Identification of Grape NRT Gene Family and Analysis of Its Expression in Leaves Under Nitrogen-Deficiency Stress

Abstract

Nitrogen, an indispensable macronutrient, significantly affects the appearance, quality, and yield of grapes (Vitis vinifera). Adequate nitrate uptake and intracellular transport, facilitated primarily by nitrate transport (NRT) proteins, are crucial for maintaining nutritional balance. However, there are no reports on the NRT gene family in grapes. In this study, we identified 53 Nitrate Transporter 1/Peptide Transporter Family (NPF), 3 nitrate transporter 2 family (NRT2), and 1 Nitrate Assimilation-Related 2 (NAR2) genes in the grapevine Pinot Noir PN40024 genome. A comprehensive analysis of these gene families, including their physicochemical properties, structural organization, chromosomal distribution, collinearity, cis-acting element distribution, and phylogenetic relationships, revealed the rich diversity and evolutionary conservation of the grapevine Nitrate Transporter (NRT) genes. Furthermore, the expression profiles of VvNRTs in different tissues demonstrated that the NRT genes possess spatio-temporal expression specificity. The expression patterns of the NRT genes were examined by transcriptome sequencing in grapevines across various tissues under nitrogen-deficiency conditions. The expression patterns in grapevine leaves under nitrogen-deficiency conditions showed upregulation of the VvNPF2.3 gene in conditions of nitrogen deficiency. This upregulation was strongly associated with a 62.2% reduction in indole-3-acetic acid (IAA) levels and a 21.3% increase in hydrogen peroxide (H₂O₂) levels, suggesting a complex regulatory response to nitrogen-induced stress. These findings emphasize the potential involvement of NRT genes in the adaptive reaction to nitrogen deficiency and set the stage for future investigations into the molecular mechanisms of nitrogen transportation in grapevines.

1. Introduction

Nitrogen, an essential nutritional element for plants, is integral to the synthesis of key biomolecules such as amino acids, proteins, enzymes, nucleic acids, and chlorophyll [1,2,3], highlighting its fundamental role in plant growth and development. While an adequate nitrogen supply fosters healthy and robust plant growth, natural ecosystems often exhibit nitrogen scarcity, prompting plants to compete with other organisms for this vital resource [4,5,6]. To overcome nitrogen resource limitations, plants have developed sophisticated strategies for nitrogen acquisition, assimilation, and recycling from diverse sources [7] by absorbing nitrate (NO₃⁻) or ammonium (NH₄⁺) [8] directly from the soil, with nitrate typically being the predominant and most accessible form of inorganic nitrogen [9]. Furthermore, through symbiosis with soil microorganisms, plants can access organic nitrogen sources such as amino acids, peptides, and proteins [10,11]. Remarkably, specific plants, including legumes in association with rhizobia and certain ferns with cyanobacteria [12], establish symbiotic relationships with nitrogen-fixing (NF) bacteria, depicting their role in converting atmospheric nitrogen (N₂) into plant-absorbable forms such as ammonium and thus ensuring a reliable nitrogen supply.

Nitrate absorption predominantly occurs in root cells, as nitrate from the soil reaches the root surface through processes such as surface runoff and is absorbed via active transport mechanisms on the root cell plasma membrane [13,14]. This process is primarily facilitated by specific nitrate transport proteins, categorized into high-affinity transport systems (HATS), which are mainly induced by lower concentrations of nitrate in the soil, and low-affinity transport systems (LATS), which adapt to varying nitrate concentrations in the environment. The dual-affinity transporter NRT1.1 is pivotal for sensing and absorbing nitrate in plants [15,16]; this transporter exhibits Km values of approximately 4 mM for LATS and 40–80 μM for HATS [17].

The NRT proteins are mainly divided into three subfamilies: NRT1/NPF, NRT2, and NRT3 [18]. The NPF gene family members are diverse and play pivotal roles in nitrate uptake, transport, and distribution within plants. Unlike the NPF family, the functional activity of NRT2 proteins often depends on the assistance of the ancillary protein NAR2 [19]. These subfamilies are conserved in various crops, including 62 AtNRT genes in Arabidopsis thaliana [20], 67 in spinach [21], 48 in pineapple [21], and 79 in poplar trees [22]. NRT family members are differentially expressed across plant tissues and organs and are responsible for nitrate sensing, absorption, and transport [13,23,24]. For instance, AtNRT1.1 in A. thaliana mediates root nitrate absorption and transports nitrate to shoots, exhibiting auxin transport activity [25]. AtNRT1.9 facilitates nitrate entry into the root phloem [26], whereas AtNRT2.5, which is predominantly expressed in the roots, is implicated in high-affinity nitrate absorption under lower external nitrate conditions. Additionally, AtNRT2.5 contributes to the transport of nitrate from underground to aboveground phloem parts, while in Oryza sativa, OsNRT2.3 expressed in the xylem is involved in nitrate transport [27]. Recent progress in nitrogen research has highlighted the functional diversity of NRT genes in crops such as tomatoes and apples. For example, MdMYB10 in red-fleshed apples regulates MdNRT2.4-1 [28], influencing nitrogen uptake and distribution, whereas overexpression of SsNRT1.1D in Suaeda salsa improves salt tolerance in transgenic tomatoes [29].

Nitrogen fertilizer application is essential for enhancing grape yield and quality, resulting in it becoming a common practice in grapevine cultivation. However, excessive use of fertilizers can lead to soil degradation, damaged grape growth, decreased yield, and deterioration of fruit quality [30,31]. The NRT gene family is crucial for nitrate uptake and assimilation and significantly influences grapevine growth, fruit development, and overall production. It may also affect grape quality by altering its response to nitrogen availability, which is associated with sugar and nutritional concentrations in berries [32,33]. It has been reported that the NRT genes, particularly VvNPF6.5, have been shown to enhance nitrogen use efficiency in grapevines [34]. Therefore, a better understanding of the mechanisms involved in plant nitrogen uptake and transport is required. The NRT gene family plays a role in this process. However, the systematic identification and functional characterization of grapevine NRT genes are lacking.

This study successfully identified 57 NRTs using a hidden Markov model (HMM). An in-depth analysis of these genes, including protein characteristics, conserved structural domains, motifs, phylogenetic relationships, cis-acting elements, and homology correlations, revealed conservation and interrelationships between orthologous and paralogous genes. This study investigated the response of NRT genes in leaves to nitrogen-deficiency stress and their impact on leaf growth. These findings provide a fundamental basis for understanding the biological functions of grapevines in nitrogen uptake, transport, and utilization.

2. Materials and Methods

2.1. Plant Material and Handling

Shine Muscat grapevine cultivar seedlings were used as the experimental material in this study. Following 3 weeks of growth in a nutrient substrate, seedlings with consistent growth were selected and cleaned, the substrate was removed from their roots, and they were then transferred to a 10% Hoagland nutrient solution for an additional week of cultivation. Subsequently, the seedlings were subjected to two separate nutritional regimes: a complete nutrient solution and a nitrogen-deficient nutrient solution. The nutrition solutions were renewed every three days to ensure consistent nutrient availability. Samples were collected one week after the initiation of the treatments for subsequent experimental analyses. The experimental design was completely randomized, and each treatment condition was replicated thrice. Detailed information regarding the specific nutrient concentrations used in the treatments is provided in Table S1.

2.2. Identification of NRTs in Grapevines and Amino Acid Characteristic Prediction

The utilization of the NCBI database (https://www.ncbi.nlm.nih.gov/, accessed on 21 June 2024) facilitated the identification of nitrate transporter genes in grapes by acquiring the genome sequence and gene annotation of Pinot Noir PN40024 [35]. In order to perform a comparative analysis, sequences of Arabidopsis genes were obtained from the TAIR database (https://www.arabidopsis.org/, accessed on 21 June 2024) to retrieve the sequences of Arabidopsis NRT gene family members, and then, conserved domains (PTR2 (PF00854), MSF_1 (PF07690), and NAR2 (PF16974)) of NRT proteins were obtained from InterPro (https://www.ebi.ac.uk/interpro/, accessed on 21 June 2024). Candidate sequences were identified using HMMER 3.0, and high-confidence sequences (E value <1 × 10⁻²⁰) were selected as candidate sequences. These sequences were further analyzed using SMART33 and the NCBI Conserved Domain Database (CDD) to confirm the conserved structure and determine the member of the grape NRT gene family. Finally, the grape NRT gene family members were described and designated according to their gene homology positions.

2.3. Phylogenetic Tree Construction and Homologous Correlation Analysis

Phylogenetic trees were constructed based on the protein sequences of NRT proteins from Arabidopsis and grapes. The alignment of gene protein sequences of NRT from Arabidopsis and grape was performed using ClustalW within the MEGA software.v11 [36] software, following default parameters. In the aftermath, phylogenetic trees were formulated utilizing the maximum likelihood (ML) method [37], with a bootstrap value of 1000. High-quality phylogenetic tree plots were constructed using TBtools [38]. An examination aiming to explore tandem and segmental duplications of NRT genes in grapes was conducted via collinearity correlation analysis with MCScanX, leveraging gene annotations and whole-genome sequences of grapes and A. thaliana. The TBtools software.v2.154 was then utilized for the visualization and annotation of NRT gene collinearity.

2.4. Analysis of Grapevine NRT Gene Structure, Conserved Motifs, and Conserved Structural Domains

The grape NRT gene structure was analyzed and mapped based on the grape NRT gene sequence, CDS sequence, and annotation files using the GSDS [39] (http://gsds.cbi.pku.edu.cn/, accessed on 24 June 2024). Conserved motifs of grape NRT proteins were analyzed using MEME (https://meme-suite.org/meme/tools/meme, accessed on 24 June 2024), with the maximum number of motifs set to 10 and other parameters set to default values. The NRT gene information was extracted according to the annotation file gene structure, and the corresponding amino acid number, molecular weight, theoretical isoelectric point, and average total water solubility were determined using TBtools. Sequencing results and annotated gene structures were visualized using TBtools software.v2.154.

2.5. Cis-Acting Element Analysis of the Promoter Region of the Grapevine NRT Gene

TBtools was used to extract the 2000 bp sequence upstream of the start codon of each grape NRT member. Cis-acting elements were predicted using PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/, accessed on 25 June 2024) [40].

2.6. Expression Pattern Analysis of Grapevine NRT Members

For quantitative real-time polymerase chain reaction (qRT-PCR) analysis, leaf and root tissues were collected from grapevines treated with full nutrients and nitrogen deficiency. Four independent biological replicates and three technical replicates were analyzed using a 2× SYBR Green qPCR Mix (Sparkjoy, corrected from sparkjade, Shandong Qingdao, China). Actin served as an internal control, and the primer list is provided in Supplementary Table S2.

2.7. Growth Hormone and Hydrogen Peroxide Content Testing

Growth hormone content was determined using an Enzyme-linked immunosorbent assay kit (Shanghai Acmec Biochemical Co., Shanghai, China). Fresh leaf samples subjected to different treatments were ground into a powder using liquid nitrogen, and the assay was completed following the manufacturer’s instructions. Hydrogen peroxide content was determined using a spectrophotometric analysis. Fresh leaves (2 g fresh weight, FW) were ground into a powder in liquid nitrogen, to which 2 mL of acetone was added at 4 °C and vortexed to mix. The mixture was centrifuged at 4000 rpm for 10 min, the precipitate was removed, and the supernatant (Vt ml) was retained. From the supernatant, 1 mL was taken, and 5% titanium sulfate (Ti(SO₄)₂) and concentrated ammonia were added, followed by centrifugation at 4000 rpm for 10 min to pellet the precipitate. The precipitate was washed with acetone to remove the pigments. An additional 5 mL of concentrated sulfuric acid was added to the precipitate to dissolve it (V1 mL), and the solution was then measured at 415 nm using a spectrophotometer. A standard curve was plotted (concentration of H₂O₂ vs. absorbance). The hydrogen peroxide concentration (mol/g FW) was calculated using the following formula:

${\mathrm{H}}_2{\mathrm{O}}_2 (\mathrm{m}\mathrm{o}\mathrm{l}/\mathrm{g} \mathrm{F}\mathrm{w})={\displaystyle \frac{\left(\mathrm{W}\times \mathrm{V}\mathrm{t}\right)}{\left(\mathrm{F}\mathrm{W}\times \mathrm{V}1\right)}}$

3. Results

3.1. Genome-Wide Characterization of the Grape NRT Gene

In the genome PN40024 of Pinot Noir (Vitis vinifera cv. Pinot Noir), the hidden Markov model was used to identify members of the NRT gene family, which were subsequently validated using a CD search by domain structure. After eliminating redundancy, 57 NRT genes were identified in grapes, including 53 VvNPFs, 3 VvNRT2s, and 1 VvNAR2, consistent with the number of other species in previous studies [41,42], which were named according to the phylogenetic relationships presented in the evolutionary tree. A comprehensive analysis of the basic physicochemical properties of the grape NRT protein family was performed (Table 1). The amino acid number (AA) of the grape VvNRTs ranges from 201 in VvNAR2.1 to 637 in VvNPF2.2, with molecular weights ranging from 22.25 kDa for VvNAR2.1 and 75.03 kDa for VvNPF2.2 The theoretical isoelectric points (pI) range from 5.27 for VvNPF5.14 to 9.49 for VvNPF5.19, indicating that most NRT proteins are alkaline, while 11 are acidic. Notably, only VvNAR2.1 has a negative GRAVY value, suggesting possible exposure to a hydrophilic region and a specific biological role.

3.2. Phylogenetic Analysis and Homologous Correlation of Grape NRT Protein Family

To clarify the functional diversity of the grapevine NRT protein family and explore the evolutionary relationships between grapevine and Arabidopsis NRTs, a phylogenetic tree was constructed using the ML method (Figure 1). Fifty-seven members of the VvNRT protein family were clearly divided into three major subfamilies (VvNPF, VvNRT2, and VvNAR2) using phylogenetic analysis. Afterwards, further division into five branches (VvNPF1 to VvNPF5) revealed that the most populous subfamily, VvNPF, had the highest number of members (25 in total) in the VvNPF5 branch (Figure 1). The VvNRT2 subfamily includes three members: VvNRT2.1, VvNRT2.2, and VvNRT2.3. In contrast, the VvNAR2 subfamily consists of a single member, VvNAR2.1. Notably, each subfamily contains groups of grapevines and A. thaliana members, suggesting possible functional similarities. These results provide valuable insights into the evolutionary history and functional diversity of the NRT protein family in grapevine and A. thaliana.

3.3. Chromosomal Localization and Collinearity Analysis of VvNRT Family Members

The chromosomal distribution of the NRT genes, as illustrated in a Circos diagram (Figure 2), revealed an uneven spread across the grapevine chromosomes. The 57 NRT genes were distributed across 14 chromosomes and were absent on chromosomes 3, 4, 10, 16, and 19. Notably, chromosome 18 exhibited the highest NRT gene count, with a total of 18 NRT genes, whereas only 1 appeared on chromosome 5. The VvNPF subfamily genes were mostly present on the same chromosomes as the NRT genes, whereas the VvNRT2 subfamily genes occurred mostly on chromosomes 6, 11, and 15. In contrast, the VvNAR2 subfamily genes were exclusively located on chromosome 17. This distribution highlights the significant variation in NRT protein density among the grapevine chromosomes.

In addition, a collinearity analysis comparing the grapevine and A. thaliana NRT genes (Figure 3) identified 21 collinear gene pairs involving 18 VvNRT and 21 AtNRT genes. This discovery indicates the retention of relatively conserved traits within the NRT family throughout its evolutionary history, offering valuable insights into the function and evolution of the NRT gene.

3.4. Analysis of Gene Structure and Conserved Motifs of the Grapevine VvNRT Family Members

The classification of VvNRT family members depends excessively on motif structure analysis, as it is essential for the interactions between different modules during transcription and signal transduction processes (Figure 3a). All members of the grapevine subfamily VvNPF had approximately seven motifs (Figure S1). Furthermore, each motif (motifs 4, 6, 2, 1, and 5) occurred once within a protein, and the motifs were arranged in a specific order along the VvNPF protein sequence. Motifs 1 and 7 persisted in each gene sequence of VvNRT2 subfamily members, whereas the majority of VvNPF members had these two motifs, suggesting a potential key role in nitrate or other substrate transport functions.

Through conserved domain analysis, various CDs (conserved domains), including MFS-NPF1-2, MFS-NPF4, and MFS-NPF7, were identified as belonging to the VvNPF family, along with the MFS superfamily, as shown in Figure 3b. The accuracy of the phylogenetic analysis was increased by the CDs of VvNRT2s and VvNAR2s, which were unique to these subfamilies and matched well with different NRT subfamilies.

In terms of gene structure (Figure 3c), the VvNPF subfamily genes have various lengths, with most having four or more exons, whereas the VvNRT2s subfamily genes are characterized by their complexity and span over ten exons. In contrast, VvNAR2.1, with its simplified gene structure of only two exons, represents an exception and highlights the remarkable diversity of gene structures in the subfamilies.

3.5. Analysis of Cis-Acting Elements of the Grape VvNRT Gene

To further clarify the role of the grape NRT gene family, we comprehensively analyzed the cis-acting elements in the 2000 bp upstream region of the grape VvNRT gene (Figure 4). The cis-acting elements of grape NRT family members can be categorized into four major types: growth and development regulatory elements, hormone response elements, environmental response elements, and light response elements. Among these, nine elements were identified as hormone response elements, including abscisic acid (ABA) response elements (ABRE) [43] and auxin response elements [44] (AuxRR core and TGA element), suggesting that NRT genes may be involved in hormone signaling pathways that affect grapevine growth and development. Ten elements were found to be related to growth and development regulation, while only four elements were associated with stress response [45], including the anaerobic and drought-inducible MBS [46] element, which may affect the stress response and adaptability of grapevine. The largest number of elements (19) were identified as participating in light response, showing the greatest diversity. For example, G-box [47] is known to play a crucial role in light-regulated gene expression, indicating that light signaling may be essential for controlling the expression of grapevine NRT genes. These results demonstrate the diverse functions that grapevine NRT genes can regulate and affect, providing important insights for further studying the functions and regulatory mechanisms of grapevine NRT genes.

3.6. Expression Analysis of Grapevine NRT Genes in Different Tissues

After analyzing the qRT-PCR data from diverse grapevine tissues, including leaves and roots, we identified distinct tissue-specific expression patterns of VvNRT genes (Figure 5), showing that members of the grapevine NRT family are commonly expressed in both roots and leaves, with higher levels observed in leaves, for instance, VvNPF3.8, VvNPF3.10, and VvNPF2.2. The increased expression of these genes suggests their involvement in important leaf functions, particularly in nitrate transport. We also observed remarkably high expression levels of certain genes in the root system, such as VvNPF5.4, VvNPF4.1, and VvNPF5.3, indicating a possible role in soil nitrate absorption.

3.7. Transcriptome Analysis of Grapevine Leaves Under Nitrogen-Deficiency Stress

To explore the role of grapevine NRT family members under nitrogen-deficiency conditions, we subjected grapevines to a one-week nitrogen deprivation treatment. This resulted in significant growth inhibition, weakened vigor, and leaf yellowing (Figure 6a,b). Subsequently, a comprehensive transcriptome analysis of the leaves of nitrogen deficiency grapevines was performed to identify responsive genes. After quality filtering, 104 GB of clean data were obtained from the six samples. Principal component analysis (PCA) revealed that the first two principal components, PC1 and PC2, accounted for 83.2% and 10.6% of the total variance, respectively (Figure 6c). The sample differences between the two treatment groups were significant, and the three biological replicates within each group demonstrated good internal consistency. Compared to the control group, 293 differentially expressed genes (DEGs) were identified in the nitrogen deficiency leaves (−N), along with 150 upregulated and 143 downregulated genes (Figure 6d).

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed the biological roles of differentially expressed genes under nitrogen-deficient conditions (Figure 7). Upregulated genes were predominantly enriched in processes related to oxidative stress response, mineral absorption, and light intensity response, underscoring their significance in facilitating plant adaptation to nitrogen deprivation. Upregulated genes associated with “response to oxidative stress” mitigate reactive oxygen species (ROS) generation and preserve cellular integrity. “Mineral absorption” enrichment signifies increased mineral uptake to mitigate nitrogen deficiencies, while “response to light intensity” genes modulate photosynthesis for maximum energy utilization.

Downregulated genes were prominent in monoatomic ion transmembrane transport, plant hormone signaling transduction, and nitrate response. The diminished demand for ion uptake, perhaps resulting from restricted nitrogen supply, accounts for the downregulation of genes involved in monoatomic ion transmembrane transport. The downregulation of genes linked to plant hormone signal transduction indicates modifications in hormonal pathways to acclimatize to nitrogen deprivation, influencing growth and stress responses. Downregulated genes associated with “response to nitrate” highlight compromised nitrogen detection and absorption (Figure 7).

It is noteworthy that both GO and KEGG analyses were enriched in functions related to mineral or nitrate absorption and response, highlighting the key role of nitrate transport proteins in the plant response to nitrate. Among the grapevine NRT family members, VvNAR2.1 and VvNPF2.3 were found to be enriched in the mentioned functions, suggesting their involvement in regulating the response to nitrogen deficiency and further confirming the significant role of grapevine NRT family members in the adaptation to nitrogen starvation.

3.8. Expression of Grapevine NRT Genes Under Nitrogen-Deficiency Stress

Under nitrogen-deficiency stress, members of the grapevine NRT family displayed distinct expression patterns (Figure 8a). Transcriptome analysis of differentially expressed genes revealed that although the majority of grapevine NRT gene expression in leaves was unaltered by nitrogen deficiency, VvNPF2.3 and VvNAR2.1 were significantly upregulated and downregulated, respectively (Figure 8b,c). To validate these findings, qRT-PCR assays were conducted on differentially expressed genes, confirming the transcriptome results (Figure 8d,e).

Further analysis was performed to examine the homology of the upregulated VvNPF2.3 with A. thaliana genes and revealed its relationship with AtNRT1.1 (AT1G12110.1) (Figure 9a). Moreover, previous studies have reported the involvement of AtNRT1.1 in nitrate transport and highlighted IAA’s role in stress adaptation in A. thaliana [48,49]. Validation through the measurement of IAA and H₂O₂ contents in grapevine leaves (Figure 9b,c) revealed a 62.2% decrease in IAA content and a 21.3% increase in H₂O₂ content under nitrogen deficiency conditions, implying that VvNPF2.3 might modulate hormone levels in response to environmental stress. The observed alterations in expression and hormone levels of grapevine NRT family members under nitrogen deficiency stress suggest a potential link to their response to nitrogen deficiency, offering a novel perspective on understanding grapevines’ adaptive mechanisms to nitrogen stress.

4. Discussion

Nitrogen is an essential element for the activities that are vital for energy metabolism in grapes. The NRT protein family, which is involved in nitrate absorption and transport, plays a significant role in plant growth, development, and stress response. The NRT gene family has been identified in various crops, e.g., 96 in rice, 79 in poplar, and 39 in potato [20,22,50,51]. Compared to grapes, the number of NRT family members exhibits significant variation among species. This study identified 57 VvNRT genes in the grapevine genome and classified the reported genes into three subfamilies based on phylogenetic analysis. The VvNPF subfamily contained 53 members as the largest component, while the VvNRT2s and VvNAR2 subfamilies contained three and one member, respectively.

Analysis of physicochemical properties revealed significant differences in molecular weight, isoelectric point, and amino acid composition among VvNRT proteins. Chromosome localization analysis indicated an uneven distribution of VvNRT genes across chromosomes, supported by a recent study in avocado under diverse nitrogen circumstances [52]. The maximum distribution is on chromosome 18 (persisting in 18 genes), and the minimum distribution is on chromosome 5 (persisting in only one gene). The clustering of some genes suggests that tandem duplication may be the primary mechanism for the expansion of grapevine NRT genes. Collinearity analysis identified 21 pairs of homologs between grapevines and Arabidopsis, indicating distinct retention and duplication events of the NRT genes across species.

Motif analysis showed that all VvNPF proteins contained conserved motifs (4, 6, 2, 1, and 5), whereas motifs 7 and 1 were ubiquitous in VvNRT2s and most VvNPF members, suggesting a close relationship with the transport function of nitrate or other substrates. These motifs are crucial for the formation of conserved structural domains in the NRT subfamilies, with different domains clearly corresponding to each subfamily. The VvNPF subfamily genes are relatively long, with most containing four or more exons; in contrast, the VvNRT2s subfamily genes are the longest and most complex, with more than ten exons, highlighting significant differences in gene structure among subfamilies. The VvNRT genes contain various hormones and stress response elements, among which there are particularly abundant photoreactive and environmental stress components, indicating potential roles in the regulation of plant hormones and abiotic stress responses [53]. The VvNRTs gene exhibit differential expression levels in various tissues due to distinct developmental regulatory elements. Additionally, its expression varies under nitrogen-deficient conditions and is driven by different stress response elements. For example, RY and MBS elements lead to VvNRTs genes that may be upregulated in different tissues in response to environmental stress (Figure 4, Figure 5, and Figure 8).

Tissue-specific expression analysis of the grapevine NRT family showed that most VvNRT subfamilies exhibited VvNAR2.1, VvNPF4.1, and VvNPF2.3 (expressed in roots and leaves), which are highly expressed in roots and potentially enhance nitrate absorption. Moreover, expression analysis highlighted the high expression of VvNPF3.8, VvNPF3.10, VvNPF2.2, and VvNPF4.2 along with other genes in the leaves that are likely involved in nitrate transport. The findings indicated that VvNRT genes exhibit tissue-specific expression patterns and undergo partial functional differentiation. The functionality of several homologous genes in A. thaliana and O. sativa was also analyzed. For instance, it was reported that the homologous gene of VvNAR2.1, designated as the NAR2gene AtNRT3.1 (AT5G50200.1), is essential for high-affinity nitrate uptake [54]. The homologous gene of VvNPF4.2, OsNPF8.1, is highly expressed in mesophyll and vascular parenchyma cells and is induced by various factors, including nitrogen deficiency, drought, NaCl, and abscisic acid [55]. Enhanced nitrate absorption aids in balancing the plant responses to salt/drought stress and nitrogen deficiency. Additionally, the homologous gene of VvNPF3.8 in tobacco (NtNPF2.11) is predominantly expressed in leaves, and under high-nitrogen conditions, overexpression of NtNPF2.11 can increase tobacco yield and the accumulation of nitrogen and potassium [56].

To explore the functions of VvNRT genes under nitrogen deficiency, the proposed methodology also examined the effects of nitrogen deprivation on grapevine growth and leaf color. The analysis revealed significant upregulation and downregulation of VvNPF2.3 and VvNAR2.1, respectively, under nitrogen deficiency conditions. VvNPF2.3 shares the highest homology with AtNRT1.1 from A. thaliana and is involved in nitrate transport and auxin regulation [25,49,57,58]. The decrease in IAA content in grapevine leaves under nitrogen deficiency conditions suggests that VvNPF2.3 might regulate hormone levels to adapt to stress. The IAA is a key phytohormone that plays a crucial role in regulation of root growth and development in plants. In roots, it has been shown that AtNRT1.1 acts as a negative regulator of auxin biosynthesis genes, and NRT1.1 negatively affects the expression of auxin influx carrier in Arabidopsis [49]. Therefore, it is potentially possible that increased expression of VvNPF2.3 in leaves allows IAA transport to the roots in grapevines under stress conditions or prevents the internal flow. The observed inverse correlation between the elevation of VvNPF2.3 and diminished IAA levels necessitates additional mechanistic exploration. The use of transport inhibitors and genetic manipulation techniques may clarify the regulatory pathways and their effects on grapevine growth and development under conditions of nitrogen deficiency.

This study provides insight into the linkage analysis between VvNRT gene expression and hormone levels during stress, offering a new perspective on grapevine stress resistance and genetic resource utilization. For example, by identifying marker genes in DEGs and detecting their expression patterns under nitrogen-deficiency conditions, farmers can adjust fertilizer application rates and timing to meet the specific nitrogen requirements of grapevines, thereby reducing fertilizer waste and environmental pollution.

The proposed pipeline provides a baseline study for the comprehensive analysis of the structural characteristics, duplication types, evolutionary features, tissue expression, and stress responses of grapevine NRT genes, leading to the establishment of a foundation for further understanding the functions and molecular mechanisms of the NRT gene family during growth and development.

5. Conclusions

In this study, 57 VvNRT genes were identified in the grape genome as unevenly distributed across 19 chromosomes. Analysis of cis-acting element suggests their involvement in grape growth, development, and stress responses. Synteny analysis underscores tandem and segmental duplications as the primary catalysts for the growth of the VvNRT family, fostering evolutionary diversity and functional distinction. Expression profiling in different tissues and under nitrogen-deficiency stress revealed significant tissue-specific expression patterns of VvNRT, with distinct expression profiles under nitrogen-deficiency conditions. Notably, the expression of VvNPF2.3 was significantly upregulated, and the level of IAA in leaves decreased by 62.2%, while the level of H₂O₂ increased by 21.3% under nitrogen-deficiency stress, suggesting its potential role in nitrogen-deficiency regulation. This study provides a basis for clarifying the biological roles of VvNRT genes in nitrogen uptake, transport, and utilization. The results hold considerable promise for grapevine breeding and agricultural methodologies, especially in pinpointing VvNRTs associated with nitrogen absorption, stress response, and root structure. These insights may inform innovative approaches to enhance grapevine growth and productivity in nitrogen-deficient conditions, meeting the demand for high-quality grapes and fostering sustainable development in the grape sector.

Author Contributions

W.J. designed the experiments; Z.Y., J.M., W.Z. and K.W. performed all experiments and analyzed all data; J.M. and W.Z. interpreted results; J.M., F.S.K., M.N.B., M.S.R. and N.A.-K. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The data that support the findings of this study are openly available in at https://doi.org/10.1093/hr/uhad061.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

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Figures and Table

Figure 1. Phylogenetic analysis of NRT members from Vitis vinifera.

Figure 2. Analysis of evolutionary relationship and gene location of NRT family members in Vitis vinifera. The red lines show the syntenic gene pairs, and the blue lines depict the syntenic gene pairs of Vitis vinifera. Chr: chromosome number. The location of NRT genes on chromosomes is also labeled.

Figure 3. Motif, conserved domain, and gene structure analysis of VvNRT genes. (a) The motif analysis using MEME tool and (b) the conserved domain analysis via NCBI Batch CD-search; (c) the yellow color pinpoints the untranslated region, and the green highlights the coding sequence. The line without color refers to introns.

Figure 4. Heat map of different cis-acting elements.

Figure 5. Differential gene expression of VvNRTs leaves and roots. The relative expression of selected VvNRT members by qRT-PCR assay. The root tissue is defined to be the reference; * represents significant differences in comparison with root group using Student’s t-test at p [less than] 0.05.

Figure 6. Phenotypic and differentially expressed genes analysis of grape under nitrogen deficiency treatment. (a) Grape growth phenotype, (b) grape leaf phenotype, and (c) PCA analysis. (d) Number of differentially expressed genes.

Figure 7. GO and KEGG pathway enrichment plot.

Figure 8. Differential gene expression of VvNRT under nitrogen deficiency. (a) TPM values of control and nitrogen-deficiency treatment groups. (b) Volcano plot of differentially expressed genes under nitrogen-deficiency stress. (c) Venn diagram including upregulated, downregulated, and VvNRT genes. (d,e) Expression of differentially expressed genes. −N: nitrogen deficiency nutrient solution treatment group; Control: complete nutrient solution control group; * indicates significant difference compared with the control group, using Student’s t-test, p [less than] 0.05.

Figure 9. Effect of endogenous hormones in grape leaves under nitrogen deficiency. (a) The relationship between the VvNPF2.3 gene and the Arabidopsis NRT gene family, (b) IAA content in leaves, and (c) H2O2 content in leaves; * indicates significant difference compared with the control group, using Student’s t-test, p [less than] 0.05.

Table 1

Characteristics of VvNRT members.

Sequence ID	No. of AA 1	MW (kDa) 2	pI 3	Instability Index 4	Aliphatic Index 5	GRAVY 6
VvNPF5.14	485	53278.46	5.27	41.67	99.36	0.307
VvNPF1.1	611	66984.42	5.7	29.41	99.23	0.259
VvNRT2.1	526	57161.99	6.02	38.52	102.53	0.386
VvNPF5.6	570	62882.97	6.1	35.04	104.7	0.373
VvNPF4.1	539	59410.09	6.16	29.55	97.37	0.365
VvNPF1.3	591	65583.95	6.36	32.79	96.04	0.246
VvNPF5.2	572	62471.14	6.45	35.48	101.12	0.384
VvNPF5.10	443	49400.41	6.51	33.08	102.12	0.305
VvNPF5.3	574	63241.35	6.67	43.86	99.02	0.302
VvNPF4.2	582	64589.45	6.86	27.65	95.5	0.191
VvNPF5.5	573	63050.07	6.93	42	99.9	0.345
VvNPF5.12	578	63347.49	7.45	38.25	102.44	0.344
VvNPF5.8	572	62965.77	7.53	35.57	96.82	0.3
VvNPF4.3	569	63438.39	8.15	24.78	99.03	0.285
VvNPF5.13	369	40945.78	8.44	31.96	106.75	0.45
VvNPF5.17	528	57812.16	8.46	43.11	102.33	0.3
VvNPF1.2	604	67469.3	8.48	32.5	94.93	0.207
VvNPF1.4	527	58488.7	8.56	35.48	94.33	0.198
VvNPF5.23	572	62943.85	8.6	39.89	94.97	0.317
VvNPF5.9	572	62794.77	8.66	37.18	101.96	0.28
VvNPF5.21	569	62616.89	8.73	33.49	105.57	0.413
VvNPF3.6	619	68522.68	8.73	40.49	98.42	0.196
VvNPF3.2	583	64498.98	8.8	29.77	99.04	0.216
VvNPF5.7	574	62775.77	8.82	38.05	99.76	0.325
VvNRT2.3	514	56847.27	8.83	39.31	99.16	0.356
VvNPF5.11	569	62591.81	8.83	35.12	106.56	0.395
VvNPF2.6	607	66814.52	8.83	38.53	94.94	0.31
VvNPF5.24	526	57979.46	8.85	35.55	104.22	0.36
VvNPF5.4	533	59809.4	8.9	40.55	105.37	0.36
VvNPF5.18	587	65303.55	8.92	35.06	107.09	0.32
VvNPF5.15	609	68124.48	8.93	38.34	96.8	0.212
VvNAR2.1	201	22252.47	8.97	30.57	84.48	−0.094
VvNPF2.1	296	33823.58	8.97	29.22	95.41	0.187
VvNPF5.25	575	63230.52	8.97	34.96	101.57	0.284
VvNPF3.5	577	63457.87	8.97	32.68	105.82	0.365
VvNPF3.8	485	54005.4	8.98	35.34	105.32	0.378
VvNPF2.5	580	63879.81	9	34.53	94.34	0.342
VvNPF3.7	585	64429.98	9.01	32.98	95.03	0.261
VvNPF5.20	597	66783.06	9.05	34.88	101.22	0.215
VvNPF2.4	587	64509.21	9.07	32.08	100.89	0.266
VvNPF3.1	603	66721.57	9.07	31.79	104.48	0.205
VvNPF3.3	622	69057.57	9.07	46.09	102.07	0.209
VvNPF4.4	568	64055.59	9.08	30.32	94.23	0.143
VvNPF3.4	577	63378.86	9.1	30.51	106.67	0.363
VvNPF2.7	609	67436.6	9.12	41.99	98.65	0.292
VvNPF2.10	433	47875.11	9.17	44.64	101.36	0.375
VvNPF2.8	559	61964.28	9.18	38.32	94.22	0.263
VvNPF2.9	585	64427.14	9.18	41.34	98.87	0.312
VvNPF5.1	467	52479.46	9.22	43.33	97.43	0.152
VvNPF3.9	586	64564.65	9.22	35.15	101.66	0.346
VvNPF5.16	592	65814.77	9.22	31.77	97.94	0.249
VvNPF2.2	673	75029.25	9.27	29.68	94.58	0.187
VvNPF3.10	577	64048.81	9.31	32.03	98.53	0.296
VvNRT2.2	615	67356.19	9.32	50.65	96.88	0.255
VvNPF2.3	658	72133.68	9.36	35.1	104.94	0.328
VvNPF5.22	585	65155.93	9.42	30.62	100.94	0.257
VvNPF5.19	580	64556.05	9.49	33.81	102.21	0.285

¹ Number of AA represents the number of the amino acids in each protein sequence. ² MW (kDa) is the mass of protein molecules. ³ The pH value at which proteins have a neutral charge under theoretical conditions. ⁴ The instability index of proteins; the higher the index, the more unstable the protein. ⁵ Describes the abundance and relative content of non-polar amino acids in proteins. ⁶ The relative proportion of hydrophilic and hydrophobic amino acids in protein molecules.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae11030252/s1. Figure S1: Detailed information about motif sequences; Table S1: Nutrition formula for plant cultivation; Table S2: Primers for qRT-PCR assay.

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