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In sweet potato, rational nitrogen (N) assimilation and distribution are conducive to inhibiting vine overgrowth. Nitrate (NO3-) is the main N form absorbed by roots, and cultivar is an important factor affecting N utilization. Herein, a hydroponic experiment was conducted that included four NO3- concentrations of 0 (N0), 4 (N1), 8 (N2) and 16 (N3) mmol L-1 with two cultivars of Jishu26 (J26, N-sensitive) and Xushu32 (X32, N-tolerant). For J26, with increasing NO3- concentrations, the root length and root surface area significantly decreased. However, no significant differences were observed in these parameters for X32. Higher NO3- concentrations upregulated the expression levels of the genes that encode nitrate reductase (NR2), nitrite reductase (NiR2) and nitrate transporter (NRT1.1) in roots for both cultivars. The trends in the activities of NR and NiR were subject to regulation of NR2 and NiR2 transcription, respectively. For both cultivars, N2 increased the N accumulated in leaves, growth points and roots. For J26, N3 further increased the N accumulation in these organs. Under higher NO3- nutrition, compared with X32, J26 exhibited higher expression levels of the NiR2, NR2 and NRT1.1 genes, a higher influx NO3- rate in roots, and higher activities of NR and NiR in leaves and roots. Conclusively, the regulated effects of NO3- supplies on root growth and NO3- utilization were more significant for J26. Under high NO3- conditions, J26 exhibited higher capacities of NO3- absorption and distributed more N in leaves and in growth points, which may contribute to higher growth potential in shoots and more easily cause vine overgrowth.

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Сoding sequences of seven housekeeping genes: actin SaACT7, ubiquitin-conjugating protein SaUBC10, glyceraldehyde-3-phosphate dehydrogenase SaGAPDH, protein of the large subunit of ribosomes SaL2, α-tubulin SaTUA, translation elongation factor SaeEF1α, and protein phosphatase SaPP2A were identified as candidate reference genes for expression analysis of target genes in the extremely salt tolerant plant Suaeda altissima (L.) Pall. The expression profiles of the genes differed. SaACT7 and SaeEF1α demonstrated the highest expression levels, while the lowest expression levels were found for SaPP2A and SaTUA. SaPP2A and SaeEF1α genes were the most stably expressed at different steady-state salinity levels and different nitrate concentrations in nutrient solutions (NSs). SaL2, SaPP2A, and SaeEF1α genes showed the greatest stability of expression when nitrate was added to nutrient solution of plants grown under conditions of nitrate deficiency. Less constant expression was demonstrated in this experiment by SaACT7 and SaTUA. SaL2, SaACT7, SaeEF1α, and SaUBC10 genes showed the smallest expression changes under salt shock. To validate the use of the most stably expressed genes for normalization of gene expression, we checked them as reference genes to study the expression of the nitrate transporter gene SaNPF6.3 in S. altissima roots under conditions of different salinity and different nitrate supply.

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Two genes of nitrate transporters SaNRT2.1 and SaNRT2.5, putative orthologs of high-affinity nitrate transporter genes AtNRT2.1 and AtNRT2.5 from Arabidopsis thaliana, were cloned from the euhalophyte Suaeda altissima. Phylogenetic bioinformatic analysis demonstrated that the proteins SaNRT2.1 and SaNRT2.5 exhibited higher levels of homology to the corresponding proteins from the plants of family Amaranthaceae; the similarity of amino acid sequences between proteins SaNRT2.1 and SaNRT2.5 was lower (54%). Both SaNRT2.1 and SaNRT2.5 are integral membrane proteins forming 12 transmembrane helices as predicted by topological modeling. An attempt to demonstrate nitrate transporting activity of SaNRT2.1 or SaNRT2.5 by heterologous expression of the genes in the yeast Hansenula (Ogataea) polymorpha mutant strain Δynt1 lacking the only yeast nitrate transporter was not successful. The expression patterns of SaNRT2.1 and SaNRT2.5 were studied in S. altissima plants that were grown in hydroponics under either low (0.5 mM) or high (15 mM) nitrate and salinity from 0 to 750 mM NaCl. The growth of the plants was strongly inhibited by low nitrogen supply while stimulated by NaCl; it peaked at 250 mM NaCl for high nitrate and at 500 mM NaCl for low nitrate. Under low nitrate supply, nitrate contents in S. altissima roots, leaves and stems were reduced but increased in leaves and stems as salinity in the medium increased. Potassium contents remained stable under salinity treatment from 250 to 750 mM NaCl. Quantitative real-time PCR demonstrated that without salinity, SaNRT2.1 was expressed in all organs, its expression was not influenced by nitrate supply, while SaNRT2.5 was expressed exclusively in roots-its expression rose about 10-fold under low nitrate. Salinity increased expression of both SaNRT2.1 and SaNRT2.5 under low nitrate. SaNRT2.1 peaked in roots at 500 mM NaCl with 15-fold increase; SaNRT2.5 peaked in roots at 500 mM NaCl with 150-fold increase. It is suggested that SaNRT2.5 ensures effective nitrate uptake by roots and functions as an essential high-affinity nitrate transporter to support growth of adult S. altissima plants under nitrogen deficiency.

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Genome co-editing technology is effective in breeding filamentous fungi for applications in the fermentation industry, achieving site-directed mutagenesis, the status of non-genetically modified organisms (non-GMOs), and wild-type-like growth phenotype. Prior to this study, thiI gene was found as a selectable marker for such genome co-editing in the filamentous fungus Aspergillus oryzae, while it cannot be reused via marker recycling. Therefore, we aimed to identify another marker gene to knock out another target gene via genome co-editing in A. oryzae. In this study, we focused on the membrane transporter gene nrtA (AO090012000623), which promotes uptake of nitrate (NO3-). It is known that, in nrtA knockout strain, chlorate (ClO3-), an analog of nitrate with antifungal activity, cannot be imported into the cytosol, which enables the mutant to grow in the presence of chlorate. Based on this information, knockout of the target gene wA was attempted using both nrtA- and wA-specific single-guide RNAs via genome co-editing with KClO3 supplementation in A. oryzae laboratory strain RIB40 and industrial strain KBN616. Resultantly, wA knockout mutant was generated, and nrtA was identified as a selectable marker. Moreover, this genome co-editing system using nrtA was compatible with that using thiI, and thus, a double knockout mutant of two target genes wA and yA was constructed in RIB40 while maintaining non-GMO status and wild-type-like growth. As nrtA homologs have been found in several industrial Aspergillus species, genome co-editing using homolog genes as selectable markers is plausible, which would contribute to the widespread breeding of industrial strains of Aspergilli.

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Modern crops exhibit diverse sensitivities to ammonium as the primary nitrogen source, influenced by environmental factors such as external pH and nutrient availability. Despite its significance, there is currently no systematic classification of plant species based on their ammonium sensitivity. We conducted a meta-analysis of 50 plant species and present a new classification method based on the comparison of fresh biomass obtained under ammonium and nitrate nutrition. The classification uses the natural logarithm of the biomass ratio as the size effect indicator of ammonium sensitivity. This numerical parameter is associated with critical factors for nitrogen demand and form preference, such as Ellenberg indicators and the repertoire of nitrogen transporters for ammonium and nitrate uptake. Finally, a comparative analysis of the developmental and metabolic responses, including hormonal balance, is conducted in two species with divergent ammonium sensitivity values in the classification. Results indicate that nitrate has a key role in counteracting ammonium toxicity in species with a higher abundance of genes encoding NRT2-type proteins and fewer of those encoding the AMT2-type proteins. Additionally, the study demonstrates the reliability of the phytohormone balance and methylglyoxal content as indicators for anticipating ammonium toxicity.

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Elucidating the mechanisms regulating nitrogen (N) deficiency responses in plants is of great agricultural importance. Previous studies revealed that decreased expression of NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1) transcriptional repressor genes upon N deficiency is involved in N deficiency-inducible gene expression in Arabidopsis thaliana. However, our knowledge of the mechanisms controlling N deficiency-induced changes in gene expression is still limited. Through the identification of Dof1.7 as a direct target of NIGT1 repressors and a novel N deficiency response-related transcriptional activator gene, we here show that NIGT1 and Dof1.7 transcription factors (TFs) differentially regulate N deficiency-inducible expression of three high-affinity nitrate transporter genes, NRT2.1, NRT2.4, and NRT2.5, which are responsible for most of the soil nitrate uptake activity of Arabidopsis plants under N-deficient conditions. Unlike NIGT1 repressors, which directly suppress NRT2.1, NRT2.4, and NRT2.5 under N-sufficient conditions, Dof1.7 directly activated only NRT2.5 but indirectly and moderately activated NRT2.1 and NRT2.4 under N-deficient conditions, probably by indirectly decreasing NIGT1 expression. Thus, Dof1.7 converted passive transcriptional activation into active and potent transcriptional activation, further differentially enhancing the expression of NRT2 genes. These findings clarify the mechanism underlying different expression patterns of NRT2 genes upon N deficiency, suggesting that time-dependent multilayered transcriptional regulation generates complicated expression patterns of N deficiency-inducible genes.

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Grain protein content (GPC) is a crucial quality trait in bread wheat, which is influenced by the key transcription factor TaNAM. However, the regulatory mechanisms of TaNAM have remained largely elusive. In this study, a new role of TaNAM was unveiled in regulating nitrogen remobilisation which impacts GPC. The TaNAM knockout mutants generated by clustered regularly interspaced short palindromic repeats/Cas9 exhibited significantly delayed senescence and lower GPC, while overexpression of TaNAM-6A resulted in premature senility and much higher GPC. Further analysis revealed that TaNAM directly activates the genes TaNRT1.1 and TaNPF5.5s, which are involved in nitrogen remobilisation. This activity aids in the transfer of nitrogen from leaves to grains for protein synthesis. In addition, an elite allele of TaNAM-6A, associated with high GPC, was identified as a candidate gene for breeding high-quality wheat. Overall, our work not only elucidates the potential mechanism of TaNAM-6A affecting bread wheat GPC, but also highlights the significance of nitrogen remobilisation from senescent leaves to grains for protein accumulation. Moreover, our research provides a new target and approach for improving the quality traits of wheat, particularly the GPC.

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Nitrogen source assimilation is important for the biological functions of fungi, and its pathway has been deeply studied. Aspergillus oryzae mutants defective in nitrogen source assimilation are known to grow poorly on Czapek-Dox (CD) medium. In this study, we found an industrial strain of A. oryzae that grew very poorly on a CD medium containing sodium nitrate as a nitrogen source. We used media with various nitrogen components to examine the steps affecting the nitrogen source assimilation pathway of this strain. The strain grew well on the CD medium supplied with nitrite salt or ammonium salt, suggesting that the strain was defective in nitrate assimilation step. To ascertain the gene causing the defect of nitrate assimilation, a gene expression vector harboring either niaD or crnA of A. oryzae RIB40 was introduced into the industrial strain. The industrial strain containing the crnA vector recovered its growth. This is the first report that a mutation of crnA causes poor growth on CD medium in an industrial strain of A. oryzae, and crnA can be used as a transformation marker for crnA deficient strains.

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In addition to absorbing nitrogen from the soil, legumes have the ability to use atmospheric N2 through symbiotic nitrogen fixation. Therefore, legumes have developed mechanisms regulating nodulation in response to the amount of nitrate in the soil; in the presence of high nitrate concentrations, nodulation is inhibited, while low nitrate concentrations stimulate nodulation and nitrogen fixation. This allows the legumes to switch from soil nitrogen acquisition to symbiotic nitrogen fixation. Recently, particular interest has been given to the nitrate transporters, such as Nitrate Transporter1/Peptide transporter Family (NPF) and Nitrate Transporter 2 (NRT2), having a role in the functioning of nodules. Nitrate transporters of the two model plants, Lotus japonicus and Medicago truncatula, shown to have a positive and/or a negative role in nodule functioning depending on nitrate concentration, are presented in this article. In particular, the following transporters were thoroughly studied: (i) members of NPF transporters family, such as LjNPF8.6 and LjNPF3.1 in L. japonicus and MtNPF1.7 and MtNPF7.6 in M. truncatula, and (ii) members of NRT2 transporters family, such as LjNRT2.4 and LjNRT2.1 in L. japonicus and MtNRT2.1 in M. truncatula. Also, by exploiting available genomic and transcriptomic data in the literature, we have identified the complete PsNPF family in Pisum sativum (69 sequences previously described and 21 new that we have annotated) and putative nitrate transporters candidate for playing a role in nodule functioning in P. sativum.

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Nitrate, the primary form of nitrogen absorbed by plants, supplies essential compounds for plant growth and development. Peas are frequently used as rotation crops to improve and stabilize soil fertility. However, the determinants of nitrate uptake and transport in peas remain largely unclear, primarily due to the pea genome's complexity and size. In this study, we utilized the complete genomic information of peas to identify three PsNRT2 family genes within the pea genome. We conducted a comprehensive examination of their protein conserved domains, physicochemical properties, gene structure, and phylogenetic evolution, revealing PsNRT2.3 as the potential key gene for high-affinity nitrate transport in peas. Subcellular localization studies indicated that PsNRT2.3 resides on the plasma membrane. Using hairy root transformation, we noted the predominant expression of PsNRT2.3 in the root stele, which is inducible by nitrate. Our experiments involving overexpression and silencing methods further confirmed that PsNRT2.3 plays a key role in enhancing nitrate uptake in peas. Additionally, our work showed that PsNAR could interact with PsNRT2.3, modulating pea nitrate uptake. After silencing PsNAR, even with the normal expression of PsNRT2.3, the ability of peas to absorb nitrate was significantly reduced. In conclusion, this study identifies the high-affinity nitrate transport gene PsNRT2.3 in peas and clarifies its critical role and regulatory network in nitrate transport, contributing to a new understanding of nitrate utilization in peas.

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NRT2.1, the major high affinity nitrate transporter in roots, can be phosphorylated at five different sites within the N- and the C-terminus. Here, we characterized the functional relationship of two N-terminal phosphorylation sites, S21 and S28, in Arabidopsis. Based on a site-specific correlation network, we identified a receptor kinase (HPCAL1, AT5G49770), phosphorylating NRT2.1 at S21 and resulting in active nitrate uptake. HPCAL1 itself was regulated by phosphorylation at S839 and S870 within its kinase domain. In the active state, when S839 was dephosphorylated and S870 was phosphorylated, HPCAL1 was found to interact with the N-terminus of NRT2.1, mainly when S28 was dephosphorylated. Phosphorylation of NRT2.1 at S21 resulted in a reduced interaction of NRT2.1 with its activator NAR2.1, but nitrate transport activity remained. By contrast, phosphorylated NRT2.1 at S28 enhanced the interaction with NAR2.1, but reduced the interaction with HPCAL1. Here we identified HPCAL1 as the kinase affecting this phospho-switch through phosphorylation of NRT2.1 at S21.

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Nitrogen fertilization can promote rice yield but decrease resistance to sheath blight (ShB). In this study, the nitrate transporter 1.1b (nrt1.1b) mutant that exhibited less susceptibility to ShB but without compromising yield under NH4+ fertilization was screened. NRT1.1B's regulation of ShB resistance was independent of the total nitrogen concentration in rice under NH4+ conditions. In nrt1.1b mutant plants, the NH4+ application modulated auxin signaling, chlorophyll content, and phosphate signaling to promote ShB resistance. Furthermore, the findings indicated that NRT1.1B negatively regulated ShB resistance by positively modulating the expression of H+-ATPase gene OSA3 and phosphate transport gene PT8. The mutation of OSA3 and PT8 promoted ShB resistance by increasing the apoplastic pH in rice. Our study identified the ShB resistance mutant nrt1.1b, which maintained normal nitrogen use efficiency without compromising yield.

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BACKGROUND: Nitrogen is one of the most important mineral nutrients for plants and is absorbed by the root system mainly in the inorganic form (NH+4 and NO-3). Plants absorb nitrogen as a food source for growth, biomass production, and development. Nitrogen is mainly absorbed as nitrate, which is the most common source of nitrogen available to higher plants. One of the unique features of nitrate transport is that NO-3 is both a substrate for transport and an inducer of NO-3 transport systems in genes and at physiological levels. METHODS: In the present study, morphological and physiological traits (chlorophyll a/b, total chlorophyll, and carotenoid, antioxidant enzymes, and protein content), correlation between traits and gene expression, and principle component analysis of traits among five barley cultivars were measured in response to nitrogen deficiency (ND). The starved plants were transferred to a nutrient solution containing 0.2 mM and 2 mM NO-3 up to 7 and 14 days after ND application and non-stressed conditions, respectively. RESULTS: Gene expression analysis revealed that the 10 HvNRT2 genes were induced in the leaf and root tissues at 7 and 14 days after ND treatments in five barley cultivars. Expression of NRT2 genes by relative quantitative qRT-PCR analysis for 10 HvNRT2 genes were determined. Based on the gene expression, HvNRT2.1, HvNRT2.2, and HvNRT2.4 were strongly induced by NO-3, peaking at 7 and 14 days after ND treatment. In contrast, the HvNRT2.4 showed only moderate induction in both leaves and roots. From our results, the Reyhan cultivar showed a significant increase in root fresh weight (RFW), protein content, and antioxidant enzyme activity in roots at 7 and 14 days after ND treatment as compared to the non-stressed condition. A highly positive correlation was observed between root catalase (CATr) and HvNRT2.2/2.5/2.6 leaves. CONCLUSION: The expression of HvNRT2.4 is increased during long-term nitrogen starvation, while the expression of HvNRT2.1 and HvNRT2.2 are transiently increased by ND. Based on physiological and morphological traits and molecular mechanisms, the Reyhan is considered a tolerant cultivar under ND condition.

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The root-colonizing endophytic fungus Piriformospora indica promotes the root and shoot growth of its host plants. We show that the growth promotion of Arabidopsis thaliana leaves is abolished when the seedlings are grown on media with nitrogen (N) limitation. The fungus neither stimulated the total N content nor did it promote 15NO3- uptake from agar plates to the leaves of the host under N-sufficient or N-limiting conditions. However, when the roots were co-cultivated with 15N-labelled P. indica, more labels were detected in the leaves of N-starved host plants but not in plants supplied with sufficient N. Amino acid and primary metabolite profiles, as well as the expression analyses of N metabolite transporter genes suggest that the fungus alleviates the adaptation of its host from the N limitation condition. P. indica alters the expression of transporter genes, which participate in the relocation of NO3-, NH4+ and N metabolites from the roots to the leaves under N limitation. We propose that P. indica participates in the plant's metabolomic adaptation against N limitation by delivering reduced N metabolites to the host, thus alleviating metabolic N starvation responses and reprogramming the expression of N metabolism-related genes.

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The bZIP (basic leucine zipper) proteins play crucial roles in various biological functions. Nitrogen (N) is an essential element for plant growth, especially in cucumber (Cucumis sativus) due to its shallow roots. However, the regulation of bZIP genes in cucumber nitrogen metabolism has not been studied yet. In this study, we identified a total of 72 bZIP genes (CsbZIPs) in the cucumber genome that could be classified into 13 groups. These genes were unevenly distributed on seven chromosomes, and synteny analysis showed that the CsbZIP genes were expanded in a segmentally duplicating manner. Furthermore, our genome-wide expression analysis suggested that CsbZIP genes had different patterns and that five CsbZIP genes were regulated by nitrogen treatment in both leaves and roots. Consistent with CsNPF, CsbZIP55 and CsbZIP65 were regulated by nitrogen treatment in leaves and roots. Moreover, the subcellular localization showed that CsbZIP55 and CsbZIP65 were specifically located in the nucleus, and the transcriptional activation assay showed that CsbZIP55 and CsbZIP65 have transcriptional activation activity. Additionally, in the CsbZIP55 and CsbZIP65 overexpression plants, most nitrogen-regulated CsNPF genes were downregulated. Taken together, our comprehensive analysis of the bZIP gene family lays a foundation for understanding the molecular and physiological functions of CsbZIPs.

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Nitrate is the main form of inorganic nitrogen that crop absorbs, and nitrate transporter 2 (NRT2) is a high affinity transporter using nitrate as a specific substrate. When the available nitrate is limited, the high affinity transport systems are activated and play an important role in the process of nitrate absorption and transport. Most NRT2 cannot transport nitrates alone and require the assistance of a helper protein belonging to nitrate assimilation related family (NAR2) to complete the absorption or transport of nitrates. Crop nitrogen utilization efficiency is affected by environmental conditions, and there are differences between varieties, so it is of great significance to develop varieties with high nitrogen utilization efficiency. Sorghum bicolor has high stress tolerance and is more efficient in soil nitrogen uptake and utilization. The S. bicolor genome database was scanned to systematically analyze the gene structure, chromosomal localization, physicochemical properties, secondary structure and transmembrane domain, signal peptide and subcellular localization, promoter region cis-acting elements, phylogenetic evolution, single nucleotide polymorphism (SNP) recognition and annotation, and selection pressure of the gene family members. Through bioinformatics analysis, 5 NRT2 gene members (designated as SbNRT2-1a, SbNRT2-1b, SbNRT2-2, SbNRT2-3, and SbNRT2-4) and 2 NAR2 gene members (designated as SbNRT3-1 and SbNRT3-2) were identified, the number of which was less than that of foxtail millet. SbNRT2/3 were distributed on 3 chromosomes, and could be divided into four subfamilies. The genetic structure of the same subfamilies was highly similar. The average value of SbNRT2/3 hydrophilicity was positive, indicating that they were all hydrophobic proteins, whereas α-helix and random coil accounted for more than 70% of the total secondary structure. Subcellular localization occurred on plasma membrane, where SbNRT2 proteins did not contain signal peptides, but SbNRT3 proteins contained signal peptides. Further analysis revealed that the number of transmembrane domains of the SbNRT2s family members was greater than 10, while that of the SbNRT3s were 2. There was a close collinearity between NRT2/3s of S. bicolor and Zea mays. Protein domains analysis showed the presence of MFS_1 and NAR2 protein domains, which supported executing high affinity nitrate transport. Phylogenetic tree analysis showed that SbNRT2/3 were more closely related to those of Z. mays and Setaria italic. Analysis of gene promoter cis-acting elements indicated that the promoter region of SbNRT2/3 had several plant hormones and stress response elements, which might respond to growth and environmental cues. Gene expression heat map showed that SbNRT2-3 and SbNRT3-1 were induced by nitrate in the root and stem, respectively, and SbNRT2-4 and SbNRT2-3 were induced by low nitrogen in the root and stem. Non-synonymous SNP variants were found in SbNRT2-4 and SbNRT2-1a. Selection pressure analysis showed that the SbNRT2/3 were subject to purification and selection during evolution. The expression of SbNRT2/3 gene and the effect of aphid infection were consistent with the expression analysis results of genes in different tissues, and SbNRT2-1b and SbNRT3-1 were significantly expressed in the roots of aphid lines 5-27sug, and the expression levels of SbNRT2-3, SbNRT2-4 and SbNRT3-2 were significantly reduced in sorghum aphid infested leaves. Overall, genome-wide identification, expression and DNA variation analysis of NRT2/3 gene family of Sorghum bicolor provided a basis for elucidating the high efficiency of sorghum in nitrogen utilization.

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Background: Nitrate is the primary type of nitrogen available to plants, which is absorbed and transported by nitrate transporter 2 (NRT2) at low nitrate conditions. Methods: Genome-wide identification of NRT2 genes in G. hirsutum was performed. Gene expression patterns were revealed using RNA-seq and qRT-PCR. Gene functions were characterized using overexpression in A. thaliana and silencing in G. hirsutum. Protein interactions were verified by yeast two-hybrid and luciferase complementation imaging (LCI) assays. Results: We identified 14, 14, seven, and seven NRT2 proteins in G. hirsutum, G. barbadense, G. raimondii, and G. arboreum. Most NRT2 proteins were predicted in the plasma membrane. The NRT2 genes were classified into four distinct groups through evolutionary relationships, with members of the same group similar in conserved motifs and gene structure. The promoter regions of NRT2 genes included many elements related to growth regulation, phytohormones, and abiotic stresses. Tissue expression pattern results revealed that most GhNRT2 genes were specifically expressed in roots. Under low nitrate conditions, GhNRT2 genes exhibited different expression levels, with GhNRT2.1e being the most up-regulated. Arabidopsis plants overexpressing GhNRT2.1e exhibited increased biomass, nitrogen and nitrate accumulation, nitrogen uptake and utilization efficiency, nitrogen-metabolizing enzyme activity, and amino acid content under low nitrate conditions. In addition, GhNRT2.1e-silenced plants exhibited suppressed nitrate uptake and accumulation, hampered plant growth, affected nitrogen metabolism processes, and reduced tolerance to low nitrate. The results showed that GhNRT2.1e could promote nitrate uptake and transport under low nitrate conditions, thus effectively increasing nitrogen use efficiency (NUE). We found that GhNRT2.1e interacts with GhNAR2.1 by yeast two-hybrid and LCI assays. Discussion: Our research lays the foundation to increase NUE and cultivate new cotton varieties with efficient nitrogen use.

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Introduction: Multi-walled nanotubes (MWCNTs) consist of multiple rolled layers of graphene. Nitrogen plays an important role in apple growth. The effect of MWCNTs on nitrogen utilization in apple needs to be further investigated. Methods: In this study, the woody plant Malus hupehensis seedlings were used as plant materials, the distribution of MWCNTs in the roots was observed, and the effects of MWCNTs on the accumulation, distribution, and assimilation of nitrate by the seedlings were explored. Results: The results showed that MWCNTs could penetrate the roots of Malus hupehensis seedlings, and the 50, 100, and 200 µg·mL-1 MWCNTs significantly promoted the root growth of seedlings, increased root number, root activity, fresh weight, and nitrate content of seedlings, and also increased nitrate reductase activity, free amino acid, and soluble protein content of roots and leaves. 15N tracer experiments indicated that MWCNTs decreased the distribution ratio of 15N-KNO3 in Malus hupehensis roots but increased its distribution ratio in stems and leaves. MWCNTs improved the utilization ratio of 15N-KNO3 in Malus hupehensis seedlings, with the values being increased by 16.19%, 53.04%, and 86.44% following the 50, 100, and 200 µg·mL-1 MWCNTs, respectively. The RT-qPCR analysis showed that MWCNTs significantly affected the expression of genes (MhNRTs) related to nitrate uptake and transport in roots and leaves, and MhNRT1.4, MhNRT1.7, MhNRT1.8, MhNRT2.1, MhNRT2.5, and MhNRT2.7 were notably up-regulated in response to 200 µg·mL-1 MWCNTs. Raman analysis and transmission electron microscopy images indicated that MWCNTs could enter the root tissue of Malus hupehensis and were distributed between the cell wall and cytoplasmic membrane. Pearson correlation analysis showed that root tip number, root fractal dimension, and root activity were the main factors affecting root uptake and assimilation of nitrate. Conclusions: These findings suggest that MWCNTs promoted root growth by entering the root, stimulated the expression of MhNRTs, and increased NR activity, thereby enhancing the uptake, distribution, and assimilation of nitrate by root, and ultimately improved the utilization of 15N-KNO3 by Malus hupehensis seedlings.

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The NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family (NPF) proteins perform an essential role in regulating plant nitrate absorption and distribution and in improving plant nitrogen use efficiency. In this study, cucumber (Cucumis sativus L.) NPF genes were comprehensively analyzed at the whole genome level, and 54 NPF genes were found to be unevenly distributed on seven chromosomes in the cucumber genome. The phylogenetic analysis showed that these genes could be divided into eight subfamilies. We renamed all CsNPF genes according to the international nomenclature, based on their homology with AtNPF genes. By surveying the expression profiles of CsNPF genes in various tissues, we found that CsNPF6.4 was specifically expressed in roots, indicating that CsNPF6.4 may play a role in N absorption; CsNPF6.3 was highly expressed in petioles, which may be related to NO3- storage in petioles; and CsNPF2.8 was highly expressed in fruits, which may promote NO3- transport to the embryos. We further examined their expression patterns under different abiotic stress and nitrogen conditions, and found that CsNPF7.2 and CsNPF7.3 responded to salt, cold, and low nitrogen stress. Taken together, our study lays a foundation for further exploration of the molecular and physiological functions of cucumber nitrate transporters.

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Although recent physiological studies demonstrate that flue-cured tobacco preferentially utilizes nitrate ( NO 3 - ) or ammonium nitrate (NH4NO3), and possesses both high- and low-affinity uptake systems for NO 3 - , little is known about the molecular component(s) responsible for acquisition and translocation in this crop. Here we provide experimental data showing that NtNRT1.1B with a 1,785-bp coding sequence exhibited a function in mediating NO 3 - transport associated with tobacco growth on NO 3 - nutrition. Heterologous expression of NtNRT1.1B in the NO 3 - uptake-defective yeast Hp△ynt1 enabled a growth recovery of the mutant on 0.5 mM NO 3 - , suggesting a possible molecular function of NtNRT1.1B in the import of NO 3 - into cells. Transient expression of NtNRT1.1B::green fluorescent protein (GFP) in tobacco leaf cells revealed that NtNRT1.1B targeted mainly the plasma membrane, indicating the possibility of NO 3 - permeation across cell membranes via NtNRT1.1B. Furthermore, promoter activity assays using a GFP marker clearly indicated that NtNRT1.1B transcription in roots may be down-regulated by N starvation and induced by N resupply, including NO 3 - , after 3 days' N depletion. Significantly, constitutive overexpression of NtNRT1.1B could remarkably enhance tobacco growth by showing a higher accumulation of biomass and total N, NO 3 - , and even NH 4 + in plants supplied with NO 3 - ; this NtNRT1.1B-facilitated N acquisition/accumulation could be strengthened by short-term 15N- NO 3 - root influx assays, which showed 15%-20% higher NO 3 - deposition in NtNRT1.1B-overexpressors as well as a high affinity of NtNRT1.1B for NO 3 - at a K m of around 30-45 µM. Together with the detection of NtNRT1.1B promoter activity in the root stele and shoot-stem vascular tissues, and higher NO 3 - in both xylem exudate and the apoplastic washing fluid of NtNRT1.1B-transgenic lines, NtNRT1.1B could be considered as a valuable molecular breeding target aiming at improving crop N-use efficiency by manipulating the absorption and long-distance distribution/transport of nitrate, thus adding a new functional homolog as a nitrate permease to the plant NRT1 family.