The expression patterns and putative function of nitrate transporter 2.5 in plants

ABSTRACT

Nitrate transporter 2.5 (NRT2.5) was originally characterized as the transporter for nitrogen (N) limitation. In Arabidopsis, NRT2.5 is expressed mainly under extremely low NO₃⁻ and N starvation conditions, and this must work in conjunction with NAR2.1. NRT2.5 is expressed both in the roots and leaves in Arabidopsis, poplars, tea trees and cassava. This is also expressed in the seeds of Arabidopsis and wheat. In wheat, NRT2.5 is expressed in the embryo and shell and plays a role in the accumulation of NO₃⁻ in the seeds. In maize, this is also expressed in silk, cobs and tassel husk leaves. In rice, OsNRT2.5 (also known as OsNRT2.3a) may help the species to remove NO₃⁻ from the roots to shoots. In addition, NRT2.5 may interact with TGA3, MYC1, LBD37, LBD38, TaNAC2 and other transcription factors and participate in the transmission of NO₃⁻ signals. The present review summarizes the functions of NRT2.5 in different plant species, which may help plant breeders and molecular biologists to improve crop yield.

Abbreviations: NRT, Nitrate transporter; NUE, nitrogen use efficiency; PTR, peptide transporter; NPF, nitrate peptide transporter family; CLC, chloride channel; LAC1/SLAH, slow anion channel-associated 1 homolog 3; LATS, low-affinity transporter systems; HATS, high-affinity transport systems; NNP, nitrate-nitrite-porter; MFS, major facilitator superfamily.

KEYWORDS:

• Low nitrogen
• nitrate transporter
• nitrogen starvation
• NRT2.5

Introduction

Nitrogen (N) is the main component of proteins, nucleic acids and phospholipids, and these substances are important components of protoplasts, cell nuclei and biofilms. Therefore, N plays an extremely important role in life activities and is called the element of life. N affects physiological metabolic processes, such as photosynthesis, respiration and antioxidant system in plants.¹

N can be available to plant roots in several different forms, including nitrate (NO₃⁻), ammonium (NH₄⁺) and organic molecules such as amino acids. NO₃⁻ is one of the most important N sources for plants. Therefore, investigating the molecular mechanisms of NO₃⁻ utilization is critical for plants,² and this also provides a vital theoretical basis for the improvement of nitrogen use efficiency (NUE) in crops, thereby contributing to sustainable agriculture.

The concentration of NO₃⁻ in soils might extremely change from 10 µM in natural soils to 100 mM in fertilized soils.³ Plants have a sophisticated network of membrane transporters for the sensing, absorption, storage and distribution of NO₃⁻ inside the plant organism. It has been reported that NO₃⁻ transporters belong to four families, that is, NRT1/PTR/NPF (nitrate transporter 1/peptide transporter/nitrate peptide transporter family), NRT2, chloride channel (CLC) and slow anion channel-associated 1 homolog 3 (SLAC1/SLAH).⁴ NO₃⁻ is absorbed from the soil by plant roots and transported to the shoots, and reused between organs, tissues and cells. These processes are mediated by NRT1 and NRT2, coupled with proton (H⁺) transport, and subjected to the plasma membrane H⁺-ATPase-regulated active transport process.^5,6

According to the difference in NO₃⁻ concentration in the soil, plants have evolved two types of NO₃⁻ transport systems, namely, low-affinity transporter system (LATS) and high-affinity transport system (HATS). LATS is activated when the external NO₃⁻ content is high, while HATS is activated when the external NO₃⁻ content is low.⁶ There are different opinions about the critical point of the NO₃⁻ absorption mode transition. Tsay et al. suggested that when Km is within 10–300 μM, HATS works, and when Km is greater than 0.5 mM, LATS works.⁷ However, Ho et al. suggested that HATS work when Km is less than 50 μM, and LATS work when Km is greater than 5 mM.⁸

In Arabidopsis, the NRT1/PTR/NPF family consists of 53 members that are mainly characterized as low-affinity transporters, and seven members have been described in the NRT2 family characterized as high-affinity nitrate transporters.⁴9 The CLC family has seven members, and the SLAC/SLAH family has five members.⁴ Overall, 35 of these 72 genes have been characterized in detail, and 24 of these have been shown to transport NO₃−. However, only the NRT1/PTR/NPF and NRT2 families are involved in root N uptake. Nine NRT1 genes and seven NRT2 genes involved in NO₃⁻ transport have been identified and confirmed in Arabidopsis.¹⁰

High-affinity NRT2 transporters belong to the Nitrate-Nitrite Porter family (NNP),¹¹ which is a member of the Major facilitator superfamily (MFS).¹² The first high-affinity nitrate transporter gene, NrtA (originally called crnA), was isolated from a filamentous fungus, Aspergillus nidulans,^13,14 and the NRT2 genes, CrNR2.1, CrNR2.2 and CrNR2.3, in Chlamydomonas reinhardtii came afterward.¹⁵ The first Nrt2 genes isolated from plants were HvNRT2.1 and HvNRT2.2 from Hordeum vulgarela,¹⁶ followed by Nicotiana plumbaginifolia.¹⁷ Glicine max¹⁸ and Arabidopsis.^19,20

All NRT2s were high-affinity transporters and only used nitrate as a specific substrate. When the available nitrate was limited, the high-affinity transport system is activated and plays a leading role.¹¹ To date, in the model plant Arabidopsis thaliana, root NO₃⁻ uptake activities have been shown for four NRT2 transporters: NRT2.1, NRT2.2, NRT2.4 and NRT2.5.^21–24 In Arabidopsis, NRT2.1, NRT2.2 and NRT2.4 are localized in plasma membranes in the roots and show more than 87% amino acid similarity.^25,26 The fourth is NRT2.5, which displays only 65% amino acid similarity, compared with NRT2.1, NRT2.2 and NRT2.4.¹⁴ Under low N conditions, NRT2.1 and NRT2.2 play an important role in the NO₃⁻ uptake. The expression level would immediately increase after receiving the low N signal. NRT2.1 is the main force for nitrate uptake and transport in the roots, NRT2.2 displays expression patterns and properties similar to those of NRT2.1, and the NRT2.1 and NRT2.2 have complementary functions.^26–28 NRT2.4 and NRT2.5 are also involved in high-affinity nitrate uptake, but their contribution is only revealed under nitrogen starvation, and NRT2.4 transporter contributes to NO₃⁻ uptake at very low external NO₃⁻ concentrations. After a long period of starvation, the expression of NRT2.5 is highly induced, and this becomes the major transporter for high-affinity uptake.^23,24 NRT2.7 is specifically expressed in seeds and is the only NRT2 transporter located in tonoplast for loading NO₃⁻ into vacuoles.²⁶ Kotur et al. demonstrated that all (expect NRT2.7) members of the NRT2 family are capable of nitrate uptake in Xenopus oocytes when co‐expressed with NAR2.1.²⁹ Recent studies have suggested that the spatio-temporal distribution of these four AtNRT2 transporters is critical for the efficient NO₃⁻ uptake to sustain growth under low N availability for plants.

NRT2.5 belongs to the NRT2 family, which widely exists across all organisms, and is initially characterized as the transporter for N limitation.²⁴ NRT2.5 is expressed in the roots and shoots, especially in the roots, and is repressed by the provision of NO₃^−24,30 However, the function of NRT2.5 has not been reported in detail. The present study summarized the multiple functions of NRT2.5 in different species, including coping with N starvation, participation in NO₃⁻ transport in the phloem of buds, seed NO₃⁻ accumulation, NO₃⁻ signal transduction guidance, etc.

Bioinformatics analysis of NRT2.5

NRT2.5 has been reported in higher plants, algae, yeast and bacteria. NRT2 belongs to the nitrate-nitrite-porter (NNP) family and is one of the major facilitator superfamily (MFS) assisting transporters.¹² The structure of a typical NRT2 protein has 12 transmembrane helixes. Every six transmembrane helixes are a group, and these groups are connected by a large hydrophilic ring. There is a conserved domain between the 2 and 3 transmembrane helixes, and a conserved domain exists within the 4 and 5 transmembrane helixes.¹²

The NRT2.5 protein sequences of 24 species were queried in NCBI. The investigators used DNAman for the multi-alignment of the amino acid sequences of NRT2.5 in different species, 23 of these had conserved MFS and NNP domains (Figure 1), with the typical domains of the NRT2 family. The number of amino acids was between 450 aa and 520 aa, except for C. reinhardtii, which had 910 aa. The prediction of transmembrane domains was performed using the TMHMM predictor. It was found that the transmembrane domains of different species were basically between 10 and 12, except that C. reinhardtii has six transmembrane domains (Table 1). The phylogenetic tree based on the entire amino acid sequence was constructed using the neighbor-joining method through the MEGA5 software after the ClustalW alignment with 1000 bootstrap trials on 24 sequences (12 sequences of dicotyledons, five sequences of monocots, two fungi, two bacteria and three algae) (Figure 2). The phylogenetic analysis revealed that monocotyledonous plants and dicotyledonous plants have a common ancestor. However, the protein sequences were different and were clustered on the two major branches, except for Suaeda salsa. This indicates that the function of NRT2.5 in halophyte and non-halophyte may be different. The NRT2.5 among the fungi is similar, but the gap is large in algae. C. reinhardtii has a half-size transporter NRT2.5. The protein sequence of Zostera marina L. as the angiosperm also changed when the sequence was compared with algae (Figure 2), indicating that its function may change. These above results indicate the co-evolutionary origin of monocotyledonous and dicotyledonous plants. However, due to the differences in species and environments, the function changed after the long-term evolution in different species.

Table 1. Summary of identified NRT2.5 in plants

Gene name	Organism	NCBI reference sequence	Amino acid	Transmembrane	Spatial locations of gene expression	Function	Reference
AtNRT2.5	Arabidopsis thaliana	Q9LPV5.1	502	12	Primary and lateral root epidermal cells, leaf phloem parenchyma cell	Loading NO3^− into phloem of N-starved adult plants	^Citation23,Citation24,Citation30
TaNRT2.5	Triticum urartu	EMS50263.1	514	12	Root, leaf, embryo, shell	Increases seed vigor, grain nitrate accumulation and yield	^Citation31–33
ZmNRT2.5	Zea mays	AQK97719.1	520	12	Root, silk. leave, cobs, tassel husk leave	Influence in the distribution of N during grain filling period	^Citation34–36
OsNRT2.3a	Oryza sativa	sp|Q94JG1-1	516	12	Root	Root to shoot NO3^− transport	^Citation37–39
OsNRT2.3b		sp|Q94JG1-2	486	11	Root, leave	Enhancing pH homeostasis; N, P uptake; grain yield and NUE
SsNRT2.5	Suaeda salsa	Unknown	508	12	leave	Unknown	^Citation40
ZmNRT2.5	Zostera marina L.	KMZ59016	517	12	Unknown	Na^+-dependent high-affinity nitrate transporter	^Citation41
CrNRT2.5	Chlamydomonas reinhardtii	AME17970.1	910	6	all	Unknown	^Citation42
PttNRT2.5A	Populus trichocarpa	XP_024437706.1	479	11	bark	High affinity transporter of nitrate in the bark	^Citation43,Citation44
PttNRT2.5B		XP_002321622.2	508	12	All vegetative organs	Unknown
PttNRT2.5 C		XP_002321624.2	508	12	Unknown	Unknown
CsNRT2.5	Camellia sinensis	Unknown	454	12	Adult leaf, root	Unknown	^Citation45
MeNRT2.5	Manihot esculenta Crantz	Unknown	492	10	Root, stem, leaf, flower	Unknown	^Citation46

Figure 1. Multi-alignment of NRT2.5 amino acid sequences in different plants. On the left is the MFS-conserved motif (MFS: G-XXX-D-XX-G-X-R), On the right is the nitrate/nitrite transporters family-conserved motif (NNP: G-W/L-G-N-M/A-G)

Figure 2. Phylogenetic analysis of NRT2.5 proteins. The amino acid sequences were aligned using ClustalW software and the phylogeny constructed using the neighbor-joining method with 1000 replicates through MEGA5 software. Database accession numbers of the amino acid sequences are as follows: Arabidopsis thaliana (OAP19210.1), Symbiodinium microadriaticum (OLQ11495.1), Ectocarpus siliculosus (CBJ31724.1), Physcomitrella patens (BAE45929.1), Chlamydomonas reinhardtii (AME17970.1), Hordeum vulgare (KAE8819762.1), Malus hupehensis (ACN22073.1), Tetrabaena socialis (PNH10816.1), Chrysanthemum ×morifolium (AMR68973.1), Gossypium arboretum (KHG03895.1), Suaeda salsa, Zostera marina L. (KMZ59016), Oryza sativa (OsNRT2.3a (sp|Q94JG1-1), OsNRT2.3b (sp|Q94JG1-2)), Lactuca sativa (XP_023762233.1), Triticum urartu (EMS50263.1), Glycine soja (KHN08612.1), Zea mays (AQK97719.1), Gossypium arboretum (XP_017636365.1), Citrus clementine (XP_024042199.1), Populus trichocarpa (A (XP_024437706.1) B (XP_002321622.2) C (XP_002321624.2)), Cucumis sativus (NP_001295862.1), Brachypodium distachyon (XP_003569637)

NRT2.5 function in Arabidopsis thaliana

In Arabidopsis, NRT2.5 is a high-affinity transporter encoded by a single gene, and AtNRT2.5 was found on isolated BACs of chromosomes I in the first half of the chromosome.²⁵

The expression and subcellular localization of AtNRT2.5

NRT2.5 is the most abundantly expressed gene in the NRT2 family in the roots and leaves and is strongly induced by N starvation in Arabidopsis.^24,47,48

The subcellular localization of NRT2.5-GFP revealed that AtNRT2.5 is localized in the plasma membrane.²⁴ In the spatial expression pattern induced by N starvation, GUS staining in the ProNRT2.5: GUS and ProNRT2.5: GUS: NRT2.5–3ʹUTR present in the root hair zone of the primary roots and lateral roots of transgenic plants.²⁴ A cross-section of the lateral root revealed that the expression was mainly located in the epidermis and the cortex, while GUS staining was limited to higher-order veins in the shoots.²⁴

AtNRT2.5 transporters is involved in NO₃⁻ uptake

AtNRT2.5 was upregulated in WT, and the atnrt2.1–1 mutant and atnar2.1–1 mutant under low N conditions (0.2 mM NO₃⁻).^47,48 When AtNRT2.5 was overexpressed in the atnrt2.1–1 mutant, part of the mutant’s ability to absorb NO₃⁻ could be restored, while part of the phenotype could be restored.²⁴ This result was also confirmed in functional studies in Xenopus oocytes.^29,49 These above results show that AtNRT2.5 plays a role in absorbing NO₃⁻.

Okamoto et al. classified the identified NRT genes in Arabidopsis into NO₃⁻ inducible, NO₃⁻ inhibitory and NO₃⁻ constitutive types.⁵⁰ The expression of the AtNRT2.5 gene was inhibited by the high concentration of NO₃⁻. After several hours of supplying NO₃⁻, its expression in the root was only 25%.^25,50,51 This means that AtNRT2.5 is a high-affinity transporter.

HATS is divided into two types: constitutive (cHATS) and inductive (iHATS). The cHATS can be expressed in the absence of NO₃⁻ in the medium, while iHATS can be expressed only in the presence of NO₃⁻ induced in the medium.⁶ At the same time, Kotur et al. reported that NO₃⁻ influx was significantly reduced in the Atnrt2.5 mutant (GK-213H10), and that the complementation of the Atnrt2.5 mutant and AtNRT2.5 cDNA led to a statistically significant increase in cHATS.³⁰ This shows that AtNRT2.5 represents the major component of cHATS influx, with the primary responsibility for NO₃⁻ uptake at low NO₃⁻ concentrations in nitrate-deprived plants.³⁰

Kotur et al. demonstrated that AtNRT2.5 is mainly expressed in the roots of nitrate-deprived WT plants and is only a 150 kDa molecular complex with AtNAR2.1.³⁰ In addition, Kotur et al. reported that the NO₃⁻ transport activity of NRT2.5 in oocytes was stimulated by the co-expression of NAR2.1 (NRT3.1).²⁹ Similarly, the activity of NRT2.1 depends on the presence of NAR2.1.^48,52 During the long-term N starvation, the expression of NAR2.1 follows that of NRT2.5, suggesting that NRT2.5 activity depends on NAR2.1.²⁴

The expression pattern of NRT2.5 in response to N starvation

Under N starvation, AtNRT2.5 is mainly expressed in adult plants, rather than in young seedlings.²⁴ NRT2.5 transcripts are mainly present in the roots, especially in the epidermal cells of primary and lateral roots, and are also expressed in the buds, mainly in the parenchymal cells of the leaf phloem,²⁴ which confirms the earlier observations.^25,50 Interestingly, Kechid et al. reported that the NRT2.5 expression in the buds was higher than that in the roots.⁵¹ In contrast, in young primary and lateral root epidermal cells, the AtNRT2.5 transcription levels during N deprivation continued to increase over time.^23,24,30 These spatial expression patterns indicate that AtNRT2.5 is responsible for the NO₃⁻ uptake from the soil, and this is predominantly dominant in adult plants during N deprivation.^23,24 The expression of NRT2.5 in the roots and shoots continued to increase from days two to 10 during the N starvation and became the highest among the analyzed NRT2 family member transcript in adult plants. However, after 10 d, the expression in the shoots reached far lower levels, when compared with that in the roots.²⁴ NRT2.5 was downregulated by exposure to NO₃⁻ after 3 h in the roots, and NO₃⁻ induction had little effect on the NRT2.5 expression in the shoot tissue.³⁰

Kechid et al. reported that Atnrt 2.5 had no difference in root weight and root growth, when compared to WT, but had a 23% reduction in root growth under low N conditions, indicating that NO₃⁻ deprivation was associated with the disruption in AtNRT2.5 and resulted in the increase in root to shoot ratio.^30,51

The plant growth of the nrt2.4 nrt2.5 double mutants also remained unaltered, but the NO₃⁻ content in the phloem sap strongly decreased and reached approximately 20% of the WT.^24,30 This indicates that the NO₃⁻ levels in the phloem are not limiting for the plant adaptation to N starvation conditions. The loss of function of NRT2.4 and NRT2.5 led to a sharp decrease in the NO₃⁻ content in the phloem exudates of the N-starved double mutant, which is specific for NO₃⁻ in phloem secretions. This occurred because the levels of amino acids in the phloem exudates and the whole leaf NO₃⁻ levels were unchanged in all mutant lines when compared with the WT.²³ It was demonstrated that NRT2.4 and NRT2.5 are complementarily involved in the phloem NO₃⁻ transport in the shoots.^23,24

The fresh weight of the triple mutant nrt2.1-nrt2.2-nrt2.5 was reduced to approximately 10% of the WT. However, the seedling weight of the nrt2.1-nrt2.2-nrt2.4-nrt2.5 tetraploid mutant was significantly reduced to 2%, and the triple mutant nrt2.1–2 nrt2.5 and quadruple mutant displayed that HATS activity decreased to 7% and 3%, respectively, when compared with the WT. However, no significant further reduction was observed for the triple mutant nrt2.1–2 nrt2.4–1, when compared to the double mutant nrt2.1–2.²⁴ At a low external NO₃⁻ concentration, the simultaneous loss of NRT2.5, NRT2.1 and NRT2.2 functions led to the further reduction in NO₃⁻ uptake and affected the growth of the shoots.²⁴ This finding led to the conclusion that the interplay between NRT2.1, NRT2.2 and NRT2.5 was required for the optimal adaptation to N limitation in plants.

NRT2.5 functions beyond nitrate transporter

Clearly, the function of AtNRT2.5 is not limited to the NO₃⁻ influx associated with cHATS activity. Kechid et al. reported that AtNRT2.5 and AtNRT2.6 play an essential role in plant growth regulation in response to the growth promoted by the rhizospheric bacterium Phyllobacterium  brassicacearum. Interestingly, this effect was NO₃⁻ independent.⁵¹

Todd et al. reported that the expression of MYB-like genes in Arabidopsis under nitrogen and phosphorus stresses decreased, when compared to the expression of AtNRT2.5, in the WT and pho2 mutants, suggesting that AtNRT2.5 may be associated with nitrogen and phosphorus interactions.⁵³

Chopin et al. studied the expression of NRT2 family members in Arabidopsis dry seeds, found only AtNRT2.7 and AtNRT2.5, and that this reaches only 6% of the EF1α (control gene) level of ATNRT2.5, while the former gene expressed 20 times more strongly than the latter. During the seed imbibition, the expression of both NRT2 genes decreased and became hardly detectable for ATNRT2.5. However, the role of AtNRT2.5 in dried seeds has not been well studied.²⁶ It was also reported that AtNRT2.5 is both induced in sugar-regulated senescence and during developmental leaf senescence.⁵⁴ Therefore, NRT2.5 may not only express in the roots and shoots in response to N starvation but also express in the seeds, and that this may be correlated to the N and P interactions. However, the specific roles of NRT2.5 have not been clearly studied.

NRT2.5 function in maize

Plett et al. divided the NRT genes of maize (Zea mays) into NRT1, NRT2 and NRT3 (NAR2) by dichotomy after sequencing the maize genome sequence. Four ZmNRT2 genes have been identified in the maize genome: ZmNRT2.1, ZmNRT2.2, ZmNRT2.3 and ZmNRT2.5.¹⁰

The research on ZmNRT2.5 has mainly focused on the transcript levels, and there has been no specific functional research. ZmNRT2.5 was the only NRT2 that was upregulated under low N conditions, supporting its possible participation in response to low N limitation.^24,34,55

The transcript levels for ZmNRT2.5 were the most N decrease responsive, and the ZmNRT2.5 expression was detectable only in plants grown after low N treatment, which had significant changes throughout its life cycle. ZmNRT2.5 reached its peak transcription level on the 25th d during N starvation.⁵⁵ The ZmNRT2.5 transcript was differently expressed after 2 h of exposure to NO₃⁻³⁵ Under 0.5 mM of NO₃⁻, ZmNRT2.5 revealed a peak expression after 29 d, which was very similar to the peak of corn NO₃⁻ uptake capacity.⁵⁶ In conclusion, ZmNRT2.5 was detected only under N starvation or low N condition, suggesting that this may play an important role in absorbing NO₃⁻³⁵ under low NO₃⁻ conditions.

Studies on ZmNRT2.5 revealed that in addition to its expression in the roots, it was also expressed during the vegetative stage throughout the growth cycle.^36,55 At the reproductive stage, no transcript could be detected in the roots or silks.³⁶ The gene was expressed in the leaves, cobs and tassel, and its expression was significantly higher in the husk leaves.³⁶ ZmNRT2.5 was the only putative NO₃⁻ transporter expressed at high levels in the husk leaves.³⁶ Since husk leaves play a central role in the distribution of N during the grain filling period, the role of ZmNRT2.5 in this process needs further study.³⁶

NRT2.5 function in wheat

To date, five nitrate transporter genes (TaNRT2.1, TaNRT2.2, TaNRT2.3, TaNRT2.4 and TaNRT2.5) have been found in wheat (Triticum urartu).^57–59 It has been reported that TaNRT2.5 not only played a role in N starvation but also played a role in seed NO₃⁻ accumulation.

In Xenopus oocyte assay, TaNRT2.5 requires the partner protein TaNAR2.1 to present NO₃⁻ transport activity.³¹ The co-localization of TaNRT2.5–3B: GFP and TaNAR2.1–6B: mRFP in the tobacco leaf transient expression system has proven that this was localized on the tonoplast. When TaNRT2.5–3B: GFP was expressed alone, the GFP signal could not be detected under the same microscope conditions, and the TaNRT2.5–3B protein was unstable.³¹ These results suggest that TaNRT2.5 might cooperate with TaNAR2.1 to play a role in NO₃⁻ uptake after anthesis. This shows that TaNRT2.5–3B plays a role in NO₃⁻ transport to the vacuole and affects intracellular NO₃⁻ distribution. Therefore, this presents an indirect function in NO₃⁻ acquisition from the soil.³¹

N starvation greatly inhibits the growth of wheat seedlings and decrease in protein and NO₃⁻ contents. The changes in phenotypic and physiological parameters may be correlated to the altered transcription levels of TaNRT1 and TaNRT2 family genes.⁵⁹ In the study of TaNRT2.5 in wheat, it was found that the transcription level of TaNRT2.5 in the roots of the control wheat seedlings (normal N) remained almost stable. However, during N starvation in wheat seedlings, the transcription level of the TaNRT2.5 gene was strongly induced throughout the N starvation process.⁵⁹ In contrast, for TaNRT2.5 in the roots, an upregulation was observed under N starvation, followed by a decline under NO₃⁻ resupply, indicating that TaNRT2.5 is a nitrate-starvation-inducible gene.⁶⁰ In response to N starvation, TaNRT2.5 plays a more important role in wheat seedlings than in other NO₃⁻ transporters.^59,60

In addition to the role of NO₃⁻ acquisition during N starvation, TaNRT2.5 was also expressed in leaf sheath, peduncle and developing seeds, especially in the husk and embryo.^59,60 It was observed that the expression of TaNRT2.5 in the roots was much higher at the grain filling stage when compared with the seedling growth stage.^31,59,60 The TaNRT2.5–3B overexpression promoted seed germination and increased the grain NO₃⁻ concentration, seed vigor and seed yield, while the RNA interference of TaNRT2.5 had the opposite effects.³¹ The TaNRT2.5 transgenic lines altered the TaAMY and TaUGPase expression and decreased in the RNAi lines, which may affect the seed germination rate.^31–33

In conclusion, TaNRT2.5 not only plays an important role during N starvation but also plays a specific role in seed NO₃⁻ accumulation, which is an important signal for seed vigor and crop establishment.

NRT2.5 function in rice

Five NRT2 genes have been identified in the rice genome. Yan et al. analyzed the rice nitrate transporters in EST, DDBJ and NCBI, and the results revealed that OsNRT2.3 has two transcripts, OsNRT2.3a and OsNRT2.3b, with 94.2% similarity in their putative amino acid sequences.^37,38 The bioinformatics analysis of the closest relationship between OsNRT2.3a/b and AtNRT2.5 revealed that OsNRT2.3a may be an ortholog of AtNRT2.5.³⁹

However, only one of the two splice variants, OsNRT2.3a in rice, requires an interaction with OsNAR2.1 to mediate the NO₃⁻ uptake, and the absorption of NO₃⁻ by OsNRT2.3b is affected by pH.^38,39,61,62 Indeed, when NAR2 was co-expressed in Xenopus oocytes, the nitrate transport activities of OsNRT2.3a were greatly enhanced. Nevertheless, NAR2 is not required for the nitrate transport activities of OsNRT2.3b.^37,39 In rice, the orthologous gene OsNRT2.5 (also known as OsNRT2.3a) is mainly expressed predominantly in xylem parenchyma cells of the root stele and has been demonstrated to play a role in the transport of NO₃⁻ from the roots to the shoots, again under low NO₃⁻ conditions that are induced by NO₃⁻ and sucrose, and inhibited by ammonium ion and aspartic acid^37,38,39,, There was no significant difference in phenotype with the overexpression of OsNRT2.3a alone. However, the OsNRT2.3b overexpression was obviously higher and bigger than WT.³⁹ In terms of function, OsNRT2.3a plays a key role in the long-distance nitrate transport from the root to the shoot at a low nitrate supply level. Furthermore, the overexpression of rice OsNRT2.3b not only enhanced the NO₃⁻ uptake but also improved the yield and nitrogen use efficiency (NUE) under both low and high nitrogen conditions in the field.^39,62 Meanwhile, OsNRT2.3b can improve the P uptake and accumulation in rice.⁶²

The differential transcriptome of rice in response to N supply revealed that under low NO₃⁻, the nitrate transporter gene OsNRT2.5 was upregulated, while under high NO₃⁻, OsNRT2.5 was downregulated.⁶³ The characterization shows that OsNRT2.5 has a specific role in transporting NO₃⁻ from the roots to the shoots in rice, and that this is different from NRT2.5 in Arabidopsis and wheat.^24,31,39

NRT2.5 function in other plants

Poplar (Populus trichocarpa) NRT2.5 is coded by three genes. Poplar NRT2.5A is expressed in the bark, and NRT2.5B is expressed in the leaves, while the expression of NRT2.5C is unknown.⁴³ In Populus tremula × tremuloides, PttNRT2.5B expression occurred in all investigated vegetative poplar organs and was the highest in fine roots.⁴⁴ Therefore, the strong PttNRT2.5B expression in the roots at low external NO₃⁻ concentrations could not be explained by N starvation. PttNRT2.5B function within the poplar roots remains obscure and need to be further analyzed in future studies.⁴⁴

Feng et al. reported that the CsNRT2.5 gene was expressed in different tissues of tea tree (Camellia sinensis), and the expression level was lower in the shoots and stems, and higher in mature leaves and roots, when induced by NO₃⁻⁴⁵

Under low concentrations of NO₃⁻, the cassava (Manihot esculenta Crantz) MeNRT2.5 gene can reach its peak within a short time in the roots, and it was speculated that MeNRT2.5 was the same as genes such as AtNRT2.1 as a NO₃⁻ receptor. However, this needs further experimental proof.⁴⁶

The function of NRT2.5 under salinity

Seagrasses are the only group of angiosperms that evolved from land plants to complete their life cycle submerged in marine environments. As far as NO₃⁻ is concerned, the concentration of NO₃⁻ in the seagrass environment is as low as 5 µM, suggesting that high-affinity transporters developed by terrestrial plants would work in seagrass, facing the challenge of salinity and alkaline conditions.⁶⁴ In terrestrial angiosperms, NRT2 encodes a high-affinity NO₃⁻ transporter that acts as an H⁺ cotransporter, but Zostera marina L. may use a Na⁺ -dependent high-affinity nitrate transporter to acquire NO₃⁻. Interestingly, only one gene (Zosma70g00300.1; NRT2.1) in the chlorella genome has annotated this function. The analysis of this sequence predicted the presence of 12 transmembrane domains, including the MFS domain of the NNP transporter family and the “nitrate tag” that appears in all members of the NNP family.⁴¹ The phylogenetic analysis revealed that the sequence had a greater relationship with NRT2.5 than with NRT2.1 and shared common ancestors with monocotyledonous and dicotyledonous plants.⁴¹ The phylogenetic analysis revealed that there was only one NRT2 gene in the Z. marina genome, which was most relevant to AtNRT2.5. This finding supports the idea that ZosmaNRT2 encodes a high-affinity NO₃⁻ transporter that localized on the plasma membrane.⁴¹ ZosmaNRT2 also requires the interaction of NAR2 to mediate NO₃⁻ transport, and the presence of NAR2 is important to stabilize the ZosmaNRT2 protein at the plasma membrane.⁴¹ These molecular data, together with the previous electrophysiological results, support that ZosmaNRT2 would have evolved to use Na⁺ as a driving ion, which could provide important insights for NO₃⁻ uptake in plants under alkaline, NO₃^– limited and salinized environments.⁶²

S. salsa is an annual herbaceous halophyte with high salt tolerance during reproductive^65–67 seed germination^68–70 and seedling stages.^71–73 The species occurs in both intertidal zones and inland saline sites in China.^74–77 Under the growth conditions of 200 mM of NaCl, and compared with the inland population of S. salsa, the expression of nitrate transporter 2.5 (SsNRT2.5) in the old and mature leaves of the intertidal population was significantly upregulated during N starvation. Therefore, SsNRT2.5 may play an important role in NO₃⁻ migration during N starvation, and this trait may make S. salsa adapts to changeable low nitrogen environments.⁴⁰ However, further investigations are needed to determine whether SsNRT2.5 plays a role in NO₃⁻ absorption during N starvation.

Transcription factors participate in modulating the expression levels of NRT2.5

At present, few transcription factors involved in NO₃⁻ signaling with NRT2.5 were reported. Ruffel et al. identified two putative transcription factors, TGA3 and MYC1, which are NRT2.5 regulators that respond to N or C signals in Arabidopsis.⁷⁸ The functional analysis of mutants combined with yeast one-hybrid experiments confirmed that both transcription factors can bind to the promoter of NRT2.5, in response to N or C signals.⁷⁸

The transcription factors, LBD37 and LBD38, also regulate NO₃⁻ response genes. Rubin et al. reported that LBD37 and LBD38 are negative regulators of NO₃⁻, which act as regulatory genes in N-free conditions.^79,80

The nitrate-induced NAC transcription factor TaNAC2-5A controls the NO₃⁻ signaling in wheat.⁸¹ ChIP-Seq and EMSA identified the downstream genes of TaNAC2, including TaNRT2.5–3B.³¹ TaNAC2 could bind to the promoter of TaNRT2.5–3B and promote the expression of TaNRT2.5, which plays a key role in seed vigor.^31,81 TaNAC2 is the transcription factor that regulates the “workhorse” TaNRT2.5 transporter, which drives NO₃⁻ accumulation and thereby seedling vigor.³¹ The TaNAC2-NRT2.5 module plays a key role in regulating the accumulation of NO₃⁻ particles and seed vigor.³¹

Concluding remarks and perspectives

The evolutionary analysis of 12 dicotyledons and five monocotyledons in the present study also obtained the same results, including the halophyte S. salsa and Z. marina (Figure 2). The most highly conserved regions of the NRT2.5 transporters are found within the predicted transmembrane domains, the NNP and MFS domain, indicating that the role of the NRT2.5 generally appears to be the NO₃⁻ and NO₂⁻ influx.¹¹

Monocotyledonous and dicotyledonous plants have different nitrogen-responsive expression patterns, leading to changes in localization. For example, in Arabidopsis, Poplar, tea tree and cassava, NRT2.5 is expressed in the roots and leaves, only in the seeds of Arabidopsis, but the expression level is extremely low. However, in monocotyledons, in addition to the roots and leaves, this was also expressed in the embryo and shell in wheat, and in silk, cobs and tassel husk leaves in maize (Table 1). In Arabidopsis, NRT2.5 mainly responds to N starvation and N absorption under very low nitrogen conditions, but it must work in conjunction with NAR2.1.^23,24,30 However, in Poplar, the NRT2.5B expression appears in all investigated vegetative organs. Hence, N starvation cannot be used to explain the strong expression of NRT2.5B in the roots at low external NO₃⁻ concentrations.^43,44 In monocotyledons, in addition to responding to N starvation, TaNRT2.5 also plays a role in seed NO₃⁻ accumulation in wheat,^31,59 and NRT2.5 in bract leaves may play a central role in N distribution during grain filling in maize.³⁶ This means that the function of NRT2.5 between monocotyledonous and dicotyledonous plants should be different. Interestingly, in halophytes such as S. salsa and Z. marina, the function of NRT2.5 may have a certain relationship with Na⁺, but the specific function of NRT2.5 in halophytes needs to be further investigated.^40,41

In summary, NRT2.5 not only responds to N starvation and NO₃⁻ absorption but also participates in the accumulation of NO₃⁻ in seeds and plays a role in seed vitality and NO₃⁻ distribution in the reproductive stage. The above results indicate that there are still many unknown functions of NRT2.5 that have not yet been tapped, and these functions may play roles in improving crop yield and participating in stress tolerance. Therefore, the physiological and molecular mechanisms of NRT2.5 involved in NO₃⁻ absorption and distribution, as well as adaptation to stress environments, such as salinity, are worthy of in-depth research.

Author contributions

Ranran Liu wrote the manuscript. Ting Jia and Bing Cui assisted in revising the manuscript. Jie Song revised and gave final approval of the manuscript.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China [U1803233].