
0.36
0.65
0.52
0.56
Anthesis stage
RDNRDN
RDN+
25%
25%
0.85
0.81
0.66
0.64
0.74
0.57
0.70
0.52
0.55
0.52
0.35
0.75
0.46
0.65
0.57
0.34
0.65
0.57
0.61
0.84
0.85
0.68
0.76
0.73
0.64
0.72
0.58
0.56
0.55
0.48
0.81
0.64
0.84
0.63
0.57
0.65
0.63
0.67
A- 0.012, B- 0.032, AB- 0.058
39
0.92
0.89
0.76
0.75
0.76
0.75
0.74
0.64
0.67
0.66
0.53
0.83
0.64
0.86
0.80
0.58
0.87
0.63
0.74
Mean
RDN50%
0.83
0.84
0.68
0.68
0.72
0.63
0.70
0.54
0.56
0.55
0.42
0.75
0.55
0.75
0.64
0.46
0.70
0.58
0.64
0.62
0.56
0.52
0.39
0.58
0.46
0.55
0.27
0.37
0.24
0.22
0.44
0.37
0.52
0.35
0.26
0.41
0.42
0.42
Post-anthesis stage
RDNRDN
RDN+
25%
25%
0.65
0.54
0.68
0.58
0.60
0.46
0.58
0.44
0.41
0.25
0.27
0.46
0.45
0.67
0.53
0.34
0.43
0.47
0.49
0.74
0.79
0.75
0.59
0.65
0.56
0.57
0.44
0.43
0.44
0.35
0.65
0.44
0.66
0.55
0.36
0.54
0.48
0.55
A- 0.024, B- 0.049, AB- 0.072
0.83
0.88
0.76
0.67
0.66
0.62
0.58
0.45
0.44
0.54
0.34
0.72
0.54
0.84
0.76
0.49
0.56
0.51
0.62
Mean
0.71
0.69
0.68
0.55
0.62
0.52
0.57
0.40
0.41
0.37
0.30
0.57
0.45
0.67
0.55
0.36
0.49
0.47
0.52
activity is more informative and facilitate monitoring of leaf senescence and remobilization
process. It has been shown that the total N content and GS activity measured in the flag leaf
alone can be used to predict the value of these two physiological traits in the whole plant
irrespective of the developmental stage of the plant.
Our results showed that GS activity gradually increased from tillering (2.79 and 3.58
μmol γ-glutamylhydroxamate formed/min/g FW during years 2009-10 and 2010-11,
respectively) to peak at anthesis (10.02 and 9.53 μmol γ-glutamylhydroxamate formed/min/g
FW during years 2009-10 and 2010-11, respectively) stages and then descended during later
stages of plant development in almost all genotypes irrespective of N dose (Fig 3). Similar
trend of GS activity has been reported in rice (Yang et al 2005) and maize (Purcino et al
2008). However, some genotypes, for instance PBW 621 and PBW 636, revealed highest
activity of GS at sub optimal doses i.e. RDN-50% and RDN-25%. Application of N had
significant effect on the activity of GS which was found to be maximum at RDN+25%.
Genotypic variation with respect to GS activity at different N levels was observed and the
interaction of N levels and genotypes was significant at all stages during both the years.
Fig. 3 Effect of different doses of nitrogen (RDN-25%, RDN-50%, RDN and
RDN+25%) on glutamine synthetase activity at tillering, anthesis and post
anthesis developmental stages of wheat genotypes during years 2009-10 and
2010-11
During year 2009-10, 36% and 17% decrease in GS activity with the application of
RDN-50% and RDN-25%, respectively as compared to optimum dose of N was observed.
However, during year 2010-11, the decrease was comparatively less as only 21% and 9%
decrease was observed at above said N doses. During year 2009-10, GS activity ranged from
1.27 to 4.80 (tillering), 6.38 to 15.04 (anthesis) and 3.50 to 9.52 μmol γ-glutamylhydroxamate
formed/min/g FW (post-anthesis) as evident from table 5.
During second year (2010-11), higher range of GS activity was detected i.e. from
1.83 to 5.47 (tillering), 4.88 to 15.47 (anthesis), 4.40 to 9.86 μmol γ-glutamylhydroxamate
40
Table 5: Effect of different doses of nitrogen on leaf glutamine synthetase activity (μmol γ-glutamylhydroxamate formed/min/g FW) at three developmental stages
of wheat during year 2009-10
Glutamine synthetase (μmol γ-glutamylhydroxamate formed/min/g FW)
Tillering stage
N Doses
Anthesis stage
Post-anthesis stage
RDN50%
RDN25%
RDN
RDN+
25%
Mean
RDN50%
RDN25%
RDN
RDN+
25%
Mean
RDN50%
RDN25%
RDN
RDN+
25%
Mean
PBW 621
1.14
5.25
5.44
6.01
4.46
10.79
15.68
15.85
17.84
15.04
5.44
9.24
10.56
11.27
9.13
PBW 636
4.29
4.53
4.74
5.64
4.80
12.34
13.65
13.88
13.00
13.22
8.94
9.21
9.39
10.55
9.52
PBW 590
1.18
3.07
4.09
7.05
3.85
5.08
10.95
10.69
10.80
9.38
5.41
7.25
8.64
14.94
9.06
DBW 17
1.22
4.53
4.61
4.07
3.61
5.79
10.13
10.28
10.29
9.12
1.94
8.25
8.31
8.97
6.87
HD 2967
2.25
1.66
5.73
5.79
3.86
10.29
10.62
11.17
13.51
11.40
6.18
7.72
8.04
8.51
7.61
PBW 509
1.96
2.02
2.41
2.54
2.23
9.50
9.13
8.19
13.03
9.96
4.02
4.20
7.69
9.13
6.26
BW 9178
0.95
1.02
1.58
3.91
1.86
4.02
4.35
8.01
12.75
7.28
1.66
1.44
6.90
8.00
4.50
BW 9183
1.13
1.33
2.19
2.03
1.67
8.86
8.43
10.00
12.00
9.82
6.08
7.32
7.13
8.83
7.34
BW 8989
0.93
0.99
1.97
2.00
1.48
3.74
4.91
7.52
9.35
6.38
1.00
2.20
6.73
6.06
4.00
BW 9022
1.11
1.36
1.40
1.98
1.46
4.73
6.24
6.94
7.79
6.43
1.18
3.52
4.13
5.15
3.50
PBW 343
1.15
1.08
1.26
1.58
1.27
7.17
7.56
8.69
9.90
8.33
5.33
5.08
6.13
7.37
5.98
PBW 550
1.14
1.38
4.29
4.92
2.93
12.10
10.98
13.07
19.38
13.88
1.32
5.08
5.92
8.02
5.08
Mean
1.54
2.35
3.31
3.96
2.79
7.87
9.39
10.36
12.47
10.02
4.04
5.88
7.46
8.90
6.57
Genotypes
41
CD (5%)
A- 0.025, B- 0.372, AB- 0.741
A- N doses, B-Genotypes, AB-Interacti
A- 0.798, B- 1.372, AB- 2.743
41
A- 0.342, B- 0.584, AB- 1.169
formed/min/g FW (post-anthesis) as compared to first year of study (2009-10) (Table 6).
Several intrinsic and extrinsic factors contributing to plant development might be responsible
for these variable effects but the overall trend during both years remained unchanged.
In the first year, genotype PBW 636 showed higher activity of GS surpassing all other
genotypes at RDN-50% during tillering (4.29 μmol γ-glutamylhydroxamate formed/min/g
FW), anthesis (12.34 μmol γ-glutamylhydroxamate formed/min/g FW) and post-anthesis
(8.94 μmol γ-glutamylhydroxamate formed/min/g FW) stages. As for other enzymes, GS
activity was also high in PBW 621 showing maximum N assimilation at RDN-25% (5.25,
15.68 and 9.24 μmol γ-glutamylhydroxamate formed/min/g FW, respectively) during all three
stages i.e. tillering, anthesis and post-anthesis. High activity of GS indicated a key role of this
enzyme in N assimilation. Heat resistant genotype BW 9022 showed lowest GS activity that
probably leads to lower protein accumulation compared to other genotypes. A rice variety
with high activity of GS and N content in leaves had also resulted in increased protein content
and grain yield (Zheng-xun et al 2007).
During second year, similar trend for GS activity in PBW 621 was observed. It
showed ample activity at RDN-25% during tillering (5.45 μmol γ-glutamylhydroxamate
formed/min/g FW), anthesis (15.87 μmol γ-glutamylhydroxamate formed/min/g FW) and
post-anthesis (9.60 μmol γ-glutamylhydroxamate formed/min/g FW) stages of plant growth
and development. GLU 1356 revealed highest activity at RDN-50% during tillering (5.04
μmol γ-glutamylhydroxamate formed/min/g FW) and post-anthesis (9.00 μmol γglutamylhydroxamate formed/min/g FW) stages. PBW 636 also showed highest activity of
GS at RDN-50% (9.00 μmol γ-glutamylhydroxamate formed/min/g FW) during post-anthesis
stage. PBW 343 and GLU 700 possessed poor GS efficiency. From study of both years, it was
observed that genotypes PBW 621 and PBW 636 showed comparable activites of GS.
GS is heavily involved in control of the N status in the form of protein in wheat. GS
plays a key role in N nutrition and grain yield also in rice (Xun et al 2007). Recent findings
indicate that tissue localization of the different forms of GS ensures optimal utilization of N,
especially during the grain-filling stages (Kichey et al 2005). Furthermore, there is a direct
relationship of grain protein and GS activity i.e. high grain protein genotypes had high GS
activity in flag leaves (Kichey et al 2005, Raghuram et al 2006, Habash et al 2007, Lochab et
al 2007). These findings were also supported by 15N-labeling experiments performed in the
field where GS and NR activities were shown as potential markers for remobilization and
estimation of N (Kichey et al 2007).
In vitro analysis of enzymes from crude extracts suggest that relatively higher specific
activities of NR, NIR and GS contribute to the higher NO3- utilizing capacity of Spirulina (Ali
et al 2008). It has also been reported that the activity of GS is positively correlated with
42
43
Table 6: Effect of different doses of nitrogen on leaf glutamine synthetase activity (μmol γ-glutamylhydroxamate formed/min/g FW) at three developmental stages
of wheat during year 2010-11
Glutamine synthetase (μmol γ-glutamylhydroxamate formed/min/g FW)
Tillering stage
Anthesis stage
Post-anthesis stage
N Doses RDN- RDN- RDN RDN+ Mean
RDNRDNRDN
RDNRDNRDN
RDN+
Mean
RDN+
Mean
50%
25%
50%
25%
50%
25%
25%
25%
25%
Genotypes
4.94
5.45
5.57
5.90
5.47
13.13
15.87 16.00
16.87
15.47
8.90
9.60
9.57
9.77
9.46
PBW 621
3.50
5.30
5.67
5.83
5.08
11.87
12.77
14.43
17.23
14.08
9.00
9.47
9.69
9.60
9.44
PBW 636
3.47
3.33
4.57
5.57
4.24
7.47
13.03 12.77
12.80
11.52
8.97
7.17
7.57
13.03
9.18
PBW 590
DBW 17
3.40
3.57
4.30
4.90
4.04
7.67
9.80
10.43
10.80
9.68
6.23
8.10
8.32
8.80
7.87
2.20
3.53
4.37
5.96
4.02
8.73
9.77
10.37
11.67
10.13
6.77
6.93
7.13
7.40
7.06
HD 2967
2.17
3.10
3.27
3.80
3.08
8.00
8.77
8.97
9.53
8.82
5.17
5.27
6.33
9.00
6.44
PBW 509
2.77
3.70
3.83
3.77
3.52
7.20
7.57
8.77
10.70
8.56
7.47
7.53
9.03
8.93
8.24
BW 9178
1.53
1.73
2.43
2.70
2.10
5.70
5.80
7.00
9.07
6.89
5.20
6.43
6.63
6.60
6.22
BW 9183
1.43
1.80
2.67
4.10
2.50
6.57
7.77
7.87
7.93
7.53
5.17
5.70
6.03
6.60
5.87
BW 8989
1.29
2.03
2.38
3.30
2.25
6.70
7.17
7.30
8.27
7.36
5.13
5.40
6.00
8.57
6.27
BW 9022
1.37
1.73
2.50
2.43
2.01
3.30
4.20
5.57
6.43
4.88
2.80
3.53
5.30
5.97
4.40
PBW 343
2.80
4.03
4.22
4.70
3.94
11.63
12.48 12.67
12.85
12.41
7.43
7.87
8.23
8.90
8.11
PBW 550
2.57
2.70
3.30
2.97
2.88
6.13
6.77
8.67
9.57
7.79
5.40
6.93
7.80
8.50
7.16
GLU 1101
5.04
5.27
5.40
6.13
5.46
8.23
10.30 16.57
16.47
12.89
9.00
8.60
10.20
11.63
9.86
GLU 1356
3.50
4.93
4.58
4.71
4.43
6.20
7.84
8.60
14.13
9.19
6.57
7.73
7.80
8.67
7.69
GLU 2001
1.30
1.63
1.73
2.67
1.83
5.10
5.57
6.70
9.93
6.82
4.37
5.17
5.43
7.37
5.59
GLU 700
1.37
3.03
3.27
3.23
2.73
5.27
6.30
7.17
10.93
7.42
5.20
5.73
6.13
6.40
5.87
PH132-4836
3.13
5.20
5.27
5.77
4.84
9.03
9.63
10.10
11.97
10.18
8.30
8.67
9.07
9.47
8.88
PH132-4840
2.65
3.45
3.85
4.36
3.58
7.66
8.97
10.00
11.51
9.53
6.50
6.99
7.57
8.62
7.42
Mean
CD (5%)
A- 0.122, B- 0.256, AB- 0.490
A- 0.143, B- 0.299, AB- 0.591
A- 0.152, B- 0.321, AB- 0.657
A- N doses, B-Genotypes, AB-Interaction
43
protein in rice (Zheng- xun et al 2007, Hakeem et al 2011). In wheat, changes in leaf total GS
activity expressed on fresh weight basis correlated well with the decline in leaf soluble protein
(Bernard et al 2008).Variation for GS activity and yield indicated that genetic differences
existed in wheat (Kichey et al 2006) which is futher supported by genetic studies in rice
(Yamaya et al 2002, Obara et al 2004) and maize (Gallais and Hirel 2004, Hirel et al 2007).
4.1.1d Glutamate synthase
The primary pathway for NH4+ conversion into amino acids involves GS and
GOGAT. Thus GS-GOGAT cycle is the main route of NH4+ assimilation in both old and
young leaves (Masclaux-Daubresse et al 2006). It has been reported that GOGAT supplies a
sole source of glutamate as the donor of amino group and this enzyme further catalyses
transfer of the amide group to α-ketoglutarate to form glutamate (Ferrario-Mery et al 2002,
Esposito et al 2005, Wickert et al 2007).
With plant development, GOGAT and GDH activities followed similar trend as
shown by GS activity. In previous investigation using maize as a model plant, it has been
shown that a general decrease in most metabolic markers and enzyme activities occurred
during leaf ageing (Hirel et al 2005b). Similar pattern was observed in our study indicating
that the changes in these markers reflect the transition state from sink to source in flag leaf
during the grain-filling period. Also with increase in N application, activities of both GOGAT
and GDH increased (Figs 4 and 5). Both N levels and genotypes showed significant variation
in GOGAT activity and their interaction was significant at all stages during both years of
study. All inorganic N is first reduced to NH4+ by NR and NIR and then to amino acids by
GS/GOGAT cycle. This metabolic system is regulated at different levels by many factors
including N source. Hence high levels of N led to an increase in the activity of N assimilating
enzymes (Forde 2000).
Fig. 4 Effect of different doses of nitrogen (RDN-25%, RDN-50%, RDN and
RDN+25%) on glutamate synthase activity at tillering, anthesis and post
anthesis developmental stages of wheat genotypes during years 2009-10 and
2010-11
44
During year 2009-10, GOGAT activity decreased by 42% and 23% with the
application of lower doses of N i.e. RDN-50% and RDN-25%, respectively compared to
RDN. However, during year 2010-11, less decrease in GOGAT activity i.e. 34% and 11%,
respectively over RDN values was observed. In the first year (2009-10), GOGAT activity
range was from 1.01 to 2.18 (tillering), 1.98 to 6.41 (anthesis) and 1.98 to 5.49 μmol NADH
oxidized/min/g FW (post-anthesis) as shown in table 7. However, during the year 2010-11,
GOGAT activity range was comparatively higher i.e. from 1.12 to 3.47 (tillering), 2.94 to
6.65 (anthesis) and 2.15 to 6.04 (post-anthesis) μmol NADH oxidized/min/g FW (Table 8).
During first year, similar genotypic behavior was observed as for other N assimilating
enzymes i.e. PBW 621 showed highest activity of 2.02 (tillering), 6.26 (anthesis) and 4.57
μmol NADH oxidized/min/g FW (post-anthesis) at RDN-50% and of 2.01 (tillering), 6.47
(anthesis) and 5.69 μmol NADH oxidized/min/g FW (post-anthesis) at RDN-25%. PBW 590
revealed maximum activity at RDN and higher doses while durable rust resistant genotype
BW 9183 showed very less activity of GS at all the stages.
During year 2010-11, it has been observed that genotype PBW 621 was better
adapted to lower doses of N i.e. RDN-50% showing maximum activity at anthesis (6.54 μmol
NADH oxidized/min/g FW) and post-anthesis (5.41 μmol NADH oxidized/min/g FW) stages
of crop development. However, genotype PBW 636 showed highest GOGAT activity at
RDN-25% during tillering (3.60 μmol NADH oxidized/min/g FW), anthesis (6.89 μmol
NADH oxidized/min/g FW) and post-anthesis (5.98 μmol NADH oxidized/min/g FW) stages.
PBW 590 revealed higher activity at optimum and higher doses of N. Among lines containing
Gpc-B1 gene, GLU 700 did not showed much activity, however, PBW 621 and PBW 636
were equally comparable showing maximum activity at all the stages.
The product of GS reaction in the form of glutamine is supplied to GOGAT so that
both enzymes work in parallel. In earlier findings, the rate of NO3- assimilation also exceeds
net flux through the GOGAT pathway by about 25% leading to accumulation of reduced N in
immediate downstream products like NH4+ and glutamine as well as in the photorespiratory
metabolites like glycine and serine (Scheible et al 2004).
4.1.1e Glutamate dehydrogenase
Glutamate dehydrogenase occupies a critical position at the junction between carbon
(2-oxoglutarate) and N (glutamate) metabolism and participates in balancing of the cellular
levels of three major components: the NH4+ ions, 2-oxoglutarate and glutamate. If GDH
operates in the aminating direction, it may assimilate excessive NH4+ ions. Conversely, if
GDH operates in the deaminating direction, it may fuel the tricarboxylic acid cycle under
conditions of carbon deficit (Aubert et al 2001, Miflin and Habash 2002, Dubois et al 2003).
The activity of GDH has always been subjected to much controversy as some
researchers reported the activity of GDH in the deaminating direction (Aubert et al 2001,
45
Table 7: Effect of different doses of nitrogen on leaf glutamate synthase activity (μmol NADH oxidized/min/g FW) at three developmental stages of wheat
during year 2009-10
Glutamate synthase (μmol NADH oxidized/min/g FW)
Tillering stage
N Doses
Anthesis stage
Post-anthesis stage
RDN50%
RDN25%
RDN
RDN+
25%
Mean
RDN50%
RDN25%
RDN
RDN+
25%
Mean
RDN50%
RDN25%
RDN
RDN+
25%
Mean
PBW 621
2.02
2.01
1.65
2.00
1.92
6.26
6.47
6.37
6.55
6.41
4.57
5.69
5.73
5.96
5.49
PBW 636
1.45
1.80
1.81
3.65
2.18
2.56
5.97
6.42
6.55
5.38
1.49
4.33
4.57
5.52
3.97
PBW 590
1.26
1.39
2.00
2.28
1.73
3.28
3.34
6.45
6.77
4.96
1.78
4.25
4.88
6.00
4.22
DBW 17
0.68
1.06
1.13
1.85
1.18
2.91
3.26
4.13
5.00
3.82
1.79
2.06
4.51
4.86
3.31
HD 2967
1.19
1.56
1.75
1.87
1.59
2.12
3.19
5.00
5.88
4.04
1.46
2.00
6.00
5.24
3.67
PBW 509
1.36
1.60
1.55
1.62
1.53
3.67
2.78
5.41
6.83
4.67
1.99
2.35
3.30
3.84
2.87
BW 9178
0.88
1.26
1.37
1.61
1.28
1.77
3.00
4.97
5.00
3.69
1.05
1.57
1.90
3.41
1.98
BW 9183
0.70
0.76
1.07
1.61
1.04
1.37
2.05
2.00
2.50
1.98
1.23
1.56
2.40
2.98
2.04
BW 8989
1.10
1.41
1.78
1.65
1.48
2.79
3.57
4.00
6.08
4.11
1.39
2.36
2.02
4.41
2.54
BW 9022
0.97
1.00
1.49
1.89
1.34
2.92
3.13
4.99
5.95
4.25
2.19
2.00
3.00
3.00
2.55
PBW 343
0.50
0.95
1.00
1.59
1.01
1.37
2.40
3.63
3.53
2.74
1.00
2.80
3.45
4.67
2.98
PBW 550
0.93
1.20
1.56
2.59
1.57
2.32
2.75
3.00
4.00
3.01
2.00
2.48
2.65
3.55
2.67
Mean
1.08
1.33
1.51
2.02
1.49
2.78
3.49
4.70
5.39
4.09
1.83
2.79
3.70
4.45
3.19
Genotypes
46
CD (5%)
A- 0.171, B- 0.304, AB- 0.598
A- N doses, B-Genotypes, AB-Interaction
A- 0.271, B- 0.473, AB- 0.949
46
A- 0.352, B- 0.609, AB- 1.205
Table 8: Effect of different doses of nitrogen on leaf glutamate synthase activity (μmol NADH oxidized/min/g FW) at three developmental stages of wheat during
year 2010-11
Glutamate synthase (μmol NADH oxidized/min/g FW)
N Doses
RDN50%
Tillering stage
RDNRDN
RDN+
25%
25%
47
Genotypes
PBW 621
2.30
2.23
2.42
PBW 636
3.59
3.60
3.51
PBW 590
1.82
2.72
3.71
DBW 17
1.48
1.53
2.30
HD 2967
1.24
1.79
1.87
PBW 509
0.78
1.65
2.56
BW 9178
1.32
1.54
1.85
BW 9183
0.79
1.40
1.40
BW 8989
0.54
1.35
1.56
BW 9022
0.70
1.48
1.51
PBW 343
0.74
1.49
1.66
PBW 550
1.40
1.74
2.56
GLU 1101
0.79
1.50
1.63
GLU 1356
1.34
2.75
3.33
GLU 2001
1.30
1.36
1.63
GLU 700
0.41
0.62
1.22
PH132-4836
0.63
1.52
2.71
PH132-4840
0.67
1.45
2.80
Mean
1.21
1.76
2.24
CD (5%)
A- 0.042, B- 0.095, AB- 0.180
A- N doses, B-Genotypes, AB-Interaction
3.12
4.25
4.42
2.33
2.83
2.68
1.81
1.45
1.53
1.80
1.68
3.50
2.24
4.75
3.37
2.25
3.46
3.50
2.83
Mean
RDN50%
Anthesis stage
RDN- RDN
RDN+
25%
25%
2.52
3.74
3.17
1.91
1.93
1.92
1.63
1.26
1.25
1.37
1.39
2.30
1.54
3.04
1.92
1.12
2.08
2.11
2.01
6.54
6.32
3.82
3.66
5.44
4.27
4.42
1.68
2.61
2.86
1.20
3.34
2.41
3.56
3.13
3.36
3.51
3.25
3.63
6.77
6.89
4.73
5.42
6.02
4.58
4.75
2.64
3.57
3.73
3.39
4.35
3.70
5.58
3.69
3.51
4.38
5.82
4.64
6.42
6.51
6.74
5.73
6.41
5.54
4.87
3.59
3.55
4.44
3.51
4.60
4.29
5.57
4.73
3.56
5.10
5.22
5.02
6.85
6.52
7.47
6.56
6.22
6.61
5.07
3.85
5.19
4.62
4.55
5.63
4.55
5.70
5.36
3.67
5.70
6.55
5.59
A- 0.040, B- 0.098, AB- 0.185
47
Mean
6.65
6.56
5.69
5.34
6.02
5.25
4.78
2.94
3.73
3.91
3.16
4.48
3.74
5.10
4.22
3.53
4.68
5.21
4.72
RDN50%
Post-anthesis stage
RDN- RDN
RDN+
25%
25%
Mean
5.41
5.33
3.41
3.86
3.55
3.60
3.39
2.09
1.65
1.32
2.67
3.22
1.27
3.75
2.23
1.19
2.86
2.10
2.94
5.76
5.98
4.13
5.27
4.72
3.91
3.60
2.47
3.29
2.32
3.32
4.50
3.75
4.70
3.79
1.80
3.69
3.67
3.92
5.38
6.04
4.43
4.77
4.75
5.17
4.21
2.65
3.35
2.71
3.28
4.42
3.68
5.16
4.05
2.15
4.02
3.61
4.10
5.27
6.33
4.78
4.29
5.01
5.55
4.26
2.49
4.25
2.68
3.32
4.69
4.41
5.66
4.49
2.36
4.01
4.12
4.33
5.07
6.51
5.40
5.64
5.72
7.64
5.59
3.56
4.20
4.51
3.80
5.24
5.30
6.56
5.68
3.24
5.53
4.54
5.21
A- 0.048, B- 0.092, AB- 0.189
Miflin and Habash 2002) while some reported its activity in the aminating direction (Kichey
et al 2005). Dubois et al (2003) suggest that its function would be more for carbohydrate
replenishment rather than N-assimilation. However, one cannot completely exclude the dual
function of the enzyme: depending on the N status of the plant it could act as a signal to
control the homeostasis of glutamate and thus the flux of reduced N (Gallais and Hirel 2004).
Reports in the literature indicated that GDH aminating and deaminating activities are
clustered together and are independent of metabolites thereby strengthening the recent view
that the enzyme may be able to sense the carbon and N balance in leaves and stems (TerceLaforgue et al 2004a).
During year 2009-10, GDH activity decreased by 39% and 13% with the application
of lower doses of N i.e. RDN-50% and RDN-25%, respectively as compared to optimum
dose. During year 2010-11, almost similar decrease in enzyme activity i.e. 34% and 15%,
respectively was observed. GDH activity was significantly affected by N treatments and
genotypes. During both years, the interaction of N levels and genotypes was found to be
significant at all stages.
Fig. 5 Effect of different doses of nitrogen (RDN-25%, RDN-50%, RDN and
RDN+25%) on glutamate dehydrogenase activity at tillering, anthesis and post
anthesis developmental stages of wheat genotypes during years 2009-10 and
2010-11
During year 2009-10, GDH activity ranged from 1.07 to 2.67 (tillering), 2.87 to 5.26
(anthesis) and 2.07 to 4.79 μmol NADH oxidized/min/g FW (post-anthesis) whereas during
2010-11, slight increase in enzyme activity was observed. Similar to GOGAT activity, GDH
activity was maximum in PBW 621 and PBW 590 as evident from table 9. PBW 621 revealed
significant activity at lower doses (RDN-50% and RDN-25%) whereas PBW 590 showed
maximum activity at RDN and at higher dose. During 2010-11, PBW 621 showed high GDH
activity of 5.33 (anthesis) and 4.75 μmol NADH oxidized/min/g FW (post-anthesis) at RDN50% and 5.68 (anthesis) and 5.45 μmol NADH oxidized/min/g FW (post-anthesis) at RDN25% (Table 10). During this year, GLU 1356 was better adapted to optimum dose at tillering
48
Table 9: Effect of different doses of nitrogen on leaf glutamate dehydrogenase activity (μmol NADH oxidized/min/g FW) at three developmental stages of wheat
during year 2009-10
Glutamate dehydrogenase (μmol NADH oxidized/min/g FW)
Tillering stage
N Doses
Genotypes
Anthesis stage
Post-anthesis stage
49
RDN50%
RDN25%
RDN
RDN+
25%
Mean
RDN50%
RDN25%
RDN
RDN+
25%
Mean
RDN50%
RDN25%
RDN
RDN+
25%
Mean
PBW 621
1.91
2.99
2.89
2.91
2.67
3.65
5.76
5.84
5.80
5.26
4.29
4.81
4.90
5.18
4.79
PBW 636
1.34
1.60
1.84
1.92
1.67
4.47
4.91
5.18
5.64
5.05
3.21
2.50
3.21
4.59
3.38
PBW 590
1.48
2.49
3.02
3.01
2.50
2.75
5.30
5.40
5.97
4.86
2.40
4.22
5.79
5.37
4.44
DBW 17
1.28
1.47
2.15
2.25
1.79
2.58
4.51
4.34
5.66
4.27
3.11
3.57
3.53
5.00
3.80
HD 2967
0.74
1.03
1.56
1.78
1.28
1.46
4.45
5.21
6.15
4.32
2.11
3.00
5.27
5.38
3.94
PBW 509
0.96
1.64
1.98
2.60
1.79
2.80
4.20
5.19
5.31
4.38
3.05
2.99
3.00
5.01
3.51
BW 9178
0.72
1.06
2.00
2.68
1.62
2.92
3.79
4.97
5.91
4.40
2.00
3.31
4.30
4.23
3.46
BW 9183
0.68
1.05
1.30
1.28
1.07
2.31
3.52
3.73
3.73
3.32
1.45
2.42
2.54
2.79
2.30
BW 8989
0.61
1.49
1.60
2.37
1.52
1.44
2.90
3.41
4.12
2.96
2.00
1.78
2.00
2.50
2.07
BW 9022
0.97
1.10
1.49
2.00
1.39
2.44
2.82
2.77
3.44
2.87
1.79
2.00
2.10
3.99
2.47
PBW 343
0.88
1.15
1.30
2.21
1.38
1.97
3.47
3.78
4.79
3.50
2.01
2.39
3.00
3.24
2.66
PBW 550
1.45
1.39
1.72
2.03
1.65
2.11
3.94
4.38
4.72
3.79
2.32
2.91
3.43
4.11
3.19
Mean
1.08
1.54
1.90
2.25
1.69
2.57
4.13
4.52
5.10
4.08
2.48
2.99
3.59
4.28
3.33
CD (5%)
A- 0.283, B- 0.484, AB- 0.631
A- N doses, B-Genotypes, AB-Interaction
A- 0.512, B- 0.899, AB- 1.130
49
A- 0.503, B- 0.862, AB- 1.058
Table 10:
Effect of different doses of nitrogen on leaf glutamate dehydrogenase activity (μmol NADH oxidized/min/g FW) at three developmental stages of
wheat during year 2009-10
Glutamate dehydrogenase (μmol NADH oxidized/min/g FW)
N Doses
RDN50%
Tillering stage
RDNRDN
RDN+
25%
25%
50
Genotypes
1.65
1.73
2.10
PBW 621
1.37
1.56
1.79
PBW 636
2.71
2.55
3.31
PBW 590
1.51
1.76
1.82
DBW 17
HD 2967
1.02
1.48
1.93
1.77
2.17
2.20
PBW 509
0.61
1.12
2.30
BW 9178
1.02
1.13
1.20
BW 9183
0.83
1.24
1.38
BW 8989
0.59
1.32
1.42
BW 9022
0.50
0.60
1.22
PBW 343
1.42
2.31
2.23
PBW 550
0.69
1.02
1.40
GLU 1101
2.13
3.28
3.46
GLU 1356
0.58
1.59
2.14
GLU 2001
0.79
1.25
1.45
GLU 700
1.63
2.26
2.28
PH132-4836
1.11
1.55
2.88
PH132-4840
1.22
1.66
2.03
Mean
CD (5%)
A- 0.041, B- 0.089, AB- 0.162
A- N doses, B-Genotypes, AB-Interaction
2.63
2.32
3.47
2.20
2.32
2.47
2.78
1.18
1.41
1.54
1.80
3.31
1.80
3.40
2.22
2.12
3.23
2.42
2.37
Mean
RDN50%
RDN25%
2.03
1.76
3.01
1.82
1.69
2.15
1.70
1.13
1.22
1.22
1.03
2.32
1.23
3.07
1.63
1.40
2.35
1.99
1.82
5.33
4.43
4.09
4.45
3.87
4.57
3.22
1.83
1.85
2.45
1.53
3.53
1.11
4.81
3.80
2.85
2.48
3.12
3.30
5.68
4.20
5.15
4.79
4.70
4.71
4.04
2.84
1.75
2.87
2.30
4.78
2.65
4.78
4.60
2.73
4.06
4.81
3.97
Anthesis stage
RDN
RDN+
25%
5.53
5.42
5.60
5.43
5.13
5.41
4.56
3.49
2.33
4.33
2.52
4.81
4.60
5.37
5.42
3.19
4.23
5.49
4.60
A- 0.047, B- 0.091, AB- 0.183
50
5.85
6.22
5.76
5.80
5.70
5.61
4.81
3.78
4.43
4.25
4.42
5.69
5.20
5.65
5.49
3.63
5.07
5.60
5.16
Mean
RDN50%
Post-anthesis stage
RDNRDN
RDN+
25%
25%
Mean
5.60
5.07
5.15
5.12
4.85
5.08
4.16
2.99
2.59
3.47
2.69
4.70
3.39
5.15
4.83
3.10
3.96
4.75
4.26
4.75
3.22
3.78
1.90
2.20
2.52
1.46
0.83
1.80
1.54
1.32
3.12
2.81
2.24
3.51
1.31
1.48
1.86
2.31
5.45
3.47
4.21
3.83
2.60
2.77
1.88
1.50
1.61
1.73
1.79
4.61
3.28
5.09
4.66
1.85
3.10
3.78
3.18
5.26
3.67
4.22
3.87
3.69
3.11
2.33
1.93
2.40
2.37
2.23
4.57
3.59
4.67
4.39
1.94
3.03
3.65
3.39
5.48
3.84
4.32
4.31
4.87
2.74
2.70
2.57
2.99
2.40
2.10
5.12
3.58
5.56
4.54
2.10
3.43
3.81
3.69
A- 0.048, B- 0.083, AB- 0.172
5.36
4.14
4.58
5.46
5.09
4.42
3.30
2.81
3.22
3.81
3.72
5.45
4.70
5.78
4.86
2.52
4.09
5.13
4.36
and post-anthesis stages. PBW 343 and BW 9183 showed poor response for GDH activity
compared to other genotypes.
An increase in the GDH activity in flag leaves of PBW 621was not substantiated with
a parallel increase of GS activity during grain filling which otherwise coincided with low
protein content in grains. It indicates that both high GS activity along with high GDH activity
is probably necessary at the time of flowering for grain filling to carry out the deamination
and transamination reactions during chloroplast hydrolysis in the flag leaves. It can be
hypothesized that the enzyme in conjunction with GS may participate in the reassimilation of
NH4+ released following protein hydrolysis during the process of N remobilization.
Previous investigations showed that high concentration of NH4+ provided externally
(Terce-Laforgue et al 2004a) or released into the sieve tube (Masclaux et al 2000, Limami et
al 2002) generally led to an increase in leaf GDH activity. It has been shown that GDH
become active in the cytosol of NH4+ treated plants and is able to assimilate NH4+ (TerceLaforgue et al 2004b). Pedicel GS and GDH activity increased 100% and 25%, respectively
in response to N supply (Seebauer et al 2004). Both GS and GDH found at the intersection of
carbon and N metabolism. It has been found that GS and GDH overexpression may improve
NUE of the plant by increasing its capacity to refix the NH4+.
4.1.2 Genotypic Variation With Respect to Metabolites in Leaves
4.1.2a Nitrogen content
Nitrogen is the most important constituent of plant proteins and is required
throughout the growth period from vegetative stage to subsequent harvesting. Delaying N
application led to lower grain yield but increased protein content (Ali 2011). N content
decreased with plant development (Fig 6) and this decrease may be linked to drastic decline
of leaf area index as also reported by Amanullah et al (2007) in maize. During year 2009-10,
N content decreased by 19% and 8% at RDN-50% and RDN-25%, respectively as compared
to optimum dose. During year 2010-11, almost similar decrease in N content was observed.
Various N treatments and genotypes had significant effect on N content present in leaves and
grains. The interaction of N levels and genotypes was significant at all three studied stages.
The range of N content was from 3.23 to 5.57 % (tillering), 1.94 to 5.60 % (anthesis)
and 1.03 to 1.63 % (post-anthesis) during 2009-10. At RDN-50%, genotype PBW621 was
found to possess maximum N content during tillering (5.27%), anthesis (5.28%) and postanthesis (1.43%) stages (Table 11). At RDN-25%, there was variation with respect to
genotypes for N content. Genotype PBW 636 expressed highest N content at optimum and
higher dose of N whereas PBW 343, BW 9022 and BW 8989 revealed low N content at postanthesis stages.
During 2010-11, N content range was from 2.89 to 6.69 % (tillering), 1.86 to 4.89 %
(anthesis) and 0.57 to 2.61 % (post-anthesis) as shown in table 12. PBW 621 showed highest
51
Fig. 6
Effect of different doses of nitrogen (RDN-25%, RDN-50%, RDN and
RDN+25%) on nitrogen content at tillering, anthesis and post anthesis
developmental stages of wheat genotypes during years 2009-10 and 2010-11
N content at RDN-50% (6.57%) and RDN-25% (6.68%) during tillering stage. As plant
developed, GLU 1356 predominated with N content of 4.87% (RDN-50%) and 5.26% (RDN25%) at anthesis stage and with 2.49% (RDN-50%) and 2.67% (RDN-25%) at post-anthesis
stage. GLU 2001 and PH132-4836 showed intermediate N content at RDN+50 %. PBW 343
showed very low N content at all three stages.
During vegetative growth, N is used mainly for growth and then assimilated to
protein pool of leaves and the stem. The decreasing content of N in seeds under certain
conditions may be due to premature leaf decay or could be due to poor partitioning of N. The
highest N content was found at 150% N application followed by 100% N and 50% N
application as reported earlier in wheat (Khaleque et al 2008) synchronising with our results
i.e. N content in a tissue increases with increase in N doses.
4.1.2b Amino acids
Amino acids in leaves increased till anthesis stage and thereafter decreased with the
crop development. Application of N dose significantly increased amino acid content at
RDN+25% over the control (Fig 7). After anthesis, leaves becomes source of N which is
partly recycled following protein hydrolysis and exported in the form of amino acids to grains
(Masclaux et al 2000). The amino acid content forms an important pool of N that gets
significantly affected by N levels. During 2009-10 and 2010-11 years, the interaction of N
levels and genotypes was significant at all stages. During first year (2009-10), lower doses of
N i.e. RDN-50% and RDN-25% decreased amino acid content by 23% and 14%, repectively.
However, during second year of study (2010-11), this decrease was 27% and 11%.
During first year (2009-10), amino acid content varied from 1.40 to 2.20 (tillering),
1.68 to 2.50 (anthesis) and 1.48 to 2.48 mg/g DW (post-anthesis) as shown in table 13. At
RDN-25%, PBW 621 showed highest amino acid content of 2.10 (tillering), 2.46 (anthesis)
and 2.59 mg/g DW (post-anthesis) during plant development. However, PBW 590 showed
52
Table 11: Effect of different doses of nitrogen on leaf nitrogen content (%) at three developmental stages of wheat during year 2009-10
Nitrogen content (%)
Tillering stage
N Doses
Genotypes
Anthesis stage
Post-anthesis stage
53
RDN50%
RDN25%
RDN
RDN+
25%
Mean
RDN50%
RDN25%
RDN
RDN+
25%
Mean
RDN50%
RDN25%
RDN
RDN+
25%
Mean
PBW 621
5.27
5.68
5.52
5.81
5.57
5.28
5.65
5.65
5.84
5.60
1.43
1.53
1.57
1.62
1.54
PBW 636
4.95
4.56
4.54
6.34
5.10
4.61
5.68
5.80
6.04
5.53
1.26
1.41
1.68
1.85
1.55
PBW 590
4.32
4.33
5.43
5.55
4.90
2.55
3.65
3.75
3.80
3.43
1.49
1.65
1.62
1.76
1.63
DBW 17
4.15
4.25
4.35
4.43
4.29
2.35
3.28
2.80
3.47
2.97
1.37
1.48
1.48
1.60
1.48
HD 2967
4.12
4.45
5.45
5.55
4.89
2.56
3.03
3.67
3.47
3.18
1.37
1.26
1.51
1.62
1.44
PBW 509
4.24
5.52
5.27
5.35
5.09
2.51
2.70
4.62
4.84
3.66
1.23
1.34
1.47
1.82
1.47
BW 9178
3.56
4.19
4.31
4.37
4.10
1.54
2.61
2.25
3.76
2.54
1.15
1.62
1.20
1.51
1.37
BW 9183
3.95
3.46
4.37
4.49
4.06
1.48
1.72
2.43
2.80
2.11
1.12
1.15
1.23
1.34
1.21
BW 8989
2.86
3.34
3.52
3.45
3.29
1.52
1.54
1.79
2.93
1.94
1.18
1.20
1.15
1.48
1.25
BW 9022
2.44
3.21
3.69
3.75
3.27
1.30
1.54
2.39
2.81
2.01
0.97
0.92
1.26
1.40
1.14
PBW 343
3.13
3.16
3.29
3.34
3.23
1.21
2.35
1.54
2.72
1.95
0.76
0.98
1.14
1.26
1.03
PBW 550
4.26
4.37
5.49
6.65
5.19
2.44
2.65
3.77
3.85
3.18
1.40
1.46
1.51
1.65
1.51
Mean
3.94
4.21
4.60
4.92
4.42
2.44
3.03
3.37
3.86
3.18
1.23
1.34
1.40
1.58
1.39
CD (5%)
A- 0.027, B- 0.044, AB- 0.088
A- N doses, B-Genotypes, AB-Interaction
A- 0.024, B- 0.047, AB- 0.082
53
A- 0.132, B- 0.234, AB- 0.198
Table 12: Effect of different doses of nitrogen on leaf nitrogen content (%) at three developmental stages of wheat during year 2010-11
N Doses
Genotypes
54
PBW 621
PBW 636
PBW 590
DBW 17
HD 2967
PBW 509
BW 9178
BW 9183
BW 8989
BW 9022
PBW 343
PBW 550
GLU 1101
GLU 1356
GLU 2001
GLU 700
RDN50%
Tillering stage
RDNRDN
RDN+
25%
25%
Mean
6.57
6.34
3.75
4.01
3.46
3.56
2.28
2.56
2.80
2.90
2.42
3.35
3.09
5.44
4.25
2.65
3.82
3.35
3.70
6.68
6.45
4.06
4.22
3.64
4.06
3.04
2.65
2.52
3.42
2.54
4.45
3.13
5.78
5.42
2.66
4.03
3.47
4.01
6.69
6.25
4.73
4.50
4.06
4.31
3.45
3.18
3.09
3.36
2.89
4.42
3.18
6.19
5.52
2.96
4.24
4.31
4.29
6.70
6.50
5.55
4.88
4.12
4.30
3.73
3.10
3.28
3.39
2.97
4.84
3.21
6.81
5.52
3.01
4.44
5.06
4.52
PH132-4836
PH132-4840
Mean
CD (5%)
A- 0.061, B- 0.125, AB- 0.248
A- N doses, B-Genotypes, AB-Interaction
6.79
5.72
5.56
4.90
5.03
5.31
4.75
4.39
3.77
3.74
3.63
5.02
3.28
6.72
6.89
3.53
4.66
5.34
4.94
Nitrogen content (%)
Anthesis stage
RDNRDNRDN
RDN+
50%
25%
25%
3.05
4.50
2.58
2.61
2.17
2.39
1.81
1.57
1.35
1.48
1.43
2.86
2.04
4.87
2.28
2.01
2.52
2.87
2.46
5.18
3.55
2.85
3.43
3.09
2.85
2.27
1.79
1.45
1.64
1.84
3.14
2.31
5.26
3.06
2.21
3.05
3.11
2.89
5.14
4.29
3.67
3.96
3.06
3.12
2.65
2.05
2.15
2.13
1.94
3.06
2.42
5.23
3.11
2.24
3.32
3.22
3.15
A- 0.053, B- 0.117, AB- 0.281
54
5.53
3.15
3.85
4.32
3.32
2.94
2.86
2.50
2.51
2.46
2.21
3.70
2.50
4.20
3.11
2.70
5.65
3.46
3.38
Mean
RDN50%
Post-anthesis stage
RDNRDN
RDN+
25%
25%
Mean
4.73
3.87
3.24
3.58
2.91
2.83
2.40
1.98
1.87
1.93
1.86
3.19
2.32
4.89
2.89
2.29
3.64
3.17
2.97
2.45
1.94
1.49
1.37
1.49
1.44
1.58
1.01
0.91
1.21
0.30
1.52
1.40
2.49
1.88
0.33
1.76
2.09
1.48
2.64
1.94
1.54
1.46
1.80
1.52
1.50
1.13
1.19
1.39
0.33
1.48
1.75
2.67
1.98
1.11
1.95
2.09
1.63
2.61
2.13
1.74
1.57
1.77
1.71
1.69
1.16
1.18
1.44
0.57
1.70
1.63
2.54
2.13
1.17
2.16
2.16
1.72
2.69
2.22
1.98
1.72
1.81
1.72
1.91
1.18
1.21
1.41
0.87
1.69
1.60
2.55
2.22
1.61
2.14
2.25
1.82
2.64
2.40
1.95
1.72
1.98
2.16
1.77
1.32
1.42
1.73
0.77
2.11
1.76
2.46
2.44
1.61
2.79
2.22
1.96
A- 0.049, B- 0.093, AB- 0.187
Fig. 7 Effect of different doses of nitrogen (RDN-25%, RDN-50%, RDN and RDN +
25%) on amino acids at tillering, anthesis and post anthesis developmental
stages of wheat genotypes during years 2009-10 and 2010-11
highest amino acid content at RDN (2.91 mg/g DW) and RDN+25% (2.96 mg/g DW) during
anthesis stage. Again widely grown cultivar PBW 343 showed lowest amino acid content at
RDN-50% (1.05, 1.28 and 1.17 mg/g DW) during all three stages.
During year 2010-11, amino acid content was in the higher range compared to first
year. Variation in amino acid content of flag leaf was from 1.28 to 2.36 (tillering), 1.76 to
2.79 (anthesis) and 1.59 to 2.47 mg/g DW (post-anthesis) (Table 14). During tillering stage,
highest amino acid content was observed in PBW 636 at RDN-50% (2.13 mg/g DW) and in
PBW 621 at RDN-25% (2.54 mg/g DW). During later stages of development, lines with GpcB1 gene predominated over other genotypes as GLU 1356 showed highest amino acid content
of 2.38 (anthesis) and 2.16 mg/g DW (post-anthesis) at RDN-50% and 2.95 (anthesis) and
2.68 (post-anthesis) mg/g DW at RDN-25%.
Widely grown cultivar PBW 343 and heat resistant cultivar BW 8989 showed low
level of amino acids indicating their poor efficiency for N use. Both glutamine and asparagine
are the preferential form in which N is assimilated and translocated due to the fact that these
molecules show a low C:N ratio representing an advantage for NH4+ incorporation to nontoxic forms (Frechilla et al 2002).
4.1.2c Soluble proteins
The major sink of the N taken from the soil is ultimately proteins (Landry and
Delhaye 2007). Soluble proteins and amino acids are N remobilzable pools for monitoring the
N status of the plant. Protein content in flag leaf increased till anthesis and decreased
thereafter at later stages of plant development. This increase was in parallel with increasing
concentration of N supplied in the field (Fig 8). Our results are in line with the earlier reports
of Diaz et al (2008) who observed that with leaf ageing both amino acids and soluble
protein concentration decreased. The progressive decrease in leaf N during grain-filling
period corresponded to the degradation of leaf proteins (Kichey et al 2006). Our results also
55
Table 13: Effect of different doses of nitrogen on amino acids (mg/g DW) at three developmental stages in leaves of wheat during year 2009-10
Amino acids (mg/g DW)
Anthesis stage
RDNRDNRDN
RDN+
50%
25%
25%
RDN50%
Tillering stage
RDNRDN
RDN+
25%
25%
Mean
Mean
RDN50%
Post-anthesis stage
RDNRDN
RDN+
25%
25%
Mean
PBW 621
1.40
2.10
2.49
2.63
2.15
1.87
2.46
2.74
2.76
2.46
1.98
2.59
2.48
2.86
2.48
PBW 636
1.80
1.96
2.49
2.55
2.20
1.95
2.12
2.68
2.86
2.40
1.88
1.87
2.33
2.77
2.22
PBW 590
1.47
1.45
2.12
2.47
1.88
2.07
2.08
2.91
2.96
2.50
1.61
1.82
2.56
2.61
2.15
DBW 17
1.61
1.91
1.56
2.13
1.80
1.86
1.87
2.07
2.65
2.11
1.86
1.87
2.48
2.47
2.17
HD 2967
1.27
2.25
2.36
2.48
2.09
1.78
2.10
2.52
2.71
2.28
1.81
2.28
2.52
2.89
2.38
PBW 509
1.32
1.41
1.88
2.38
1.75
1.85
2.11
2.11
2.86
2.23
1.46
1.41
1.72
2.56
1.79
BW 9178
1.21
1.20
1.54
2.10
1.51
1.75
1.78
2.00
2.67
2.05
1.18
1.36
1.90
2.26
1.68
BW 9183
1.18
1.31
1.37
2.17
1.51
1.51
1.54
1.76
2.43
1.81
1.55
1.58
1.57
2.19
1.72
BW 8989
1.11
1.11
1.20
2.17
1.40
1.74
1.56
1.76
2.49
1.89
1.27
1.38
1.62
2.36
1.66
BW 9022
1.05
1.25
1.31
2.20
1.45
1.56
1.78
2.01
2.53
1.97
1.34
1.48
1.37
2.27
1.61
PBW 343
1.05
1.16
1.26
2.14
1.40
1.28
1.54
1.75
2.14
1.68
1.17
1.21
1.32
2.20
1.48
PBW 550
1.45
1.50
1.64
2.11
1.68
1.83
1.68
2.60
2.87
2.26
1.67
1.83
1.93
2.64
2.02
Mean
1.33
1.55
1.77
2.29
1.74
1.75
1.89
2.24
2.66
2.14
1.56
1.72
1.98
2.51
1.94
N Doses
Genotypes
56
CD (5%)
A- 0.255, B- 0.432, AB- 0.864
A- N doses, B-Genotypes, AB-Interaction
A- 0.190, B- 0.339, AB- 0.662
56
A- 0.192, B- 0.332, AB- 0.671
Table 14: Effect of different doses of nitrogen on amino acids (mg/g DW) at three developmental stages in leaves of wheat during year 2010-11
N Doses
RDN50%
Tillering stage
RDNRDN
RDN+
25%
25%
Mean
1.70
2.13
1.65
1.51
1.04
1.07
1.37
1.11
1.96
1.07
1.08
1.24
1.84
1.64
1.28
1.59
1.38
1.39
1.45
2.54
2.25
1.74
1.65
1.35
1.41
1.63
1.25
1.14
1.54
1.15
1.87
1.95
2.44
1.61
1.54
1.54
1.60
1.68
2.34
2.34
2.06
1.81
1.46
1.58
1.68
1.35
1.63
1.37
1.28
1.93
1.90
2.36
1.83
1.81
1.78
1.82
1.80
Amino acids (mg/g DW)
Anthesis stage
RDNRDNRDN
RDN+
50%
25%
25%
Mean
RDN50%
2.55
2.68
2.68
2.48
2.00
2.34
2.35
2.03
1.76
1.91
1.84
2.41
2.52
2.79
2.43
2.14
2.58
2.39
2.33
1.58
1.58
1.56
1.97
1.55
1.49
1.64
1.54
1.14
1.28
1.09
1.60
1.96
2.16
1.46
1.61
1.58
1.79
1.58
Post-anthesis stage
RDNRDN
25%
RDN+
25%
Mean
2.75
2.74
2.45
2.73
1.83
2.17
2.21
1.77
2.15
1.75
2.08
2.57
2.19
2.40
2.86
2.46
2.54
2.50
2.34
2.38
2.08
2.13
2.30
1.72
1.75
1.89
1.68
1.66
1.61
1.59
2.07
2.09
2.47
2.01
2.06
2.09
2.10
1.98
Genotypes
PBW 621
PBW 636
PBW 590
DBW 17
HD 2967
PBW 509
BW 9178
57
BW 9183
BW 8989
BW 9022
PBW 343
PBW 550
GLU 1101
GLU 1356
GLU 2001
GLU 700
PH132-4836
PH132-4840
2.50
2.38
2.57
1.85
1.71
1.77
1.76
1.11
1.54
1.18
1.17
2.11
1.74
2.67
1.67
1.62
1.63
2.37
1.85
Mean
CD (5%)
A- 0.075, B- 0.143, AB- 0.291
A- N doses, B-Genotypes, AB-Interaction
2.63
2.61
2.27
2.21
1.73
2.06
1.95
1.94
1.86
1.70
1.70
2.51
2.08
2.68
2.76
2.48
2.56
1.91
2.20
1.71
2.25
2.10
1.95
1.79
1.91
1.84
1.58
1.35
1.48
1.25
1.84
2.05
2.38
1.95
1.39
2.03
1.85
1.82
2.53
2.54
2.57
2.27
2.03
2.36
2.13
1.87
1.39
1.81
1.97
2.43
2.81
2.95
2.14
2.04
2.36
2.15
2.24
2.98
2.89
2.96
2.67
1.99
2.46
2.57
2.38
1.99
2.15
2.11
2.57
2.57
3.08
2.57
2.48
2.72
2.72
2.55
A- 0.078, B- 0.152, AB- 0.301
57
2.97
3.04
3.08
3.04
2.16
2.62
2.87
2.28
2.32
2.22
2.04
2.78
2.68
2.76
3.05
2.66
3.21
2.86
2.70
2.44
1.97
2.18
1.95
1.60
1.55
1.73
1.61
1.48
1.57
1.50
2.08
2.04
2.68
1.78
1.98
1.59
1.92
1.87
2.76
2.04
2.33
2.55
1.88
1.78
1.96
1.81
1.86
1.85
1.85
2.02
2.18
2.63
1.96
2.18
2.66
2.19
2.14
A- 0.065, B- 0.142, AB- 0.275
Fig. 8
Effect of different doses of nitrogen (RDN-25%, RDN-50%, RDN and
RDN+25%) on soluble proteins at tillering, anthesis and post anthesis
developmental stages of wheat genotypes during years 2009-10 and 2010-11
indicated a similar decrease in protein content with increasing dose of N.
During year 2009-10, protein content decreased by 10% and 4% at RDN-50% and
RDN-25%, respectively as compared to RDN whereas during year 2010-11, this decrease was
enhanced to 25% and 11%, respectively. Both N treatments and genotypes significantly
affected soluble proteins and their interaction was significant at all stages during both years of
study. During year 2009-10, protein content varied from 143.5 to 149.2 (tillering), 151.2 to
158.4 (anthesis) and 147.6 to 155.0 mg/g DW (post-anthesis) as shown in table 15. Protein
content was highest in PBW 621 at RDN-50% and RDN-25% during tillering (148.3, 149.3
mg/g DW), anthesis (152.3, 159.6 mg/g DW) and post-anthesis (150.8, 156.0 mg/g DW)
stages. At higher doses of N, PBW 636 revealed higher protein content which was in parallel
to amino acid.
During second year i.e. 2010-11, protein content was found to vary in the range of
137.4 to 152.4 (tillering), 145.0 to 168.7 (anthesis) and 140.6 to 158.4 mg/g DW (postanthesis) (Table 16). As discussed earlier, genotype PBW 621 showed highest protein content
at lower doses of N i.e. RDN-50% (145.3 mg/g DW) and RDN-25% (155.3 mg/g DW) at
tillering stage. During tillering and post-anthesis stages, soluble protein content was highest in
GLU 1356 at RDN-50% (164.5 and 148.5 mg/g DW) and RDN-25% (170.2 and 160.0 mg/g
DW), respectively. During later stages also, lines with Gpc-B1 gene had higher protein
content. Infact, lines with Gpc-B1 gene were far more superior to other genotypes on the basis
of protein content which is due to the presence of Gpc-B1 gene. Infact, this gene is
responsible for accumulation, translocation and assimilation of N from flag leaf to grain in the
form of amino acids and proteins. Compared to other genotypes protein content was low in
widely grown cultivar PBW 343.
Reports in the literature also indicate that regardless of the N fertilization regime
Larger amounts of protein present in the flag leaf is due to photosynthetic apparatus that
58
Table 15: Effect of different doses of nitrogen on soluble proteins (mg/g DW) at three developmental stages in leaves of wheat during year 2009-10
Soluble proteins (mg/g DW)
Tillering stage
N Doses
Anthesis stage
Post-anthesis stage
RDN50%
RDN25%
RDN
RDN+
25%
Mean
RDN50%
RDN25%
RDN
RDN+
25%
Mean
RDN50%
RDN25%
RDN
RDN+
25%
Mean
PBW 621
148.3
149.3
148.7
150.3
149.2
152.3
159.6
150.2
161.6
158.4
150.8
156.0
155.3
157.9
155.0
PBW 636
146.0
148.6
149.4
149.1
148.3
151.6
155.6
159.8
162.0
157.2
146.8
152.3
156.7
158.1
153.5
PBW 590
143.3
144.1
143.8
150.8
145.5
148.5
154.3
151.7
157.7
153.1
147.1
150.6
157.7
156.9
153.1
DBW 17
144.0
144.4
145.1
147.2
145.2
149.7
153.2
155.1
154.5
153.1
146.6
147.9
148.5
152.2
148.8
HD 2967
144.2
144.8
146.8
147.0
145.7
150.6
151.2
152.3
155.5
152.4
148.9
149.8
149.9
156.9
151.4
PBW 509
144.9
144.6
145.9
147.5
145.7
150.4
154.5
156.7
158.1
154.9
148.6
151.0
153.7
152.0
151.3
BW 9178
143.5
145.1
147.3
147.0
145.7
149.2
155.7
151.6
159.3
153.9
147.3
150.5
151.3
150.7
150.0
BW 9183
143.2
144.1
146.8
147.6
145.4
150.2
151.4
155.7
158.8
154.1
144.1
147.3
148.4
149.8
147.4
BW 8989
142.0
143.6
143.8
144.6
143.5
146.9
149.1
151.6
157.4
151.3
146.1
146.7
149.2
149.5
147.9
BW 9022
142.1
144.8
144.9
148.5
145.1
149.1
150.6
152.6
157.5
152.4
145.8
147.9
146.9
150.1
147.7
PBW 343
143.8
145.5
145.4
145.4
145.0
147.9
149.7
150.6
156.5
151.2
145.0
146.6
149.1
149.8
147.6
PBW 550
143.5
144.5
144.5
147.6
145.0
147.4
150.8
156.1
159.5
153.4
146.5
149.0
156.0
154.5
151.5
Mean
144.1
145.3
146.0
147.7
145.8
149.5
153.0
154.5
158.2
153.8
147.0
149.6
151.9
153.2
150.4
Genotypes
59
CD (5%)
A- 0.236, B- 0.403, AB- 0.801
A- N doses, B-Genotypes, AB-Interaction
A- 0.754, B- 1.303, AB- 2.609
59
A- 0.452, B- 0.791, AB- 1.597
Table 16: Effect of different doses of nitrogen on soluble proteins (mg/g DW) at three developmental stages in leaves of wheat during year 2010-11
N Doses
RDN50%
Tillering stage
RDNRDN
RDN+
25%
25%
Mean
145.3
138.5
139.0
132.0
138.0
133.0
134.0
133.0
130.3
136.0
131.1
132.1
140.7
141.0
133.0
133.4
131.4
132.0
135.2
155.3
155.0
147.3
146.0
142.4
140.5
143.4
144.0
141.0
136.0
139.0
146.3
148.8
147.2
145.1
143.7
143.1
142.4
143.6
152.4
150.3
147.2
143.1
142.6
140.1
145.0
142.4
141.0
141.5
137.4
142.6
148.9
150.4
146.5
146.8
147.9
143.3
144.6
Soluble proteins (mg/g DW)
Anthesis stage
RDNRDNRDN
RDN+
50%
25%
25%
Mean
RDN50%
Post-anthesis stage
RDNRDN
RDN+
25%
25%
Mean
163.6
162.6
161.7
149.8
160.5
152.3
151.9
156.2
155.9
154.8
145.6
145.0
147.1
168.7
167.4
168.0
161.6
155.1
157.2
146.3
146.2
143.7
136.0
139.5
136.5
136.0
135.4
136.8
136.5
134.0
140.0
143.0
148.5
142.0
141.5
137.0
144.5
139.4
147.2
145.0
145.0
142.0
139.0
140.0
146.0
146.0
145.0
133.1
139.0
144.0
149.0
160.0
145.0
149.0
146.0
147.0
144.9
155.1
151.9
149.3
144.8
145.7
143.8
147.8
146.6
146.7
145.3
140.6
149.1
151.8
158.4
152.4
155.0
150.8
150.0
148.7
Genotypes
PBW 621
PBW 636
PBW 590
DBW 17
HD 2967
PBW 509
BW 9178
60
BW 9183
BW 8989
BW 9022
PBW 343
PBW 550
GLU 1101
GLU 1356
GLU 2001
GLU 700
PH132-4836
PH132-4840
153.0
153.3
149.5
144.0
144.0
144.0
148.5
144.0
144.5
143.0
139.0
146.1
150.5
161.0
147.0
152.0
158.0
145.3
147.9
Mean
CD (5%)
A- 0.982, B- 2.085, AB- 4.161
A- N doses, B-Genotypes, AB-Interaction
156.0
154.3
153.0
150.4
146.1
143.0
154.0
148.5
148.0
150.9
140.5
146.0
155.5
152.4
161.0
158.0
159.2
153.5
151.7
162.0
156.0
156.0
141.0
157.0
147.5
146.0
147.2
145.9
149.1
139.5
149.0
145.0
164.5
160.4
162.0
151.0
158.0
151.8
161.5
160.0
158.0
149.0
158.0
150.7
150.0
153.0
152.5
151.2
141.0
155.9
144.2
170.2
168.0
168.1
158.0
150.2
154.9
165.0
165.4
163.1
151.0
160.0
153.2
154.2
159.0
159.0
159.0
147.0
156.0
144.3
172.0
170.2
169.7
163.0
154.0
158.4
A- 0.913, B- 1.945, AB- 3.881
60
166.0
169.0
169.5
158.2
167.0
157.8
157.5
165.6
166.2
160.0
155.0
159.0
155.0
168.0
171.0
172.0
174.2
158.1
163.7
165.0
154.3
149.3
146.2
151.1
146.1
152.0
151.0
153.4
153.0
143.0
153.2
155.0
162.0
156.0
163.5
158.3
153.0
153.1
A- 0.982, B- 2.074, AB- 4.142
162.0
162.2
159.2
155.0
153.0
152.5
157.0
154.0
151.5
158.5
146.4
159.0
160.0
163.0
166.7
166.0
162.0
155.4
157.3
maximizes carbon assimilation and N availability (Gastal and Lemaire 2002). The increase in
total soluble proteins may be the result of enhanced amino acid formation during N
assimilation.
4.1.2d Total chlorophyll content
Nitrogen is a structural component of chlorophyll. The increase in chlorophyll
concentration with increasing dose of N is important for the plant to enhance CO2 assimilation
and thereby achieving a greater leaf area and yield (Leon et al 2007). Leaf becomes more
green with increasing dose of N as observed in the present study and the maximum content of
chlorophyll was found at tillering stage (Fig 9). The highest chlorophyll content was obtained
at RDN+25% during pre- and post-anthesis stages, respectively which was significantly
higher over control. Hence higher levels of N promote chlorophyll synthesis (Lawlor 2002).
Interaction between various levels of N and genotypes was significant at all stages during the
both years.
Fig. 9 Effect of different doses of nitrogen (RDN-25%, RDN-50%, RDN and RDN+25%)
on chlorophyll content at tillering, anthesis and post anthesis developmental
stages of wheat genotypes during years 2009-10 and 2010-11
During year 2009-10, chlorophyll content decreased by 9% and 5% with the
application of RDN-50% and RDN-25%, respectively as compared to RDN. However, during
year 2010-11, only 4% and 2% reduction in chlorophyll content was observed. Increase in N
level produced more number of leaves/plant. An increase in leaf area with increasing N level
is attributed to better crop growth with higher N availability (Vadivel et al 2001). Chlorophyll
content varied during tillering (2.36 to 3.09 mg/g FW), anthesis (2.13 to 2.82 mg/g FW) and
post-anthesis (2.07 to 2.53 mg/g FW) stages as evident from table 17. PBW 636 showed
highest chlorophyll content of 2.95 (tillering), 2.73 (anthesis) and 2.57 mg/g FW (postanthesis) at RDN-25% and 3.17 (tillering), 2.97 (anthesis) and 2.63 mg/g FW (post-anthesis)
at RDN. At RDN-50%, there was variation with respect to genotypes. For instance PBW 636
showed highest chlorophyll content at tillering (2.73 mg/g FW), DBW 17 at anthesis (2.57
mg/g FW) and PBW 590 at post-anthesis (2.44 mg/g FW) stages. Durable rust resistant BW
61
Table 17: Effect of different doses of nitrogen on chlorophyll content (mg/g) at three developmental stages in leaves of wheat during year 2009-10
Chlorophyll content (mg/g)
Tillering stage
N Doses
Anthesis stage
Post-anthesis stage
RDN50%
RDN25%
RDN
RDN+
25%
Mean
RDN50%
RDN25%
RDN
RDN+
25%
Mean
RDN50%
RDN25%
RDN
RDN+
25%
Mean
PBW 621
2.55
2.73
3.50
3.57
3.09
2.51
2.68
2.94
2.83
2.74
2.32
2.47
2.52
2.54
2.46
PBW 636
2.73
2.95
3.17
3.33
3.05
2.49
2.73
2.97
3.10
2.82
2.32
2.57
2.63
2.60
2.53
PBW 590
2.55
2.73
2.72
2.98
2.74
2.45
2.52
2.60
2.74
2.58
2.44
2.52
2.48
2.54
2.50
DBW 17
2.67
2.76
2.84
3.33
2.90
2.57
2.62
2.64
2.67
2.63
2.27
2.53
2.56
2.31
2.42
HD 2967
2.62
2.59
2.80
2.93
2.73
2.50
2.47
2.51
2.77
2.56
2.30
2.37
2.38
2.53
2.39
PBW 509
2.60
2.63
2.87
2.90
2.75
2.44
2.51
2.54
2.85
2.58
2.30
2.32
2.34
2.63
2.40
BW 9178
2.13
2.50
2.67
2.60
2.47
2.32
2.44
2.53
2.59
2.47
2.15
2.30
2.34
2.40
2.30
BW 9183
2.11
2.41
2.49
2.45
2.36
1.93
2.26
2.06
2.30
2.13
1.91
2.09
2.19
2.26
2.11
BW 8989
2.34
2.46
2.53
2.61
2.49
2.10
2.11
2.53
2.59
2.33
2.00
2.11
2.18
2.23
2.13
BW 9022
2.38
2.32
2.61
2.63
2.48
2.15
2.36
2.53
2.50
2.39
1.94
2.13
2.00
2.23
2.07
PBW 343
2.50
2.57
2.56
2.69
2.58
2.42
2.38
2.44
2.62
2.46
2.15
2.15
2.30
2.23
2.21
PBW 550
2.39
2.73
3.00
2.89
2.75
2.33
2.31
2.59
2.80
2.51
2.35
2.31
2.50
2.67
2.46
Mean
2.46
2.61
2.81
2.91
2.70
2.35
2.45
2.57
2.70
2.52
2.20
2.32
2.37
2.43
2.33
Genotypes
62
CD (5%)
A- 0.036, B- 0.198, AB- 0.763
A- N doses, B-Genotypes, AB-Interaction
A- 0.048, B- 0.265, AB- 0.843
62
A- 0.073, B- 0.246, AB- 0.728
9183 genotype showed very low chlorophyll content.
During second year (2010-11), chlorophyll content ranged between 2.65 to 3.00
(tillering), 2.49 to 2.77 (anthesis) and 2.35 to 2.65 (post-anthesis) mg/g FW (Table 18).
Genotype HD 2967 showed highest content at RDN-50% during tillering (3.03 mg/g FW) and
anthesis (2.71 mg/g FW) stages. During tillering stage, genotype PBW 621 had maximum
chlorophyll content at RDN-25%, RDN and RDN+25% doses. However, during later stages,
there was variation with respect to genotypes. There was more decrease in chlorophyll content
in genotypes with Gpc-B1 gene. This showed that these genotypes senescence early as
compared to other genotypes.
Previous investigations in maize revealed a decreased level of most metabolic
markers and enzyme activities during leaf ageing and this decrease was more pronounced
when plants were N- deficit (Hirel et al 2005b). Similar results were obtained in the wheat
where these markers accurately reflect the transition from sink to source leaves during the
grain-filling period (Kichey et al 2005). Flag leaf chlorophyll, soluble protein and total GS
activity increased from booting to anthesis stage. After anthesis, these parameters gradually
declined showing statistically significant changes in flag leaves to support grain filling
processes from early milky stage onwards (Bernerd et al 2008). Anthesis triggers the start of
global changes in wheat leaf metabolism with soluble protein, chlorophyll, GS enzyme
activity in a well co-ordinated way. These changes are characteristic to the onset of
senescence and further remobilization of assimilate to the developing grain (Bernard et al
2008, Bernard and Habash 2009). Results of both years showed that genotypes PBW 621,
PBW 636 and GLU 1356 showed higher assimilation of N at lower doses i.e. RDN-50% and
RDN-25% while PBW 590 was effective at higher doses in metabolizing N.
4.1.3 Genotypic Variation With Respect to Metabolites in Mature Grains
4.1.3a Nitrogen content
Grain N appears to be regulated by N source. In wheat, the kinetics of grain N build
up suggest that during grain filling translocation of N from the vegetative organs is mainly
limited by the availability of the substrate from the source organs (Bertheloot et al 2008).
During both years, N content in grains as well as in straw increased with increasing dose of N
(Fig 10). N content in grains and straw was significantly affected by various N levels and
genotypes and their interaction was significant. During year 2009-10, N content in grains
decreased by 18% and 6% at RDN-50% and RDN-25%, respectively as compared to optimum
dose. However, during year 2010-11, more decrease in N content was observed i.e. 23% and
12%, respectively.
During 2009-10, N content in grains varied from 1.88 to 3.58 % as shown in table 19.
PBW 621 had highest level of N in grains at RDN-50% (3.40%) and RDN-25% (3.59%).
However, in leaves, similar trend with respect to genotypes was observed which indicates that
63
Table 18: Effect of different doses of nitrogen on chlorophyll content (mg/g) at three developmental stages in leaves of wheat during year 2010-11
Chlorophyll content (mg/g)
N Doses
64
Genotypes
PBW 621
PBW 636
PBW 590
DBW 17
HD 2967
PBW 509
BW 9178
BW 9183
BW 8989
BW 9022
PBW 343
PBW 550
GLU 1101
GLU 1356
GLU 2001
GLU 700
PH132-4836
PH132-4840
RDN50%
RDN25%
2.87
2.74
2.80
2.78
3.03
2.74
2.63
2.80
2.83
2.66
2.66
2.75
2.62
2.58
2.60
2.61
2.69
2.53
2.72
3.07
2.80
2.96
2.87
2.78
2.81
2.63
2.90
2.87
2.72
2.68
2.86
2.84
2.60
2.64
2.77
2.78
2.82
2.80
Tillering stage
RDN
RDN+
25%
3.10
2.91
3.04
2.86
2.82
2.89
2.76
2.85
2.89
2.77
2.73
2.81
2.75
2.79
2.67
2.80
2.88
2.90
2.85
Mean
CD (5%)
A- 0.051, B- 0.104, AB- 0.558
A- N doses, B-Genotypes, AB-Interaction
3.13
3.01
2.99
3.03
2.81
2.93
2.89
2.94
3.04
2.88
2.71
2.95
2.84
2.73
2.70
2.88
2.90
2.99
2.91
Mean
RDN50%
RDN25%
3.00
2.87
2.95
2.89
2.86
2.84
2.73
2.92
2.91
2.76
2.69
2.84
2.76
2.67
2.65
2.77
2.81
2.81
2.82
2.60
2.61
2.70
2.66
2.71
2.69
2.50
2.57
2.52
2.72
2.49
2.65
2.46
2.45
2.52
2.48
2.60
2.61
2.58
2.72
2.67
2.75
2.66
2.72
2.64
2.55
2.74
2.66
2.59
2.59
2.67
2.50
2.51
2.57
2.55
2.62
2.66
2.63
Anthesis stage
RDN
RDN+
25%
2.80
2.73
2.81
2.73
2.82
2.63
2.63
2.70
2.78
2.59
2.59
2.71
2.50
2.60
2.59
2.57
2.83
2.72
2.68
2.89
2.80
2.81
2.81
2.83
2.64
2.79
2.77
2.83
2.50
2.58
2.74
2.52
2.62
2.63
2.64
2.87
2.81
2.73
A- 0.064, B- 0.129, AB- 0.674
64
Mean
RDN50%
Post-anthesis stage
RDNRDN
RDN+
25%
25%
Mean
2.75
2.70
2.77
2.71
2.77
2.65
2.62
2.70
2.70
2.60
2.56
2.69
2.49
2.54
2.58
2.56
2.73
2.70
2.66
2.58
2.59
2.45
2.48
2.50
2.50
2.42
2.50
2.44
2.30
2.54
2.44
2.32
2.36
2.40
2.19
2.42
2.48
2.44
2.50
2.60
2.51
2.48
2.62
2.58
2.43
2.63
2.44
2.40
2.40
2.57
2.39
2.41
2.43
2.36
2.50
2.49
2.48
2.62
2.65
2.56
2.57
2.63
2.59
2.50
2.62
2.49
2.42
2.47
2.58
2.43
2.38
2.47
2.35
2.52
2.51
2.52
2.52
2.66
2.53
2.59
2.72
2.50
2.55
2.64
2.51
2.41
2.49
2.65
2.45
2.36
2.48
2.45
2.55
2.51
2.53
A- 0.068, B- 0.125, AB- 0.825
2.88
2.75
2.75
2.74
2.67
2.79
2.61
2.72
2.59
2.56
2.46
2.68
2.57
2.38
2.59
2.41
2.60
2.57
2.63
N content in leaves determine the N status of grains. During year 2010-11, N content
in PBW 621 was found to be 3.47% at RDN-50% and 3.58% at RDN-25% (Table 20) but at
higher doses genotypes with Gpc-B1 predominated having maximum N content over other
genotypes. N content in grain was on the lower side in BW 9183, BW 8989 and BW 9022
during both years.
During year 2009-10, N content in straw decreased by 31% and 23% with application
of lower
Fig. 10 Effect of different doses of nitrogen (RDN-25%, RDN-50%, RDN and
RDN+25%) on nitrogen content in grains and straw during years 2009-10
and 2010-11
N doses i.e. RDN-50% and RDN-25%, respectively compared to optimum dose. During
second year (2010-11), almost similar decrease was observed and N content was significantly
affected by various N levels and genotypes. The interaction between N levels and genotypes
was significant during both years. N content in straw varied from 0.25 to 0.38 % during year
2009-10 (Table 19) and was 0.23 to 0.44 % during year 2010-11 (Table 20). Genotype PBW
636 showed highest content of N (0.44%).
Most of the N utilized by developing seeds of wheat is mobilized from vegetative
tissues of the plant. During grain filling stages, NO3- uptake capacity declines sharply
(Triková and Kamínek 2000, Stehno et al 2005) and leaves, stalks act as sources of N while
grains serve as sink (Hirel et al 2001). With the use of labeled N fertilizer it was found
that N absorbed at post-anthesis stages is allocated directly to the grains (Gallais and Coque
2005). Accumulation of N in the grain is related to the accumulation of C:N through the
65
Table 19: Effect of different doses of nitrogen on nitrogen content (%) in wheat grains and straw samples during year 2009-10
Grains
N Doses
Straw
RDN50%
RDN25%
RDN
RDN+
25%
Mean
RDN50%
RDN25%
RDN
RDN+
25%
Mean
PBW 621
3.40
3.59
3.60
3.72
3.58
0.34
0.36
0.40
0.41
0.38
PBW 636
2.38
2.50
3.65
3.86
3.10
0.25
0.33
0.34
0.48
0.35
PBW 590
1.94
2.32
2.76
3.24
2.57
0.27
0.29
0.32
0.34
0.31
DBW 17
2.38
2.56
2.64
2.80
2.60
0.28
0.35
0.42
0.40
0.36
HD 2967
2.26
2.50
2.62
2.97
2.59
0.22
0.27
0.32
0.42
0.31
PBW 509
1.98
2.06
2.4
2.84
2.32
0.22
0.28
0.33
0.36
0.30
BW 9178
1.64
1.72
1.96
2.18
1.88
0.24
0.3
0.34
0.38
0.32
BW 9183
1.86
2.36
2.56
2.92
2.43
0.25
0.28
0.34
0.36
0.31
BW 8989
1.82
2.24
2.65
2.83
2.39
0.22
0.23
0.25
0.29
0.25
BW 9022
1.82
2.30
2.48
2.86
2.37
0.22
0.25
0.28
0.31
0.27
PBW 343
1.54
2.22
2.82
3.14
2.43
0.25
0.26
0.28
0.35
0.29
PBW 550
1.72
1.94
2.58
2.92
2.29
0.25
0.35
0.35
0.39
0.34
Mean
2.06
2.36
2.73
3.02
2.54
0.25
0.30
0.33
0.37
0.31
Genotypes
66
CD (5%)
A- 0.563, B- 0.979, AB- 1.107
A- N doses, B-Genotypes, AB-Interaction
A- 0.024, B- 0.038, AB- 0.066
66
67
Table 20: Effect of different doses of nitrogen on nitrogen content (%) in wheat grains and straw samples during year 2010-11
Nitrogen content (%)
Grains
Straw
N Doses
RDNRDNRDN
RDN+
Mean
RDNRDNRDN
RDN+
50%
25%
50%
25%
25%
25%
Genotypes
PBW 621
3.47
3.58
3.66
3.72
3.61
0.22
0.25
0.30
0.35
PBW 636
2.00
2.52
2.70
3.52
2.68
0.32
0.45
0.46
0.53
PBW 590
2.08
2.56
2.60
3.12
2.59
0.22
0.27
0.34
0.45
DBW 17
2.26
2.58
2.80
2.92
2.64
0.28
0.30
0.29
0.36
HD 2967
2.10
2.46
2.60
2.86
2.51
0.24
0.31
0.38
0.45
PBW 509
2.14
2.42
2.56
2.70
2.46
0.22
0.25
0.31
0.30
BW 9178
2.05
2.22
2.60
2.86
2.43
0.27
0.26
0.29
0.36
BW 9183
1.98
2.18
2.40
2.74
2.33
0.22
0.23
0.28
0.26
BW 8989
1.80
2.10
2.54
2.64
2.27
0.20
0.24
0.29
0.29
BW 9022
1.98
2.22
2.20
2.70
2.28
0.20
0.21
0.25
0.27
PBW 343
1.92
2.10
3.54
2.62
2.55
0.20
0.22
0.26
0.32
PBW 550
1.86
2.22
2.70
2.84
2.41
0.27
0.32
0.34
0.37
GLU 1101
1.90
2.28
2.36
3.44
2.50
0.20
0.25
0.27
0.36
GLU 1356
3.30
3.24
3.38
3.96
3.47
0.29
0.35
0.36
0.41
GLU 2001
2.38
2.96
3.74
3.81
3.22
0.23
0.24
0.33
0.41
GLU 700
1.96
2.42
2.68
3.02
2.52
0.18
0.16
0.47
0.45
PH132-4836
2.04
2.22
3.04
3.20
2.63
0.19
0.28
0.36
0.43
PH132-4840
2.04
2.16
2.68
3.04
2.48
0.23
0.20
0.28
0.41
Mean
2.18
2.47
2.82
3.10
2.64
0.23
0.27
0.33
0.38
CD (5%)
A- 0.052, B- 0.114, AB- 0.225
A- 0.018, B- 0.036, AB- 0.066
A- N doses, B-Genotypes, AB-Interaction
67
Mean
0.28
0.44
0.32
0.31
0.35
0.27
0.29
0.25
0.26
0.23
0.25
0.33
0.27
0.35
0.31
0.31
0.31
0.28
0.30
enzymes involved in carbon metabolism and N assimilation (Below et al 2000). According to
Gallais and Coque (2005) some grain proteins like zein and globulin also play the role of sink
for N.
4.1.3b Amino acids and soluble proteins
The end products of the assimilation of NO3− by the plants are amino acids and
proteins (Sanchez et al 2004). Amino acids in grains decreased by 43% and 18% with
application of lower doses of N i.e. RDN-50% and RDN-25%, respectively as compared to
optimum dose. Application of increasing levels of N led to an increased content of amino
acid. Significant variation was observed in N levels and genotypes. The interaction of N
levels and genotypes was non-significant. Amino acid range varied from 0.83 to 1.23 mg/g
(Table 21). Amino acids vary with respect to genotypes but were comparatively higher in
genotypes with Gpc-B1 gene and lower in PBW 343 as compared to other genotypes. During
grain filling, an increased trend of newly synthesized amino acids was found in the grains. As
reported by Souza et al (1999), increase in N fertilization resulted in enhanced amino acid
content.
Soluble proteins decreased by 24% and 12% at RDN-50% and RDN-25%,
respectively compared to optimum dose and were significantly affected by various N levels
and genotypes. Interaction between N levels and genotypes was significant. Soluble proteins
in the mature grains was found to vary in the range of 101.1 to 135.1 mg/g. GLU 1356 had
highest protein content at RDN-50% (126.9 mg/g) and RDN-25% (136.2 mg/g) (Table 21).
Higher amino acid and protein content in lines with Gpc-B1 gene at lower doses confirmed
the presence of this gene which is responsible for better remobilization of N and amino acids
from leaves resulting in higher grain protein content. PBW 621 showed highest protein
content at RDN while GLU 2001 at RDN+25%. N fertilization was found to result in
enhanced protein content and yield/plant as reported by Farooq et al (2012).
Nitrogenous compounds are carried from vegetative organs to grains in the form of
amino acids and synthesized into proteins within grains (Zheng-xun et al 2007). The total N,
amino acid and protein concentrations decreased with ageing. This trend was attributed to the
N dilution process as already described for many plant species (Plenet and Lemaire 2000).
The large N availability causes higher build up of free amino-N, but this N is only assimilated
when energy is spent in its reduction and assimilation through the enzymes of N metabolism
(Santos et al 2007).
4.1.3c Sugars and starch
Carbohydrate distribution within plant is also affected by N supply which strongly
influences the processes of carbon assimilation, allocation and partitioning. There was 34%
and 12% decrease in total sugars at RDN-50% and RDN-25%, respectively compared to
optimum dose. Total sugars were significantly affected by various N levels and genotypes and
68
Table 21: Effect of different doses of nitrogen on amino acids and soluble proteins (mg/g) in mature wheat grains during year 2010-11
Amino acids (mg/g)
N Doses
Soluble proteins (mg/g)
RDN50%
RDN25%
RDN
RDN+
25%
Mean
RDN50%
RDN25%
RDN
RDN+
25%
Mean
0.67
0.41
0.58
0.39
0.40
0.29
0.40
0.38
0.39
0.25
0.29
0.25
0.38
0.53
0.52
0.48
0.59
0.55
0.47
0.68
0.54
0.58
0.59
0.59
0.70
0.78
0.64
0.61
0.50
0.55
0.68
0.60
0.99
0.59
0.51
0.63
0.82
0.68
1.11
0.98
1.03
0.80
0.79
0.75
0.68
0.69
0.72
0.63
0.72
0.63
0.20
0.98
0.86
0.68
0.86
1.11
0.83
2.45
2.36
2.23
1.92
2.31
2.4
2.04
2.13
1.89
2.14
1.93
2.05
2.15
2.14
2.50
2.01
2.06
2.39
2.17
1.23
1.07
1.11
0.93
1.02
1.04
0.98
0.96
0.90
0.88
0.87
0.90
0.83
1.16
1.12
0.92
1.04
1.22
1.04
106.2
118.4
102.7
94.5
104.2
101.2
89.1
103.0
93.1
91.9
105.7
92.5
106.0
126.9
109.5
105.7
101.3
96.5
103.2
114.2
120.3
110.6
135.7
110.8
105.4
90.8
103.7
96.6
106.2
98.4
101.5
110.0
136.2
107.8
119.2
113.5
119.3
111.7
139.3
118.7
122.3
111.4
118.7
114.0
106.7
106.7
116.1
110.6
114.7
101.5
126.1
136.5
120.0
122.2
116.5
123.5
120.3
139.4
122.2
126.4
112.6
112.8
136.4
121.3
116.7
121.0
111.3
120.2
108.9
126.8
140.9
141.8
124.3
136.1
125.3
126.4
124.8
119.9
115.5
113.6
111.6
114.3
102.0
107.5
106.7
105.0
109.8
101.1
117.2
135.1
119.8
117.9
116.9
116.2
115.4
Genotypes
69
PBW 621
PBW 636
PBW 590
DBW 17
HD 2967
PBW 509
BW 9178
BW 9183
BW 8989
BW 9022
PBW 343
PBW 550
GLU 1101
GLU 1356
GLU 2001
GLU 700
PH132-4836
PH132-4840
Mean
CD (5%)
A- 2.045, B- 4.337, AB- 8.670
A- N doses, B-Genotypes, AB-Interaction
A- 0.140, B- 0.306, AB- NS
69
their interaction was significant. Sugars ranged from 13.8 to 19.3 mg/g in mature grains and
highest content was found in genotype BW 9178 (18.6 mg/g) at RDN-50% (Table 22). Higher
sugar and starch content reflects adequate partitioning of assimilates to the sink leading to
higher grain yield.
Starch content decreased by 34% and 9% with the application of lower doses of N i.e.
RDN-50% and RDN-25%, respectively as compared to optimum dose. Starch content was
significantly affected by various N levels and genotypes and their interaction was significant.
Highest starch content was recorded in BW 8989 at RDN-50% (63.98%) and RDN25%(66.36%). BW 9022 and PBW 343 revealed highest starch content at RDN and
RDN+25% (Table 22). These genotypes maintained higher sugar and starch content while
protein content was low as compared to other genotypes thus showing their antagonist effect.
Earlier studies showed that there is slight increase in sugar content as N rate was increased. N
fertilizer led to an increase in the level of soluble carbohydrate in sweet sorghum (Almodares
et al 2008).
4.2 PHYSIOLOGICAL STUDIES ON FIELD RAISED CROP UNDER DIFFERENT
NITROGEN LEVELS
4.2.1 Genotypic Variation With Respect to Yield Attributes and Nitrogen Use Efficiency
4.2.1a Yield
Grain yield is the cumulative effect of many components and is significantly
influenced by N application (Singh et al 2000, Sial et al 2005). An increase in grain yield and
its attributes supplied with higher doses of N was observed (Fig 11). During both years,
interaction of various N levels and genotypes was significant for yield and its attributes. The
variation in grain yield due to different levels of N was related to the differences in size of
photosynthetic surface and to the relative efficiency of total sink activity. During year 200910, 39% and 8% decrease in yield was observed with the application of lower doses of N i.e.
RDN-50% and RDN-25%, respectively compared to RDN. Yield was in the range of 0.293 to
0.545 Kg/sqm (Table 23) during 2009-10 and almost similar range was observed in the
second year also. Ample nutrient supply resulted in enhanced growth and production of more
reproductive structures per plant thereby increasing overall yield of the crop (Lawlor 2002,
Valerol et al 2005).
During first year (2009-10), highest yield was recorded in genotype PBW 621 at
RDN-50% (0.380 Kg/sqm), RDN-25% (0.590 Kg/sqm) and RDN (0.600 Kg/sqm) while at
RDN+50% genotype PBW 636 gave maximum yield (0.630 Kg/sqm). Genotype BW 8989
gave very low yield (0.293 Kg/sqm) during first year of study. At a given level of N,
differences in yield means that there were differences in NUE among different genotypes.
During second year (2010-11), there was 20% and 5% decrease in yield with the application
70
Table 22: Effect of different doses of nitrogen on sugars (mg/g) and starch (%) content in mature wheat grains during year 2010-11
N Doses
Genotypes
PBW 621
PBW 636
PBW 590
DBW 17
HD 2967
PBW 509
BW 9178
BW 9183
BW 8989
71
BW 9022
PBW 343
PBW 550
GLU 1101
GLU 1356
GLU 2001
GLU 700
PH132-4836
PH132-4840
RDN-50%
RDN-25%
13.7
13.8
14.9
13.5
14.3
14.1
18.6
15.9
16.1
15.8
18.4
14.6
14.8
13.7
14.4
13.6
14.5
14.0
14.9
14.5
14.3
15.2
15.1
15.6
15.6
18.8
17.8
18.5
16.9
19.3
17.9
15.1
14.0
14.8
13.8
14.5
14.2
15.9
Sugars (mg/g)
RDN
RDN+ 25%
Mean
CD (5%)
A- 0.390, B- 0.836, AB- 1.668
A- N doses, B-Genotypes, AB-Interaction
14.6
14.6
15.7
15.4
16.6
15.9
19.1
18.1
19.2
19.1
19.5
18.2
15.4
14.4
15.4
13.8
15.0
14.6
16.4
14.7
15.2
15.8
15.5
16.8
16.4
19.6
18.9
19.8
19.3
20.0
19.5
15.6
14.6
15.6
14.1
15.4
14.7
16.8
Mean
RDN-50%
RDN-25%
Starch (%)
RDN
RDN+ 25%
Mean
14.4
14.5
15.4
14.9
15.8
15.5
19.0
17.7
18.4
17.8
19.3
17.5
15.2
14.2
15.1
13.8
14.8
14.4
16.0
61.10
61.92
62.50
62.95
63.00
63.28
63.76
63.88
63.98
63.84
63.96
63.16
62.60
61.27
61.90
62.74
62.23
62.44
62.81
63.94
64.50
64.59
64.79
65.13
65.16
65.70
65.67
66.36
65.60
65.85
66.30
64.58
64.08
64.20
64.90
64.85
65.10
65.07
66.50
66.70
66.48
67.30
67.28
68.07
67.88
68.30
68.34
68.90
68.36
68.28
66.80
66.24
66.58
67.39
67.32
67.04
67.43
69.15
69.40
69.72
69.88
70.60
71.62
72.50
72.35
73.26
72.10
73.64
71.90
71.40
69.20
69.76
70.00
69.30
71.20
70.94
65.17
65.63
65.82
66.23
66.50
67.03
67.46
67.55
67.99
67.61
67.95
67.41
66.35
65.20
65.61
66.26
65.93
66.45
66.56
A- 0.412, B- 0.883, AB- 1.766
71
of RDN-50% and RDN - 25%, respectively as compared to optimum N dose. Highest yield
was recorded in PBW 621 at all doses of N (0.390, 0.620, 0.650 and 0.660 Kg/sqm at RDN50%, RDN-25%, RDN and RDN+25%, respectively) and it was low in genotypes with GpcB1 gene (Table 24). This study confirmed that recently released cultivar PBW 621 is high
yielding and is also efficient in metabolizing N. Yielding ability including chlorophyll
content, photosynthesis and starch content are some of the most important quantitative
parameters foro detecting grain yield (Sahoo and Guru 1998). Previous studies have shown
that the supply of N facilitates the utilization of carbohydrates by the grains thus influencing
grain yield (Below et al 2000). Total grain proteins generally increased with increasing N rate
(Halvorson et al 2004). Genetic variance for NUE has been observed both at low and high N
fertilization levels (Gallais and Coque 2005). The enhancement of spikelet number and seed
yield/plant can be explained on the basis of the fact that under optimal N nutrition CO2
assimilation is favorably up regulated (Shah 2008).
4.2.1b Thousand grain weight
Thousand grain weight is an important parameter contributing towards yield as it
plays decisive role in presenting the potential of a genotype. The low N supply decreases
grain weight due to less supply of the grain with carbohydrates and amino compounds during
the lag phase when the number of storage cells and starch granules are being formed as
reported by Paponov et al (2005). During year 2009-10, thousand grain weight was in the
range of 25.57 to 33.40 g and the maximum thousand grain weight of 33.39 g (RDN-50%)
and 33.50 g (RDN-25%) was recorded in BW 9022 as depicted in table 23. During year 201011, higher grain weight was observed in comparision to previous year and it ranged from
36.27 to 40.28 g. BW 9022 showed maximum grain weight of 37.89 g (RDN-50%) and 39.78
g (RDN-25%) and GLU 1356 also showed similar value of 37.89 g at RDN-50% as shown in
table 24. Reports in the literature indicate that thousand grain weight increases with increasing
dose of N in wheat (Gouis et al 2000, Guarda et al 2004) and corn (Hokamlipour et al 2010).
4.2.1c Biomass
Biomass is one of the important parameters reflecting the growth of the crop. During
year 2009-10, decrease in biomass was 28% with the application of RDN-50% while there
was slight increase at RDN-25% as compared to optimum dose (RDN). Biomass varied in the
range of 1.18 to 1.92 Kg/plant (Table 23). Maximum biomass was recorded in genotype PBW
621 at RDN-50% (1.20 Kg/plant) and in genotype PBW 636 at RDN-25%, RDN and
RDN+25%.
During year 2010-11, compared to optimum N dose the average decrease in biomass
at RDN-50% and RDN-25% was 19% and 7%, respectively. Variation in biomass was in the
range of 1.25 to 1.77 Kg/plant (Table 24). Maximum biomass was found in genotype GLU
1356 at RDN-50% (1.67 Kg/plant) and genotype PBW 636 at RDN-25% (1.73 Kg/plant).
72
Similar to our observations biomass increased significantly with increasing N fertilizer
level as reported in corn and sweet sorghum indicated that by (Almodares et al 2009). Results
indicated that decrease in biomass was proportional with the decrease in sub optimal
dose of N.
Fig. 11 Effect of different doses of nitrogen (RDN-25%, RDN-50%, RDN and
RDN+25%) on yield, thousand grain weight, biomass, plant height, tiller
number and spikelet number during years 2009-10 and 2010-11
4.2.1d Plant height
Plant height is an important index of growth and development which is an important
parameter for yield and its attributes. During years 2009-10 and 2010-11, there was almost
similar decrease in plant height at RDN-50% and RDN-25%, respectively. During year 200910, plant height varied from 74 to 94 cm (Table 25) and the tallest genotypes were HD 2967
(85 cm) and BW 9183 (85 cm). Significant increase in plant height and thousand grain weight
with the application of N were mainly responsible for improvement in grain yield. Singh et al
(2003) have also observed a significant increase in grain yield at high N levels.
During year 2010-11, plant height varied from 80 to 93 cm (Table 26). GLU 700 was
the tallest among all genotypes. Growth and yield attributes of sweet sorghum showed
marked improvement with successive increase in N level (Miri et al 2012) as also reported by
other researchers (Tripathi et al 2004, Hossain et al 2004 and Sukartono et al 2004).
Increased grain yield due to N application could be ascribed to increased biomass production,
improved harvest index and increased seed set with N fertilization. Positive effect of N on
73
Table 23: Effect of different doses of nitrogen on yield (Kg/sqm), thousand grain weight (g) and biomass (Kg/plant) during year 2009-10
Yield (Kg/sqm)
N Doses
Thousand grain weight (g)
Biomass (Kg/plant)
74
RDN50%
RDN25%
RDN
RDN+
25%
Mean
RDN50%
RDN25%
RDN
RDN+
25%
Mean
RDN50%
RDN25%
RDN
RDN+
25%
Mean
Genotypes
PBW 621
0.380
0.590
0.600
0.610
0.545
27.72
28.50
29.89
28.89
28.75
1.20
1.45
1.70
2.10
1.61
PBW 636
0.330
0.520
0.540
0.630
0.505
28.06
28.56
29.50
30.22
29.09
1.17
1.85
2.05
2.60
1.92
PBW 590
0.330
0.430
0.450
0.460
0.418
29.89
31.39
32.89
33.94
32.03
1.10
1.30
1.40
1.55
1.34
DBW 17
0.250
0.400
0.460
0.470
0.395
27.33
27.83
29.61
29.67
28.61
1.10
1.10
1.35
1.70
1.31
HD 2967
0.270
0.420
0.430
0.450
0.393
30.33
33.11
33.67
34.00
32.78
0.83
1.35
1.35
2.00
1.38
PBW 509
0.280
0.320
0.460
0.490
0.388
31.39
32.22
32.56
34.28
32.61
1.10
1.33
1.50
1.70
1.41
BW 9178
0.200
0.380
0.370
0.480
0.358
28.89
29.50
30.61
32.67
30.42
0.80
1.40
1.45
1.80
1.36
BW 9183
0.220
0.320
0.350
0.400
0.323
29.89
29.94
30.06
30.89
30.20
1.13
1.85
1.90
2.10
1.75
BW 8989
0.190
0.290
0.320
0.370
0.293
31.94
32.83
33.22
35.61
33.40
0.90
1.00
1.10
1.70
1.18
BW 9022
0.200
0.350
0.380
0.410
0.335
33.39
33.50
34.22
34.44
33.89
0.97
1.25
1.75
2.00
1.49
PBW 343
0.200
0.310
0.340
0.420
0.318
23.56
25.89
26.33
26.50
25.57
1.07
1.20
1.50
1.50
1.32
PBW 550
0.240
0.380
0.400
0.450
0.368
28.89
30.94
32.22
32.44
31.12
1.13
1.70
1.70
2.20
1.68
Mean
0.258
0.393
0.425
0.470
0.386
29.27
30.35
31.23
31.96
30.70
1.04
1.40
1.56
1.91
1.48
CD (5%)
A- 0.038, B- 0.066, AB- 0.105
A- N doses, B-Genotypes, AB-Interaction
A- 0.014, B- 0.027, AB- 0.083
74
A- 0.149, B- 0.242, AB- 0.478
Table 24: Effect of different doses of nitrogen on yield (Kg/sqm), thousand grain weight (g) and biomass (Kg/plant) during year 2010-11
N Doses
75
Genotypes
PBW 621
PBW 636
PBW 590
DBW 17
HD 2967
PBW 509
BW 9178
BW 9183
BW 8989
BW 9022
PBW 343
PBW 550
GLU 1101
GLU 1356
GLU 2001
GLU 700
PH132-4836
PH132-4840
RDN50%
RDN25%
0.390
0.370
0.310
0.310
0.320
0.200
0.190
0.210
0.210
0.180
0.210
0.190
0.210
0.260
0.230
0.200
0.250
0.230
0.248
0.620
0.540
0.440
0.430
0.400
0.280
0.370
0.340
0.330
0.370
0.340
0.410
0.330
0.310
0.350
0.270
0.340
0.310
0.376
Yield (Kg/sqm)
RDN
RDN+
25%
0.650
0.550
0.460
0.480
0.470
0.500
0.410
0.370
0.350
0.370
0.370
0.430
0.380
0.360
0.320
0.310
0.370
0.360
0.417
Mean
CD (5%)
A- 0.050, B- 0.126, AB- 0.203
A- N doses, B-Genotypes, AB-Interaction
0.660
0.640
0.480
0.500
0.510
0.530
0.490
0.420
0.390
0.400
0.460
0.480
0.420
0.390
0.370
0.390
0.410
0.430
0.465
Mean
RDN50%
Thousand grain weight (g)
RDNRDN
RDN+
25%
25%
Mean
RDN50%
Biomass (Kg/plant)
RDNRDN
RDN+
25%
25%
Mean
0.580
0.525
0.423
0.430
0.425
0.378
0.365
0.335
0.320
0.330
0.345
0.378
0.335
0.330
0.318
0.293
0.343
0.333
0.377
37.56
37.67
36.68
36.11
36.67
36.67
36.56
37.11
37.79
37.89
35.11
36.67
35.56
37.89
35.00
33.78
36.56
37.00
36.57
38.11
38.33
37.67
38.00
38.79
38.33
37.82
39.00
38.11
39.78
37.11
36.89
37.11
38.67
37.77
35.89
36.93
37.33
37.87
38.61
38.57
38.20
39.01
39.57
38.16
38.46
39.24
37.89
40.28
37.45
37.94
37.22
39.09
37.53
36.27
37.35
38.56
38.30
1.33
1.40
1.23
1.17
1.33
1.43
1.53
1.20
1.13
1.53
1.50
1.30
1.07
1.67
1.20
1.30
1.03
1.13
1.30
1.43
1.73
1.57
1.70
1.53
1.53
1.67
1.60
1.40
1.53
1.53
1.43
1.27
1.70
1.50
1.37
1.43
1.17
1.51
1.58
1.77
1.53
1.67
1.60
1.54
1.76
1.59
1.47
1.62
1.67
1.48
1.35
1.74
1.56
1.42
1.41
1.25
1.56
38.10
39.22
38.56
38.91
40.03
38.44
38.00
39.22
38.11
41.44
38.44
39.10
37.22
38.78
38.22
37.30
37.89
38.92
38.66
A- 0.980, B- 2.032, AB- 2.863
75
40.67
39.06
39.89
43.00
41.78
39.18
41.44
41.64
37.56
43.00
39.12
39.11
39.00
41.00
39.11
38.11
38.00
41.00
40.09
1.70
1.87
1.60
1.73
1.60
1.57
1.90
1.70
1.50
1.60
1.60
1.57
1.40
1.77
1.67
1.50
1.57
1.27
1.62
A- 0.152, B- 0.274, AB- 0.525
1.87
2.07
1.73
2.07
1.93
1.63
1.93
1.87
1.83
1.80
2.03
1.63
1.67
1.83
1.87
1.50
1.60
1.43
1.79
grain yield and yield attributes of sweet sorghum was reported by Hugar et al (2010).
4.2.1e Tiller number and Spikelet number
During year 2009-10 and 2010-11, an average of 17% and 4% decrease in tiller
number for RDN-50% and RDN-25, respectively was observed. The increase in number of
fertile tillers with the increasing levels of N can be attributed to the reduction in mortality of
tillers and enabling the production of more tillers from the main stem (Warraich et al 2002).
During year 2009-10, tiller number was found to lie in the range of 70 to 91 (Table 25). Tiller
number was highest in BW 9183 at RDN-50% (86) and at RDN-25% (92), respectively.
During year 2010-11, tiller number was highest in BW 9178 at RDN-50% (86) and at RDN25% (92), respectively (Table 26).
There was marginal decrease in spikelet number at RDN-50% and RDN-25%,
respectively. In the year 2009-10, maximum spikelet number was recorded in BW 9183 (20)
and BW 9022 (20) at RDN-50% and in BW 9183 (21) at RDN-25% as shown in table 25.
During year 2010-11, maximum spikelet number was recorded in PBW 621 (17) and GLU
1356 (17) at RDN-50% and in PBW 636 and GLU 1356 (18) at RDN-25% (Table 26).
Previous results showed that all studied traits except NUE significantly increased
with increasing N fertilization rates. The increasing level of N was more effective in
improving yield traits. Application of 50, 75 and 100 kg N /fed increased grain yield over the
25 kg N / fed by 38.6, 50.9 and 58.7%, respectively (El-Gizawy 2005). This may be due to
the maximum expression of important yield attributes like number of tillers per plant,
spikelets per spike, grains per spike and 1000-grain weight.
The favorable effect of N fertilization on plant height and number of spikes/m2 which
are associated with number of tillers/plant may explain the role of N in stimulating cell
division, elongation and development. However, the role of N in encouraging metabolic
processes in wheat plants, consequently their growth, spike initiation and grain filling is
responsible for the increase of spike length, number of spikelets and grains/spike, thousandgrain weight and ultimately grain yield/fed. Similar results were reported by Sawires (2000)
and El-Gizawy (2005) in wheat.
4.2.1f Nitrogen use efficiency (NUE)
During both years (2009-10 and 2010-11) of study, genotypic variation for NUE was
observed. NUE was based on yield performance i.e. on grain yield per N input. NUE
decreased with increasing dose of N during both years. A decrease in NUE with increasing
fertilizer rates is due to less increase in grain yield in comparison to N supply as observed by
Lopez-Bellido and Lopez-Bellido (2001) in wheat. Similar results were also reported by
Limon-Ortega et al (2000), Camara et al (2003) and Staggenborg et al (2003) in wheat and
Zhao et al (2006) in sorghum. Application of high dose of N resulted in poor uptake of N and
low NUE due to excessive N losses which in result decreases N utilization efficiency (grain
76
Table 25: Effect of different doses of nitrogen on on plant height (cm), tiller number (per m row length) and spikelet number during year 2009-10
Plant height (cm)
N Doses
Tiller number (per m row length)
Spikelet number
RDN50%
RDN25%
RDN
RDN+
25%
Mean
RDN50%
RDN25%
RDN
RDN+
25%
Mean
RDN50%
RDN25%
RDN
RDN+
25%
Mean
PBW 621
85
88
92
101
92
81
84
86
92
86
19
20
20
21
20
PBW 636
81
82
88
90
85
78
85
95
101
90
18
19
19
20
19
PBW 590
77
83
87
89
84
57
68
79
82
72
17
18
18
19
18
DBW 17
75
83
89
89
84
66
66
70
76
70
19
19
19
19
19
HD 2967
85
90
96
100
93
55
75
84
88
76
19
20
20
21
20
PBW 509
75
83
85
88
83
70
75
75
86
77
18
19
19
19
19
BW 9178
81
88
88
90
87
58
81
96
98
83
17
19
20
20
19
BW 9183
85
91
96
97
92
86
92
93
93
91
20
21
21
21
21
BW 8989
72
73
74
75
74
68
69
71
92
75
19
20
20
20
20
BW 9022
81
88
88
89
87
65
82
95
100
86
20
20
21
21
21
PBW 343
83
96
97
98
94
75
76
78
79
77
18
18
19
19
19
PBW 550
76
76
79
85
79
77
79
79
86
80
18
19
19
20
19
80
85
88
91
Mean
CD (5%)
A- 2.617, B- 4.529, AB- 6.752
A- N doses, B-Genotypes, AB-Interaction
86
70
78
83
89
80
19
19
20
20
19
Genotypes
77
A- 6.306, B- 10.92, AB- 15.21
77
A- 1.059, B- 1.194, AB- 1.287
Table 26: Effect of different doses of nitrogen on plant height (cm), tiller number (per m row length) and spikelet number during year 2010-11
N Doses
Genotypes
PBW 621
PBW 636
PBW 590
DBW 17
HD 2967
PBW 509
BW 9178
78
BW 9183
BW 8989
BW 9022
PBW 343
PBW 550
GLU 1101
GLU 1356
GLU 2001
GLU 700
PH132-4836
PH132-4840
RDN50%
83
83
79
78
86
79
82
78
80
80
81
77
85
84
80
86
81
85
82
Plant height (cm)
RDN- RDN
RDN+
25%
25%
92
84
81
83
90
88
86
92
80
87
93
78
82
87
84
87
85
81
86
95
90
81
85
96
88
86
84
81
85
95
79
91
83
80
96
88
86
87
100
86
86
86
101
89
90
97
83
95
98
88
90
88
87
98
89
88
91
Mean
CD (5%)
A- 3.797, B- 5.280, AB- 7.544
A- N doses, B-Genotypes, AB-Interaction
Mean
92
86
82
83
93
86
86
88
81
87
92
80
87
86
83
92
86
85
86
Tiller number (per m row length)
RDN- RDNRDN
RDN+
Mean
50%
25%
25%
81
81
65
71
68
65
86
52
63
72
68
64
57
80
50
62
69
55
67
83
82
82
97
85
87
92
81
63
82
75
73
83
80
93
71
84
76
82
91
103
89
85
92
91
95
93
86
74
92
79
86
100
94
97
94
77
90
A- 6.550, B- 11.17, AB- 16.85
78
98
103
89
114
93
102
98
105
97
89
95
87
102
109
105
102
99
82
98
88
92
81
92
85
86
93
83
77
79
83
76
82
92
86
83
87
73
84
RDN50%
14
17
13
16
16
16
14
16
16
16
16
14
15
17
16
15
15
16
15
Spikelet number
RDN- RDN
RDN+
25%
25%
18
17
17
17
16
16
16
16
17
16
16
16
15
18
17
15
16
16
16
18
20
16
17
17
16
16
17
18
16
17
16
17
18
17
16
17
16
17
A- 1.102, B- 1.637, AB- 1.373
18
21
18
19
19
16
16
17
18
17
17
17
18
18
18
19
17
17
18
Mean
17
19
16
17
17
16
16
17
17
16
17
16
16
18
17
16
16
16
17
weight produced per unit plant N).
During year 2009-10, NUE varied from 28.8 to 54.9 Kg Kg-1 as shown in table 27.
Highest NUE was recorded in PBW 621 at both RDN-50% (63.3 Kg Kg-1) and RDN-25%
(65.6 Kg Kg-1). During year 2010-11, similar results were obtained as of first year i.e.
genotype PBW 621 showed highest NUE at RDN-50% (65.0 Kg Kg-1) and at RDN-25% (68.9
Kg Kg-1) (Table 28). Medium low dose (RDN-25%) gave better results than lower-most dose
(RDN-50%) during both years. Our main focus of the study was not to deprive plants for
optimum nutrients but to find N dose upto which N fertilization can be reduced with high
yield and NUE.
Both N capture (uptake) and N conversion (utilization) plays an important role in
improving NUE. It appears that the NUE of barley genotypes grown in field depends on the
level of N supplied (Beatty et al 2010, Rahimizadeh et al 2010). Experiments conducted with
low N supply indicated genetic variation in NUE of maize which was related to N utilization
efficiency while at high N supply genetic variation in NUE was due to N uptake and N
utilization efficiencies. So in order to improve NUE, both N uptake and N utilization need to
be efficient (Moose and Below 2009).
Several studies on different crops have shown genetic variability for NUE at a given
level of N (Bertin and Gallais 2000, Presterl et al 2002). Field studies on barley have shown
differences in the NUE (Sinebo et al 2004, Abeledo et al 2008, Anbessa et al 2009). A
positive link between NUE and GS activity was demonstrated in many crops (Obara et al
2001, Yamaya et al 2002, Gallais and Hirel 2004, Habash et al 2007). GS is critical for
cycling and assimilation of N throughout the growth cycle of plants. GS also plays the most
important role in remobilisation of N from senescing leaves to protein sinks (Good et al 2004,
Hirel et al 2007).
4.2.2 Correlation study and cluster analysis
4.2.2a Correlation study
A pooled correlation analysis was done for various biochemical and physiological
parameters studied at different stages during years 2009-10 and 2010-11. From the correlation
studies we have interpreted the relationship between biochemical parameters and NUE with
grain yield.
During both years of study, NR, NIR and GS activities as well as soluble proteins,
amino acids and N content in leaves at tillering, anthesis and post-anthesis were found to be
positively correlated with NUE, yield and N content in mature grains at 1% and 5% level of
significance, respectively (Table 29 and 30). Amongst various enzymes studied, the activities
of NR and GS showed highest positive correlation with NUE (r=0.857**, r=0.908**,
respectively) and grain yield (r=0.848**, r=0.899**, respectively). This indicated that NR and
GS could serve as marker enzymes associated with NUE. This finding also confirms that in
79
Table 27: Effect of different nitrogen doses on variation in nitrogen use efficiency (NUE) (Kg Kg-1) during year 2009-10
2009-10 Year
N Doses
RDN50%
RDN25%
RDN
RDN+
25%
Mean
63.3
55.0
55.0
41.7
45.0
46.7
33.3
36.7
33.3
33.3
31.7
40.0
42.9
65.6
57.8
47.8
44.4
46.7
35.6
42.2
35.6
34.4
38.9
32.2
42.2
43.6
50.0
45.0
37.5
38.3
35.8
38.3
30.8
29.2
28.3
31.7
26.7
33.3
35.4
40.7
42.0
30.7
31.3
30.0
32.7
32.0
26.7
28.0
27.3
24.7
30.0
31.3
54.9
49.9
42.7
38.9
39.4
38.3
34.6
32.0
31.0
32.8
28.8
36.4
38.3
Genotypes
PBW 621
PBW 636
PBW 590
DBW 17
HD 2967
PBW 509
BW 9178
80
BW 9183
BW 8989
BW 9022
PBW 343
PBW 550
Mean
80
Table 28: Effect of different nitrogen doses on variation in nitrogen use efficiency (NUE) (Kg Kg-1) during year 2010-11
2010-11 Year
N Doses
RDN50%
RDN25%
RDN
RDN+
25%
Mean
65.0
61.7
51.7
51.7
53.3
33.3
31.7
35.0
35.0
30.0
35.0
31.7
35.0
43.3
38.3
33.3
41.7
38.3
41.4
68.9
60.0
48.9
47.8
44.4
31.1
41.1
37.8
37.8
41.1
36.7
45.6
36.7
34.4
38.9
30.0
37.8
34.4
41.9
51.7
45.8
38.3
40.0
39.2
41.7
34.2
30.8
30.8
30.8
29.2
35.8
31.7
30.0
26.7
25.8
30.8
30.0
34.8
43.3
42.7
32.0
33.3
34.0
35.3
32.7
28.0
30.7
26.7
26.0
32.0
28.0
26.0
24.7
26.0
27.3
28.7
31.0
58.0
52.5
42.7
43.2
42.7
35.4
34.9
32.9
33.6
32.2
31.7
36.3
32.8
33.4
32.1
28.8
34.4
32.9
37.3
Genotypes
81
PBW 621
PBW 636
PBW 590
DBW 17
HD 2967
PBW 509
BW 9178
BW 9183
BW 8989
BW 9022
PBW 343
PBW 550
GLU 1101
GLU 1356
GLU 2001
GLU 700
PH132-4836
PH132-4840
Mean
81
Table 29: Correlation coefficients between biochemical and physiological traits during year
2009-10
Parameters (Stages)
NUE
Yield
1000 grain Biomass
wt
%N
(Grains)
%N
(Straw)
(T) 0.599*
0.586*
0.171
0.469
0.592*
0.398
(A) 0.857**
0.848**
0.442
0.255
0.827**
0.447
(P) 0.843**
0.808**
0.011
0.021
0.776**
0.521
(T) 0.474
0.462
0.587
-0.015
0.421
0.423
(A) 0.819**
0.831**
-0.024
0.346
0.799**
0.826**
(P) 0.643*
0.868**
-0.261
0.294
0.782**
0.659*
(T) 0.908**
0.899**
-0.139
0.401
0.890**
0.784**
(A) 0.758**
0.753**
-0.279
0.67
0.742**
0.788**
(P) 0.782**
0.754**
-0.359
0.437
0.757**
0.566
(T) 0.340
0.341
0.173
0.434
0.808**
0.507
(A) 0.337
0.336
0.136
0.083
0.775**
0.431
(P) 0.376
0.350
-0.231
0.171
0.798**
0.659*
0.436
0.512
-0.096
-0.036
0.534
0.364
(A) 0.575
0.570
-0.279
0.273
0.505
0.624*
(P) 0.595*
0.572
-0.138
0.036
0.664*
0.678*
0.893**
0.902**
-0.365
0.626
0.884**
0.611*
(A) 0.860**
0.878**
-0.22
0.678
0.851**
0.550
(P) 0.913**
0.901**
-0.035
0.379
0.808**
0.655*
(T) 0.916**
0.908**
-0.09
0.426
0.928**
0.806**
(A) 0.863**
0.848**
0.193
0.293
0.754**
0.616*
(P) 0.354
0.339
-0.034
0.306
0.136
0.905**
Chlorophyll content (T) 0.391
0.293
-0.311
0.336
0.186
0.505
(A) 0.326
0.335
-0.262
0.211
0.111
0.557
(P) 0.777**
0.769**
-0.194
0.271
0.906**
0.428
(T) 0.811**
0.800**
-0.007
0.472
0.744**
0.735**
(A) 0.968**
0.974**
-0.204
0.55
0.953**
0.670*
(P) 0.774**
0.764**
0.126
0.235
0.684*
0.684*
NR activity
NIR activity
GS activity
GOGAT activity
GDH activity
Soluble proteins
Amino acids
N content (Leaves)
(T)
(T)
** - Significant at 1%, * - Significant at 5%
T – Tillering stage, A – Anthesis stage, P – Post-anthesis stage
82
Table 30:
Correlation coefficients between biochemical and physiological traits during year
2010-11
Parameters (Stages)
NUE
Yield
(T) 0.716**
0.613**
0.201
0.307
0.575*
0.557*
(A) 0.653**
0.644**
0.277
0.336
0.651**
0.694**
(P) 0.632**
0.508*
0.119
0.28
0.569*
0.558*
(T) 0.679**
0.560*
0.396
0.036
0.522*
0.562*
(A) 0.419
0.417
0.328
0.35
0.407
0.677**
(P) 0.383
0.408
0.348
0.345
0.446
0.416
(T) 0.637**
0.653**
0.493*
0.22
0.691**
0.585*
(A) 0.747**
0.661**
0.326
0.204
0.612**
0.617**
(P) 0.637**
0.607**
0.327
0.215
0.555*
0.556*
(T) 0.598**
0.372
0.205
0.262
0.487*
0.776**
(A) 0.389
0.268
0.473*
0.212
0.449
0.623**
(P) 0.318
0.218
0.381
0.399
0.507*
0.645**
(T) 0.412
0.557**
0.096
0.046
0.446
0.470*
(A) 0.458*
0.476*
0.462
0.220
0.572*
0.544*
(P) 0.561
0.399
0.351
0.032
0.714**
0.424
(T) 0.449
0.697**
0.329
0.106
0.828**
0.422
(A) 0.623**
0.827**
0.246
0.393
0.534*
0.482*
(P) 0.370
0.619**
0.439
0.096
0.481*
0.573*
(T) 0.670**
0.728**
0.27
0.202
0.604**
0.714**
(A) 0.540*
0.775**
0.297
0.26
0.587*
0.715**
(P) 0.601**
0.620**
0.393
0.174
0.679**
0.530*
Chlorophyll content (T) 0.378
0.395
0.37
0.165
0.418
0.402
(A) 0.457
0.437
0.347
0.102
0.375
0.383
(P) 0.428
0.37
0.383
0.342
0.459
0.427
N content (Leaves) (T) 0.686**
0.699**
0.357
0.313
0.787**
0.634**
(A) 0.657**
0.664**
0.281
0.219
0.659**
0.681**
(P) 0.380
0.784**
0.46
0.05
0.715**
0.492*
NR activity
NIR activity
GS activity
GOGAT activity
GDH activity
Soluble proteins
Amino acids
1000 grain
wt
Biomass
** - Significant at 1%, * - Significant at 5%
T – Tillering stage, A – Anthesis stage, P – Post-anthesis stage
83
%N
(Grains)
%N
(Straw)
higher plants generally (Oliveria et al 2002) and in cereals particularly (Tabuchi et al 2005)
these enzymes are the main components that contribute through N metabolite assimilation and
translocation to plant biomass production and yield. Since NR is the first enzyme in the Nassimilation process, its correlation is quite obvious. It has also been observed that the
reaction catalysed by GS is one of the main checkpoints controlling the N status of the plant
in cereals such as rice (Obara et al 2001) and maize (Hirel et al 2001, Hirel et al 2005b). The
amount of N taken up after anthesis is tightly correlated to NR activity (Kichey et al 2006). In
our study, NR activity was also found to be correlated to N content in grains.
A positive correlation was observed between NR activity and protein content in
soybean seed (Keresi et al 2008). It has been reported that N content was shown to be
positively correlated with NR activity (Maighany and Ebrahimzadeh 2004, Kichey et al
2007). In wheat and maize, leaf GS activity and several agronomic traits related to yield
showed positive correlation (Habash et al 2007 and Hirel et al 2007). These studies highlight
the importance of cytosolic GS genes in determining several aspects of N use traits in cereal
crops, with potential implications for breeding (Andrews et al 2004, Hirel et al 2007).
Correlation between GDH activity and the physiological traits was not obtained at all
confirming that the enzyme does not play a direct role during N assimilation and recycling as
also observed in earlier studies (Hirel et al 2005b, Kichey et al 2006). Grain number is
usually determined before flowering and thousand grain weight is dependent on carbohydrate
availability rather than N assimilate supply. It is therefore not surprising that we did not find
any correlations between thousand grain weight and biochemical traits.
4.2.2b Cluster analysis
Twelve genotypes during first year (2009-10) and eighteen genotypes during second
year (2010-11) were grouped into clusters on the basis of all studied biochemical parameters
for N metabolism. Cluster tree was generated using NTSYS software (Rohlf 1998). Cluster
analysis of first year showed that 12 genotypes were divided into three clusters (I, II and III)
(Fig 12). In the cluster of this year, PBW 621 and PBW 636 were clustered together. Second
year cluster analysis showed that 18 genotypes were also divided into three clusters (I, II and
III) (Fig 13). During this year, PBW 621 and GLU 1356 were grouped in cluster I. In Cluster
II, PBW 636, PBW 550, DBW 17, HD 2967, PBW 509, PH132-4836, BW 9178, PBW 590,
PH132-4840, GLU 2001 while in cluster III BW 9183, BW 8989, PBW 343, BW 9022, GLU
1101 and GLU 700 were grouped together. Genotypes clustered together showed similar
biochemical behaviour for N metabolism. From cluster analysis of both years, it has been
observed that PBW 621, PBW 636 and GLU 1356 share biochemical similarity.
84
I
II
III
Fig 12. Cluster of 12 wheat genotypes by UPGMA clustering method for year 2009-10
Fig. 12 Cluster of twelve wheat genotypes by UPGMA clustering method for the year 2009-10
I
II
III
Fig 13. Cluster of 18 wheat genotypes by UPGMA clustering method for year 2010-11
Fig. 13 Cluster of eighteen wheat genotypes by UPGMA clustering method for the year 2010-11
85
4.3
LABORATORY
STUDIES
(TILLER
AND
HYDROPONIC
CULTURE
TECHNIQUE) AT DIFFERENT NITROGEN DOSES
4.3.1 Liquid Culture Technique
The effect of different doses of N (glutamine) on the setting and filling of grains was
investigated using detached tillers of wheat grain cultured in liquid media for 5 days. NR and
NIR showed maximum activity at optimum N concentration (17 mM) in developing grains of
PBW 621 and PBW 343 whereas GS, GOGAT and GDH activities increased with increasing
concentration of N (Table 31). Similar increase in enzyme activities with increasing N has
been reported by Hakeem et al (2011). PBW 343 showed higher NR, NIR, GS and GOGAT
activities than PBW 621 while GDH activity was high in PBW 621.
Protein and amino acid concentration also increased with increasing concentration of
N in both the cultivars. PBW 343 maintained higher content of these metabolites at all N
concentrations and responded better to exogenously fed glutamine in the culture medium.
Higher activities of NR and GS in PBW 343 are probably responsible for higher build up of
amino acids and proteins and thus facilitating higher metabolism of N over PBW621. NR and
GS activities has been reported as marker enzymes for NUE in wheat as reported by Kichey et
al (2007). Our results indicated that higher N metabolism at mid-milky stage of grain
development is responsible for assimilating inorganic N efficiently to organic form of N.
The results indicated that N affected the wheat quality traits mainly through the
protein content. However, direct application of N to the detached panicle neutralized the
genotypic differences as both genotypes showed marked differences under field conditions.
Table 31: Effect of different doses of nitrogen on nitrogen metabolism in developing
grains using tiller culture technique
Activities
(μmol product formed or released/min or h/g FW)
Contents
(mg/g FW)
Glutamine (mM) Nitrate
Nitrite Glutamine Glutamate Glutamate Amino Soluble
Genotypes
reductase reductase synthetase synthase dehydro- acids proteins
genase
PBW 621
PBW 343
CD (5%)
5
0.41
0.22
2.37
0.35
1.19
0.32
12.23
17
1.89
0.44
4.07
0.80
1.45
0.59
12.40
25
0.83
0.31
5.00
1.24
1.54
0.84
13.49
5
0.56
0.24
2.87
0.50
0.77
0.45
12.47
17
1.96
0.59
4.90
0.89
0.88
0.65
13.29
25
0.98
0.33
5.40
1.66
1.01
0.90
14.21
A-NS
A-0.069
B-0.031
B-0.071
AB-NS
AB-0.103
A – N doses, B – Genotypes, AB - Interaction
A-0.060
B-0.105
AB-0.187
86
A-0.057
A-NS
B-0.062
B-NS
AB-0.088 AB-NS
A-0.807 A-0.094
B-0.982 B-0.119
AB-1.216 AB-NS
4.3.2 Hydroponic Culture Technique
Hydroponics experiment was carried out on six wheat genotypes (PBW 621, PBW
636, GLU 1356, BW 8989, GLU 700 and PBW 343) to study early stage genotypic
differences and also to analyze the response of different tissues (shoot and root) to different N
doses. Results indicates that in shoots as well as in roots with increasing concentration of N
the enzyme activities (NR and GS) as well as other biochemical parameters (soluble proteins,
amino acids, N content and N uptake) increased in all genotypes (Table 32 and 33).
In the present study, NR and GS activities and soluble proteins, amino acids and N
content were higher in shoots than roots whereas converse was true for N uptake. Similar
results were obtained in Arabidopsis (Molard et al 2008). Results obtained complemented our
earlier observations from field studies where PBW 621, PBW 636 and GLU 1356 were better
adapted to lower doses of N.
NR play an important role in the absorption of NO3-, regulating the levels of NO3- and
amino acids in root cells. Since NO3- induces NR, the rate of NO3- uptake to the site of
induction is the main controlling factor for the level of NR activity (Hakeem et al 2011).
From our results the differences in NR activity in cultivars is possibly connected to
differences in the rate of uptake and accumulation of NO3- ions in the leaves and roots.
Genetically different wheat genotypes differed in the amount of N assimilated during the
entire lifespan of crop (Abrol 1990).
An increase in GS activity was observed with an increasing concentration of N.
Previous studies revealed that a substantial portion of N is not re-translocated to the harvested
structures resulting in low GS activity (Habash et al 2006). The end products of the
assimilation of NO3- by the plants are amino acids and proteins (Sanchez et al 2004). In all the
studied genotypes, there was increase in protein content with increase in the level of N.
Application of increasing levels of N led to an increase in the content of N in rice (Santos et
al 2007). In the present experiment, the amino acid and protein concentrations were higher in
shoots than in roots. This is normal since the N assimilation occurring in the leaves could
explain the predominance of these nitrogenous compounds in this organ.
In conclusion, higher NUE and grain yield in PBW 621 and PBW 636 at sub optimal
doses of N is probably linked to higher activities of NR and GS enzymes while widely grown
cultivar PBW 343 and advanced breeding lines BW 9178, BW 9183, BW 8989 and BW 9022
showed low efficiency for N assimilating enzymes. NR and GS enzymes showed positive
correlation with NUE and yield indicating that these might be the rate limiting steps in N
metabolism. Although 120 kg/ha is recommended dose but considering environmental and
economic issues, it was observed that decrease in N dose to RDN-25% is tolerated by N
efficient genotypes without marked loss in yield but its further reduction to RDN-50% may
cause N starvation. It would be particularly interesting to further investigate the differential N
87
Table 32: Effect of different doses of nitrogen on nitrogen metabolism in wheat shoots
grown hydroponically
Activities (μmol product
formed or released/min or
h/g FW)
Potassium
nitrate
(mM)
Genotype
Contents (mg/g FW)
%
mg/L
Nitrate
reductase
Glutamine
synthetase
Soluble
proteins
Amino
acids
Nitrogen
content
Nitrogen
uptake
0
1.28
5.86
125.3
14.58
0.49
15
2
1.49
7.46
136.2
17.86
0.53
18
6
1.42
8.43
138.2
19.20
0.63
20
0
1.27
5.43
126.5
16.82
0.46
13
2
1.32
6.54
139.4
17.30
0.54
17
6
1.47
5.92
141.8
18.56
0.56
22
0
1.27
5.28
129.6
15.70
0.42
14
2
1.35
6.60
147.6
18.54
0.56
18
6
1.38
8.69
136.8
16.28
0.74
21
0
0.63
2.64
106.2
11.70
0.34
08
2
0.74
2.67
111.0
12.90
0.39
11
6
0.87
3.70
121.6
13.58
0.47
15
0
0.58
2.68
81.0
10.86
0.30
07
2
0.64
2.89
118.0
11.45
0.34
10
6
0.67
4.20
122.0
12.60
0.35
13
0
0.53
2.80
109.4
11.32
0.24
05
2
0.62
3.58
113.7
11.48
0.27
09
6
0.69
4.28
121.0
13.04
0.34
12
A-0.031
A-0.069
B-0.042
B-0.097
AB-0.078
AB-0.159
A – N doses, B – Genotypes, AB – Interaction
A-6.369
B-9.009
AB-11.62
PBW 621
PBW 636
GLU 1356
BW 8989
Glu 700
PBW 343
CD (5%)
88
A-1.026
B- 2.938
AB-4.266
A-0.026
B-0.032
AB-0.066
A-0.097
B-0.060
AB-0.121
Table 33: Effect of different doses of nitrogen on nitrogen metabolism in wheat roots grown
hydroponically
Activities (μmol product
formed or released/min or
h/g FW)
Potassium
nitrate
(mM)
Genotype
PBW 621
PBW 636
GLU 1356
BW 8989
Glu 700
PBW 343
CD (5%)
Contents (mg/g FW)
%
mg/L
Nitrate
reductase
Glutamine
synthetase
Soluble
proteins
Amino
acids
Nitrogen
content
Nitrogen
uptake
0
0.27
2.42
38.0
4.54
0.28
27
2
0.28
3.16
48.2
4.86
0.47
48
6
0.30
2.77
54.6
5.14
0.54
58
0
0.25
2.57
37.6
5.05
0.21
33
2
0.28
2.81
49.0
4.92
0.30
43
6
0.28
2.64
53.0
5.24
0.37
48
0
0.21
2.45
35.8
4.79
0.25
22
2
0.27
2.73
43.9
5.28
0.33
43
6
0.32
3.34
53.0
5.46
0.46
50
0
0.17
1.39
15.0
2.80
0.16
10
2
0.20
1.49
22.0
3.90
0.18
16
6
0.21
1.66
27.6
4.30
0.22
36
0
0.16
1.28
21.0
2.18
0.15
11
2
0.17
1.27
24.8
3.48
0.17
17
6
0.19
1.37
38.6
3.26
0.20
24
0
0.11
1.27
16.0
2.70
0.13
15
2
0.16
1.34
22.0
3.13
0.16
21
6
0.18
1.47
24.0
3.62
0.21
27
A-0.027
A-0.076
B-0.030
B-0.102
AB-0.085
AB-0.177
A – N doses, B – Genotypes, AB - Interaction
A-2.259
B-3.104
AB-3.771
89
A-0.548
B-0.781
AB-1.528
A-0.022
B-0.032
AB-0.069
A-1.632
B-1.728
AB-2.436
responsiveness of contrasting genotypes in terms of complex regulatory network involved in
N uptake, assimilation and remobilization. These N efficient genotypes may be used as donor
stocks in wheat breeding programme.
90
CHAPTER-V
SUMMARY
Nitrogen (N) plays a very important role for growth and development of the plant and
is extensively used to maximize yield. However, its over-use leads to economic loss as well as
several environmental problems. To address these issues, there is need to identify genotypes
with high nitrogen use efficiency (NUE). In this two years study (2009-10 and 2010-11) 18
wheat genotypes (PBW 621, PBW 636, PBW 590, DBW 17, HD 2967, PBW 509, BW 9178,
BW 9183, BW 8989, BW 9022, PBW 343, PBW 550, GLU 1101, GLU 1356, GLU 2001,
GLU 700, PH132-4836, PH132-4840) selected on the basis of their commercial relevance or
distinct genetic background were used for studying N metabolism during plant growth and
development so that NUE of the crop can be maximized. The effect of different doses of N
[RDN-50% (60 kg/ha), RDN-25% (90 kg/ha), RDN (120 kg/ha) and RDN+25% (150 kg/ha)]
on biochemical processes associated with NUE and their correlation was established. A
number of enzymes involved in N assimilation in relation to deposition of amino acids,
proteins and starch were assayed at tillering (30 days after sowing; DAS), anthesis (about 90100 DAS depending on the genotype) and post-anthesis (15 days post anthesis) stages.
Activities of N metabolizing enzymes viz. nitrate reductase (NR), nitrite reductase
(NIR), glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate
dehydrogenase (GDH) as well as amino acids and soluble proteins increased from tillering till
anthesis and thereafter declined at post-anthesis stage in all the genotypes whereas the
contents of chlorophyll and N peaked around tillering stage and declined thereafter. Contents
of amino acids, soluble proteins, N and chlorophyll increased with increase in dose of N.
Grain yield and its attributes namely thousand grain weight, biomass, plant height, tiller
number and spikelet number were reduced significantly at RDN-50% and RDN-25%.
During both years, genotypic variation was observed with respect to N metabolizing
enzymes. As observed, PBW 621 showed high activities of NR (4.52 μmol NO2- formed/h/g
FW at anthesis stage), NIR (0.49 and 0.85 μmol NO2- released/h/g FW at tillering and postanthesis stages, respectively) and GS (15.87 μmol γ-glutamylhydroxamate formed/min/g FW
at anthesis stage) at RDN-25%. At RDN-50%, GS activity was high in PBW 636 (9.0 μmol γglutamylhydroxamate formed/min/g FW) and GLU 1356 (9.0 μmol γ-glutamylhydroxamate
formed/min/g FW) during post-anthesis stage. This is in correspondance with high protein
content in GLU 1356 (2.49 mg/g DW) indicating that GS is heavily involved in control of the
N status in form of protein. HD 2967 and PBW 590 revealed maximum activities of these
enzymes at RDN and RDN+25%. At suboptimal doses of N, PBW 590, HD 2967, PBW636
and DBW 17 had high chlorophyll content exhibiting that prolonged photosynthetic capacity
is beneficial for fixing high amount of carbon. However, lines with Gpc-B1 gene possessed
91
less chlorophyll content indicating that these genotypes senescence early. Interestingly,
widely grown cultivar PBW 343 and advanced breeding lines BW 9178, BW 9183, BW 8989
and BW 9022 exhibited low activity of N assimilating enzymes showing their poor NUE.
In grains of GLU 1356, high protein content (126.9 mg/g and 136.2 mg/g at RDN50% and RDN-25%, respectively) was observed indicating higher translocation of N from the
vegetative organs to sink. Sugar and starch content was high in the PBW 343, BW 9178, BW
8989 and BW 9022 genotypes in which amino acid and protein content was less showing
inverse relation. With decreasing application of N fertilizer, NUE showed an increasing trend
and was higher in PBW 621 (68.9 Kg Kg-1 at RDN-25%) compared to other genotypes. PBW
621 also revealed high grain yield (0.620 Kg/sqm at RDN-25%). Both NR and GS enzymes
showed positive correlation with NUE (r=0.857**, r=0.908**, respectively) and grain yield
(r=0.848**, r=0.899**, respectively). Apparently, these two enzymes can be used as marker
enzymes associated with NUE. Cluster analysis revealed biochemical similarity between
PBW 621, PBW 636 and GLU 1356 genotypes. Through tiller culture technique, the effect of
different doses of N in grains were investigated. It appeared that direct application of N to the
detached panicle did not reveal much differences in the genotypes. However, study on
hydroponically raised seedling showed results parallel to the field studies. It has been
observed that decrease in N dose from RDN to RDN-25% has no considerable effect on
productivity in N efficient genotypes but its further reduction to RDN-50% may cause N
starvation. Due to stable performance of PBW 621, PBW 636 and GLU 356 at suboptimal
doses over two years, these genotypes hold future potential for developing new cultivars with
improved NUE.
92
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