GM CROPS & FOOD

2021, VOL. 12, NO. 2, 627–646

https://doi.org/10.1080/21645698.2021.1921545

REVIEW

Novel plant breeding techniques to advance nitrogen use efficiency in rice: A

review
Sajid Fiaz a, Xiukang Wangb, Sher Aslam Khana, Sunny Ahmarc, Mehmood Ali Noor d
, Aamir Riaze, Kazim Alif,
Farhat Abbasg, Freddy Mora-Pobletec, Carlos R. Figueroac, and Badr Alharthi h,i
a
Department of Plant Breeding and Genetics, The University of Haripur 22620, Khyber, Pakhtunkhwa, Pakistan; bCollege of Life Sciences, Yan’an
University, Yan’an, Shaanxi, China; cInstitute of Biological Sciences, Campus Talca, Universidad deTalca, Talca, Chile; dInstitute of Crop Sciences,
Chinese Academy of Agricultural Sciences, Key Laboratory of Crop Physiology and Ecology, Ministry of Agriculture, Beijing, China; eState Key
Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou, Zhejiang, China; fNational Institute for Genomics and Advanced
Biotechnology, National Agricultural Research Centre, Islamabad, Pakistan; gResearch Center for Ornamental Plants, College of Forestry and
Landscape Architecture, South China Agricultural University, Guangzhou, China; hCollege of Khurma, Taif University, Taif, Saudi Arabia; iCollege
of Science and Engineering, Flinders University, Adelaide, South Australia

ABSTRACT ARTICLE HISTORY

Recently, there has been a remarkable increase in rice production owing to genetic improvement Received 25 January 2021
and increase in application of synthetic fertilizers. For sustainable agriculture, there is dire need to Revised 4 April 2021
maintain a balance between profitability and input cost. To meet the steady growing demands of Accepted 20 April 2021
the farming community, researchers are utilizing all available resources to identify nutrient use KEYWORDS
efficient germplasm, but with very little success. Therefore, it is essential to understand the under­ Green revolution; synthetic
lying genetic mechanism controlling nutrients efficiency, with the nitrogen use efficiency (NUE) fertilizers; genome
being the most important trait. Information regarding genetic factors controlling nitrogen (N) engineering; resource use
transporters, assimilators, and remobilizers can help to identify candidate germplasms via high- efficiency; food security
throughput technologies. Large-scale field trials have provided morphological, physiological, and
biochemical trait data for the detection of genomic regions controlling NUE. The functional aspects
of these attributes are time-consuming, costly, labor-intensive, and less accurate. Therefore, the
application of novel plant breeding techniques (NPBTs) with context to genome engineering has
opened new avenues of research for crop improvement programs. Most recently, genome editing
technologies (GETs) have undergone enormous development with various versions from Cas9,
Cpf1, base, and prime editing. These GETs have been vigorously adapted in plant sciences for novel
trait development to insure food quantity and quality. Base editing has been successfully applied to
improve NUE in rice, demonstrating the potential of GETs to develop germplasms with improved
resource use efficiency. NPBTs continue to face regulatory setbacks in some countries due to
genome editing being categorized in the same category as genetically modified (GM) crops.
Therefore, it is essential to involve all stakeholders in a detailed discussion on NPBTs and to
formulate uniform policies tackling biosafety, social, ethical, and environmental concerns. In the
current review, we have discussed the genetic mechanism of NUE and NPBTs for crop improvement
programs with proof of concepts, transgenic and GET application for the development of NUE
germplasms, and regulatory aspects of genome edited crops with future directions considering
NUE.

1 Introduction of the ever-increasing human population.2 Rice

Over the last three decades, global rice production production increased remarkably during and after
has increased by three fold despite the increases in the green revolution period due to the development
rice production constraints and input costs. Rice of high input-responsive rice germplasm. However,
ensures food and nutritional security to more than the improved germplasm requires more synthetic
half of the world’s population.1 As such, rice fertilizers, pesticides, and a frequent supply of irri­
demands a great effort for the development of high- gation water. Nitrogen (N) is an integral nutrient
yield, nutritious, climate-resilient, and resource for plant growth and development.3 N is primarily
use-efficient varieties to meet the caloric demands found in photosynthetic metabolites, proteins, and

CONTACT Sajid Fiaz sfiaz@uoh.edu.pk; wangxiukang@yan.edu.cn Department of Plant Breeding and Genetics, The University of Haripur 22620, Khyber,
Pakhtunkhwa, Pakistan; College of Life Sciences, Yan’an University, Yan’an, Shaanxi, China
This article has been corrected with minor changes. These changes do not impact the academic content of the article.
© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
628 S. FIAZ ET AL.

Figure 1. Illustration explaining various traits essential for the identification of NUE genotypes and different OMICS for the functional
characterization of genes controlling NUE in rice.

nucleic acids and plays a key role in metabolic and however, it has been unanimously accepted that
growth-related activities.4,5 Cultural and agro­ NUE is the result of N uptake efficiency (NUpE)
nomic management practices can help to achieve and N utilization efficiency (NUtE).6–8 Improving
efficient utilization of N fertilizers. The nitrogen use NUE enhances crop economics i.e., grain quality,
efficiency (NUE) of several agronomically impor­ yield, and biomass.9 10 N is primarily applied as
tant crops is of great interest for academia and synthetic fertilizer while a smaller portion is con­
research. NUE is based on the economic benefit tributed by grain legumes from N fixation.
from per unit application of N fertilizer. Several Plants uptake N through their roots in the form
researchers have suggested definitions for NUE; of nitrate (NO3−) or ammonium (NH4+) and it is
 GM CROPS & FOOD 629

actively utilized to complete metabolic processes.11 obtained. Moreover, NPBTs have enabled the pro­
Being mobile in nature, N losses from soil are duction of transgene-free plants that are categor­
greater than any other element; moreover, crop ized as non-genetically modified (GM) crops. The
species differ in their N uptake ability. Despite the transgene-free plants do not contain exogenous
positive influence of N on yield and yield related genes and therefore escape the strict regulatory
components, plants can only uptake 30–50% of the framework of GM crops. The extraordinary
supplied N depending on the soil type, environ­ NPBTs with reference to GETs are now available
mental condition, and plant population.9 N loss and can be utilized for various crop improvement
from soil is caused by volatilization, denitrification, programs to ensure food and nutritional security
and leaching, ultimately polluting the air and for the ever-increasing human population.
water12–14 while simultaneously increasing the Acknowledging the importance of NUE and recent
cost of production.15 It has recently been reported developments in NPBTs, we provide a non-
that 24–39% of wheat, rice, soybean, and maize comprehensive review highlighting the multiple
production areas have demonstrated stagnation or factors influencing NUE and explore how genetic
collapse of yield.13,16 Therefore, it is crucial to opti­ understanding will improve our knowledge of the
mize N fertilizer application or improve crop NUE utility of genetic factors to enhance NUE through
to achieve high production while also reducing various GETs, with a focus on how these GETs can
environmental pollution and production cost. be applied to modify genes to improve NUE in rice.
Several studies have developed germplasms with Moreover, detailed information has been provide
improved NUE through classical plant breeding, on the regulatory policies for genome edited crops
molecular techniques, and genetic engineering around the world and future directions with per­
methods.17 Moreover, genetic engineering spective to NUE.
approaches have not yet been extensively adopted
compared to traditional breeding techniques for the
2 Genetics Mechanism for NUE
development of germplasm with improved NUE.15
Identifying NUE rice genotypes requires a detailed Advances in marker-assisted selection, biotechno­
survey of different morphological, physiological, logical tools, and genomics have helped to reveal
and biochemical traits, along with functional that NUE is multigenic in nature. Genomic regions
genetic studies based on various -omics associated with NUE have been investigated in
approaches18 (Fig. 1). Arabidopsis, rice, maize, and wheat.21–23 The agro­
Genome-editing technology (GET) is a reliable, nomic attributes, namely grain weight, yield, pro­
cost-effective, and versatile approach that has been tein content, and NUE characteristics, namely
widely adopted by plant science researchers. The N harvest index, grain N content, and
associated efficiency in generating genetic modifi­ N remobilization, are considered to be indicators
cations for desirable phenotypes has opened new for NUE in plants. In rice, four quantitative trait
avenues of research.19 However, traditional plant loci (QTLs) responsible for grain N content have
breeding tools and classical GETs have been unable been identified, with two being for shoot N content
to meet the demands of high precision, efficiency, on chromosomes 8, 9, and 10 under both low and
and timeliness, leading researchers to adapt novel normal N levels. Similarly, two QTLs controlling
plant breeding techniques (NPBTs). These NPBTs harvest index and one QTL for physiological NUE
include clustered regularly interspaced short palin­ on chromosomes 5 and 7 were identified, respec­
dromic repeats and CRISPR-associated protein tively. In wheat, major QTLs for root NUE, shoot
(CRISPR/Cas), CRISPR-CRISPR from Prevotella dry weight, and grain yield have been identified.24
and Francisella 1 (Cpf1), base editing (BE), and Moreover, in barley fifteen QTLs influencing NUE
prime editing (PE), and have proven to be powerful have been identified.25 The results of studies per­
tools for successfully modifying genomic sequences formed on different crops are challenging to inter­
in a simple and precise manner.20 The use of these pret and inconsistent, ultimately requiring larger
GETs gas been reported in several crop plants plant populations, the existence of genetic diversity,
where desirable phenotypes were successfully and conducting multiple trials across growing
630 S. FIAZ ET AL.

seasons.2 Moreover, the identified QTLs must be 22 Genetic Mechanisms of N Assimilation

functionally characterized to understand their key
The NH4+ incorporation into plant cells is reduced
roles associated with NUE. The genetic mechan­
to nitrite by the nitrate reductase enzyme in the
isms of N transporters, assimilators, and remobili­
cytosol.35 From there, nitrite is translocated to the
zers are discussed in detail below.
plastids and chloroplasts where it is then converted
to ammonium by the nitrite reductase (NiR)
2.1 Genetic Mechanisms of N Transporters enzyme. This ammonium derived from nitrate, or
that produced by photorespiration or amino acid
N in soil is typically available in the form of NO3−
recycling, is generally assimilated in the plastids by
under aerobic conditions or as NH4+ in a flooded
the glutamate synthase (GS/GOGAT) cycle.10 GS/
situation. Mechanistically, these two forms (NO3−ˍ
GOGAT, a vital enzyme for N assimilation and
and NH4+) are uptaken from the soil by specialized
remobilization, has two isoforms, namely GS1,
N transporters involving two physiological phenom­
which is responsible for primary ammonium
ena, namely the high-affinity transport system
assimilation in roots or re-assimilation of ammo­
(HATS) and low-affinity transport system (LATS).
nium in leaves, and GS2, which regulates ammo­
HATS works under low N concentrations (<250 μM)
nium assimilation in chloroplasts. Among the three
by employing Nitrate Transporter 2 (NRT2) and
GS members in rice, OsGS1.1 and OsGS1.2 are
Ammonium Transporter 1 (AMT1) for the uptake of
reportedly expressed in all organs and a reciprocal
NO3− and NH4+, respectively. Whereas, under the
response to ammonium supply in the rice roots has
LATS system, NPF (NRT1/PTR) works under ele­
been observed. GOGAT is divided into two types,
vated N concentrations (>250 μM) to uptake NO3−
which differ in their electron donor specificity,
and NH4+.26,27 In rice, ammonium transporters
namely ferredoxin-dependent (Fd-GOGAT) and
(AMT) grouped into four subgroups from
NADH-dependent (NADH-GOGAT). Among
OsAMT1 to OsAMT4 among three AMT from
these, one ferredoxin and two NADH-dependent
OsAMT1 involved in high-affinity transport, and
enzymes have been identified in rice plants.36 To
seven members of OsAMT2, OsAMT3, and
increase the overall NUE of a crop plant, it is
OsAMT4 act as low-affinity NH4+ transporters.28
essential to increase the N assimilation efficiency.
AMT transporter proteins are reportedly more effec­
In addition to improvements in N assimilation,
tive in improving NUE than nitrate transporters for
carbon (C) assimilation is also a critical factor
ammonium-preferring rice cultivars. Given that
involving several enzymes; therefore, detecting
NO3− uptake in rice is much lower than NH4+,
enzyme activity is essential for the development of
results have suggested that NO3− and NH4+ efficient
rice varieties with high NUE.37
uptake has the potential to increase NUE along with
the improving rice grain production.15 In the root
zone of rice, ammonium oxidation involves root 2.3 Genetic Mechanisms of N Remobilization
aerenchyma, thus releasing oxygen which leads to
the formation of NO3− which is then taken up by N remobilization is an important process that
plants.29 Nitrate transporter1/peptide transporter involves the translocation of N from old senescing
(NRT1/PTR) family genes are recognized as the plant parts to younger parts during a vegetative
main transporters for nitrate, amino acids, peptides, stage or into storage organs during the reproductive
glucosinolates, indole-3-acetic acid, abscisic acid, stage.38 Although N remobilization is comprised of
etc.27 Genes from this family regulate the transport various metabolic events, it is a vital process to
and allocation of NO3− within the plant body enhance NUE in crop plants.10 In cereal crops,
organs30,31 The NRT1 and NRT2 gene families are a major contribution of the grain N comes from
known as the main regulators of low and high affi­ remobilized N from vegetative organs, and generally
nity transporters in low nitrate environments.32 this contribution ranges 60–92% depending upon
Over 80 genes have been identified in the NRT1 the rate of N remobilization and total
and NRT2 families, but only a select few have were N remobilization efficiency. Seed N content is
found to belong to the NRT1 family.33,34 important for proper germination and subsequent
 GM CROPS & FOOD 631

germinated seedling growth and establishment. N remobilization process.36 Studies have shown
Coordination between N remobilization and senes­ that delayed leaf senescence increases grain yields
cence-induced protein degradation is required to due to an extended photosynthesis duration which
improve N remobilization efficiency because the contributes more photosynthates to the final grain
onset of senescence triggers the translocation of yields; however, this elongation reduces the rate of
N to reproductive organs. In rice plants, N remobilization and subsequently grain
N translocation from senescing organs accounts N content.10 Several genomics studies and QTL
for 80% of N in the rice panicle. GS and GOGAT analyses have highlighted GS involvement in
enzymes are known to regulate this N translocation N remobilization efficiency in various crops.39
to reproductive organs.36 GS1.1 and NADH- Moreover, Fig. 2 shows several genes/gene families
GOGAT1 play an important role during the that play key roles in controlling components of

Figure 2. Genes/Gene families involved in plant NUE (modified from.31,40

Table 1. A list of genes available in rice genome controlling NUE.

Gene Function Source Reference
41
NRT1.1 [NPF 6.3,CHL1) Nitrate transporter Oryza sativa L.
42
NRT2.1 Nitrate transporter Oryza sativa L.
43
NRT3.1 [NAR2.1] Nitrate transport component Oryza sativa L.
44
AMT1.1 Ammonium transporter Oryza sativa L.
45
GS1 Glutamine synthetase [cytosolic] Oryza sativa L.
46
GS2 Glutamine synthetase [plastidic] Oryza sativa L.
47
GOGAT Glutamate synthase Oryza sativa L.
48
AlaAT Aminotransferase Oryza sativa L.
49
ENOD93–1 Early nodulin Oryza sativa L.
50
OsGOGAT1, OsAMT1 Increase NUpE in low N conditions Oryza sativa L.
51
glnA Grain yield in high, moderate and low N conditions Oryza sativa L.
52
ASN1 N content in grains Oryza sativa L.
53
SHMT1 Photosynthesis and grain number per panicle Oryza sativa L.
54
AAP1 Tiller number and grain yield Oryza sativa L.
55
AAP3 Tiller number and grain yield Oryza sativa L.
56
AAP5 Tiller number and grain yield Oryza sativa L.
57
AAP6 Amino acid uptake from roots, Amino acid transport Oryza sativa L.
and grain protein content
58
Pup1/OsPupK46 2/PSTOL1 Tissue P concentration and relative tiller Oryza sativa L.
number
59
qNGR9/DEP1 Plant height response to N Oryza sativa L.
60
TOND1 Relative plant dry weight under N-deficient Oryza sativa L.
to N-sufficient conditions
61
qNGR2/GRF4 Ammonium uptake Oryza sativa L.
62
DRO1 N uptake and leaf N concentration after heading Oryza sativa L.
MADS25 Increase the expressions of NO3 – transporter genes Oryza sativa L. 63
59
DEP1 Ammonium uptake and assimilation Oryza sativa L.
64
NAP NAC transcription factor positively regulating leaf senescence Oryza sativa L.
632 S. FIAZ ET AL.

NUE, such as N remobilization, transporters, 32 CRISPR/Cpf1 Genome Editing System

uptake, and assimilation, which may prove to be
The CRISPR system from Prevotella and Francisella1
valuable targets for GETs. Moreover, a list of genes
is known as Cpf1, heretofore Cas12a. The CRISPR/
available in rice genome has controlling NUE been
Cpf1 gained researchers attention owing to the sig­
given in Table 1. Few genes enlisted in Table 1 have
nificant benefits of efficiency and accuracy in genome
been studied through transgenic approaches how­
manipulation.75 The Cpf1 endonuclease is compara­
ever, there is need to utilize NPBTs to achieve
tively smaller to Cas9 therefore, needs shorter
precision.
CRISPR RNA (crRNA) with more working
efficiency.76 Cpf1 binds upstream of the protospacer
adjacent motif (PAM) guided by single RNA and
3 NPBTs for Targeted Mutagenesis
cleave the DNA at a distance from the seed region,
NPBTs enable plant scientists to make precise mod­ proximal end of the PAM by introducing staggered
ifications to the genome. Classical GETs induce cuts of 5 base pair (bp).77 The Cpf1 system bypass the
double-strand breaks (DSBs) at a particular geno­ need of trans-activating crRNA (tracrRNA) during
mic location and harnesses non-homologous end processing of Cpf1-associated CRISPR repeats to
joining (NHEJ) and homology-directed repair mature into crRNAs.78 This mechanism efficiently
(HDR) pathways for repair. However, the BE and cut the target region to a short T-rich PAM, however
PE system does not require DSBs for genetic Cas9 system require G-rich PAM sequence. The Cpf1
manipulation. These both system holds ability to system keep PAM sequence intact which may vary
generated single base pair mutation with more pre­ based on its origin of ortholog whereas, create tar­
cision. NPBTs are paving the way for further devel­ geted mutagenesis into the desired DNA. There are
opments to modify the genome for broader several online tools especially Cpf1-database which
objectives, especially for food and nutritional helps to find the potential target site and design the
security. gRNA in a fast, easy and simple way. Moreover, the
online Cpf1-database helps to identify Cpf1 and
LbCpf1 through recognition of DNA sequence.79
3.1 CRISPR/Cas9 Genome Editing System
GETs have revolutionized the field of genetic
33 BE Genome Editing System
through efficient and precise manipulation of
genomic DNA.65 The GETs categorized into first Base editing (BE) is a worthy addition to GETs for
generation i.e., meganucleases, zinc finger achieving more efficient genome manipulation with
nucleases (ZFNs), second generation transcription irreversible based conversion at target site. BE is
activator like effector nucleases (TALENs) and much simpler and precise in nature allowing con­
third generation includes CRISPR (clustered reg­ version of nucleotides without formation of DSBs
ulatory interspaced short palindromic repeats)/ within target DNA.80,81 The conversion of cytosine
Cas9 (CRISPR-associated proteins) and related (C) to thymine (T) called cytosine BE (CBE) was
CRISPR/Cas systems.66 In comparison to other firstly developed demonstrated high efficiency.82,83
GETs, CRISPR/Cas9 system is widely exploited The CBE system consist of four elements i) single
by researchers owing to efficient, accurate, easy sgRNA, ii) dCas9, iii) C deaminase and iv) uracil
in handling and cost effective.67 The Cas9 system DNA glycosylase inhibitor (UGI). With the in-
requires short guide sequence (sgRNA) to direct depth molecular understanding of deaminases,
Cas9 nuclease to cleave the target site.68 The Cas9 another system called adenine BE (ABE) developed
holds ability to cleave the double stranded DNA with conversion efficiency of adenine (A) to gua­
target site complementary to sgRNA and success­ nine (G).82,84,85 The BEs restrict indels formation
fully deployed various living backgrounds e.g., both at target and off-target site/s without require­
bacteria,69 eukaryotic cells,70 animal cells, mam­ ment of DSBs DNA modification,86,87 further
malian system71,72 and plants.73,74 allowing single bp conversion i.e., bp substitutions
 GM CROPS & FOOD 633

without depending on donor DNA.80 Recently, sev­ and point mutations with more precision and
eral other BEs have been developed other than CBE efficiency. The investigated nine rice and seven
and ABE e.g., RBEs (conversion from C to U). In wheat lines at protoplasts showed mutation effi­
comparison to the previous GETs, BEs proved ciency of approximately 19.2%.89 The hybridi­
being more efficient, precise and less time consum­ zation of target DNA-pegRNA PBS and target
ing to achieve nucleotide/s substitution in different DNA-reverse transcript) resulting minimum
plant species. off-target effects. The following technique ham­
pered the modification in promoter/introns
easier, allowing the allelic replacement in target
3.4 PE Genome Editing System site feasible. It is noteworthy, the mutation effi­
The recent development in GET has been taken ciency of PE is similar to BE system however,
place with the addition of new technique called showed specificity much higher than previously
prime editing (PE). PE technique allows the discussed GETs. The PE system is at foundation
manipulation of all 12 base-to-base conversion stage further developments and application for
(transition and transversion) bypassing DSBs in crop improvement program will took place with
targeted DNA.88 PE utilize Cas9 nickase bind the passage of time. Lastly, the available litera­
with reverse transcriptase and PE guide RNA ture on GETs application for rice crop
(peg RNA), consist of primer binding site improvement has proved it a viable approach
(PBS), target sequence and a sequence to iden­ to achieve an objective in a shortest period of
tify the targeted site. The PE system has time. The schematic illustration for the applica­
achieved indels from (approximately 44–80bp), tion of GETs for crop improvement program

Figure 3. The basic flow chart of genome editing scheme for rice NUE improvement. (1) Selection of desirable germplasm. (2) The
extraction of genomic DNA from selected germplasm. (3) Primarily analysis of genome through bioinformatics techniques to identify
genes controlling NUE. (4) Selection of gene/genes of interest identified through bioinformatics analysis, available literature/online
database. (5) Selection of target site based on GETs and availability/selection of vector. (6) Construction of vector holding gene of
interest/target site. (7) Vector transformation through different transformation techniques (protoplast, agrobacterium transformation,
and particle bombardment etc. (8) Utilization of Cas genome engineering machinery for targeted modification and extraction of
genomic DNA from transgenic plants for mutation identification analysis. (9) The utilization of designed primers for PCR amplification
of the target gene site to get Sanger sequencing results. (10) Screening of transgenic mutant plants based on Sanger sequencing
results (type of mutation) and phenotypic changes. (11) Selection of transgene-free mutant plants for further collection of (morpho­
logical, physiological and biochemical) phenotypic data and interpretation of results.
634 S. FIAZ ET AL.

has been described in Fig. 3. The GETs panicle size28; OsNPF7.3 (OsPTR6) is involved in
mechanism described and proof of concepts in glutamine synthetase and N uptake30; OsNPF6.3
crop plants established facts to adopt for (OsNRT1.1A) modulates N utilization within the
enhance NUE in rice. rice plant103 ; and OsNRT1.1B regulates nitrate
uptake and translocation.104 Regarding the high-
affinity transporter group, four NRT2 and two
4 Transgenic Approaches for Improving NUE
NAR2 genes have been identified in rice, among
Genetic engineering approaches have been under­ which OsNRT2.3b and OsNRT2.4 work indepen­
taken to enhance NUE in various crops; however, dently. In contrast, OsNRT2.1, OsNRT2.2, and
transgenic plants have proven unable to make any OsNRT2.3a interact with OsNAR2.1 to regulate
significant improvement in NUE for multiple nitrate uptake.105,106 Moreover, numerous studies
reasons90,7 Recently, it has been demonstrated attempted to improve NUE through the overex­
that a specific enzyme, GS, is essential for the pression of N assimilation genes,40 but have had
synthesis of the Gln gene, which is responsible for limited success and inconsistent results.
nitrogen recycling and further influences the reduc­ Additionally, the transgenic plants must be evalu­
tion of nitrogen in pholeum sap in rice.91 Over ated under both high and low N conditions. Some
expression of the GS1 enzyme provides studies have demonstrated that over expression of
a significant improvement in grain yield per plant OsGS1.1 and/or OsGS1.2 enhances GS activities,
in rice.92 In maize, the knockout of gln1-3 and gln1- but there was no significant fluctuation in the rice
4 resulted in a reduced number of kernels and grain yield,107 while another study found that over
kernel yield; however, overexpression of Gln1-3 expression of OsGS1.2 caused an increase in NUE
resulted in a 30% increase in yield.93 When GS1 only under the controlled conditions of a growth
was over expressed in wheat, a significant improve­ chamber.45 Mutation of OsNADH-GOGAT2 (like
ment in root biomass, along with the number of OsGS1.1) was found to cause a reduction in spikelet
ears and grains per plant was observed.94 Based on number, growth rate, and grain filling rate108.
these observations in rice, maize, and wheat, it can Additionally, OsNADH-GOGAT1 (like OsGS1.2)
be assumed that the GS1 enzyme holds significant mutants have been found to have reduced levels
importance for crop improvement and transgenic of amino acids and ammonium ions, along with
approaches can be employed for future studies. The a reduced tiller number.109 Over expression of
phenotypic expression is largely based on transcrip­ NADH-GOGAT has been reported to cause
tion factors involving regulatory networks,95 a significant increase in Indica rice grain weight,47
enzymes, transporters, and genes related to NUE whereas OsGS1.3 regulates ammonium assimila­
that influence nutrient uptake, redistribution, tion in rice grains.36 In rice, three Gln1 genes were
assimilation, and storage.96 identified that encode GS1.39 These Gln1 genes are
Previously, OsAMT1.1 transporter mutant rice differentially expressed within the plant body and
were used to increase the NUE in ammonium- have different isoforms and functions in different
preferring rice.97 In another study, the ammonium plant tissues.110 In this regard, several studies have
transporter OsAMT2.1 was expressed under vary­ identified the genes encoding proteins involved in
ing nitrogen sources and OsAMT3.1 was found to the processes of senescence and N remobilization.21
exhibit weak expression under the same In a study by,111 it was found that cytosolic GS
conditions.98 Several studies have investigated (GS1) re-assimilates ammonium released from pro­
ammonium transport in rice via OsAMT genes tein hydrolysis, which thereby regulates Gln synth­
but have had limited success.99,100 In rice, esis in phloem sap and influences the
OsNPF8.9 (OsNRT1) was characterized as a low- remobilization efficiency in rice.91 Rice mutants
affinity transporter gene responsible for N uptake lacking OsGS1.1 exhibited reduced growth and
through root epidermis,101 and an increase in had a decreased rate of grain filling.121 Similarly,
N content in rice has been reported in response to OsGS1.1 has also been found to be involved in
its over expression.102 Similarly, the PTR gene mediating glutamine generation, including during
OsNPF4.1 (SP1) is responsible for controlling the N remobilization process.112 OsGS1.1 is
 GM CROPS & FOOD 635

essential for rice growth and yield, while OsGS1.2 transgenic rice enhanced the N responsiveness,
and OsGS1.3 are unable to compensate for the loss resulting high grain yield.116 Several studies have
of OsGS1.1.36 documented the key role of the G-protein pathway
The signaling molecule in plants is NO3−, but for N consumption during rice plant development.
signaling is also influenced by genes such as A major genomic region, Dense and Erect Panicles 1
AtNPF6.3/NRT1.1 and protein kinases (e.g., (DEP1), controls the number of panicles ultimately
AtCIPK8 and AtCIPK23).113,114 studied the poten­ yielded.117 The mutant allele, dep1, was found to be
tial role of AtNPF6.3/NRT1.1 in N assimilation and associated with the ammonium transporter
plant growth in rice and reported that overexpres­ OsAMT1.1, ultimately increasing N uptake.59,118
sion of AtNPF6.3/NRT1.1 elevated the reported a transcription factor, AtHY5, responsible
N assimilation under low N concentration. for light regulation. Moreover,,119revealed the
Transcription of DNA-binding One Zinc Finger potential role of AtHY5 in N uptake. In rice, culti­
(DOF) controls hormone signaling, tissue differen­ var with high GS activity to recycle NH3 leave less
tiation, and other biological process in plants.115 NH3 compared to cultivars with less GS activity.120
Transgenic rice for Zea mays Dof1 (ZmDof1) has Another gene, DOF18, induces the ammonium
been developed, and these mutant plants demon­ transporters AMT1, AMT2, and AMT3 to influence
strated increased assimilation of both N and C in ammonium uptake from rice root tissue.53
the roots, along with an increased photosynthesis Transgenic approaches have been successfully uti­
rate.114 Similar findings were reported by,17 where lized with foreign DNA to develop GM crops that
the FERREDOXIN-NADP+ REDUCTASE gene was have gone on to pass through strict ethical, social,
introduced in rice and maize, with the transgenic and biosafety-related regulatory frameworks.
rice showing increased kernel weight while the Genetic engineering mechanism manipulating
transgenic maize displayed improved cob size. genes available in rice genome to improve NUE
The overexpression of the Dof OsRDD1 gene in are listed in Table 2.

Table 2. Transgenic approaches manipulating genes controlling amino acid metabolism and transport to improve nitrogen use
efficiency in rice.
Promoter
Gene Source used Phenotype observed Reference
30
PTR6 Oryza sativa L. Ubiquitin Increased plant growth
121
AMT1.1 Oryza sativa L. Ubiquitin Increased ammonium, uptake and seed yield
98
AMT2.1 Oryza sativa L. CaMV 35S Increased Ammonium uptake
107
GS1 Oryza sativa L. CaMV 35S Increased N, decreased seed yield
46
GS2 Oryza sativa L. CaMV 35S Photorespiration capacity up
47
GOGAT Oryza sativa L. CaMV 35S Increased grain weight
122
AlaAT Hordeum OsAnt1 Increased biomass and seed yield
vulgare L.
51
GDHA Aspergillus CaMV 35S Increased DW, N, yield in field
123
DOF1 Zea mays L. CaMV 35S Increase nitrogen content 30%, enhance growth rate under low N, reduced glucose level
49
ENOD93–1 Oryza sativa L. Ubiquitin Increased shoot biomass and seed yield
124
glnA Escherichia col CaMV 35S Increase grain yield under high, moderate and low N conditions
125
GS1.1, GS2 Oryza sativa L. CaMV 35S Increase N assimilation and plant biomass
126
OsGOGAT1 Oryza sativa L. Activation Increase NUpE in low N conditions; increase N content of grains
tagging
lines
127
ASN1 Oryza sativa L. Ubiquitin Increase N content of grains; no impact on grain yield
51
gdhA Aspergillus CaMV 35S Increase ammonia assimilation and plant biomass under high N conditions
niger
128
GDH Trichurus Ubiquitin Increase N assimilation, thousand grain
weight, grain number and seed protein content under high, moderate and low N field conditions
53
SHMT1 Oryza sativa L. Actin Increased photosynthesis and grain number per panicle
122
ALAAT Hordeum Ant1 Increase plant biomass, NUpE and
vulgare L. final seed yield under high N conditions independently of soil N source [ammonia/nitrate]
129
ALAAT2 Cucumis sativa Ant1 Increase NUpE and grain yield in high and moderate N conditions
L.
54
AAP1 Oryza sativa L. CaMV 35S Increase tiller number and grain yield
55
AAP3 Oryza sativa L. CaMV 35S Decrease tiller number and grain yield
130
AAP5 Oryza sativa L. CaMV 35S Decrease tiller number and grain yield
57
AAP6 Oryza sativa L. CaMV 35S Increase amino acid uptake from roots, amino acid transport and grain protein content at final
harvest; maintain grain yield
636 S. FIAZ ET AL.

5 NPBTs for Improving NUE in Rice technique in plant sciences. The application of var­
ious genome editing techniques targeting various
The green revolution has proven to be
traits in different plant species has been described
a breakthrough in agricultural production to ensure
in detail, and enhancing NUE is no exception.
food and nutritional security. However, the germ­
A CRISPR/Cas9 APOBEC1 BE system has been
plasm production potential remains dependent on
used to target one site each from the NRT1.1B
fertilizer application,131 requiring resources to use
and SLR1 genes. The results demonstrated 1.4–­
efficient germplasms.132 The recent advances in
11.5% C/T substitution while 1.6–3.9% of the edi­
marker-assisted selection, omics approaches, next-
ted plants accounted for C/G replacement. In
generation sequencing, validation of candidate
another study, the BE technology using the rat
genes, gene expression analyses, and GETs have
cytidine deaminase enzyme (APOBEC1) has been
aided in the development and screening of potential
successfully employed to induce point mutations in
germplasms to achieve NUE genotypes. Sustainable
two agriculturally important genes, NRT1.1B and
agriculture requires crop germplasms with premium
SLR1, in rice.135 NRT1.1B encodes a nitrogen trans­
yield, resistance to biotic and abiotic stresses, envir­
porter and SLR1 encodes a DELLA protein. As
onmental resilience, resource use efficiency, and less
previously reported, a C/T replacement
dependency on artificial fertilizers. The NUE is
(Thr327Met) in NRT1.1B could increase NUE in
a ratio of yield to N supply, indicating whether
rice,104 and an amino acid substitution in or near its
the developed germplasm is lacking in NUE.
TVHYNP motif results in reduced plant
Conversely, in many parts of the world, especially
height.104,136 The successful application of GETs
in developing countries, low-nutrients soils are
has demonstrated the potential for improving
common and there is often neither the funds nor
NUE not only in rice but in many other crops
the infrastructure to provide N-based fertilizers to
important for food and nutritional security. The
small farmers. Therefore, geneticists are selecting
availability of genomics data can be further
genotypes/hybrids with the ability for high yield
exploited to achieve desirable phenotypic manipu­
under low N for small farmers. The strong associa­
lation to support sustainable agricultural develop­
tion between yield under high and low N allows
ment. Based on the successful utilization, it can be
breeders to select for broad adaptability in nutrient-
assumed that GETs hold the potential for a second
replete soils. The classical example of selecting for
green revolution to achieve the United
a plant’s ability to utilize N efficiently is Norman
Nations second sustainable development goal of
Borlaug’s introduction and selection of dwarfing
zero hunger. The successful application of genetic
genes that resulted in semi-dwarf high yield culti­
engineering approaches and GETs in crop plants
vars. These genes (Rht-B1 and Rht-D1), which were
are dealt same in several countries and strict reg­
originally derived from a cross between a Japanese
ulatory regimes are enforced thus require discus­
variety of dwarf wheat (Norin 10) and a high-
sion among all stakeholder involving researchers,
yielding American variety (Brevor), became the
policy makers and farming community.
model for the use of dwarfing genes to produce
plants that use higher levels of N without the lod­
ging that is common in tall varieties.133 The dwarf­ 6 Regulatory Aspects for Genome Edit Crops
ing genes altered stem strength and plant
The NPBTs is widely adapted for genome alterna­
architecture and indirectly generated plants that
tion of crop plants. The GETs are a valuable
could produce much higher yield under high (stan­
resource for the improvement of agricultural
dardized) levels of fertilizer and hence had
crops to withstand biotic, abiotic stresses and to
enhanced NUE.133
develop environmentally resilient crops.20 The
To resolve the regulatory concerns of trans­ application of NPBTs in plant sciences has raised
genics, NPBTs that are faster, more predictable, regulatory concerns both at national and interna­
and can be utilized in a wide range of plant species tional arena to ensure biological, ecological safety,
have been developed.134 Genome editing through associated risk management, and legal guidelines
endonucleases is the most widely adopted on misuse of such sophisticated technologies.137
 GM CROPS & FOOD 637

The NPBTs holds potential to resolve global nutri­ state council of China formulated “Regulation on
tional and food security concern however, there is Administration of Agricultural Genetically
a need of discussion among various stakeholders to Modified Organisms Safety”, categorized genome
differentiate among transgenic i.e., GM and gen­ edit with GM crops.146 Similarly, the Indian,
ome edit crop plants. The anti-GM campaign is Japanese and New Zealand regulatory bodies cate­
based on, i) the insertion of foreign DNA to plant gorize the genome edit crops similar to GM apply­
genome causing harmful impact on human health ing strict biosafety guidelines.147,148 Therefore, the
and ii) the insertion of T-DNA with antibiotic already existing regulatory framework in particular
resistance genetic factors e.g., Golden Rice and Bt countries are applied on genome edit crops.
Cotton. These arguments are perfectly resolved Moreover, the advancements in GETs to produce
through NPBTs, GETs modify the endogenous transgene free plants may help avoid the enforced
genes similar to natural variations furthermore, biosafety related regulations as followed in conven­
GETs holds ability to introduce the point mutations tional transgenic plants.142 In nutshell, it’s the
in any gene of interest, not possible to achieve responsibility of all stakeholders to debate the reg­
through classical GETs.1 The non-target mutation ulatory framework and came up with uniform reg­
effects are reduced to minimum level through ulations promoting the safety of humans, animals,
employing Cas9 variants e.g., Cpf1, base editing plants and the environment.
and prime editing.138 The gene transformation
methods in GETs have made these techniques reli­
7 Future Directions
able and bio-safe e.g., Agrobacterium tumefaciens,
a soil-borne bacteria used for gene transformation Based on the revolution of molecular biology and
contain natural DNA, allowing to obtain transgene the discovery of CRISPR sequences in the microbial
clean plants to bypass strict GM regulations.139 immune system, biotechnologists are now able to
The large debate on GM and Genome edit crops manipulate the any genome of interest in a specific
require governmental intervention to formulate and precise way. These NPBTs have provided abil­
clear and uniform regulatory policies. The ity to plant scientists for the precise and quick
Cartagena Protocol on Biosafety advanced under­ insert/manipulation of desirable traits than conven­
standing for the international trade of GM organ­ tional breeding.
isms/plants however, still several governments have
a divergent opinion on development, commerciali­
7.1 Gene Regulation
zation, production and consumption.140 Presently,
the genome edit crops are dealt with under two The genome editing techniques has been utilized
regulatory guidelines, i) process-based and, ii) pro­ not only for gene knockout and knockin but also
duct based.141,142 Moreover, the regulation for gen­ for genetic regulations. The genome regulations
ome edit crops varies among countries as few primarily consist of activation or repression of
nations deal with genome edit crops same as GM genes achieved through fusion of transcriptional
others deal with such crops as non-GM.141 For activators or repressors with DNA-domains of vec­
instance, United States of America and Brazilian tor constructs i.e., dCas9, targeting only the regu­
government agreed to regulate genome edit crops latory domain of endogenous genes.73 Cas9
similar to developed through conventional technology has successfully edited SlCLV3 promo­
breeding,143 Canadian regulatory guidelines states ters in Solanum lycopersicum generating regulatory
any plant based technology to develop new attri­ mutation.149 To modulate the translation of
butes require to go through Canadian Food mRNAs the upstream open reading frame of
Inspection Agency regulations.144 The Court of LsGGP2 resulted tolerant Lactuca sativa for oxida­
Justice of the European Union (ECJ) has declared tive stress and elevated ascorbate content.81 The
crops produced via NPBTs regulated the same as GETs influence the transcript level, and hold ability
GMOs however, traditional mutagenic techniques to manipulate the normal function of non-
with established biosafety records are exempted.145 canonical RNAs for crop improvement. The GETs
To ensure management and risk assessment, the can engineer transcription mechanism of such
638 S. FIAZ ET AL.

RNAs directly to understand their underlying func­ Therefore, the available genetic information
tion. Based on these observation, the gene regula­ for resource use efficiency in different crop
tion mechanism can be exploited for activation/ species can be easily manipulated to achieve
repression of genomic regions controlling NUE in food security.
rice.
7.4 Targeted Epigenetic Modification
7.2 Mutant Libraries
The advancement in technologies had provided
The complete genome sequence of several crops opportunities to investigate chromatin modifi­
e.g., Oryza sativa, Triticum aestivum, Zea mays, cations, gene expression, and genome
Gossypium hirsutum, Glycine max is available how­ structure.155 Plants are heavily dependent on
ever, to analyze the functional aspects of genes is epigenetic modifications to respond to environ­
challenging in post-genomic era. The 3 K rice gen­ mental stimuli therefore, these modifications
ome project has enabled to get genome sequence are crucial. The alternation in epigenome can
data of rice mega-varieties grown across large areas elevate the activities of promoters for genes
and under different ecosystems.150 To validate the related to biotic and abiotic stresses moreover,
functional aspects of genes influencing NUE in can activate the silent genes to generate novel
plant species through GETs are considered as an traits for crop improvement. The epigenetics
effective strategy therefore, the high throughput modification activate the endogenous gene
mutant libraries at whole genome level can proved expression through targeting a fusion protein
to be a useful resource for elevation of NUE in crop of dCas9 and DNA methyl transferase oracetyl
improvement programs. transferase to plant promoters utilizing gRNAs.
The methyl transferase function can be altered
via dCas9 and gRNA in plant genome target
7.3 Multiplexing and Gene Stacking
site to modify epigenetic makeup to achieve
In plants, the metabolic pathways are responsi­ desirable gene expression. There is few litera­
ble for traits with economic importance. These ture describing exploitation of targeted genetic
metabolic pathways are controlled by complex modifications for NUE and it can be recom­
genetic networks within a cellular system. mended for generation of germplasm with
Therefore, molecular techniques holding ability improved NUE.
to manipulate several genes altogether are of
great importance in both basic and applied
7.5 Transgene Free Editing
research.32 The GETs allow the genetic manip­
ulation of several genes through multiplexing, The introduction of foreign DNA into the plant
editing multiple target sites.70 The application genome has arisen regulatory concerns and
of Golden Gate cloning or Gibson Assembly regarded as GMOs.156 Following the development
method, multiple gRNAs were assembled driven of precision genome editing the researchers have
by different promoters.151,152developed a simple focused on the generation of transgene free genome
strategy to engineer endogenous tRNA through edit plants. The removal of Cas9 gene would also
simple and robust method expanding targeting help to reduce the off-target mutation.157 Earlier
and multiplex editing through CRISPR/Cas9 the percentage of transgene clean plants was much
system. The CRISPR/Cpf1 system had dual lower however, the novel developments e.g., BE and
nuclease to cleave targeted DNA and its own PE systems enabled to generate higher number of
CRISPR RNA.153,154 demonstrated feasibility of transgene clean plants. A combination of BE system
multiplex editing in rice through Cpf1 system. and DNA-free editing successfully deployed in
Moreover, multiple sgRNAs can also utilized to wheat with C-to T conversion of 1.8%.158, 159 The
elevate genome editing in model and non- ability to generate plants without transgene can
model crop plants with low gene transforma­ help to skip strict regulatory regime adapted by
tion or induced mutation rate percentage. several countries.
 GM CROPS & FOOD 639

8 Conclusion Acknowledgments
The elevation in global rice production at low cost is The authors are thankful to the Department of Plant Breeding
vital for sustainable food and nutritional security. and Genetics, The University of Haripur for providing
The improvement in NUE is a key constituent for research conducive environment.
agronomic, economic and environmental aspects
therefore, plant breeders and molecular biologists
are taking it as challenge. The NUE being polygenic Disclosure Statement
and complex in nature is a hotspot for dissecting the The authors declare that the research was conducted in the
genetic mechanism through classical and NPBTs in absence of any commercial or financial relationships that
rice. So far, several genomic regions have been iden­ could be construed as a potential conflict of interest.
tified playing their integral role in controlling NUE.
The availability of 3000 rice genome project database
can further be utilized to understand the underlying Funding
genetic factors influencing the N transportation, The publication of the present work is supported by the
assimilation and remobilization. Previously, the National Key Research and Development Program of China
transgenic approaches successfully exploited (grant no. 2017YFC0504704) and the National Natural
through over expression of genes controlling NUE, Science Foundation of China (51669034, 41761068,
thus providing the opportunity to explore negatively 51809224).
regulation genes for the development of resource use
efficient crops with better agronomic traits. The
recent developments in NPBTs have enabled plant ORCID
scientists to modify the genome of a model and non- Sajid Fiaz http://orcid.org/0000-0001-9097-4359
model plant species through targeted engineering of Mehmood Ali Noor http://orcid.org/0000-0003-2459-2357
attributes essential for biotic, abiotic stress-resistant, Badr Alharthi http://orcid.org/0000-0003-4515-1171
environmentally resilient and resource use efficient
crops. The GETs have revolutionized biological
research, from novel traits developments, epigenetic References
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