Genome-Wide Analysis and Functional Characterization of the NRT1 Gene Family in Grapevine (Vitis vinifera L.) Reveals Roles of VvNRT1.19 and VvNRT1.47 in Salt Stress and Nitrogen Response

Genome-Wide Analysis and Functional Characterization of the NRT1 Gene Family in Grapevine (Vitis vinifera L.) Reveals Roles of VvNRT1.19 and VvNRT1.47 in Salt Stress and Nitrogen Response | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-Wide Analysis and Functional Characterization of the NRT1 Gene Family in Grapevine (Vitis vinifera L.) Reveals Roles of VvNRT1.19 and VvNRT1.47 in Salt Stress and Nitrogen Response Chen Zhou, Yingchun Chen, Kai Liu, Xiujie Li, Xuehui Zhao, Bo Li, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8769993/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The NRT1 (nitrate transporter 1) family is referred to as the NPF (nitrate/peptide transporter family). It constitutes the largest and most functionally diverse group of nitrate transporters in plants and plays an essential role in nitrate translocation and allocation. These are fundamental to plant growth, development, and stress adaptation. However, a comprehensive genome-wide characterization of this family in grapevine ( Vitis vinifera L.) remains unexplored. Results A total of 65 NRT1 genes were identified in the grapevine genome and phylogenetically classified into five distinct clades. Their physicochemical properties, as well as gene and protein structural features, were analyzed in detail. Homology analyses revealed that both segmental and tandem duplication events played major roles in driving the expansion of the VvNRT1 gene family. In addition, analysis of tissue-specific expression patterns and cis-regulatory elements suggested that VvNRT1 genes participated in the regulation of grapevine development and adaptive responses to diverse environmental stresses. Nitrate treatment and salt stress induced the expression of multiple VvNRT1s , among which VvNRT1.19 and VvNRT1.47 exhibited the most pronounced transcriptional upregulation. The expression levels of these two genes were positively associated with nitrogen uptake capacity and salt tolerance in grapevine. Furthermore, heterologous overexpression of VvNRT1.19 and VvNRT1.47 in Arabidopsis significantly enhanced nitrogen use efficiency and salt tolerance, thereby promoting plant growth and improving stress resistance. Conclusion This study presents a comprehensive characterization of the VvNRT1 gene family and provides insights into its evolutionary history in grapevine. VvNRT1.19 and VvNRT1.47 are proposed to function as positive regulators of plant responses to stress. Collectively, these findings establish a solid foundation for future functional investigations of NRT1 genes and highlight their potential roles in improving nitrogen regulation and salt stress tolerance in grapevine. Genome-wide analysis Nitrate Transporters Grapevine Gene expression Gene function Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Nitrogen is an essential mineral element required for plant growth and development and can be involved in the synthesis of key biological molecules, including proteins, nucleic acids, chlorophyll, and hormones [ 1 ]. The efficiency of nitrogen absorption, transport, and assimilation directly determines plant growth rate, yield formation, and quality regulation. Nitrate (NO 3 − ) and ammonium (NH 4 + ) are the major inorganic nitrogen (N) sources available to plants. Owing to its high mobility in soil and ready accessibility to roots, NO 3 − is the predominant nitrogen form taken up by most plant species [ 2 ]. NO 3 − uptake mainly occurs in plant roots and is mediated by nitrate transporter (NRT) proteins, which are widely recognized as essential for N acquisition in plants. Transmembrane NO 3 − transport depends on the precise regulation of NRTs, which are classified into three families, NRT1 , NRT2 , and NRT3 , according to their functional characteristics and sequence homology. Among these families, NRT1 , referred to as the Nitrate Peptide Transporter Family (NPF), contains the largest number of members and exhibits the broadest functional diversity [ 3 ]. Therefore, it has become the central focus of recent plant nitrogen metabolism research. NRT proteins are pivotal for nitrate transport and distribution, with their primary function being the mediation of root NO 3 ⁻ uptake. Based on the differences in external nitrate concentrations, NRT family members are divided into two transport systems: the Low-Affinity Transport System (LATS) and the High-Affinity Transport System (HATS). Under low NO 3 ⁻ conditions, typically below 1 mM, NO 3 ⁻ uptake is dominated by HATS, which is mainly mediated by the NRT2 family. Under high NO 3 ⁻ concentrations, generally above 1 mM, LATS dominated by the NRT1 family takes precedence [ 4 – 5 ]. However, certain NRT1 members (e.g., NRT1.1 ( AtNPF6.3 ) in Arabidopsis thaliana ) exhibit “dual-affinity” characteristics, enabling roots to absorb NO 3 ⁻ from the soil while also functioning as NO 3 ⁻ sensors that coordinate N uptake and carbon metabolism through the regulation of downstream gene expression [ 6 ]. AtNPF7.2 ( NRT1.8 ) is specifically localized in xylem parenchyma cells and is responsible for reloading NO 3 ⁻ from xylem sap, thereby regulating NO 3 ⁻ allocation to shoots and influencing leaf photosynthesis and nitrogen remobilization [ 7 ]. Furthermore, AtNPF4.6 participates in drought stress responses by transporting abscisic acid (ABA), whereas AtNPF5.5 regulates storage substance accumulation during seed development, highlighting the central role of the NRT1 family within the “nitrogen metabolism-growth and development-stress adaptation” network [ 8 – 9 ]. With the development of genome sequencing technologies, studies on the NRT1 family have expanded from model plants to diverse crops and economically important species. In rice, natural variation in OsNRT1.1B is associated with nitrogen use efficiency, with cultivars containing specific alleles exhibiting higher yields under low-nitrogen conditions [ 10 ]. OsNPF6.5 influences grain protein content by regulating root NO 3 ⁻ uptake and N allocation to panicles [ 11 ]. In maize, ZmNRT1.1 expression is induced by low-nitrogen stress, with overexpression significantly increasing plant biomass in N-deficient soils. This demonstrates the breeding potential of this gene family for N-efficient crops [ 12 ]. In fruit trees, MdNPF6.5 functions as a key low-nitrogen-inducible nitrate transporter that plays a critical role in significantly enhancing apple tolerance under low-nitrogen conditions [ 13 ]. Collectively, these studies indicate that the NRT1 family exhibits both functional conservation, such as core nitrate transport activity across plant lineages, and species-specific characteristics shaped by evolutionary history. Salt stress is a major abiotic stressor that constrains global crop production and severely disrupts N metabolic balance. Numerous studies have demonstrated that salt stress markedly inhibits plant NO 3 ⁻ uptake and assimilation capacity, leading to a decline in leaf nitrate content. In peanut, salt stress reduces NO 3 ⁻ accumulation in leaves, with the inhibitory effect intensifying as the salt concentration increases, a response closely associated with the suppressed expression of nitrate transporters [ 14 ]. Similarly, in rice, treatment with 0.3% NaCl significantly decreases NO 3 ⁻ content and the activities of nitrate reductase (NR), glutamine synthetase (GS), and NADH-dependent glutamate synthase (NADH-GOGAT) in both roots and leaves, while increasing ammonium accumulation and the activities of glutamate dehydrogenase (GDH) and aspartate aminotransferase (AST). These changes indicate a metabolic shift from the NO 3 ⁻ assimilation pathway to alternative nitrogen metabolic routes under salt stress conditions [ 15 ]. Under salt stress, NRT1 family proteins enhance plant adaptability through multiple mechanisms, including the maintenance of N uptake, regulation of ion homeostasis, participation in signal transduction, and facilitation of root architecture remodeling. For instance, in Suaeda salsa under high-salinity and low-N conditions, N uptake is promoted by the induction of SsNRT2.1/2.5 expression acting in concert with NRT1, along with the activation of plasma membrane H + -ATPases that drive proton gradient–dependent active NO 3 ⁻ transport [ 16 – 17 ]. In Arabidopsis , NRT1.5 ( NPF7.3 ) functions as an H + /K + antiporter that facilitates K + transport to shoots, and the NRT1.5 ( lks2 ) knockout mutant exhibits leaf chlorosis under salt stress, indicating its role in alleviating Na + toxicity by maintaining K + homeostasis [ 18 ]. In rice, NRT1.1 ( OsNRT1.1B ) acts as a NO 3 ⁻ sensor and activates transcription factors such as ANR1 and NLP7 to regulate downstream gene expression [ 19 ]. Ectomycorrhizal fungi, such as Paxillus involutus , mitigate salt stress–induced nitrate efflux by activating host poplar genes PcNRT1.1/1.2 and PcHA4 , lowering rhizosphere pH, and promoting H + -ATPase activity [ 20 ]. Grapevine ( Vitis vinifera L.) is one of the world’s most economically important fruit crops and is characterized by a long growth cycle and complex synchrony of organ development, resulting in pronounced stage-specific N demands. High nitrogen availability is required at budburst to support shoot growth, whereas insufficient nitrogen supply from flowering to fruit enlargement leads to reduced fruit set, and excessive N input during ripening inhibits fruit coloration and flavor compound accumulation[ 21 – 22 ]. With ongoing changes in the global ecological environment, grapevine growth is increasingly affected by environmental pressures, s uch as salt stress. Our previous research indicated that salt stress adversely affected N absorption and assimilation in grapevine and ultimately constrained plant growth [ 23 ]. Therefore, elucidating the molecular mechanisms underlying nitrogen uptake and transport in grapevine is of practical significance for N management and fruit quality optimization. The recent completion of grapevine whole-genome sequencing projects, including the PN40024 reference genome and high-quality assemblies such as the CHR_T2T2 telomere-to-telomere genome, can provide crucial support for functional gene mining [ 24 ]. However, research on the grapevine NRT1 gene family remains limited [ 25 ]. Early studies primarily predicted a small number of potential members through homology-based sequence alignment and focused mainly on expression pattern analyses of genes related to root nitrate uptake, such as VvNRT1.1 [ 26 ]. Core questions regarding the genome-wide identification of family members, phylogenetic relationships, gene structural variations, tissue-specific expression patterns, and associations with stress responses remain largely unresolved. To address these issues, the present study performed a genome-wide analysis and identified 65 full-length NRT1 genes in grapevine. Using a multifaceted bioinformatic framework, this study characterized the physicochemical properties, gene and protein architectures, chromosomal distributions, phylogenetic relationships, and tissue-specific expression patterns of the identified genes. These findings advance our understanding of the functional landscape of the grapevine NRT1 gene family and highlight its potential involvement in salt stress response mechanisms. Results VvNRT1s gene identification To identify potential NRT1 homologs in the grapevine genome (PN_T2T2) [ 27 ], Arabidopsis NRT1 protein sequences were used as queries for BLASTP searches. This initial screening retrieved approximately 80 candidate proteins. Subsequent validation using the SMART and NCBI-CDD databases confirmed the presence of characteristic MFS and extensin domains. After this filtering step, 65 VvNRT1 proteins were identified (Fig. S1 ), representing a number higher than that reported in Arabidopsis. These genes were systematically designated VvNRT1.1 – VvNRT1.65 according to their linear positions on the chromosomes. The predicted molecular weights of the 65 VvNRT1 proteins ranged from 10.6 kDa for VvNRT1.40 to 162.42 kDa for VvNRT1.63 . Consistent with this variation, the protein lengths differed markedly, spanning from 96 amino acids ( VvNRT1.40 ) to 1466 amino acids ( VvNRT1.63 ). In addition, the theoretical isoelectric points of these proteins varied from 4.82 for VvNRT1.55 to 9.8 for VvNRT1.6 (Table 1 ). Table 1 Characteristics of NRT1s in Grapevine ( Vitis vinifera L.). Gene name Gene ID Chromosome location Exon Size (amino acids) MW (kDa) pI VvNRT1.1 VitviT2T_000270 chr1:3035085 − 308496 4 583 64.50 8.8 VvNRT1.2 VitviT2T_000553 chr1:6408849–6416804 5 586 64.61 5.47 VvNRT1.3 VitviT2T_000867 chr1:10897334–10901324 4 619 68.52 8.73 VvNRT1.4 VitviT2T_000875 chr1:10997314–11001502 4 585 64.42 9.18 VvNRT1.5 VitviT2T_001641 chr1:27439472–27442560 4 640 70.83 9.01 VvNRT1.6 VitviT2T_002188 chr2:5135546–5146196 7 786 86.01 9.8 VvNRT1.7 VitviT2T_002412 chr2:8626578–8631608 4 591 65.58 6.36 VvNRT1.8 VitviT2T_002882 chr2:20036564–20037454 1 296 33.82 8.97 VvNRT1.9 VitviT2T_004646 chr4:2928810–2931535 4 589 65.19 6.1 VvNRT1.10 VitviT2T_004647 chr4:2933378–2935949 4 592 65.11 7.87 VvNRT1.11 VitviT2T_006768 chr5:9116491–9121269 4 592 65.81 9.22 VvNRT1.12 VitviT2T_007807 chr6:1599766–1602508 5 611 66.98 5.7 VvNRT1.13 VitviT2T_007840 chr6:1993924–1996770 3 534 60.21 8.78 VvNRT1.14 VitviT2T_007842 chr6:2057074–2064811 6 667 76.65 8.36 VvNRT1.15 VitviT2T_008029 chr6:4410373–4410825 1 150 16.85 8.82 VvNRT1.16 VitviT2T_008030 chr6:4427501–4430493 4 577 63.38 9.1 VvNRT1.17 VitviT2T_008032 chr6:4458253–4460909 4 577 63.46 8.97 VvNRT1.18 VitviT2T_008280 chr6:6896959–6901242 4 582 64.59 6.86 VvNRT1.19 VitviT2T_009857 chr7:7407053–7410478 4 597 66.78 9.05 VvNRT1.20 VitviT2T_009858 chr7:7415929–7419696 4 609 68.12 8.93 VvNRT1.21 VitviT2T_012005 chr8:12945007–12949683 4 599 66.23 9.08 VvNRT1.22 VitviT2T_012348 chr8:16570040–16573831 4 587 65.30 8.92 VvNRT1.23 VitviT2T_012404 chr8:17138913–17142492 4 568 64.06 9.08 VvNRT1.24 VitviT2T_012405 chr8:17144873–17147263 4 569 63.44 8.15 VvNRT1.25 VitviT2T_012415 chr8:17228720–17245294 5 444 50.42 8.72 VvNRT1.26 VitviT2T_013400 chr9:3797543–3801577 4 622 69.06 9.07 VvNRT1.27 VitviT2T_015272 chr10:8758372–8760963 5 596 65.70 8.45 VvNRT1.28 VitviT2T_016500 chr11:2931120–2933169 4 598 66.42 8.66 VvNRT1.29 VitviT2T_016501 chr11:2935712–2937182 4 276 31.13 9.62 VvNRT1.30 VitviT2T_016502 chr11:2937227–2937730 1 167 17.84 4.99 VvNRT1.31 VitviT2T_016649 chr11:4416724–4425469 7 623 68.53 8.89 VvNRT1.32 VitviT2T_016651 chr11:4443282–4446833 5 587 64.51 9.07 VvNRT1.33 VitviT2T_016652 chr11:4447461–4448228 3 143 15.54 9.12 VvNRT1.34 VitviT2T_017823 chr12:6204846–6208077 4 594 66.15 9.18 VvNRT1.35 VitviT2T_018872 chr12:24010632–24013783 5 607 66.81 8.83 VvNRT1.36 VitviT2T_019055 chr13:1289308–1293528 5 628 70.23 9.43 VvNRT1.37 VitviT2T_019317 chr13:3795593–3798375 4 597 67.00 8.93 VvNRT1.38 VitviT2T_020247 chr13:23151898–23154691 4 575 63.23 8.97 VvNRT1.39 VitviT2T_020250 chr13:23182256–23187154 3 499 56.11 9.43 VvNRT1.40 VitviT2T_020251 chr13:23189064–23189462 1 96 10.60 8.93 VvNRT1.41 VitviT2T_020253 chr13:23198483–23204010 3 538 59.28 8.86 VvNRT1.42 VitviT2T_020751 chr14:295133–297492 5 620 68.37 7.55 VvNRT1.43 VitviT2T_021566 chr14:13040883–13041776 3 169 19.08 8.95 VvNRT1.44 VitviT2T_021877 chr14:20772346–20774714 3 198 21.18 6.8 VvNRT1.45 VitviT2T_022515 chr14:28488931–28491946 4 533 59.81 8.9 VvNRT1.46 VitviT2T_022572 chr14:28916994–28979694 4 585 64.43 9.01 VvNRT1.47 VitviT2T_023196 chr15:12838567–12843593 5 604 67.47 8.48 VvNRT1.48 VitviT2T_023463 chr15:17551952–17554126 6 529 58.63 8.79 VvNRT1.49 VitviT2T_026074 chr17:6447730–6450309 4 577 64.05 9.31 VvNRT1.50 VitviT2T_026075 chr17:6451581–6454340 4 586 64.99 8.78 VvNRT1.51 VitviT2T_026076 chr17:6456777–6459464 4 586 64.56 9.22 VvNRT1.52 VitviT2T_026077 chr17:6463343–6465728 5 570 62.82 8.01 VvNRT1.53 VitviT2T_026078 chr17:6465936–6466590 3 132 13.98 6.25 VvNRT1.54 VitviT2T_026087 chr17:6540785–6543752 4 579 63.57 8.81 VvNRT1.55 VitviT2T_026703 chr17:16544148–16544727 2 141 15.37 4.82 VvNRT1.56 VitviT2T_027631 chr18:9572337–9575747 6 587 64.75 8.53 VvNRT1.57 VitviT2T_027795 chr18:11322484–11324487 5 617 68.27 8.34 VvNRT1.58 VitviT2T_027796 chr18:11327097–11329870 5 581 64.58 8.76 VvNRT1.59 VitviT2T_028874 chr18:30296518–30344687 6 1035 114.29 8.86 VvNRT1.60 VitviT2T_028876 chr18:30380900–30411853 4 894 99.07 8.77 VvNRT1.61 VitviT2T_028878 chr18:30435965–30446252 3 531 59.04 8.97 VvNRT1.62 VitviT2T_028880 chr18:30447236–30447676 1 146 16.50 7.74 VvNRT1.63 VitviT2T_028881 chr18:30468432–30497044 9 1466 162.42 6.8 VvNRT1.64 VitviT2T_028883 chr18:30527824–30544755 9 1139 126.20 6.98 VvNRT1.65 VitviT2T_029282 chr18:36195994–36199645 5 609 67.44 9.12 Phylogenetic and synteny analyses of VvNRT1s To clarify the evolutionary and phylogenetic relationships, an unrooted phylogenetic tree was constructed using 32 AtNRT1 and 65 VvNRT1 protein sequences (Fig. 1 a). The resulting topology grouped the NRT1 proteins into five primary clades (Ⅰ–Ⅴ). Group Ⅰ represented the largest clade, containing twenty-six members, whereas Groups Ⅱ and Ⅳ each comprised 17 proteins. In contrast, Groups Ⅲ and Ⅴ were much smaller, consisting of two and three proteins, respectively. Notably, this five-clade classification differed from the three-group pattern commonly reported for most other plant species. The VvNRT1 genes were unevenly distributed across the 16 grapevine chromosomes (Fig. 1 b). Only one gene was located on chromosomes 5, 9, and 10, whereas chromosomes 4, 7, 12, and 15 each harbored two VvNRT1 members. In contrast, most VvNRT1 genes were preferentially clustered on chromosomes 6, 11, 13, 17, and 18. Among these, chromosome 18 contained the highest number of VvNRT1 genes, suggesting a potentially prominent role for this chromosome in nitrate transport and metabolism in grapevine. To explore the evolutionary mechanisms underlying the expansion of the VvNRT1 gene family, gene duplication events were analyzed. Based on sequence homology, 20 VvNRT1 genes (30.77%) formed 10 segmental duplication pairs, whereas 42 VvNRT1 genes (64.62%) formed 29 tadem duplication pairs (Fig. 1 b and Table S2). Conserved motif and gene structure analyses of VvNRT1s The conserved protein motifs and exon–intron structures of VvNRT1 genes were examined using MEME and GSDS 2.0, respectively. A maximum-likelihood phylogenetic tree was generated to assess the relationships among evolutionary clades, motif composition, and gene structural features (Fig. 2 a), which was consistent with the clustering pattern observed in Fig. 1 a. The identification of conserved motifs provides crucial evidence for understanding evolutionary relationships. In total, 10 conserved motifs were identified, with motif lengths ranging from 17 to 41 amino acids (Figs. 2 b and S2). In groups Ⅰ and Ⅳ, VvNRT1.62 , VvNRT1.43 , VvNRT1.15 , VvNRT1.30 , and VvNRT1.53 each contained only two motifs, whereas VvNRT1.40 , VvNRT1.55 , VvNRT1.33 , and VvNRT1.44 in groups I, Ⅱ, and Ⅳ possessed a single motif. Members within the same clade exhibited similar motif positions and distribution patterns, which were largely conserved across groups I–Ⅳ. Conversely, group V contained markedly fewer motifs than the other groups. The exon–intron architecture is a key component of the gene structural architecture. Accordingly, the structural features of all 65 VvNRT1 genes were analyzed, including the number, length, and position of exons and introns (Fig. 2 c). Genes belonging to the same phylogenetic subfamily displayed highly conserved gene structures, further supporting the established classification of grapevine NRT1s . In groups I, Ⅱ, and Ⅳ, VvNRT1.62 , VvNRT1.40 , VvNRT1.8 , VvNRT1.15 , and VvNRT1.30 each consisted of a single exon. Most group I genes contained five or more exons, with VvNRT1.63 and VvNRT1.64 each containing nine exons. Genes in group Ⅱ generally possessed four to six exons, whereas those in subgroups Ⅲ and Ⅳ typically contained four exons. In contrast, group V genes exhibited a wider range of exon numbers, from three to six, and their gene lengths were notably shorter than those of other groups. All members of group Ⅲ had contained both 3’-UTR and 5’-UTR regions, whereas group V genes lacked one or both UTRs. These variations in gene structure may underlie the functional divergence observed among VvNRT1 family members. Cis-regulatory element analysis of VvNRT1s The regulatory element composition of VvNRT1 genes was analyzed, and the genes were classified into distinct clusters according to their response patterns to different regulatory signals. Each cluster exhibited characteristic regulatory profiles associated with specific environmental and developmental cues (Fig. 3 a). In cluster Ⅰ, most VvNRT1 genes exhibited strong responsiveness to stress-related elements, including defense and anaerobic induction elements (green, red, and purple squares), indicating their potential involvement in stress responses. Cluster Ⅱ was mainly characterized by VvNRT1 genes responsive to hormone-related regulatory elements, such as auxin and salicylic acid (orange, brown, and light pink squares). Cluster Ⅲ displayed broad associations with developmental regulation, particularly seed-specific regulation and meristem-related elements (blue squares), suggesting an important role in plant growth regulation. The genes in cluster Ⅳ exhibited the most diverse response patterns, with the largest proportion being responsive to methyl jasmonate (MeJA; 36 genes), highlighting a prominent role in stress and hormone signaling crosstalk. In contrast, cluster V presented a relatively limited responsiveness, while some genes were associated with low-temperature and circadian control elements (light blue squares), and overall contained fewer VvNRT1 genes, especially those related to seed-specific regulation and gibberellin responses. Consistent with these observations, heatmap analysis (Fig. 3 b) revealed distinct regulatory distributions of VvNRT1 genes across the examined biological processes. In cluster Ⅳ, a substantial number of VvNRT1 genes (46) were responsive to MeJA, whereas cluster Ⅱ exhibited strong responsiveness to auxin-related elements (41 genes). In contrast, cluster Ⅴ contained fewer VvNRT1 genes responsive to low-temperature and anaerobic induction signals. Overall, these results indicate that VvNRT1 genes are differentially regulated by environmental stress, hormone-related signals, and developmental cues. The clustering patterns based on cis-regulatory elements further underscored the functional complexity of VvNRT1 genes in grapevine growth regulation and stress response. Tissue-specific expression profile of VvNRT1s To further clarify the potential roles of VvNRT1s , expression profiles across 19 different organs or tissues at multiple developmental stages were obtained from the BAR database (Fig. 4 ). The VvNRT1 genes exhibited widespread expression across nearly all examined tissues. Evident expression differences were observed among phylogenetic groups, where members of Groups I and II demonstrated broader expression ranges across diverse tissues than those belonging to other groups. Several genes showed significantly elevated expression levels in specific tissues or developmental stages. For example, VvNRT1.7 and VvNRT1.21 showed the significantly higher expression in roots than other tissues or organs, whereas VvNRT1.34 and VvNRT1.38 reached peak expression during the flowering stage. Moreover, VvNRT1.1 and VvNRT1.60 exhibited the highest expression levels at the bud swell and mature fruit stages, respectively. In addition, some genes, such as VvNRT1.34 , were highly expressed in multiple tissues, whereas others, including VvNRT1.9 and VvNRT1.10 , consistently maintained low expression levels in most tissues. Furthermore, all genes in group Ⅲ demonstrated uniformly lower expression across all examined tissues and organs than those in other subgroups. Based on these expression patterns, VvNRT1 genes may exhibit functional differentiation that supports coordinated nitrogen uptake and transport across grapevine tissues. Collectively, these genes represent potential targets for improving nitrogen use efficiency and may serve as integral regulators of grapevine growth and responses to environmental stress. Expression profiles of VvNRT1s after KNO 3 treatment and salt stress Based on the above analyses, numerous hormone- and stress-related cis-acting elements were identified in the promoters of VvNRT 1 genes. In addition, previous studies have shown that NPF5.15 and NPF7.3 play important roles in salt tolerance in Arabidopsis [ 23 ]. To examine the responses of VvNRT1 genes to NO 3 ⁻ treatment and salt stress, their expression levels were analyzed in grape leaves and roots at 10 min, 0.5 h, 2 h, and 8 h after NO 3 ⁻ treatment, and at 6, 12, 24, and 48 h after salt treatment. The experiment was conducted using own-rooted seedlings of the ‘ Shine Muscat ’ cultivar, which was selected for its wide cultivation, economic value, and strong stress tolerance, thereby enabling the assessment of expression patterns of selected VvNRT1s under stress conditions. Four grape genes homologous to Arabidopsis AtNPF4.3 , AtNPF5.10 , AtNPF5.15 , and AtNPF7.3 were examined, corresponding to VvNRT1.35 , VvNRT1.59 , VvNRT1.19 , and VvNRT1.47 , respectively. The expression levels of VvNRT1.19 and VvNRT1.47 were higher than those of the 0 h control under both NO 3 ⁻ treatment and salt stress. Under NO 3 ⁻ treatment, a progressive increase in expression was observed in the roots over time, with the expression in both roots and leaves reaching a maximum at 8 h (Fig. 5 a). Under salt stress, the expression in both roots and leaves peaked at 6 h, after which the expression of VvNRT1.19 in the roots gradually declined (Fig. 5 b). Although VvNRT1.35 and VvNRT1.59 demonstrated increased expression in roots under salt stress compared with the 0 h control, their expression in leaves under both nitrate and salt treatments displayed time points at which transcript levels were reduced relative to the control (Fig. 5 ). VvNRT1.19 and VvNRT1.47 overexpression enhanced nitrogen use efficiency and salt tolerance in Arabidopsis Both VvNRT1.19 and VvNRT1.47 were transcriptionally induced in grape roots and leaves at 10 min, 0.5 h, 2 h, and 8 h following NO 3 ⁻ treatment, as well as at 6, 12, 24, and 48 h after salt treatment. VvNRT1.19 and VvNRT1.47 were highly homologous to AtNPF5.15 and AtNPF7.3 , respectively, which are key members of the NO 3 ⁻ transporter family known to contribute to nitrogen use efficiency, although their roles in plant salt tolerance remain unclear. To examine the functions of VvNRT1.19 and VvNRT1.47 in responses to NO 3 ⁻ availability and salt stress, 12 VvNRT1.19 -overexpressing ( VvNRT1.19-OE ) lines and 10 VvNRT1.47 -overexpressing ( VvNRT1.47-OE ) Arabidopsis lines were generated via Agrobacterium -mediated transformation. qPCR analysis confirmed the markedly elevated transcript levels of both genes in the leaves of the transgenic lines (Fig. 6 a and b). Based on these results, the lines showing the highest expression levels of VvNRT1.19 (#11) and VvNRT1.47 (#10) were selected for further analyses. To determine whether the overexpression of VvNRT1.19 and VvNRT1.47 influenced nitrogen use efficiency and salt tolerance, transgenic and wild-type (WT) plants were grown under 0, 1, and 5 mM KNO 3 conditions and subjected to 200 mM NaCl stress. Under all tested KNO 3 concentrations, the WT seedlings displayed inhibited root development, cotyledon yellowing, and retarded growth, whereas the overexpression lines maintained well-developed root systems (Fig. 6 c) and vigorous seedling growth (Fig. 6 d). At 1 and 5 mM KNO 3 , the average root length of the overexpression lines was significantly greater than that of WT plants (Fig. 6 f). Under 0 and 1 mM KNO 3 conditions, both the number of lateral roots and cotyledon area were also significantly increased in the overexpression lines compared with WT plants (Fig. 6 g and h). These results indicated that the overexpression of VvNRT1.47 and VvNRT1.19 enhanced NO 3 ⁻ uptake and utilization, thereby promoting plant growth under both nitrate-deficient and nitrate-sufficient conditions. In addition, the performance of overexpression lines was assessed under salt stress (200 mM NaCl). The WT seedlings exhibited severe growth inhibition and pronounced leaf yellowing, whereas VvNRT1.47-OE and VvNRT1.19-OE seedlings maintained comparatively better growth and more normal morphology (Fig. 6 e). Moreover, the survival rates of the overexpression lines were significantly higher than those of WT plants at both day 8 and day 14 (Fig. 6 i), suggesting that VvNRT1.47 and VvNRT1.19 contributed to improved survival under salt stress conditions. Discussion NRT1 proteins represent the largest and most functionally diverse NO 3 ⁻ transporter group in plants and are central to plant growth regulation, stress responses, and NO 3 ⁻ uptake and metabolism. Genome-wide analyses of NRT1 gene families have been reported in several species, including Sorghum bicolor [ 28 ], Eucalyptus grandis [ 29 ], Setaria italica [ 30 ], Brassica rapa [ 31 ], and Cocos nucifera L. [ 32 ]. However, a systematic genome-wide investigation of the NRT1 family in grapevine ( Vitis vinifera ) has not been performed. Here, 65 VvNRT1 genes were identified in grapevine and further characterized by phylogenetic classification, gene structure analysis, and expression profiling. These results provide evidence for the evolutionary features of grapevine NRT1s and support their potential involvement in nitrogen utilization and stress adaptation. The 65 VvNRT1 genes identified in this study exceeded the 53 NRT1 members reported in Arabidopsis [ 33 ], indicating an expanded NRT1 repertoire in grapevine. Phylogenetic analysis divided VvNRT1s into five groups (Ⅰ–Ⅴ), differing from the eight-group classification commonly reported in Arabidopsis and rice [ 33 ], which may reflect the grapevine-specific evolutionary history, including polyploidization and lineage-specific duplication. Chromosomal mapping revealed an enrichment of VvNRT1s on chromosome 18 (Fig. 1 b), where multiple tandemly duplicated genes (e.g., VvNRT1.59 – VvNRT1.65 ) were concentrated, suggesting that tandem duplication contributed substantially to this clustering. Such clustering may facilitate the coordinated regulation of genes related to N transport or stress responses in grapevine. The duplication analysis indicated that 64.62% of VvNRT1s were associated with segmental duplication, whereas 30.77% formed tandem duplication pairs (Fig. 1 b), indicating that both duplication modes contributed to family expansion, similar to the pattern reported for the apple NRT1 family [ 34 ]. Tandem duplicates often retain similar functions, whereas segmental duplicates more frequently diverge functionally [ 35 ]. Consistent with this tendency, tandemly duplicated genes, such as VvNRT1.49 – VvNRT1.51 , displayed similar motif compositions and tissue expression patterns, whereas the segmentally duplicated pair VvNRT1.1 and VvNRT1.6 differed markedly in cis-regulatory element profiles (Fig. 3 ), implying possible divergence associated with environmental adaptation. Conserved motif and gene structure analyses further indicated that genes within the same phylogenetic group generally shared similar motif compositions and exon–intron architectures (Fig. 2 ), supporting the reliability of the classification. For instance, Group Ⅰ members VvNRT1.63 and VvNRT1.64 both contained nine exons and motifs 1–10, whereas Group Ⅴ members carried fewer motifs, potentially reflecting their functional specialization. Notably, VvNRT1.40 and VvNRT1.55 contained a single exon and one motif. Comparable compact NRT1 genes have also been reported in Arabidopsis (e.g., AtNPF2.9 ) and proposed to enable rapid responses to environmental signals via a simplified regulatory architecture [ 36 ]. Cis-element analysis suggested that VvNRT1 promoters were enriched in elements related to hormone responses (e.g., MeJA and ABA), stress responses (e.g., low temperature and anoxia), and growth and developmental regulation (Fig. 3 ), which can support a role in multi-signal integration during grapevine growth and stress adaptation. Notably, Group Ⅴ contained abundant stress-responsive elements and was enriched in hormone-related elements, suggesting a contribution to hormone–stress crosstalk, consistent with reports that rice NPFs participate in the coordinated regulation of salt stress responses and ABA signaling [ 37 – 38 ]. Tissue-specific expression profiling revealed distinct expression patterns among VvNRT1s . VvNRT1.7 and VvNRT1.21 were predominantly expressed in roots, suggesting roles in nitrate uptake, whereas VvNRT1.34 and VvNRT1.38 showed elevated expression during flowering, indicating their potential involvement in the nitrogen supply to reproductive organs. In addition, VvNRT1.1 exhibited high expression during bud swelling, implying a role in bud development (Fig. 4 ). This expression specificity resembled the tissue-based functional differentiation reported for Arabidopsis NRT1s , such as AtNPF6.3 , which functions in root NO 3 ⁻ uptake, and AtNPF7.2 , which mediates NO 3 ⁻ redistribution in leaves [ 39 – 40 ]. These observations suggest that the grapevine NRT1 family retained conserved functional patterns while acquiring specialized expression profiles in newly expanded members to accommodate the perennial growth habit of fruit trees. The functions of NRT genes in Arabidopsis thaliana have been extensively characterized, with different subfamilies participating in N uptake, transport, signaling, and stress adaptation. AtNRT genes primarily act as molecular carriers that mediate NO 3 ⁻ uptake and internal allocation. The two major subfamilies operate under distinct nitrogen regimes. The NRT1 (NPF) subfamily mainly mediates low-affinity transport under high nitrogen conditions (soil NO 3 − > 1 mmol/L), facilitating NO 3 ⁻ uptake or its translocation within the plant (root → stem → leaf; source organ → sink organ) [ 41 ]. For instance, AtNRT1.5 [ 40 ] mediates root-to-shoot NO 3 − translocation, whereas AtNRT1.4 [ 42 ] is involved in NO 3 − storage in leaves. Notably, certain members, such as AtNRT1.1 [ 43 ], act as “dual-affinity transporters”, enabling NO 3 − absorption across a broad range of external concentrations. In contrast, the NRT2 subfamily specializes in high-affinity nitrate transport under low nitrogen availability (soil NO 3 − < 0.5 mmol/L) and plays a critical role in nitrogen deficiency adaptation [ 41 ]. Representative genes, such as AtNRT2.1 and AtNRT2.2 , are predominantly expressed in root epidermal cells and root hairs, where they directly absorb NO 3 − from the soil [ 44 ]. In this study, VvNRT1.19 and VvNRT1.47 , which showed the highest homology to Arabidopsis AtNPF5.15 and AtNPF7.3 , respectively, were significantly upregulated under high-N conditions (Fig. 5 a). Both genes were strongly induced by salt stress (Fig. 5 b), indicating that they responded to N signaling and participated in salt stress responses. Subsequently, VvNRT1.19 and VvNRT1.47 were cloned to generate 12 VvNRT1.19-OE and 10 VvNRT1.47-OE Arabidopsis lines. Lines exhibiting the highest expression levels were selected for the validation of nitrogen use efficiency and salt tolerance (Fig. 6 a and b). Overexpression of VvNRT1.19 or VvNRT1.47 resulted in improved nitrogen use efficiency and enhanced salt tolerance under nitrate treatment (Fig. 6 c–i), demonstrating that both genes contributed to the coordinated regulation of nitrogen utilization and salt tolerance. In summary, this study presents a comprehensive analysis of the NRT1 gene family in grapevine. By integrating bioinformatics and experimental approaches, this study defined the key structural, evolutionary, and functional features of VvNRT1s and established a framework for further investigation. This study provides the first evidence that VvNRT1.19 and VvNRT1.47 function as N-responsive genes involved in both N metabolism and salt stress responses, offering insights for future functional studies of NRT1 genes in grapevine and other plant species. Conclusion In this study, 65 intact NRT1 genes were systematically identified in the grapevine genome and classified into five distinct phylogenetic groups. Duplication pattern analyses indicated that both segmental and tandem duplication events contributed to the expansion of the VvNRT1 gene family. Combined analyses of promoter cis-elements and tissue-specific expression patterns suggested that VvNRT1 genes participated in grapevine growth, environmental stress responses, and nitrogen regulation. Moreover, several VvNRT1 members exhibited pronounced transcriptional induction in roots and leaves in response to NO 3 − and salt treatments. Functional assays using transgenic Arabidopsis plants further demonstrated that ectopic expression of VvNRT1.19 and VvNRT1.47 significantly enhanced nitrogen use efficiency and salt tolerance. In summary, these results extend the current understanding of the functional diversity of the VvNRT1 family and highlight its potential value in improving nutrient acquisition and stress adaptability in plants. Materials and methods Identification of VvNRT1 Genes Arabidopsis NRT1 protein sequences were obtained from the TAIR 1 database and used as queries to screen the grapevine genomes. BLASTP [ 45 ] searches were performed against the Phytozome2 2 and Winberige 3 databases to identify putative VvNRT1 members, and only candidates with E-values below 1 × 10 − 10 were retained. To confirm domain composition, conserved LRR and extensin domains were examined using the NCBI Conserved Domain Database and SMART. Basic physicochemical properties, including protein length, molecular weight, and theoretical isoelectric point, were predicted using the ExPASy ProtParam server 4 . Phylogenetic and Collinearity Analysis Full-length NRT1 protein sequences from grapevine and Arabidopsis were aligned using ClustalW. A maximum-likelihood phylogenetic tree was constructed using MEGA 7.0, and branch support was assessed using 1000 bootstrap replicates [ 46 ]. Collinearity and gene duplication events were analyzed using MCScanX [ 47 ]. Chromosomal location data were obtained from the Winberge database, and syntenic relationships were visualized using TBtools [ 48 ]. Gene Structure and Motif Analysis The exon–intron organization of VvNRT1 genes was analyzed using the Gene Structure Display Server 5 [49]. Gene structure diagrams were generated using TBtools. Conserved protein motifs were identified using MEME 6 [50], and motif distribution patterns among VvNRT1 proteins were examined to evaluate their structural conservation. Promoter Analysis Upstream sequences (2 kb) of each VvNRT1 gene were extracted using TBtools. Cis-regulatory elements were predicted with PlantCARE 7 , and their distribution patterns were visualized. Tissue Expression Analysis Transcriptome datasets were retrieved from the BAR database 8 , covering roots, stems, leaves, buds, flowers, fruits, seedlings, and pollen. After removing low-abundance transcripts, expression values were log₂-transformed and normalized. Heatmaps were generated using TBtools. Plant Materials and Treatments One-year-old own-rooted grapevine seedlings ( Vitis vinifera L. cv. Shine Muscat) were obtained from Shandong Zhichang Agricultural Science and Technology Development Co., Ltd. Uniform seedlings were selected and grown under greenhouse conditions (12 h light/12 h dark; 25–28°C day/5–10°C night; 55–65% relative humidity). Seedlings were cultivated in plastic pots containing washed sand and treated with 15 mM KNO 3 or 200 mM NaCl to evaluate nitrate and salt responses. Leaves and roots were collected at designated time points, immediately frozen in liquid nitrogen, and stored at − 80°C. qRT-PCR Gene-specific primers were designed using SnapGene. Total RNA was extracted using the QuickRNA kit, followed by genomic DNA removal. RNA quality was assessed using NanoDrop and Bioanalyzer platforms. cDNA synthesis was performed using PrimeScript RT Master Mix. qRT-PCR was conducted on a CFX96 system using TB Green chemistry, with VvUBI serving as the internal reference gene [ 44 ]. Each reaction contained 2 µL of cDNA. Amplification was performed for 40 cycles following initial denaturation, and melting curve analysis was used to verify primer specificity. Relative gene expression levels were calculated using the 2⁻ΔΔCt method [ 51 ]. Vector Construction and Plant Transformation Full-length coding sequences (CDS) of VvNRT1.47 (1815 bp) and VvNRT1.19 (324 bp) were amplified from cDNA synthesized from leaves of own-rooted ‘Shine Muscat’ grapevine seedlings and verified by sequencing. Validated CDS fragments were cloned into the plant expression vector pBWA(V)BS under the control of the CaMV 35S promoter using the MultiF Seamless Assembly system (ABclonal). Recombinant plasmids pBWA(V)BS-VvNRT1.47 and pBWA(V)BS-VvNRT1.19 were introduced into Agrobacterium tumefaciens strain GV3101. Primer sequences used for gene amplification are listed in Table S1 . Transgenic Arabidopsis thaliana plants harboring 35S::VvNRT1.47 and 35S::VvNRT1.19 constructs were generated via Agrobacterium -mediated transformation. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details 1 College of Horticulture and Landscape Architecture, Yangzhou University, Yangzhou 225009, China 2 Shandong Academy of Grape, Shandong Academy of Agricultural Sciences, Jinan 250100, China 3 Weifang University, Weifang 261061, China Funding This study was supported by the Natural Science Foundation of Shandong Province (Grant No. ZR2023MC101) and Agricultural Science and Technology Innovation Project of Shandong Academy of Agricultural Sciences(Grant No. CXGC2026A47), and Science and Technology Support Plan for Youth Innovation of Colleges and Universities of Shandong Province of China (2024KJI023) and Jinan Comprehensive Experiment Station of National Grape Industry Technology System (CARS-29-16). Author Contribution Chen Zhou : Conceptualization, Sample preparation and analysis, Methodology, Data curation, Writing-original draft, Supervision. Yingchun Chen : Data curation, Methodology, Formal analysis. Kai Liu : Methodology, Conceptualization, Supervision. Xiujie Li : Conceptualization, Supervision. Xuehui Zhao : Data curation, Conceptualization. Bo Li : Conceptualization, Supervision. Li Liu : Conceptualization, Writing – review & editing, Supervision. Zhaosen Xie : Conceptualization, Methodology, Supervision. Data Availability All data generated or analyzed during this study are included in this published article. The genome-wide sequence data of *Vitis vinifera* L. supporting the findings of this study are publicly available in the NCBI database ( [https://www.ncbi.nlm.nih.gov/](https:/www.ncbi.nlm.nih.gov) ) and Winberige database (http://www.winberige.cc/index.php). References Wang Q, Li S, Li J, Huang D. The Utilization and Roles of Nitrogen in Plants. Forests 2024, 15(7). Hachiya T, Sakakibara H. Interactions between nitrate and ammonium in their uptake, allocation, assimilation, and signaling in plants. J Exp Bot 2016. Nedelyaeva OI, Khramov DE, Balnokin YV, Volkov VS. Functional and Molecular Characterization of Plant Nitrate Transporters Belonging to NPF (NRT1/PTR) 6 Subfamily. Int J Mol Sci 2024, 25(24). TONG Y-p CAIC, LIU, Q-y. LI J-y, LI Z-s: Recent advances in molecular biology of nitrate transporters in higher plants. J Plant Nutr Fertilizers. 2004;10(4):433–40. Orsel M, Filleur S, Fraisier V, Daniel-Vedele F. Nitrate transport in plants: which gene and which control? J Exp Bot. 2002;53(370):825–33. Wen Z, Kaiser BN. Unraveling the functional role of NPF6 transporters. Front Plant Sci. 2018;9:973. Rahmat Z, Sohail MN, Perrine-Walker F, Kaiser BN. Balancing nitrate acquisition strategies in symbiotic legumes. Planta. 2023;258(1):12. Kanno Y, Kamiya Y, Seo M. Nitrate does not compete with abscisic acid as a substrate of AtNPF4. 6/NRT1. 2/AIT1 in Arabidopsis. Plant Signal Behav. 2013;8(12):e26624. Léran S, Garg B, Boursiac Y, Corratgé-Faillie C, Brachet C, Tillard P, Gojon A, Lacombe B. AtNPF5. 5, a nitrate transporter affecting nitrogen accumulation in Arabidopsis embryo. Sci Rep. 2015;5(1):7962. Siangliw JL, Ruangsiri M, Theerawitaya C, Cha-Um S, Poncheewin W, Songtoasesakul D, Thunnom B, Ruanjaichon V, Toojinda T. Contrasting Alleles of OsNRT1. 1b Fostering Potential in Improving Nitrogen Use Efficiency in Rice. Plants. 2024;13(20):2932. Duan D, Zhang H. A single SNP in NRT1. 1B has a major impact on nitrogen use efficiency in rice. Sci China Life Sci. 2015;58(8):827. Cao H, Liu Z, Guo J, Jia Z, Shi Y, Kang K, Peng W, Wang Z, Chen L, Neuhaeuser B. ZmNRT1. 1B (ZmNPF6. 6) determines nitrogen use efficiency via regulation of nitrate transport and signalling in maize. Plant Biotechnol J. 2024;22(2):316–29. Wang Q, Liu C, Dong Q, Huang D, Li C, Li P, Ma F. Genome-wide identification and analysis of apple NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family (NPF) genes reveals MdNPF6. 5 confers high capacity for nitrogen uptake under low-nitrogen conditions. Int J Mol Sci. 2018;19(9):2761. Zhang H, Zhao X, Sun Q, Yan C, Wang J, Yuan C, Li C, Shan S, Liu F. Comparative transcriptome analysis reveals molecular defensive mechanism of Arachis hypogaea in response to salt stress. Int J Genomics 2020, 2020(1):6524093. Yang D-c, Xu Q, Zhao F-g, Liu M, Liu X. Effects of NaCI stress on nitrogen metabolism and related gene expression of rice with different resistance. 2020. Liu R, Cui B, Jia T, Song J. Role of Suaeda salsa SsNRT2. 1 in nitrate uptake under low nitrate and high saline conditions. Plant Physiol Biochem. 2021;159:171–8. Liu R, Cui B, Lu X, Song J. The positive effect of salinity on nitrate uptake in Suaeda salsa. Plant Physiol Biochem. 2021;166:958–63. Sena F, Kunze R. The K+ transporter NPF7. 3/NRT1. 5 and the proton pump AHA2 contribute to K+ transport in Arabidopsis thaliana under K + and NO3-deficiency. Front Plant Sci. 2023;14:1287843. Wang X, Liu Y, Li W, Ma X, Wang W, Jiang Z, Wang Y, Li L, Hu B, Chu C. OsNRT1. 1B-OsCNGC14/16‐Ca2+‐OsNLP3 Pathway: Phosphorylation‐Mediated Maintenance of Nitrogen Homeostasis. Adv Sci. 2025;12(43):e07919. Sa G, Yao J, Deng C, Liu J, Zhang Y, Zhu Z, Zhang Y, Ma X, Zhao R, Lin S. Amelioration of nitrate uptake under salt stress by ectomycorrhiza with and without a Hartig net. New Phytol. 2019;222(4):1951–64. Keller M, Koblet W. Dry matter and leaf area partitioning, bud fertility and second season growth of Vitis vinifera L.: Responses to nitrogen supply and limiting irradiance. Vitis. 1995;34(2):77–83. Stefanello LO, Schwalbert R, Marques AR, Tarouco CP, Vitto BB, Krug A, dos Santos JJ, Galarza BP, Zalamena J, dos Santos HP. Photosynthetic activity and grape yield of'Alicante Bouschet'(Vitis vinifera L.) grapevines submitted to nitrogen supply methods and doses. Vitis. 2020;59:133–40. Zhang C, Lu X, Yan H, Gong M, Wang W, Chen B, Ma S, Li S. Nitrogen application improves salt tolerance of grape seedlings via regulating hormone metabolism. Physiol Plant. 2023;175(2):e13896. Altemose N, Logsdon GA, Bzikadze AV, Sidhwani P, Langley SA, Caldas GV, Hoyt SJ, Uralsky L, Ryabov FD, Shew CJ. Complete genomic and epigenetic maps of human centromeres. Science. 2022;376(6588):eabl4178. Yang Z, Mei J, Zheng W, Khan FS, Bhuiyan MN, Wang K, Rhaman MS, Abe-Kanoh N, Ji W. Identification of Grape NRT Gene Family and Analysis of Its Expression in Leaves Under Nitrogen-Deficiency Stress. Horticulturae. 2025;11(3):252. Monte R, Tomasi N, Cesco S, Zamboni A, Varanini Z, Pinton R. Physiological and molecular characterization of nitrate uptake mechanisms in roots of grapevine (Vitis vinifera L.) cultivars. 2009. Shi X, Cao S, Wang X, Huang S, Wang Y, Liu Z, Liu W, Leng X, Peng Y, Wang N. The complete reference genome for grapevine (Vitis vinifera L.) genetics and breeding. Hortic Res. 2023;10(5):uhad061. Zhiqiang G, Yuexiu L, Fan F, Lixun Z, Jiali F, Xiaodong J, Jinhui L, Chunlai Z. Genome-wide Identification of NRT1 Gene, Expression Profiling and DNA Variation Analysis in Sorghum. J Laser Biology/Acta Laser Biology Sinica 2021, 30(5). Li G, Yang D, Hu Y, Xu J, Lu Z. Genome-wide identification and expression analysis of nitrate transporter (NRT) gene family in Eucalyptus grandis. Genes. 2024;15(7):930. Cheng J, Tan H, Shan M, Duan M, Ye L, Yang Y, He L, Shen H, Yang Z, Wang X. Genome-wide identification and characterization of the NPF genes provide new insight into low nitrogen tolerance in Setaria. Front Plant Sci. 2022;13:1043832. Zhang Y, Ritonga FN, Zhang S, Wang F, Li J, Gao J. Genome-Wide Identification of the NRT1 Family Members and Their Expression under Low-Nitrate Conditions in Chinese Cabbage (Brassica rapa L. ssp. pekinensis). Plants. 2023;12(22):3882. Liu X, Li J, Luo D, Ding H, Zhang M, Gao P, Mehvish A, Sun X, Tong C, Wu Q. Genome-wide characterization of the NRT1 family members under cold stress in Coconut (Cocos nucifera L). Sci Hort. 2025;341:113959. Léran S, Varala K, Boyer J-C, Chiurazzi M, Crawford N, Daniel-Vedele F, David L, Dickstein R, Fernandez E, Forde B. A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants. Trends Plant Sci. 2014;19(1):5–9. Tahir MM, Wang H, Ahmad B, Liu Y, Fan S, Li K, Lei C, Shah K, Li S, Zhang D. Identification and characterization of NRT gene family reveals their critical response to nitrate regulation during adventitious root formation and development in apple rootstock. Sci Hort. 2021;275:109642. Laha SD, Dutta S, Schäffner AR, Das M. Gene duplication and stress genomics in Brassicas: Current understanding and future prospects. J Plant Physiol. 2020;255:153293. Wang Y-Y, Tsay Y-F. Arabidopsis nitrate transporter NRT1. 9 is important in phloem nitrate transport. Plant Cell. 2011;23(5):1945–57. Hang J, Wu B, Qiu D, Yang G, Fang Z, Zhang M. OsNPF3. 1, a nitrate, abscisic acid and gibberellin transporter gene, is essential for rice tillering and nitrogen utilization efficiency. J Integr Agric. 2024;23(4):1087–104. Islam MS. Sensing and uptake of nitrogen in rice plant: a molecular view. Rice Sci. 2019;26(6):343–55. LIU H, LIU Q, GAO X, FU X. Role of nitrogen sensing and its integrative signaling pathways in shaping root system architecture. Eng Agric. 2022;9(3):316–32. Li J-Y, Fu Y-L, Pike SM, Bao J, Tian W, Zhang Y, Chen C-Z, Zhang Y, Li H-M, Huang J. The Arabidopsis nitrate transporter NRT1. 8 functions in nitrate removal from the xylem sap and mediates cadmium tolerance. Plant Cell. 2010;22(5):1633–46. Chiba Y, Shimizu T, Miyakawa S, Kanno Y, Koshiba T, Kamiya Y, Seo M. Identification of Arabidopsis thaliana NRT1/PTR FAMILY (NPF) proteins capable of transporting plant hormones. J Plant Res. 2015;128(4):679–86. Krapp A, David LC, Chardin C, Girin T, Marmagne A, Leprince A-S, Chaillou S, Ferrario-Méry S, Meyer C, Daniel-Vedele F. Nitrate transport and signalling in Arabidopsis. J Exp Bot. 2014;65(3):789–98. Bouguyon É, Brun F, Meynard D, Kubeš M, Pervent M, Léran S, Lacombe B, Krouk G, Guiderdoni E, Zažímalová E. Multiple mechanisms of nitrate sensing by Arabidopsis nitrate transceptor NRT1. 1. Nat plants. 2015;1(3):1–8. Ho C-H, Lin S-H, Hu H-C, Tsay Y-F. CHL1 functions as a nitrate sensor in plants. Cell. 2009;138(6):1184–94. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215(3):403–10. Thompson JD, Gibson TJ, Higgins DG. Multiple sequence alignment using ClustalW and ClustalX. Curr protocols Bioinf 2003(1):2.3. 1-2.3. 22. Wang Y, Tang H, DeBarry JD, Tan X, Li J, Wang X, Lee T-h, Jin H, Marler B, Guo H. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012;40(7):e49–49. Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020;13(8):1194–202. Guo A-Y, Zhu Q-H, Chen X, Luo J-C. GSDS: a gene structure display server. Yi chuan= Hereditas. 2007;29(8):1023–6. Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, Li WW, Noble WS. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 2009;37(suppl2):W202–8. Liu K, Yang A, Yan J, Liang Z, Yuan G, Cong P, Zhang L, Han X, Zhang C. MdAIL5 overexpression promotes apple adventitious shoot regeneration by regulating hormone signaling and activating the expression of shoot development-related genes. Hortic Res. 2023;10(11):uhad198. Footnotes https://www.arabidopsis.org/browse/gene_family https://phytozome-next.jgi.doe.gov/blast-search http://www.winberige.cc/ftp.html https://web.expasy.org/protparam/ http://gsds.cbi.pku.edu.cn/ https://meme-suite.org/meme/tools/meme http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ https://bar.utoronto.ca/efp_grape/cgi-bin/efpWeb.cgi Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8769993","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":608164969,"identity":"cc25890f-bc4b-411c-a05e-4ba203e9bd08","order_by":0,"name":"Chen Zhou","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Zhou","suffix":""},{"id":608164970,"identity":"13470a28-ed11-4ff2-a133-24f8e9e9677f","order_by":1,"name":"Yingchun Chen","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yingchun","middleName":"","lastName":"Chen","suffix":""},{"id":608164971,"identity":"a97c9108-c2b4-4860-804f-8bc8d2a8aac9","order_by":2,"name":"Kai Liu","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Liu","suffix":""},{"id":608164972,"identity":"71bf6d1a-01d3-4650-8e52-e99156d14440","order_by":3,"name":"Xiujie Li","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiujie","middleName":"","lastName":"Li","suffix":""},{"id":608164973,"identity":"cbc99884-c673-4768-a83b-7c251c09338e","order_by":4,"name":"Xuehui Zhao","email":"","orcid":"","institution":"Weifang University","correspondingAuthor":false,"prefix":"","firstName":"Xuehui","middleName":"","lastName":"Zhao","suffix":""},{"id":608164974,"identity":"2829b1d4-e262-45aa-9eec-984dc011d709","order_by":5,"name":"Bo Li","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Li","suffix":""},{"id":608164975,"identity":"a1dd0458-ab7c-4863-b0a3-47d430e749b7","order_by":6,"name":"Li Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIie3PMYrCQBTG8QcDsXma9olBrzAgZFlY8AIeYgZtBSuZwiKSZabQPcFewkosHYRUY5/CIrmBdna6vZJkO4v51e8P3wPwvDcUDKy5XtQdRya1hVDL+qRDTDNyLOKYTXjhsvqkTy0Nbc2+OI3jbvnNGgzrrXRBLsAPwljJJIDQrEV1ElnD5wrx81cvcrmPgNxpW52ATIgcIZyPu1y6ADjNGiRtzRFyEc+lZg0SkvovEcjzaQzNErTpkNwBu5tsQsJlWPvLwJiyvKjDKGyl9npTy35ofqqTJ/i/c8/zPO+lBwwiSs9cZtW0AAAAAElFTkSuQmCC","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Li","middleName":"","lastName":"Liu","suffix":""},{"id":608164976,"identity":"42d4602e-e453-41a9-af48-56818ab25d6e","order_by":7,"name":"Zhaosen Xie","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Zhaosen","middleName":"","lastName":"Xie","suffix":""}],"badges":[],"createdAt":"2026-02-03 02:23:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8769993/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8769993/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105035653,"identity":"40a85656-61e8-4690-a408-dff6e147714a","added_by":"auto","created_at":"2026-03-20 07:26:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":733550,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic and synteny analyses of the NRT1 gene family. \u003cstrong\u003ea\u003c/strong\u003e Phylogenetic relationship between NRT1 proteins from grapevine and \u003cem\u003eArabidopsis\u003c/em\u003e. \u003cstrong\u003eb\u003c/strong\u003e Duplication events and chromosomal distribution of \u003cem\u003eVvNRT1s\u003c/em\u003e. Segmentally duplicated gene pairs are linked by red lines, and tandemly duplicated genes are linked by green lines.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8769993/v1/60c936b6bcdbb70935de3c18.png"},{"id":105005447,"identity":"032fec22-b5db-4459-a934-42188639d7b3","added_by":"auto","created_at":"2026-03-19 18:19:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":409919,"visible":true,"origin":"","legend":"\u003cp\u003eConserved motifs and structures of \u003cem\u003eVvNRT1\u003c/em\u003e genes.\u003cstrong\u003e a \u003c/strong\u003ePhylogenetic tree of VvNRT1s.\u003cstrong\u003e b \u003c/strong\u003eConserved motifs within \u003cem\u003eVvNRT1s\u003c/em\u003e, represented by colored squares for motifs 1–10.\u003cstrong\u003ec \u003c/strong\u003eGene structures of \u003cem\u003eVvNRT1s\u003c/em\u003e, with UTRs and CDSs shown as green and yellow squares, respectively, and introns are indicated by black lines.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8769993/v1/8a20332fe33be2ef650b6fd5.png"},{"id":105005448,"identity":"64fa831f-ed5f-4e99-ae8a-53569334011c","added_by":"auto","created_at":"2026-03-19 18:19:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":669823,"visible":true,"origin":"","legend":"\u003cp\u003eMajor cis-acting elements in \u003cem\u003eVvNRT1\u003c/em\u003e promoters. \u003cstrong\u003ea\u003c/strong\u003e Location of various cis-element types on chromosomes. Different types of cis-elements are represented by circles of different colors. \u003cstrong\u003eb\u003c/strong\u003e Heatmap of the abundance of different cis-elements. The red and blue boxes indicate higher and lower numbers of each cis-element type, respectively.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8769993/v1/c007d5004e00669bd608c692.png"},{"id":105005450,"identity":"9d83e689-538d-47ac-954e-c5be8fab1353","added_by":"auto","created_at":"2026-03-19 18:19:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":728322,"visible":true,"origin":"","legend":"\u003cp\u003eTissue-specific expression patterns of \u003cem\u003eVvNRT1\u003c/em\u003e genes\u003cstrong\u003e.\u003c/strong\u003e The heatmap illustrates the expression profiles of \u003cem\u003eVvNRT1s\u003c/em\u003e across different grapevine tissues, derived from in silico analysis using data retrieved from the BAR database and mapped to the \u003cem\u003eVitis vinifera\u003c/em\u003e T2T2 genome. The expression values were normalized, log\u003csub\u003e2\u003c/sub\u003e-transformed, and hierarchically clustered. The color scale from blue to red represents the low to high expression levels of individual \u003cem\u003eVvNRT1\u003c/em\u003e genes.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8769993/v1/2c4bdb9774106c05536e9af1.png"},{"id":105005449,"identity":"c431ed22-f6ef-46c7-95ec-4e018b1a3ed4","added_by":"auto","created_at":"2026-03-19 18:19:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":653291,"visible":true,"origin":"","legend":"\u003cp\u003eExpression profiles of four \u003cem\u003eVvNRT1s \u003c/em\u003ein Shine Muscat leaves at various time points after treatment. \u003cstrong\u003ea \u003c/strong\u003eExpression levels under nitrate treatment. \u003cstrong\u003eb\u003c/strong\u003e Expression levels following salt treatment. Error bars represent the standard deviation (SD). Different lowercase letters indicate significant differences according to Duncan’s multiple range test at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, whereas identical letters indicate no significant differences. The leaves and roots are highlighted in green and brown, respectively.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8769993/v1/9af8602838f960b262b5502f.png"},{"id":105005452,"identity":"0576cee0-1433-4be7-9e7b-9ff322ba8052","added_by":"auto","created_at":"2026-03-19 18:19:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":729301,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of \u003cem\u003eVvNRT1.19\u003c/em\u003e and \u003cem\u003eVvNRT1.47\u003c/em\u003e enhances nitrogen utilization and salt tolerance in Arabidopsis. \u003cstrong\u003ea\u003c/strong\u003e Relative expression levels of \u003cem\u003eVvNRT1.19\u003c/em\u003e in overexpression (OE) lines and wild-type (WT) plants. \u003cstrong\u003eb\u003c/strong\u003e Relative expression levels of \u003cem\u003eVvNRT1.47\u003c/em\u003e in OE lines and WT plants. \u003cstrong\u003ec\u003c/strong\u003e Root morphology of WT, \u003cem\u003eVvNRT1.47-OE\u003c/em\u003e, and \u003cem\u003eVvNRT1.19-OE\u003c/em\u003e seedlings cultivated on half-strength MS (1/2 MS) medium supplemented with 0, 1, or 5 mM KNO\u003csub\u003e3\u003c/sub\u003e as the sole nitrogen source. Scale bar = 1 cm. \u003cstrong\u003ed\u003c/strong\u003e Whole-seedling phenotypes corresponding to the treatments shown in (c). Scale bar = 1 cm. \u003cstrong\u003ee\u003c/strong\u003e Phenotypic comparison of WT and transgenic seedlings grown on control (1/2 MS) or salt-stressed medium (1/2 MS + 200 mM NaCl). Scale bar = 1 cm. \u003cstrong\u003ef\u003c/strong\u003e Mean primary root length of WT and OE lines under different nitrate concentrations (0, 1, and 5 mM KNO\u003csub\u003e3\u003c/sub\u003e). \u003cstrong\u003eg\u003c/strong\u003e Lateral root numbers of WT and OE lines under the same nitrate treatment. \u003cstrong\u003eh\u003c/strong\u003e Total cotyledon area of WT and OE seedlings grown under varying nitrate concentrations. \u003cstrong\u003ei\u003c/strong\u003e Survival rates of WT and OE plants exposed to 200 mM NaCl. Error bars represent the standard deviation (SD). Different lowercase letters indicate statistically significant differences (P \u0026lt; 0.05, Student’s t-test).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8769993/v1/1b7a6e3d9ed5045ec63fda0b.png"},{"id":108007355,"identity":"67e3b3ba-d7ea-4cad-8a40-f9d2a41e8fee","added_by":"auto","created_at":"2026-04-28 12:59:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4671985,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8769993/v1/e1963b72-439b-400f-8936-d0d15e84bb0a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-Wide Analysis and Functional Characterization of the NRT1 Gene Family in Grapevine (Vitis vinifera L.) Reveals Roles of VvNRT1.19 and VvNRT1.47 in Salt Stress and Nitrogen Response","fulltext":[{"header":"Background","content":"\u003cp\u003eNitrogen is an essential mineral element required for plant growth and development and can be involved in the synthesis of key biological molecules, including proteins, nucleic acids, chlorophyll, and hormones [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The efficiency of nitrogen absorption, transport, and assimilation directly determines plant growth rate, yield formation, and quality regulation. Nitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) and ammonium (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e) are the major inorganic nitrogen (N) sources available to plants. Owing to its high mobility in soil and ready accessibility to roots, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e is the predominant nitrogen form taken up by most plant species [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e uptake mainly occurs in plant roots and is mediated by nitrate transporter (NRT) proteins, which are widely recognized as essential for N acquisition in plants. Transmembrane NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e transport depends on the precise regulation of NRTs, which are classified into three families, \u003cem\u003eNRT1\u003c/em\u003e, \u003cem\u003eNRT2\u003c/em\u003e, and \u003cem\u003eNRT3\u003c/em\u003e, according to their functional characteristics and sequence homology. Among these families, \u003cem\u003eNRT1\u003c/em\u003e, referred to as the Nitrate Peptide Transporter Family (NPF), contains the largest number of members and exhibits the broadest functional diversity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, it has become the central focus of recent plant nitrogen metabolism research.\u003c/p\u003e \u003cp\u003eNRT proteins are pivotal for nitrate transport and distribution, with their primary function being the mediation of root NO\u003csub\u003e3\u003c/sub\u003e⁻ uptake. Based on the differences in external nitrate concentrations, \u003cem\u003eNRT\u003c/em\u003e family members are divided into two transport systems: the Low-Affinity Transport System (LATS) and the High-Affinity Transport System (HATS). Under low NO\u003csub\u003e3\u003c/sub\u003e⁻ conditions, typically below 1 mM, NO\u003csub\u003e3\u003c/sub\u003e⁻ uptake is dominated by HATS, which is mainly mediated by the NRT2 family. Under high NO\u003csub\u003e3\u003c/sub\u003e⁻ concentrations, generally above 1 mM, LATS dominated by the NRT1 family takes precedence [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, certain NRT1 members (e.g., \u003cem\u003eNRT1.1\u003c/em\u003e (\u003cem\u003eAtNPF6.3\u003c/em\u003e) in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e) exhibit \u0026ldquo;dual-affinity\u0026rdquo; characteristics, enabling roots to absorb NO\u003csub\u003e3\u003c/sub\u003e⁻ from the soil while also functioning as NO\u003csub\u003e3\u003c/sub\u003e⁻ sensors that coordinate N uptake and carbon metabolism through the regulation of downstream gene expression [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. \u003cem\u003eAtNPF7.2\u003c/em\u003e (\u003cem\u003eNRT1.8\u003c/em\u003e) is specifically localized in xylem parenchyma cells and is responsible for reloading NO\u003csub\u003e3\u003c/sub\u003e⁻ from xylem sap, thereby regulating NO\u003csub\u003e3\u003c/sub\u003e⁻ allocation to shoots and influencing leaf photosynthesis and nitrogen remobilization [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Furthermore, \u003cem\u003eAtNPF4.6\u003c/em\u003e participates in drought stress responses by transporting abscisic acid (ABA), whereas \u003cem\u003eAtNPF5.5\u003c/em\u003e regulates storage substance accumulation during seed development, highlighting the central role of the NRT1 family within the \u0026ldquo;nitrogen metabolism-growth and development-stress adaptation\u0026rdquo; network [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. With the development of genome sequencing technologies, studies on the NRT1 family have expanded from model plants to diverse crops and economically important species. In rice, natural variation in \u003cem\u003eOsNRT1.1B\u003c/em\u003e is associated with nitrogen use efficiency, with cultivars containing specific alleles exhibiting higher yields under low-nitrogen conditions [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. \u003cem\u003eOsNPF6.5\u003c/em\u003e influences grain protein content by regulating root NO\u003csub\u003e3\u003c/sub\u003e⁻ uptake and N allocation to panicles [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In maize, \u003cem\u003eZmNRT1.1\u003c/em\u003e expression is induced by low-nitrogen stress, with overexpression significantly increasing plant biomass in N-deficient soils. This demonstrates the breeding potential of this gene family for N-efficient crops [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In fruit trees, \u003cem\u003eMdNPF6.5\u003c/em\u003e functions as a key low-nitrogen-inducible nitrate transporter that plays a critical role in significantly enhancing apple tolerance under low-nitrogen conditions [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Collectively, these studies indicate that the NRT1 family exhibits both functional conservation, such as core nitrate transport activity across plant lineages, and species-specific characteristics shaped by evolutionary history.\u003c/p\u003e \u003cp\u003eSalt stress is a major abiotic stressor that constrains global crop production and severely disrupts N metabolic balance. Numerous studies have demonstrated that salt stress markedly inhibits plant NO\u003csub\u003e3\u003c/sub\u003e⁻ uptake and assimilation capacity, leading to a decline in leaf nitrate content. In peanut, salt stress reduces NO\u003csub\u003e3\u003c/sub\u003e⁻ accumulation in leaves, with the inhibitory effect intensifying as the salt concentration increases, a response closely associated with the suppressed expression of nitrate transporters [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Similarly, in rice, treatment with 0.3% NaCl significantly decreases NO\u003csub\u003e3\u003c/sub\u003e⁻ content and the activities of nitrate reductase (NR), glutamine synthetase (GS), and NADH-dependent glutamate synthase (NADH-GOGAT) in both roots and leaves, while increasing ammonium accumulation and the activities of glutamate dehydrogenase (GDH) and aspartate aminotransferase (AST). These changes indicate a metabolic shift from the NO\u003csub\u003e3\u003c/sub\u003e⁻ assimilation pathway to alternative nitrogen metabolic routes under salt stress conditions [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Under salt stress, NRT1 family proteins enhance plant adaptability through multiple mechanisms, including the maintenance of N uptake, regulation of ion homeostasis, participation in signal transduction, and facilitation of root architecture remodeling. For instance, in \u003cem\u003eSuaeda salsa\u003c/em\u003e under high-salinity and low-N conditions, N uptake is promoted by the induction of SsNRT2.1/2.5 expression acting in concert with NRT1, along with the activation of plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPases that drive proton gradient\u0026ndash;dependent active NO\u003csub\u003e3\u003c/sub\u003e⁻ transport [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eNRT1.5\u003c/em\u003e (\u003cem\u003eNPF7.3\u003c/em\u003e) functions as an H\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e antiporter that facilitates K\u003csup\u003e+\u003c/sup\u003e transport to shoots, and the \u003cem\u003eNRT1.5\u003c/em\u003e (\u003cem\u003elks2\u003c/em\u003e) knockout mutant exhibits leaf chlorosis under salt stress, indicating its role in alleviating Na\u003csup\u003e+\u003c/sup\u003e toxicity by maintaining K\u003csup\u003e+\u003c/sup\u003e homeostasis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In rice, \u003cem\u003eNRT1.1\u003c/em\u003e (\u003cem\u003eOsNRT1.1B\u003c/em\u003e) acts as a NO\u003csub\u003e3\u003c/sub\u003e⁻ sensor and activates transcription factors such as \u003cem\u003eANR1\u003c/em\u003e and \u003cem\u003eNLP7\u003c/em\u003e to regulate downstream gene expression [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Ectomycorrhizal fungi, such as \u003cem\u003ePaxillus involutus\u003c/em\u003e, mitigate salt stress\u0026ndash;induced nitrate efflux by activating host poplar genes PcNRT1.1/1.2 and \u003cem\u003ePcHA4\u003c/em\u003e, lowering rhizosphere pH, and promoting H\u003csup\u003e+\u003c/sup\u003e-ATPase activity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGrapevine (\u003cem\u003eVitis vinifera\u003c/em\u003e L.) is one of the world\u0026rsquo;s most economically important fruit crops and is characterized by a long growth cycle and complex synchrony of organ development, resulting in pronounced stage-specific N demands. High nitrogen availability is required at budburst to support shoot growth, whereas insufficient nitrogen supply from flowering to fruit enlargement leads to reduced fruit set, and excessive N input during ripening inhibits fruit coloration and flavor compound accumulation[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. With ongoing changes in the global ecological environment, grapevine growth is increasingly affected by environmental pressures, s uch as salt stress. Our previous research indicated that salt stress adversely affected N absorption and assimilation in grapevine and ultimately constrained plant growth [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, elucidating the molecular mechanisms underlying nitrogen uptake and transport in grapevine is of practical significance for N management and fruit quality optimization. The recent completion of grapevine whole-genome sequencing projects, including the PN40024 reference genome and high-quality assemblies such as the CHR_T2T2 telomere-to-telomere genome, can provide crucial support for functional gene mining [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, research on the grapevine \u003cem\u003eNRT1\u003c/em\u003e gene family remains limited [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Early studies primarily predicted a small number of potential members through homology-based sequence alignment and focused mainly on expression pattern analyses of genes related to root nitrate uptake, such as \u003cem\u003eVvNRT1.1\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Core questions regarding the genome-wide identification of family members, phylogenetic relationships, gene structural variations, tissue-specific expression patterns, and associations with stress responses remain largely unresolved. To address these issues, the present study performed a genome-wide analysis and identified 65 full-length \u003cem\u003eNRT1\u003c/em\u003e genes in grapevine. Using a multifaceted bioinformatic framework, this study characterized the physicochemical properties, gene and protein architectures, chromosomal distributions, phylogenetic relationships, and tissue-specific expression patterns of the identified genes. These findings advance our understanding of the functional landscape of the grapevine \u003cem\u003eNRT1\u003c/em\u003e gene family and highlight its potential involvement in salt stress response mechanisms.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eVvNRT1s\u003c/b\u003e \u003cb\u003egene identification\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo identify potential NRT1 homologs in the grapevine genome (PN_T2T2) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], \u003cem\u003eArabidopsis\u003c/em\u003e NRT1 protein sequences were used as queries for BLASTP searches. This initial screening retrieved approximately 80 candidate proteins. Subsequent validation using the SMART and NCBI-CDD databases confirmed the presence of characteristic MFS and extensin domains. After this filtering step, 65 \u003cem\u003eVvNRT1\u003c/em\u003e proteins were identified (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), representing a number higher than that reported in Arabidopsis. These genes were systematically designated \u003cem\u003eVvNRT1.1\u003c/em\u003e\u0026ndash;\u003cem\u003eVvNRT1.65\u003c/em\u003e according to their linear positions on the chromosomes.\u003c/p\u003e \u003cp\u003eThe predicted molecular weights of the 65 \u003cem\u003eVvNRT1\u003c/em\u003e proteins ranged from 10.6 kDa for \u003cem\u003eVvNRT1.40\u003c/em\u003e to 162.42 kDa for \u003cem\u003eVvNRT1.63\u003c/em\u003e. Consistent with this variation, the protein lengths differed markedly, spanning from 96 amino acids (\u003cem\u003eVvNRT1.40\u003c/em\u003e) to 1466 amino acids (\u003cem\u003eVvNRT1.63\u003c/em\u003e). In addition, the theoretical isoelectric points of these proteins varied from 4.82 for \u003cem\u003eVvNRT1.55\u003c/em\u003e to 9.8 for \u003cem\u003eVvNRT1.6\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of NRT1s in Grapevine (\u003cem\u003eVitis vinifera\u003c/em\u003e L.).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChromosome location\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eExon\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSize\u003c/p\u003e \u003cp\u003e(amino acids)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eMW\u003c/p\u003e \u003cp\u003e(kDa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003epI\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_000270\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1:3035085\u0026thinsp;\u0026minus;\u0026thinsp;308496\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e583\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e8.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_000553\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1:6408849\u0026ndash;6416804\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e586\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e5.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_000867\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1:10897334\u0026ndash;10901324\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e619\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e68.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e8.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_000875\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1:10997314\u0026ndash;11001502\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e585\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e9.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_001641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1:27439472\u0026ndash;27442560\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e640\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e70.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e9.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_002188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2:5135546\u0026ndash;5146196\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e786\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e86.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e9.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_002412\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2:8626578\u0026ndash;8631608\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e591\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e65.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e6.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_002882\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2:20036564\u0026ndash;20037454\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e296\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e33.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e8.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_004646\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr4:2928810\u0026ndash;2931535\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e589\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e65.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e6.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_004647\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr4:2933378\u0026ndash;2935949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e592\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e65.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e7.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_006768\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr5:9116491\u0026ndash;9121269\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e592\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e65.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e9.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_007807\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr6:1599766\u0026ndash;1602508\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e611\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e66.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e5.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_007840\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr6:1993924\u0026ndash;1996770\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e534\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e60.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e8.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_007842\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr6:2057074\u0026ndash;2064811\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e667\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e76.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e8.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.15\u003c/p\u003e \u003c/td\u003e 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colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e577\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e63.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e9.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_008032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr6:4458253\u0026ndash;4460909\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e577\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e63.46\u003c/p\u003e \u003c/td\u003e 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colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e609\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e68.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e8.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_012005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr8:12945007\u0026ndash;12949683\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e599\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e66.23\u003c/p\u003e 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colname=\"c3\"\u003e \u003cp\u003echr11:2931120\u0026ndash;2933169\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e598\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e66.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e8.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_016501\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr11:2935712\u0026ndash;2937182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e276\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e31.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e9.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_016502\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr11:2937227\u0026ndash;2937730\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e17.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e4.99\u003c/p\u003e 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colname=\"c5\"\u003e \u003cp\u003e143\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e9.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_017823\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr12:6204846\u0026ndash;6208077\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e594\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e66.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e 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align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e8.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_020250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr13:23182256\u0026ndash;23187154\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e499\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e56.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e9.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.40\u003c/p\u003e \u003c/td\u003e 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align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e538\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e59.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e8.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_020751\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr14:295133\u0026ndash;297492\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e620\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e68.37\u003c/p\u003e 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\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e9.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_023196\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr15:12838567\u0026ndash;12843593\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e604\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e67.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e8.48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_023463\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr15:17551952\u0026ndash;17554126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e529\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e58.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e8.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_026074\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr17:6447730\u0026ndash;6450309\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e577\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e9.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_026075\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr17:6451581\u0026ndash;6454340\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e586\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e8.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_026076\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr17:6456777\u0026ndash;6459464\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e586\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e9.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_026077\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr17:6463343\u0026ndash;6465728\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e570\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e62.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e8.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_026078\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr17:6465936\u0026ndash;6466590\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_026087\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr17:6540785\u0026ndash;6543752\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e579\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e63.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e8.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_026703\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr17:16544148\u0026ndash;16544727\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e141\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e4.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_027631\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr18:9572337\u0026ndash;9575747\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e587\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e8.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_027795\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr18:11322484\u0026ndash;11324487\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e617\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e68.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e8.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_027796\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr18:11327097\u0026ndash;11329870\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e581\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e8.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_028874\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr18:30296518\u0026ndash;30344687\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1035\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e114.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e8.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_028876\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr18:30380900\u0026ndash;30411853\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e894\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e8.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_028878\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr18:30435965\u0026ndash;30446252\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e531\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e59.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e8.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_028880\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr18:30447236\u0026ndash;30447676\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e7.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_028881\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr18:30468432\u0026ndash;30497044\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1466\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e162.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e6.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_028883\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr18:30527824\u0026ndash;30544755\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1139\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e126.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e6.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVvNRT1.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitviT2T_029282\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr18:36195994\u0026ndash;36199645\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e609\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e67.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e9.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePhylogenetic and synteny analyses of\u003c/b\u003e \u003cb\u003eVvNRT1s\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo clarify the evolutionary and phylogenetic relationships, an unrooted phylogenetic tree was constructed using 32 AtNRT1 and 65 VvNRT1 protein sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The resulting topology grouped the NRT1 proteins into five primary clades (Ⅰ\u0026ndash;Ⅴ). Group Ⅰ represented the largest clade, containing twenty-six members, whereas Groups Ⅱ and Ⅳ each comprised 17 proteins. In contrast, Groups Ⅲ and Ⅴ were much smaller, consisting of two and three proteins, respectively. Notably, this five-clade classification differed from the three-group pattern commonly reported for most other plant species.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eVvNRT1\u003c/em\u003e genes were unevenly distributed across the 16 grapevine chromosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Only one gene was located on chromosomes 5, 9, and 10, whereas chromosomes 4, 7, 12, and 15 each harbored two \u003cem\u003eVvNRT1\u003c/em\u003e members. In contrast, most \u003cem\u003eVvNRT1\u003c/em\u003e genes were preferentially clustered on chromosomes 6, 11, 13, 17, and 18. Among these, chromosome 18 contained the highest number of \u003cem\u003eVvNRT1\u003c/em\u003e genes, suggesting a potentially prominent role for this chromosome in nitrate transport and metabolism in grapevine. To explore the evolutionary mechanisms underlying the expansion of the \u003cem\u003eVvNRT1\u003c/em\u003e gene family, gene duplication events were analyzed. Based on sequence homology, 20 \u003cem\u003eVvNRT1\u003c/em\u003e genes (30.77%) formed 10 segmental duplication pairs, whereas 42 \u003cem\u003eVvNRT1\u003c/em\u003e genes (64.62%) formed 29 tadem duplication pairs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and Table S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eConserved motif and gene structure analyses of\u003c/b\u003e \u003cb\u003eVvNRT1s\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe conserved protein motifs and exon\u0026ndash;intron structures of \u003cem\u003eVvNRT1\u003c/em\u003e genes were examined using MEME and GSDS 2.0, respectively. A maximum-likelihood phylogenetic tree was generated to assess the relationships among evolutionary clades, motif composition, and gene structural features (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), which was consistent with the clustering pattern observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. The identification of conserved motifs provides crucial evidence for understanding evolutionary relationships. In total, 10 conserved motifs were identified, with motif lengths ranging from 17 to 41 amino acids (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and S2). In groups Ⅰ and Ⅳ, \u003cem\u003eVvNRT1.62\u003c/em\u003e, \u003cem\u003eVvNRT1.43\u003c/em\u003e, \u003cem\u003eVvNRT1.15\u003c/em\u003e, \u003cem\u003eVvNRT1.30\u003c/em\u003e, and \u003cem\u003eVvNRT1.53\u003c/em\u003e each contained only two motifs, whereas \u003cem\u003eVvNRT1.40\u003c/em\u003e, \u003cem\u003eVvNRT1.55\u003c/em\u003e, \u003cem\u003eVvNRT1.33\u003c/em\u003e, and \u003cem\u003eVvNRT1.44\u003c/em\u003e in groups I, Ⅱ, and Ⅳ possessed a single motif. Members within the same clade exhibited similar motif positions and distribution patterns, which were largely conserved across groups I\u0026ndash;Ⅳ. Conversely, group V contained markedly fewer motifs than the other groups.\u003c/p\u003e \u003cp\u003eThe exon\u0026ndash;intron architecture is a key component of the gene structural architecture. Accordingly, the structural features of all 65 \u003cem\u003eVvNRT1\u003c/em\u003e genes were analyzed, including the number, length, and position of exons and introns (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Genes belonging to the same phylogenetic subfamily displayed highly conserved gene structures, further supporting the established classification of grapevine \u003cem\u003eNRT1s\u003c/em\u003e. In groups I, Ⅱ, and Ⅳ, \u003cem\u003eVvNRT1.62\u003c/em\u003e, \u003cem\u003eVvNRT1.40\u003c/em\u003e, \u003cem\u003eVvNRT1.8\u003c/em\u003e, \u003cem\u003eVvNRT1.15\u003c/em\u003e, and \u003cem\u003eVvNRT1.30\u003c/em\u003e each consisted of a single exon. Most group I genes contained five or more exons, with \u003cem\u003eVvNRT1.63\u003c/em\u003e and \u003cem\u003eVvNRT1.64\u003c/em\u003e each containing nine exons. Genes in group Ⅱ generally possessed four to six exons, whereas those in subgroups Ⅲ and Ⅳ typically contained four exons. In contrast, group V genes exhibited a wider range of exon numbers, from three to six, and their gene lengths were notably shorter than those of other groups. All members of group Ⅲ had contained both 3\u0026rsquo;-UTR and 5\u0026rsquo;-UTR regions, whereas group V genes lacked one or both UTRs. These variations in gene structure may underlie the functional divergence observed among VvNRT1 family members.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCis-regulatory element analysis of\u003c/b\u003e \u003cb\u003eVvNRT1s\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe regulatory element composition of \u003cem\u003eVvNRT1\u003c/em\u003e genes was analyzed, and the genes were classified into distinct clusters according to their response patterns to different regulatory signals. Each cluster exhibited characteristic regulatory profiles associated with specific environmental and developmental cues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eIn cluster Ⅰ, most \u003cem\u003eVvNRT1\u003c/em\u003e genes exhibited strong responsiveness to stress-related elements, including defense and anaerobic induction elements (green, red, and purple squares), indicating their potential involvement in stress responses. Cluster Ⅱ was mainly characterized by \u003cem\u003eVvNRT1\u003c/em\u003e genes responsive to hormone-related regulatory elements, such as auxin and salicylic acid (orange, brown, and light pink squares). Cluster Ⅲ displayed broad associations with developmental regulation, particularly seed-specific regulation and meristem-related elements (blue squares), suggesting an important role in plant growth regulation. The genes in cluster Ⅳ exhibited the most diverse response patterns, with the largest proportion being responsive to methyl jasmonate (MeJA; 36 genes), highlighting a prominent role in stress and hormone signaling crosstalk. In contrast, cluster V presented a relatively limited responsiveness, while some genes were associated with low-temperature and circadian control elements (light blue squares), and overall contained fewer \u003cem\u003eVvNRT1\u003c/em\u003e genes, especially those related to seed-specific regulation and gibberellin responses.\u003c/p\u003e \u003cp\u003eConsistent with these observations, heatmap analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) revealed distinct regulatory distributions of \u003cem\u003eVvNRT1\u003c/em\u003e genes across the examined biological processes. In cluster Ⅳ, a substantial number of \u003cem\u003eVvNRT1\u003c/em\u003e genes (46) were responsive to MeJA, whereas cluster Ⅱ exhibited strong responsiveness to auxin-related elements (41 genes). In contrast, cluster Ⅴ contained fewer \u003cem\u003eVvNRT1\u003c/em\u003e genes responsive to low-temperature and anaerobic induction signals.\u003c/p\u003e \u003cp\u003eOverall, these results indicate that \u003cem\u003eVvNRT1\u003c/em\u003e genes are differentially regulated by environmental stress, hormone-related signals, and developmental cues. The clustering patterns based on cis-regulatory elements further underscored the functional complexity of \u003cem\u003eVvNRT1\u003c/em\u003e genes in grapevine growth regulation and stress response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTissue-specific expression profile of\u003c/b\u003e \u003cb\u003eVvNRT1s\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further clarify the potential roles of \u003cem\u003eVvNRT1s\u003c/em\u003e, expression profiles across 19 different organs or tissues at multiple developmental stages were obtained from the BAR database (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The \u003cem\u003eVvNRT1\u003c/em\u003e genes exhibited widespread expression across nearly all examined tissues. Evident expression differences were observed among phylogenetic groups, where members of Groups I and II demonstrated broader expression ranges across diverse tissues than those belonging to other groups. Several genes showed significantly elevated expression levels in specific tissues or developmental stages. For example, \u003cem\u003eVvNRT1.7\u003c/em\u003e and \u003cem\u003eVvNRT1.21\u003c/em\u003e showed the significantly higher expression in roots than other tissues or organs, whereas \u003cem\u003eVvNRT1.34\u003c/em\u003e and \u003cem\u003eVvNRT1.38\u003c/em\u003e reached peak expression during the flowering stage. Moreover, \u003cem\u003eVvNRT1.1\u003c/em\u003e and \u003cem\u003eVvNRT1.60\u003c/em\u003e exhibited the highest expression levels at the bud swell and mature fruit stages, respectively. In addition, some genes, such as \u003cem\u003eVvNRT1.34\u003c/em\u003e, were highly expressed in multiple tissues, whereas others, including \u003cem\u003eVvNRT1.9\u003c/em\u003e and \u003cem\u003eVvNRT1.10\u003c/em\u003e, consistently maintained low expression levels in most tissues. Furthermore, all genes in group Ⅲ demonstrated uniformly lower expression across all examined tissues and organs than those in other subgroups. Based on these expression patterns, \u003cem\u003eVvNRT1\u003c/em\u003e genes may exhibit functional differentiation that supports coordinated nitrogen uptake and transport across grapevine tissues. Collectively, these genes represent potential targets for improving nitrogen use efficiency and may serve as integral regulators of grapevine growth and responses to environmental stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression profiles of\u003c/b\u003e \u003cb\u003eVvNRT1s\u003c/b\u003e \u003cb\u003eafter KNO\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e \u003cb\u003etreatment and salt stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBased on the above analyses, numerous hormone- and stress-related cis-acting elements were identified in the promoters of \u003cem\u003eVvNRT\u003c/em\u003e1 genes. In addition, previous studies have shown that \u003cem\u003eNPF5.15\u003c/em\u003e and \u003cem\u003eNPF7.3\u003c/em\u003e play important roles in salt tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. To examine the responses of \u003cem\u003eVvNRT1\u003c/em\u003e genes to NO\u003csub\u003e3\u003c/sub\u003e⁻ treatment and salt stress, their expression levels were analyzed in grape leaves and roots at 10 min, 0.5 h, 2 h, and 8 h after NO\u003csub\u003e3\u003c/sub\u003e⁻ treatment, and at 6, 12, 24, and 48 h after salt treatment. The experiment was conducted using own-rooted seedlings of the \u0026lsquo;\u003cem\u003eShine Muscat\u003c/em\u003e\u0026rsquo; cultivar, which was selected for its wide cultivation, economic value, and strong stress tolerance, thereby enabling the assessment of expression patterns of selected \u003cem\u003eVvNRT1s\u003c/em\u003e under stress conditions. Four grape genes homologous to \u003cem\u003eArabidopsis AtNPF4.3\u003c/em\u003e, \u003cem\u003eAtNPF5.10\u003c/em\u003e, \u003cem\u003eAtNPF5.15\u003c/em\u003e, and \u003cem\u003eAtNPF7.3\u003c/em\u003e were examined, corresponding to \u003cem\u003eVvNRT1.35\u003c/em\u003e, \u003cem\u003eVvNRT1.59\u003c/em\u003e, \u003cem\u003eVvNRT1.19\u003c/em\u003e, and \u003cem\u003eVvNRT1.47\u003c/em\u003e, respectively.\u003c/p\u003e \u003cp\u003eThe expression levels of \u003cem\u003eVvNRT1.19\u003c/em\u003e and \u003cem\u003eVvNRT1.47\u003c/em\u003e were higher than those of the 0 h control under both NO\u003csub\u003e3\u003c/sub\u003e⁻ treatment and salt stress. Under NO\u003csub\u003e3\u003c/sub\u003e⁻ treatment, a progressive increase in expression was observed in the roots over time, with the expression in both roots and leaves reaching a maximum at 8 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Under salt stress, the expression in both roots and leaves peaked at 6 h, after which the expression of \u003cem\u003eVvNRT1.19\u003c/em\u003e in the roots gradually declined (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Although \u003cem\u003eVvNRT1.35\u003c/em\u003e and \u003cem\u003eVvNRT1.59\u003c/em\u003e demonstrated increased expression in roots under salt stress compared with the 0 h control, their expression in leaves under both nitrate and salt treatments displayed time points at which transcript levels were reduced relative to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eVvNRT1.19\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eVvNRT1.47\u003c/b\u003e \u003cb\u003eoverexpression enhanced nitrogen use efficiency and salt tolerance in\u003c/b\u003e \u003cb\u003eArabidopsis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBoth \u003cem\u003eVvNRT1.19\u003c/em\u003e and \u003cem\u003eVvNRT1.47\u003c/em\u003e were transcriptionally induced in grape roots and leaves at 10 min, 0.5 h, 2 h, and 8 h following NO\u003csub\u003e3\u003c/sub\u003e⁻ treatment, as well as at 6, 12, 24, and 48 h after salt treatment. \u003cem\u003eVvNRT1.19\u003c/em\u003e and \u003cem\u003eVvNRT1.47\u003c/em\u003e were highly homologous to \u003cem\u003eAtNPF5.15\u003c/em\u003e and \u003cem\u003eAtNPF7.3\u003c/em\u003e, respectively, which are key members of the NO\u003csub\u003e3\u003c/sub\u003e⁻ transporter family known to contribute to nitrogen use efficiency, although their roles in plant salt tolerance remain unclear. To examine the functions of \u003cem\u003eVvNRT1.19\u003c/em\u003e and \u003cem\u003eVvNRT1.47\u003c/em\u003e in responses to NO\u003csub\u003e3\u003c/sub\u003e⁻ availability and salt stress, 12 \u003cem\u003eVvNRT1.19\u003c/em\u003e-overexpressing (\u003cem\u003eVvNRT1.19-OE\u003c/em\u003e) lines and 10 \u003cem\u003eVvNRT1.47\u003c/em\u003e-overexpressing (\u003cem\u003eVvNRT1.47-OE\u003c/em\u003e) \u003cem\u003eArabidopsis\u003c/em\u003e lines were generated via \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation. qPCR analysis confirmed the markedly elevated transcript levels of both genes in the leaves of the transgenic lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and b). Based on these results, the lines showing the highest expression levels of \u003cem\u003eVvNRT1.19\u003c/em\u003e (#11) and \u003cem\u003eVvNRT1.47\u003c/em\u003e (#10) were selected for further analyses. To determine whether the overexpression of \u003cem\u003eVvNRT1.19\u003c/em\u003e and \u003cem\u003eVvNRT1.47\u003c/em\u003e influenced nitrogen use efficiency and salt tolerance, transgenic and wild-type (WT) plants were grown under 0, 1, and 5 mM KNO\u003csub\u003e3\u003c/sub\u003e conditions and subjected to 200 mM NaCl stress.\u003c/p\u003e \u003cp\u003eUnder all tested KNO\u003csub\u003e3\u003c/sub\u003e concentrations, the WT seedlings displayed inhibited root development, cotyledon yellowing, and retarded growth, whereas the overexpression lines maintained well-developed root systems (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec) and vigorous seedling growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). At 1 and 5 mM KNO\u003csub\u003e3\u003c/sub\u003e, the average root length of the overexpression lines was significantly greater than that of WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). Under 0 and 1 mM KNO\u003csub\u003e3\u003c/sub\u003e conditions, both the number of lateral roots and cotyledon area were also significantly increased in the overexpression lines compared with WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg and h). These results indicated that the overexpression of \u003cem\u003eVvNRT1.47\u003c/em\u003e and \u003cem\u003eVvNRT1.19\u003c/em\u003e enhanced NO\u003csub\u003e3\u003c/sub\u003e⁻ uptake and utilization, thereby promoting plant growth under both nitrate-deficient and nitrate-sufficient conditions. In addition, the performance of overexpression lines was assessed under salt stress (200 mM NaCl). The WT seedlings exhibited severe growth inhibition and pronounced leaf yellowing, whereas \u003cem\u003eVvNRT1.47-OE\u003c/em\u003e and \u003cem\u003eVvNRT1.19-OE\u003c/em\u003e seedlings maintained comparatively better growth and more normal morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Moreover, the survival rates of the overexpression lines were significantly higher than those of WT plants at both day 8 and day 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei), suggesting that \u003cem\u003eVvNRT1.47\u003c/em\u003e and \u003cem\u003eVvNRT1.19\u003c/em\u003e contributed to improved survival under salt stress conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eNRT1 proteins represent the largest and most functionally diverse NO\u003csub\u003e3\u003c/sub\u003e⁻ transporter group in plants and are central to plant growth regulation, stress responses, and NO\u003csub\u003e3\u003c/sub\u003e⁻ uptake and metabolism. Genome-wide analyses of \u003cem\u003eNRT1\u003c/em\u003e gene families have been reported in several species, including \u003cem\u003eSorghum bicolor\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], \u003cem\u003eEucalyptus grandis\u003c/em\u003e [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], \u003cem\u003eSetaria italica\u003c/em\u003e [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], \u003cem\u003eBrassica rapa\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and \u003cem\u003eCocos nucifera L.\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, a systematic genome-wide investigation of the NRT1 family in grapevine (\u003cem\u003eVitis vinifera\u003c/em\u003e) has not been performed. Here, 65 \u003cem\u003eVvNRT1\u003c/em\u003e genes were identified in grapevine and further characterized by phylogenetic classification, gene structure analysis, and expression profiling. These results provide evidence for the evolutionary features of grapevine NRT1s and support their potential involvement in nitrogen utilization and stress adaptation.\u003c/p\u003e \u003cp\u003eThe 65 \u003cem\u003eVvNRT1\u003c/em\u003e genes identified in this study exceeded the 53 NRT1 members reported in \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], indicating an expanded \u003cem\u003eNRT1\u003c/em\u003e repertoire in grapevine. Phylogenetic analysis divided \u003cem\u003eVvNRT1s\u003c/em\u003e into five groups (Ⅰ\u0026ndash;Ⅴ), differing from the eight-group classification commonly reported in \u003cem\u003eArabidopsis\u003c/em\u003e and rice [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], which may reflect the grapevine-specific evolutionary history, including polyploidization and lineage-specific duplication. Chromosomal mapping revealed an enrichment of \u003cem\u003eVvNRT1s\u003c/em\u003e on chromosome 18 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), where multiple tandemly duplicated genes (e.g., \u003cem\u003eVvNRT1.59\u003c/em\u003e\u0026ndash;\u003cem\u003eVvNRT1.65\u003c/em\u003e) were concentrated, suggesting that tandem duplication contributed substantially to this clustering. Such clustering may facilitate the coordinated regulation of genes related to N transport or stress responses in grapevine.\u003c/p\u003e \u003cp\u003eThe duplication analysis indicated that 64.62% of \u003cem\u003eVvNRT1s\u003c/em\u003e were associated with segmental duplication, whereas 30.77% formed tandem duplication pairs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), indicating that both duplication modes contributed to family expansion, similar to the pattern reported for the apple \u003cem\u003eNRT1\u003c/em\u003e family [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Tandem duplicates often retain similar functions, whereas segmental duplicates more frequently diverge functionally [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Consistent with this tendency, tandemly duplicated genes, such as \u003cem\u003eVvNRT1.49\u003c/em\u003e\u0026ndash;\u003cem\u003eVvNRT1.51\u003c/em\u003e, displayed similar motif compositions and tissue expression patterns, whereas the segmentally duplicated pair \u003cem\u003eVvNRT1.1\u003c/em\u003e and \u003cem\u003eVvNRT1.6\u003c/em\u003e differed markedly in cis-regulatory element profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), implying possible divergence associated with environmental adaptation.\u003c/p\u003e \u003cp\u003eConserved motif and gene structure analyses further indicated that genes within the same phylogenetic group generally shared similar motif compositions and exon\u0026ndash;intron architectures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), supporting the reliability of the classification. For instance, Group Ⅰ members \u003cem\u003eVvNRT1.63\u003c/em\u003e and \u003cem\u003eVvNRT1.64\u003c/em\u003e both contained nine exons and motifs 1\u0026ndash;10, whereas Group Ⅴ members carried fewer motifs, potentially reflecting their functional specialization. Notably, \u003cem\u003eVvNRT1.40\u003c/em\u003e and \u003cem\u003eVvNRT1.55\u003c/em\u003e contained a single exon and one motif. Comparable compact \u003cem\u003eNRT1\u003c/em\u003e genes have also been reported in \u003cem\u003eArabidopsis\u003c/em\u003e (e.g., \u003cem\u003eAtNPF2.9\u003c/em\u003e) and proposed to enable rapid responses to environmental signals via a simplified regulatory architecture [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCis-element analysis suggested that \u003cem\u003eVvNRT1\u003c/em\u003e promoters were enriched in elements related to hormone responses (e.g., MeJA and ABA), stress responses (e.g., low temperature and anoxia), and growth and developmental regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), which can support a role in multi-signal integration during grapevine growth and stress adaptation. Notably, Group Ⅴ contained abundant stress-responsive elements and was enriched in hormone-related elements, suggesting a contribution to hormone\u0026ndash;stress crosstalk, consistent with reports that rice NPFs participate in the coordinated regulation of salt stress responses and ABA signaling [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTissue-specific expression profiling revealed distinct expression patterns among \u003cem\u003eVvNRT1s\u003c/em\u003e. \u003cem\u003eVvNRT1.7\u003c/em\u003e and \u003cem\u003eVvNRT1.21\u003c/em\u003e were predominantly expressed in roots, suggesting roles in nitrate uptake, whereas \u003cem\u003eVvNRT1.34\u003c/em\u003e and \u003cem\u003eVvNRT1.38\u003c/em\u003e showed elevated expression during flowering, indicating their potential involvement in the nitrogen supply to reproductive organs. In addition, \u003cem\u003eVvNRT1.1\u003c/em\u003e exhibited high expression during bud swelling, implying a role in bud development (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This expression specificity resembled the tissue-based functional differentiation reported for \u003cem\u003eArabidopsis NRT1s\u003c/em\u003e, such as \u003cem\u003eAtNPF6.3\u003c/em\u003e, which functions in root NO\u003csub\u003e3\u003c/sub\u003e⁻ uptake, and \u003cem\u003eAtNPF7.2\u003c/em\u003e, which mediates NO\u003csub\u003e3\u003c/sub\u003e⁻ redistribution in leaves [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. These observations suggest that the grapevine \u003cem\u003eNRT1\u003c/em\u003e family retained conserved functional patterns while acquiring specialized expression profiles in newly expanded members to accommodate the perennial growth habit of fruit trees.\u003c/p\u003e \u003cp\u003eThe functions of \u003cem\u003eNRT\u003c/em\u003e genes in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e have been extensively characterized, with different subfamilies participating in N uptake, transport, signaling, and stress adaptation. \u003cem\u003eAtNRT\u003c/em\u003e genes primarily act as molecular carriers that mediate NO\u003csub\u003e3\u003c/sub\u003e⁻ uptake and internal allocation. The two major subfamilies operate under distinct nitrogen regimes. The NRT1 (NPF) subfamily mainly mediates low-affinity transport under high nitrogen conditions (soil NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e \u0026gt; 1 mmol/L), facilitating NO\u003csub\u003e3\u003c/sub\u003e⁻ uptake or its translocation within the plant (root \u0026rarr; stem \u0026rarr; leaf; source organ \u0026rarr; sink organ) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. For instance, \u003cem\u003eAtNRT1.5\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] mediates root-to-shoot NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e translocation, whereas \u003cem\u003eAtNRT1.4\u003c/em\u003e [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] is involved in NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e storage in leaves. Notably, certain members, such as \u003cem\u003eAtNRT1.1\u003c/em\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], act as \u0026ldquo;dual-affinity transporters\u0026rdquo;, enabling NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e absorption across a broad range of external concentrations. In contrast, the NRT2 subfamily specializes in high-affinity nitrate transport under low nitrogen availability (soil NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e \u0026lt; 0.5 mmol/L) and plays a critical role in nitrogen deficiency adaptation [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Representative genes, such as \u003cem\u003eAtNRT2.1\u003c/em\u003e and \u003cem\u003eAtNRT2.2\u003c/em\u003e, are predominantly expressed in root epidermal cells and root hairs, where they directly absorb NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e from the soil [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, \u003cem\u003eVvNRT1.19\u003c/em\u003e and \u003cem\u003eVvNRT1.47\u003c/em\u003e, which showed the highest homology to \u003cem\u003eArabidopsis AtNPF5.15\u003c/em\u003e and \u003cem\u003eAtNPF7.3\u003c/em\u003e, respectively, were significantly upregulated under high-N conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Both genes were strongly induced by salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), indicating that they responded to N signaling and participated in salt stress responses.\u003c/p\u003e \u003cp\u003eSubsequently, \u003cem\u003eVvNRT1.19\u003c/em\u003e and \u003cem\u003eVvNRT1.47\u003c/em\u003e were cloned to generate 12 \u003cem\u003eVvNRT1.19-OE\u003c/em\u003e and 10 \u003cem\u003eVvNRT1.47-OE Arabidopsis\u003c/em\u003e lines. Lines exhibiting the highest expression levels were selected for the validation of nitrogen use efficiency and salt tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and b). Overexpression of \u003cem\u003eVvNRT1.19\u003c/em\u003e or \u003cem\u003eVvNRT1.47\u003c/em\u003e resulted in improved nitrogen use efficiency and enhanced salt tolerance under nitrate treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec\u0026ndash;i), demonstrating that both genes contributed to the coordinated regulation of nitrogen utilization and salt tolerance.\u003c/p\u003e \u003cp\u003eIn summary, this study presents a comprehensive analysis of the \u003cem\u003eNRT1\u003c/em\u003e gene family in grapevine. By integrating bioinformatics and experimental approaches, this study defined the key structural, evolutionary, and functional features of \u003cem\u003eVvNRT1s\u003c/em\u003e and established a framework for further investigation. This study provides the first evidence that \u003cem\u003eVvNRT1.19\u003c/em\u003e and \u003cem\u003eVvNRT1.47\u003c/em\u003e function as N-responsive genes involved in both N metabolism and salt stress responses, offering insights for future functional studies of \u003cem\u003eNRT1\u003c/em\u003e genes in grapevine and other plant species.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, 65 intact NRT1 genes were systematically identified in the grapevine genome and classified into five distinct phylogenetic groups. Duplication pattern analyses indicated that both segmental and tandem duplication events contributed to the expansion of the \u003cem\u003eVvNRT1\u003c/em\u003e gene family. Combined analyses of promoter cis-elements and tissue-specific expression patterns suggested that \u003cem\u003eVvNRT1\u003c/em\u003e genes participated in grapevine growth, environmental stress responses, and nitrogen regulation.\u003c/p\u003e \u003cp\u003eMoreover, several \u003cem\u003eVvNRT1\u003c/em\u003e members exhibited pronounced transcriptional induction in roots and leaves in response to NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e and salt treatments. Functional assays using transgenic Arabidopsis plants further demonstrated that ectopic expression of \u003cem\u003eVvNRT1.19\u003c/em\u003e and \u003cem\u003eVvNRT1.47\u003c/em\u003e significantly enhanced nitrogen use efficiency and salt tolerance.\u003c/p\u003e \u003cp\u003eIn summary, these results extend the current understanding of the functional diversity of the \u003cem\u003eVvNRT1\u003c/em\u003e family and highlight its potential value in improving nutrient acquisition and stress adaptability in plants.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of VvNRT1 Genes\u003c/h2\u003e \u003cp\u003eArabidopsis NRT1 protein sequences were obtained from the TAIR\u003csup\u003e1\u003c/sup\u003e database and used as queries to screen the grapevine genomes. BLASTP [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] searches were performed against the Phytozome2\u003csup\u003e2\u003c/sup\u003e and Winberige\u003csup\u003e3\u003c/sup\u003e databases to identify putative \u003cem\u003eVvNRT1\u003c/em\u003e members, and only candidates with E-values below 1 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e were retained.\u003c/p\u003e \u003cp\u003eTo confirm domain composition, conserved LRR and extensin domains were examined using the NCBI Conserved Domain Database and SMART. Basic physicochemical properties, including protein length, molecular weight, and theoretical isoelectric point, were predicted using the ExPASy ProtParam server\u003csup\u003e4\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhylogenetic and Collinearity Analysis\u003c/h3\u003e\n\u003cp\u003eFull-length NRT1 protein sequences from grapevine and Arabidopsis were aligned using ClustalW. A maximum-likelihood phylogenetic tree was constructed using MEGA 7.0, and branch support was assessed using 1000 bootstrap replicates [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Collinearity and gene duplication events were analyzed using MCScanX [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Chromosomal location data were obtained from the Winberge database, and syntenic relationships were visualized using TBtools [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGene Structure and Motif Analysis\u003c/h2\u003e \u003cp\u003eThe exon\u0026ndash;intron organization of \u003cem\u003eVvNRT1\u003c/em\u003e genes was analyzed using the Gene Structure Display Server\u003csup\u003e5\u003c/sup\u003e [49]. Gene structure diagrams were generated using TBtools. Conserved protein motifs were identified using MEME\u003csup\u003e6\u003c/sup\u003e [50], and motif distribution patterns among VvNRT1 proteins were examined to evaluate their structural conservation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePromoter Analysis\u003c/h3\u003e\n\u003cp\u003eUpstream sequences (2 kb) of each VvNRT1 gene were extracted using TBtools. Cis-regulatory elements were predicted with PlantCARE\u003csup\u003e7\u003c/sup\u003e, and their distribution patterns were visualized.\u003c/p\u003e\n\u003ch3\u003eTissue Expression Analysis\u003c/h3\u003e\n\u003cp\u003eTranscriptome datasets were retrieved from the BAR database\u003csup\u003e8\u003c/sup\u003e, covering roots, stems, leaves, buds, flowers, fruits, seedlings, and pollen. After removing low-abundance transcripts, expression values were log₂-transformed and normalized. Heatmaps were generated using TBtools.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePlant Materials and Treatments\u003c/h2\u003e \u003cp\u003eOne-year-old own-rooted grapevine seedlings (\u003cem\u003eVitis vinifera\u003c/em\u003e L. cv. Shine Muscat) were obtained from Shandong Zhichang Agricultural Science and Technology Development Co., Ltd. Uniform seedlings were selected and grown under greenhouse conditions (12 h light/12 h dark; 25\u0026ndash;28\u0026deg;C day/5\u0026ndash;10\u0026deg;C night; 55\u0026ndash;65% relative humidity). Seedlings were cultivated in plastic pots containing washed sand and treated with 15 mM KNO\u003csub\u003e3\u003c/sub\u003e or 200 mM NaCl to evaluate nitrate and salt responses. Leaves and roots were collected at designated time points, immediately frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eqRT-PCR\u003c/h2\u003e \u003cp\u003eGene-specific primers were designed using SnapGene. Total RNA was extracted using the QuickRNA kit, followed by genomic DNA removal. RNA quality was assessed using NanoDrop and Bioanalyzer platforms. cDNA synthesis was performed using PrimeScript RT Master Mix. qRT-PCR was conducted on a CFX96 system using TB Green chemistry, with \u003cem\u003eVvUBI\u003c/em\u003e serving as the internal reference gene [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Each reaction contained 2 \u0026micro;L of cDNA. Amplification was performed for 40 cycles following initial denaturation, and melting curve analysis was used to verify primer specificity. Relative gene expression levels were calculated using the 2⁻ΔΔCt method [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eVector Construction and Plant Transformation\u003c/h2\u003e \u003cp\u003eFull-length coding sequences (CDS) of \u003cem\u003eVvNRT1.47\u003c/em\u003e (1815 bp) and \u003cem\u003eVvNRT1.19\u003c/em\u003e (324 bp) were amplified from cDNA synthesized from leaves of own-rooted \u0026lsquo;Shine Muscat\u0026rsquo; grapevine seedlings and verified by sequencing. Validated CDS fragments were cloned into the plant expression vector pBWA(V)BS under the control of the CaMV 35S promoter using the MultiF Seamless Assembly system (ABclonal). Recombinant plasmids pBWA(V)BS-VvNRT1.47 and pBWA(V)BS-VvNRT1.19 were introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101. Primer sequences used for gene amplification are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Transgenic \u003cem\u003eArabidopsis thaliana\u003c/em\u003e plants harboring \u003cem\u003e35S::VvNRT1.47\u003c/em\u003e and \u003cem\u003e35S::VvNRT1.19\u003c/em\u003e constructs were generated via \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eAuthor details\u003c/h2\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003e College of Horticulture and Landscape Architecture, Yangzhou University, Yangzhou 225009, China\u003c/p\u003e \u003cp\u003e \u003csup\u003e2\u003c/sup\u003e Shandong Academy of Grape, Shandong Academy of Agricultural Sciences, Jinan 250100, China\u003c/p\u003e \u003cp\u003e \u003csup\u003e3\u003c/sup\u003e Weifang University, Weifang 261061, China\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was supported by the Natural Science Foundation of Shandong Province (Grant No. ZR2023MC101) and Agricultural Science and Technology Innovation Project of Shandong Academy of Agricultural Sciences(Grant No. CXGC2026A47), and Science and Technology Support Plan for Youth Innovation of Colleges and Universities of Shandong Province of China (2024KJI023) and Jinan Comprehensive Experiment Station of National Grape Industry Technology System (CARS-29-16).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eChen Zhou : Conceptualization, Sample preparation and analysis, Methodology, Data curation, Writing-original draft, Supervision. Yingchun Chen : Data curation, Methodology, Formal analysis. Kai Liu : Methodology, Conceptualization, Supervision. Xiujie Li : Conceptualization, Supervision. Xuehui Zhao : Data curation, Conceptualization. Bo Li : Conceptualization, Supervision. Li Liu : Conceptualization, Writing \u0026ndash; review \u0026amp; editing, Supervision. Zhaosen Xie : Conceptualization, Methodology, Supervision.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this published article. The genome-wide sequence data of *Vitis vinifera* L. supporting the findings of this study are publicly available in the NCBI database ( [https://www.ncbi.nlm.nih.gov/](https:/www.ncbi.nlm.nih.gov) ) and Winberige database (http://www.winberige.cc/index.php).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang Q, Li S, Li J, Huang D. The Utilization and Roles of Nitrogen in Plants. Forests 2024, 15(7).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHachiya T, Sakakibara H. Interactions between nitrate and ammonium in their uptake, allocation, assimilation, and signaling in plants. J Exp Bot 2016.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNedelyaeva OI, Khramov DE, Balnokin YV, Volkov VS. Functional and Molecular Characterization of Plant Nitrate Transporters Belonging to NPF (NRT1/PTR) 6 Subfamily. Int J Mol Sci 2024, 25(24).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTONG Y-p CAIC, LIU, Q-y. LI J-y, LI Z-s: Recent advances in molecular biology of nitrate transporters in higher plants. J Plant Nutr Fertilizers. 2004;10(4):433\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrsel M, Filleur S, Fraisier V, Daniel-Vedele F. Nitrate transport in plants: which gene and which control? J Exp Bot. 2002;53(370):825\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWen Z, Kaiser BN. Unraveling the functional role of NPF6 transporters. Front Plant Sci. 2018;9:973.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahmat Z, Sohail MN, Perrine-Walker F, Kaiser BN. Balancing nitrate acquisition strategies in symbiotic legumes. Planta. 2023;258(1):12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKanno Y, Kamiya Y, Seo M. Nitrate does not compete with abscisic acid as a substrate of AtNPF4. 6/NRT1. 2/AIT1 in Arabidopsis. Plant Signal Behav. 2013;8(12):e26624.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL\u0026eacute;ran S, Garg B, Boursiac Y, Corratg\u0026eacute;-Faillie C, Brachet C, Tillard P, Gojon A, Lacombe B. AtNPF5. 5, a nitrate transporter affecting nitrogen accumulation in Arabidopsis embryo. Sci Rep. 2015;5(1):7962.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiangliw JL, Ruangsiri M, Theerawitaya C, Cha-Um S, Poncheewin W, Songtoasesakul D, Thunnom B, Ruanjaichon V, Toojinda T. Contrasting Alleles of OsNRT1. 1b Fostering Potential in Improving Nitrogen Use Efficiency in Rice. Plants. 2024;13(20):2932.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuan D, Zhang H. A single SNP in NRT1. 1B has a major impact on nitrogen use efficiency in rice. Sci China Life Sci. 2015;58(8):827.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao H, Liu Z, Guo J, Jia Z, Shi Y, Kang K, Peng W, Wang Z, Chen L, Neuhaeuser B. ZmNRT1. 1B (ZmNPF6. 6) determines nitrogen use efficiency via regulation of nitrate transport and signalling in maize. Plant Biotechnol J. 2024;22(2):316\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Q, Liu C, Dong Q, Huang D, Li C, Li P, Ma F. Genome-wide identification and analysis of apple NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family (NPF) genes reveals MdNPF6. 5 confers high capacity for nitrogen uptake under low-nitrogen conditions. Int J Mol Sci. 2018;19(9):2761.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H, Zhao X, Sun Q, Yan C, Wang J, Yuan C, Li C, Shan S, Liu F. Comparative transcriptome analysis reveals molecular defensive mechanism of Arachis hypogaea in response to salt stress. Int J Genomics 2020, 2020(1):6524093.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang D-c, Xu Q, Zhao F-g, Liu M, Liu X. Effects of NaCI stress on nitrogen metabolism and related gene expression of rice with different resistance. 2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu R, Cui B, Jia T, Song J. Role of Suaeda salsa SsNRT2. 1 in nitrate uptake under low nitrate and high saline conditions. Plant Physiol Biochem. 2021;159:171\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu R, Cui B, Lu X, Song J. The positive effect of salinity on nitrate uptake in Suaeda salsa. Plant Physiol Biochem. 2021;166:958\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSena F, Kunze R. The K+ transporter NPF7. 3/NRT1. 5 and the proton pump AHA2 contribute to K+ transport in Arabidopsis thaliana under K\u0026thinsp;+\u0026thinsp;and NO3-deficiency. Front Plant Sci. 2023;14:1287843.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Liu Y, Li W, Ma X, Wang W, Jiang Z, Wang Y, Li L, Hu B, Chu C. OsNRT1. 1B-OsCNGC14/16‐Ca2+‐OsNLP3 Pathway: Phosphorylation‐Mediated Maintenance of Nitrogen Homeostasis. Adv Sci. 2025;12(43):e07919.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSa G, Yao J, Deng C, Liu J, Zhang Y, Zhu Z, Zhang Y, Ma X, Zhao R, Lin S. Amelioration of nitrate uptake under salt stress by ectomycorrhiza with and without a Hartig net. New Phytol. 2019;222(4):1951\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeller M, Koblet W. Dry matter and leaf area partitioning, bud fertility and second season growth of Vitis vinifera L.: Responses to nitrogen supply and limiting irradiance. Vitis. 1995;34(2):77\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStefanello LO, Schwalbert R, Marques AR, Tarouco CP, Vitto BB, Krug A, dos Santos JJ, Galarza BP, Zalamena J, dos Santos HP. Photosynthetic activity and grape yield of'Alicante Bouschet'(Vitis vinifera L.) grapevines submitted to nitrogen supply methods and doses. Vitis. 2020;59:133\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang C, Lu X, Yan H, Gong M, Wang W, Chen B, Ma S, Li S. Nitrogen application improves salt tolerance of grape seedlings via regulating hormone metabolism. Physiol Plant. 2023;175(2):e13896.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAltemose N, Logsdon GA, Bzikadze AV, Sidhwani P, Langley SA, Caldas GV, Hoyt SJ, Uralsky L, Ryabov FD, Shew CJ. Complete genomic and epigenetic maps of human centromeres. Science. 2022;376(6588):eabl4178.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Z, Mei J, Zheng W, Khan FS, Bhuiyan MN, Wang K, Rhaman MS, Abe-Kanoh N, Ji W. Identification of Grape NRT Gene Family and Analysis of Its Expression in Leaves Under Nitrogen-Deficiency Stress. Horticulturae. 2025;11(3):252.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMonte R, Tomasi N, Cesco S, Zamboni A, Varanini Z, Pinton R. Physiological and molecular characterization of nitrate uptake mechanisms in roots of grapevine (Vitis vinifera L.) cultivars. 2009.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi X, Cao S, Wang X, Huang S, Wang Y, Liu Z, Liu W, Leng X, Peng Y, Wang N. The complete reference genome for grapevine (Vitis vinifera L.) genetics and breeding. Hortic Res. 2023;10(5):uhad061.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhiqiang G, Yuexiu L, Fan F, Lixun Z, Jiali F, Xiaodong J, Jinhui L, Chunlai Z. Genome-wide Identification of NRT1 Gene, Expression Profiling and DNA Variation Analysis in Sorghum. J Laser Biology/Acta Laser Biology Sinica 2021, 30(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi G, Yang D, Hu Y, Xu J, Lu Z. Genome-wide identification and expression analysis of nitrate transporter (NRT) gene family in Eucalyptus grandis. Genes. 2024;15(7):930.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng J, Tan H, Shan M, Duan M, Ye L, Yang Y, He L, Shen H, Yang Z, Wang X. Genome-wide identification and characterization of the NPF genes provide new insight into low nitrogen tolerance in Setaria. Front Plant Sci. 2022;13:1043832.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Ritonga FN, Zhang S, Wang F, Li J, Gao J. Genome-Wide Identification of the NRT1 Family Members and Their Expression under Low-Nitrate Conditions in Chinese Cabbage (Brassica rapa L. ssp. pekinensis). Plants. 2023;12(22):3882.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu X, Li J, Luo D, Ding H, Zhang M, Gao P, Mehvish A, Sun X, Tong C, Wu Q. Genome-wide characterization of the NRT1 family members under cold stress in Coconut (Cocos nucifera L). Sci Hort. 2025;341:113959.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL\u0026eacute;ran S, Varala K, Boyer J-C, Chiurazzi M, Crawford N, Daniel-Vedele F, David L, Dickstein R, Fernandez E, Forde B. A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants. Trends Plant Sci. 2014;19(1):5\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTahir MM, Wang H, Ahmad B, Liu Y, Fan S, Li K, Lei C, Shah K, Li S, Zhang D. Identification and characterization of NRT gene family reveals their critical response to nitrate regulation during adventitious root formation and development in apple rootstock. Sci Hort. 2021;275:109642.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaha SD, Dutta S, Sch\u0026auml;ffner AR, Das M. Gene duplication and stress genomics in Brassicas: Current understanding and future prospects. J Plant Physiol. 2020;255:153293.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y-Y, Tsay Y-F. Arabidopsis nitrate transporter NRT1. 9 is important in phloem nitrate transport. Plant Cell. 2011;23(5):1945\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHang J, Wu B, Qiu D, Yang G, Fang Z, Zhang M. OsNPF3. 1, a nitrate, abscisic acid and gibberellin transporter gene, is essential for rice tillering and nitrogen utilization efficiency. J Integr Agric. 2024;23(4):1087\u0026ndash;104.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIslam MS. Sensing and uptake of nitrogen in rice plant: a molecular view. Rice Sci. 2019;26(6):343\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLIU H, LIU Q, GAO X, FU X. Role of nitrogen sensing and its integrative signaling pathways in shaping root system architecture. Eng Agric. 2022;9(3):316\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J-Y, Fu Y-L, Pike SM, Bao J, Tian W, Zhang Y, Chen C-Z, Zhang Y, Li H-M, Huang J. The Arabidopsis nitrate transporter NRT1. 8 functions in nitrate removal from the xylem sap and mediates cadmium tolerance. Plant Cell. 2010;22(5):1633\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChiba Y, Shimizu T, Miyakawa S, Kanno Y, Koshiba T, Kamiya Y, Seo M. Identification of Arabidopsis thaliana NRT1/PTR FAMILY (NPF) proteins capable of transporting plant hormones. J Plant Res. 2015;128(4):679\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrapp A, David LC, Chardin C, Girin T, Marmagne A, Leprince A-S, Chaillou S, Ferrario-M\u0026eacute;ry S, Meyer C, Daniel-Vedele F. Nitrate transport and signalling in Arabidopsis. J Exp Bot. 2014;65(3):789\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBouguyon \u0026Eacute;, Brun F, Meynard D, Kubeš M, Pervent M, L\u0026eacute;ran S, Lacombe B, Krouk G, Guiderdoni E, Zaž\u0026iacute;malov\u0026aacute; E. Multiple mechanisms of nitrate sensing by Arabidopsis nitrate transceptor NRT1. 1. Nat plants. 2015;1(3):1\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHo C-H, Lin S-H, Hu H-C, Tsay Y-F. CHL1 functions as a nitrate sensor in plants. Cell. 2009;138(6):1184\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAltschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215(3):403\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThompson JD, Gibson TJ, Higgins DG. Multiple sequence alignment using ClustalW and ClustalX. Curr protocols Bioinf 2003(1):2.3. 1-2.3. 22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Tang H, DeBarry JD, Tan X, Li J, Wang X, Lee T-h, Jin H, Marler B, Guo H. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012;40(7):e49\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020;13(8):1194\u0026ndash;202.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo A-Y, Zhu Q-H, Chen X, Luo J-C. GSDS: a gene structure display server. Yi chuan= Hereditas. 2007;29(8):1023\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, Li WW, Noble WS. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 2009;37(suppl2):W202\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu K, Yang A, Yan J, Liang Z, Yuan G, Cong P, Zhang L, Han X, Zhang C. MdAIL5 overexpression promotes apple adventitious shoot regeneration by regulating hormone signaling and activating the expression of shoot development-related genes. Hortic Res. 2023;10(11):uhad198.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Footnotes","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org/browse/gene_family\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org/browse/gene_family\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phytozome-next.jgi.doe.gov/blast-search\u003c/span\u003e\u003cspan address=\"https://phytozome-next.jgi.doe.gov/blast-search\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.winberige.cc/ftp.html\u003c/span\u003e\u003cspan address=\"http://www.winberige.cc/ftp.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gsds.cbi.pku.edu.cn/\u003c/span\u003e\u003cspan address=\"http://gsds.cbi.pku.edu.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/tools/meme\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/tools/meme\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bar.utoronto.ca/efp_grape/cgi-bin/efpWeb.cgi\u003c/span\u003e\u003cspan address=\"https://bar.utoronto.ca/efp_grape/cgi-bin/efpWeb.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Genome-wide analysis, Nitrate Transporters, Grapevine, Gene expression, Gene function","lastPublishedDoi":"10.21203/rs.3.rs-8769993/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8769993/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eNRT1\u003c/em\u003e (nitrate transporter 1) family is referred to as the \u003cem\u003eNPF\u003c/em\u003e (nitrate/peptide transporter family). It constitutes the largest and most functionally diverse group of nitrate transporters in plants and plays an essential role in nitrate translocation and allocation. These are fundamental to plant growth, development, and stress adaptation. However, a comprehensive genome-wide characterization of this family in grapevine (\u003cem\u003eVitis vinifera\u003c/em\u003e L.) remains unexplored.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA total of 65 \u003cem\u003eNRT1\u003c/em\u003e genes were identified in the grapevine genome and phylogenetically classified into five distinct clades. Their physicochemical properties, as well as gene and protein structural features, were analyzed in detail. Homology analyses revealed that both segmental and tandem duplication events played major roles in driving the expansion of the \u003cem\u003eVvNRT1\u003c/em\u003e gene family. In addition, analysis of tissue-specific expression patterns and cis-regulatory elements suggested that \u003cem\u003eVvNRT1\u003c/em\u003e genes participated in the regulation of grapevine development and adaptive responses to diverse environmental stresses. Nitrate treatment and salt stress induced the expression of multiple \u003cem\u003eVvNRT1s\u003c/em\u003e, among which \u003cem\u003eVvNRT1.19\u003c/em\u003e and \u003cem\u003eVvNRT1.47\u003c/em\u003e exhibited the most pronounced transcriptional upregulation. The expression levels of these two genes were positively associated with nitrogen uptake capacity and salt tolerance in grapevine. Furthermore, heterologous overexpression of \u003cem\u003eVvNRT1.19\u003c/em\u003e and \u003cem\u003eVvNRT1.47\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e significantly enhanced nitrogen use efficiency and salt tolerance, thereby promoting plant growth and improving stress resistance.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study presents a comprehensive characterization of the \u003cem\u003eVvNRT1\u003c/em\u003e gene family and provides insights into its evolutionary history in grapevine. \u003cem\u003eVvNRT1.19\u003c/em\u003e and \u003cem\u003eVvNRT1.47\u003c/em\u003e are proposed to function as positive regulators of plant responses to stress. Collectively, these findings establish a solid foundation for future functional investigations of \u003cem\u003eNRT1\u003c/em\u003e genes and highlight their potential roles in improving nitrogen regulation and salt stress tolerance in grapevine.\u003c/p\u003e","manuscriptTitle":"Genome-Wide Analysis and Functional Characterization of the NRT1 Gene Family in Grapevine (Vitis vinifera L.) Reveals Roles of VvNRT1.19 and VvNRT1.47 in Salt Stress and Nitrogen Response","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-19 18:19:20","doi":"10.21203/rs.3.rs-8769993/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"eba8eb93-f197-402e-96d8-664fb4261cba","owner":[],"postedDate":"March 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-28T05:25:52+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-19 18:19:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8769993","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8769993","identity":"rs-8769993","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}