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. 2014 Apr 22;9:e28501. doi: 10.4161/psb.28501

Nitrate regulation of AFB3 and NAC4 gene expression in Arabidopsis roots depends on NRT1.1 nitrate transport function

Elena A Vidal 1, José M Álvarez 1, Rodrigo A Gutiérrez 1,*
PMCID: PMC4091544  PMID: 24642706

Abstract

Nitrogen is an essential macronutrient for plants and its availability is a major limiting factor for plant growth and crop production. Nitrate is the main source of inorganic N for plants in aerobic soils and can act as a potent signal to control global gene expression. We found that gene expression in response to nitrate treatment of the AFB3 auxin receptor and its target, the NAC4 transcription factor depends on the nitrate transport function of NRT1.1. This gene regulatory function of NRT1.1 on AFB3 and NAC4 differs from the previously described signaling function controlling NRT2.1, NIA1 and NIA2 transcript levels and root colonization of nitrate-rich patches. Our work suggests two different signaling pathways may exist to control gene expression in response to nitrate downstream of NRT1.1.

Keywords: nitrate, NRT1.1, auxin, roots, Arabidopsis

Nitrogen (N) is an essential macronutrient present in aerobic soils mainly as nitrate.1,2 Nitrate is able to induce morphological changes in plants by controlling plant metabolism, physiology, growth and development.3-6 These changes are due partly by nitrate regulation of transcript levels of a myriad of genes involved in different processes.7-12 Understanding the structure of regulatory networks that control plant responses to nitrate is an important step toward engineering traits of interest, such as enhanced growth under nitrate-limiting conditions.

We have recently described a gene regulatory network controlled by the miR393 microRNA and the AFB3 auxin receptor that is able to control root growth in response to external and internal nitrate availability.13,14 The NAC4 transcription factor acts downstream of AFB3 in pericycle cells and mediates changes in lateral root density in response to nitrate treatments. Although we have made progress describing the regulatory networks acting downstream of AFB3, the regulatory factors acting upstream of AFB3 remain largely uncharacterized.

One of the main components of the nitrate signaling pathway is the NRT1.1 nitrate transporter/ sensor. NRT1.1 is a dual affinity nitrate transporter able to switch from low-affinity to high-affinity by phosphorylation of its Thr101 by CIPK23 kinase.15-17 Thr101 phosphorylation has also been shown to be important for the nitrate sensor function of NRT1.1.17 Mutation of NRT1.1 affects the expression of more than 100 nitrate-responsive genes.9,18,19 Analysis of the chl1–9 mutant in which the nitrate uptake and nitrate sensing functions are decoupled showed that NRT1.1 is a nitrate sensor.17 Moreover, the response of T101D and T101A transgenic plants, in which NRT1.1 is locked in high-affinity and low-affinity respectively, showed that transcript levels in response to nitrate of the NRT2.1 high-affinity nitrate transporter depend on the phosphorylation of T101.17

The response of two other nitrate primary response genes, NIA1 and NIA2 has also been shown to depend on a transporter-independent function of NRT1.1.17 As such, NRT1.1 has been proposed to act as a nitrate sensor, coordinating the expression of primary response genes to the external availability of nitrate.17 However, it is not known whether regulation of other nitrate responsive genes depends on a signaling function of NRT1.1. Since NRT1.1 has been shown to control lateral root development in response to nitrate,20-23 this transporter represents an attractive candidate to control the nitrate response of the AFB3 regulatory network.

In order to determine if NRT1.1 acts upstream of AFB3 and NAC4, we analyzed the nitrate response of AFB3 and NAC4 in three different NRT1.1 mutants: the NRT1.1 deletion mutant chl1–5 24, the chl1–9 mutant, defective in nitrate transport but not on nitrate signaling15,17 and the T101D mutant, that mimics a constitutively phosphorylated transporter, locking it in the high affinity mode.17 These mutants have been extensively used for functional characterization of the NRT1.1 nitrate transporter.15,17,24-27 We grew plants in hydroponic medium consisting of 1X Murashige and Skoog (MS) salts without N, supplemented with 0.5 mM ammonium succinate and 3 mM sucrose for two weeks. Plants were treated at the onset of the 15th day with 5 mM KNO3 or 5 mM KCl as control for 30, 60, 120 and 240 min. As shown in Figure 1, nitrate regulation of the NRT2.1 transporter was altered in the chl1–5 mutant (Fig. 1A) and in the T101D mutant (Fig. 1C) but not in the chl1–9 mutant (Fig. 1B), indicating that NRT2.1 induction depends on a signaling function of NRT1.1 as has been previously shown.17 Interestingly, shorter exposure to nitrate (30 min) causes a reduced accumulation of the NRT2.1 transcript in T101D as compared with WT, as shown in previous studies17 while longer exposure to nitrate (1, 2 and 4 h) causes a higher accumulation of the NRT2.1 transcript in T101D (Fig. 1C). Since T101D plants mimic a phosphorylated NRT1.1 transporter, higher expression of the high-affinity transporter NRT2.1 in response to longer exposure to nitrate could represent the correct adaptation to an apparent low nitrate availability based on the NRT1.1-T101D function.

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Figure 1. The expression of AFB3 and NAC4 in response to nitrate depends on the nitrate transport function of NRT1.1. Wild-type Col-0, chl1–5, chl1–9 and T101D plants were grown in ammonium succinate for 2 wk and were treated with 5 mM KNO3 or 5 mM KCl for 30, 60, 120 and 240 min. We measured RNA levels of NRT2.1, AFB3 and NAC4 using real-time qPCR. Filled bars represent wild-type (Col-0) and dashed bars represent mutant plants. A-C: transcript levels of NRT2.1 in chl1–5 (A), chl1–9 (B) and T101D (C). D-F: transcript levels of AFB3 in chl1–5 (D), chl1–9 (E) and T101D (F). G-I: transcript levels of NAC4 in chl1–5 (G), chl1–9 (H), and T101D (I). We show the results of three biological replicates. Bars represent standard error. The asterisks represent means that statistically differ between genotypes (t test, P ≤ 0.05).

In contrast to NRT2.1 response, we found that the nitrate response of AFB3 and NAC4 was altered in the chl1–5 (Fig. 1D and 1G) and in the chl1–9 mutant (Fig. 1E and 1H). This result suggests that nitrate induction of AFB3 and NAC4 depends on the nitrate transport function of NRT1.1. Furthermore, nitrate response of AFB3 and NAC4 was not affected in the T101D mutant (Fig. 1F and 1I), indicating that the gene expression response of AFB3 and NAC4 is independent of the signaling function of NRT1.1. NRT1.1 along with NRT1.2 and NRT2.1 and NRT2.2 are the main nitrate uptake transporter expressed in Arabidopsis roots.28 In order to determine if transcript accumulation in response to nitrate of AFB3 and NAC4 depended on the nitrate transport function of NRT1.1, we analyzed gene expression of AFB3 and NAC4 in nrt1.2 and nrt2.1/nrt2.2 mutants. As shown in Figure 2, transcript levels of AFB3 and NAC4 in response to nitrate are not affected in the nrt1.2–1 mutant20 (Fig. 2A, C) or in the nrt2.1–2 mutant (Fig. 2B, D), which has a deletion in both NRT2.1 and NRT2.2 genes.29 These results suggest that the nitrate transport function of NRT1.1 is crucial for AFB3 and NAC4 expression.

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Figure 2. Nitrate regulation of AFB3 and NAC4 is not affected in nrt1.2 and nrt2.1/nrt2.2 mutants. Wild-type Ws, nrt1.2–1 and nrt2.1–1 plants were grown in ammonium succinate for 2 wk and were treated with 5 mM KNO3 or 5 mM KCl for 60, 120 and 240 min. We measured RNA levels of AFB3 and NAC4 using real-time qPCR. Filled bars represent wild-type (Col-0) and dashed bars represents mutant plants. A-B: transcript levels of AFB3 in nrt1.2–1 (A) and nrt2.1–1 (B). C-D: transcript levels of NAC4 in nrt1.2–1 (C) and nrt2.1–1 (D). We show the results for three biological replicates. Bars represent standard error. The asterisks represent means that statistically differ between genotypes (t test, P ≤ 0.05).

To date, only a handful of molecular actors involved in nitrate perception/signaling have been identified in plants. Transcription factors such as NPL7,30-32 LBD37/38/3933 and SPL934 have been shown to be important for normal gene expression in response to nitrate of nitrate transporter and nitrate metabolism genes, among other genes. However, the miR393/AFB3-dependent regulatory network acts in a pathway that is independent of these regulatory factors, indicating that multiple signaling pathways can act at the root level to control plant responses to nitrate.32 Previous reports have shown that a significant fraction of nitrate responsive genes is dependent on NRT1.1,9 however it is unknown whether the response of these genes (besides NRT2.1, NIA1 and NIA2) depends on the signaling or transporter functions of NRT1.1. In this work, we determined that the nitrate transport function of NRT1.1 is required for expression of AFB3 and NAC4 in response to nitrate. We found that the expression of AFB3 and NAC4 is not affected in the T101D mutant, which is affected in nitrate transport in the low-affinity range.16,17 This indicates that AFB3 and NAC4 expression is not dependent on the total amount of nitrate transported by NRT1.1, but is dependent on nitrate transport itself. Thus, nitrate transport by NRT1.1 could trigger an alternative signaling pathway different from NRT1.1 phosphorylation that regulates gene expression in response to nitrate.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We wish to thank Dr. Yi-Fang Tsay, Academia Sinica, Taiwan, for kindly providing the chl1–5, chl1–9 and T101D seeds and Dr. Gabriel Krouk, INRA Montpellier for providing the nrt1.2–1 and nrt2.1–1 seeds. This work was funded by the International Early Career Scientist program from Howard Hughes Medical Institute, Fondo de Desarrollo de Areas Prioritarias (FONDAP) Center for Genome Regulation (15090007), Millennium Nucleus Center for Plant Functional Genomics (P10–062-F), Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) 1100698 and Comisión Nacional de Investigación Científica y Tecnológica (CONICYT)-ANR-007. E.A.V. is funded by FONDECYT 11121225 and Proyecto de Inserción en la Academia (PSD74).

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