Molecular approaches for improving nitrogen use efficiency (NUE) in cereal crops. 1. NUE is important to improve as only 30-50% of applied nitrogen is taken up by plants, with the rest lost to the environment. 2. Studies have identified genes involved in nitrogen uptake, assimilation, remobilization and utilized QTL mapping to find genes controlling NUE traits. 3. Approaches to improve NUE include conventional breeding, quantitative trait loci mapping, genome-wide selection, and transgenic methods targeting genes such as glutamine synthetase, asparagine synthetase, and alanine aminotransferase.

1. NUE
welcome

2. WELCOME

3. Molecular approaches for
improving nitrogen use
efficiency(NUE) in cereal crops
SEMINAR -II
ON

4. •Introduction
•Need for improvement of NUE
•Global Status of N fertilizer use and consumption
•What is NUE ?
•Components of NUE
•Molecular basis of NUE
•Approaches for improving NUE
•Case studies
•Patents
•Conclusion
CONTENTS WHAT WILL
I LEARN
TODAY?

5. Essential elements in plants

8. Why is NUE Improvement is Important ?
In the last 40 years, the amount of synthetic nitrogen (N) applied to crops
has risen dramatically, from 12 to 104 Tg/ year (Mulvaney et al., 2009)
 N is the most important nutrient in a plant and a limiting factor in
plant growth and development (Kraiser et al., 2011).
 Only 30%–50% being taken up by the plant .
Lost to surface run-off, leaching of nitrates, ammonia (NH3) volatilization or
bacterial competition (Garnett et al., 2009)

9. Global Status of N fertilizer consumption & NUE
•There is 20-fold increase in the global use of N fertilizer applications during the
past five decades (Glass, 2003) and this is expected to increase at least threefold
by 2050 (Good et al., 2004).
•The current average NUE in the field is approximately 33% and substantial
proportion of the remaining 67% is lost into the environment, especially in the
intensively cropped area.
•It is estimated that 1% increase in NUE could save $1.1 billion annually.

10. Fischer et al., 2015
The United Nations: world population -11 billion by 2050.
The FAO of United Nations:
• Food price index 9%
• Cereal Price index 21%
Between 2010 and 2015,
• Fertilizer price index 28%, further cost to the producer.
. Additionally,
Nutrient fertilizer consumption 2–2.5% / year up to 2015
Between June and July 2015

11. Cornimer et al., 2010
• Most cereal crops take up roughly 40% of applied N, the remaining
60% is lost to the environment .
Approximately,
• 19% of the applied N -- denitrification or bacterial uptake,
• 20% is leached into groundwater,
• 21% is volatilized into N2O .
• Eutrophication of aquatic ecosystems .
• Greenhouse gas nitrous oxide Increased 5–7% per decade since 1979
• Haber–Bosch process (N fertilizer production)
Requires approximately 1% of the world’s annual energy supply
Nitrogen (N) is a key limiting nutrient in the growth
of staple crops.

12. Where, When and Why NUE traits are required?
• It’s required in all environmental condition where yield is required,
because NUE ⍺ crop yield.
• For abiotic stress improvement in crops, NUE has become the second
priority after drought both in the private and in the public sector.
• To minimize N loss, maximize N uptake & reduce environmental
pollution.
• The recovery of the applied N is low, with only 33% of the applied N
ending up in the grain

13. • Nitrogen use efficiency is the grain production per unit of N available
in soil and applied fertilizer.
• N use efficiency (NUE) is the weight of the gains divided by the
amount of N available in the soil
• Physiological NUE =Eefficiency with which the plant uses N from
acquired available N to total plant dry matter
• Agronomic NUE =N imported from the field to the crop product per N
applied
What is NUE ?
Moll et al. (1982)

14. Definition Formula
Usage index (UI) UI = SDM x [(SDM) / (N content of shoot)]
Uptake efficiency (UpE) UpE = (plant (or shoot) N content) / (N supply)
Grain nitrogen use
efficiency (NUE)
NUEgrain = (grain mass) / (N supply)
Utilization efficiency (UtE) UtE = (grain mass) / (plant (or shoot) N content)
NHI As N in grain/total N uptake
Agronomic efficiency (AE)
AE = [(grain mass with fertilizer) – (grain weight of
unfertilized control)] / (N supply)
SLN
the amount
of N per unit of leaf area called specific leaf N
(Good et al., 2004)
Common terminology used to describe nitrogen use
efficiency (NUE) in plants

15. Soil
Atmosphere
Atmosphere
N2
N2
Soil
Nitrogen-fixing bacteria
H+
(from soil)
Ammonifying
bacteria
NH3
(ammonia)
NH4
+
(ammonium)
Nitrifying
bacteria
Organic material (humus)
N2
NO3
–
(nitrate)
Denitrifying
bacteria
NH4
+
Nitrate and
nitrogenous
organic
compounds
exported in
xylem to
shoot system
Root
Pathway of nitrogen from rhizosphere to plants

16. 1. Nitrogen Uptake Efficiency
2. Nitrogen Utilization Efficiency
Components of NUE
Mathew et al., 2009

17. “The amount of N taken up by the crop as a fraction of the amount
available to the crop from all sources”
N uptake efficiency drives biomass production and depend on
Amount of N uptake
Storage
Assimilation
Nitrogen uptake efficiency

18. (Kant & Rothstein., 2010)
N uptake
• For plant N is mainly available in the form
of NO3
- and NH4
+
• There are 3 type of NO3
- transporter -
LATS, cHATS & iHATS
• Several structural gene involve in N uptake
• NRT1 families member (NRT1.1 and
NRT1.2) are characterized as LAT
• NRT2 families member (NRT2.1 and
NRT2.2) are characterized as HAT
• AMT families member are involved in
NH4
+ (AMT1, AMT2 & AMT3)

19. • NO3
- after entering into the plant cell, assimilation is begin .
No3
- No2
- NH4
- Amino
acid
• Absorbed N may stored in the vacoule or directly assimilate into amino
acid
N Storage
N Assimilation

20. Schematic diagram of nitrate assimilation pathway Abrol et al., 2008

21. 2. Nitrogen Utilization Efficiency
• Utilization efficiency involved N remobilization.
• At the reproductive stage both N assimilation & N remobalization become
critical.
• Leaves & shoot act as a source for amino acid to the reproductive and
storage organ.
• Upto 80% of the grain N content is derived from leaves in rice and wheat.
• During leaf senescence N release via protease activities.
Martin et al., 2006

22. Amino acid
The predominant gene families which involved in phloem
loading process is AAP (AAP1 - AAP8)
Loading
into
Phloem
NH4
+
Stored Protein

23. Molecular basis of NUE
• NUE is a complex quantitative traits which involves many genes.
• Expression of multiple gene depend on a number of internal and
external factors.
• There are 100s of nitrate responsive gene.
• For their transcription require regulatory sequence i.e. NRE
(Nitrate responsive element).
Han et al., 2012

24. Conti..
• One of the such sequence originally reported to be comprised of an
A[G/C]TCA sequence.
• These sequence is randomly distributed throughout the genome.
• QTL mapping is a powerful tool for analysis of complex NUE.
• By using QTL mapping in some model spp. such as rice, arabidopsis
and maize, candidate genes encoding enzyme that involved in N uptake,
assimilation and utilization have been identified.
Han et al., 2012

25. GENE GENE PRODUCT FUNCTION
Nrt 1.1
Dual affinity nitrate
transporter
Nitrate uptake from
external environment
Nrt1.2
Low affinity nitrate
transporter
-
Nrt 2.1 &
Nrt 2.2
High affinity nitrate
transporter
-
CLCa - -
Amt 1, Amt 2
High affinity NH4
+
transporter
Ammonium uptake
Amt3
Low affinity NH4
+
transporter
-
Gene involved in Nitrogen uptake
(Kant et al., 2011)

26. GENE GENE PRODUCT FUNCTION
NR Nitrate reductase
Convert
NO3
- to NO2
-Nia
Nia2
NiR Nitrite reductase
Convert NO2
-
to NH4
-
GS2
Chloroplastic
glutamine synthetase
Glutamine
Synthesis
Fd- GOGAT
Ferredoxin dependent
glutamate synthase
Glutamate synthesis
(Good et al., 2004)
Gene involved in Nitrate Assimilation

27. Contd.
GENE GENE PRODUCT FUNCTION
GS1
Cytosolic glutamine
synthetase
Glutamine synthesis
NADH-GOGAT
NADH-dependent
glutamate synthase
Glutamate synthesis
GDH
Glutamate
dehydrogenase
Still controversial
ASN1
Glutamine dependent
Asparagine synthetase
Asparagine synthesis
ASNI Asparagine synthetase -
Good et al., 2004

28. Moose et al., 2009

29. Nitrogen management in various crops
(A)During vegetative growth,
(B) The relative contribution (%) of N remobilization and post flowering
N uptake in different crops Hirel et al., 2007

32. Strategies to improve nitrogen use efficiency
 Conventional breeding approaches
 Molecular and genetic engineering approaches
 Quantitative Trait Loci and Candidates Genes
 Genome-Wide Selection
 Transgenes for NUE

33. Quantitative trait loci (QTL)
Many studies have been identified QTL controlling NUE and some of their
component traits (Agrama et al. 1999; Bertin and Gallais 2001; Hirel et al.
2001; Gallais and Hirel 2004)
QTLs for N-uptake and N utilization efficiency (at high N input five QTLs
explained 39% of phenotypic variance)
QTLs for leaf nitrate content
QTLs for glutamine synthetase (GS) activity
QTLs for glutamate dehydrogenase (GDH)
Quantitative trait loci (QTL) mapping provides the best understanding of
the genetic control and inheritance of NUE and can be used to indicate the
best selection strategy.

34. Main QTL studies on NUE in plants

35. A set of RIL s X A tester populations were used
Studied at
 low input (N±)
 high input (N+)
OBJECTIVE
To study the genetic variability and the genetic
basis of nitrogen (N) use efficiency in maize.
Trait studied : Grain yield and its components, grain protein content,
post-anthesis nitrogen uptake and remobilization.

36. Locations of the detected QTLs for agronomic traits

37. Locations of the detected QTLs for physiological trait

38. • Role of the gene encoding cytosolic GS (gln4 locus) located on chromosome 5
as a candidate gene for which the corresponding enzyme activity influences
grain
filling.
• In conclusion,
These results clearly show that genetic and physiological bases of NUE can
be studied in a integrated manner by means of a quantitative genetic
approach using molecular markers, genomics, and combining both
agronomic and physiological studies.
• Such an approach leads to,
The identification of candidate genes to validate other approaches such as
gene transfer or mutagenesis.
Major breakthroughs from these studies

39. .
Meuwissen et al., 2001
The Genome-Wide Selection
• Once the markers are identified, their effects are estimated from phenotypic
data that are obtained from a population that is known as the estimation
population.
• Once the effects are estimated, they are tested in a validation population.
• After this step, the markers that explain most of the genetic variance of a
trait are selected.
Thus, this information is effectively incorporated into the selection stage of the
breeding program (RESENDE, 2008)

40. • The 41 single-crosses -evaluated under experiment low nitrogen availability (LN),
• Genotyping the estimation population 80 (SSR)
• These markers were chosen for their informational content, location on
chromosomes and, mainly, for their association with agronomic traits and
abiotic stresses tolerance (MAIZEGDB, 2009)

41. The Genome-Wide Selection method may significantly increased the genetic gains in
maize root trait breeding programs that investigate nutritional stress conditions
• The GWS was also compared to the phenotypic selection method in
terms of the gain per unit time

42. Transgenic approach
• For improve NUE its is largely revolved around manipulation and
over-expression of many crucial candidate genes. Transgenic efforts have
concentrated on diverse targets that includes genes belonging to N uptake
translocation, remobilization, signaling and metabolism.
• Glutamine synthetase (GS) is the most studied gene aiming to improve
NUE both in monocot. In some cases transgenic lines overexpressing GS
displayed improvements in NUE, resulting in increased biomass and
grain yield.
• There is currently an existing transgenic technology – alanine
aminotransferase that is being evaluated in Australian cereals as part of a
commercialisation process.

43. Chandra et.al., 2012
Gene
Gene Source
(Gene/promoter)
Engineered
Plant
NUE Improvement
(Percent)
Grown in
the Field?
Glutamine
synthetase (GS)
Bean/rice Wheat 10 No
Glutamine
synthetase (GS)
Corn/plant virus Corn 30 No
Glutamate synthase
(GOGAT)
Rice/rice Rice 80 No
Asparagine
synthetase (AS)
Arabidopsis/plant
virus
Arabidopsis 21 No
Glutamate
dehydrogenase
E. coli/plant virus Tobacco 10 Yes
Dof1 Corn/plant virus Arabidopsis
Nitrogen content:
30; growth: ~65
No
Alanine
aminotransferase
(ALA)
Barley/canola Canola 40 Yes
Alanine
aminotransferase
(ALA)
Barley/rice Rice 31–54 Yes
Genes Used to Improve NUE through Genetic Engineering

44. MicroRNA
• Under N limiting conditions, miRNAs can be up- or down regulated.
Expression profiles of different miRNA families have been observed in various
crop species such as maize,rice.
• Maize reproductive tissues during grain filling period only two miRNAs,
miR395 and miR399, have been identified to be important in nutrient stresses
responses
• Several miRNAs have been reported to play vital roles in root growth and
development under N deficiency
• MiR164 accelerate senescence, N remobilization, miR172 and 173 regulates the
root development and nitrogen homeostasis, N uptake in maize.
Chiou et al. (2007)Nguyen et al., 2015,

45. Model regulatory network of miRNA responses to nitrogen limiting
condition
Fischer et al., 2010

46. Case studies

47. OBJECTIVE
Whether Over-expression of TOND1 increases the tolerance
to N deficiency in the TOND1-deficient rice cultivars.
• Identified a major QTL (TOND1) on the long arm of chromosome
12, confer tolerance to nitrogen deficiency in Teqing

49. • The identification of TOND1 provides a molecular basis for
breeding rice varieties with improved grain yield despite decreased
input of N fertilizers.
Conclusion
• Identified TOND1, a major QTL in rice controlling tolerance of N deficiency.
Increased expression of the TOND1 gene led to a remarkable increase in the N
deficiency tolerance and grain yield of rice plants grown under N-deficient
conditions

50. Case study
To map QTLs for N uptake (NUP) in wheat and to
investigate factors influencing NUP.
OBJECTIVE
• Two treatments with low N (LN) and high N(HN)
• 120 DH lines derived from winter wheat varieties Hanxuan
10 and Lumai 14

51. Totally 17 QTLs were detected for NUP in the two field trials, among the
17 NUP QTLs, nine and eight were detected under LN and HN conditions,
respectively.

52. The 17 NUP QTLs distributed at 13 loci on 12 chromosomes, with the chromosomes of B
genome having the highest number (9) of QTLs.
Conclusion : Early vigorous growth has also been suggested as a major factor
influencing N uptake in wheat. The higher N uptake of vigorous wheat
genotype has been associated with vigorous early root and shoot growth.

53. 3.Maize
OBJECTIVE
Whether the overexpression of Gln1-3 and Gln1-4 in
maize improves yields and enhances nitrogen using
efficiency

55. • Over-expression of Gln1-3 and Gln1-4 genes enhanced the yields of
transgenic plants compared with the WT and increased GS activity and
photosynthesis rates in the transgenic lines indicated that N uptake was
enhanced.
Conclusion

56. Some Patents

57. A nitrate transporter (NT) from yeast Pichia angusZa,YNT1, has been shown to
be involved in nitrogen uptake When expressed in vivo in Arabidopsis and
maize plants as Well as in in vitro assays. The present invention provides NT
polynucleotides, codon optimized NT gene coding sequences, related
polypeptides, and all conservatively modified variants of the present NT
sequences.

58. The invention relates to monocot plants having enhanced nitrogen
utilization efficciency (NUE), to methods for enhancing NUE in monocot
plants, and to methods of increasing biomass and seed yield in monocot
plants grown under nitrogen limiting conditions. This invention also relates
to monocot antiquitin promoters.

59. This article was published on July 30, 2013

60. CONCLUSION

61. T
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