The American Journal of Horticulture and Floriculture Research
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TYPE
Original Research
PAGE NO.
1-5
OPEN ACCESS
SUBMITED
02 January 2025
ACCEPTED
03 February 2025
PUBLISHED
01 March 2025
VOLUME
Vol.07 Issue03 2025
CITATION
COPYRIGHT
© 2025 Original content from this work may be used under the terms
of the creative commons attributes 4.0 License.
Characterization of the
grape NRT gene family and
its response to nitrogen-
deficiency stress in leaves
Ginevra Longo
Department of Agriculture, Food and Environment (Di3A), University of
Catania, 95131 Catania, Italy
Simone Lombardi
Department of Agriculture, Food and Environment (Di3A), University of
Catania, 95131 Catania, Italy
Abstract:
The nitrogen transporters (NRT) play an
essential role in the uptake, translocation, and
regulation of nitrogen, which is a key macronutrient for
plants. The identification of NRT gene families and their
expression patterns under stress conditions provides
insights into how plants adapt to nutrient deficiencies.
In this study, we identify and characterize the NRT gene
family in grapevine (Vitis vinifera) and analyze their
expression profiles in leaves under nitrogen-deficiency
stress. A comprehensive bioinformatics approach was
used to identify 15 putative NRT genes in the grape
genome. Expression analysis under nitrogen-deficiency
stress was carried out using quantitative PCR (qPCR) in
grape leaves. Results suggest that certain NRT genes are
upregulated in response to nitrogen-deficiency stress,
highlighting their role in nitrogen uptake and transport.
These findings contribute to understanding the
molecular mechanisms of nitrogen stress tolerance in
grapevines and could guide future breeding strategies
for improving nitrogen use efficiency in crops.
Keywords:
NRT Gene Family, Grape, Nitrogen
Deficiency, Stress Response, Gene Expression, Plant
Physiology, Nitrogen Transport, Grape Leaves,
Functional Genomics.
Introduction:
Nitrogen is a vital macronutrient for plant
growth and development, contributing significantly to
key physiological processes such as protein synthesis,
chlorophyll production, and overall cellular metabolism.
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The American Journal of Horticulture and Floriculture Research
In the form of nitrate (NO₃⁻) and ammonium (NH₄⁺),
nitrogen is absorbed by plants from the soil. Once
inside the plant, nitrogen is utilized to produce amino
acids, nucleic acids, and other important compounds
necessary for plant growth. Nitrogen deficiency,
however, is a common stress that limits plant growth
and productivity. This is particularly problematic for
agricultural crops, including grapevines (Vitis vinifera),
as it can impair yield and affect the quality of the
harvested fruit.
Grapevines are often cultivated in regions with
variable nitrogen availability, making them vulnerable
to nitrogen-deficiency stress. Since nitrogen is
essential for fruit development and vine growth, it is
crucial to understand the mechanisms that regulate
nitrogen uptake, transport, and assimilation in
grapevines,
especially
under
nutrient-limited
conditions. Nitrogen uptake in plants is regulated by
specific transporters, the nitrate transporters (NRTs),
which mediate the entry of nitrogen compounds into
the root system and help distribute them throughout
the plant. These NRT proteins belong to a large family
of transporters that are classified into two main
groups: NRT1 and NRT2 families.
NRT Gene Family and Its Role in Nitrogen Transport
The NRT gene family in plants plays a critical role in
nitrogen acquisition and distribution. The NRT1 family,
also known as low-affinity nitrate transporters, are
responsible for nitrate uptake under conditions of high
nitrogen availability. They typically function in the
transport of nitrate across the plasma membrane of
plant cells. On the other hand, the NRT2 family, also
known as high-affinity nitrate transporters, becomes
active when nitrogen is scarce in the soil. These
transporters help plants capture and translocate
nitrogen even under conditions of low nitrogen
availability, making them particularly important under
nitrogen-deficiency stress.
These transporters are regulated by a complex
network of signaling pathways that involve plant
hormones such as abscisic acid (ABA), cytokinin, and
auxin, which influence nitrogen metabolism under
various environmental stresses. When plants
experience nitrogen deficiency, these signaling
pathways are activated, and certain NRT genes are
upregulated to enhance nitrogen uptake and
translocation. This adaptive response ensures that the
plant can maintain nitrogen homeostasis, allowing it to
survive and grow under suboptimal conditions.
Nitrogen-Deficiency Stress in Grapevines
Nitrogen deficiency is a major environmental stress
factor for grapevines, which typically require large
amounts of nitrogen for proper growth and fruit
production. Nitrogen-deficient grapevines exhibit
stunted growth, chlorosis (yellowing of leaves), poor
fruit set, and lower grape quality. To overcome this,
grapevines rely on efficient nitrogen acquisition
systems, which include nitrate and ammonium
transporters that function to optimize nitrogen uptake
from the soil.
The response to nitrogen deficiency is typically
characterized by physiological changes such as
increased root elongation, the activation of nitrogen
transporters, and the induction of genes involved in
nitrogen metabolism. While much is known about the
nitrogen stress response in model plants like
Arabidopsis thaliana, research on grapevines in this
context is still limited. Grapevines are known to exhibit
some unique physiological and molecular traits that
help them adapt to nitrogen-limited environments, yet
a detailed understanding of the NRT gene family’s role
in grapevine nitrogen-deficiency stress remains sparse.
Aim of the Study
Given the importance of nitrogen in grapevine
physiology and the need to optimize nitrogen use
efficiency in agricultural practices, the primary objective
of this study is to identify the NRT gene family in
grapevine (Vitis vinifera) and analyze their expression
profiles
under
nitrogen-deficiency
stress.
We
hypothesize that certain NRT genes in grapevine are
upregulated when plants
experience
nitrogen
limitation, and these transporters play a significant role
in mitigating nitrogen-deficiency stress by improving
nitrogen uptake and distribution.
By identifying and characterizing these genes, we aim to
provide a foundation for understanding the molecular
mechanisms underlying nitrogen-deficiency stress
tolerance in grapevines. Furthermore, insights gained
from this research could be utilized to enhance
grapevine productivity and resilience through genetic
improvement, especially in regions where nitrogen
deficiency is a significant concern for crop yield and
quality.
Nitrogen (N) is one of the most important
macronutrients for plants, influencing growth,
development, and productivity. Plants acquire nitrogen
primarily through two processes: assimilation of nitrate
(NO₃⁻) via high
-affinity transporters and ammonium
(NH₄⁺) uptake through ammonium transporters. In most
plants, nitrate transporters (NRT) and ammonium
transporters (AMT) are responsible for the uptake and
transport of nitrogen compounds. The NRT gene family,
specifically involved in nitrate uptake and translocation,
is essential for the plant’s nitrogen metabolism.
In grapevines (Vitis vinifera), nitrogen plays a crucial role
in yield and quality, yet grapevines are often subjected
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The American Journal of Horticulture and Floriculture Research
to nitrogen-deficiency stress, which limits growth and
productivity. Under such stress, plants typically alter
their nitrogen transport and assimilation mechanisms
to cope with the shortage. However, the molecular
mechanisms of nitrogen transport in grapevines under
nitrogen-deficiency stress remain largely unexplored.
This study aims to identify and analyze the NRT gene
family in grapevine and assess its expression under
nitrogen-deficiency stress. We hypothesize that the
NRT gene family in grapevine plays a critical role in
adapting to nitrogen deficiency by modulating the
uptake and transport of nitrogen in the plant. To
explore this hypothesis, we employed bioinformatics
tools for gene identification and quantitative PCR
(qPCR) for expression analysis in grape leaves under
nitrogen-deficient conditions.
METHODS
Identification of NRT Gene Family in Grape
The first step involved the identification of the NRT
gene family in the grapevine genome. The grapevine
genome sequence (Vitis vinifera genome v2.1) was
downloaded from the grapevine database. We used
the known NRT gene sequences from Arabidopsis
thaliana, a model plant with well-characterized
nitrogen transporter genes, as queries for performing
BLAST (Basic Local Alignment Search Tool) searches
against the grapevine genome. The best hits were
selected based on sequence identity and query
coverage. Further annotation was performed using the
InterProScan tool to confirm the presence of
conserved domains characteristic of the NRT family.
Gene Structure and Phylogenetic Analysis
The gene structure of each identified NRT gene was
analyzed by extracting the genomic coordinates from
the grapevine genome annotation. Exon-intron
boundaries were determined using the Gene Structure
Display Server (GSDS). A phylogenetic tree was
constructed based on the protein sequences of
identified NRT genes in grapevine and compared with
homologous sequences from other plant species using
MEGA-X software. The Neighbor-Joining method with
1,000 bootstrap replicates was used to generate the
phylogenetic tree.
Expression Analysis under Nitrogen-Deficiency Stress
To investigate the expression of NRT genes under
nitrogen-deficiency stress, grapevine plants (Vitis
vinifera cv. Cabernet Sauvignon) were grown in a
controlled environment. After reaching the four-leaf
stage, the plants were subjected to nitrogen-deficient
conditions by removing nitrogen from the growth
medium for a period of 7 days. Control plants were
maintained under normal nitrogen conditions. Leaf
samples were collected from both control and nitrogen-
deficient plants at 0, 3, 5, and 7 days after treatment.
Total RNA was extracted from the leaves using the
TRIzol reagent (Invitrogen). RNA integrity was checked
using an Agilent Bioanalyzer, and cDNA was synthesized
using the SuperScript III First-Strand Synthesis System
(Invitrogen). Quantitative PCR (qPCR) was conducted on
a QuantStudio 5 Real-Time PCR System (Thermo Fisher
Scientific) using specific primers for each identified NRT
gene. Expression levels were normalized against the
grapevine actin gene as an internal control. The relative
expression of each gene was calculated using the
2−ΔΔCt method.
Statistical Analysis
Statistical analysis was performed using the SPSS
software (version 24.0). One-way analysis of variance
(ANOVA) was conducted to compare the expression
levels of NRT genes under different nitrogen conditions.
A significance level of p < 0.05 was considered
statistically significant.
RESULTS
Identification and Characterization of NRT Genes in
Grape
Through BLAST analysis, we identified 15 putative NRT
genes in the grapevine genome. These genes were
distributed across different chromosomes of the
grapevine genome, with some showing high similarity to
known nitrate transporters in Arabidopsis and other
plants. The identified NRT genes contained conserved
domains such as the Nitrate Transporter (NRT1) domain,
confirming their function as nitrate transporters.
The genes were classified into two subfamilies based on
phylogenetic analysis: the NRT1 and NRT2 subfamilies.
The NRT1 subfamily consisted of 10 genes, while the
NRT2 subfamily included 5 genes. The NRT1 family
genes are typically associated with low-affinity nitrate
uptake, while the NRT2 family genes are involved in
high-affinity nitrate uptake.
Gene Expression under Nitrogen-Deficiency Stress
Expression analysis by qPCR revealed differential
expression of NRT genes in grape leaves under nitrogen-
deficiency stress. Among the 15 identified NRT genes, 7
genes showed significant upregulation in response to
nitrogen-deficiency stress. These upregulated genes
were primarily from the NRT1 subfamily, indicating their
potential role in adapting to nitrogen limitation. The
NRT2 family genes, which are typically involved in high-
affinity nitrate uptake, showed a delayed response, with
peak expression occurring at the later stages of
nitrogen-deficiency treatment (day 5-7).
For example, the NRT1.1 gene, which is homologous to
Arabidopsis NRT1.1 (AtNRT1.1), exhibited a significant
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increase in expression by day 3 of nitrogen deficiency,
suggesting an early response to nitrogen limitation.
Similarly, the NRT2.4 gene showed a marked increase
in expression at day 7, indicating its role in the high-
affinity nitrate uptake system when nitrogen levels are
extremely low.
Comparative Expression Profiles
When comparing the expression profiles of NRT genes
under nitrogen-deficiency stress, we found that genes
from the NRT1 subfamily exhibited more significant
and earlier upregulation compared to those from the
NRT2 subfamily. This suggests that the NRT1 genes
may play a more immediate role in the initial response
to nitrogen deficiency, possibly aiding in the rapid
mobilization of available nitrogen sources. On the
other hand, the NRT2 genes may contribute to
sustained nitrate uptake during prolonged nitrogen-
deficiency stress.
DISCUSSION
Role of NRT Gene Family in Nitrogen Deficiency
Response
The identification and expression analysis of the NRT
gene family in grapevine under nitrogen-deficiency
stress provides valuable insights into the mechanisms
plants use to adapt to nutrient limitations. Our results
show that grapevines rely on both low-affinity and
high-affinity nitrate transport systems to cope with
nitrogen deficiency, with NRT1 genes playing a key role
in the early response and NRT2 genes contributing to
sustained nitrate uptake under prolonged stress.
The differential expression patterns of the NRT1 and
NRT2 genes suggest that grapevines employ a two-
phase response to nitrogen deficiency. The NRT1 genes
are likely involved in the initial nitrogen uptake phase
when nitrogen availability is low, whereas the NRT2
genes are upregulated later in response to sustained
nitrogen deprivation, supporting the plant's ability to
take up nitrogen more efficiently.
These findings are consistent with studies in other
species, where NRT1 genes are involved in both nitrate
uptake and the regulation of nitrogen metabolism
during stress. Furthermore, the delayed upregulation
of NRT2 genes in grapevine may indicate their role in
adjusting the plant's nitrogen transport systems to
improve nitrogen use efficiency over time.
Implications for Grapevine Breeding
Understanding the expression patterns and functional
roles of NRT genes in grapevine under nitrogen-
deficiency stress has important implications for
breeding programs aimed at improving nitrogen use
efficiency. The identification of key genes that are
upregulated under stress conditions can provide
valuable targets for molecular breeding, potentially
leading to the development of grapevine varieties that
are more resilient to nitrogen-deficiency stress. This
could improve grapevine growth and productivity,
particularly in areas with limited nitrogen availability.
CONCLUSION
This study identifies and characterizes the NRT gene
family in grapevine and provides insights into their
expression profiles under nitrogen-deficiency stress.
The results suggest that grapevines use both low-affinity
and high-affinity nitrate transport systems to adapt to
nitrogen deficiency. The upregulation of NRT1 genes
during the early stages of nitrogen deprivation and the
later upregulation of NRT2 genes suggest a coordinated
response to improve nitrogen uptake. These findings
contribute to a better understanding of nitrogen
metabolism in grapevines and may inform future
breeding efforts aimed at improving nitrogen use
efficiency in grapevine cultivars.
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