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Beyond nitrate transport: AtNRT2.4 responds to local and systemic nitrogen signaling in Arabidopsis

BMC Plant Biology volume 25, Article number: 655 (2025) Cite this article

Abstract

Background

Plants have evolved the ability to detect nitrogen fluctuations to optimize their nitrogen acquisition. However, the mechanisms of nitrogen perception and signaling still need to be well characterized.

Results

Through split-root experiments, this study demonstrated that nitrate transporter 2.4 (AtNRT2.4) can respond to both local and systemic nitrate signals, modulating the transcription of genes such as AtANR1 and AtCIPK23, thereby altering root architecture. Beyond merely detecting the fluctuations of environmental nitrate concentrations, AtNRT2.4 was actively engaged in the dual-affinity transition of AtNRT1.1 and suppressed the expression of AtNLP7, which is crucial for responding to intracellular nitrate signals. Notably, AtNRT2.4 did not participate in the CEP-mediated systemic nitrogen stress signaling pathway and also did not require AtNRT3.1 as a chaperone protein. The knockout of AtNRT2.4 did not affect the growth of Arabidopsis thaliana under low nitrate conditions. However, its overexpression significantly enhanced biomass accumulation and seed yield under normal nitrate concentrations. Furthermore, under nitrate deficiency stress, AtNRT2.4 induced the expression of key genes involved in anthocyanin synthesis and accumulation, thereby promoting anthocyanin accumulation in leaves.

Conclusions

In summary, AtNRT2.4 plays a crucial role in local and systemic nitrate signals sensing, adjustment of root architecture, and anthocyanin accumulation, providing new insights into how plants respond to nitrogen deprivation.

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Background

Nitrogen (N) is an essential element for plant growth, development, and reproduction. Plants have evolved sophisticated transport mechanisms to optimize N uptake and utilization, thereby adapting to fluctuations in environmental availability [1]. Nitrate and ammonium are the primary inorganic N forms absorbed and utilized by higher plants [2], with nitrate typically being the predominant N source in aerobic soils. In Arabidopsis thaliana, Nitrate transporters 1 and 2 (NRT1 s and NRT2 s) are vital in nitrate absorption and transportation. NRT1 belongs to the low-affinity nitrate transport system (LATS) and primarily performs functions when external nitrate concentrations are high; in contrast, NRT2 belongs to the high-affinity nitrate transport system (HATS) and mediates N uptake when external nitrate concentrations are low [3, 4].

Seven NRT2 family members have been identified in the A. thaliana. AtNRT2.1, AtNRT2.2, AtNRT2.4, and AtNRT2.5 exclusively mediate high-affinity nitrate influx. However, external nitrate availability does not affect AtNRT2.3, AtNRT2.6, or AtNRT2.7 [5,6,7]. AtNRT2.1 is a central component of HATS, and internal HATS activity is reduced by 72% in the nrt2.1 mutant. AtNRT2.4 is primarily upregulated in the epidermis of lateral roots and is specifically localized to the outer layer of the plasma membrane under severe N deficiency [6], and demonstrated the greater sensitivity to high N inhibition compared to AtNRT2.1 [8]. Nitrate transport by multiple NRT2 s requires the chaperone protein NRT3.1 (NAR2.1), and they may function as tetramer through an as-yet-unknown mechanism [9,10,11]. The Xenopus oocyte experiments demonstrated that AtNRT2.4, unlike AtNRT2.1, can transport nitrate independently of AtNRT3.1 [5, 6]. Furthermore, the nrt2.4 mutant does not affect plant biomass, nitrate influx, or total plant nitrate content but specifically reduces nitrate levels in the leaf phloem [7]. According to these studies, AtNRT2.4 might have unknown functional roles than those that have been identified.

Nitrate serves a dual role as an essential nutrient and a signaling molecule that triggers numerous physiological and developmental responses, including primary nitrate responses (PNR) [12, 13]. Plants perceive nitrate signals through several pathways: (i) AtNRT1.1, which mediates the perception of local N signals [14, 15]; (ii) C-terminally encoded peptides (CEPs), which are involved in long-distance systemic signaling [16]; and (iii) NIN-like protein 7 (NLP7), which is responsible for intracellular nitrate sensing [17, 18]. AtNRT1.1 is a dual-affinity nitrate transporter and sensor. Its phosphorylation state, which is regulated by the Calcineurin B-like protein 9 (CBL9) and the CBL-interacting protein kinase23 (CIPK23), affects its affinity for nitrate transport [14, 19]. Plant roots initiate CEP peptides in response to N starvation, which are transported to the shoot via xylem, detected by CEP receptors (CEPRs) on shoot, leading to the synthesis of CEP-derived peptides (CEPDs), which were then transported back to the roots, where they increased AtNRT2.1 expression and pushed nitrate uptake [16]. NLP7 rapidly binds to nitrate within the cell, and its N-terminal HLY residues form coordination bonds with nitrate molecules, inducing conformational changes that enable nuclear translocation and subsequent activation of nitrate-responsive genes [18, 20, 21]. Additionally, under N-deficient conditions, AtNLP7 interacts with teosinte branched1/cycloidea/proliferating cell factor 1–20 (AtTCP20) and upregulate the expression levels of AtNRT1.1 [22].

The root system architecture (RSA) of A. thaliana has been significantly influenced by both local and systemic N signaling pathways [23]. Under severe N deficiency, root elongation and proliferation are suppressed, while mild N deficiency stimulates the elongation of lateral roots (LR). High nitrate, ammonium, and organic N sources (such as glutamate) restrict root growth [24,25,26]. Under N-deficient conditions, plants use"root foraging"to allocate resources to areas with abundant nitrate by promoting LR elongation and proliferation [27,28,29]. Under uneven nitrate distribution, transcription factors AtTCP20 and AtANR1 respond to systemic nitrate signals, activate LR foraging behavior, and regulate the expression of genes related to primary nitrate uptake, transport, and assimilation [30,31,32]. Furthermore, specific kinases (such as CIPK8, CIPK23, and CPK10/30/32) modulate RSA by influencing the phosphorylation status of AtNRT1.1 [20, 33].

Anthocyanins, flavonoid compounds derived from the phenylpropanoid pathway, protect plants from adverse environmental conditions by scavenging reactive oxygen species (ROS), thereby serving as an autoprotective mechanism against adversity [34, 35]. These pigments accumulated in different plant tissues in response to multiple abiotic stresses such as ultraviolet light [36, 37], drought [38], cold [39], and N deficiency [36, 40, 41]. Typically, low N availability facilitates the accumulation of anthocyanins, whereas high N levels suppress this process [42,43,44]. Early biosynthesis genes (like CHS and F3H), late biosynthesis genes (like DFR and ANS), and regulatory factors (like PAP1/2 and TT8) have all worked together to control the biosynthesis of anthocyanins [45, 46]. An accumulation of anthocyanins was reported by the induction of AtPAP1, AtPAP2, and AtGL3 expression under N-deficient conditions [40]. It has been reported that under N-limited conditions, members of the MYB transcription factor family, such as PAP1 in A. thaliana and MdMYB1 in Malus domestica, regulate anthocyanin biosynthesis by forming MBW complexes with bHLH and WD40-type transcription factors [47, 48].

In A. thaliana, mutations in AtNRT2.4 have been reported not to affect nitrate transport and the normal growth and development of the plant [6, 7], yet the underlying mechanisms have not been fully understood. Through split-root experiments conducted in this study, we demonstrated that AtNRT2.4 can sense both local and systemic nitrate signals, thereby modulating RSA. Comparing key mutants with the Col-0, we found that the nitrate sensing mechanism of AtNRT2.4 is distinct from the known sensors: the local N signal sensor AtNRT1.1, the systemic signal sensor AtCEP, and the intracellular nitrate sensor AtNLP7. Furthermore, AtNRT2.4 plays a significant role in the biosynthesis and accumulation of anthocyanins under N-limited conditions. These findings suggest that AtNRT2.4, in addition to its role as a high-affinity nitrate transporter, may also serve as a nitrate sensor.

Results

AtNRT2.4 can respond to local N-deficiency signal and systemic nitrate signal

It has been reported that the nrt2.4 mutant exhibits growth performance similar to that of Col-0 under low nitrate conditions [6, 7]. To further investigate whether AtNRT2.4 can sense nitrate deficiency stress and elucidate its response mechanisms, we designed two homogeneous control treatments (C.NaCl and C.NaNO3) and a split-root system (Sp.NaCl/Sp.NaNO3) to characterize the nrt2.4 mutant in detail (Fig. 1a). Based on the classification by Ruffel et al. [13], these signals were categorized into local N-deficiency signals, systemic N-deficiency signals, and systemic N-sufficiency signals, with the latter two collectively referred to as systemic nitrate signals.

Fig. 1
figure 1

AtNRT2.4 participates in root system architecture and systemic nitrate signal transduction. a-c Root morphology, total root length, and lateral root number in homogeneous treatments (C.NaCl, C.NaNO3) and split-root system (Sp.NaCl/Sp.NaNO3). The diameter of the Petri dish amounts to 9 cm. The data represents mean ± SE (n = 4), and the letters above the horizontal lines are based on Tukey's HSD test (p < 0.05)

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In this study, it was found that Col-0 is capable of perceiving local N-deficiency signals, promoting root growth under C.NaCl conditions. Additionally, Col-0 can detect systemic N-deficiency signals, thereby facilitating LR growth on Sp.NaNO3 side. Conversely, it responds to systemic N-sufficiency signals by inhibiting LR growth on Sp.NaCl side. These root responses of Col-0 to the external nitrate status were consistent with previous reports [13, 49]. As expected, in the homogeneous controls, the nrt2.4 mutant displayed inhibited root elongation under C.NaCl conditions and stimulated LR growth under C.NaNO3 conditions. In the split-root system, the nrt2.4 mutant showed reduced LR growth on Sp.NaNO3 side (Fig. 1a - c), contrasting sharply with the behavior observed in Col-0. It indicates that the nrt2.4 mutant cannot perceive local N-deficiency and systemic nitrate signals, ultimately affecting RSA.

We comprehensively elaborated the effect of local N-deficiency and systemic nitrate signals on the RSA, nitrate uptake and transport, and root development related genes expression. The specific data are detailed in Table 1 and illustrated in Additional file 1: Fig. S1. We found that the AtNRT2.4 gene responded to local N-deficiency signal, promoted root growth under C.NaCl treatment and up-regulated root development, nitrate uptake, and transport related genes. Additionally, AtNRT2.4 could sense systemic N-deficiency signals, primarily by promoting root growth on the Sp.NaNO3 side and up-regulated the genes involved in root development and the N starvation response. In parallel, AtNRT2.4 also perceives a systemic N-sufficiency signal, resulting in inhibited root growth on the Sp.NaCl side and down-regulated the transcription levels of genes linked to root development and nitrate uptake. Significantly, the gene expression pattern of AtANR1 responsive to local N-deficiency and systemic nitrate signals was most analogous to RSA, with the most significant alterations in transcriptional activity, indicating a stronger correlation between the AtANR1 transcriptional activity and AtNRT2.4 presence or absence (Table 1, Additional file 1: Fig. S1).

Table 1 AtNRT2.4 responds to local N-deficiency and systemic nitrate signals
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In conclusion, AtNRT2.4 can perceive both local N-deficiency and systemic nitrate signals. It modulates root growth in response to rapid fluctuations in local nitrate availability by regulating the expression of genes associated with nitrogen sensing and root architecture development.

AtNRT2.4 is not involved in CEP-mediated N stress response pathways and does not require AtNRT3.1 as a chaperone

As described above, AtNRT2.4 can sense both local N-deficiency and systemic nitrate signals. To elucidate the relationship between the specific sensing mechanism of AtNRT2.4 with the currently known nitrate signal transduction pathways, we selected Col-0, nrt2.4, cepr2, and nrt3.1 mutants and subjected them to short-term N starvation and nitrate resupply treatments. This experimental design enabled us to analyze the patterns of transcriptional activity of essential genes involved in these mechanisms. The transcriptional activity changes in all mutants were compared with those in Col-0 throughout our rest investigation.

AtNRT2.4 is not regulated by AtCEPRs as the expression pattern of AtCEPR2 remained consistent between Col-0 and nrt2.4 mutant under low nitrate (LN) conditions. However, it was markedly upregulated in nrt2.4 mutant under normal nitrate (NN) conditions (Fig. 2a). AtNRT2.4 might negatively regulate the expression level of AtCEPR2 under NN conditions. The expression of AtNRT2.1 was significantly down-regulated in both nrt2.4 and cepr2 mutants under LN conditions and up-regulated significantly in these mutants under NN conditions (Fig. 2b). Quite differently, the expression of AtNRT2.4 was not significantly different between cepr2 mutant under LN and NN conditions (Fig. 2c), indicating that the absence of AtCEPR2 does not considerably influence on the transcription of AtNRT2.4. These results suggest that under LN conditions, AtNRT2.1 is induced and up-regulated by AtCEPR2, while AtCEPR2 does not influence AtNRT2.4.

Fig. 2
figure 2

Gene expression patterns of Col-0, nrt2.4, cepr2, and nrt3.1 mutants under short-term N starvation and nitrate resupply conditions. a-c Expression patterns of genes AtCEPR2, AtNRT2.1, and AtNRT2.4 in Col-0, nrt2.4, and cepr2 mutants under LN and NN resupply conditions. d Expression patterns of genes AtNRT3.1 in Col-0, nrt2.4, and nrt3.1 mutants under LN and NN resupply conditions. e,f Expression patterns of genes AtNRT2.1 and AtNRT2.4 in Col-0 and nrt3.1 mutant under LN and NN resupply conditions. The analysis of gene expression levels was conducted using whole seedling samples. The data represents mean ± SE (n = 4), and the letters above the horizontal lines are based on Tukey's HSD test (p < 0.05)

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Transcriptional activity of AtNRT2.4 is not affected by AtNRT3.1 as the expression of AtNRT3.1 was significantly down-regulated in nrt2.4 mutant under LN conditions and showed no significant difference from Col-0 under NN conditions (Fig. 2d), suggesting that the transcription of AtNRT3.1 is suppressed due to an inhibitory effect of AtNRT2.4 under LN conditions. Furthermore, AtNRT2.1 expression was down-regulated as nitrate concentration increased in Col-0, while it was not affected as nitrate concentration increased in nrt2.4 and nrt3.1 mutants (Fig. 2e), indicating that the response of AtNRT2.1 to external nitrate concentration is dependent on the assistance of AtNRT2.4 and AtNRT3.1. In contrast, the expression pattern of AtNRT2.4 remained consistent between Col-0 and nrt3.1 mutant under LN and NN conditions (Fig. 2f), indicating that the transcriptional activity of AtNRT2.4 is independent of AtNRT3.1. As a pivotal member of the HATS, AtNRT2.1 relies not only on AtNRT3.1 as a chaperone but also has its transcription influenced by AtNRT2.4, whereas AtNRT2.4 itself is not affected by AtNRT3.1.

In summary, AtNRT2.4 does not participate in the CEP-mediated systemic N stress signaling pathway, and its function does not require AtNRT3.1 to be a chaperone protein. In addition to functioning as a nitrate transporter, AtNRT2.4 is involved in the nitrate sensing pathway that is distinct from that of AtNRT2.1.

AtNRT2.4 is involved in the dual-affinity transition of AtNRT1.1

To further elucidate the nitrate sensing pathway involving AtNRT2.4, we introduced an additional key mutant, nrt1.1, and subjected it to the same experimental conditions as described above. Our results demonstrated that the expression of AtNRT2.4 was significantly down-regulated exclusively in the nrt1.1 mutant under LN condition (Fig. 3a), indicating that under LN conditions, the transcriptional activity of AtNRT2.4 is up-regulated under the influence of AtNRT1.1. Surprisingly, the expression of AtNRT1.1 was up-regulated in nrt2.4 mutant under both LN and NN conditions (Fig. 3b), suggesting that AtNRT2.4 may apply an unknown negative regulatory effect on the transcriptional activity of AtNRT1.1. These findings indicate that AtNRT2.4 and AtNRT2.1 exhibit reciprocal regulatory interactions under different nitrate concentrations, which may be attributed to their distinct roles in nitrate sensing and transport.

Fig. 3
figure 3

Gene expression patterns of Col-0, nrt2.4, and nrt1.1 mutants under short-term N starvation and nitrate resupply. a-e Differential expression patterns of the genes AtNRT2.4, AtNRT1.1, AtCBL9, AtCIPK23, and AtNLP7 across the whole plant under LN and NN resupply conditions. The data represents mean ± SE (n = 4), and the letters above the horizontal lines are based on Tukey's HSD test (p < 0.05)

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Previous work has demonstrated that the CBL1/9-CIPK23 complex's phosphorylation of the T101 residue on AtNRT1.1 is essential for effective nitrate uptake in low N conditions [14, 50]. The detailed transcriptional activities of AtCBL9 and AtCIPK23 were analyzed in different mutants. We observed that AtCBL9 and AtCIPK23 expression levels were both up-regulated in nrt2.4 mutant, with no significant difference in nrt1.1 mutant (Fig. 3c, d). It was suggested that AtNRT2.4 may suppress the transcriptional activity of AtCBL9 and AtCIPK23. These results suggest that AtNRT2.4 may participate in the dual-affinity transition of AtNRT1.1 by downregulating the transcriptional levels of AtCBL9, AtCIPK23, and AtNRT1.1.

AtNRT2.4 constrains AtNLP7-mediated perception of intracellular nitrate signaling

AtNLP7 has been identified as a principal transcription factor, that directly binds to nitrate ions, sense the intracellular nitrate levels and govern the expression of nitrate signaling genes such as AtNRT1.1, AtNRT2.1, and AtCEPR2, either directly or indirectly [17]. Interestingly, further detailed investigation of the attachment data of Alvarez's research [17] discovered that AtNRT2.4 expression was consistently unaffected among all AtNLP7 genetically modified materials. This finding pushed us to investigate the unknown but potential relationship between AtNLP7 and AtNRT2.4 in nitrate signaling transduction. Therefore, this study analyzed the differential expression patterns of AtNLP7 in Col-0, nrt2.4, and nrt1.1 mutants under LN and NN conditions. In contrast to Col-0, the expression level of AtNLP7 was significantly elevated in nrt2.4 mutant under both LN and NN conditions, there was no significant difference in the nrt1.1 mutant (Fig. 3e). These outcomes suggest that AtNRT2.4 might play a critical role in regulating intracellular nitrate signaling transduction by inhibiting the transcriptional activity of AtNLP7.

The overexpression of AtNRT2.4 can boost the growth of A. thaliana

To elucidate the mechanism by which the deletion of AtNRT2.4 does not adversely affect the growth of A. thaliana and to identify the compensatory genes that functionally substitute for its loss, we successfully produced overexpression lines (OE3, OE16) in the Col-0 background and created complementation lines (Com4, Com9) in the nrt2.4 mutant background (Fig. 4a). In line with previous studies, we subjected Col-0, nrt2.4 mutant, and these transgenic lines to short-term N starvation and nitrate resupply experiments.

Fig. 4
figure 4

Gene expression patterns, ammonium accumulation, and phenotypic traits in AtNRT2.4 transgenic lines under short-term N starvation and nitrate resupply conditions. a-f Comparative gene expression patterns of AtNRT2.4, AtNRT2.1, AtNRT2.2, AtNRT2.5, AtNRT1.1, and AtNLP7 in Col-0, nrt2.4 mutant, complementation lines (Com4, Com9), and overexpression lines (OE3, OE16) across the whole plant under LN and NN resupply conditions. g Accumulation of ammonium in the shoot parts of these lines under LN and NN pot conditions. h,i Biomass of shoots and seeds yield of these lines under LN and NN pot conditions. Data represents mean ± SE. The letters above the horizontal lines are based on Tukey's HSD test (p < 0.05), with LN and NN groups comparisons

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The expression levels of the AtNRT2 family members are typically induced by LN concentrations and suppressed by NN concentrations, as exemplified by AtNRT2.4, AtNRT2.1, AtNRT2.2, and AtNRT2.5 (Fig. 4a - d). In contrast, AtNRT1.1 is predominantly induced by NN concentrations (Fig. 4e). AtNLP7 primarily detects the presence of nitrate and is less influenced by the changes in its concentration (Fig. 4f). Compared to Col-0, the AtNRT2.1 expression was significantly reduced in nrt2.4 mutant under LN conditions (Fig. 4b). However, the expression levels of AtNRT2.2, AtNRT2.5, AtNRT1.1, and AtNLP7 were considerably higher in nrt2.4 mutant under LN conditions (Fig. 4c, f). Moreover, these five genes'expression patterns in the complementation and overexpression lines were largely in accordance with those observed in Col-0. These findings suggest that under LN conditions, the functional deficiency of AtNRT2.4 as a high-affinity nitrate transporter is primarily compensated by the upregulation of AtNRT2.2 and AtNRT2.5 in the nrt2.4 mutant. Meanwhile, AtNLP7 quickly initiates the transduction of intracellular nitrate signaling.

To further determine the influence AtNRT2.4 on A. thaliana growth and seed yield, we conducted long-term pot experiments under LN and NN conditions (Additional file 2: Fig. S2). The accumulation of nitrate and ammonium in the above-ground parts of various lines, and phenotypic traits were assessed. Under both LN and NN conditions, the rosette leaf diameter of nrt2.4 mutant was significantly larger than that of the transgenic lines, but leaf area did not show remarkable differences among the lines (Additional file 3: Fig. S3b-d). Under LN conditions, the nitrate and ammonium accumulation were comparable across all lines, indicating that the presence or absence of AtNRT2.4 has little effect on nitrate absorption and transport under LN conditions. However, under NN conditions, the overexpression lines accumulated significantly more ammonium than other lines (Fig. 4g, Additional file 3: Fig. S3a). Under NN conditions, the overexpression lines displayed significantly better phenotypic traits, including plant height, number of siliques, biomass, and seed yield, compared to other lines. In contrast, under LN conditions, the nrt2.4 mutant showed a reduced plant height, while no significant differences were observed in silique numbers, biomass, or seed yield (Fig. 4h, i, Additional file 3: Fig. S3e, f). These results suggest that the overexpression of AtNRT2.4 enhances ammonium accumulation under NN conditions, thereby promoting plant growth and increasing silique number, biomass, and seed yield.

In conclusion, the absence of AtNRT2.4 does not affect nitrate absorption and transport in A. thaliana, but the overexpression of AtNRT2.4 enhances N uptake and accumulation, thereby promoting A. thaliana growth and development.

AtNRT2.4 promote anthocyanin biosynthesis in response to N deficiency

In our preliminary experiments, we observed that under LN conditions, the leaves of the nrt2.4 mutant remained green. In contrast, the leaves of in Col-0 exhibited a purple coloration and contained significantly higher levels of anthocyanins than in nrt2.4 mutant (Additional file 4: Fig. S4). Based on these observations, it is hypothesized that the absence of AtNRT2.4 may impair the plant's ability to perceive external nitrate deficiency signals, thereby reducing the anthocyanin accumulation. To test this hypothesis, we conducted short-term N starvation and nitrate resupply experiments on the previously mentioned transgenic lines. Under LN conditions, a significant purple coloration was evident in Col-0 leaves but absent in the nrt2.4 mutant; the leaf color of the complementation lines was intermediate between that of Col-0 and nrt2.4 mutant, while the overexpression lines exhibited leaf colors similar to or more intense than those of Col-0 (Fig. 5a). These reproducible phenotypes further validate the potential connection between AtNRT2.4 and the process of anthocyanin biosynthesis and accumulation.

Fig. 5
figure 5

AtNRT2.4 enhances anthocyanin synthesis and accumulation in the leaves of A. thaliana under low nitrate conditions. a Leaf phenotypes of diverse A. thaliana lines following short-term N starvation and nitrate resupply. Scale bar = 1 cm. b-e Expression levels of anthocyanin biosynthesis and accumulation-related genes AtDFR, AtANS, AtTT8, and AtPAP2 in whole A. thaliana plants following short-term N starvation and nitrate resupply. Data represents mean ± SE (n = 4). The letters above the horizontal lines are based on Tukey's HSD test (p < 0.05), with LN and NN groups comparisons

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This observation quantified the expression levels of key genes involved in anthocyanin biosynthesis and accumulation (AtDFR, AtANS, AtTT8, AtPAP2, AtF3H). Under LN conditions, the expression of these genes declined in nrt2.4 mutant and significantly increased in both the overexpression and complementation lines (Fig. 5b - e, Additional file 4: Fig. S4). These data strongly support our hypothesis that AtNRT2.4 responds to low nitrate/nitrogen deficiency stress and initiates the biosynthesis and accumulation of anthocyanins in leaves.

Discussion

Through an in-depth investigation of the nitrate perception mechanisms in plants, we can better understand nitrogen uptake and utilization under varying nitrogen conditions, thereby enhancing nitrogen use efficiency. Although several key genes involved in nitrate sensing have been identified in recent years [14, 17], the complete mechanistic details of nitrate perception remain elusive. This study elucidates the novel role of AtNRT2.4 in perceiving both local and systemic nitrate signals and modulating root architecture, suggesting that AtNRT2.4 functions as a nitrate sensor.

In fluctuating N environments, plants have evolved intricate foraging strategies to optimize N acquisition, guaranteeing their proper growth and development [12, 13, 28]. In this study, the Col-0 could detect local nitrate-deficiency signals, which led to the induction of root elongation and lateral root formation on C.NaCl and the inhibition effects of root growth on C.NaNO3. Under heterogeneous split-root treatment, Col-0 could also perceive the systemic N-deficiency signal to trigger the root growth on Sp.NaNO3 side.. In parallel, Col-0 senses N-sufficient signals to suppress root growth on Sp.NaCl side (Fig. 1a - c, Col-0), consistent with previous research [13, 49, 51]. In contrast, the nrt2.4 mutant exhibits impaired responsiveness to both local N-deficiency and systemic nitrate signals, leading to significant changes in root morphology and gene expression patterns (Fig. 1, Table 1, and Additional file 1: Fig. S1). AtTCP20 and AtANR1 are pivotal root development regulators sensitive to local and systemic nitrate signals. They promote lateral root proliferation on nitrate-rich sides and inhibit lateral root development on nitrate-deficient sides [30, 52, 53], as further confirmed in this study. Notably, the expression patterns of AtANR1 and AtTCP20 are significantly altered in the nrt2.4 mutant, which correlates with the observed changes in RSA (Table 1, Additional file 1: Fig. S1b, f). Furthermore, the RSA of nrt2.4 mutant in the split-root system (Fig. 1) is highly similar to that of the tcp20 mutant [30]. By integrating these findings, we propose that AtNRT2.4 may be part of the same foraging signaling pathway as AtTCP20, potentially perceiving the foraging signal upstream of AtTCP20. These results prove that AtNRT2.4 can sense local N-deficiency and systemic nitrate signals, influencing RSA.

AtNRT2.1 is believed to be induced by long-distance N starvation signals mediated by CEPs [16, 54] and is further subject to feedback regulation by assimilation products [14, 33, 55]. In this study, we observed that the expression of AtNRT2.1 is downregulated in cepr2 and nrt3.1 mutants (Fig. 2b, e), reinforcing the notion that CEP-mediated N starvation signals regulate AtNRT2.1 and that its functional execution depends on the presence of AtNRT3.1. However, the transcription of AtNRT2.4 remains unaffected in cepr2 and nrt3.1 mutants (Fig. 2c, f), indicating that the function of AtNRT2.4 is independent of AtCEPR2 and does not require AtNRT3.1 as a chaperone protein. Therefore, AtNRT2.4 exhibits significant functional differences from AtNRT2.1, suggesting that AtNRT2.4 may play a more substantial role in signal perception rather than merely acting as a nitrate transporter.

It is widely recognized that the AtCBL1/9-AtCIPK23 complex modulates the phosphorylation status of AtNRT1.1, enabling its transition between high- and low-affinity states for nitrate uptake [14, 50]. In this study, we found that AtNRT2.4 suppresses the expression of AtCBL9, AtCIPK23, and AtNRT1.1 under both LN and NN conditions (Fig. 3b - d), indicating that AtNRT2.4 is involved in the nitrate sensing pathway associated with AtNRT1.1. Further investigation revealed that AtNRT1.1 is negatively regulated only by local N-deficiency signals (Additional file 5:Table S1, Additional file 1: Fig. S1i), while AtCIPK23 and AtCBL1 are activated by local N-deficiency signals and repressed by systemic nitrate signals (Table 1, Additional file 1: Fig. S1c, g) in an AtNRT2.4-dependent manner. The intimate relationship between AtNRT2.4 and AtNRT1.1-mediated sensing pathway indicates that AtNRT2.4 plays an integral role in modulating the response to nitrate availability.

Studies have established that AtNRT1.1 functions not only as a nitrate transporter but also as a nitrate sensor [14]. However, our research indicates that AtNRT2.4 can perceive nitrate signals, initiate nitrate response reactions, and influence the expression of several PNR genes, including AtNRT1.1, AtNRT2.1, AtANR1, AtCIPK23, and AtNLP7. Therefore, we propose that AtNRT2.4 perceives both local and systemic nitrate signals, possibly acting upstream of AtNRT1.1 in the nitrate sensing cascade. Although this hypothesis requires further validation, our study sheds new light on the roles of AtNRT2.4 and AtNRT1.1 in nitrate signal perception and the regulation of LR development.

In the complex regulatory network of plant N response, AtNLP7 is widely recognized as an intracellular nitrate sensor that can directly bind to nitrate ion and translocate to the nucleus. This process subsequently triggers the transcription of a range of early response genes involved in nitrate signaling [17, 18]. However, a more detailed analysis of the findings from Alvarez et al. [17] revealed that AtNRT1.1 and AtNRT2.1 are transient target genes of AtNLP7, whereas no evidence was found for the regulation of AtNRT2.4 by AtNLP7 at any time point. Additionally, the expression of AtNRT2.4 is not influenced by any member of the AtNLP family under nitrate supply, while AtNRT2.1 and AtNRT3.1can be induced by all AtNLP members [18]. Under non-limiting nitrate supply, AtNRT2.4 is not a direct target gene of AtNLP2. Nevertheless, the expression level of AtNRT2.4 is slightly enhanced in the nlp2-1 nlp7-1 double mutant [56]. Notably, the expression of AtNLP7 is significantly upregulated in nrt2.4 mutant, especially under LN condition (Fig. 3e, 4f). Based on these observations, we propose that AtNRT2.4 primarily detects changes in external nitrate concentrations, Then, intracellular AtNLP7 decides whether or not it translocate to the nucleus to initiate the transcription of a series of genes.

Aligning with previous findings [6, 7], we observed that the accumulation of nitrate and ammonium, biomass, and seed yield in nrt2.4 mutant were all similar to those of Col-0 under both LN and NN conditions (Fig. 6g - i, Additional file 3: Fig. S3a). Our research further revealed that in the nrt2.4 mutant, the upregulation of AtNRT2.2 and AtNRT2.5 could make up for the mutant's loss of nitrate uptake function (Fig. 4c, d). Notably, the expression of AtNLP7 were elevated in nrt2.4 mutant (Fig. 3e, 4f). Furthermore, we observed that anthocyanin accumulation in the leaves of nrt2.4 mutant was less prominent compared to Col-0 under LN condition (Fig. 5a, Additional file 4: Fig. S4). It is widely recognized that nutrient deficiencies, including nitrogen and phosphorus deficiencies, can induce the accumulation of anthocyanins in plants [36, 41, 57]. Moreover, our study found that AtNRT2.4 can upregulate key genes involved in anthocyanin biosynthesis and accumulation (Fig. 5b - e). It is further confirmed that AtNRT2.4 may sense the nitrogen deficiency signal to induce anthocyanin biosynthesis and accumulation to combat the damage caused by low nitrogen stress. However, the underlying molecular mechanisms remain to be elucidated.

Fig. 6
figure 6

Schematic model of AtNRT2.4 involvement in the regulation of root development by sensing the local and systemic nitrate signaling. AtNRT2.4 perceives local N-deficiency signal and activates the AtANR1 and AtTCP20 mediated signaling pathways, thereby promoting LR elongation and proliferation (left). Conversely, when external nitrate is abundant, it inhibits root elongation by suppressing this cascade (right). Additionally, AtNRT2.4 can sense low nitrate signals to activate the pathway of AtPAP2/AtTT8 regulating the AtF3H-AtDFR/AtANS genes, leading to anthocyanin accumulation (left). AtNRT2.4 also involved the phosphorylation of AtNRT1.1 by the AtCBL1/9-AtCIPK23 complex in response to external nitrate concentration, facilitating the switch between high and low-affinity nitrate transport. Furthermore, systemic N-sufficiency signals inhibit the AtTCP20 and AtANR1-mediated signaling pathways in an AtNRT2.4-dependent manner, thereby suppressing the elongation of the primary root and proliferation of LR on the -N side. In contrast, the systemic N-deficiency signals enable AtNRT2.4 to promote AtANR1, enhancing the elongation of the primary root and proliferation of lateral roots on the + NO3− side (middle). Gradient-colored dots represent nitrate; arrows indicate positive regulation, and flat ends indicate negative regulation. Blue and red dashed lines represent systemic nitrate sufficiency and deficiency signaling, respectively

Full size image

Conclusions

This study elucidates the pivotal role of AtNRT2.4 in A. thaliana in nitrate signal perception and nitrogen response regulation. AtNRT2.4 not only detects local nitrate deficiency signals and systemic nitrate signals but also coordinates plant adaptive responses to nitrogen environmental changes by modulating root architecture, including lateral root development and primary root elongation, as well as anthocyanin synthesis (Fig. 6). These findings expand our understanding of the molecular framework underlying plant nitrate perception and provide new insights into the hierarchical organization of the nitrogen signaling network. Future studies should explore the specific mechanisms underlying the role of AtNRT2.4 in nitrate signaling and its interactions with other key transcription factors.

Methods

Plant materials

Acquisition of mutant materials. The three mutants, including nrt2.4 (SALK_205302), nrt1.1 (SALK_097431 C), and nrt3.1 (SALK_043672) were all procured from the AraShare Science platform (https://www.arashare.cn/index/). The cepr2 mutant (SALK_014533) was generously provided by Professor Lei Zhang from Shandong University of Traditional Chinese Medicine. This mutant has been previously characterized [58]. Homozygosity verification of the nrt2.4, nrt3.1 and nrt1.1 mutants were presented in Additional file 6: Fig. S5, and the primers used for this verification are listed in Additional file 7: Table S2.

Transformation and screening of transgenic lines. The coding sequence of AtNRT2.4 was amplified using polymerase chain reaction (PCR) and then cloned into a modified overexpression vector derived from pCAMBIA1300. This vector, which was regulated by the CaMV 35S promoter, fused a green fluorescent protein (GFP) tag, and with a hygromycin (Hyg) resistance selection marker gene. This overexpression construct was introduced into Agrobacterium tumefaciens strain GV3101 to facilitate plant transformation. The nrt2.4 mutant and Col-0 plants were transformed by using the floral dip method. Seeds from each generation were sown on half-strength Murashige and Skoog (1/2 MS) medium containing Hyg for root selection, followed by individual plant harvesting. In the T2 generation, single-copy transgenic lines were identified based on Mendelian inheritance principles. Seeds from the T2 generation that exhibited uniform rooting ability on the selection medium were designated as homozygous transgenic lines in the T3 generation. After two generations of stringent selection, we successfully isolated transgenic homozygous lines in the T3 generation. The two overexpression lines were designated as OE3 and OE16, and the two complementation lines were designated as Com4 and Com9.

Plant cultivation and nitrate treatments

Col-0 and nrt2.4 mutant seeds were sown on vermiculite and cultivated under controlled environment with a 14-h photoperiod and a 10-h scotoperiod at 22℃. Seedlings were transplanted to vermiculite after 15 days of germination and subsequently irrigated with the nutrient solution of Hoagland containing either 0.1 mM NaNO3 (low nitrate, LN) or 5 mM NaNO3 (normal nitrate, NN) [56, 59]. Irrigation with the Hoagland nutrient solution was performed once a month, while water alone was used for irrigation during the remaining periods. Anthocyanin content in leaves was quantified 40 days after transplantation (n = 4).

The Col-0, nrt2.4 mutant, and four transgenic lines (OE3, OE16, Com4, and Com9) were cultivated on vermiculite under the previously described conditions. Plant growth parameters were assessed at the following time point after transplantation. The rosette diameter was measured at 30 days (n = 6). Leaf number and area were quantified 40 days, coinciding with the determination of nitrate (NO3−) and ammonium (NH4+) accumulation in the leaves (n = 4). The number of siliques and the height of the plants were recorded at 65 days (n = 9). Furthermore, biomass and seed yield were evaluated for 80 days (n = 9—10). ImageJ software was employed to quantify rosette diameter, leaf number, and area.

Split-root experiments

Following the protocol detailed by Ruffel et al. [13], seeds of nrt2.4 mutant and Col-0 were cleaned on the surface with 70% ethanol containing 0.2% Tween for 10 min, followed by a 1-min rinse with anhydrous ethanol, and then rinsed with sterile water five or six times. Following sterilization, the seeds were planted and cultivated for seven days on 1/2 MS medium. The primary roots were excised and returned to 1/2 MS medium for an additional 4 days to proliferate lateral roots. The generated roots were evenly divided and transferred to split-agar plates comprising three types: two homogeneous control (C.NaCl and C.NaNO3) and one heterogeneous split-root treatment (Sp.NaCl/Sp.NaNO3). The split-root treatment utilized 9-cm circular Petri dishes divided into two compartments. Both compartments contained nitrogen-free MS medium (PhytoTech Labs, M531) supplemented with either 5 mM NaCl or 5 mM NaNO3 on each side, respectively. For detailed compositions, please refer to Table S3. The C.NaCl control plates were filled with N-free medium in both compartments, while the C.NaNO3 control plates contained medium supplemented with 5 mM NaNO3 on both sides. In contrast, the heterogeneous split-root plates (Sp.NaCl/Sp.NaNO3) featured one compartment with 5 mM NaCl (Sp.NaCl) and the other with 5 mM NaNO3 (Sp.NaNO3). After 8 days of growth on these media, the length of the total root and the number of LR were quantified using the Win RHIZO STD4800 LA2400 root scanner. Throughout the experiment, the culture conditions were maintained under a photoperiod of 14 h of light and 10 h of darkness at 22 °C. RNA was taken out of 8-day-old root samples for succeeding real-time PCR (qRT-PCR) and reverse transcription analysis to assess gene expression associated with root architecture, N absorption, and transport. To ensure the reliability of the experimental results, each sample group has 4 biological replicates.

To discriminate different types of signals, we referred to the classification of nitrate signals reported in Ruffel et al. [13]: the significant difference in a trait between N-free and nitrate homogeneous media (C.NaCl/C.NaNO3, ratio ≥ 2 or ≤ 0.5) defined as being influenced by the local N-deficiency signal. The significant difference in a trait between N-free split-root side and N-free homogeneous media (Sp.NaCl/C.NaCl, ratio ≥ 2 or ≤ 0.5) is termed as being influenced by the systemic N-sufficiency signal; likewise, the significant difference in a trait between nitrate split-root side and nitrate homogeneous media (Sp.NaNO3/C.NaNO3, ratio ≥ 2 or ≤ 0.5) is termed as being influenced by the systemic N-deficiency signal. The later both are collectively designated to as systemic nitrate signals. For ease of characterization, we logarithmically transformed these ratios with base 2, where a negative effect of AtNRT2.4 is indicated by values < −1, and a positive effect is indicated by values > 1, and values within the interval (−1, 1) indicate no significant impact.

Short-term N-starvation and nitrate resupply experimentation

Col-0, mutants (nrt2.4, nrt1.1, nrt3.1, and cepr2), and four transgenic lines (OE3, OE16, Com4, and Com9) were subjected to short-term N-starvation and nitrate resupply experiments. Seeds were sterilized, rinsed and grown as previously described, for 10 days. After this initial growth period, seedlings were transferred to N-free 1/2 MS medium for a 7-day N starvation treatment to deplete internal N reserves. The seedlings were resupplied with different nitrate concentrations: LN (0.1 mM NaNO3) and NN (5 mM NaNO3). The whole plant samples were collected 3 days after the LN and NN treatments to evaluate gene expression using real-time quantitative PCR (qRT-PCR).

Gene expression analysis

Complementary DNA (cDNA) was created by reverse transcription of total RNA extracted from the previously mentioned samples. To extract RNA, we utilized the Plant Total RNA Isolation Kit Plus (Foregene, RE-05024). To ensure complete removal of genomic DNA contamination, we employed the HiScript III RT SuperMix for qPCR with gDNA wiper (Vazyme, R323). Subsequently, qRT-PCR analysis was performed using the Taq SYBR® Green qPCR Premix (BestEnzymes, EG20117M) on the Roche LightCycler 96 system following a three-step protocol. A. thaliana glyceraldehyde-3-phosphate dehydrogenase (AtGAPDH) was employed as a reference control by Czechowski et al. [60] and the Col-0 as the control group to normalize the relative expression levels of the target genes. The results of the preliminary experiments showed that AtGAPDH could be consistently expressed across whole-plant samples treated with various nitrogen sources and is therefore suitable as an internal reference control. The 2−∆∆ct method was applied to determine the relative expression levels. All of the primers used in this investigation were designed by NCBI website (https://www.ncbi.nlm.nih.gov/) and listed in Additional file 7: Table S2.

Anthocyanin, nitrate and ammonium content determination

The anthocyanin content in A. thaliana leaves was quantified using the modified citrate-ethanol method as proposed by Hussain et al. [61]. For the determination of the nitrate content of plant tissues, a modified salicylic acid-sulfuric acid method was used by Xu et al. [62]. A certain level of ammonium has been determined using the ZATD plant ammonium test kit from Comin, which utilizes the classic phenol-hypochlorous acid spectrophotometric method. To ensure the reliability of the experimental results, each sample group was subjected to 3 to 4 biological replicates.

Statistical analysis

One-way ANOVA was used to analyze data from several groups, and Tukey's HSD test was utilized to evaluate the significance of differences among groups. Significant differences are indicated at the 95% confidence level by letters a, b, and c. For comparisons involving fewer than ten samples, Col-0 under the LN conditions served as the control; for comparisons involving ten or more samples, Col-0 under both LN and NN conditions was used as the control. Two-tailed t-tests were used to directly compare the two groups, with'*'and'**'denoting p-values less than 0.05 and 0.01, respectively, to signify statistically significant significance.

Data availability

The data supporting the findings of this study are available in the supplementary material of this article.

Abbreviations

N:

Nitrogen

LN:

Low nitrate

HN:

High nitrate

NN:

Normal nitrate

NRTs:

Nitrate transporters

LATS:

Low-affinity nitrate transport system

HATS:

High-affinity nitrate transport system

A. thaliana :

Arabidopsis thaliana

PNR:

Primary nitrate responses

CEPs:

C-terminally encoded peptides

CEPRs:

CEP receptors

CEPDs:

CEP-derived peptides

NLP7:

NIN-like protein 7

CBLs:

Calcineurin B-like proteins

CIPKs:

CBL-interacting protein kinases

TCP20:

Teosinte branched1/ cycloidea/ proliferating cell factor 1-20

RSA:

Root system architecture

LR:

Lateral root

ROS:

Reactive oxygen species

qRT‒PCR :

Quantitative real-time PCR analyses

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Acknowledgements

We would like to thank Prof. Lei Zhang (Shandong University of Traditional Chinese Medicine, China) for supplying the cepr2 mutant, and all members in the laboratory for insightful discussions and advice on the manuscript. We also acknowledge the editor and reviewers for their critiques and constructive comments on the drafted manuscript.

Funding

The work was collectively supported by the grants from National Natural Science Foundation of China (32060714), Hainan Province Science and Technology Special Fund (ZDYF2023XDNY179), Project of National Key Laboratory for Tropical Crop Breeding (NKLTCBCXTD07), Hainan University Research Star-up Funding (RZ2100003186, KYQD20016), and Graduate Innovation Project of Hainan Province (Qhyb2022-57).

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Authors and Affiliations

  1. Sanya Institute of Breeding and Multiplication/School of Tropical Agriculture and Forestry, National Key Laboratory for Tropical Crop Breeding, Hainan University, Sanya, China

    Huaifang Zhang, Yanmei Li, Jinling Zhao, Weitao Mai, Luqman Khan, Wenquan Wang, Changying Zeng & Xin Chen

  2. Institute of Tropical Bioscience and Biotechnology/Sanya Research Institute, Chinese Academy of Tropical Agricultural Science, Haikou, China

    Huaifang Zhang, Jinxia Huang, Yanmei Li, Jinling Zhao, Weitao Mai & Xin Chen

  3. Liaoning Academy of Agricultural Sciences, Shenyang, China

    Meng Zhang

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  1. Huaifang Zhang
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Contributions

CZ, XC and WW conceived the idea and supervised the overall project. HZ, JH and CZ wrote and revised the manuscript. HZ, JH designed and performed the experiments. JZ, WM, YL, LK and MZ participated in the experimental assay, phenotype measurements and data analysis. All authors read and approved the final manuscript.

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Correspondence to Changying Zeng or Xin Chen.

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Supplementary Information

Additional file 1: Fig. S1 Differential gene expression of Col-0 and nrt2.4 mutant in the split-root system

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Additional file 2: Fig. S2 Phenotypic traits of Col-0, nrt2.4 mutant, and transgenic lines under low nitrate (LN) and normal nitrate (NN) pot conditions

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Additional file 3: Fig. S3 Physiological and phenotypic traits of Col-0, nrt2.4 mutant, and transgenic lines under LN and NN pot culture conditions

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Additional file 4: Fig. S4 Leaf phenotypes and anthocyanin accumulation in Col-0 and nrt2.4 mutant under LN pot conditions

Additional file 5: Table S1. Other nitrate-related genes respond to local N-deficiency and systemic nitrate signals

Additional file 6: Fig. S5 Identification of homozygous mutants for nrt2.4, nrt1.1 and nrt3.1

Additional file 7: Table S2 All primers used in the experiments

Additional file 8: Table S3 The constituents of the culture medium utilized in the experiments

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Zhang, H., Huang, J., Li, Y. et al. Beyond nitrate transport: AtNRT2.4 responds to local and systemic nitrogen signaling in Arabidopsis. BMC Plant Biol 25, 655 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12870-025-06695-4

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BMC Plant Biology

ISSN: 1471-2229

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