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. 2022 Jul 3;12:11227. doi: 10.1038/s41598-022-15202-w

Genome-wide identification and in silico analysis of NPF, NRT2, CLC and SLAC1/SLAH nitrate transporters in hexaploid wheat (Triticum aestivum)

Aman Kumar 1, Nitika Sandhu 1,✉, Pankaj Kumar 1, Gomsie Pruthi 1, Jasneet Singh 1, Satinder Kaur 1, Parveen Chhuneja 1
PMCID: PMC9250930  PMID: 35781289

Abstract

Nitrogen transport is one of the most important processes in plants mediated by specialized transmembrane proteins. Plants have two main systems for nitrogen uptake from soil and its transport within the system—a low-affinity transport system and a high-affinity transport system. Nitrate transporters are of special interest in cereal crops because large amount of money is spent on N fertilizers every year to enhance the crop productivity. Till date four gene families of nitrate transporter proteins; NPF (nitrate transporter 1/peptide transporter family), NRT2 (nitrate transporter 2 family), the CLC (chloride channel family), and the SLAC/SLAH (slow anion channel-associated homologues) have been reported in plants. In our study, in silico mining of nitrate transporter genes along with their detailed structure, phylogenetic and expression analysis was carried out. A total of 412 nitrate transporter genes were identified in hexaploid wheat genome using HMMER based homology searches in IWGSC Refseq v2.0. Out of those twenty genes were root specific, 11 leaf/shoot specific and 17 genes were grain/spike specific. The identification of nitrate transporter genes in the close proximity to the previously identified 67 marker-traits associations associated with the nitrogen use efficiency related traits in nested synthetic hexaploid wheat introgression library indicated the robustness of the reported transporter genes. The detailed crosstalk between the genome and proteome and the validation of identified putative candidate genes through expression and gene editing studies may lay down the foundation to improve nitrogen use efficiency of cereal crops.

Subject terms: Genetics, Molecular biology, Plant sciences, Structural biology

Introduction

Nitrogen is one of the essential elements required by plants. It is a constituent of nucleic acids, amino acids and proteins and therefore is of great importance in plant physiology and metabolic processes. Though N2 is abundant in atmosphere, only legumes are able to fix atmospheric N2 with the help of Rhizobium bacteria. All other plants mainly absorb N in the form of inorganic ions (ammonium (NH4+) and nitrate (NO3−)) from soil. Nitrate is mostly absorbed in aerobic soils, while ammonium is mostly absorbed in acidic soils and wet lands. After uptake, NO3− and NH4+ are assimilated, transformed and mobilized through various processes within plant system.

The agricultural systems focussed on the high-yield crop production remove nitrogen from the soil and depends mostly on the application of large quantities of nitrogenous fertilizers such as urea for the sustained productivity over time. Unfortunately, a large fraction of the applied nitrogen is not directly absorbed by the plants and is lost by the leaching1. Despite significant efforts made by the scientific community in the last 50 years, the nitrogen-use efficiency for the cereal crops has not been improved2. Beyond this, the economic losses and detrimental environmental consequences caused by the use of large quantities of fertilizers in agriculture are critical issues to be considered3,4. Unravelling the genomic regions or the putative candidate genes improving nitrogen-use efficiency will be the first step toward developing nutrient-efficient crop varieties.

To transport N from soil to roots and to other parts of plants, plasma membrane localized proteins known as transporters are essential. They are involved in regulation of N root uptake, root to shoot and leaf to sink transport5,6. Plants have evolved two systems for N uptake to cope with changes in N availability. These two systems are the low-affinity transport system (LATS) and high-affinity transport system (HATS). A low-affinity transport system (LATS) is involved where adequate amounts of nitrogen levels are present. A high-affinity transport system (HATS) is involved where limited amounts of N are present. Plants have two low-affinity and two high affinity N transport systems,for nitrate (NRT1- low-affinity NO3− transporters and NRT2-high-affinity NO3− transporters) and ammonium (AMT1-low-affinity NH4+ transporters and AMT2-high affinity NH4+ transporters). Majority of N in cereal crops such as wheat is taken up in form of nitrate (NO3−). Therefore, nitrate transporters are of great importance.

In plants four families of NO3- transporters have been identified named NPF (NRT1/PTR), NRT2, CLC (chloride channel) and SLAC1/SLAH (slow type anion channel associated homologs)7. NRT1.1 was first NO3- transporter to be identified in Arabidopsis8. The NRT1 transporter family which has been renamed as NPF family is the largest family of nitrate transporters and can further be classified into eight subfamilies9. In Arabidopsis NPF transporters have been well characterized and contain 53 members divided into eight subfamilies9. In rice (Oryza sativa) NPF transporters contain 93 members10. The majority of NPF transporters are involved in LATS with few exceptions of NRT1.1/NPF6.3 in Arabidopsis and MtNRT1.3 in Medicago truncatula, which are involved in both HATS and LATS11,12. Although majority of NPFs are involved in nitrate transport, several studies have suggested their role in transport of other substrates such as nitrite13, peptides14, amino acids15 and several plant hormones16–20. The second family known as NRT2 contains high affinity nitrate transporters. A total of seven NRT2 transporters in Arabidopsis21 and five NRT2 transporters in rice have been reported22,23. Most of NRT2 transporters require a partner protein—NAR2 (nitrate assimilation related protein) to function as high affinity nitrate transporters22–25. Third family of nitrate transporters, CLC (chloride channel) family is mainly associated with vacoular transport of NO3−26. In Arabidopsis, six CLC genes have been reported and are responsible for nitrate and chloride homoeostasis, thereby regulating stomatal movement and salt tolerance26–28. The fourth family—SLAC1/SLAH (slow type anion channel associated homologs) is anion channel family. In Arabidopsis this family contains four members-SLAC1, SLAH1, SLAH2 and SLAH3 which are involved in the nitrate transport in guard cells and roots and in chloride acquisition29. Together these four transporter families are involved in efficient nitrate uptake and utilization in plants.

To the best of our knowledge, the nitrate transporters in hexaploid wheat have not been characterized and explored completely. There are some studies conducted to access the effect of different nitrogen conditions on some of NPF and NRT2 genes30. Most of the studies in wheat have been conducted on members of TaNRT2 gene family. Overexpression of TaNRT2.5 has been associated with increased grain nitrate uptake and yield31. TaNRT2.1 has been associated with post flowering nitrate uptake in wheat32. Expression of TaNRT2.1 can be induced by nitrogen starvation and abscisic acid (ABA)33–37. Some phylogenetic studies and expression-based studies have been conducted on NPF and NRT2 genes recently34–36,38 but CLC and SLAC1/SLAH genes still remain uncharacterized. Structure of proteins play very important role in the functionality of transporter proteins but still no studies have been conducted on structure prediction of any of NPF, NRT2, CLC and SLAC1/SLAH genes in wheat. In our study we have identified and characterized genes belonging to all the four families of nitrate transporters. Our analysis includes gene composition, chromosomal location, phylogenetic relations with members of rice and Arabidopsis and expression analysis. We adopted a new nomenclature for identified genes as the earlier nomenclature systems do not include complete information about subgenome and homoeologs. We have classified the genes based on phylogeny and identified homoeologous pairs of the gene. Expression profiles of all the genes were studied for different developmental stages and different tissues. Further the structures of all the members of gene families were investigated.

Methodology

Sequence search and annotation of nitrate transporter genes

Two methods were used for the identification of NRT1, NRT2 genes in wheat. In the first method, the CDD IDs (conserved domain database IDs) specific to TaNPF, TaCLC, TaSLAC/TaSLAH and TaNRT2 genes (Table 1) were used as identifiers to retrieve genes from the wheat reference genome (IWGSC RefSeq V2.0) from the Ensembl Plants (https://plants.ensembl.org/index.html). In the second method, protein sequences were downloaded from the NCBI database using Nitrate/Nitrogen transporters, and NRT as queries. Incomplete, partial sequences, hypothetical, and predicted protein sequences were filtered out. The downloaded sequences were manually curated to remove duplicate sequences and incomplete sequences. The remaining protein sequences (1687 genes) were aligned using Clustal Omega, and the output Stockholm file was used to create the HMMER profile. The HMMER profile was used to search similar protein sequences in the wheat protein database downloaded from IWGSC. A total of 403 high confidence and 38 low confidence proteins were obtained. Separate searches were performed for TaCLC and TaSLAC1/TaSLAH genes using the same method. A total of 41 TaCLC and 43 TaSLAC1/TaSLAH high confidence genes and 10 TaCLC and 7 TaSLAC1/TaSLAH low confidence genes were obtained. The sequences from both the methods were combined, followed by the removal of low confidence proteins and duplicate sequences, and after manual curation, a final set of 412 genes belonging to all four nitrate transporter families were selected. The same methodology was used to identify sequences for Triticum dicoccoides (AABB), T. turgidum (AABB), T. urartu (AA), and Aegilops tauschii (DD) for comparative analysis.

Table 1.

Summary of nitrate transporter gene numbers in wheat, rice, Arabidopsis and wheat progenitors.

Conserved domain (CDD/ Pfam Id) Gene Family Triticum aestivum (AABBDD) (2n = 42) Arabidopsis thaliana (2n = 10) Oryza sativa (2n = 24) Triticum dicoccoides (AABB) (2n = 28) Triticum turgidum (AABB) (2n = 28) Triticum urartu (AA) (2n = 14) Aegilops tauchii (DD) (2n = 14)
cd17413 NPF6 22 3 6 15 15 7 7
cd17414 NPF4 33 7 12 23 22 9 9
cd17415 NPF3 12 1 5 8 8 3 3
cd17416 NPF1 &2 47 17 13 39 32 17 16
cd17417 NPF5 97 16 32 73 79 31 29
cd17418 NPF8 70 5 16 47 47 20 24
cd17419 NPF7 11 3 4 10 8 5 3
Cd17341 NRT2 46 7 4 20 20 15 18
PF00654 CLC 34 6 7 23 22 10 11
PF03595 SLAC1/ SLAH 40 5 8 21 21 12 10
Total 412 71 107 253 239 121 120
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Maximum likelihood phylogeny of nitrate transporter genes

The alignments of TaNRT1/TaNPF, TaCLC, TaSLAC1/TaSLAH and TaNRT2 sequences were created separately using wheat, rice, and Arabidopsis sequences by MAFT (E-INS-I algorithm). The evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based model. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbour-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model and then selecting the topology having superior log-likelihood value. Evolutionary studies were conducted in MEGA X. The consistency of the phylogenetic estimate was evaluated by bootstraps (1000 replicates). The resulting tree was visualized using FIGTREE v.1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/).

Gene structure prediction and identification of homoeologs

The genomic and CDS sequences of genes were downloaded from the Ensembl plants database. The sequence information was utilized to predict the intron/exon positions by using the GSDS server (Gene Structure Display Server, http://gsds.cbi.pku.edu.cn39). Separate phylogenies were generated for members of each subfamily to resolve the relationship between them. The analysis was performed in MEGA X by the method described previously. Homoeologous genes were identified based on the phylogenetic relationship between the members of subfamilies. The information regarding physical positions of genes were obtained from Ensembl Plants database. Genome wide distribution map of nitrate transporter genes was developed by web based online visualization tool PhenoGram (http://visualization.ritchielab.org/phenograms/plot).

Naming of TaNPF, TaNRT2, TaCLC and TaSLAC1/SLAH genes

We adopted the method proposed by Schilling et al.40 for the naming of NRT genes. The genes were named based on their phylogenetic relationships and subgenome location (A, B, or D). Each gene name started with the abbreviation for the species name Triticum aestivum (Ta), followed by the most closely related Arabidopsis gene name (i.e., NPF1-NPF8, NRT2), which was followed by the subgenome identifier (A, B, and D). Putative homoeologs were given identical gene names except for the subgenome identifier (TaNPF4-A1, TaNPF4-B1 and TaNPF4-D1). The genes belonging to the same subfamily in the same subgenome were consecutively numbered (Table 2).

Table 2.

Grouping and Naming of nitrate transporter genes identified in wheat genome Refeq v2.0.

IWGSC RefSeq ID Name
Triad/Tetrad/Diad/ Singleton A B D Un A B D Un
TaNPF1-T1 TraesCS3A02G304400 TraesCS3B02G332100 TraesCS3D02G297600 TaNPF1-3A1 TaNPF1-3B1 TaNPF1-3D1
TaNPF1-T2 TraesCS3A02G304500 TraesCS3B02G332000 TraesCS3D02G297400 TaNPF1-3A2 TaNPF1-3B2 TaNPF1-3D2
TaNPF2-T1 TraesCS2A02G045500 TraesCS2B02G057700 TraesCS2D02G044200 TaNPF2-2A1 TaNPF2-2B1 TaNPF2-2D1
TaNPF2-T2 TraesCS3A02G418700 TraesCS3B02G454000 TraesCS3D02G414300 TaNPF2-3A1 TaNPF2-3B1 TaNPF2-3D1
TaNPF2-T3 TraesCS3A02G418800 TraesCS3B02G454100 TraesCS3D02G414400 TaNPF2-3A2 TaNPF2-3B2 TaNPF2-3D2
TaNPF2-T4 TraesCS4A02G283900 TraesCS4B02G029600 TraesCS4D02G026800 TaNPF2-4A1 TaNPF2-4B1 TaNPF2-4D1
TaNPF2-S1 TraesCS4A02G440300 TaNPF2-4A2
TaNPF2-S2 TraesCS4A02G440400 TaNPF2-4A3
TaNPF2-S3 TraesCS4A02G440500 TaNPF2-4A4
TaNPF2-S4 TraesCS4A02G440600 TaNPF2-4A5
TaNPF2-S5 TraesCS4A02G440700 TaNPF2-4A6
TaNPF2-T5 TraesCS5A02G004400 TraesCS5B02G001100 TraesCS5D02G012500 TaNPF2-5A1 TaNPF2-5B1 TaNPF2-5D1
TaNPF2-T6 TraesCS5A02G037900 TraesCS5B02G039100 TraesCS5D02G045300 TaNPF2-5A2 TaNPF2-5B2 TaNPF2-5D2
TaNPF2-T7 TraesCS5A02G153200 TraesCS5B02G152000 TraesCS5D02G158500 TaNPF2-5A3 TaNPF2-5B3 TaNPF2-5D3
TaNPF2-D1 TraesCS7A02G054000 TraesCS7D02G049300 TaNPF2-7A1 TaNPF2-7D1
TaNPF2-D2 TraesCS7A02G054100 TraesCS7D02G049400 TaNPF2-7A2 TaNPF2-7D2
TaNPF2-T8 TraesCS7A02G121600 TraesCS7B02G020200 TraesCS7D02G119800 TaNPF2-7A3 TaNPF2-7B3 TaNPF2-7D3
TaNPF2-T9 TraesCS7A02G121700 TraesCS7B02G020500 TraesCS7D02G120200 TaNPF2-7A4 TaNPF2-7B4 TaNPF2-7D4
TaNPF2-D3 TraesCS2B02G057600 TraesCS2D02G044000 TaNPF2-7B5 TaNPF2-7D5
TaNPF2-D4 TraesCS7B02G020300 TraesCS7D02G119900 TaNPF2-7B6 TaNPF2-7D6
TaNPF2-S6 TraesCS7D02G076900 TaNPF2-7D7
TaNPF3-T1 TraesCS1A02G257400 TraesCS1B02G267900 TraesCS1D02G256700 TaNPF3-1A1 TaNPF3-1B1 TaNPF3-1D1
TaNPF3-T2 TraesCS1A02G257800 TraesCS1B02G268200 TraesCS1D02G257100 TaNPF3-1A2 TaNPF3-1B2 TaNPF3-1D2
TaNPF3-T3 TraesCS1A02G257900 TraesCS1B02G268300 TraesCS1D02G257200 TaNPF3-1A3 TaNPF3-1B3 TaNPF3-1D3
TaNPF3-T4 TraesCS7A02G206400 TraesCS7B02G113600 TraesCS7D02G209200 TaNPF3-7A1 TaNPF3-7B1 TaNPF3-7D1
TaNPF4-T1 TraesCS2A02G264500 TraesCS2B02G277600 TraesCS2D02G259400 TaNPF4-2A1 TaNPF4-2B1 TaNPF4-2D1
TaNPF4-T2 TraesCS2A02G309100 TraesCS2B02G326200 TraesCS2D02G307400 TaNPF4-2A2 TaNPF4-2B2 TaNPF4-2D2
TaNPF4-T3 TraesCS2A02G350000 TraesCS2B02G368500 TraesCS2D02G348400 TaNPF4-2A3 TaNPF4-2B3 TaNPF4-2D3
TaNPF4-T4 TraesCS2A02G350100 TraesCS2B02G368600 TraesCS2D02G348500 TaNPF4-2A4 TaNPF4-2B4 TaNPF4-2D4
TaNPF4-D1 TraesCS2A02G350200 TraesCS2B02G368400 TaNPF4-2A5 TaNPF4-2B5
TaNPF4-T5 TraesCS2A02G350300 TraesCS2B02G368700 TraesCS2D02G348600 TaNPF4-2A6 TaNPF4-2B6 TaNPF4-2D6
TaNPF4-S1 TraesCS3A02G272600 TaNPF4-3A1
TaNPF4-T6 TraesCS4A02G225400 TraesCS4B02G090800 TraesCS4D02G087900 TaNPF4-4A1 TaNPF4-4B1 TaNPF4-4D1
TaNPF4-T7 TraesCS5A02G056100 TraesCS5B02G060800 TraesCS5D02G067100 TaNPF4-5A1 TaNPF4-5B1 TaNPF4-5D1
TaNPF4-T8 TraesCS5A02G056200 TraesCS5B02G060500 TraesCS5D02G067400 TaNPF4-5A2 TaNPF4-5B2 TaNPF4-5D2
TaNPF4-T9 TraesCS5A02G388000 TraesCS5B02G393100 TraesCS5D02G398000 TaNPF4-5A3 TaNPF4-5B3 TaNPF4-5D3
TaNPF4-T10 TraesCS7A02G365100 TraesCS7B02G262200 TraesCS7D02G357300 TaNPF4-7A1 TaNPF4-7B1 TaNPF4-7D1
TaNPF5-T1 TraesCS1A02G150200 TraesCS1B02G168000 TraesCS1D02G147200 TaNPF5-1A1 TaNPF5-1B1 TaNPF5-1D1
TaNPF5-T2 TraesCS1A02G150400 TraesCS1B02G168100 TraesCS1D02G147400 TaNPF5-1A2 TaNPF5-1B2 TaNPF5-1D2
TaNPF5-T3 TraesCS1A02G269400 TraesCS1B02G279900 TraesCS1D02G269500 TaNPF5-1A3 TaNPF5-1B3 TaNPF5-1D3
TaNPF5-T4 TraesCS1A02G269500 TraesCS1B02G280000 TraesCS1D02G269600 TaNPF5-1A4 TaNPF5-1B4 TaNPF5-1D4
TaNPF5-T5 TraesCS1A02G269600 TraesCS1B02G280100 TraesCS1D02G269700 TaNPF5-1A5 TaNPF5-1B5 TaNPF5-1D5
TaNPF5-T6 TraesCS2A02G565600 TraesCS2B02G626000 TraesCS2D02G576000 TaNPF5-2A1 TaNPF5-2B1 TaNPF5-2D1
TaNPF5-D1 TraesCS2A02G571800 TraesCS2D02G583300 TaNPF5-2A2 TaNPF5-2D2
TaNPF5-T7 TraesCS2A02G571900 TraesCS2B02G615500 TraesCS2D02G583400 TaNPF5-2A3 TaNPF5-2B3 TaNPF5-2D3
TaNPF5-D2 TraesCS2A02G572000 TraesCS2B02G615400 TaNPF5-2A4 TaNPF5-2B4
TaNPF5-S1 TraesCS2A02G572100 TaNPF5-2A5
TaNPF5-T8 TraesCS2A02G572200 TraesCS2B02G615300 TraesCS2D02G583500 TaNPF5-2A6 TaNPF5-2B6 TaNPF5-2D6
TaNPF5-T9 TraesCS2A02G572300 TraesCS2B02G615200 TraesCS2D02G583600 TaNPF5-2A7 TaNPF5-2B7 TaNPF5-2D7
TaNPF5-T10 TraesCS3A02G185600 TraesCS3B02G215200 TraesCS3D02G189500 TaNPF5-3A1 TaNPF5-3B1 TaNPF5-3D1
TaNPF5-T11 TraesCS3A02G382100 TraesCS3B02G414800 TraesCS3D02G375900 TaNPF5-3A2 TaNPF5-3B2 TaNPF5-3D2
TaNPF5-T12 TraesCS3A02G382200 TraesCS3B02G414900 TraesCS3D02G375800 TaNPF5-3A3 TaNPF5-3B3 TaNPF5-3D3
TaNPF5-T13 TraesCS3A02G382300 TraesCS3B02G415200 TraesCS3D02G375700 TaNPF5-3A4 TaNPF5-3B4 TaNPF5-3D4
TaNPF5-T14 TraesCS3A02G382400 TraesCS3B02G415300 TraesCS3D02G375600 TaNPF5-3A5 TaNPF5-3B5 TaNPF5-3D5
TaNPF5-D3 TraesCS3A02G382600 TraesCS3D02G375500 TaNPF5-3A6 TaNPF5-3D6
TaNPF5-D4 TraesCS3A02G382700 TraesCS3D02G375400 TaNPF5-3A7 TaNPF5-3D7
TaNPF5-D5 TraesCS3A02G382800 TraesCS3D02G375300 TaNPF5-3A8 TaNPF5-3D8
TaNPF5-D6 TraesCS3A02G382900 TraesCS3D02G375200 TaNPF5-3A9 TaNPF5-3D9
TaNPF5-T15 TraesCS3A02G383200 TraesCS3B02G415600 TraesCS3D02G376200 TaNPF5-3A10 TaNPF5-3B10 TaNPF5-3D10
TaNPF5-T16 TraesCS3A02G383300 TraesCS3B02G415700 TraesCS3D02G376300 TaNPF5-3A11 TaNPF5-3B11 TaNPF5-3D11
TaNPF5-T17 TraesCS5A02G485000 TraesCS5B02G498400 TraesCS5D02G498500 TaNPF5-5A1 TaNPF5-5B1 TaNPF5-5D1
TaNPF5-T18 TraesCS5A02G485200 TraesCS5B02G498500 TraesCS5D02G498700 TaNPF5-5A2 TaNPF5-5B2 TaNPF5-5D2
TaNPF5-T19 TraesCS5A02G485300 TraesCS5B02G498700 TraesCS5D02G498800 TaNPF5-5A3 TaNPF5-5B3 TaNPF5-5D3
TaNPF5-S2 TraesCS5A02G508500 TaNPF5-5A4
TaNPF5-T20 TraesCS6A02G041300 TraesCS6B02G056500 TraesCS6D02G047600 TaNPF5-6A1 TaNPF5-6B1 TaNPF5-6D1
TaNPF5-T21 TraesCS7A02G196100 TraesCS7B02G101800 TraesCS7D02G197600 TaNPF5-7A1 TaNPF5-7B1 TaNPF5-7D1
TaNPF5-T22 TraesCS7A02G461200 TraesCS7B02G362700 TraesCS7D02G449400 TaNPF5-7A2 TaNPF5-7B2 TaNPF5-7D2
TaNPF5-D7 TraesCS7A02G504300 TraesCS7D02G491400 TaNPF5-7A3 TaNPF5-7D3
TaNPF5-S3 TraesCS2B02G013000 TaNPF5-2B8
TaNPF5-S4 TraesCS2B02G248000 TaNPF5-2B9
TaNPF5-S5 TraesCS2B02G401000 TaNPF5-2B10
TaNPF5-S6 TraesCS2B02G626100 TaNPF5-2B11
TaNPF5-S7 TraesCS2B02G626600 TaNPF5-2B12
TaNPF5-S8 TraesCS2B02G626700 TaNPF5-2B13
TaNPF5-S9 TraesCS3B02G304500 TaNPF5-3B12
TaNPF5-S10 TraesCS3B02G415000 TaNPF5-3B13
TaNPF5-S11 TraesCS3B02G415100 TaNPF5-3B14
TaNPF5-S12 TraesCS4B02G057000 TaNPF5-4B1
TaNPF5-S13 TraesCS4B02G338600 TaNPF5-4B2
TaNPF5-S14 TraesCS4D02G335100 TaNPF5-4D1
TaNPF5-D8 TraesCS7B02G040100 TraesCS7D02G139600 TaNPF5-7B4 TaNPF5-7D4
TaNPF5-S15 TraesCS7B02G312500 TaNPF5-7B5
TaNPF6-T1 TraesCS1A02G031300 TraesCS1B02G038700 TraesCS1D02G032700 TaNPF6-1A1 TaNPF6-1B1 TaNPF6-1D1
TaNPF6-T2 TraesCS1A02G210900 TraesCS1B02G224900 TraesCS1D02G214200 TaNPF6-1A2 TaNPF6-1B2 TaNPF6-1D2
TaNPF6-T3 TraesCS1A02G211000 TraesCS1B02G225000 TraesCS1D02G214300 TaNPF6-1A3 TaNPF6-1B3 TaNPF6-1D3
TaNPF6-T4 TraesCS2A02G335800 TraesCS2B02G346100 TraesCS2D02G327000 TaNPF6-2A1 TaNPF6-2B1 TaNPF6-2D1
TaNPF6-S1 TraesCS4B02G371000 TaNPF6-4B1
TaNPF6-S2 TraesCS4B02G375800 TaNPF6-4B2
TaNPF6-S3 TraesCS4D02G361500 TaNPF6-4D3
TaNPF6-T5 TraesCS5A02G409600 TraesCS5B02G414000 TraesCS5D02G419200 TaNPF6-5A1 TaNPF6-5B1 TaNPF6-5D1
TaNPF6-S4 TraesCS5A02G537100 TaNPF6-5A2
TaNPF6-T6 TraesCS7A02G301700 TraesCS7B02G201900 TraesCS7D02G297000 TaNPF6-7A1 TaNPF6-7B1 TaNPF6-7D1
TaNPF7-S1 TraesCS4A02G284300 TaNPF7-4A1
TaNPF7-S2 TraesCS5A02G546200 TaNPF7-5A1
TaNPF7-T1 TraesCS6A02G263500 TraesCS6B02G290500 TraesCS6D02G251500 TaNPF7-6A1 TaNPF7-6B1 TaNPF7-6D1
TaNPF7-T2 TraesCS6A02G280200 TraesCS6B02G309200 TraesCS6D02G260500 TaNPF7-6A2 TaNPF7-6B2 TaNPF7-6D2
TaNPF7-S3 TraesCS7A02G413200 TaNPF7-7A1
TaNPF7-S4 TraesCS4B02G380000 TaNPF7-4B2
TaNPF7-S5 TraesCSU02G130200 TaNPF7-Un1
TaNPF8-T1 TraesCS2A02G416800 TraesCS2B02G000500 TraesCS2D02G413900 TaNPF8-2A1 TaNPF8-2B1 TaNPF8-2D1
TaNPF8-T2 TraesCS3A02G056400 TraesCS3B02G069100 TraesCS3D02G056300 TaNPF8-3A1 TaNPF8-3B1 TaNPF8-3D1
TaNPF8-T3 TraesCS3A02G057000 TraesCS3B02G070200 TraesCS3D02G056700 TaNPF8-3A2 TaNPF8-3B2 TaNPF8-3D2
TaNPF8-T4 TraesCS3A02G392800 TraesCS3B02G424700 TraesCS3D02G385600 TaNPF8-3A3 TaNPF8-3B3 TaNPF8-3D3
TaNPF8-T5 TraesCS3A02G392900 TraesCS3B02G424800 TraesCS3D02G385700 TaNPF8-3A4 TaNPF8-3B4 TaNPF8-3D4
TaNPF8-T6 TraesCS4A02G075700 TraesCS4B02G231500 TraesCS4D02G232900 TaNPF8-4A1 TaNPF8-4B1 TaNPF8-4D1
TaNPF8-T7 TraesCS4A02G075900 TraesCS4B02G231700 TraesCS4D02G233000 TaNPF8-4A2 TaNPF8-4B2 TaNPF8-4D2
TaNPF8-T8 TraesCS4A02G076000 TraesCS4B02G231800 TraesCS4D02G233100 TaNPF8-4A3 TaNPF8-4B3 TaNPF8-4D3
TaNPF8-T9 TraesCS4A02G076100 TraesCS4B02G232000 TraesCS4D02G233000 TaNPF8-4A4 TaNPF8-4B4 TaNPF8-4D4
TaNPF8-T10 TraesCS4A02G076200 TraesCS4B02G232100 TraesCS4D02G233400 TaNPF8-4A5 TaNPF8-4B5 TaNPF8-4D5
TaNPF8-T11 TraesCS4A02G262700 TraesCS4B02G052200 TraesCS4D02G052400 TaNPF8-4A6 TaNPF8-4B6 TaNPF8-4D6
TaNPF8-S1 TraesCS4A02G287300 TaNPF8-4A7
TaNPF8-S2 TraesCS4A02G287900 TaNPF8-4A8
TaNPF8-T12 TraesCS6A02G142600 TraesCS6B02G171000 TraesCS6D02G132100 TaNPF8-6A1 TaNPF8-6B1 TaNPF8-6D1
TaNPF8-D1 TraesCS7A02G095200 TraesCS7D02G091600 TaNPF8-7A1 TaNPF8-7D1
TaNPF8-T13 TraesCS7A02G381500 TraesCS7B02G283400 TraesCS7D02G377800 TaNPF8-7A2 TaNPF8-7B2 TaNPF8-7D2
TaNPF8-S3 TraesCS7A02G381600 TaNPF8-7A3
TaNPF8-T14 TraesCS7A02G381700 TraesCS7B02G283800 TraesCS7D02G377900 TaNPF8-7A4 TaNPF8-7B4 TaNPF8-7D4
TaNPF8-T15 TraesCS7A02G381800 TraesCS7B02G284300 TraesCS7D02G378300 TaNPF8-7A5 TaNPF8-7B5 TaNPF8-7D5
TaNPF8-D2 TraesCS7A02G412100 TraesCS7B02G311400 TaNPF8-7A6 TaNPF8-7B6
TaNPF8-T16 TraesCS7A02G413100 TraesCS7B02G312600 TraesCS7D02G406200 TaNPF8-7A7 TaNPF8-7B7 TaNPF8-7D7
TaNPF8-T17 TraesCS7A02G413300 TraesCS7B02G312700 TraesCS7D02G406400 TaNPF8-7A8 TaNPF8-7B8 TaNPF8-7D8
TaNPF8-S4 TraesCS7A02G531000 TaNPF8-7A9
TaNPF8-D3 TraesCS3B02G069900 TraesCS3D02G057000 TaNPF8-3B5 TaNPF8-3D5
TaNPF8-D4 TraesCS4B02G026700 TraesCS4D02G024400 TaNPF8-4B9 TaNPF8-4D9
TaNPF8-S5 TraesCS4B02G398100 TaNPF8-4B10
TaNPF8-S6 TraesCS5B02G245300 TaNPF8-5B1
TaNPF8-D5 TraesCS6B02G406100 TraesCS6D02G353500 TaNPF8-6B2 TaNPF8-6D2
TaNPF8-S7 TraesCS7D02G518900 TaNPF8-7D9
TaNPF8-S8 TraesCSU02G207500 TaNPF8-Un1
TaNPF8-S9 TraesCSU02G115500 TaNPF8-Un2
TaNRT2-D1 TraesCS2A02G074800 TraesCS2D02G073500 TaNRT2-2A1 TaNRT2-2D1
TaNRT2-T1 TraesCS3A02G254000 TraesCS3B02G285900 TraesCS3D02G254900 TaNRT2-3A1 TaNRT2-3B1 TaNRT2-3D1
TaNRT2-D2 TraesCS6A02G030700 TraesCS6B02G044100 TaNRT2-6A1 TaNRT2-6B1
TaNRT2-T2 TraesCS6A02G030800 TraesCS6B02G044400 TraesCS6D02G035900 TaNRT2-6A2 TaNRT2-6B2 TaNRT2-6D2
TaNRT2-T3 TraesCS6A02G030900 TraesCS6B02G044300 TraesCS6D02G035800 TaNRT2-6A3 TaNRT2-6B3 TaNRT2-6D3
TaNRT2-T4 TraesCS6A02G031000 TraesCS6B02G044200 TraesCS6D02G035700 TaNRT2-6A4 TaNRT2-6B4 TaNRT2-6D4
TaNRT2-D3 TraesCS6A02G031100 TraesCS6B02G044500 TaNRT2-6A5 TaNRT2-6B5
TaNRT2-T5 TraesCS6A02G031200 TraesCS6B02G044000 TraesCS6D02G035600 TaNRT2-6A6 TaNRT2-6B6 TaNRT2-6D6
TaNRT2-T6 TraesCS6A02G032400 TraesCS6B02G045600 TraesCS6D02G037200 TaNRT2-6A7 TaNRT2-6B7 TaNRT2-6D7
TaNRT2-T7 TraesCS6A02G032500 TraesCS6B02G045700 TraesCS6D02G037300 TaNRT2-6A8 TaNRT2-6B8 TaNRT2-6D8
TaNRT2-T8 TraesCS6A02G032800 TraesCS6B02G046500 TraesCS6D02G037800 TaNRT2-6A9 TaNRT2-6B9 TaNRT2-6D9
TaNRT2-D4 TraesCS6A02G032900 TraesCS6D02G037900 TaNRT2-6A10 TaNRT2-6D10
TaNRT2-TT1 TraesCS6A02G033000 TraesCS6B02G046600 TraesCS6D02G038100 TraesCS6D02G038000 TaNRT2-6A11 TaNRT2-6B11 TaNRT2-6D11x TaNRT2-6D11y
TaNRT2-D5 TraesCS6A02G033100 TraesCS6D02G038300 TaNRT2-6A12 TaNRT2-6D12
TaNRT2-T9 TraesCS6A02G033200 TraesCS6B02G046700 TraesCS6D02G038200 TaNRT2-6A13 TaNRT2-6B13 TaNRT2-6D13
TaNRT2-T10 TraesCS7A02G428500 TraesCS7B02G328700 TraesCS7D02G420900 TaNRT2-7A1 TaNRT2-7B1 TaNRT2-7D1
TaNRT2-D6 TraesCS1D02G035700 TraesCSU02G002800 TaNRT2-1D1 TaNRT2-Un1
TaCLC-T1 TraesCS2A02G309900 TraesCS2B02G326900 TraesCS2D02G308100 TaCLC-2A1 TaCLC-2B1 TaCLC-2D1
TaCLC-T2 TraesCS2A02G517500 TraesCS2B02G546000 TraesCS2D02G519000 TaCLC-2A2 TaCLC-2B2 TaCLC-2D2
TaCLC-T3 TraesCS3A02G253600 TraesCS3B02G285500 TraesCS3D02G254500 TaCLC-3A1 TaCLC-3B1 TaCLC-3D1
TaCLC-TT1 TraesCS3A02G125300 TraesCS3B02G144700 TraesCS3D02G126700 TraesCS3D02G126600 TaCLC-3A2 TaCLC-3B2 TaCLC-3D2x TaCLC-3D2y
TaCLC-T4 TraesCS3A02G390100 TraesCS3B02G418700 TraesCS3D02G379600 TaCLC-3A3 TaCLC-3B3 TaCLC-3D3
TaCLC-T5 TraesCS4A02G277600 TraesCS4B02G035500 TraesCS4D02G033500 TaCLC-4A1 TaCLC-4B1 TaCLC-4D1
TaCLC-T6 TraesCS5A02G449500 TraesCS5B02G457100 TraesCS5D02G456000 TaCLC-5A1 TaCLC-5B1 TaCLC-5D1
TaCLC-T7 TraesCS6A02G098500 TraesCS6B02G126400 TraesCS6D02G084300 TaCLC-6A1 TaCLC-6B1 TaCLC-6D1
TaCLC-T8 TraesCS6A02G098600 TraesCS6B02G126800 TraesCS6D02G084000 TaCLC-6A2 TaCLC-6B2 TaCLC-6D2
TaCLC-T9 TraesCS6A02G283600 TraesCS6B02G312100 TraesCS6D02G264100 TaCLC-6A3 TaCLC-6B3 TaCLC-6D3
TaCLC-T10 TraesCS7A02G240700 TraesCS7B02G136300 TraesCS7D02G239700 TaCLC-7A1 TaCLC-7B1 TaCLC-7D1
TaSLAC-T1 TraesCS1A02G127500 TraesCS1B02G147400 TraesCS1D02G126500 TaSLAC-1A1 TaSLAC-1B1 TaSLAC-1D1
TaSLAC-D1 TraesCS1A02G423000 TraesCS1B02G455100 TaSLAC-1A2 TaSLAC-1B2
TaSLAC-T2 TraesCS1A02G423900 TraesCS1B02G456000 TraesCS1D02G432500 TaSLAC-1A3 TaSLAC-1B3 TaSLAC-1D3
TaSLAC-D2 TraesCS1A02G424400 TraesCSU02G204200 TaSLAC-1A4 TaSLAC-Un1
TaSLAC-T3 TraesCS1A02G424500 TraesCS1B02G456500 TraesCS1D02G433100 TaSLAC-1A5 TaSLAC-1B5 TaSLAC-1D5
TaSLAC-T4 TraesCS2A02G398000 TraesCS2B02G416100 TraesCS2D02G395700 TaSLAC-2A1 TaSLAC-2B1 TaSLAC-2D1
TaSLAC-T5 TraesCS3A02G028100 TraesCS3B02G018300 TraesCS3D02G017800 TaSLAC-3A1 TaSLAC-3B1 TaSLAC-3D1
TaSLAC-T6 TraesCS3A02G151400 TraesCS3B02G178600 TraesCS3D02G159600 TaSLAC-3A2 TaSLAC-3B2 TaSLAC-3D2
TaSLAC-T7 TraesCS3A02G167000 TraesCS3B02G199200 TraesCS3D02G174800 TaSLAC-3A3 TaSLAC-3B3 TaSLAC-3D3
TaSLAC-T8 TraesCS3A02G225100 TraesCS3B02G254700 TraesCS3D02G228400 TaSLAC-3A4 TaSLAC-3B4 TaSLAC-3D4
TaSLAC-D3 TraesCS1B02G456100 TraesCS1D02G432700 TaSLAC-1B6 TaSLAC-1D6
TaSLAC-S1 TraesCS1B02G388600 TaSLAC-1B7
TaSLAC-T9 TraesCS1B02G456200 TraesCS1D02G432900 TraesCSU02G001500 TaSLAC-1B8 TaSLAC-1D8 TaSLAC-Un2
TaSLAC-T10 TraesCS1B02G456400 TraesCS1D02G432800 TraesCSU02G001400 TaSLAC-1B9 TaSLAC-1D9 TaSLAC-Un3
TaSLAC-T11 TraesCS1B02G456300 TraesCS1D02G433200 TraesCSU02G001600 TaSLAC-1B10 TaSLAC-1D10 TaSLAC-Un4
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Structure prediction of nitrate transporter proteins

Due to the unavailability of crystal structures, gene homology modelling was carried out to predict their three-dimensional (3D) structure. The sequences of TaNRT1, TaCLC, TaSLAC1/TaSLAH and TaNRT2 genes were submitted to web-based server Phyre241. Briefly, Phyre2 used PSI-BLAST to detect sequence homologues which was followed by Psi-pred and Diso-pred to predict secondary structure and disorder. Then Hidden Markov models (HMM) of sequences were generated based on homologues detected before. HMMs of query proteins were scanned against library of HMMs of proteins with experimentally solved structures to construct 3D models of query proteins. Transmembrane helix and topology prediction was carried by memsat-svm41.

Expression analysis of nitrate transporter genes

The RNAseq data of TaNPF, TaNRT2, TaCLC and TaSLAC1/TaSLAH genes of various tissues (root, shoot/leaf, spike, grain) at three developmental stages (seedling, vegetative and reproductive) for Chinese spring and Azhurnaya (cv) was downloaded from the wheat expression database (www.wheat-expression.com). Expression levels were downloaded as log2(transcripts per million) (log2tpm) for different tissues at different time points. Several tissue-specific (root, shoot, leaf, grain) genes were identified based on expression patterns. For triad expression analysis, a method described by Ramírez-González et al.42 was used. Briefly, the expression data from spring wheat (CS) and Azhurnaya was downloaded from the wheat expression database as TPM for root, leave, shoot spike and grain. For analysis, the triads with expression below one tpm were excluded. Expression values were normalized, triads were assigned balanced, A/B/D suppressed or A/B/D dominant profiles. To elucidate the role of Nitrate transporter genes towards N starvation and N recovery, the gene expression data set34–36 from wheat omics 1.0 database (http://wheatomics.sdau.edu.cn/) was analysed. The dataset contained expression data in roots of 10-day old wheat plants (Chinese Spring) treated for N-starvation for 5 days and then subjected for N-recovery34–36.

Development of validation panel to check the efficacy of the identified nitrate transporter genes

The nested synthetic hexaploid wheat (N-SHW) introgression library constituting a set of 352 breeding lines derived from four sub-populations (Pop1: 75 lines from PDW233/Ae. tauschii acc. pau 14,135 amphiploid //2*BWL4444; Pop2: 106 lines from PDW233/Ae. tauschii acc. pau 14,135 amphiploid //2*BWL3531; Pop3: 88 lines from PBW114/Ae. tauschii acc. pau 14,170 amphiploid //2*BWL4444; Pop4: 83 lines from PBW114/Ae. tauschii acc. pau 14,170 amphiploid //2*BWL3531) were developed43. These N-SHW library, six parents and two synthetic hexaploid wheats were assessed over 2 years in 2018 and 2019 at 3 nitrogen levels [i.e., zero N (0 kg ha−1), half N (60 kg ha-1) and full N (recommended, 120 kg ha−1]. The detailed phenotyping of the N-SHW introgression libraries for the nitrogen-use efficiency related traits was carried out across years and treatments43. High-density genotyping was performed using the 35 K Axiom® Wheat Breeder’s Array (Affymetrix UK Ltd., United Kingdom). The population structure of the 352 N-SHW lines was assessed on the basis of 9,474 SNPs distributed across all 21 wheat chromosomes. The most appropriate K explaining the population structure was K = 3 at MAF ≥ 5% (Supplementary Fig. 4A). The kinship heatmap suggested a weak relatedness in the panel (Supplementary Fig. 4B). The first three principal components (PCs) were most informative gradually decreasing (Supplementary Fig. 4C,D) until the tenth PC. The kinship and PCs were considered during the GWAS analysis to correct for population structure. The appropriate number of sub-populations was determined from the largest delta K value of 3 (Supplementary Fig. 4E). The kinship and PCs were considered during the GWAS analysis to identify population structure. Significant marker-trait associations were identified using CMLM (compressed mixed linear model)/P3D (population parameters previously defined) in GAPIT (Genome Association and Prediction Integrated Tool) executed in R. Over 322 marker trait associations for NUE were compared to nitrate transporter genes.

Results

The wheat genome consists of 412 nitrate transporter genes belonging to four different families

A total of 412 nitrate transporter sequences excluding splice variants were identified in IWGSC wheat genome assembly (RefSeq V2.0). The wheat genome consists of 292 TaNPF genes, 34 TaCLC genes, 40 TaSLAC1/TaSLAH genes and 46 TaNRT2 genes. The TaNPF genes could be divided into eight subgroups (TaNPF1 to TaNPF8) based on the presence of conserved domains (Table 1). TaNPF5 subgroup was the largest group consisting of 97 genes followed by TaNPF8 (70 genes), TaNPF2 (41 genes), TaNPF4 (33 genes), TaNPF6 (22 genes), TaNPF3 (12 genes) and TaNPF7 (11 genes). The NPF1 subgroup was the smallest one consisting of 6 genes present on homoeologous group chromosomes 3A, 3B and 3D. TaNRT1/TaNPF genes were present throughout the genome (Fig. 1). The location of genes across chromosomes varied according to the size of the subfamily. The genes belonging to larger subfamilies (e.g., TaNPF5, TaNPF8, TaNPF2) were predominantly located in tandem positions on the distal region of chromosomes. The genes belonging to smaller subfamilies (TaNPF1, TaNPHF7, TaNPF3) were located on proximal regions of chromosomes. The genes present near distal ends of chromosomes were found to be in the form of clusters in close vicinity to each other. The majority of TaNRT2 genes were present in the clusters on the distal end of homoeologous chromosomes 6A, 6B and 6D. TaCLC genes were distributed across the wheat genome. TaSLAC1/TaSLAH genes were only distributed on homoeologous chromosomes 1A,1B, 1D, 2A, 2B, 2D, 3A, 3B and 3D. The predicted gene structures contained several intron regions (Supplementary Fig. 1a–c) for many genes in TaNPF, TaCLC and TaSLAC1/TaSLAH families. All the TaNRT2 genes were intron less. The size of predicted genes ranged between 1 and 25 Kb. Several truncated and duplicated genes were also predicted.

Figure 1.

Figure 1

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Genome wide distribution of TaNPF, TaNRT2, TaCLC and TaSLAC1/TaSLAH genes in hexaploid wheat. Figure was generated by web-based software tool-Phenogram from Ritchie Lab44 (http://visualization.ritchielab.org/phenograms/plot).

Phylogenetic relationships among nitrate transporter genes

The maximum likelihood phylogenetic tree of all the nitrate transporter genes predicted that wheat contains all the major subfamilies present in Arabidopsis and rice (Oryza sativa) (Fig. 2a). The TaNRT1/TaNPF and TaNRT2 genes could be classified into five subclades. The subclades in the phylogenetic tree followed species phylogeny with Arabidopsis genes displaying sister group relationship with wheat genes. Based on the phylogenetic relationship, TaNRT1/TaNPF genes fitted well into eight subfamilies (TaNPF1 to TaNPF8) following the Arabidopsis model. The topology of larger subclades (TaNPF5, TaNPF8, TaNPF2) was more complex than smaller subclades as they were more expanded in wheat than Arabidopsis and rice (Fig. 2a, Supplementary Fig. 2). TaNRT2 genes were present as a separate subclade and were closely related to the TaNPF2 subfamily. The phylogenetic analysis of TaCLC and TaSLAC1/TaSLAH genes was carried out separately. The results showed TaCLC genes could be classified into 6 groups according to phylogenetic relation with Arabidopsis and rice genes (Fig. 2b). TaSLAC1/TaSLAH genes were divided into 4 subclades. The largest subclade in TaSLAC1/TaSLAH genes showed close relationship with rice SLAC1/SLAH genes but not with Arabidopsis genes (Fig. 2c).

Figure 2.

Figure 2

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Phylogenetic tree depicting relationship between (a) TaNPF and TaNRT2 genes in hexaploid wheat and Arabidopsis thaliana (b) TaCLC genes in wheat, rice and Arabidopsis thaliana (c) TaSLAC1/SLAH genes in hexaploid wheat, rice and Arabidopsis thaliana. Phylogenetic analysis was performed by MEGA X software45 and the results were edited and visualized by FIGTREE software v1.4.4. (http://tree.bio.ed.ac.uk/software/figtree/) to generate final images.

Homoeologs retention and gene duplication in nitrate transporter genes

The number of nitrate transporter genes in each family were significantly higher than those in Arabidopsis and rice (Table 1, Supplementary Table 1). The comparison with T. dicoccoides (AABB), T. turgidum (AABB), T. urartu (AA) and Ae. tauschii (DD) suggested that most of the homoeologs in hexaploid wheat were retained during evolution (Fig. 3, Supplementary Table 1). There was also evidence of gene duplications in tetraploids and hexaploid wheat, reflected in gene number and phylogenetic data (Fig. 2, Supplementary Fig. 1a–c). Most duplicated genes were present in subfamilies with a larger number of genes (TaNPF5, TaNPF8, TaNPF2 and TaNRT2). Nitrate transporters could be grouped into 13 triads, 26 diads, 2 tetrads and 48 singleton genes based on phylogeny (Table 3). Out of a total of 292 TaNPF genes, about 74% of TaNPF genes could be grouped into 72 triads of homoeologous genes (A, B, D) based on phylogenetic relationships. Similarly, 71% of TaNRT2 genes, 97% of TaCLC genes and 80% of TaSLAC1/TaSLAH genes could be grouped into homoeologous triads.

Figure 3.

Figure 3

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Synteny relationships of wheat nitrate transporter genes orthologous with (A) A. thaliana, (B) O. sativa, (C) T. urartu, (D) Ae. tauschii, (E) T. dicoccoides and, (F) T. turgidum. Circos plots were generated by web-based application- shinyCircos (https://venyao.xyz/shinycircos/)46.

Table 3.

Number of triads, tetrads, diads and singletons detected in nitrate transporter families in hexaploid wheat genome.

Family/ Subfamily No. of Triads No. of diads No. of Tetrads Singletons
A:B:D = (1:1:1) A:B:D = (1:1:0) A:B:D = (1:0:1) A:B:D = (0:1:1) A:B:D = (1:1:2) A:B:D = (1:2:1) A:B:D = (2:1:1) A:B:D = (1:0:0) A:B:D = (0:0:1) A:B:D = (0:1:0) Un
TaNPF1 2 0 0 0 0 0 0 0 0 0 1
TaNPF2 9 0 2 2 0 0 0 5 1 0 0
TaNPF3 4 0 0 0 0 0 0 0 0 0 0
TaNPF4 10 1 0 0 0 0 0 1 0 0 0
TaNPF5 22 1 1 6 2 12 1 0
TaNPF6 6 0 0 0 0 0 0 1 2 1 0
TaNPF7 2 0 0 0 0 0 0 3 1 1 1
TaNPF8 17 1 1 3 4 2 1 2
TaNRT2 10 2 3 0 1 0 0 0 0 0 1
TaCLC 10 0 0 0 1 0 0 0 0 0 0
TaSLAC1/TaSLAH 11 1 1 1 0 0 0 0 0 1 4
Total 103 6 8 12 2 0 0 16 18 5 9
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Nitrate transporter proteins contain multiple transmembrane helices

To study the structural features of nitrate transporters, we predicted the 3D structures of all 412 protein sequences. All nitrogen transporters were predicted to be transmembrane proteins containing multiple transmembrane segments (Fig. 4i). The majority of proteins comprised of 12–14 transmembrane helices (TMs) with some variation. The basic structure of TaNRT/TaNPF proteins included N and C terminal segments followed by multiple transmembrane helices (TMs). The transmembrane helices were connected by alternating cytoplasmic and extracellular loop segments (Fig. 4ii). In TaNRT1/TaNPF family, approximately 67% of the proteins contained 14 TMs, 21% contained 13 TMs, 7% of proteins contained 12 TMs while 4% of proteins contained less than 12 TMs (Supplementary Table 2). Subfamily wise studies showed TaNPF1 proteins contained only 13 TMs and TaNPF7 contained only 14 TMs. In rest of subfamilies (TaNPF2-6, TaNPF8) majority of proteins contained 14 TMs but variation existed. Proteins with even number of TMs had both C and N terminals in cytoplasmic side of membrane. Proteins with odd number of TMs had one end in cytoplasmic side and other in extracellular side (Fig. 4ii). All TaNRT2 family members contained only 12 TMs (Supplementary Table 2) (Fig. 4ii). Both C and N terminals of TaNRT2 proteins were present in cytoplasmic side of the membrane. Both TaCLC and TaSLAC1/SLAH proteins contained 10 TMs with both N and C terminals in cytoplasmic side of membrane. TaCLC genes were characterized by presence of a 30–40 amino acids long re-entrant helix in cytoplasmic side (Fig. 4 ii) which was not observed in the proteins of other nitrate transporter gene families.

Figure 4.

Figure 4

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Protein structure prediction: (i) representative structures of TaNPF genes (A–H), TaNRT2 genes (I) TaCLC genes (J) and TaSLAC1/TaSLAH genes (K). (ii) Representative TMs structures of nitrate transporters containing (A) 14 TMs, (B) 13 TMs (C) 12 TMs and (D) CLC proteins containing 10 TMs and a re-entrant helix. Figures were developed by homology-based modelling by Phyre2 server41.

Expression patterns of nitrate transporter genes in development stages of wheat

To elucidate the expression patterns of nitrate transporter genes, we studied and compared the expression data of Chinese spring and Azhurnaya for different developmental stages. Approximately 77% of TaNPF genes, 30% of TaNRT2, 85% of TaCLC genes and 36% of TaSLAC1/TaSLAH genes were expressed at least at one developmental stage in wheat with a wide expression range of 1–103 tpm (Supplementary Table 3, Supplementary Fig. 3). The remaining genes showed very low or no expression (tpm < 1). Overall, we identified 20 triads in which 48 genes were showing tissue specific expression, out of which 8 triads were root specific, 5 triads were leaf/shoot specific and 7 triads were showing grain/ spike specific expression (Supplementary table 4). Tissue and developmental stage-specific expression were observed in TaNPF1 genes, which were only expressed in spike and grain at the reproductive stage (Fig. 5A). Similarly, TaNRT2 genes were predominantly expressed in roots in both vegetative and reproductive stages (Fig. 5A). TaSLAC1/TaSLAH genes were predominately expressed in roots and leaves with some genes showing expression in spikes also (Fig. 5B). TaCLC genes showed mostly ubiquitous expression (Fig. 5B). For the rest of the subfamilies, the genes within one subfamily differed considerably in their expression patterns. In TaNPF2 genes, spike/grain specific (3 genes), leaf, spike and grain specific (5 genes) and ubiquitous expression (6 genes) were observed (Fig. 5A). TaNPF3 genes showed spike/grain, leaf specific expression, TaNPF4 genes showed leaf/root-specific (4 genes) and ubiquitous expression (10 genes) (Fig. 5A). TaNPF5 and TaNPF8 genes mostly showed ubiquitous expression though the root-specific expression was observed in a few genes (Fig. 5A). TaNPF6 showed ubiquitous (6 genes), leaf and root-specific (6 genes), spike specific (3 genes) and root-specific expression (Fig. 5A). TaNPF7 showed ubiquitous expression in three genes, grain specific expression in two genes and root-specific expression in one gene (Fig. 5A).

Figure 5.

Figure 5

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Expression patterns of nitrate transporter gene triads in wheat (a) Tissue and development stage specific expression profiles of TaNPF and TaNRT2 genes (b) Tissue and development stage specific expression profiles of TaCLC and TaSLAC1/SLAH genes. The heat maps were generated by heatmap tool from wheat expression database42 (http://wheat-expression.com/).

To find out up to what extent homoeologs differ in the expression patterns, triad expression analysis was performed. Most of the triads showed balanced expression ranging from 55.6 to 65.2% in all the tissues (Fig. 6A). In roots, a total of 54 triads were showing expression out of total 83 triads. Out of which 55.6% showed balanced expression, 18.5% showed A suppressed, 11.1% showed D suppressed, 9.3% showed B suppressed expression. Three triads showed A, B and D dominant expression (1 each) (Fig. 6B). In leaf/shoot out of 51 triads, 64.7% showed balanced expression, 9.8% showed A suppressed and B suppressed each, 3.9% triads showed D suppressed expression. 5.8% triads showed A and D dominant expression each while no B dominant expression was observed (Fig. 6B). In spikes, 61.9% triads out of 42 triads showed balanced expression. Only D dominant expression was observed in 9.5% of triads while A suppressed, B suppressed, and D suppressed expressions were in about 16.7, 7.1% 4.7% triads (Fig. 6B). Only 23 triads were expressing in grains at the reproductive stage, out of which 65.2% showed balanced expression, 8.7% triads showed A, B, and D suppressed each and 4.3% triads showed B and D dominant expression (Fig. 6B).

Figure 6.

Figure 6

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Triad expression of nitrate transporters in wheat (A) Overall triad expression of all nitrate transporter genes (B) Tissue specific triad expression of nitrate transporter genes. Normalized expression values were used to generate ternary plots using online web-based tool (https://www.ternaryplot.com/).

Nitrate transporter genes are located in close proximity to the NUE associated SNPs

In a parallel study in our laboratory, the nested synthetic wheat introgression (N-SHW) libraries capturing novel genetic variation from wild wheat for the nitrogen use efficiency related traits were developed and genotyped using a high-density SNP array43. These libraries were phenotypically assessed for the root traits and agronomic performance under three nitrogen input conditions (N: 0 kg ha−1; N: 60 kg ha−1 and N:120 kg ha−1) in the field over two years in 2018 and 2019. Genome-wide association mapping was used to identify marker-trait associations for the root and agronomic traits to identify the marker-trait associations for traits improving nitrogen use efficiency in wheat (Supplementary Table 5). We compared 322 marker trait associations for NUE identified in this study43 to nitrate transporter genes identified during genome wide analysis. We identified 67 SNPs, which were in close proximity to nitrate transporter genes in the wheat genome. A total of 93 nitrate transporter genes could be located near NUE linked SNPs, out of which, 63 genes belonged to TaNPF family, 15 genes belonged to TaNRT2 family, 11 genes belonged to TaCLC and 4 genes belonged to TaSLAC1/TaSLAH family (Table 4, Supplementary Fig. 5).

Table 4.

Proximity of nitrogen use efficiency (NUE) linked SNPs43 to nitrate transporters detected in present study.

SNP related to NUE Chromosome SNP Position Nearby nitrate transporters Nitrate transporter Position Distance (in Mb)
AX94950355 1A 12918698 TaNPF6-1A1 14519757 1.601059
AX94815202 1A 14468156 TaNPF6-1A1 14519757 0.051601
AX94665912 1B 624080881 TaSLAC-1B10 622365197 1.715684
AX94923560 2A 729858424 TaCLC-2A2 740847366 10.988942
AX94906008 2A 737049474 TaCLC-2A2 740847366 3.797892
AX95162328 2A 745066946 TaCLC-2A2 740847366 4.21958
AX94601746 2B 745715147 TaCLC-2B2 742813858 2.901289
AX95203088 2B 748700718 TaCLC-2B2 742813858 5.88686
AX95190948 2B 752830609 TaCLC-2B2 742813858 10.016751
AX95189671 2D 394797805 TaNPF4-2D2, TaCLC-2D1 394118961 395130092 0.332287, 0.678844
AX94829391 2D 601212191 TaCLC-2D2 608915455 7.703264
AX95142803 2D 601600533 TaCLC-2D2 608915455 7.314922
AX94799671 2D 608756380 TaCLC-2D2 608915455 0.159075
AX95142189 2D 609577225 TaCLC-2D2 608915455 0.66177
AX94786006 2D 610277424 TaCLC-2D2 608915455 1.361969
AX95148777 2D 641963392 TaNPF5-2D1-TaNPF5-2D5 639677529–643761743 1.798351–2.285863
AX95238274 3A 429463868 TaSLAC-3A4 421719078 7.74479
AX94593608 3A 671144035 TaNPF2-3A1, TaNPF2-3A2 660436466 660507764 10.636271, 10.707569
AX95237615 3B 6378879 TaSLAC-3B1 7598907 1.220028
AX95259763 3B 229302401 TaSLAC-3B3 227663976 1.638425
AX95136655 3B 235865416 TaSLAC-3B3 227663976 8.20144
AX94723497 3B 236511642 TaSLAC-3B3, TaNPF3B1 227663976 8.847666
AX94561045 3B 642481079 TaNPF5-3B3–TaNPF5-3B10, TaCLC-3B3 651425224–655435367 8.944145–12.954288
AX94539428 3B 657947249 TaCLC-3B3, TaNPF5-3B3–TaNPF5-3B10, TaNPF-3B4, TaNPF-3B5 651425224–662795946 2.511882–6.522025
AX94386613 3B 658604225 TaCLC-3B3, TaNPF5-3B3–TaNPF5-3B10, TaNPF-3B4, TaNPF-3B5 651425224–662795946 3.168858–7.179001
AX94418180 3B 659275308 TaCLC-3B3, TaNPF5-3B3–TaNPF5-3B10, TaNPF-3B4, TaNPF-3B5 651425224–662795946 3.839941–7.850084
AX94429243 3B 659787974 TaCLC-3B3, TaNPF5-3B3–TaNPF5-3B10, TaNPF-3B4, TaNPF-3B5 651425224–662795946 4.352607–8.36275
AX94910184 3D 352948426 TaCLC-3D1, TaNRT2-3D1 355885478 356623041 2.937052, 3.674615
AX94514369 4A 544201715 TaNPF4-4A1 533257983 10.943732
AX94926692 4A 544202284 TaNPF4-4A1 533257983 10.944301
AX94766675 4A 575009572 TaNPF8-4A6 575006132 0.00344
AX94400142 4A 581754986 TaCLC-4A1, TaNPF2-4A1, TaNPF7-4A1, TaNPF8-4A7, TaNPF8-4A8 585431883–593113134 3.676897–11.358148
AX94414780 4B 25929732 TaCLC-4B1, TaNPF2-4B1, TaNPF4B1 20278828–25842359 5.650904
AX94478236 4B 28716503 TaCLC-4B1, TaNPF2-4B1, TaNPF4B1 20278828–25842359 2.874144–8.437675
AX94997694 4B 34789538 TaCLC-4B1, TaNPF2-4B1, TaNPF4B1 20278828–25842359 8.947179–14.51071
AX94517352 4D 21886662 TaCLC-4D1, TaNPF2-4D1, TaNPF8-4D1 10764927–15356868 6.529794–11.121735
AX94586364 4D 22947854 TaCLC-4D1, TaNPF2-4D1, TaNPF8-4D1 10764927–15356868 7.590986–12.182927
AX94914919 4D 28974006 TaNPF8-4D2 28481269 0.492737
AX94738199 5D 10899555 TaNPF2-5D1 6820550 4.079005
AX95110067 5D 467774783 TaNPF4-5D3 464415850 3.358933
AX95002541 5D 468689841 TaNPF4-5D3 464415850 4.273991
AX95132327 5D 472234562 TaNPF4-5D3 464415850 7.818712
AX94631745 5D 528728651 TaNPF5-5D1—TaNPF5-5D3 528294425–528587054 0.141597–0.43423
AX94803288 6A 14353974 TaNRT2-6A1- TaNRT2-6A13 15727844–16408185 1.37387–2.054211
AX95017906 6A 23433182 TaNRT2-6A14 21634811 1.798371
AX94983341 6A 28412753 TaNRT2-6A14 21634811 6.777942
AX95210745 6A 29967076 TaNRT2-6A14 21634811 8.332265
AX94510892 6A 112585030 TaNPF8-6A1 117412062 4.827032
AX94534539 6A 497462168 TaNPF7-6A1 486547388 10.91478
AX94573487 6D 27978202 TaNPF5-6D1 22172543 5.805659
AX94415776 6D 28700804 TaNPF5-6D1 22172543 6.528261
AX94978974 6D 29876083 TaNPF5-6D1 22172543 7.70354
AX94737868 6D 29876631 TaNPF5-6D1 22172543 7.704088
AX95250225 6D 29928065 TaNPF5-6D1 22172543 7.755522
AX94461279 6D 451183032 TaNPF8-6D2 449226044 1.956988
AX94665619 7A 222939896 TaCLC-7A1 216343576 6.59632
AX94566038 7A 683488235 TaNPF5-7A3 692626752 9.138517
AX95178548 7B 112337703 TaNPF5-7B2 116046396 3.708693
AX94532247 7B 524619772 TaNPF8-7B1- TaNPF8-7B3 517623485–518338133 6.281639–6.996287
AX94424632 7B 562740463 TaNPF8-7B4–TaNPF8-7B6 556686657- 558639959 4.100504–6.053806
AX94880654 7B 592345313 TaNRT2-7B1 583923053 8.42226
AX94553632 7B 633318727 TaNPF5-7B4 624468356 8.850371
AX94781629 7D 206695834 TaCLC-7D1 204246408 2.449426
AX94678472 7D 487753483 TaNPF8-7D2–TaNPF8-7D4 489153269–489673028 1.39978- 1.91954
AX95080011 7D 592191388 TaNPF5-7D4 600836846 8.645458
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Response of nitrate transporter genes during N-starvation and N-recovery

The response of all N transporter genes towards N starvation and N recovery was analysed from WheatOmics database34–36,47,48. The results suggested that the expression of N transporter genes towards N starvation and N recovery was variable. We specifically identified the genes whose expression patterns changed significantly in response to N starvation or N recovery. The expression values of TaNPF1 and TaNPF3 genes were not significant (Fig. 7A,C). Three genes in TaNPF2 showed increased expression in N starvation and their expression values returned to normal during N recovery (Fig. 7B). The expression values of most of TaNPF5 genes were slightly reduced during N starvation and increased significantly during N recovery (Fig. 7E,F). TaNPF6 genes expression reduced during both N starvation and N recovery (1 h) but their expression returned to normal 24 h after recovery (Fig. 7G). The expression of most of TaNPF7 genes was upregulated during N starvation and N recovery (1 h) and downregulated after 24 h of N recovery (Fig. 7H). The expression of TaNPF4 and TaNPF8 genes was variable (Fig. 7D,I,J). The expression of most of TaNRT2 and TaCLC genes was upregulated during N recovery (1 h) phase (Fig. 7K,L,M,N). The expression values of some TaSLAC1/TaSLAH genes were reduced in response to N starvation and increased during N recovery (24 h) (Fig. 7O,P). Specifically looking into the expression pattern of 93 genes in close proximity of NUE associated SNPs, we could identify 32 genes whose expression pattern changed in response to N starvation and N recovery (Supplementary Fig. 6, Supplementary Table 6). These genes can serve as candidate genes and may be further utilized in genomics-assisted breeding programs targeting improved nitrogen-use efficiency in wheat.

Figure 7.

Figure 7

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Expression profiles of nitrate transporter genes in response to Nitrogen starvation and Nitrogen recovery. The graphs were generated by GeneExpression tool from WheatOmics 1.0 database47,48.

Discussion

The main aim of this study was to identify and analyse nitrate transporters belonging to all the four families and study their dynamics in wheat. The number of nitrate transporter genes detected in wheat was higher as compared to other plant species. This could be explained by a large genome (~ 18 Gb) and hexaploid nature of wheat. Presence of three homoeologous sub-genomes in wheat could allow multiple copies of nitrate transporters resulting in higher number of transporter genes. When comparing with diploid progenitors (Ae. tauschii and T. urartu) and tetraploid wheats (T. dicoccoides and T. turgidum) the number of genes in each subfamily were approximately proportional (Table 1). The genes were distributed randomly in the genome except for TaNRT2 genes which were predominantly present on group 6 homoeologous chromosome. Many genes were present in form of clusters and showed high percentage of similarity indicating gene-duplication events. There were genes with deleted segments present in the genome. The phylogenetic relationships with orthologues in other plants could be used to classify the genes in subfamilies. All the major subclades were conserved in wheat in comparison to other plant species indicating biological importance of the subfamilies. Based on phylogeny the genes could be grouped in homoeologous triads. Almost 73% of the genes could be assigned to 1:1:1 homoeologous groups which is very much above the average homoeologous retention rate (35.8%) in wheat (IWGSC 2018). Many genes were also grouped into tetrads and diads based on homology indicating gene duplication and deletion events in the genome. The overall results revealed that wheat nitrogen transporter families are much more complex than in other plant species. This complexity arises mostly due to presence of three sub-genomes (A B D) and gene duplication and deletion events.

The complexity of wheat genome also affects the expression patterns of genes. Due to presence of multiple sets of homoeologs on A, B and D genomes the buffering effects are observed in expression of genes. To study up to what extent these interactions affect the expression of nitrate transporters, triad expression analysis was performed. More than 55% of genes showed balanced expression in all the tissues which is comparable to genome-wide assessment of all transcripts in wheat42. The expression profiles of the genes identified in this study were in accordance to the previous studies in other plants. The expression patterns of nitrate transporter genes were similar to expression patterns of close orthologs in rice and Arabidopsis indicating the conservation of gene functions. CLC genes in previous studies in Arabidopsis showed ubiquitous expression which was observed in this study for wheat as well27,28. Several tissue specific nitrate transporter genes were identified which can be targeted for gene manipulation for wheat improvement. Several TaNRT2 and TaSLAC1/TaSLAH genes showed root specific expression suggesting their role in root nitrate uptake. Root specific expression of NRT2 and TaSLAC1/TaSLAH genes has already been reported in rice and Arabidopsis29,49. TaNPF1 genes and some TaSLAC1/SLAH genes showed grain and spike specific expression suggesting their role in nitrate transfer in developing seeds.

Structure plays a very important role in the function of transporter proteins. X-ray crystallographic structures of eukaryotic nitrate transporters have been elucidated50. All the nitrate transporter families belong to a much larger major facilitator superfamily (MFS) according to transporter classification database51. All the nitrate transporter proteins were predicted to have a typical MFS protein structure with multiple TMs. To the best of our knowledge our study is the first one to report homology-based models of nitrate transporter proteins belonging to all four families in wheat. The number of transmembrane segments play very important role in the optimal functioning MFS transporter proteins52. For an MFS transporter protein to have optimal transport properties pseudosymmetry is important which is provided by even number of TMs50. According to previous studies most of MFS proteins required 12 TMs to have optimal function53. In our study we predicted nitrate transporter families having variation in the number of TMs. TaNPF family being the largest of all showed most variation in the number of TMs with number ranging from 12 to 14. Several proteins with odd number of TMs were also observed. For example, all the members of TaNPF1 subfamily contain 13 TMs. All TaNRT2 proteins were highly conserved and contained 12 TMs. Most of the TaCLC and TaSLAC1/TaSLAH genes contained only 10 TMs. The variation in number of TMs between and within subfamilies and presence of odd number of TMs could not be corelated with expression data suggesting that a much more flexible criteria exists for the function of nitrate transporter proteins. The structural information presented in this study offer foundation for future work to identify molecular mechanisms responsible for functioning of nitrate transporters in wheat.

Previously in many studies overexpression of nitrate transporter genes has been linked to improved nitrogen use efficiency and yield in many plants54–57 and58. Overexpression of OsNRT2.1, OsNRT2.3b, OsNPF6.3 in rice and ZmNRT1.1A in maize has resulted in increased grain yield25,34–36,57,57,59. In wheat TaNRT2.1 is reported to be involved in post-flowering N uptake32 and is an important gene for improvement of nitrogen use efficiency. The CLC genes have been reported to be involved in nitrate accumulation in plants26 and many CLC genes have been reported to have role in stress responses. SLAC1 is a key player in regulation of stomatal closure. SLAH genes are involved in root nitrate and chloride acquisition and translocation to shoot. SLAC1/SLAH genes have also been reported to have important role in drought responses49. The genome wide analysis of TaCLC and TaSLAC1/TaSLAH genes in this study is the first reported study of these genes in wheat to the best of our knowledge. Nitrate transporters identified in this study can be promising candidates for gene manipulation to enhance productivity and nitrogen use efficiency in wheat. The identification of nitrate transporter genes in the close proximity to the marker-traits associations indicated the robustness of genome wide association mapping studies and the reliability of the reported transporter genes. The identified nitrate transporters could deepen the understanding of genetic and molecular mechanism behind improving nitrogen-use efficiency in wheat crop. The nutrient efficient improved breeding lines/accessions possessing identified potential nitrate transporters in the present study may have an effective and strong coordinated signal transduction network involving nitrate transceptor, nitrate response regulator and the master response regulator.

The in-silico mining of nitrate transporter genes along with their detailed structure, phylogenetic and expression studies reported a total of 412 nitrate transporter genes including 20 root specific, 11 leaf/shoot specific and 17 grain/spike specific putative candidate genes. The identification of nitrate transporter genes in the close proximity to the previously identified 67 marker-traits associations associated with the nitrogen use efficiency related traits in nested synthetic hexaploid wheat introgression library43 indicated the robustness of the reported transporter genes. The detailed crosstalk between the genome and proteome and the validation of identified putative candidate genes through expression and gene editing studies may lay down the foundation to improve nitrogen use efficiency of cereal crops. The existing genetic variability for 48 tissue specific genes and 93 genes in close proximity to NUE associated SNPs identified in the present study in different wild and cultivated wheat accessions/varieties may be further utilized in genomics-assisted breeding programs targeting improved nitrogen-use efficiency in wheat. A total of 32 genes out of these 93 genes show significant changes in expression patterns in response to N starvation and/ or N recovery suggesting their involvement in N uptake and assimilation. These genes can serve as initial candidates for targeting N use efficiency in wheat. The identification of improved breeding lines or the wild accessions possessing the potential nitrate transporters may serve as novel donors to be used in genomics-assisted introgression program developing nitrogen-efficient wheat varieties. The identified nitrate transporters may have potential for efficient nitrogen uptake and its transport from source to sink.

Once validated, the candidate genes may further be deployed in genomics-assisted breeding program to develop nutrient efficient wheat varieties. The present study provides important information on potential nitrate transporters that may lay foundation to develop a new breeding strategy for the sustainable agricultural development of cereal crops with less input—more output and the environmental protection. The identified nitrate transports may be of great significance both in the theory and in the genomics-assisted breeding application39–26.

Supplementary Information

Supplementary Information 1. (5.1MB, docx)
Supplementary Information 2. (893.1KB, xlsx)
Supplementary Information 3. (1.4MB, xlsx)

Acknowledgements

We are thankful to IWGSC and Wheat-OMICS database for the data availability. We are thankful to the Department of Biotechnology, Govt. of India for providing DBT RA fellowship.

Author contributions

N.S. and A.K. designed this study; A.K. conducted the in-silico studies and drafted the manuscript; N.S. conducted the field and genome wide association mapping studies and contributed to manuscript draft; P.K. helped in the in-silico analysis; G.P. and J.S. helped in visualisation; N.S., S.K. and P.C. provided resources and contributed to the critical revision of the manuscript.

Funding

The work was compiled under projects funded by the Department of Biotechnology, Govt. of India (Grant No. BT/IN/UK-VNC/42/RG/2015–16 and BT/PR30871/BIC/101/1159/2018).

Data availability

All data used in this research are included in this published article and its supplementary information files.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Footnotes

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-022-15202-w.

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