Characterization of the Nitrate Transporter gene family and functional identification of HvNRT2.1 in barley (Hordeum vulgare L.).

Nitrogen use efficiency (NUE) is the efficiency with which plants acquire and use nitrogen. Plants have high-affinity nitrate transport systems, which involve certain nitrate transporter (NRT) genes. However, limited data are available on the contribution of the NRT2/3 gene family in barley nitrate transport. In the present study, ten putative NRT2 and three putative NRT3 genes were identified using bioinformatics methods. All the HvNRT2/3 genes were located on chromosomes 3H, 5H, 6H or 7H. Remarkably, the presence of tandem repeats indicated that duplication events contributed to the expansion of the NRT2 gene family in barley. In addition, the HvNRT2/3 genes displayed various expression patterns at selected developmental stages and were induced in the roots by both low and high nitrogen levels. Furthermore, the overexpression of HvNRT2.1 improved the yield related traits in Arabidopsis. Taken together, the data generated in the present study will be useful for genome-wide analyses to determine the precise role of the HvNRT2/3 genes during barley development, with the ultimate goal of improving NUE and crop production.

Introduction

Nitrogen, as a function of nutrient availability, plays an essential role in controlling plant growth and development [1]. Limited nitrogen supplies decrease crop production. However, the excessive use of nitrogen fertilizers results in severe pollution and environmental deterioration [2]. Therefore, it is important to improve nitrogen use efficiency for cereal production with a low nitrogen supply [3].

Improving nitrogen use efficiency (NUE) in plants requires a more complete understanding of the transport of NO3- from the soil to the plant and within the plant itself. To date, two kinetically distinct nitrate uptake systems have been identified by physiological and molecular studies in plant roots, including the low-affinity transport system (LATS), which is encoded by the NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER (NRT1/PTR) family (NPF), and the high-affinity transport system (HATS), which is encoded by the nitrate transporter 2 (NRT2) family [4-8]. The functions of some NRT genes have been analyzed in plants. In Arabidopsis, AtNRT1.1 (CHL1), a dual-affinity nitrate transporter, functions as high-affinity and low-affinity nitrate transporters, when it is phosphorylated and dephosphorylated, respectively [9]. OsNRT1.1B (OsNPF6.5) is a putative homolog of CHL1, and it contributes to grain yield and NUE in rice [10]. The overexpression of OsNRT1.1A (OsNPF6.3) greatly improves N utilization and grain yield, as well as shortens the maturation time [11]. There is a lesser number of NRT2 genes than NRT1, and they are expressed predominantly in the root [8, 12]. In rice, OsNRT2.3 is transcribed into two spliced isoforms (OsNRT2.3a and OsNRT2.3b), and the overexpression of OsNRT2.3b leads to an improvement in grain yield and NUE by 40% [13]. However, when OsNRT2.3a expression is decreased, xylem loading of nitrate is impaired, and there is decreased plant growth at low nitrate levels [14]. This indicates that both alternative splices execute different functions in rice development.

NRT3 (also named NAR2) proteins are partner proteins that interact with most NRT2 proteins and contribute to high-affinity nitrate uptake [15]. In Atnrt3.1 mutant plant, the expression levels of AtNRT1.1 and AtNRT2.1 are reduced in response to nitrate induction, and constitutive high-affinity influx and high-affinity nitrate-inducible influx are dramatically decreased [15]. Similarly, OsNRT2.1, OsNRT2.2 and OsNRT2.3a are also synchronously suppressed in Osnar2.1 mutant, while high- and low-affinity nitrate transport is greatly impaired. A further analysis revealed that OsNAR2.1 not only interacts with OsNRT2.1/OsNRT2.2 but also with OsNRT2.3a [16]. Thus, the NRT2 genes combine with the NRT3 genes to enable the plant to cope with variable nitrate supplies.

In barley, four HvNRT2 (HvNRT2.1-2.4) and three HvNRT3 (HvNAR2.1-2.3) genes were isolated from the roots. The expression pattern of the HvNRT2 genes was also characterized under various nitrogen sources, and a highly specific interaction is suggested between HvNRT2.1 and HvNAR2.3 by using 15N-enriched nitrate uptake into Xenopus oocytes. However, until now, none of the HvNRT2/3 gene family have been described at the barley genome level and their functions are still unclear [17-19]. In the present study, we identified HvNRT2 and HvNRT3 genes in barley using bioinformatics and constructed a phylogenetic tree. Then, the expression patterns of these genes in response to low and high nitrate levels were analyzed. In addition, the HvNRT2.1 gene was overexpressed to determine its function in Arabidopsis. These results will be useful in further investigations into the functions of the NRT family in high-affinity nitrate uptake in plants.

Materials and methods

Plant materials

Barley cv. Morex was used in the present study. The seeds were surface-sterilized and germinated on wet filter paper at 25°C for 3 days. The germinated seeds were transferred into 60-well plastic containers (25 L) with aerated and nitrogen-free one-tenth-strength modified Johnson’s solution for 4 days [20]. The plants were then supplied with 0.2 mM NO3- (Low nitrogen, LN) or 5 mM NO3- (High nitrogen, HN). The plants were grown in a growth chamber at 25°C/22°C (day/night) under a 16-h/8-h light/dark cycle and 70% relative humidity. At 0 h, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h after the treatment, the total roots were harvested from ten plants and were immediately frozen in liquid nitrogen for RNA extraction, with three biological replicates at each time point. For each replicate, the RNA from four plants of each genotype was analyzed. The Arabidopsis thaliana Columbia-0 (Col-0) ecotype was used as the wild type. The plants were grown in a greenhouse in soil at 20°C under long-day conditions (16 h light/8 h dark). For the in vitro seedling assays, the seeds were surface sterilized and cold treated at 4°C for 3 days in the dark and were then exposed to white light. The plants were grown at 20°C on horizontal or vertical plates containing Murashige and Skoog (MS) medium, 3% sucrose, and 0.9% agarose (Merck).

Sequence database searches

The amino acid sequences of the NRT2 and NRT3 genes generated from Arabidopsis [15, 21], rice [22], and maize [23, 24] were used as query sequences. The barley sequence data were sourced from the Morex (http://webblast.ipk-gatersleben.de/barley/) [25], Gramene (http://ensembl.gramene.org/Hordeum_vulgare/Info/Index), and NCBI databases (http://www.ncbi.nlm.nih.gov/). BLAST programs (TBLASTN and BLASTN) were available for the IPK barley genome database and the NCBI barley EST database. Multiple database searches were performed to collect all members of the barley NRT2 and NRT3 genes. Domain searches (PF07690.14 and PF16974.3) were performed using SignalP (http://www.cbs.dtu.dk/services/SignalP/) [26], HMMSCAN (https://www.ebi.ac.uk/Tools/hmmer/search/hmmscan) [27], and SMART (Simple Modular Architecture Research Tool: http://smart.embl-heidelberg.de/) [28], with the default cutoff parameters. The isoelectric points and protein molecular weights were obtained with the help of the proteomics and sequence analysis tools on the ExPASy proteomics server (http://expasy.org/) [29]. The names of the HvNRT2 genes were designated according to their ascending order in barley chromosomes. However, the names of the HvNRT3 genes were given according to the definition provided by Tong et al. [30].

Chromosomal location, gene structure, and duplication events of the NRT2/3 genes

The chromosomal locations were retrieved from the IPK database (http://webblast.ipk-gatersleben.de/barley/). All the genes were mapped to the chromosomes with MapDraw software [31]. The exon/intron structures were constructed using GSDS (http://gsds.cbi.pku.edu.cn/) [32]. Tandem duplication genes were identified manually if they were within the 10 predicted genes or within 30 kb of each other on the physical barley map [33]. Segmental duplications were identified by BLASTP in ten predicted proteins upstream and downstream of each of the HvNRT2/3 genes [34].

Phylogenetic tree analysis

Full-length amino acid sequences of the HvNRT2/3 genes identified in barley were aligned using the Clustal X 1.83 program with the default pairwise and multiple alignment parameters. The phylogenetic tree was constructed based on this alignment using the neighbor joining (NJ) method in MEGA version 6 with the following parameters: Poisson correction; pairwise deletion; uniform rates; and bootstrap (1000 replicates) [35]. Conserved motifs were investigated by multiple alignment analyses using MEME version 3.0 [36].

NRT2 and NRT3 gene expression analyses

Gene expression data from eight tissues of the cultivar ‘Morex’ were obtained from the barley genome database (http://apex.ipk-gatersleben.de/apex/f?p=284:10:6281639160219::NO). Deep RNA sequencing (RNA-seq) was carried out on fifteen Morex tissues from almost all stages of the barley life cycle, which was comprised 4-day embryos derived from a germinated seed, the roots and shoots from a 10 cm seedling (10 cm shoot stage), inflorescences (5 cm and 1-1.5 cm), developing grains at 5 and 15 days after pollination (DAP), an etiolated seedling (10 DAP), the epidermal strips and roots (28 DAP), rachis of inflorescences (35 DAP), lemma, lodicule, and palea (42 DAP), developing tillers at the 3rd internode (42 DAP), and senescing leaves (56 DAP) [25]. The expression patterns are presented as heat maps in green/yellow/red coding, which reflects the FPKM (Fragments Per Kilobase of transcript per Million mapped reads), with red indicating a high expression level, yellow indicating a moderate expression level, and green indicating a low expression level.

Isolation of total RNA and quantitative real-time PCR

Total RNA was isolated using an RNA extraction kit (TRIzol reagent, Invitrogen, USA), and the isolated RNA was incubated with RNase-free DNase I (TaKaRa, Japan) to remove any contaminating DNA. The RNA quality and yield were analyzed by agarose gel electrophoresis and a NanoDrop 1000 Spectrophotometer V 3.7. First strand cDNA was generated from 2 μg of total RNA with M-MLV reverse transcriptase (TaKaRa, Japan) using random primers. The specific primers used for the quantitative real-time PCR analysis are listed in S1 Table. The reactions were carried out in 20 μl reaction systems containing 10 mM Tris-HCl (pH 8.5), 50 mM KCl, 2 mM MgCl2, 0.4 μl DMSO, 200 mM dNTPs, 10 pmol specific PCR primers, 1 U Taq DNA polymerase, and 0.5 μl SYBR GREEN I fluorescence dye. Quantitative real-time PCR was performed using a ViiA™ 7 Real-Time PCR System (Applied Biosystems, USA). The running protocol was as follows: 94°C for 3 min, followed by 40 cycles at 94°C for 30 s; 58°C for 30 s; 72°C for 30 s; and a final extension of 72°C for 5 min. The amplification of HvActin (Accession number HORVU1Hr1G002840) was employed as an internal standard. All the reactions were run in triplicate. The Ct values were determined by ViiA™ 7 software using the default settings. The relative expression levels of the target genes were determined using the 2-ΔΔCt method [37]. For each sample, the PCR was performed with three biological replicates.

Generation of Arabidopsis transgenic plants

The full-length coding sequence of HvNRT2.1 was cloned into the Gateway entry vector pDNOR221 (S1 Table). After confirmation by DNA sequencing, the HvNRT2.1 fragments were recombined into the pB2GW7 destination vector, which expresses under the control of the CaMV35S promoter. The resulting construct was introduced into the Agrobacterium tumefaciens strain GV3101 and was used to transform the Arabidopsis plants using the floral dip method. The transgenic plants were selected by herbicide treatment, and three representative T3 homozygous lines were obtained for the phenotypic analysis. The statistical analysis of the differences between the wide type and transgenic plants was performed by using a Student’s t-test.

Silique and seed size measurements

Mature siliques were harvested, and their lengths were recorded using vernier calipers. The seed size measurement was performed as described previously [38]. Briefly, the siliques were harvested once they were dry. The seeds were allowed to dry completely in centrifuge tubes for at least seven days before the measurement. Next, the seeds were spread and taped onto a piece of white paper, so that none of the seeds were touching each other. Using a flatbed scanner (Canon CanoScan 9000F), images of each accession was obtained at a resolution of 300 dpi with transmitted light. The “particle analysis” feature of ImageJ software was used to measure the seed length, width, and area. Three biological replicates were undertaken with 10 plants per transformed plant.

Nitrate and nitrogen content measurements

To determine the tissue nitrate content at maturity, Col-0 and transgenic plants were weighed and boiled in distilled water for 5 min, and the nitrate content was determined by the Cataldo method [39]. Whole plants were dried to a constant weight at 80°C and were ground in a Cyclotec 1093 sample mill (Hoganas City, Sweden) before being sieved through a 0.5 mm screen. The total nitrogen content in the plants was quantified according to the Kjeldahl method by using FOSS Kjeltec TM 2300 (Foss Analytical AB, Sweden) [40]. The statistical analysis of the differences in aerial part traits between the Col-0 and transgenic plants was performed using a Student’s t-test.

Results

Identification of NRT2/3 genes in the barley genome

A total of ten putative HvNRT2 and three putative HvNRT3 genes from the entire barley genome were identified (Table 1). The HvNRT2 genes encoded proteins ranging from 483 (HvNRT2.10) to 514 (HvNRT2.2) amino acids, with protein masses that ranged from 50.40 kD to 55.38 kD and protein pI values ranging from 7.01 (HvNRT2.10) to 8.98 (HvNRT2.1) (Table 1). However, the HvNRT3 genes encoded low molecular weight proteins compared to the HvNRT2 genes, with the protein masses ranging from 21.13 kD to 21.27 kD. These were also basic proteins (the pI values ranged from 9.00 to 9.23). A further analysis revealed that HvNRT3.2 was located on the end of 5HL, HvNRT2.2-9 was on the end of 6HS, HvNRT3.1 on the centromeric regions of 6H, and the remaining genes were distributed on the long arm of chromosomes 3, 6, and 7 (Fig 1). Remarkably, the duplication event analysis indicated that 8 (8/10, 80%) of the HvNRT2 genes were tandem repeated (Fig 1). The tandem duplicated genes contained two clusters, and each cluster contained four genes (HvNRT2.2-5 and HvNRT2.6-9 gene pairs). The gene structural analyses showed that the HvNRT2 genes contained one exon. However, the HvNRT3 genes shared three exons (S1 Fig).

Table 1

The information of HvNRT2/3 genes in barley.

Gene Name	Gene ID	Chr.	Physical Position on Barley Genome (start position (bp)-end position (bp))	Coding Sequence Length (bp)	Amino Acid Length (aa)	Mass (Da)	pI
HvNRT2.1	HORVU3Hr1G066090.1	3	503310429-503312717	1542	514	55376.14	8.98
HvNRT2.2	HORVU6Hr1G005570.1	6	12363356-12364512	1521	507	54703.55	8.39
HvNRT2.3	HORVU6Hr1G005580.1	6	12371132-12373090	1521	507	54673.52	8.39
HvNRT2.4	HORVU6Hr1G005590.1	6	12378589-12380579	1521	507	54627.44	8.39
HvNRT2.5	HORVU6Hr1G005600.2	6	12385842-12387748	1527	509	54974.94	8.53
HvNRT2.6	HORVU6Hr1G005720.1	6	12654106-12655479	1524	508	54658.86	8.67
HvNRT2.7	HORVU6Hr1G005770.1	6	12754340-12756226	1527	509	54553.54	8.14
HvNRT2.8	HORVU6Hr1G005780.1	6	12765305-12766893	1521	507	54367.15	7.9
HvNRT2.9	HORVU6Hr1G005930.1	6	13075246-13077142	1524	508	54636.74	8.17
HvNRT2.10	HORVU7Hr1G098550.4	7	598494068-598496471	1449	483	50402.35	7.01
HvNAR3.1	MLOC_3053.1	6	268,053,711-268,054,907	591	197	21129.3	9.11
HvNAR3.2	HORVU5Hr1G115500.3	5	646684461-646686179	777	259	21268.37	9
HvNAR3.3	HORVU6Hr1G053710.1	6	336809019-336810586	594	198	21137.32	9.23

Fig 1

The chromosomal location of the HvNRT2/3 genes in the barley physical map.

The red lines represent tandemly duplicated gene pairs.

Conserved amino acid sequence and phylogenetic tree for the HvNRT2/3 proteins in plants

Bioinformatic methods were employed to analyze the HvNRT2 and HvNRT3 proteins in the present study, revealing that an MFS domain and an NAR domain were present in the HvNRT2 and HvNRT3 proteins, respectively (S2 Fig). The alignment and comparison of the HvNRT2 full length protein sequences illustrated that the proteins had ten (HvNRT2.10), eleven (HvNRT2.1/6/8), or twelve (HvNRT2.2/3/4/5/7/9) (S2 Fig) transmembrane domains, while only one transmembrane region was found in the HvNRT3 proteins.

According to the phylogenetic tree of the NRT2 and NRT3 proteins in barley, rice, maize, and Arabidopsis, the NRT2 proteins could clearly be divided into three distinct clusters (Ⅰ, Ⅱ, and Ⅲ). A total of 18 NRT2 genes belonged to cluster Ⅰ, including five NRT2 genes derived from dicotyledonous Arabidopsis and 13 NRT2 genes from monocotyledonous plants (8, 2, and 3 genes from barley, rice, and maize, respectively). AtNRT2.5 and AtNRT2.7 represented clusters Ⅱ and Ⅲ, containing four and three genes, according to the phylogenetic tree, respectively (Fig 2A). In the NRT3 phylogenetic tree, the proteins derived from the dicotyledonous and monocotyledonous plants clustered together (Fig 2B), indicating an evolutionary dichotomy of the NRT2/3 genes between the dicot and monocot plants. In addition, the number of NRT2 and NRT3 genes in barley was larger than in other plants.

Fig 2

Phylogenetic analysis of the NRT2/3 proteins in Arabidopsis, maize, rice, and barley.

A, the phylogenetic analysis of the NRT2 genes; B, the phylogenetic analysis of the NRT3 genes. NRT2 gene sequence of Arabidopsis, maize, and rice generated from the report by Plett et al. [20].

HvNRT2/3 gene expression patterns at different developmental stages

An exploration of the expression patterns of genes contributes to our understanding of their molecular function [41]. Thus, we searched the deep RNA sequencing (RNA-seq) data from the cultivar ‘Morex’ [25, 42]. A heat map of the FPKM signal values for the 13 HvNRT2/3 genes in the selected Morex tissues is presented in Fig 3. All of the HvNRT2/3 genes displayed relatively high expression levels in the 4-day embryos dissected from germinating seeds (except HvNRT2.10) and in roots (10 cm shoot stage and 28 DAP). Conversely, a limited expression of the HvNRT2/3 genes was observed in the developing inflorescences (0.5-1.5 cm) and grains (5 and 15 DAP). In addition, HvNRT2.10 had a higher expression level than other genes in the shoots (10 cm shoot stage), while HvNRT3.2 had a relatively high expression level in the senescing leaves. Remarkably, HvNRT3.2 showed an exceptionally high expression level compared to the other genes in the developing tillers (the third internode at 42 DAP), etiolated seedlings (10 DAP), lemmas (42 DAP), lodicules (42 DAP), palea (42 DAP), epidermis (28 DAP), and rachises (35 DAP). This expression pattern analysis may contribute to our understanding of the function of the HvNRT2/3 genes in barley.

Fig 3

Expression profile analysis of the HvNRT2/3 genes in barley.

The expression patterns are presented as heat maps in green/yellow/red coding, which reflects the FPKM, with red indicating high expression levels, yellow indicating moderate expression levels, and green indicating low expression levels. The data on the expression of the HvNRT3.1 gene were only gathered from eight selected tissues [39]. The default values are shown in white.

HvNRT2/3 gene expression analysis under low and high nitrogen levels

The expression pattern of the HvNRT2 and HvNRT3 genes in barley roots was investigated using quantitative real-time PCR. As shown in Fig 4, when the barley roots were exposed to low (0.2 mM) and high (5 mM) nitrogen levels, the number of transcripts of seven HvNRT2 and two HvNRT3 genes increased, with peaks at 1 h (HvNRT2.1/10 genes), 3 h (HvNRT2.2/5 genes), and 6 h (HvNRT2.3/4/9 and HvNRT3.1/2 genes), before gradually decreasing. However, the mRNA levels of HvNRT2.6/7/8 rapidly increased and peaked at 6 h under the low nitrogen conditions and at 72 h under the high nitrogen conditions. In addition, the expression of HvNRT3.3 displayed two peaks at 3 h and 24 h and responded more quickly to high rather than low nitrogen conditions.

Fig 4

Expression patterns of the HvNRT2/3 genes in the roots pretreated with low and high nitrogen levels.

A, expression patterns of the HvNRT2 genes; B, expression patterns of the HvNRT3 genes.

Morphological characterization of the HvNRT2.1 overexpression plants

In order to investigate the functional roles of the barley NRT genes, HvNRT2.1, which has a high homology to OsNRT2.3, and was overexpressed in Arabidopsis. In the transgenic lines, HvNRT2.1 was introduced into the genomic DNA and was expressed at the transcription level in three independent overexpression plants (S3 Fig). Phenotypic changes were observed between the Col-0 and overexpression plants at the mature stage (Fig 5). There were no obvious differences in the plant height, dry weight, and number of siliques per plant between the Col-0 and transgenic lines (Fig 6A, 6B and 6D). However, 7.9%, 18.5%, and 23.3% increments in the length of siliques, yield per plant and grain number per siliques, respectively, were observed in the transgenic lines compared with Col-0 (Fig 6C, 6E and 6F). The size of the seeds, including the seed length, width, and thousand grain weight, was higher in the three independent HvNRT2.1 transgenic lines compared to the wild type (Fig 6G, 6H and 6I). The seed length and width significantly increased by 11.5% and 24.5% compared to the wild type, respectively. The thousand grain weight dramatically increased by 37.0%, suggesting that the barley HvNRT2.1 gene positively regulated seed development. In addition, the seeds from the overexpression plant accumulated significantly more nitrate and nitrogen, and these increased by 47.2% and 41.4%, respectively, compared with the wide type for the mature plants (Fig 7).

Fig 5

Phenotype of the overexpression and Col-0 plants.

A, Whole plant; B, Silique; C, Seed.

Fig 6

Phenotype analysis of the Col-0 and overexpression plants.

A, plant height; B, dry weight; C, yield per plant; D, number of silique per plant; E, length of silique; F, grain number per plant; G, seed length; H, seed width; I, thousand grain weight. ∗p < 0.05; ∗∗p < 0.01.

Fig 7

Nitrate and nitrogen content in the Col-0 and overexpression seeds.

A, nitrate content; B, nitrogen content. ∗p < 0.05; ∗∗p < 0.01.

Discussion

Both NRT2 and NRT3 proteins are involved in high-affinity nitrate uptake in plants [15]. Due to the sequencing of plant genomes, the NRT2/3 genes have been widely analyzed in plants. For example, a total of 4, 7, and 4 NRT2 genes have been analyzed in rice, Arabidopsis, and maize, respectively, as well as two NRT3 genes [8, 15, 23–24, 43]. Although several HvNRT2 and HvNRT3 genes have been cloned in barley [30, 44], there is no information about barley HvNRT2/3 genes at the genome level. The present study identified ten putative NRT2 and three putative NRT3 genes that encode components of the high-affinity nitrate transport system (HATS) in barley. The number of NRT2/3 genes in barley is greater than those in rice, Arabidopsis, and maize. A duplication event analysis indicated that tandem duplication played an important role in the expansion of the NRT2 genes and may have contributed to the difference in the number of NRT genes in barley, rice, Arabidopsis, and maize [45]. In addition, SignalP and SMART predicted that three HvNRT3 proteins had signal peptides with 21 (HvNRT3.2/3) and 25 (HvNRT3.1) amino acids (S2 Table), which was similar to that of the AtNRT3 genes in Arabidopsis.

Phylogenetic analyses and evolutionary relationships are used to investigate the functions of genes in plants [46]. In rice, OsNRT2.1 and OsNRT2.2 share identical amino acid sequences and are clustered together with other NRT2 genes. Rice is a monocotyledon plant known to have undergone tandem duplication [8]. Interestingly, similar events were detected at the end of 6HS, where the HvNRT2.2/3/4/5 and HvNRT2.6/7/8/9 genes were clustered. Both HvNRT2.1 and HvNRT2.10 were closely related to OsNRT2.3 and OsNRT2.4 based on the phylogenetic tree. It is worth noting that barley has one exon in common with rice (except OsNRT2.4) but not Arabidopsis, suggesting that these NRT2 genes diverged before the monocotyledon-dicotyledon separation in higher plants [8, 12]. The structure of the NRT3 genes with two exons is conserved in plants, while divergence is observed in the protein homology between monocotyledon and dicotyledon plants [8, 15].

Expression analyses of organs is employed to predict the potential role of specific genes in plant growth and development, as well as their response to different environments [47]. NRT2 genes, as members of the high-affinity transport system, combined with NRT3 genes, were detected in the roots with high abundance levels, indicating that both gene families are responsible for induction either at low or high nitrate levels in the root tissue [8, 12, 15]. All of the thirteen genes investigated had high expression levels in the root tissue and displayed obvious changes following low and high nitrogen treatments. Remarkably, HvNRT2.10 is a homologous of AtNRT2.7 and OsNRT2.4, with 45% and 51% amino acid identity, respectively. It has a similar pattern of expression compared to its homologs, with higher expression levels in the shoots than the roots, and is also relatively highly expressed in seeds, which is consistent with the AtNRT2.7 gene [48, 49]. In rice, a decreased expression of OsNRT2.3a is related to the accumulation of significantly higher levels of nitrate and total nitrogen in the root and lower levels in the shoot [14]. However, enhancing the expression of OsNRT2.3b improves the growth, yield, and NUE in rice [13]. The HvNRT2.1 protein had a similar amino acid length and showed 83.3% identity with OsNRT2.3a, but only 78.4% identity with OsNRT2.3b genes, indicating that HvNRT2.1 plays an important role in long-distance nitrate transport, from the roots to shoots, under low nitrate supply levels in barley. These results will be useful in further investigations of HvNRT2.1 and HvNRT2.10 in barley.

NRT3 genes encode a partner protein that interacts with some members of NRT2 in plants [16, 50]. For example, AtNRT2.1 combined with AtNRT3.1 forms a 150-kDa plasma membrane complex. Both genes were thought to constitute the high-affinity nitrate transporter of Arabidopsis roots [50]. OsNRT3.1 (OsNAR2.1) was induced by low and high nitrogen levels, and in vitro experiments demonstrated that OsNAR2.1 interacts with OsNRT2.1 and OsNRT2.2, as well as OsNRT2.3a [16]. Remarkably, eight HvNRT2 genes and two HvNRT3 genes displayed co-expression patterns under low nitrogen conditions. Therefore, the interactions between the HvNRT2 and HvNRT3 genes should be further investigated to clarify the functions of the NRT genes in barley.

Several NRT2 genes have been characterized in plants. For example, an Atnrt2.1-1 mutant was specifically deficient in HATS, and a further analysis showed that when the ATNRT2.1 protein combined with ATNAR2.1, it forms a complex that transports nitrate efficiently [15, 51, 52]. ATNRT2.7, which had a high expression level in the dry seeds, was localized to the vacuolar membrane and displayed a positive regulation of nitrate content in the seeds. In the present study, the overexpression of HvNRT2.1 led to high nitrate and nitrogen contents and improved the yield-related traits of Arabidopsis. However, both a biomass and plant height increase were not observed in the overexpression plants, indicating that the HvNRT2.1 gene plays a specific role in nitrate and total nitrogen accumulation in plants.

Conclusion

In summary, ten putative NRT2 and three putative NRT3 genes were identified from barley sequencing using bioinformatic methods. A duplication event analysis indicated that tandem repeats contributed to the expansion of the NRT2 gene family in barley. A phylogenetic tree revealed that the NRT2/3 proteins displayed a clear divergence between the monocot and dicot plants. In addition, the HvNRT2/3 genes displayed various expression patterns at selected development stages and were induced in the roots under low and high nitrogen levels. Remarkably, HvNRT2.1, as a homologous gene of OsNRT2.3, significantly improved the yield-related traits in Arabidopsis. Thus, the data generated in the present study will be powerful for genome-wide analyses to determine the precise role of the HvNRT2/3 genes during barley development, with the ultimate goal of improving NUE and crop production.

Gene structure analysis of the HvNRT2/3 genes.

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Alignments of the HvNRT2/3 proteins.

Amino acid sequence alignments of the HvNRT2 (A) and HvNRT3 (B) proteins. The conserved transmembrane sequence regions are indicated by the black lines above the sequences. The red frame indicates the MFS and NAR domains in the HvNRT2 and HvNRT3 proteins, respectively.

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PCR analysis of the overexpression and wild-type (Col-0) plants.

A, PCR analysis at the genomic level; B, RT-PCR analysis at the transcription level.

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Primers used in this study.

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Summary of the functional domains present in the NRT proteins.

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31 Jan 2020

PONE-D-19-33678

Characterization of the Nitrate Transporter gene family and functional identification of HvNRT2.1 in barley (Hordeum vulgare L.)

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript described a genome-wide characterization of high-affinity nitrate transporter NRT2/3 family members by taking advantage of the publically-released barley reference sequence. The gene duplication, transcriptional pattern and chromosomal assignment as well as the phylogenetic analysis were further represented. One NRT2-type gene HvNRT2.1 was over-expressing in transgenic Arabidopsis plants that showed the increases on the parameters of grains, as well as the accumulation of nitrate and total nitrogen uptake in plant tissues.

1, A short introduction referring to the current understanding of individual HvNRT2/3 gene in cultivated barley is suggested.

2, Fig. 7 showing the increases of nitrate and total nitrogen content should be described in Results. This might be important in order to support the story of this study.

3, Use Arabidopsis thaliana or A. thaliana. Refer the variety “Col-0” in Materials.

4, Fig. 4, statistical analysis is recommended (e.g. Tukey’s HSD).

5, The language editing is suggested.

Reviewer #2: The authors mapped the NRT2/3 gene family in the barley genome and analysed their expression patterns under N treatments. Besides, one member from the family HvNRT2.1 was exotically expressed in Arabidopsis for function characterisation. The work provides new insights into the barley NUE research and I have the following concerns before it could be accepted for publishing.

Major issues

1. The English language needs professional proofreading as there are massive typos and grammar errors in the main document.

2. Why the HvNRT2.1 but not other NRT transporters were specifically investigated in Arabidopsis?

3. Line 223, information missing in the sentence

4. Line 256, peaks at ??

5. Line 261, how much similar are they (% identity)?

6. Line 264, was Arabidopsis ecotype Col-0 used? Should not be in italic.

7. Line 267, how much increments?

8. Line 278, two NRT3 from which species?

9. Line 282, what does “components” mean?

10. Line 307-309, please specify the details in the changes.

11. Line 313-318, state one or two functions in rice so that it can be more evidently related to barley.

12. Line 335, the statement has been repeated a couple of times in the ms.

13. Line 338, increase of biomass and plant height in Arabidopsis or ?? If so could be rephrased as “In the present study, overexpression of HvNRT2.1 led to high nitrate and nitrogen content, increased biomass and plant height and improved the yield-related traits in Arabidopsis indicating that….

14. The authors should provide either qualitative or quantitive information regarding the HvNRT2.1 transcript levels in transgenic Arabidopsis lines.

15. HvNRT2.1 expression in barley responded to different N levels so why not investigate the growth performance of transgenic Arabidopsis lines under N treatments?

Minor issues

1. Line 20, rectify the grammar issue

2. Line 23, in barley “nitrate transport”

3. Line 25, “chromosomes”

4. Line 29, replace “notably” with “furthermore”

5. Line 55, should be “they are” but not “it is”

6. Line 61, under low N condition?

7. Line 86-89, confusing with the experimental layout. Indeed how many plants were sampled and used for RNA extraction?

8. Line 137, “0.5 cm” would be better.

9. Line 224, according “to”

10. Line 228, “clustered”

11. Line 320, “encode”

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Yong Han

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

10 Mar 2020

Responses to reviewer 1:

Comment 1: A short introduction referring to the current understanding of individual HvNRT2/3 gene in cultivated barley is suggested.

Response 1: According to the reviewer’s suggestion, we have added the sentence in the revised manuscript. In barley, four HvNRT2 (HvNRT2.1-2.4) and three HvNRT3 (HvNAR2.1-2.3) genes were isolated from the roots. The expression pattern of the HvNRT2 genes was also characterized under various nitrogen sources, and a highly specific interaction is suggested between HvNRT2.1 and HvNAR2.3 by using 15N-enriched nitrate uptake into Xenopus oocytes. However, until now, none of the HvNRT2/3 gene family have been described at the barley genome level and their functions are still unclear.

Comment 2: Fig. 7 showing the increases of nitrate and total nitrogen content should be described in Results. This might be important in order to support the story of this study.

Response 2: We agree with this suggestion and have added the result in the “Morphological characterization of the HvNRT2.1 gene overexpression plants” paragraph as follows: In addition, seeds of overexpression plant accumulated significantly more nitrate and nitrogen, and increased by 47.2% and 41.4% compared with wide type in mature plants (Fig 7).

Comment 3: Use Arabidopsis thaliana or A. thaliana. Refer the variety “Col-0” in Materials.

Response 3: Thank the reviewer for this content. The Arabidopsis thaliana Columbia-0 (Col-0) ecotype was used as in materials. We have revised this in the new manuscript (see “Plant materials” part)

Comment 4: Fig. 4, statistical analysis is recommended (e.g. Tukey’s HSD).

Response: Thank the reviewer’s suggestion, statistical analysis of the differences between wide type (Col-0) and transgenic plants was performed by using Student’s t-test. We have added the result of Student’s t-test in Fig 6 and Fig 7.

Comment 5: The language editing is suggested.

Response: The statements have been corrected. We will be happy to edit the MS further base on helpful comments from reviewers.

Responses to reviewer 2:

Major issues

Comment 1: The English language needs professional proofreading as there are massive typos and grammar errors in the main document.

Response 1: The statements have been corrected. We really hope the flow and language level have been improved.

Comment 2: Why the HvNRT2.1 but not other NRT transporters were specifically investigated in Arabidopsis?

Response 2: HvNRT2.1 gene was the first to respond to low nitrogen with peaks at 1 h, which has high homology to OsNRT2.3 (a gene derived from rice which has been contribute to improvement grain yield and NUE by 40%). Therefore, HvNRT2.1 was specifically investigated in Arabidopsis.

Comment 3: Line 223, information missing in the sentence.

Response 3: Thank the reviewer for this content. The sentence was as follows: A total of 18 NRT2 genes belonged to cluster Ⅰ, including five NRT2 genes derived from dicotyledonous Arabidopsis and 13 NRT2 genes from monocotyledonous plants (8, 2, and 3 genes from barley, rice, and maize, respectively). AtNRT2.5 and AtNRT2.7 represented clusters Ⅱ and Ⅲ, containing four and three genes, according to the phylogenetic tree, respectively (Fig 2A). We have revised this in the new manuscript.

Comment 4: Line 256, peaks at ??

Response 4: Thank the reviewer’s for this content. We have revised this as follows: The expression of HvNRT3.3 displayed two peaks at 3 h and 24 h and responded more quickly to high rather than low nitrogen conditions.

Comment 5. Line 261, how much similar are they (% identity)?

Response 5: According to the reviewer’s suggestion. We analyzed the homology identity between HvNRT2.1 and OsNRT2.3. The detail information was as follow: The HvNRT2.1 protein had a similar amino acid length and showed 83.3% identity with OsNRT2.3a, but only 78.4% identity with OsNRT2.3b genes, indicating that HvNRT2.1 plays an important role in long-distance nitrate transport, from the roots to shoots, with low nitrate supply levels in barley. We have added this in the new manuscript (see “Discussion” part).

Comment 6: Line 264, was Arabidopsis ecotype Col-0 used? Should not be in italic.

Response 6: Thank the reviewer for this content. The Arabidopsis thaliana Columbia-0 (Col-0) ecotype was used as in materials. We have revised this in the new manuscript (see “Plant materials” part)

Comment 7: Line 267, how much increments?

Response 7: According to the reviewer’s suggestion, the increments were calculated in the new revised manuscript. The detail information was as follows: 7.9%, 18.5%, and 23.3% increments in the length of siliques, yield per plant and grain number per siliques, respectively, were observed in the transgenic lines compared with Col-0 (Fig 6C, E, and F).

Comment 8. Line 278, two NRT3 from which species?

Response 8: Two NRT3 have been analyzed from rice, Arabidopsis and maize. Thus, we have revised this sentence in the manuscript as follows: a total of 4, 7, and 4 NRT2 genes have been analyzed in rice, Arabidopsis, and maize, respectively, as well as two NRT3 genes.

Comment 9. Line 282, what does “components” mean?

Response 9: For HATS activity in plants, NRT2/NRT3 is a two-component high-affinity nitrate transport system, each of them is part of high-affinity nitrate transport system and interact at the protein level.

Comment 10: Line 307-309, please specify the details in the changes.

Response 10: According to the reviewer’s suggestion, we have added the results in the “HvNRT2/3 gene expression patterns at different developmental stages” and “HvNRT2/3 gene expression analysis under low and high nitrogen levels” parts.

Comment 11: Line 313-318, state one or two functions in rice so that it can be more evidently related to barley.

Response 11: According to the reviewer’s suggestion, we have added the discussion in the revised manuscript as follows: In rice, a decreased expression of OsNRT2.3a is related to the accumulation of significantly higher levels of nitrate and total nitrogen in the root and lower levels in the shoot. However, enhancing the expression of OsNRT2.3b improves the growth, yield, and NUE in rice.

Comment 12. Line 335, the statement has been repeated a couple of times in the ms.

Response 12: Thank the reviewer for this content, we have deleted the repeat sentence about this in the revised manuscript.

Comment 13. Line 338, increase of biomass and plant height in Arabidopsis or ?? If so could be rephrased as “In the present study, overexpression of HvNRT2.1 led to high nitrate and nitrogen content, increased biomass and plant height and improved the yield-related traits in Arabidopsis indicating that….

Response 13: Thank the reviewer for this content. We check the result, and adjust the discussion in the new revised manuscript. The detail information as follows: In the present study, the overexpression of HvNRT2.1 led to high nitrate and nitrogen contents and improved the yield-related traits of Arabidopsis. However, both a biomass and plant height increase were not observed in the overexpression plants, indicating that the HvNRT2.1 gene plays a specific role in nitrate and total nitrogen accumulation in plants.

Comment 14. The authors should provide either qualitative or quantitive information regarding the HvNRT2.1 transcript level in transgenic Arabidopsis lines.

Response 14: According to the reviewer’s suggestion. We have added this information in S3 Fig in the reviewed manuscript (see “Morphological characterization of the HvNRT2.1 overexpression plants” part). The detail information as follows: In the transgenic lines, HvNRT2.1 was introduced into the genomic DNA and was expressed at the transcription level in three independent overexpression plants

Comment 15: HvNRT2.1 expression in barley responded to different N levels so why not investigate the growth performance of transgenic Arabidopsis lines under N treatments?

Response 15: We agree with this suggestion. We have plant wide type and transgenic Arabidopsis lines on the MS (without NH4NO3) with different KNO3 level, unfortunately, all plants cannot survive. Thus, the growth performance of wide type and transgenic lines was observed on the normal conditions.

Comment Minor issues

Comment 1: Line 20, rectify the grammar issue

Response 1: Thank the reviewer for this comment. We have corrected the sentence as follow: Nitrogen use efficiency (NUE) is the efficiency with which plants acquire and use nitrogen.

Comment 2. Line 23, in barley “nitrate transport”

Response 2: Thank the reviewer for this content. We have added the word “nitrate transport” in the new manuscript.

Comment 3: Line 25, “chromosomes”

Response 3: Thank the reviewer for this comment. We have replaced the “chromosome” with “chromosomes”.

Comment 4: Line 29, replace “notably” with “furthermore”

Response 4: Thank the reviewer for this comment. We have replaced the “notably” with “furthermore”.

Comment 5: Line 55, should be “they are” but not “it is”

Response 5: Thank the reviewer for this comment. We have replaced the “it is” with “they are”.

Comment 6: Line 61, under low N condition?

Response 6: Nitrate supply was 0.5 mM in the reference (Tang et al., 2012), which was consider as low nitrate supply.

Comment 7: Line 86-89, confusing with the experimental layout. Indeed, how many plants were sampled and used for RNA extraction?

Response 7: According to the reviewer’s suggestion. We have revised this sentence as follow: At 0 h, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h and 72 h after treatment, total roots were harvested from ten plants and immediately frozen in liquid nitrogen for RNA extraction with three biological replicates at each time point.

Comment 8: Line 137, “0.5 cm” would be better.

Response 8: Thank the reviewer for this comment. We have replaced the “mm” with “cm”.

Comment 9: Line 224, according “to”

Response 9: Thank the reviewer for this comment. We have added this word in the revised manuscript.

Comment 10. Line 228, “clustered”

Response 10: Thank the reviewer for this comment. We have a replaced the “cluster” with “clustered”

Comment 11: Line 320, “encode”

Response 11: Thank the reviewer for this comment. The word “encoding” has been replace by “encode”.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

3 Apr 2020

PONE-D-19-33678R1

Characterization of the Nitrate Transporter gene family and functional identification of HvNRT2.1 in barley (Hordeum vulgare L.)

PLOS ONE

Dear Dr. Xu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by May 18 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Guoping Zhang

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I appreciated that the authors have addressed to all of my concerns. I have no further comments for this manuscript.

Reviewer #2: The revised manuscript has been improved and I have the following comments in regard to typos or grammar errors. In addition, I suggest the authors adding the gene accession numbers or gene IDs such as HORVU.. as the last section of Methods, for a better understanding and traceback with other researches.

Line 37: Replace “serious” with “severe/intense”

Line 48: Replace “shows” with “functions as”

Line 53: Can be rephrased as “There is a lesser number of NRT2 genes than NRT1”

Line 177: “herbicide treatment”

Line 227: Check the 3 distinct clusters

Line 230: It has to be cluster II and III?

Line 330: Replace “with” with “under”

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Ping Yang

Reviewer #2: Yes: Yong Han

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

5 Apr 2020

Responses to reviewer 2

Comment 1: The revised manuscript has been improved and I have the following comments in regard to typos or grammar errors. In addition, I suggest the authors adding the gene accession numbers or gene IDs such as HORVU.. as the last section of Methods, for a better understanding and traceback with other researches.

Response 1: Thank the reviewer for this comment. We have listed the gene IDs, chromosome position, coding sequence length, amino acid length, mass and pI in the Table 1 in the revised manuscript.

Comment 2: Line 37: Replace “serious” with “severe/intense”

Response 2: Thank the reviewer for this comment. We have replaced the “serious” with “severe”.

Comment 3: Line 48: Replace “shows” with “functions as”

Response 3: Thank the reviewer for this comment. We have replaced the “shows” with “functions as”.

Comment 4: Line 53: Can be rephrased as “There is a lesser number of NRT2 genes than NRT1”.

Response 4: We agreed with the reviewer’s suggestion, and rephrased as “There is a lesser number of NRT2 genes than NRT1” in the revised manuscript.

Comment 5: Line 177: “herbicide treatment”

Response 5: We agreed with the reviewer’s suggestion. we have added the word “treatment” after “herbicide”

Comment 6: Line 227: Check the 3 distinct clusters

Response 6: Thank the reviewer for this comment. The words“Ⅰ, Ⅱ, and Ⅲ”have not displayed properly in the PDF version. The sentence as follows: According to the phylogenetic tree of the NRT2 and NRT3 proteins in barley, rice, maize, and Arabidopsis, the NRT2 proteins could clearly be divided into three distinct clusters (Ⅰ, Ⅱ, and Ⅲ).

Comment 7: Line 230: It has to be cluster II and III?

Response 7: Thank the reviewer for this comment. The words“Ⅱ, Ⅲ”have not displayed properly in the PDF version. The sentence as follows: AtNRT2.5 and AtNRT2.7 represented clusters Ⅱ and Ⅲ, containing four and three genes, according to the phylogenetic tree, respectively.

Comment 8: Line 330: Replace “with” with “under”

Response 8: We agreed with the reviewer’s suggestion. We have replaced the “with” with “under”.

Submitted filename: Response to reviewers.doc

Click here for additional data file.

7 Apr 2020

Characterization of the Nitrate Transporter gene family and functional identification of HvNRT2.1 in barley (Hordeum vulgare L.)

PONE-D-19-33678R2

Dear Dr. Xu,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Guoping Zhang

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

10 Apr 2020

PONE-D-19-33678R2

Characterization of the Nitrate Transporter gene family and functional identification of HvNRT2.1 in barley (Hordeum vulgare L.)

Dear Dr. Xu:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof Guoping Zhang

Academic Editor

PLOS ONE