Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. DOI 10.1007/s40011-015-0661-3
RESEARCH ARTICLE
Enzymatic Efficiency and Genotypic Differences for Nitrogen Assimilation in Wheat Balraj Kaur1 • Bavita Asthir1 • Navtej Singh Bains2
Received: 23 February 2015 / Revised: 10 August 2015 / Accepted: 31 August 2015 Ó The National Academy of Sciences, India 2015
Abstract The present study was conducted to evaluate the potential of nitrogen applied in soil (90, 120 and 150 kg/ha) on activities of nitrogen assimilating enzymes viz. nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) in relation to protein, amino acid and nitrogen content with respect to source-sink relationship (flag leaf and grains) at different developmental stages of six wheat genotypes (PBW 621, PBW 636, PBW 343, PBW 550, HD 2967 and GLU 1356). GLU 1356, HD 2967 and PBW 621 genotypes responded better at suboptimal dose of N (90 and 120 kg/ha N), while PBW 636, PBW 343 and PBW 550 at recommended dose of N. Significant increase in the activities of NR, GS, GOGAT, GOT and GPT was noted under 150 kg/ha N application, which caused an increase in protein and amino acid contents in all the genotypes. Activity pattern of studied enzymes revealed an increasing trend from tillering to anthesis stage and thereafter declined in parallel with decrease in protein and amino acid contents. Conversely, nitrogen and chlorophyll content showed a consistent decline with leaf growth. In crux, sub-optimal N doses had significant influence on nitrogen metabolism especially in GLU 1356 due to high activities of GS and GOGAT. GOT and GPT regulated protein and amino acid & Bavita Asthir
[email protected] Balraj Kaur
[email protected] 1
Department of Biochemistry, Punjab Agricultural University, Ludhiana, India
2
Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana, India
biosynthesis. GLU 1356, HD 2967 and PBW 621 were observed as nitrogen efficient genotypes for enhancing wheat crop productivity under low dose of nitrogen to save environment and input cost. Keywords Nitrogen assimilation Wheat Yield Protein Amino acid Abbreviations NR Nitrate reductase GS Glutamine synthetase GOGAT Glutamate synthase NUE Nitrogen use efficiency RDN Recommended dose of N
Introduction Nitrogen (N) is one of the main nutrients responsible for improving crop productivity of cereals. Nitrogen metabolism is a complex process and varies with species [1]. Uptake and assimilation of N encompasses several metabolic steps which include different organelles and organs of the plant. Excess of N applied to the soil is lost to the environment, with an average of only 30–50 % being taken up by the plant depending on the species and cultivar, with the remainder being lost to surface run-off, leaching of nitrate (NO3-), ammonia (NH3) volatilization or bacterial competition [2, 3]. Nitrogen is taken by the roots of plant in the form of nitrate which is then reduced to nitrite by nitrate reductase in cytosol and is transported into chloroplasts, where nitrite is further reduced by the action of nitrite reductase to
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ammonium ions. Ammonia is then assimilated into organic form as glutamine and glutamate by glutamine synthetase and glutamate synthase cycle, which serves as the N donor for the biosynthesis of essentially all amino acids, nucleic acids and other N containing compounds such as chlorophyll [4, 5]. At anthesis, leaves change from ‘sinks’ to ‘sources’ as remobilization ‘loads’ N into the developing grain during grain filling. The developing grains are a strong sink for N, and monopolize the nutrients that are remobilized from senescing parts of the plant, from storage sources and from root uptake [6]. The amino acids derived from leaves are transformed within the grain to provide appropriate types of amino acids in the correct proportions for the synthesis of grain proteins. The accumulation of proteins into endosperm of developing grain depends upon enzymatic activities of aminotransferase such as glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) [7, 8]. The GOT catalyzes the reaction to produce aspartate which is the precursor of aspartate family of amino acids, whereas GPT catalyzes the reaction to produce alanine. The metabolic efficiency exhibited by the steps in nitrogen assimilation pathway outlined above have great relevance for economic as well as ecological aspects of agriculture. These concerns have become serious in recent years in view of sustainability of agricultural systems. The present study investigates efficiency of metabolic steps in N assimilation pathway in the context of a diverse set of six wheat genotypes, three soil N regimes and two plant parts (flag leaf and grains) representative of source and sink tissues. Amino acid and protein content as well as productivity in terms of yield serve as output parameters of the system. The aim of the study is to demarcate key/bottleneck steps in the pathway, evaluate genotypic differences for each step and explore the possibility of efficient N use particularly in low N-input regimes.
Material and Methods Soil Characterization The soil samples were evaluated for fertilizer recommendation from the Soil Testing Laboratory at Punjab Agricultural University, Ludhiana, India. The soil was sandy loam in texture and tested near neutral to pH 7.4, nonsaline, low in organic carbon (0.090 %), available nitrogen (120.4 kg/ha) and medium in Olsen P (38.25 kg/ha) and exchangeable K (405 kg/ha) (at the beginning of experimentation, 2012–2013). Therefore, based on soil test report, 150 kg N/ha was used as RDN. The standard recommendation to the farmers of the region stands at 120 kg N/ha. Plant Material and Nitrogen Treatments Six wheat (Triticum aestivum L.) genotypes viz.: PBW 621, PBW 636, PBW 343, PBW 550, HD 2967 and GLU 1356 were raised in the experimental fields of Department of Plant Breeding and Genetics, PAU, Ludhiana during 2012–2013. Table 1 summaries the important features of six wheat genotypes used for biochemical analysis at different N doses. For each wheat variety, three N treatments were given, namely, sub-optimal N doses (90 and 120 kg N/ha) as well as recommended dose of N (RDN, 150 kg N/ha). Nitrogen was applied as urea in two equal splits: half at the time of sowing and half at the time of first irrigation. The crop was grown in plots of 4 rows of 2 m length each at a row to row spacing of 20 cm (0.80 m 9 2 m) in three replications. The plots supplied with RDN were served as control. Collection of Samples Samples (flag leaf and grains) were collected at 9 am on the day of sampling for various biochemical analyses. Fresh
Table 1 Important features of six wheat genotypes used for the study of nitrogen use efficiency Genotype
Parentage
Important features
PBW 621
KAUZ//ALTAR84/AOS/3 MILAN/KAUZ/4/HUITES
Released variety for timely sown irrigated conditions of north western plains of India
PBW 636
KSWW1/PBW 552
Winter wheat 9 spring wheat derivative
PBW 343
ND/VG9144//KAL/BB/3/YACO’S’/4/Vee#5
Widely sown released variety for timely sown irrigated conditions of north western eastern plains of India
PBW 550
WH594/RAJ3856//W485
Good grain released variety for timely sown irrigated conditions of north western plains of India
HD 2967
ALD/COC//URES/3/HD2160 M/HD2278
Released variety for timely sown irrigated conditions of north western plains of India
GLU 1356
GLUPRO/3*PBW554
Line with high grain protein content conferred by Gpc-B1 gene originally introgressed from Triticum dicoccoides (tetraploid wild wheat)
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Enzymatic Efficiency and Genotypic Differences for Nitrogen Assimilation in Wheat
tissue was used for enzyme assays whereas dried tissue was stored for N, amino acid and soluble protein content analysis. 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, DPA) for flag leaf and at two stages i.e. 15 DPA and 30 DPA for grains. Enzyme Assays The nitrate reductase activity was estimated by the method described by Jaworski [9]. Glutamine synthatase (GS) and glutamate synthase (GOGAT) were extracted by the method of Mohanty and Fletcher [10]. The GS activity was assayed following the method of Kanamori and Matsumoto [11] and GOGAT according to procedure of Bulen [12]. Glutamate oxaloacetate transaminase and glutamate pyruvate transaminase was determined following the procedure of Tonhazy [13, 14]. The procedure for assaying GPT activity was same as described for GOT except that the addition of aniline citrate step was omitted [14]. Extraction and Estimation of Soluble Protein, Amino Acid, Nitrogen Content and Chlorophyll Content Total soluble protein and amino acids were extracted by the method as described by Singh et al. [15]. Proteins were assayed according to the method of Lowry et al. [16] and amino acids were determined employing the method of Lee and Takahashi [17]. Nitrogen content was estimated by the method of McKenzie and Wallace [18]. Chlorophyll content was determined as described by Arnon [19].
Results and Discussion Genotypic variations among crop plants provide a valuable tool in the selection of genotypes with desirable traits as they respond differentially to various conditions [21]. Six genotypes studied include 4 released genotypes spanning a period from 1995 (PBW 343) to 2011 (HD 2967). The two advanced lines add genetic diversity to this set as PBW 636 is a winter wheat 9 spring wheat derivative and GLU 1356 carries a grain protein content enhancing gene Gpc-B1, originally derived from Triticum dicoccoides, a wild variety. The Gpc-B1 gene is known to accelerate remobilization of nutrients from leaves to grain thereby improving protein content [22]. As for the N doses used in the study two suboptimal doses were employed besides RDN. Earlier report using maize as a model plant, revealed a decreasing trend of N uptake and assimilation at leaf ageing and that the decrease was enhanced when plants were N starved [23]. However, reverse trend was observed in crops raised under higher levels of N, which revealed coordinated increase in the activities of all N assimilating enzymes [24]. Further, regulatory bottlenecks seem to become more pronounced as one moves from source to sink in the N assimilation pathway. In wheat, assimilate supply in the form of N is less of a limiting factor and N assimilation depends more on its utilization to grain activity as sink [25]. In this study, the authors elaborated 5 stage assay (three-vegetative/flag leaf based, two- grain based) for full set of enzymes (NR, GS, GOGAT, GOT and GPT) to understand biochemical basis of source sink transition of wheat in relation to their variability. Activity of Nitrogen Metabolizing Enzymes Nitrate Reductase
Yield and Nitrogen Use Efficiency (NUE) Grain yield was determined by recording the seed yield after threshing. The grain yield of each plot was recorded and expressed as t/m2. NUE was calculated by the formula given by Vukovic et al. [20]: NUE ¼ ðYF YC Þ=N applied ðYyield; Ffertilized; CcontrolÞ NUE1 ¼ YC ð150 kg N=haÞ YF ð120 kg N=haÞ=N applied NUE2 ¼ YC ð150 kg N=haÞ YF ð90 kg N=haÞ=N applied
Statistical Analysis All the values reported in the present paper are the means of three replicates (LSD at 5 % level of significance). Data obtained was subjected to analysis of variance for factorial experiments in randomized block design.
In the present study, the range of NR activity in flag leaf at tillering (1.33–2.89 lmol NO2- formed/h/g FW), anthesis (2.58–4.97 lmol NO2- formed/h/g FW) and post-anthesis (1.45–4.65 lmol NO2- formed/h/g FW) stages, indicated maximum activity at anthesis stage (Table 2). On comparison within sub-optimal dose, low activity was found in PBW 636 at tillering and post-anthesis stage i.e. 1.33 lmol NO2- formed/h/g FW and 2.45 lmol NO2- formed/h/g FW respectively, whereas at anthesis stage low activity of NR was observed in PBW 343 (2.58 lmol NO2- formed/h/ g FW) (Table 2). In grains, NR activity ranged from 1.61 to 2.92 lmol NO2- formed/h/g FW at 15 DPA and 1.26 to 2.35 lmol NO2- formed/h/g FW at 30 DPA (Table 3). In the present study, the NR activity maintained high level with the enhancement of nitrogen fertilizer level as reported by Lemaitre et al. [26]. But the higher activity of
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B. Kaur et al. Table 2 Activities of nitrate reductase, glutamine synthetase and glutamate synthase in flag leaf at three developmental stages of six wheat genotypes grown in various nitrogen regimes Tillering stage 90 kg N/ha
Anthesis stage
120 kg N/ha 150 kg N/ha 90 kg N/ha
Post anthesis stage
120 kg N/ha 150 kg N/ha 90 kg N/ha
120 kg N/ha 150 kg N/ha
Nitrate reductase (lmol NO2 formed/h/g FW) PBW 621
2.25 ± 0.01 2.45 ± 0.03
2.49 ± 0.04
2.65 ± 0.02 4.38 ± 0.05
4.41 ± 0.06
2.49 ± 0.02 4.01 ± 0.04
4.21 ± 0.05
PBW 636 PBW 343
1.33 ± 0.06 2.51 ± 0.05 1.69 ± 0.04 2.35 ± 0.05
2.54 ± 0.02 2.45 ± 0.03
2.69 ± 0.04 4.61 ± 0.03 2.58 ± 0.05 3.96 ± 0.05
4.68 ± 0.04 3.99 ± 0.02
2.45 ± 0.03 4.16 ± 0.06 2.53 ± 0.02 3.15 ± 0.06
4.58 ± 0.01 3.68 ± 0.08
PBW 550
1.61 ± 0.03 2.49 ± 0.03
2.53 ± 0.04
2.61 ± 0.05 3.79 ± 0.03
3.81 ± 0.09
2.55 ± 0.05 3.53 ± 0.03
3.71 ± 0.05
HD 2967
2.15 ± 0.07 2.46 ± 0.04
2.55 ± 0.03
2.72 ± 0.05 4.15 ± 0.06
4.92 ± 0.06
2.51 ± 0.08 4.21 ± 0.02
4.67 ± 0.07
GLU 1356 2.48 ± 0.07 2.56 ± 0.04
2.89 ± 0.04
4.63 ± 0.05 4.75 ± 0.06
4.97 ± 0.03
3.85 ± 0.04 4.26 ± 0.04
4.65 ± 0.04
CD (5 %)
A: 0.17 9 10-2, B: 0.12 9 10-2, C: 0.12 9 10-2
Glutamine synthetase (lmol c-glutamylhydroxamate formed/min/g FW) PBW 621
5.78 ± 0.04 5.82 ± 0.05
6.03 ± 0.03
10.5 ± 0.06 10.6 ± 0.05
11.8 ± 0.02
9.79 ± 0.04 9.92 ± 0.03
10.3 ± 0.06
PBW 636
5.48 ± 0.04 5.59 ± 0.06
5.98 ± 0.03
11.3 ± 0.02 11.3 ± 0.04
11.5 ± 0.06
9.65 ± 0.07 9.88 ± 0.02
10.8 ± 0.09
PBW 343
4.12 ± 0.06 4.26 ± 0.05
4.96 ± 0.06
7.96 ± 0.05 8.01 ± 0.03
8.19 ± 0.04
5.64 ± 0.02 5.86 ± 0.02
5.94 ± 0.05
PBW 550
4.65 ± 0.03 4.72 ± 0.05
4.98 ± 0.03
9.65 ± 0.04 9.74 ± 0.07
9.94 ± 0.03
7.05 ± 0.05 7.15 ± 0.08
7.52 ± 0.04
HD 2967
5.16 ± 0.03 5.25 ± 0.07
5.85 ± 0.01
10.4 ± 0.06 10.5 ± 0.09
11.6 ± 0.07
9.88 ± 0.06 9.98 ± 0.07
10.2 ± 0.05
GLU 1356 5.62 ± 0.04 5.71 ± 0.04
6.01 ± 0.05
12.3 ± 0.05 12.4 ± 0.03
13.1 ± 0.07
9.56 ± 0.03 9.69 ± 0.01
10.6 ± 0.04
6.15 ± 0.17 6.06 ± 0.13
5.50 ± 0.08 5.65 ± 0.06 4.02 ± 0.03 4.62 ± 0.02
5.81 ± 0.16 5.01 ± 0.12
CD (5 %)
A: 0.21 9 10-5, B: 0.14 9 10-5, C: 0.15 9 10-5
Glutamate synthase (lmol NADH oxidized/min/g FW) PBW 621 PBW 636
2.40 ± 0.16 2.60 ± 0.05 2.81 ± 0.09 2.84 ± 0.02
2.95 ± 0.15 3.41 ± 0.18
5.56 ± 0.09 5.82 ± 0.07 5.72 ± 0.02 5.96 ± 0.03
PBW 343
1.52 ± 0.09 1.83 ± 0.01
2.72 ± 0.02
4.05 ± 0.08 4.15 ± 0.09
5.09 ± 0.09
3.65 ± 0.06 3.92 ± 0.05
4.25 ± 0.08
PBW 550
1.55 ± 0.02 1.93 ± 0.07
2.40 ± 0.17
4.01 ± 0.06 4.62 ± 0.06
4.96 ± 0.11
3.34 ± 0.08 3.96 ± 0.06
3.98 ± 0.16
HD 2967
1.29 ± 0.13 1.33 ± 0.07
2.20 ± 0.09
5.15 ± 0.01 5.20 ± 0.09
5.86 ± 0.13
4.08 ± 0.02 4.11 ± 0.17
4.99 ± 0.11
GLU 1356 2.53 ± 0.42 2.67 ± 0.13
2.87 ± 0.09
5.58 ± 0.09 6.57 ± 0.17
6.70 ± 0.16
5.06 ± 0.08 5.56 ± 0.19
5.66 ± 0.11
CD (5 %)
A: 0.33 9 10-2, B: 0.23 9 10-2, C: 0.23 9 10-2
Values are mean ± SD of three replications A, genotypes; B, treatments; C, stages
NR in GLU 1356 at low N dose probably can be correlated with higher assimilation and utilization efficiency of N over other genotypes. Reports in the literature reveal that translocation of N in the roots become efficient at lower doses of N due to presence of various carriers or transport systems [27]. Therefore it appears that genotypic variation exists for NR activity and is possibly connected to differences in the rate of uptake and accumulation of NO3- ions in the tissues [28, 29].
was found to be highly efficient for GS activity at anthesis while PBW 621 was more efficient at tillering and PBW 636 showed superiority at post-anthesis (Table 2). In grains, lowest GS activity value was found in PBW 343 (2.82 lmol c-glutamylhydroxamate formed/min/g FW) and highest value in GLU 1356 (9.99 lmol c-glutamylhydroxamate formed/min/g FW) (Table 3).
Glutamine Synthatase
The GOGAT activity varied from 1.29 to 2.95 lmol NADH oxidized/min/g FW at tillering, 4.01 to 6.70 lmol NADH oxidized/min/g FW at anthesis and 3.34 to 5.81 lmol NADH oxidized/min/g FW at post anthesis in flag leaf. Highest activity at 150 kg N/ha dose was found in PBW 636 at tillering, GLU 1356 at anthesis and PBW 621 at post anthesis (Table 2). In grains, lowest value was observed in PBW 550 (1.67 lmol NADH oxidized/min/g FW), highest in GLU 1356 (4.80 lmol NADH oxidized/ min/g FW). These results revealed that GLU 1356 showed
The GS activity increased with increase in N dose but activity at 150 kg N/ha was statistically at par with 120 kg N/ha depicting that 120 kg N/ha gave comparable results to RDN. It ranged from 4.12 to 6.03 lmol c-glutamylhydroxamate formed/min/g FW at tillering, 7.96 to 13.1 lmol c-glutamylhydroxamate formed/min/g FW at anthesis and 5.64 to 10.8 lmol c-glutamylhydroxamate formed/min/g FW at post anthesis in flag leaf. GLU 1356
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Glutamate Synthase
Enzymatic Efficiency and Genotypic Differences for Nitrogen Assimilation in Wheat Table 3 Activities of nitrate reductase, glutamine synthetase and glutamate synthase activities in grains at 15 and 30 days post-anthesis (DPA) of six wheat genotypes grown in various nitrogen regimes 15 DPA 90 kg N/ha
30 DPA 120 kg N/ha
150 kg N/ha
90 kg N/ha
120 kg N/ha
150 kg N/ha
Nitrate reductase (lmol NO2 formed/h/g FW) PBW 621
2.56 ± 0.04
2.61 ± 0.07
2.63 ± 0.04
1.75 ± 0.03
2.00 ± 0.05
2.11 ± 0.04
PBW 636 PBW 343
2.41 ± 0.03 1.86 ± 0.04
2.49 ± 0.09 1.90 ± 0.03
2.50 ± 0.05 1.93 ± 0.07
1.72 ± 0.05 1.26 ± 0.03
2.08 ± 0.04 1.57 ± 0.10
2.29 ± 0.03 1.84 ± 0.03
PBW 550
1.61 ± 0.07
1.63 ± 0.04
1.69 ± 0.04
1.27 ± 0.09
1.51 ± 0.04
1.59 ± 0.10
HD 2967
2.52 ± 0.04
2.56 ± 0.04
2.61 ± 0.08
1.75 ± 0.05
2.10 ± 0.08
2.35 ± 0.09
GLU 1356
2.81 ± 0.05
2.89 ± 0.03
2.92 ± 0.07
1.65 ± 0.06
2.26 ± 0.04
2.32 ± 0.06
CD (5 %)
A: 0.26, B: 0.18, C: NS
Glutamine synthetase (lmol c-glutamylhydroxamate formed/min/g FW) PBW 621
9.72 ± 0.17
9.80 ± 0.16
9.89 ± 0.13
4.89 ± 0.12
4.96 ± 0.17
5.18 ± 0.02
PBW 636
9.68 ± 0.12
9.70 ± 0.15
9.72 ± 0.12
4.82 ± 0.17
4.94 ± 0.19
5.43 ± 0.19
PBW 343
8.61 ± 0.13
8.68 ± 0.19
8.80 ± 0.19
2.82 ± 0.14
2.93 ± 0.12
2.97 ± 0.15
PBW 550
8.53 ± 0.16
8.57 ± 0.12
8.60 ± 0.16
3.52 ± 0.13
3.57 ± 0.15
3.76 ± 0.14
HD 2967
9.61 ± 0.17
9.64 ± 0.16
9.69 ± 0.17
4.94 ± 0.19
4.99 ± 0.13
5.13 ± 0.18
GLU 1356
9.91 ± 0.14
9.96 ± 0.13
9.99 ± 0.12
4.78 ± 0.29
4.84 ± 0.19
5.32 ± 0.16
CD (5 %)
A: 0.49, B: 0.34, C: NS 2.90 ± 0.09 2.50 ± 0.08
Glutamate synthase (lmol NADH oxidized/min/g FW) PBW 621 PBW 636
4.52 ± 0.12 4.33 ± 0.08
4.59 ± 0.14 4.36 ± 0.09
4.60 ± 0.13 4.38 ± 0.11
2.75 ± 0.10 2.01 ± 0.15
2.82 ± 0.07 2.31 ± 0.07
PBW 343
3.65 ± 0.13
3.70 ± 0.09
3.78 ± 0.08
1.82 ± 0.14
1.96 ± 0.08
2.12 ± 0.09
PBW 550
3.48 ± 0.05
3.51 ± 0.11
3.54 ± 0.14
1.67 ± 0.16
1.98 ± 0.08
1.99 ± 0.10
HD 2967
4.42 ± 0.06
4.49 ± 0.08
4.52 ± 0.12
2.04 ± 0.12
2.05 ± 0.09
2.49 ± 0.20
GLU 1356
4.72 ± 0.15
4.79 ± 0.07
4.80 ± 0.09
2.53 ± 0.08
2.78 ± 0.09
2.83 ± 0.08
CD (5 %)
A: 0.44, B: 0.31, C: NS
Values are mean ± SD of three replications A, genotypes; B, treatments; C, stages
higher NR, GS and GOGAT activity at anthesis stage in flag leaf and at 15 DPA in grains under 150 kg N/ha dose (Table 3). Glutamate Oxaloacetate Transaminase and Glutamate Pyruvate Transaminase The present results showed that increase in N dose significantly increased the activities of aminotransferase viz. GOT and GPT indicating that nitrogen nutrition increases aminotransferase activities [1, 15] (Tables 4, 5). The reports in literature also showed positive link between N and aminotransferase, indicating that GPT plays a major role in transfer of C3 units to maintain a nitrogen–carbon balance through the interaction between alanine aminotransferase (AlaAT) and pyruvate; while GOT might be responsible in N-metabolism and participate in the consumption of glutamic acid for further metabolism [16]. It is
also probable that the resulting amino acids were substrates for GOT and GPT, thus inducing the enzyme activities which suggested the role of enzymes in nitrate assimilation, translocation and reassimilation between source and sink. Since the activity of transaminases is stimulated under the condition of 150 kg/ha dose of N that is otherwise deleterious to protein synthesis, it is speculated that aminotransferase might be acting in the direction of deamination to provide amino-acids, especially glutamic acid for common N-pool [17]. In conclusion reaction catalysed by transaminases are simply reversible and strongly depend on the concentration of the substrate (high N dose increases glutamate concentration which results in high GOT and GPT activities; and thereby, facilitates the biosynthesis of various amino acids). Glutamate is synthesised by GSGOGAT pathway and GOT/GPT activities (N assimilation or N - transport system) would be interesting for effective use of nitrogen.
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B. Kaur et al. Table 4 Activities of glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) activities in flag leaf at three developmental stages of six wheat genotypes grown in various nitrogen regimes Tillering stage 90 kg N/ha
Anthesis stage
120 kg N/ha 150 kg N/ha 90 kg N/ha
Post anthesis stage
120 kg N/ha 150 kg N/ha 90 kg N/ha
120 kg N/ha 150 kg N/ha
GOT (lmol oxaloacetate min-1 g-1 FW) PBW 621
1.14 ± 0.27 1.37 ± 0.42
1.56 ± 0.12
1.66 ± 0.13 1.89 ± 0.25
1.92 ± 0.36
1.03 ± 0.24 1.26 ± 0.35
1.32 ± 0.16
PBW 636 PBW 343
1.02 ± 0.19 1.25 ± 0.34 0.78 ± 0.16 1.01 ± 0.17
1.45 ± 0.08 1.15 ± 0.14
1.48 ± 0.16 1.71 ± 0.22 0.93 ± 0.08 1.16 ± 0.17
1.82 ± 0.41 1.49 ± 0.25
0.92 ± 0.26 1.15 ± 0.16 0.61 ± 0.18 0.84 ± 0.08
1.25 ± 0.11 1.32 ± 0.10
PBW 550
0.93 ± 0.28 1.16 ± 0.26
1.28 ± 0.16
1.19 ± 0.13 1.42 ± 0.11
1.56 ± 0.26
0.91 ± 0.15 1.14 ± 0.14
1.16 ± 0.14
HD 2967
0.99 ± 0.20 1.22 ± 0.25
1.39 ± 0.17
1.45 ± 0.17 1.68 ± 0.09
1.82 ± 0.47
0.93 ± 0.11 1.16 ± 0.11
1.19 ± 0.19
GLU 1356 1.35 ± 0.34 1.58 ± 0.32
1.69 ± 0.24
1.83 ± 0.15 2.06 ± 0.38
2.27 ± 0.51
1.23 ± 0.23 1.46 ± 0.19
1.62 ± 0.25
CD (5 %)
A: 0.41, B: 0.38, C: 0.18
GPT (lmol pyruvate min-1 g-1 FW) PBW 621
1.10 ± 0.15 1.29 ± 0.16
1.80 ± 0.19
1.57 ± 0.16 1.76 ± 0.16
2.27 ± 0.26
0.85 ± 0.19 1.04 ± 0.19
1.55 ± 0.13
PBW 636
1.14 ± 0.11 1.33 ± 0.13
1.84 ± 0.22
1.47 ± 0.14 1.66 ± 0.18
2.17 ± 0.21
0.83 ± 0.17 1.02 ± 0.15
1.53 ± 0.14
PBW 343
0.94 ± 0.13 1.13 ± 0.09
1.64 ± 0.18
1.23 ± 0.16 1.42 ± 0.15
1.93 ± 0.19
0.53 ± 0.15 0.72 ± 0.09
1.23 ± 0.11
PBW 550
1.04 ± 0.14 1.23 ± 0.21
1.74 ± 0.17
1.36 ± 0.12 1.55 ± 0.13
2.06 ± 0.25
1.49 ± 0.12 1.68 ± 0.07
2.19 ± 0.23
HD 2967
1.20 ± 0.16 1.39 ± 0.16
1.90 ± 0.24
1.53 ± 0.15 1.72 ± 0.18
2.23 ± 0.36
0.92 ± 0.23 1.11 ± 0.11
1.62 ± 0.25
GLU 1356 1.27 ± 0.13 1.46 ± 0.14
1.97 ± 0.26
1.65 ± 0.19 1.84 ± 0.21
2.35 ± 0.16
0.93 ± 0.20 1.12 ± 0.16
1.63 ± 0.21
CD (5 %)
A: 0.39, B: 0.51, C: 0.62
Values are mean ± SD of three replications A, genotypes; B, treatments; C, stages Table 5 Activities of glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) in grains at 15 and 30 days postanthesis (DPA) of six wheat genotypes grown in various nitrogen regimes 15 DPA 90 kg N/ha
30 DPA 120 kg N/ha
150 kg N/ha
90 kg N/ha
120 kg N/ha
150 kg N/ha
GOT (lmol oxaloacetate min-1 g-1 FW) PBW 621
1.08 ± 0.23
1.23 ± 0.13
1.36 ± 0.23
0.60 ± 0.13
0.63 ± 0.14
0.55 ± 0.19
PBW 636
1.01 ± 0.19
1.19 ± 0.09
1.33 ± 0.21
0.51 ± 0.16
0.54 ± 0.11
0.61 ± 0.07
PBW 343
0.79 ± 0.09
1.01 ± 0.17
1.19 ± 0.15
0.09 ± 0.02
0.18 ± 0.09
0.22 ± 0.06
PBW 550
0.89 ± 0.11
1.16 ± 0.16
1.19 ± 0.17
0.14 ± 0.04
0.27 ± 0.13
0.39 ± 0.05
HD 2967
1.19 ± 0.13
1.28 ± 0.17
1.35 ± 0.16
0.52 ± 0.06
0.71 ± 0.18
0.81 ± 0.09
GLU 1356 CD (5 %)
1.21 ± 0.17 1.36 ± 0.20 A: 0.44, B: 0.62, C: 0.59
1.47 ± 0.11
0.71 ± 0.16
0.85 ± 0.20
0.99 ± 0.11
GPT (lmol pyruvate min-1 g-1 FW) PBW 621
1.21 ± 0.26
1.26 ± 0.20
1.68 ± 0.13
0.51 ± 0.11
0.63 ± 0.19
0.73 ± 0.12
PBW 636
1.13 ± 0.19
1.20 ± 0.16
1.69 ± 0.15
0.39 ± 0.13
0.53 ± 0.13
0.66 ± 0.19
PBW 343
0.92 ± 0.18
1.03 ± 0.14
1.27 ± 0.16
0.24 ± 0.15
0.39 ± 0.06
0.43 ± 0.09
PBW 550
1.09 ± 0.16
1.15 ± 0.16
1.33 ± 0.12
0.29 ± 0.14
0.47 ± 0.08
0.51 ± 0.15
HD 2967
1.37 ± 0.28
1.45 ± 0.26
1.74 ± 0.11
0.67 ± 0.09
0.81 ± 0.14
0.98 ± 0.11
GLU 1356
1.42 ± 0.31
1.56 ± 0.36
1.88 ± 0.13
0.76 ± 0.08
0.92 ± 0.12
1.11 ± 0.18
CD (5 %)
A: 0.82, B: 0.73, C: 0.55
Values are mean ± SD of three replications A, genotypes; B, treatments; C, stages
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Enzymatic Efficiency and Genotypic Differences for Nitrogen Assimilation in Wheat Table 6 Protein, amino acid and nitrogen content in flag leaf at three developmental stages of six wheat genotypes grown in various nitrogen regimes Tillering stage 90 kg N/ha
Anthesis stage 120 kg N/ha
Post anthesis stage
150 kg N/ha
90 kg N/ha
120 kg N/ha
150 kg N/ha
90 kg N/ha
120 kg N/ha
150 kg N/ha
Protein (% 9 0.25) PBW 621
9.31 ± 0.09
9.91 ± 0.13
10.03 ± 0.09
13.57 ± 0.65
16.08 ± 0.12
17.19 ± 0.12
10.31 ± 0.11
11.46 ± 0.31
11.92 ± 0.24
PBW 636
9.30 ± 0.04
9.72 ± 0.14
10.33 ± 0.07
12.17 ± 0.26
12.43 ± 0.14
16.05 ± 0.13
11.74 ± 0.19
12.07 ± 0.05
12.23 ± 0.31
PBW 343
9.60 ± 0.21
10.91 ± 0.23
11.98 ± 0.06
12.72 ± 0.27
13.63 ± 0.13
14.01 ± 0.11
10.28 ± 0.19
12.54 ± 0.35
12.64 ± 0.25
PBW 550
9.00 ± 0.11
9.41 ± 0.34
11.48 ± 0.07
10.13 ± 0.11
12.63 ± 0.21
13.19 ± 0.14
10.95 ± 0.27
11.37 ± 0.19
11.90 ± 0.15
HD 2967
6.80 ± 0.13
9.61 ± 0.28
10.23 ± 0.02
10.41 ± 0.23
14.68 ± 0.34
15.01 ± 0.09
10.71 ± 0.36
11.23 ± 0.21
11.38 ± 0.13
GLU 1356
10.50 ± 0.12
11.71 ± 0.23
11.88 ± 0.10
15.17 ± 0.24
16.38 ± 0.27
19.00 ± 0.11
13.78 ± 0.41
13.27 ± 0.11
13.91 ± 0.17
CD (5 %)
A: 0.024, B: 0.026, C: 0.046
Amino acids (%) PBW 621
3.09 ± 0.03
3.92 ± 0.05
4.13 ± 0.03
3.35 ± 0.06
4.54 ± 0.21
4.71 ± 0.03
4.95 ± 0.06
5.44 ± 0.03
5.56 ± 0.09
PBW 636
3.18 ± 0.04
4.07 ± 0.02
4.29 ± 0.10
3.35 ± 0.04
4.38 ± 0.13
4.59 ± 0.13
4.80 ± 0.12
5.54 ± 0.11
5.73 ± 0.10
PBW 343
2.61 ± 0.01
2.64 ± 0.01
3.21 ± 0.06
2.91 ± 0.02
3.75 ± 0.11
4.38 ± 0.08
3.72 ± 0.06
4.71 ± 0.10
5.11 ± 0.11
PBW 550
2.76 ± 0.01
3.68 ± 0.02
4.02 ± 0.04
3.05 ± 0.02
4.15 ± 0.13
4.69 ± 0.13
3.75 ± 0.07
4.26 ± 0.05
4.84 ± 0.08
HD 2967
3.36 ± 0.02
4.15 ± 0.04
4.37 ± 0.02
3.55 ± 0.01
4.38 ± 0.12
4.72 ± 0.14
4.98 ± 0.05
5.56 ± 0.12
5.72 ± 0.03
3.41 ± 0.03
3.52 ± 0.03
3.59 ± 0.03
3.94 ± 0.06
4.03 ± 0.09
4.65 ± 0.13
4.95 ± 0.04
5.59 ± 0.06
5.77 ± 0.04
GLU 1356 CD (5 %)
A: 0.023, B: 0.045, C: 0.031
Nitrogen (%) PBW 621
7.37 ± 0.02
7.61 ± 0.02
7.85 ± 0.03
6.70 ± 0.12
6.75 ± 0.12
6.92 ± 0.08
2.79 ± 0.01
2.85 ± 0.01
2.93 ± 0.11
PBW 636
6.47 ± 0.01
6.58 ± 0.03
6.97 ± 0.04
6.01 ± 0.09
6.07 ± 0.02
6.29 ± 0.12
2.74 ± 0.03
2.92 ± 0.02
3.05 ± 0.03
PBW 343
5.18 ± 0.03
5.48 ± 0.04
5.65 ± 0.01
4.05 ± 0.07
4.54 ± 0.01
4.87 ± 0.04
2.18 ± 0.01
2.35 ± 0.03
2.42 ± 0.07
PBW 550
6.11 ± 0.02
6.25 ± 0.01
6.31 ± 0.02
4.81 ± 0.02
5.07 ± 0.01
5.17 ± 0.01
2.67 ± 0.01
2.79 ± 0.04
2.85 ± 0.04
HD 2967
6.47 ± 0.02
6.51 ± 0.01
6.62 ± 0.04
5.40 ± 0.01
5.58 ± 0.03
5.79 ± 0.03
2.49 ± 0.02
2.74 ± 0.02
2.86 ± 0.03
GLU 1356
7.57 ± 0.01
7.68 ± 0.03
7.82 ± 0.03
7.03 ± 0.03
7.12 ± 0.01
7.25 ± 0.02
2.79 ± 0.01
2.95 ± 0.02
3.09 ± 0.01
CD (5 %)
A: 0.007, B: 0.066, C: 0.054
Values are mean ± SD of three replications A, genotypes; B, treatments; C, stages
Total Soluble Protein, Free Amino Acid and Nitrogen Content High protein content was found in GLU 1356 i.e. 4.75 % at anthesis stage in flag leaf (Table 6) and 8.32 % in grains at 15 DPA (Table 7), whereas high amino acid content was noted in GLU 1356 (5.77 %) at post anthesis stage in flag leaf (Table 6) and 4.56 % in grains at 30 DPA (Table 7). Apparently, in flag leaf, high N content was observed in PBW 621 (7.85 %) at tillering stage (Table 6) and in grains, higher content was found in GLU 1356 (4.91 %) (Table 7). Amino acid content was lowest at tillering followed by rapid increase in its content from anthesis to post anthesis stages in flag leaf and 15 DPA to 30 DPA in grains (Tables 6, 7). The content of soluble protein and N was high at the beginning of anthesis till tillering then decreased towards post anthesis stages in flag leaf and after 15 DPA in grains. The total soluble protein, free amino acids and nitrogen content were maximum in GLU 1356 followed by HD 2967, PBW 621, PBW 636, PBW 343, PBW 550. In all treatments, amino acid, soluble protein content and N content was in accordance with increasing amount of nitrogen and its sequence was 150 [ 120 [ 90 kg N/ha.
Changes in the content of soluble metabolites could reflect the ability of the plant osmotic adjustment. Present studies showed that during growth and development of wheat, the level of soluble protein content not only reflected the level of plant nitrogen metabolism, but also was regarded as an important indicator of the degree of leaf senescence, especially in wheat grain filling stage. The increasing soluble protein content in flag leaf was conducive to the maintenance of the flag leaf growth and extending the photosynthetic function (as in GLU 1356, HD 2967 and PBW 621), so as to lay the metabolite basis for the accumulation of grain carbonitrides [30]. Results of this study showed that soluble protein content was high during anthesis and after anthesis it started to decline, but maintained a high level with respect to increasing amount of nitrogen fertilizer indicated that the increase in nitrogen fertilizer is conducive to delaying flag leaf senescence. High GS/GOGAT activities can be correlated with increase in protein content with respect to increasing N dose which reflects an enhancement of operation of nitrogen metabolism to promote the synthesis and conversion of amino acids [24]. PBW 343 and PBW 550
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B. Kaur et al. Table 7 Protein, amino acid and nitrogen content in grains at 15 and 30 days post-anthesis (DPA) of six wheat genotypes grown in various nitrogen regimes 15 DPA
30 DPA
90 kg N/ha
120 kg N/ha
150 kg N/ha
90 kg N/ha
PBW 621
8.04 ± 2.03
8.08 ± 1.08
8.09 ± 1.02
1.67 ± 0.04
1.72 ± 0.13
1.78 ± 0.02
PBW 636 PBW 343
7.95 ± 1.09 6.98 ± 1.06
7.99 ± 1.16 7.04 ± 1.05
8.10 ± 1.03 7.08 ± 1.07
1.43 ± 0.06 1.23 ± 0.01
1.59 ± 0.14 1.25 ± 0.01
1.64 ± 0.04 1.32 ± 0.03
PBW 550
6.88 ± 1.04
6.93 ± 1.08
6.97 ± 1.11
1.12 ± 0.04
1.19 ± 0.09
1.24 ± 0.04
Protein (%)
120 kg N/ha
150 kg N/ha
(% 9 10)
HD 2967
8.12 ± 1.16
8.16 ± 1.03
8.18 ± 1.16
1.98 ± 0.09
2.02 ± 0.24
2.07 ± 0.07
GLU 1356
8.15 ± 1.31
8.20 ± 1.07
8.32 ± 0.99
2.13 ± 0.11
2.22 ± 0.13
2.34 ± 0.08
CD (5 %)
A: 0.025, B: 0.035, C: 0.035
Amino acids (%) PBW 621
2.14 ± 0.01
2.19 ± 0.01
2.26 ± 0.01
3.96 ± 0.01
4.11 ± 0.02
4.16 ± 0.01
PBW 636
2.11 ± 0.02
2.17 ± 0.04
2.21 ± 0.01
3.77 ± 0.03
4.09 ± 0.01
4.15 ± 0.01
PBW 343
2.01 ± 0.01
2.07 ± 0.03
2.11 ± 0.02
3.47 ± 0.04
3.54 ± 0.01
3.57 ± 0.03
PBW 550
1.96 ± 0.03
2.04 ± 0.02
2.07 ± 0.06
3.34 ± 0.03
3.47 ± 0.01
3.54 ± 0.02
HD 2967
2.06 ± 0.01
2.15 ± 0.01
2.18 ± 0.01
4.32 ± 0.02
4.41 ± 0.06
4.47 ± 0.04
GLU 1356
2.24 ± 0.03
2.33 ± 0.01
2.39 ± 0.03
4.47 ± 0.02
4.54 ± 0.01
4.56 ± 0.03
CD (5 %)
A: 0.094, B: 0.076, C: 0.111
Nitrogen (%) PBW 621 PBW 636
4.65 ± 0.03 4.55 ± 0.04
3.70 ± 0.02 3.59 ± 0.01
3.74 ± 0.06 3.65 ± 0.07
0.44 ± 0.06 0.37 ± 0.02
0.51 ± 0.02 0.39 ± 0.02
0.52 ± 0.01 0.42 ± 0.01
PBW 343
4.08 ± 0.02
3.19 ± 0.06
3.24 ± 0.03
0.24 ± 0.03
0.31 ± 0.03
0.34 ± 0.02
PBW 550
3.90 ± 0.03
2.98 ± 0.04
3.01 ± 0.01
0.22 ± 0.01
0.24 ± 0.01
0.29 ± 0.01
HD 2967
4.82 ± 0.01
3.85 ± 0.05
3.89 ± 0.02
0.18 ± 0.03
0.23 ± 0.03
0.26 ± 0.06
GLU 1356
4.91 ± 0.03
3.98 ± 0.01
4.04 ± 0.02
0.27 ± 0.03
0.31 ± 0.04
0.33 ± 0.04
CD (5 %)
A: 0.036, B: 0.035, C: 0.043
Values are mean ± SD of three replications A: genotypes, B: treatments, C: stages
showed lowest GS and GOGAT activity leading to lower protein accumulation while GLU 1356, HD 2967, PBW 621 had higher level of protein content. Likewise, in rice, high activity of GS and N content also resulted in increase in protein content [31]. It is well known phenomenon that higher NH4? contents lead to activation of GS and GOGAT [32]. The end products of the assimilation of NO3- by the plants are amino acids and proteins which increased with increasing level of N. Nitrogenous compounds are carried from vegetative organs to grains in the form of amino acids and synthesized into proteins within grains [27, 33]. The total N and protein concentrations decreased with ageing. This trend was attributed to the N dilution process as already described for many plant species [28]. 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 [29]. But with decrease in protein content soluble amino acids increased which concluded that hydrolysis of proteins
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might be responsible for the accumulation of amino acids [24, 34]. Chlorophyll Content, Grain Yield and NUE Significant increase in chlorophyll and grain yield was observed in crop supplied with 150 kg N/ha over 120 and 90 kg N/ha (Table 8). However, yielding ability of the crop at 90 kg N/ha was statistically at par with application of 120 kg N/ha. There was variation with respect to genotypes at all three doses. GLU 1356, HD 2967 showed higher yield whereas PBW 621, PBW 636 showed moderate and PBW 343, PBW 550 showed low yield under present experimental conditions (Table 8). 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 in all genotypes [35]. Mobilisation of chloroplast N has a central role in leaf lamina metabolic activity and canopy senescence [36], and grain N acquisition is linked to senescence patterns in durum wheat [37].
Enzymatic Efficiency and Genotypic Differences for Nitrogen Assimilation in Wheat Table 8 Chlorophyll content in flag leaf and yield of six wheat genotypes grown in various nitrogen regimes Tillering stage 90 kg N/ha
Anthesis stage
120 kg N/ha 150 kg N/ha 90 kg N/ha
Post anthesis stage
120 kg N/ha 150 kg N/ha 90 kg N/ha
120 kg N/ha 150 kg N/ha
Chlorophyll content (mg/g) PBW 621
2.67 ± 0.09 3.61 ± 0.06
3.77 ± 0.14
2.52 ± 0.07 2.56 ± 0.08
2.63 ± 0.09
1.46 ± 0.06 1.52 ± 0.07
1.64 ± 0.15
PBW 636
2.74 ± 0.14 3.74 ± 0.15
3.83 ± 0.13
2.60 ± 0.15 2.72 ± 0.08
2.69 ± 0.08
1.43 ± 0.14 1.63 ± 0.06
1.69 ± 0.08
PBW 343
2.47 ± 0.06 3.56 ± 0.10
3.71 ± 0.14
2.44 ± 0.09 2.53 ± 0.09
2.53 ± 0.13
1.38 ± 0.07 1.52 ± 0.14
1.61 ± 0.08
PBW 550
2.51 ± 0.08 3.67 ± 0.09
3.71 ± 0.16
2.40 ± 0.16 2.43 ± 0.15
2.44 ± 0.12
1.35 ± 0.06 1.42 ± 0.07
1.49 ± 0.09
HD 2967
2.75 ± 0.12 3.75 ± 0.15
3.71 ± 0.12
2.63 ± 0.14 2.65 ± 0.10
2.72 ± 0.13
1.56 ± 0.08 1.65 ± 0.08
1.79 ± 0.14
GLU 1356 2.77 ± 0.14 3.67 ± 0.09
3.71 ± 0.15
2.64 ± 0.15 2.69 ± 0.13
2.74 ± 0.17
1.63 ± 0.12 1.66 ± 0.11
1.84 ± 0.15
CD (5 %)
A: 1.212, B: 0.181, C: 0.253 Yield (t/sqm) 90 kg N/ha
120 kg N/ha
150 kg N/ha
PBW 621
0.00096 ± 0.17
0.00101 ± 0.04
0.00113 ± 0.11
PBW 636
0.00068 ± 0.19
0.00069 ± 0.02
0.00070 ± 0.23
PBW 343
0.00054 ± 0.02
0.00056 ± 0.01
0.00058 ± 0.06
PBW 550
0.00045 ± 0.04
0.00049 ± 0.05
0.00051 ± 0.02
HD 2967
0.00098 ± 0.08
0.00101 ± 0.11
0.00111 ± 0.07
GLU 1356 CD (5 %)
0.00110 ± 0.13 A: 0.326, B: 0.197, C: 1.231
0.00117 ± 0.10
0.00125 ± 0.03
Values are mean ± SD of three replications A: genotypes, B: treatments, C: stages
Delayed senescence and ‘stay-green’ traits could therefore have an important role in increasing yield of wheat crops grown under moderate N supply [1]. Significant increase in grain yield and NUE was observed in crop supplied with higher dose of N as observed in the present study [38, 39]. High value of NUE was found in GLU 1356 (1.22) followed by PBW 621 (1.11), HD 2967 (1.09), PBW 636 (0.68), PBW 550 (0.57) and PBW 343 (0.50) (Table 9). The new breeding stock in the form of advanced lines had significant increase in N assimilation thereby depicting better N metabolism and hence improved yield over preexisting one such as PBW 343 and PBW 550. Likewise, Gpc-B1 gene present in GLU 1356 not only improves remobilization from stem reserve but also contributed in N assimilation which ultimately results in high protein content. Correlation Analysis The correlation was analyzed between biochemical parameters and grain yield. The activity of NR, GS and GOGAT was positively correlated with metabolites (soluble protein, amino acids and nitrogen) as well as with grain yield (Table 10). In flag leaf, NR was positively correlated with protein (0.855), chlorophyll (0.827), yield
Table 9 Nitrogen use efficiency (NUE) (kg kg-1) of six wheat genotypes Genotypes
NUE
Mean
NUE1
NUE2
PBW 621
1.11
1.1
1.11
PBW 636
0.69
0.68
0.68
PBW 343 PBW 550
0.57 0.5
0.56 0.49
0.57 0.50
HD 2967
1.09
1.08
1.09
GLU 1356
1.23
1.21
1.22
NUE1 = YC (150 kg N/ha) - YF (120 kg N/ha)/N applied NUE
2
= YC (150 kg N/ha) - YF (90 kg N/ha)/N applied
(0.936) and GS with GOGAT (0.849), protein (0.945), nitrogen (0.904). Amino acid content was negatively correlated with protein (-0.007), non-significant with nitrogen (0.179) and yield (0.080) in flag leaf whereas all parameters were positively correlated to each other in grains (Table 10). It was found that the content of nitrate nitrogen in flag leaf as well as in grains have significant positive correlation with the activities of NR, GS and GOGAT.
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B. Kaur et al. Table 10 Correlation coefficients between biochemical traits and grain yield (Tables 2, 3, 6–8) at RDN -25 % in flag leaf (A) and grains (B) NR
GS
GOGAT
Amino acid
Protein
Nitrogen
Chlorophyll
(A) Flag leaf GS
0.746
GOGAT
0.710
0.849*
Amino acid
0.197
0.057
Protein
0.855*
0.945**
0.797
Nitrogen
0.734
0.904**
0.912**
0.179
0.810*
Chlorophyll
0.827*
0.792
0.637
0.052
0.928**
0.560
Yield
0.936**
0.794
0.800
0.080
0.911*
0.797
0.445 -0.007
0.833*
(B) Grain GS
0.964**
GOGAT Amino acid
0.948** 0.977**
0.920** 0.936**
0.918**
Protein
0.909*
0.842*
0.874*
0.969**
Nitrogen
0.997**
0.949**
0.962**
0.968**
0.905*
Chlorophyll
0.889*
0.834*
0.756*
0.936**
0.939**
0.869*
Yield
0.918**
0.839*
0.948**
0.947**
0.962**
0.930**
0.833*
,
* ** Significantly different at 5 and 1 % level, respectively
Conclusion Results indicated that GLU 1356 possessed higher N assimilation rate (over HD 2967, PBW 621 and PBW 636), while PBW 343 and PBW 550 acted as benchmark for this study. Higher N used in this study (150 kg N/ha instead of 120 kg N/ha) correlated more with enhanced activity pattern of N metabolizing enzymes and metabolite contents under the prevailing environmental conditions. It would be particularly interesting further to investigate the differential N responsiveness of contrasting genotypes in terms of complex regulatory network involved in N assimilation. GLU 1356 and HD 2967 could be exploited further as donor stocks in wheat breeding programme and biotechnology for high yield potential. Acknowledgments The authors are grateful for financial support from Maulana Azad National Fellowship for minority students, granted by UGC, India. Compliance with Ethical Standards Conflict of interest of interest.
The authors declare that they have no conflict
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