TaNAR2.1 and TaNAR2.2 differ in influencing nitrogen uptake and growth of wheat (Triticum aestivum L.).

NAR2 (Nitrate assimilation related protein) is a protein chaperone involved in transporting nitrate across membranes. However, the expression pattern and function of NAR2 genes in wheat are still largely unknown. Here, we cloned two TaNAR2 genes (TaNAR2.1 and TaNAR2.2). To assess and compare the functional differences of TaNAR2.1 and TaNAR2.2, we analyzed the subcellular localization and expression pattern of the two genes in wheat under low nitrogen (LN) and high nitrogen (HN) conditions, as well as the nitrate influx and root system architecture of TaNAR2.1 and TaNAR2.2 overexpression wheat under LN and HN. Additionally, we investigated the effects of TaNAR2.1 and TaNAR2.2 overexpression on the growth phenotype, nitrogen uptake and yield of wheat throughout the growth period. There are significant differences in the expression patterns and functions of TaNAR2.1 and TaNAR2.2. TaNAR2.1 is located in the cytoplasm, nucleus and the plasma membrane, whereas TaNAR2.2 is a cytoplasm-specific protein. TaNAR2.1 appears to exhibit larger changes in expression levels and a higher capacity for nitrate influx than TaNAR2.2 under external nitrate supply. Overexpression of TaNAR2.1 significantly improves grain nitrogen use efficiency and increases grain yield, whereas overexpression of TaNAR2.2 enhances vegetative and reproductive growth of wheat roots. These findings indicate that TaNAR2.1 plays a crucial role in wheat nitrogen accumulation and yield, while TaNAR2.2 is pivotal for wheat root growth.

[Identification, expression and DNA variation analysis of high affinity nitrate transporter NRT2/3 gene family in Sorghum bicolor].

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.

Identity and functional characterisation of the transporter supporting the Na+ -dependent high-affinity NO3 - uptake in Zostera marina L.

Zostera marina is a seagrass, a group of angiosperms that evolved from land to live submerged in seawater, an environment of high salinity, alkaline pH and usually very low NO3 - . In 2000, we reported the first physiological evidence for the Na+ -dependent high-affinity NO3 - uptake in this plant. Now, to determine the molecular identity of this process, we searched for NO3 - transporters common to other vascular plants encoded in Z. marina's genome. We cloned two candidates, ZosmaNPF6.3 and ZosmaNRT2 with its partner protein ZosmaNAR2. ZosmaNAR2 expression levels increase up to 4.5-fold in Z. marina leaves under NO3 - -deficiency, while ZosmaNRT2 and ZosmaNPF6.3 expressions were low and unaffected by NO3 - . NO3 - transport capacity, kinetic properties and H+ or Na+ -dependence were examined by heterologous expression in the Hansenula polymorpha high-affinity NO3 - transporter gene disrupted strain (∆ynt1). ZosmaNPF6.3 functions as a H+ -dependent NO3 - transporter, without functionality at alkaline pH and apparent dual kinetics (KM = 11.1 µM at NO3 - concentrations below 50 µM). ZosmaNRT2 transports NO3 - in a H+ -independent but Na+ -dependent manner (KM = 1 mM Na+ ), with low NO3 - affinity (KM = 30 µM). When ZosmaNRT2 and ZosmaNAR2 are co-expressed, a Na+ -dependent high-affinity NO3 - transport occurs (KM = 5.7 µM NO3 - ), mimicking the in vivo value. These results are discussed in the physiological context, providing evidence that ZosmaNRT2 is a Na+ -dependent high-affinity NO3 - transporter, the first of its kind to be functionally characterised in a vascular plant, that requires ZosmaNAR2 to achieve the necessary high-affinity for nitrate uptake from seawater.

Identification, expression and DNA variation analysis of high affinity nitrate transporter NRT2/3 gene family in Sorghum bicolor / 生物工程学报

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.

EgNRT2.3 and EgNAR2 expression are controlled by nitrogen deprivation and encode proteins that function as a two-component nitrate uptake system in oil palm.

Oil palm (Elaeis guineensis Jacq.) is an important crop for oil and biodiesel production. Oil palm plantations require extensive fertilizer additions to achieve a high yield. Fertilizer application decisions and management for oil palm farming rely on leaf tissue and soil nutrient analyses with little information available to describe the key players for nutrient uptake. A molecular understanding of how nutrients, especially nitrogen (N), are taken up in oil palm is very important to improve fertilizer use and formulation practice in oil palm plantations. In this work, two nitrate uptake genes in oil palm, EgNRT2.3 and EgNAR2, were cloned and characterized. Spatial expression analysis showed high expression of these two genes was mainly found in un-lignified young roots. Interestingly, EgNRT2.3 and EgNAR2 were up-regulated by N deprivation, but their expression pattern depended on the form of N source. Promoter analysis of these two genes confirmed the presence of regulatory elements that support these expression patterns. The Xenopus oocyte assay showed that EgNRT2.3 and EgNAR2 had to act together to take up nitrate. The results suggest that EgNRT2.3 and EgNAR2 act as a two-component nitrate uptake system in oil palm.

NAR2.1/NRT2.1 functional interaction with NO3(-) and H(+) fluxes in high-affinity nitrate transport in maize root regions.

Spatial and temporal fluctuations in nitrate (NO3(-)) availability are very common in agricultural soils. Therefore, understanding the molecular and physiological mechanisms involved in regulating NO3(-) uptake in regions along the primary root is important for improving the NO3(-) uptake efficiency (NUpE) in crops. Different regions of maize primary root, named R1, R2 and R3, NO3(-) starved for 3 days, were exposed to 50 µM NO3(-). Electrophysiological measurements (membrane potential and H(+) and NO3(-) fluxes) and NPF6.3, NRT2.1, NAR2.1, MHA1, MHA3 and MHA4 gene expression analyses were carried out. The results confirmed variable spatial and temporal patterns in both NO3(-) and H(+) fluxes and gene expression along the primary maize root. A significant correlation (P = 0.0023) between nitrate influx and gene transcript levels was observed only when NAR2.1 and NRT2.1 co-expression were considered together, showing for the first time the NRT2.1/NAR2.1 functional interaction in nitrate uptake along the root axis. Taken together these results suggest differing roles among the primary root regions, in which the apical part seem to be involved to sensing and signaling in contrast with the basal root which appears to be implicate in nitrate acquisition.

A 150 kDa plasma membrane complex of AtNRT2.5 and AtNAR2.1 is the major contributor to constitutive high-affinity nitrate influx in Arabidopsis thaliana.

In plants that have been deprived of nitrate for a significant length of time, a constitutive high-affinity nitrate transport system (cHATS) is responsible for initial nitrate uptake. This absorbed nitrate leads to the induction of the major nitrate transporters and enzymes involved in nitrate assimilation. By use of (13) NO3 (-) influx measurements and Blue Native polyacrylamide gel electrophoresis we examined the role of AtNRT2.5 in cHATS in wild type (WT) and various T-DNA mutants of Arabidopsis thaliana. We demonstrate that AtNRT2.5 is predominantly expressed in roots of nitrate-deprived WT plants as a 150 kDa molecular complex with AtNAR2.1. This complex represents the major contributor to cHATS influx, which is reduced by 63% compared with WT in roots of Atnrt2.5 mutants. The remaining cHATS nitrate influx in these mutants is due to a residual contribution by the inducible high-affinity transporter encoded by AtNRT2.1/AtNAR2.1. Estimates of the kinetic properties of the NRT2.5 transporter reveal that its low Km for nitrate makes this transporter ideally suited to detect and respond to trace quantities of nitrate in the root environment.

Identification and functional assay of the interaction motifs in the partner protein OsNAR2.1 of the two-component system for high-affinity nitrate transport.

A partner protein, NAR2, is essential for high-affinity nitrate transport of the NRT2 protein in plants. However, the NAR2 motifs that interact with NRT2s for their plasma membrane (PM) localization and nitrate transporter activity have not been functionally characterized. In this study, OsNAR2.1 mutations with different carbon (C)-terminal deletions and nine different point mutations in the conserved regions of NAR2 homologs in plants were generated to explore the essential motifs involved in the interaction with OsNRT2.3a. Screening using the membrane yeast two-hybrid system and Xenopus oocytes for nitrogen-15 ((15)N) uptake demonstrated that either R100G or D109N point mutations impaired the OsNAR2.1 interaction with OsNRT2.3a. Western blotting and visualization using green fluorescent protein fused to either the N- or C-terminus of OsNAR2.1 indicated that OsNAR2.1 is expressed in both the PM and cytoplasm. The split-yellow fluorescent protein (YFP)/BiFC analyses indicated that OsNRT2.3a was targeted to the PM in the presence of OsNAR2.1, while either R100G or D109N mutation resulted in the loss of OsNRT2.3a-YFP signal in the PM. Based on these results, arginine 100 and aspartic acid 109 of the OsNAR2.1 protein are key amino acids in the interaction with OsNRT2.3a, and their interaction occurs in the PM but not cytoplasm.