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Article

Genome-Wide Identification and Functional Analysis of Nitrate Transporter Genes (NPF, NRT2 and NRT3) in Maize

by
Lihua Jia
1,2,
Desheng Hu
1,
Junbo Wang
1,
Yuanyuan Liang
1,
Fang Li
1,
Yi Wang
1,* and
Yanlai Han
1,*
1
State Key Laboratory of Wheat and Maize Crop Science, College of Resources and Environment, Henan Agricultural University, Zhengzhou 450046, China
2
College of Agronomy, Henan Agricultural University, Zhengzhou 450046, China
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2023, 24(16), 12941; https://doi.org/10.3390/ijms241612941
Submission received: 19 July 2023 / Revised: 9 August 2023 / Accepted: 16 August 2023 / Published: 18 August 2023
(This article belongs to the Section Molecular Plant Sciences)
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Abstract

:
Nitrate is the primary form of nitrogen uptake in plants, mainly transported by nitrate transporters (NRTs), including NPF (NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER FAMILY), NRT2 and NRT3. In this study, we identified a total of 78 NPF, seven NRT2, and two NRT3 genes in maize. Phylogenetic analysis divided the NPF family into eight subgroups (NPF1-NPF8), consistent with the results in Arabidopsis thaliana and rice. The NRT2 family appears to have evolved more conservatively than the NPF family, as NRT2 genes contain fewer introns. The promoters of all NRTs are rich in cis-acting elements responding to biotic and abiotic stresses. The expression of NRTs varies in different tissues and developmental stages, with some NRTs only expressed in specific tissues or developmental stages. RNA-seq analysis using Xu178 revealed differential expression of NRTs in response to nitrogen starvation and nitrate resupply. Moreover, the expression patterns of six key NRTs genes (NPF6.6, NPF6.8, NRT2.1, NRT2.5 and NRT3.1A/B) varied in response to alterations in nitrogen levels across distinct maize inbred lines with different nitrogen uptake rates. This work enhances our understanding of the structure and expression of NRTs genes, and their roles in nitrate response, paving the way for improving maize nitrogen efficiency through molecular breeding.

1. Introduction

Nitrogen is an essential element for plant growth, metabolism and heredity, and its nutritional status directly affects crop yield. While the application of nitrogen fertilizer increases crop yield, it also gives rise to a range of environmental issues. Exploring the genetic potential of nitrogen use efficiency (NUE) in crops represents one effective strategy for improving NUE and reducing nitrogen fertilizer application rates. Plant NUE is mainly affected by N uptake efficiency (NUpE), N utilization (assimilation) efficiency (NUtE), N transport efficiency, and N remobilization efficiency [1]. In general, NUpE and NUtE are the main contributors to plant NUE. Nitrate transporters (NRTs) play a crucial and extensive role in uptaking nitrate from the soil and transporting it to different organs for plant growth and development. An in-depth analysis of NRTs genes can advance our understanding of the mechanism behind efficient nitrogen utilization and help in identifying NUE genes.
Arabidopsis thaliana and rice (Oryza sativa) possess 53 and 93 NPF genes respectively [2,3], the majority of which are low-affinity nitrate transporters except for AtNPF6.3 (NRT1.1/CHL1), which has double affinity for nitrate uptake [4]. NRT2 family, in contrast to the NPF family, has seven and five members in Arabidopsis thaliana and rice, respectively [2], with the majority exhibiting high-affinity activity. NPF gene family members have diverse and pivotal roles in nitrate uptake, transportation and distribution within plants [2]. For example, AtNPF6.3/NRT1.1 and OsNPF6.5/NRT1.1B are responsible for nitrate uptake in Arabidopsis thaliana and rice [5,6,7,8], while seven genes (AtNPF7.3/NRT1.5, AtNPF7.2/NRT1.8, AtNPF2.9/NRT1.9, AtNPF2.3, OsNPF2.2, OsNPF2.4 and OsNPF6.5/NRT1.1B) are involved in the nitrate transportation from root to shoot [9,10,11,12,13,14]. AtNPF1.1/NRT1.11, AtNPF1.2/NRT1.12 and AtNPF2.13/NRT1.7 genes participate in the remobilization of nitrate from source to sink [1]. Unlike the NPF family, the functional activity of NRT2 transporters often depends on assistance from the ancillary proteins NAR2 (or NRT3) interacting with NRT2 [15]. Seven NRT2 genes, including AtNRT2.1, AtNRT2.2, AtNRT2.4, AtNRT2.5, OsNRT2.1, OsNRT2.2, and OsNRT2.4, are involved in nitrate uptake in Arabidopsis thaliana and rice. Furthermore, AtNRT2.4 and AtNRT2.5 are also involved in the remobilization of nitrate from source to sink. Genetic improvement of some of these NPF and NRT2 genes, such as OsNRT1.1B [7], OsNRT2.3b [16], OsNPF6.1HapB [17] and OsNRT2.3a [18], can significantly increase crop yield and NUE, highlighting the crucial roles of these genes in nitrate uptake and transport in plants.
Although extensive research in Arabidopsis thaliana and rice has been conducted, studies on the NRTs gene family in maize have primarily focused on analyzing their expression patterns and conducting electrophysiological studies of selected NRTs. For instance, Garnett T. et al. (2013) and Dechorgnat J. et al. (2019) investigated tissue-specific and nitrogen-linked expression profiles of certain nitrate transporters [19,20]. Furthermore, ZmNPF6.4 and ZmNPF6.6 have been modified to exhibit different selectivity to nitrate and chloride, with ZmNPF6.6 acting as a pH-dependent non-biphasic high-affinity nitrate-specific transporter [21]. NAR2.1/NRT2.1 has improved functional interaction in nitrate uptake along the root axis of maize, as evidenced by gene expression results [22]. Although NPF genes had been identified and named as family members based on version 3 of the maize B73 genome, phylogenetic analysis, gene structure and cis-elements have yet to be studied in-depth [3]. Additionally, there have been no studies exploring the contribution of key NPF or NRT2 genes to plant NUE. Hence, in our study, we identified and characterized all members of the NPF, NRT2 and NRT3 gene family based on the maize B73 version 5 genome. We conducted RNA-seq analysis to investigate the expression patterns of all NRTs genes in response to changes in external nitrate levels. Based on the RNA-seq results, we identified six NRTs genes that were responsive to nitrate and potentially linked to nitrate uptake in maize. We subsequently examined the expression patterns of these candidate genes in four maize inbred lines with differing nitrogen uptake efficiency using quantitative real-time RT-PCR (RT-qPCR). Through the above work, we aim to obtain a comprehensive understanding of NRTs in maize, which will provide valuable information and serve as a reference for identifying key NRTs that control nitrate uptake and transport in maize.

2. Results

2.1. Identification of NPF, NRT2 and NRT3 Genes in Maize

A previous study named 79 NPF genes in maize based on the phylogenetic relationship of NPF genes in 31 fully sequenced plant genomes [3]. However, this analysis was conducted based on the V3 version of the B73 genome, which has since been updated to version V5 in 2021 with improved sequence accuracy and gene annotations. In this study, we identified 78 NPF genes (Table S1) in the maize B73 V5 genome by aligning all protein sequences to those of the 53 NPF genes in Arabidopsis thaliana and filtering for the PTR2 domain (Section 4). Two new NPF genes were identified, and four previously identified NPF genes were eliminated. Furthermore, one NPF gene identified previously was annotated as two adjacent genes in the maize B73 V5 genome. The nomenclature of NPF genes from the previous study was used for the commonly identified genes, while the newly identified NPF genes were named based on their subfamily, inferred from a tree constructed using all the protein sequences of NPF genes in maize and Arabidopsis thaliana. In addition, we identified seven NRT2 genes and two NRT3 genes in the maize B73 V5 genome using the same approach. The names of five NRT2 genes and two NRT3 genes provided in MaizeGDB were adopted, whereas the remaining NRT2 genes were named NRT2.4 and NRT2.6, respectively.

2.2. Phylogenetic Analysis of NPF, NRT2 and NRT3 Genes

Being present in many plants including Arabidopsis thaliana [3], NPF genes can be categorized into eight subfamilies based on their protein sequences. To determine the phylogenetic relationships of NPF, NRT2 and NRT3 genes in maize, we constructed a phylogenetic tree (as outlined in Section 4). Our results revealed that members of the same subfamily in both maize and Arabidopsis thaliana were clustered together (Figure 1). NRT2 genes formed a distinct clade from NPF and NRT3 genes, while NRT3 genes were clustered together with the NPF1 and NPF2 subfamilies.

2.3. Chromosomal Localization of NPF, NRT2 and NRT3 Genes

A total of 87 NRTs genes were observed to be unevenly distributed across all ten chromosomes, with most genes tending to be localized toward the distal regions (Figure 2). Among the ten chromosomes, chromosome 1 possessed the greatest number of NRTs genes, with 23 genes, followed by chromosomes 3 and 5, each with 11 genes. On the other hand, chromosome 7 had the fewest NRTs genes, with only two genes identified (NPF7.9 and NPF8.5). Members of the same subfamily were often distributed on multiple chromosomes. For example, we observed that the 23 NPF8 genes were distributed across six different chromosomes, while the seven NRT2 genes existed across six different chromosomes as well. Nevertheless, we also detected several NPF, NRT2 and NRT3 gene clusters in close genomic regions.

2.4. Gene Structure and Protein Domain Analysis of NPF, NRT2 and NRT3 Genes

In order to examine variations in the structures of NRTs genes, we utilized the Visualize Gene Structure (Basic) module of TBtools [23] to visualize their exon and intron structures. As depicted in Figure 3A, genes belonging to the same subgroup exhibited similar exon-intron structures. Notably, nearly all NPF genes contained intron structures, with their coding sequences being interrupted by 1–5 introns. In contrast, except for NRT2.4, all NRT2 genes featured a single exon and lacked introns.
We used Pfam and the Gene Structure View (Advanced) modules of TBtools [23,24] to annotate functional domains in the proteins encoded by NRTs genes, as illustrated in Figure 3. The proteins encoded by NPF, NRT2, and NRT3 genes contained distinct domains, namely PTR2, MFS_1, and NAR2, respectively (Figure 3B). Notably, an HSP70 domain was identified in the protein sequence of NPF8.5.

2.5. Putative Cis-Acting Regulatory Elements (CREs) in the Promoters of NPF, NRT2 and NRT3 Genes

To investigate the biological functions of NRTs genes, we conducted a thorough analysis of cis-acting elements by extracting the 2.0-kb fragment upstream of the transcription start site in the promoter region of each gene. Our analysis revealed a diverse array of functional elements within the promoter regions of NRTs genes, including light-responsive elements, hormone-responsive elements, anaerobic-responsive elements, and meristem expression elements (Figure 4). Among these elements, the Me-JA responsive and abscisic acid-responsive elements were found to occur at a higher frequency than other elements across all subfamilies. Particularly noteworthy was the NRT3 subfamily, which exhibited the highest number of auxin and abscisic acid-responsive elements, while the NRT2 subfamily displayed the lowest occurrence of these elements. Furthermore, the NRT3 subfamily displayed the lowest frequency of meristem expression and anaerobic responsive elements, while the NPF and NRT2 subfamilies had similar higher frequencies. Interestingly, the frequency occurrence of the NRT2 subfamily’s response to gibberellin and drought was located in the middle.

2.6. Co-Functional Network Analysis of NPF, NRT2 and NRT3 Genes

To identify candidate genes that potentially interact with NRTs genes to regulate nitrogen assimilation or transportation, we employed the 87 NRTs genes as guide genes to construct a co-functional network using MaizeNet [25]. Among all 87 guide genes, no candidate genes were obtained for 15 guide genes including ZmNPF1.3, ZmNPF3.3, ZmNPF4.2, ZmNPF4.4, ZmNPF4.11, ZmNPF5.18, ZmNPF7.11, ZmNPF7.13, ZmNPF8.4, ZmNPF8.6, ZmNPF8.17, ZmNRT2.4, ZmNRT2.6, ZmNRT2.7 and ZmNRT3.1A. A total of 762 candidate genes were obtained for the remaining 72 genes.
Subsequently, we conducted a GO enrichment analysis utilizing both NRTs genes and their candidate interacting genes, employing the geneset analysis functionality implemented in MaizeNet. Enriched GO terms were visualized by Revigo with default parameters. This analysis revealed a significant enrichment of genes involved in tripeptide transport, tube development, nitrate assimilation, chlorophyll catabolic process, and phenylalanine response in the co-functional network of NRTs genes (Figure 5). Specifically, NPF genes and their interacting genes were mainly involved in the response to biological hormones, bacterium, wounding and chlorophyll catabolic process. On the other hand, NRT2 genes and their interacting genes played significant roles in various biological processes such as lateral root development, cellular response to nitrate, nitrite transport, transmembrane transport, and response to Karrikin.

2.7. Spatial and Temporal Expression Analysis of NPF, NRT2 and NRT3 Genes

To gain insights into the spatiotemporal expression patterns of NRTs genes in maize, we extracted their gene expression data of different tissues/stages from qTeller (Table S2) [26]. The tissues were categorized into six groups, including roots, leaves, stem and shoot apical meristem (SAM), internode, reproductive tissues, and seeds. All NRTs genes were clustered into five distinct groups based on their expression patterns (Figure 6). Group I comprised five NPF8 genes (NPF8.17, NPF8.18, NPF8.10, NPF8.1 and NPF8.24) and two NPF2 genes (NPF2.2 and NPF2.3), which exhibited high expression levels in almost all tissues. In Group II, NPF genes were expressed at a moderate level across all tested tissues. Group III genes displayed relatively high expression levels in leaves, while their expression in seeds, reproductive tissues, internodes, and stems was lower. Group IV was the largest group, with the majority of genes showing low expression levels and distinct tissue-specific expression patterns. Among these genes, ZmNRT2.1 and ZmNRT2.2 were specially expressed in roots. Group V was the smallest, containing only three genes—ZmNRT3.1A, ZmNPF4.1, and ZmNPF5.18—all of which showed high expression levels primarily in roots.

2.8. Expression Profiles of NRTs Genes in Response to Nitrogen Starvation and Nitrate Resupply

RNA sequencing was conducted using the maize inbred line Xu178 to investigate the expression patterns of NRTs genes in response to nitrogen levels (Section 4, Table S3). The third leaves and roots of Xu178 seedlings were sampled at various time points during nitrogen starvation and nitrate resupply. More than 20 million clean reads were obtained for each sample. Based on the nitrate-response expression profiles in roots, NRTs genes were classified into four distinct groups (Figure 7). Genes in group I demonstrated the highest expression levels among all time points, indicating their significant roles in nitrate uptake in maize. Notably, NRT2.1, NRT2.2 and NRT3.1A displayed decreased expression levels during prolonged nitrogen starvation but exhibited a rapid increase in expression after nitrate resupply, highlighting their responsiveness to changes in nitrogen levels. The similar expression trend of NRT2.1/2.2 and NRT3.1A indicated that NRT3.1A might be involved in nitrate transport activity of NRT2.1/2.2 in maize. Genes in group II had low expression levels in roots and exhibited no evident response to changes in nitrogen levels. Group III comprised five NPF genes with relatively high expression levels, and among them, NPF6.6 exhibited a clear nitrate response, similar to NRT2.1/2.2. Finally, genes in group IV had moderate expression levels and were found to be responsive to nitrogen starvation and nitrate resupply. For instance, the expression of NRT2.5 was upregulated with prolonged nitrogen starvation and reduced after nitrate resupply. Its expression level was restored to DN0 when the recovery of nitrate supply was 12 h (RN12).
The expression patterns of NRTs genes in leaves, in response to nitrogen levels, differed significantly from those in roots and were classified into five groups. Genes in group I displayed the highest expression levels in leaves, which varied with nitrogen starvation and resupply. Notably, this group included NPF8.23, NPF8.24, and NPF6.4 genes, which were also highly expressed in roots. The expression of NPF6.8 in leaves continued to increase with prolonged nitrogen starvation, peaking at DN96, but decreased after nitrate resupply, exhibiting a response pattern similar to NRT2.5 in roots. However, the response pattern of NRT2.5 to nitrogen levels in leaves was opposite to that in roots, as its expression continuously decreased under nitrogen starvation stress and rapidly increased after nitrate resupply. Approximately half of the NRTs genes were clustered in group II, with low or no expression in leaves, and they did not significantly respond to changes in nitrogen levels, including most NRT2 genes (except NRT2.5 and NRT2.6). NRT3.1A, in group III, showed low expression levels during nitrogen starvation but increased after nitrate resupply, reaching its peak at RN12. Genes in groups IV and V had moderate to high expression levels in leaves, exhibiting a clear response to changes in nitrogen levels. The response pattern of NPF6.6 was similar to that of NRT2.5, as its expression decreased during nitrogen starvation but was upregulated after nitrate resupply. These results imply that different NRTs are responsible for nitrate uptake and transport in the roots and leaves of maize. Moreover, their responses to nitrogen starvation and nitrate supply significantly vary between these two tissues, highlighting the intricate and tissue-specific regulation of nitrogen utilization in maize.

2.9. NRTs Expression Patterns Exhibit Variations among Diverse Inbred Lines with Different Nitrate Uptake Rates

To explore the differences in the response patterns of NRT genes among different maize inbred lines, we focused on six key genes (NPF6.6, NPF6.8, NRT2.1, NRT2.5, NRT3.1A and NRT3.1B) that showed varying nitrate response patterns based on RNA-seq results. These genes were subjected to RT-qPCR analysis to assess their nitrate response patterns in four maize inbred lines (Figure 8). We observed a remarkable difference in nitrate uptake rates among the four maize inbred lines (Section 4, Figure S1). Particularly, the inbred line B73 exhibited significantly higher nitrate uptake efficiency compared to the other inbred lines. Furthermore, the time points at which the four inbred lines reached their maximum uptake efficiency varied.
The expression of NRTs genes in the roots of the four inbred lines exhibited varied responses to nitrate supply, with Zong3 showing the highest gene expression level (Figure 8A). ZmNRT2.1 and ZmNRT2.5 exhibited similar expression patterns in response to changes in nitrate levels in the roots of all four inbred lines. ZmNRT2.1 had the lowest expression at RN1h in all four inbred lines, but the time at which it reached its peak differed. It reached its highest peak at RN3h in B73 and Xu178, while in Zong3 and Mo17, it peaked at RN6h. In contrast to ZmNRT2.1, the expression level of ZmNRT2.5 peaked at RN1h in all four inbred lines and then decreased with prolonged nitrate resupply time. The expression level of ZmNPF6.6 in Zong3 decreased at RN3h, while in the other three inbred lines, it decreased at RN6h. The expression patterns of ZmNPF6.8 in response to changes in nitrate levels were alike in B73 and Xu 178, but they exhibited a significant reduction at RN1h in Zong3 and Mo17. Regarding ZmNRT3.1A, its expression in the four inbred lines showed similar response patterns to changes in nitrate levels, with the differences mainly reflected in the expression levels. On the other hand, the expression of ZmNRT3.1B in Mo17, B73, and Xu178 was consistent, contrasting with its expression in Zong3.
In leaves (Figure 8B), the response patterns of the six key NRTs genes to changes in nitrate levels in the four inbred lines were similar, which were significantly different from the results observed in roots. The highest expression levels of these genes were observed in Zong3, consistent with the results in roots.

3. Discussion

3.1. Diversity in Structure and Function of NRTs Gene Families

Nitrate transporter genes play a crucial role in the uptake and transportation of nitrate in plants, significantly contributing to improving plant nitrogen efficiency (NUE). In addition to Arabidopsis thaliana, studies have been carried out on the NRTs gene family in crops such as rice, barley and wheat [3,27,28,29]. While previous research had identified NRTs family members in maize through homologous sequence alignment [3] and analyzed expression patterns and electrophysiological characteristics of some NRTs genes in different tissues and developmental stages [19,21], a systematic analysis of NRTs family members, including genetic evolution, gene structure, and functional element analysis, has been lacking. Moreover, no analysis has been conducted on nitrate response and its contribution to improving crop NUE and yield.
In our study, we used the newly published and more accurate maize genome sequence to identify 78 NPF genes, seven NRT2 genes, and two NRT3/NAR2 genes, through conserved functional domains analysis of NRTs. The distribution of NPF and NRT2 family genes on chromosomes was uneven, and genetic evolution analysis identified eight subfamilies of NPF family genes, similar to Arabidopsis thaliana and rice [3].
NPF family members were found to have a range of two to five introns, while all NRT2 genes, except for NRT2.4, were intron less, indicating the conservation of NRT2 gene during evolution. Cis-acting elements play critical roles in gene response to environment changes and the regulation of their expression in various biological processes [30] The promoter sequences of NRTs family members contain a diverse range of cis-acting elements, with abscisic acid responsiveness and Me-JA responsiveness elements being particularly abundant. Similarly, NRTs genes of Arabidopsis thaliana and rice contain various plant hormones (IAA, ABA, JAs, and/or GAs) cis-acting elements [31], providing the foundation for NRTs genes to respond to stresses such as drought, cold, heat, UV, and salt.
In Arabidopsis thaliana, AtNPF6.3 was highly induced by IAA treatment under low nitrogen conditions [32], AtNPF2.10 was upregulated by Me-JA treatment, thereby accelerating the transport of gibberellin [33], while AtNPF3.1 was transcriptionally regulated by both GA and ABA [34,35]. Additionally, auxin may also mediate nitrogen uptake and transport, which is derived from the changes in the expression of many NRTs genes under IAA treatment [36]. In rice, it has been reported that OsNRT2.4 is up-regulated by exogenous auxin and JA [37].
We integrated and analyzed publicly available transcriptome data from maize and found that genes belonging to the NPF and NRT2 families displayed varying degrees of responsiveness to stress conditions (Figure S2). According to the analysis of cis-acting regulatory elements of NRTs genes (Figure 4), NPF8.15 and NPF8.23 were found to respond to drought stress, NPF1.1 responded to Me-JA, NRT2.4 and NRT3.1B responded to ABA. Function analysis of the NRTs family members revealed their broad functional scope, with NPF genes potentially involved in even more diverse physiological processes.

3.2. Variation in the Expression Patterns of NRTs Genes

Gene expression patterns in plant tissues and developmental stages offer valuable insights into potential gene functions. Our integrative analysis of published RNA-seq data revealed specific patterns for a few NRTs genes, indicating their crucial roles in plant and organ development. In roots, seven genes (ZmNPF8.17, ZmNPF8.18, ZmNPF2.2, ZmNPF2.3, ZmNPF8.10, ZmNPF8.1, and ZmNPF8.24) showed consistently high expression levels that were minimally affected by developmental stages. The functions of some homologous genes in rice have been analyzed. For example, OsNPF8.1 have been reported to contribute to dimethyl arsenate accumulation in rice grains [38], and OsNPF2.2 plays a role in unloading nitrate from the xylem and influencing nitrate transport and plant development [9]. Along with the seven genes mentioned above, genes ZmNRT3.1A, ZmNPF4.1, and ZmNPF5.18A also exhibit high expression levels in the roots, however, their expression levels are significantly influenced by the developmental period. The expression pattern of NRT2.1 and NRT2.2 genes in roots resembled that of NRT3.1A. Several genes have been identified that are involved in root nitrate uptake in Arabidopsis thaliana, including AtNRT2.1/2.2/2.4/2.5, AtNPF6.3, and AtNPF4.6 [32,39,40,41]. Similarly, in rice, genes such as OsNRT2.1/2.2/2.4 and OsNPF2.4/6.5/8.9 have been found to be involved in root nitrate uptake [7,8,13,37,42,43]. Research conducted in maize has revealed that ZmNPF6.6 plays a role in nitrate transport [21]. The NRT genes, which exhibit high expression levels in roots, are likely to be involved in the process of nitrate uptake by the roots.
In leaves, NRT gene expression is more markedly influenced by developmental stages and organ tissues. For instance, the expression levels of ZmNPF2.2 and ZmNPF2.3 genes peak at the V9 stage, whereas the expression of ZmNPF6.8, ZmNRT2.5, and ZmNRT3.1B are higher during the pollination period than before, and their expression in leaves are considerably higher than that in roots. Following nitrate uptake by the plant roots, specific NRTs genes are responsible for nitrate transport and distribution in leaves. For example, the OsNPF2.4 gene in rice plays a role in nitrate redistribution in leaves, while in Arabidopsis thaliana, AtNPF1.1/1.2/2.13, AtNRT2.4, and AtNRT2.5 genes regulate nitrate redistribution between sources and sinks in leaves.
Adequate nitrate is crucial for seed development in plants, and specialized NRT genes are responsible for nitrate transport to the grains. For instance, the OsNPF2.4 gene in rice regulates seed vigor [13], while overexpression of the TaNRT2.5 in wheat can increase nitrate accumulation, seed vigor, and yield [44]. In Arabidopsis thaliana, AtNRT2.7 controls the nitrate content in grains [45]. In maize, we observed that ZmNRT2.5 had peak expression in maize seeds at 2.5 days after pollination, while the expression of ZmNPF8.17 and ZmNPF8.18 genes was highest at eight days after pollination but decreased as pollination time was delayed. However, it remains unclear whether these two genes are involved in nitrate transport during seed development.
In internodes, NRTs gene expression varied with developmental stage. ZmNRT2.5, ZmNPF5.1, and ZmNPF7.9 exhibited significantly higher expression during pollination than during vegetative growth, while ZmNPF2.2 and ZmNPF2.3 had lower expression levels at the V9 stage than at other stages. ZmNPF6.6 expression gradually decreased during growth and during development. In rice, OsNPF2.4 and OsNPF6.5 were responsible for nitrate transport from roots to shoots, suggesting that internode genes primarily participate in nitrate transport, and their expression varies with developmental stage.

3.3. Maize NRT Gene Expression Is Regulated by Nitrate Levels and Genetic Background

To investigate the impact of nitrogen levels on the expression of NRTs genes in maize, RNA-seq was performed to analyze the expression patterns of different NRTs genes in the roots and leaves of maize under nitrogen-deprived and nitrogen-resupply conditions. The results showed that nearly half of the NRTs genes in roots did not exhibit significant changes in response to alternations in nitrogen levels. However, ZmNRT2.1, ZmNRT2.2, and ZmNRT3.1A displayed high expression levels in the roots and showed a considerable response to nitrate levels. During an extended period of nitrogen deprivation, the expression of ZmNRT2.1 declined but significantly increased after three hours of nitrogen resupply, and then decreased once again with continued nitrogen supply. The NRT2s are high-affinity nitrate transporters, and their transport activity frequently requires the support of NRT3. A previous study conducted in Arabidopsis thaliana has demonstrated that the transcript abundance of AtNRT2.1 was significantly correlated with the high-affinity nitrate influx after the provision of nitrate to nitrate-deprived plants [46]. Increased expression of OsNRT2.1 was associated with enhanced nitrogen-use efficiency and nitrate-dependent root elongation in rice [42,43]. Transcript levels of ZmNRT2.1 and ZmNRT2.2 were observed to be correlated with high-affinity root nitrate uptake capacity and also nitrogen availability [19]. In both nitrogen-starved and nitrogen-resupply states, ZmNPF6.6 displayed higher expression, with a significant increase observed after nitrogen resupply (Figure 7A). Studies conducted on Arabidopsis thaliana and rice revealed that the ZmNPF6.6 homologous, AtNPF6.3, and OsNPF6.5, function as double-affinity nitrate transporters [7,32]. Increased expression of OsNRT1.1B leads to an accumulation of N in plants, promoting rice growth under low N conditions [47]. In contrast, ZmNRT2.5 in roots exhibited a different response pattern with its expression continuing to increase under nitrogen-starved conditions and returning to pre-starvation condition levels following a prolonged period of nitrogen-resupply (Figure 7A). This indicates that ZmNRT2.5 may play an important role in mediating nitrate uptake by plants under long-term low nitrogen stress. It has demonstrated that AtNRT2.5 displayed the highest expression level in roots among the seven Arabidopsis thaliana NRT2 genes under long-term nitrogen starvation stress [41]. Overexpression of the homologous gene OsNRT2.3a, which is identical to NRT2.5 in rice, significantly improves nitrogen use efficiency and increases the yield of rice under low nitrogen conditions [48]. The homologous gene TaNRT2.5 in wheat is expressed in the embryo and shell and plays an important role in the accumulation of nitrate in seed [44].
The response patterns of genes in leaves differ significantly from those in roots, with most of the genes in leaves showing no significant response to nitrate levels. In our study, the expression level of ZmNPF6.8 in leaves is upregulated with the extension of nitrogen starvation time (Figure 7B). The previous study found that ZmNPF6.8 have a higher expression pattern in old leaves than in root or young leaves during vegetative and reproductive development stages [20]. According to the existing results, it is speculated that the ZmNPF6.8 gene is responsible for leaf nitrogen remobilization. ZmNRT2.5 exhibits an expression pattern opposite to that in roots, increasing the expression level in shoots when nitrogen is sufficient and decreasing the expression when nitrogen is deficient (Figure 7B). In rice, the corresponding gene OsNRT2.3a has a specific role in transporting nitrate from the roots to the shoots [18]. In addition to ZmNRT2.5, ZmNPF6.6 also has relatively high expression levels in both roots and leaves. Previous research has reported that nitrate-induced upregulation of ZmNPF6.6 in leaves is rapid and homologous genes, such as AtNPF6.3, have been shown to enhance expression in shoots and improve growth under nitrogen deficiency stress [49]. This suggests that ZmNPF6.6 responds quickly to external nitrate in leaves, promoting growth under nitrogen deficiency stress.
To investigate the relationship between NRTs gene expression levels and plant nitrate uptake efficiency, four inbred maize lines with varying nitrate uptake efficiencies were selected to analyze the expression patterns of different NRTs. The results indicated that the expression patterns of candidate NRTs (NRT2.1, NRT2.5, NPF6.6, NPF6.8, NRT3.1A, NRT3.1B) in response to nitrate changes differed among different genetic backgrounds. Studies have shown that different genotypes of the same gene contribute differently to the phenotype of the plant. It has been demonstrated that variations in the NRT1.1B (OsNPF6.5) affect nitrate use efficiency in different subspecies of rice. OsNRT1.1B-indica transgenic plants exhibited better growth performance and greater NUE than OsNRT1.1B-japonica transgenic plants [7]. Tang et al. (2019) identified a rare natural allele, OsNPF6.1HapB, which enhances nitrate uptake and improves NUE in rice [17]. The findings imply that identifying and harnessing beneficial allelic variations of key NRTs genes in crops can be a promising approach to enhancing crop NUE through genetic means. Our findings reveal variations in the response strength of NRTs to nitrate among distinct inbred lines of maize, suggesting the presence of allelic diversity in NRTs across different materials that may impact the NUE of maize.

4. Materials and Methods

4.1. Identification of NRTs Genes in Maize

To identify all the NPF genes in the maize genome, we performed a protein sequence alignment of 53 NPF genes annotated in Arabidopsis thaliana against all protein sequences of the maize B73 genome (version 5, http://www.maizegdb.org (accessed on 20 June 2022)) using BLASTP [3,50,51]. Candidate NPF genes were selected by filtering the BLASTP results according to specific criteria (e-value ≤ 1 × 10−3, identity ≥ 30%, query coverage ≥30%). We then used HMMER (v3.0) to search for the “Proton-dependent oligopeptide transporter family” (IPR000109) domain in the protein sequences encoded by all candidate NPF genes, and excluded proteins without this domain (e-value ≤ 1 × 10−5) [52]. The remaining genes were identified as NPF genes in maize. The same approach was used to identify NRT2 and NRT3 genes in the maize genome.

4.2. Phylogenetic Analysis

We performed a multiple sequence alignment using the protein sequences encoded by the 78 NPF, seven NRT2 and two NRT3 genes using the MUSCLE algorithm implemented in MEGA7 [53]. A phylogenetic tree was subsequently constructed based on the alignment results using the neighbor-joining (NJ) method implemented in MEGA7 with 1000 bootstrap replicates. The tree was further enhanced using the iTOL software (https://itol.embl.de/ (accessed on 17 August 2022)) [54].

4.3. Chromosomal Localization of NPF, NRT2 and NRT3 Genes

We obtained the chromosomal locations of the 87 NPF/NRT1, NRT2 and NRT3 genes in the maize B73 genome (V5) from MaizeGDB (http://www.maizegdb.org (accessed on 20 Jun 2022)), and subsequently visualized them by creating circular plots using shinyCircos (https://venyao.xyz/shinyCircos/ (accessed on 10 July 2022)) [55].

4.4. Gene Structure Analysis

The exon/intron structures of the NPF, NRT2, and NRT3 genes were extracted from the gene annotation file of the maize B73 genome (V5), and visualized using the gene structure view module (advanced) of TBtools [23].

4.5. Identification of Putative Cis-Acting Regulatory Elements

For the identification of putative cis-acting regulatory elements, we obtained a 2.0-kb genomic sequence upstream of the transcription start sites for all NPF, NRT2, and NRT3 genes in the maize B73 reference genome (V5). These sequences were analyzed using Plant CARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 15 November 2022)) [56].

4.6. Functional Domain Analysis

To annotate functional domains in all NPF, NRT2 and NRT3 genes, we utilized Pfam and visualized the domains using the “Visualize Domain Pattern (from Pfam search)” module of TBtools [23,24].

4.7. Expression Analysis with Public RNA-Seq Data

The expression levels of NPF, NRT2, and NRT3 genes in various tissues of B73 at diverse developmental stages were retrieved from qTeller (http://qteller.maizegdb.org (accessed on 20 March 2023)) [26].

4.8. Plant Materials and Growth Conditions

Surface sterilization of seeds from four maize inbred lines (Xu178, Zong3, Mo17, and B73) was carried out using a 10% (v/v) hydrogen peroxide solution for 30 min, followed by rinsing with distilled water and soaking in distilled water at room temperature for 12–16 h. After germinating in distilled sand, uniform seedlings were transferred to plastic containers and grown in a half-strength nutrient solution for two days, with the residual endosperm removed before transfer. Subsequently, the seedlings were transferred to a full-strength nutrient solution with high nitrogen (HN, 4 mM NO3−). Once the third leaf emerged from the leaf collar (approximately 7 days cultured in HN solution), the uniform seedlings were transferred to nitrogen-deprived (DN) nutrient solution for 96 h (DN96), followed by nitrogen resupply (RN, 4 mM NO3−) for 24 h (RN24). Each treatment was conducted with three biological replicates. The full-strength nutrient solution consisted of 2 mM Ca(NO3)2, 0.75 mM K2SO4, 0.65 mM MgSO4·7H2O, 0.1 mM KCl, 0.3 mM KH2PO4, 0.025 mM K2HPO4·3H2O, 0.1 mM Fe-EDTA, and micronutrients (1 µM H3BO3, 1 µM MnSO4·H2O, 0.01 µM CuSO4·5H2O, 1 µM ZnSO4·7H2O and 0.05 µM (NH4)6Mo7 O24·4H2O) at a pH of 6.0, as previously described by Guo et al. [28]. In the nitrogen-deprived treatment, 2 mM CaCl2 was used to compensate for the reduction in Ca2+ concentration.

4.9. Sample Collection for RT-qPCR and Transcriptome Sequencing

The third leaves and roots were collected at various time points after the onset of nitrogen deprivation and resupply, and all samples were immediately frozen in liquid nitrogen and stored at −80 °C. For RT-qPCR, samples of four maize inbred lines were collected at 0 h and 96 h after nitrogen deprivation, and 1 h, 3 h, 6 h, 12 h, and 24 h after nitrogen resupplied. For RNA sequencing, 48 samples of Xu178 were collected at 0 h, 3 h, 12 h, 24 h, and 96 h after nitrogen deprivation, and 3 h, 12 h, and 24 h after nitrogen resupplied.

4.10. RNA Extraction, cDNA Synthesis, and RT-qPCR

Total RNAs were extracted using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. First-strand cDNAs were synthesized from the total RNA using HiScript II Q RT Super Mix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China). Subsequently, RT-qPCR was conducted using the Eva Green 2X qPCR Master Mix-Low ROX Kit (abm) on the Quant Studio 3 System (Applied Biosystems of Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s protocol. The relative expression levels of the target genes were determined by normalizing them to the abundance of ZmActin1 detected in the same sample and presented as 2−ΔCT. The RT-qPCR primers used are listed in Table S4.

4.11. Transcriptome Sequencing and Data Processing

The extracted total RNAs were initially subjected to a thorough quality assessment. Only the high-quality RNA samples were selected to create sequencing libraries, which were prepared using the Illumina TruSeq RNA Library Preparation Kit v2. Subsequently, these libraries underwent sequencing using 150-bp paired-end reads on the Illumina HiSeq X Ten (GSA (Genome Sequence Archive) database Accession Number: CRA012157).
For each set of RNA sequencing data, we first removed the adapters and low-quality reads using Fastp (version v0.20.1) [57] and then aligned clean reads to the maize B73 reference genome (version 5, http://www.maizegdb.org (accessed on 20 June 2022)) using STAR (version 2.7.5a) [58] with default parameters. Based on the alignment results, we then calculated the read counts for each gene using htseq-count from HTSeq (version 1.0) [59] and obtained their expression levels using edgeR (version 3.28.1) [60].

4.12. Determination of Nitrate Uptake Rate

To measure the nitrate uptake rate, we employed the depletion method with a sheltered plastic bowl. First, seedlings from four inbred lines were cultured in HN nutrient solution until the third leaf fully expanded. After 96 h of nitrogen starvation treatment, uniform seedlings were selected from each line, and their roots were submerged in an aerated uptake solution containing 1 mM NO3−. Samples were collected at various time intervals (0, 0.5, 1, 1.5, 2, 2.5, 3, 6, 9, 12, and 24 h), and the fresh weight of the root was recorded. The concentration of NO3− was measured using the sulfuric acid salicylic method at 410 nm, employing a Thermo Scientific Microplate Reader. Kinetic parameters of NO3− uptake in the root system, including Imax (maximum uptake rate), Km (the concentration of NO3− ions in uptake solution when the uptake rate is equal to 1/2 Imax), and Cmin (the concentration of NO3− ions in the uptake solution when the uptake rate is equal to zero), were calculated based on the curve y = ax2 + bx + c, where x is the concentration of NO3− in uptake solution and y is the rate of NO3− ions uptake per unit of fresh root weight, according to the formula Imax = (4ac − b2)/4a. The calculated results of the kinetic parameters are listed in Table S5.

5. Conclusions

Nitrate transporters play critical roles in facilitating the uptake, translocation, and distribution of nitrate in plants. Identification of key contributing NRTs can significantly improve crop grain yield and NUE. Our study consisted of a comprehensive analysis of all NRTs in maize, involving investigations into their phylogenetic relationships, gene structure, and expression patterns. By employing RNA-seq, we systematically assessed the response patterns of NRTs to variations in nitrate levels and analyzed the expression patterns of several potential NRTs candidates in diverse maize inbred lines. These results establish a foundation for deepening our understanding of the molecular functions of NRTs in maize and provide insights into the identification of crucial NRTs related to maize nitrogen utilization efficiency for promoting the development of nitrogen-efficient maize varieties.

Supplementary Materials

The supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms241612941/s1.

Author Contributions

Y.W. and Y.H. designed the research; L.J. and D.H. conducted the experiment and data analysis; J.W. and Y.L. performed experiments related to qPCR; L.J. and Y.W. wrote the manuscript; F.L., Y.W. and Y.H. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 31601812), the National Key R&D Program of China (Grant No. 2018YFD0200600), the Open Project of the State Key Laboratory of Wheat and Maize Crop Science (Grant No. SKL2020KF04), and Key Scientific Research Projects of Higher Education Institutions in Henan Province (Grant No. 23A210011).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within this article and Supplementary Material.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogenetic tree of NRTs from maize and Arabidopsis thaliana. The unrooted neighbor-joining (NJ) tree was constructed using MEGA7.0 with 1000 bootstrap replicates. Rings of subtrees are colored, indicating different NRTs subgroups.
Figure 1. Phylogenetic tree of NRTs from maize and Arabidopsis thaliana. The unrooted neighbor-joining (NJ) tree was constructed using MEGA7.0 with 1000 bootstrap replicates. Rings of subtrees are colored, indicating different NRTs subgroups.
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Figure 2. Chromosomal location of NRTs genes. Members of different subfamilies are indicated with varying colors.
Figure 2. Chromosomal location of NRTs genes. Members of different subfamilies are indicated with varying colors.
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Figure 3. Schematic diagram of gene structure and functional protein domain of NRT genes in maize. (A) Gene structure. The green boxes, yellow boxes and gray lines represent the UTR, CDS and introns, respectively. (B) Protein domain of NRT genes. The protein domains are represented with a box. Box size indicates the length of the domain. The gray lines represent the non-functional sequences.
Figure 3. Schematic diagram of gene structure and functional protein domain of NRT genes in maize. (A) Gene structure. The green boxes, yellow boxes and gray lines represent the UTR, CDS and introns, respectively. (B) Protein domain of NRT genes. The protein domains are represented with a box. Box size indicates the length of the domain. The gray lines represent the non-functional sequences.
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Figure 4. Analysis of cis-acting regulatory elements in the promoters of NRT genes in maize. (A) Schematic diagram of cis-elements in the promoters of NRT genes. (B) Frequency of representative cis-acting elements in the promoters of NRT genes.
Figure 4. Analysis of cis-acting regulatory elements in the promoters of NRT genes in maize. (A) Schematic diagram of cis-elements in the promoters of NRT genes. (B) Frequency of representative cis-acting elements in the promoters of NRT genes.
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Figure 5. The TreeMap visualization of enriched GO terms associated with NPF/NRT genes and their interacting genes. The size of the rectangles indicates the number of genes involved in the corresponding biological progress.
Figure 5. The TreeMap visualization of enriched GO terms associated with NPF/NRT genes and their interacting genes. The size of the rectangles indicates the number of genes involved in the corresponding biological progress.
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Figure 6. Expression profile of NRT genes. Gene expression data were normalized for each gene and are shown as log2- transformed data of normalized data + 1. Each column represents a tissue sampled at specific developmental stage, while each row represents a NRT gene. The subfamily of all NRT genes are indicated by colored boxes on the left. Tissues are represented by colored boxes on the top.
Figure 6. Expression profile of NRT genes. Gene expression data were normalized for each gene and are shown as log2- transformed data of normalized data + 1. Each column represents a tissue sampled at specific developmental stage, while each row represents a NRT gene. The subfamily of all NRT genes are indicated by colored boxes on the left. Tissues are represented by colored boxes on the top.
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Figure 7. Expression profile of NRT genes in the root and leaf of Xu178 in response to nitrogen starvation and nitrate resupply. (A) Each column represents the root of Xu178 sampled at a specific time point. (B) Each column represents the leaf of Xu178 sampled at a specific time point. DN3, treated with nitrogen-deprived (DN) nutrient solution for 3 h. RN3, nitrogen resupply (RN, 4 mM NO3−) for 3 h. Representative genes of multiple groups are highlighted with underlines.
Figure 7. Expression profile of NRT genes in the root and leaf of Xu178 in response to nitrogen starvation and nitrate resupply. (A) Each column represents the root of Xu178 sampled at a specific time point. (B) Each column represents the leaf of Xu178 sampled at a specific time point. DN3, treated with nitrogen-deprived (DN) nutrient solution for 3 h. RN3, nitrogen resupply (RN, 4 mM NO3−) for 3 h. Representative genes of multiple groups are highlighted with underlines.
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Figure 8. Expression profile of six NRT genes in four maize inbred lines in response to nitrogen starvation and nitrate resupply. (A) Each column represents the root of four maize inbred lines sampled at specific time point. (B) Each column represents the leaf of four maize inbred lines sampled at specific time point. DN3, treated with nitrogen-deprived (DN) nutrient solution for 3 h. RN3, nitrogen resupply (RN, 4 mM NO3−) for 3 h.
Figure 8. Expression profile of six NRT genes in four maize inbred lines in response to nitrogen starvation and nitrate resupply. (A) Each column represents the root of four maize inbred lines sampled at specific time point. (B) Each column represents the leaf of four maize inbred lines sampled at specific time point. DN3, treated with nitrogen-deprived (DN) nutrient solution for 3 h. RN3, nitrogen resupply (RN, 4 mM NO3−) for 3 h.
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MDPI and ACS Style

Jia, L.; Hu, D.; Wang, J.; Liang, Y.; Li, F.; Wang, Y.; Han, Y. Genome-Wide Identification and Functional Analysis of Nitrate Transporter Genes (NPF, NRT2 and NRT3) in Maize. Int. J. Mol. Sci. 2023, 24, 12941. https://doi.org/10.3390/ijms241612941

AMA Style

Jia L, Hu D, Wang J, Liang Y, Li F, Wang Y, Han Y. Genome-Wide Identification and Functional Analysis of Nitrate Transporter Genes (NPF, NRT2 and NRT3) in Maize. International Journal of Molecular Sciences. 2023; 24(16):12941. https://doi.org/10.3390/ijms241612941

Chicago/Turabian Style

Jia, Lihua, Desheng Hu, Junbo Wang, Yuanyuan Liang, Fang Li, Yi Wang, and Yanlai Han. 2023. "Genome-Wide Identification and Functional Analysis of Nitrate Transporter Genes (NPF, NRT2 and NRT3) in Maize" International Journal of Molecular Sciences 24, no. 16: 12941. https://doi.org/10.3390/ijms241612941

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