Integration of shoot-derived polypeptide signals by root TGA transcription factors is essential for survival under fluctuating nitrogen environments

Kobayashi, Ryutaro; Ohkubo, Yuri; Izumi, Mai; Ota, Ryosuke; Yamada, Keiko; Hayashi, Yoko; Yamashita, Yasuko; Noda, Saki; Ogawa-Ohnishi, Mari; Matsubayashi, Yoshikatsu

doi:10.1038/s41467-024-51091-5

Download PDF

Article
Open access
Published: 23 August 2024

Integration of shoot-derived polypeptide signals by root TGA transcription factors is essential for survival under fluctuating nitrogen environments

Nature Communications volume 15, Article number: 6903 (2024) Cite this article

1402 Accesses
13 Altmetric
Metrics details

Subjects

Abstract

Unlike plants in the field, which experience significant temporal fluctuations in environmental conditions, plants in the laboratory are typically grown in controlled, stable environments. Therefore, signaling pathways evolved for survival in fluctuating environments often remain functionally latent in laboratory settings. Here, we show that TGA1 and TGA4 act as hub transcription factors through which the expression of genes involved in high-affinity nitrate uptake are regulated in response to shoot-derived phloem mobile polypeptides, CEP DOWNSTREAM 1 (CEPD1), CEPD2 and CEPD-like 2 (CEPDL2) as nitrogen (N) deficiency signals, and Glutaredoxin S1 (GrxS1) to GrxS8 as N sufficiency signals. CEPD1/2/CEPDL2 and GrxS1-S8 competitively bind to TGA1/4 in roots, with the former acting as transcription coactivators that enhance the uptake of nitrate, while the latter function as corepressor complexes together with TOPLESS (TPL), TPL-related 1 (TPR1) and TPR4 to limit nitrate uptake. Arabidopsis plants deficient in TGA1/4 maintain basal nitrate uptake and exhibit growth similar to wild-type plants in a stable N environment, but are impaired in regulation of nitrate acquisition in response to shoot N demand, leading to defective growth under fluctuating N environments where rhizosphere nitrate ions switch periodically between deficient and sufficient states. TGA1/4 are crucial transcription factors that enable plants to survive under fluctuating and challenging N environmental conditions.

Shoot-to-root mobile CEPD-like 2 integrates shoot nitrogen status to systemically regulate nitrate uptake in Arabidopsis

Article Open access 31 January 2020

Gibberellin and abscisic acid transporters facilitate endodermal suberin formation in Arabidopsis

Article 06 April 2023

A CYBDOM protein impacts iron homeostasis and primary root growth under phosphate deficiency in Arabidopsis

Article Open access 11 January 2024

Introduction

Nitrogen (N), which is mostly present as nitrate in soils, is an essential macronutrient that plays a crucial role in plant development. However, due to the high mobility of nitrate ions through soil with water, marked spatiotemporal fluctuations can occur with respect to soil nitrate availability. To cope with such fluctuations in the external N environment, plants have evolved regulatory mechanisms that enable them to modulate the efficiency of root N acquisition in response to their internal N demand and rhizosphere N availability^1,2. This systemic adaptive response is mediated by shoot-root communication via phloem-mobile descending CEP DOWNSTREAM 1 (CEPD1), CEPD2 and CEPD-like 2 (CEPDL2) polypeptides³. Among these, CEPD1 is induced in shoots in response to the C-TERMINALLY ENCODED PEPTIDE (CEP), which is a root-derived N deficiency signal^4,5, while CEPDL2 is induced in response to the shoot’s own N deficiency⁶. CEPD2 is induced in response to both root-derived CEP and shoot N deficiency.

CEPD1/2/CEPDL2 promote nitrate uptake in roots by both transcriptional upregulation of high-affinity nitrate transporter genes, such as NRT2.1, and dephosphorylation-mediated post-translational activation of NRT2.1 by the protein phosphatase, CEPD-INDUCED PHOSPHATASE (CEPH), which is also induced by CEPD1/2/CEPDL2⁷. Accordingly, the cepd1,2 cepdl2 triple mutant showed severely reduced shoot growth, which is characterized by a significant reduction in high-affinity nitrate influx in roots compared with WT.

CEPD1/2/CEPDL2 are non-secreted polypeptides that are composed of 99 to 102 amino acids and belong to a large protein family comprising 21 members in Arabidopsis. Currently, these polypeptides are assigned to the plant-specific class III glutaredoxin (Grx) family⁸, although from a structural point of view, there is controversy as to whether individual members are enzymatically active in redox regulation⁹. Of these, ROXY1 and ROXY2 have been shown to regulate petal morphogenesis and microspore formation¹⁰. Conversely, ROXY18 has been shown to play roles in biotic and abiotic stresses^11,12. The Grx family also includes members that are upregulated in shoots under nitrate-sufficient conditions^13,14,15, which is opposite to the response associated with CEPD1/2/CEPDL2. Ectopic overexpression of one such gene, GrxS8, caused downregulation of nitrate transporter genes accompanied with repression of lateral root development¹⁶.

There is increasing, but fragmentary, evidence suggesting a possible link among CEPDs, Grxs, TGA family transcription factors, TOPLESS family transcriptional co-repressors¹⁷ and nitrate signaling. (i) Combining yeast two-hybrid screening data from multiple independent experiments, showed that all 21 members of the class III Grx family are likely to interact with all 10 TGA transcription factors in vitro, albeit with varying affinities^{11,18,19,20,21,22,23}. (ii) Most of the Grxs, except for 4 members in the CEPD1/2/CEPDL2 clade, have been shown to interact with the TOPLESS family of transcriptional co-repressors in the yeast two-hybrid assay²⁴. (iii) Co-expression network analysis or transcriptome under varying nitrogen conditions identified TGA1 as a key regulatory factor of the nitrate response^25,26. The double mutant of TGA1 and its closest homolog TGA4 exhibited a decrease in lateral root initiation and a reduction in root hair development in response to nitrate treatment^25,27. The tga1 tga4 double mutant, however, showed no observable changes in nitrate uptake activity in roots under normal growth conditions²⁵. It has also not yet been definitively established whether TGA1 directly binds to promoters of genes involved in high-affinity nitrate uptake, such as NRT2.1 and CEPH^25,26.

To piece together these fragmentary observations, here we analyzed in planta signaling network by co-immunoprecipitation-mass spectrometry (CoIP-MS) and chromatin immunoprecipitation followed by sequencing (ChIP-Seq). CEPDs and subset of Grxs competitively bind to TGA1/4, with the former acting as transcription coactivators that enhance the uptake of nitrate, while the latter function as corepressor complexes together with TOPLESS family of transcriptional corepressors to limit nitrate uptake. Arabidopsis plants deficient in TGA1/4 were impaired in demand-dependent regulation of nitrate acquisition, resulting in defective growth under a temporally fluctuating N environment.

Results

CEPDL2 acts through interaction with TGA1 and TGA4 transcription factors

We first searched for signaling targets that physically interact with CEPD family polypeptides in roots using an unbiased proteomic approach. Here, GFP-tagged CEPDL2, which has the same signaling properties as untagged CEPDL2⁶, was used as bait for CoIP-MS. Root extracts were prepared from 14-day-old seedlings expressing GFP-CEPDL2 in cepd1,2 cepdl2 mutant (GFP-CEPDL2/cepd1,2 cepdl2) under the control of its own promoter, and GFP-CEPDL2 was immunoprecipitated from the extracts using immobilized anti-GFP antibodies. Nano liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) was performed on three replicate immunoprecipitations from GFP-CEPDL2/cepd1,2 cepdl2 seedling roots as well as three control immunoprecipitations from NRT2.1pro:GFP seedling roots. NRT2.1 expression is detected in epidermis, cortex and vasculature throughout the root and overlaps with the localization of CEPDL2²⁸.

Volcano plots of enrichment and significance showed that TGA1 and TGA4 were the most enriched transcription factors (Fig. 1a). We confirmed direct interaction between CEPDL2 and TGA1 by the yeast two-hybrid assay (Fig. 1b). Overexpression of CEPDL2 in a tga1,4 double mutant did not upregulate neither of CEPDL2 target genes such as NRT2.1 and CEPH (Fig. 1c, Supplementary Fig. 1a), indicating that TGA1 and TGA4 are epistatic to, and act downstream of, CEPDL2.

**Fig. 1: CEPDs function as coactivators for TGA1/4-mediated transcription.**

Genes involved in nitrate acquisition are enriched in TGA1 ChIP-seq target genes

To determine the binding sites of TGA1 across the genome in vivo, we used chromatin immunoprecipitation followed by sequencing (ChIP-Seq). For the ChIP-Seq analysis, we used transgenic Arabidopsis plants expressing TGA1-GFP using their own promoter. TGA1-GFP localized in the nucleus of endodermal, cortical, and epidermal cell layers in roots, which overlaps with the localization of CEPDL2 (Fig. 1d, e). We performed ChIP-Seq on 16-day-old roots and identified 1105 TGA1 in vivo target loci (q < 10⁻⁴⁰) (Supplementary Data 1). The TGA1 targets included genes related to high-affinity nitrate uptake, such as NRT2.1 and CEPH (Fig. 1f, g), while NRT1.1, which is involved in low-affinity nitrate uptake, was not detected. We confirmed binding motifs for TGA1 enriched in the ChIP-Seq reads as “TGACG” (e-value = 4.0 × 10⁻⁷) and the most enriched GO term as “response to nitrate” (Supplementary Fig. 1b, c). We further examined the overlap between the 296 differentially expressed genes (DEGs) (fold-change ≥ 2) induced in 35 S:CEPDL2 plant roots⁶ and the TGA1 ChIP-Seq target genes. We identified 48 genes that overlapped (Supplementary Data 2), including NRT2.1, NRT2.2, NRT3.1, NRT1.5 and CEPH, all of which have been reported to play key roles in nitrate acquisition.

CEPDs function as coactivators for TGA1/4-mediated transcription

Given that CEPDL2 directly interacts with transcription factor TGA1/4 and upregulates the expression of a set of TGA target genes related to nitrate acquisition, we hypothesized that CEPDs function as coactivators in TGA1/4-mediated pathway. To test this possibility, we performed transient dual-luciferase reporter assay in Arabidopsis mesophyll cells to assess the transcriptional activity of TGA1/4 in the presence or absence of CEPDL2. Arabidopsis mesophyll protoplasts prepared from WT or tga1,4 double mutant were transformed with a CEPH promoter-driven firefly luciferase (LUC) reporter plasmid and either GFP expression vector or GFP-CEPDL2 expression vector, together with Renilla LUC (REN) expression vector as an internal standard. Cotransformation of reporter vector with GFP-CEPDL2 expression vector in WT protoplasts showed ≈1.5-fold induction in the expression of the luciferase reporter gene compared with the transformation with GFP expression vector. However, no such induction was observed when GFP-CEPDL2 expression vector was cotransformed with reporter vector in tga1,4 protoplasts (Fig. 1h). These results indicate that CEPDs act as coactivators for TGA1/4-mediated transcription.

We also tested whether CEPDs are required for the binding of TGA1 to target gene promoters. We produced transgenic Arabidopsis plants expressing TGA1-GFP using the own promoter in WT plants and the cepd1,2 cepdl2 triple mutant. ChIP-qPCR analysis showed that TGA1 binds to the promoters of NRT2.1 and CEPH genes in the cepd1,2 cepdl2 triple mutant with a comparable affinity to those in WT, even though the expression level of NRT2.1 and CEPH decreased to 48% and 52% of those in WT, respectively (Fig. 1i, j). These results indicate that CEPDs act as coactivators for TGA-mediated transcriptional activation of the target genes, but that they are not required for the binding of TGAs to the target-binding regions.

Loss of TGA1/4 counteracted the growth defects observed in the cepd1,2 cepdl2 triple mutant

We previously reported that the cepd1,2 cepdl2 triple mutant shows severe growth defects, such as a decrease in shoot fresh weight and reduced expression of the NRT2.1 and CEPH genes⁶. However, it has been reported that the tga1,4 double mutant shows no such growth defects, which we also confirmed in this study (Fig. 2a–c). To test the genetic interactions between CEPDs and TGAs, we crossed the cepd1,2 cepdl2 triple mutant with the tga1,4 double mutant. Unexpectedly, the introduction of the tga1,4 mutation counteracted the growth defects in the phenotypes of the cepd1,2 cepdl2 triple mutant, restoring them to WT levels (Fig. 2a–c). These results indicate that TGA1/4 have a positive effect on regulating the transcription of nitrate uptake genes after binding to coactivator CEPD1/2/CEPDL2 but exhibits a negative effect on the transcription of nitrate uptake genes in the absence of CEPDs.

**Fig. 2: Loss of *TGA1/4* counteracted the growth defects of the *cepd1,2 cepdl2* triple mutant.**

GrxS1-S8 family polypeptides have roles opposite to those of CEPDs

Given the repressive effect of TGA1/4 on the transcription of nitrate uptake genes in the absence of coactivator CEPDs, we considered that a plausible explanation for this transcriptional repression would be the presence of a corepressor interacting with TGA1/4. We focused on the GrxS1/S2/S3/S4/S5/S6/S7/S8 family polypeptides as potential candidates because, despite having a sequence that is highly similar to CEPDs, GrxS8 has been reported to suppress the transcription of nitrate transporter genes when overexpressed¹⁶.

GrxS1 to GrxS8 genes are expressed almost exclusively in the phloem tissues of leaves (Fig. 3a, Supplementary Fig. 2a, b) and upregulated in leaves under nitrate-sufficient conditions, a response that is opposite to that observed with CEPDs (Supplementary Fig. 2c). When a GFP-GrxS5 construct driven by the native GrxS5 promoter was introduced into WT plants, we observed GFP-GrxS5 signals in the nucleus of endodermal, cortical, and epidermal cell layers in roots (Fig. 3b), indicating that the GrxS5 functions as a phloem-mobile shoot-to-root signal. This GFP-GrxS5 transgene, which caused over-accumulation of GrxS5 transcripts, suppressed the expression of 414 genes, including both high- and low-affinity nitrate uptake genes (Fig. 3c, Supplementary Fig. 2d, e, Supplementary Data 3). Notably, among the genes that were downregulated by GrxS5, 11 genes, including NRT2.1, NRT2.2, NRT3.1, NRT1.5 and CEPH, were direct targets regulated by the CEPD/TGA pathway (Supplementary Data 4).

When ectopically overexpressed, all genes from GrxS1 to GrxS8 suppressed the expression of NRT2.1 (Supplementary Fig. 2f). Conversely, in the octuple mutant grxs1-s8 (i.e., grxs1-1, grxs2-1, grxs3-1, grxs4-1, grxs5-1, grxs6-1, grxs7-1, grxs8-1) generated by T-DNA and CRISPR/Cas9-mediated mutation (Supplementary Fig. 2g), the mutations had positive effects on the expression levels of nitrate uptake genes (Fig. 3c, Supplementary Fig. 2e), increasing both high- and low-affinity nitrate uptake activities (Fig. 3d, Supplementary Fig. 2h). When the concentration of NO₃^– in the medium was increased from 0.3 mM to 3 or 50 mM, the basal expression of CEPH in the WT plants was significantly reduced. In contrast, this N supply-dependent down-regulation of CEPH was alleviated in the grxs1-s8 octuple mutant (Supplementary Fig. 2i). Similarly, the expression level of NRT2.1 under 50 mM nitrate was markedly increased in grxs1-s8 mutant compared to WT (Supplementary Fig. 2j). The grxs1-s8 octuple mutant showed a decrease in survival rate under excess nitrate (120 mM) conditions (Supplementary Fig. 2k), likely due to the lack of negative feedback on excess nitrate uptake. These findings indicate that while CEPD1/2/CEPDL2 and GrxS1-S8 function as phloem-mobile shoot-to-root signals, they play opposite roles in regulating nitrate acquisition.

GrxS1-S8 form a corepressor complex with TPL/TPR1/4

We searched for signaling targets that physically interact with the GrxS1-S8 family of polypeptides in roots by CoIP-MS using GFP-GrxS5 as a bait. Co-IP data showed the interactions between GFP-GrxS5 and TGA1/4 in addition to TOPLESS (TPL), TOPLESS-related 1 (TPR1) and TPR4 (Fig. 3e), which suggests that GrxS5 interacts with TOPLESS family corepressors to inhibit TGA-mediated transcription. Supporting this model, NRT2.1 and CEPH were upregulated in the tpl tpr1 tpr4 triple mutant (Fig. 3f). Overexpression of GrxS5 in a tga1,4 double mutant background failed to downregulate NRT2.1 and CEPH, confirming that TGA1 and TGA4 acts downstream of GrxS5 (Fig. 3g, Supplementary Fig. 3a). In contrast, loss of TGA1/4 had no effect on GrxS5-dependent downregulation of NRT1.1, suggesting that GrxS5 represses low affinity nitrate uptake in a pathway independent of the TGA1/4 (Supplementary Fig. 3b).

We confirmed by yeast three-hybrid assay that TGA1 interacts with TPL only when co-expressed with GrxS5, but not when coexpressed with CEPDL2, indicating that GrxS5 bridges the interaction between TGA1 and TPL (Fig. 3h). To further verify whether GrxS-TPL complex indeed functions as a corepressor for TGA1/4 transcription factors, we performed luciferase reporter assay in Arabidopsis mesophyll cells to assess the transcriptional activity of TGA1/4 in the presence or absence of GrxS5. Co-transformation of GFP-GrxS5 expression vector and CEPH promoter-driven LUC reporter vector in WT protoplasts resulted in ≈30% reduction in the expression of the luciferase reporter gene compared with the transformation of reporter vector alone (Fig. 3i). In contrast, no such reduction was observed when GFP-GrxS5 expression vector was co-transformed with the reporter vector in tga1,4 protoplasts. These results indicate that GrxS-TPL complex acts as a corepressor on TGA1/4-mediated pathway.

CEPDL2 and GrxS5 competitively interact with TGA1/4

We hypothesized that, due to their striking sequence similarity, CEPDL2 and GrxS5 might bind to TGA in a competitive manner. To test this possibility, we overexpressed GrxS5 using an estradiol-inducible XVE system in GFP-CEPDL2/cepd1,2 cepdl2 plants and immunoprecipitated the GFP-CEPDL2/TGA complex using an anti-GFP antibody. CoIP-MS data showed that, under conditions where GFP-CEPDL2 was immunoprecipitated with comparable efficiency in estradiol-treated and mock-treated XVE:GrxS5/GFP-CEPDL2 plants, the amount of co-immunoprecipitated TGA1 and TGA4 decreased in estradiol-treated GrxS5-overexpressing plants compared to mock plants (Fig. 3j, Supplementary Fig. 3c), indicating that GrxS5 competes with CEPDL2 for binding to both TGA1 and TGA4. Based on these results, we propose a model in which CEPD1/2/CEPDL2 and GrxS1-S8 competitively bind to TGA1/4, with the former acting as transcription coactivators that enhance the uptake of nitrate, while the latter function as corepressor complexes together with TOPLESS to limit nitrate uptake. We note that, based on the fold-change values in the Co-IP data, the binding affinity of GrxS5 to TGA1/4 appears to be weaker than that of CEPDL2 to TGA1/4, suggesting that N deficiency signals are recognized in preference to N sufficiency signals.

TGA1/4 are critical for plant survival under a temporally fluctuating N environment

Despite the presumed mechanistic importance of TGA1/4 in N acquisition, the tga1,4 double mutant did not exhibit any visible phenotypes under either N sufficient (3 mM NO₃^–) (Fig. 2a) or low N (0.1 mM NO₃^–) conditions (Fig. 4a). These results raise the question of how a system that integrates N deficiency signals and N sufficiency signals from aboveground parts through root TGA1/4 transcription factors contributes to plant survival. Since this long-range signaling system should regulate root nitrate uptake in response to aboveground N demand, we hypothesize that TGA1/4 are particularly important in environments where shoot N demand and rhizosphere N availability fluctuate over time.

**Fig. 4: TGA1/4 are critical for plant survival under temporally fluctuating nitrogen environment.**

To investigate this possibility, we cultured WT and tga1,4 mutant plants in a hydroponic system in which N-deficient (0 mM NO₃^–) and N-sufficient (1 mM NO₃^–) conditions were alternated for 18 h of and 6 h of each day over 7 d, respectively. Under this fluctuating N environment, a clear difference in phenotypes between the WT and tga1,4 mutant plants was observed, with the tga1,4 mutant showing stunted growth characterized by smaller rosettes and reduced shoot biomass compared with WT plants (Fig. 4b, c). The tga1,4 mutant also exhibited a significant reduction in nitrate content in shoots (Fig. 4d), accompanied by early leaf yellowing that is typically induced by N deprivation. Since root nitrate content of the tga1,4 mutant was comparable to that of the WT under this condition, the tga1,4 mutation appears to affect both nitrate uptake and nitrate root-to-shoot translocation (Fig. 4e). These results indicate that TGA1/4 transcription factors are indispensable for plant survival under temporally fluctuating N conditions.

Demand-dependent regulation of nitrate acquisition was impaired in tga1,4 mutants

Finally, we explored the reasons why the tga1,4 mutant failed to adapt to fluctuating N environments by analyzing the time-course expression levels of N uptake-related genes during repetitive cycles of N deficiency and sufficiency. Since the nitrate content in the shoots of the tga1,4 mutant was already lower than that of the WT at 72 h after the beginning of the repetitive cycles (Supplementary Fig. 3d), we examined the expression of CEPDs and GrxS5 in leaves, as well as nitrate uptake genes in roots from 24 h to 72 h using RT-qPCR.

In shoots of both the tga1,4 mutant and the WT, the expression of CEPDL2, CEPD2, and CEPD1 were induced under N deficient-conditions and repressed under N-sufficient conditions (Fig. 5a). Conversely, in both the tga1,4 mutant and WT plants, the expression of GrxS5 was repressed under N-deficient conditions and induced under N-sufficient conditions (Fig. 5a).

**Fig. 5: The *tga1,4* mutant was impaired in demand-dependent regulation of nitrate acquisition.**

In roots, during N-deficient conditions, the induction of CEPDL2 and CEPD1/2 in leaves and their subsequent translocation to the roots led to gradual upregulation of CEPH and NRT1.5 at the late stage of nitrogen deficient periods in WT plants (Fig. 5b). NRT1.5 is known to be involved in root-to-shoot translocation of nitrate²⁹. However, such N demand-dependent responses were not observed in the roots of the tga1,4 mutant. The transcription of NRT2.1 was strongly repressed during N-deficient conditions and recovered to the basal levels during N-sufficient conditions in both the tga1,4 mutant and the WT plants (Fig. 5b), which corresponds to the nitrate-inducible nature of NRT2.1 and inability of CEPDs to induce NRT2.1 under N-deficient conditions³. During N-sufficient conditions, CEPH also showed rapid induction in both WT and tga1,4 plants, possibly reflecting its cell-autonomous nitrate-inducible nature.

Using ¹⁵N-metabolic labeled quantitative proteomic profiling, we further analyzed the level of Ser⁵⁰¹-dephosphorylated active form of NRT2.1, a residue that functions as a negative phospho-switch. The WT and tga1,4 plants were cultured on medium containing ¹⁴NO₃^– or ¹⁵NO₃^– in a reciprocal manner for biological duplicates (¹⁴N WT/¹⁵N tga1,4 [forward experiment] and ¹⁵N WT/¹⁴N tga1,4 [reciprocal experiment]). At 66 h after the beginning of the repetitive cycles, which corresponds to the end of the third N-deficient period, the Ser⁵⁰¹-dephosphorylated active form of NRT2.1 in the tga1,4 mutant decreased to 70% and 45% of that in WT plants in the forward and reciprocal experiments, respectively, without any alteration in NRT2.1 protein expression levels (Fig. 5c, Supplementary Fig. 3e–i). Consequently, tga1,4 plants exhibited only 55% of the high affinity nitrate uptake activity of WT plants at 66 h (Fig. 5d). These findings suggest that the inability of tga1,4 mutants to adapt to fluctuating N environments is due to a decrease in both CEPH-mediated activation (dephosphorylation) of NRT2.1 high affinity nitrate uptake and the NRT1.5-mediated translocation of nitrate ions from roots to shoots.

Discussion

Our findings elucidated the crucial roles played by TGA1/4 transcription factors under fluctuating N conditions. These roles have been overlooked because tga1,4 mutants do not show distinct phenotypes under either continuous N-deficient or continuous N-sufficient conditions, despite their presumed involvement in N responses. Under continuous N-deficient conditions, N demand signals derived from leaves are strongly upregulated. However, due to the absence of rhizospheric N, long-distance signaling through CEPD1/2/CEPDL2 and TGA1/4 does not exert its intrinsic effect. The environmental conditions under which the CEPD-TGA system is most effective are when N availability is temporally fluctuating, i.e., where shoots remain in a N-deficient state while nitrate becomes available again in the rhizosphere.

TGA1/4 transcription factors not only interact with the shoot-derived N demand signals CEPD1/2/CEPDL2, but also integrate the N sufficiency signals GrxS1 to GrxS8 in order to optimize the expression of nitrate uptake-related genes in roots. The grxs1-s8 octuple mutant generated in this study showed a decrease in survival rate under excess nitrate conditions. However, since such high concentrations of nitrate ions are rare in nature, the GrxS1-S8 pathway is likely a feedback mechanism that prevents the CEPD1/2/CEPDL2 pathway from becoming overactive which leads to uptake of more nitrate than needed, rather than an adaptation to extreme environments. In fact, the binding of GrxS5 to TGA1/4 was considerably weaker compared to CEPDL2. Therefore, the function of TGA1/4, which is suppressed by GrxS1-S8 during N sufficiency, would be switched rapidly to the transcriptional activation of high-affinity nitrate uptake genes by competitive binding with CEPD1/2/CEPDL2 (Supplementary Fig. 3j).

Nitrate in soils is an essential macronutrient that plays a crucial role throughout plant development; however, due to its high mobility through soil with water, soil nitrate availability fluctuates over time. Plants take up dissolved nitrate ions in the soil pore water, which is the water located between the soil particles. Real-time measurement of the nitrate concentration in the soil pore water revealed rapid changes in nitrate concentration following irrigation or precipitation cycles on a timescale of hours to days³⁰. The rapid increase in soil water content leads to a temporary decrease in nitrate concentration in the pore water. In contrast, as the soil water content decreases, the nitrate concentration in the pore water increases again due to the re-diffusion of nitrate from soil particles.

It is also known that water-deficient conditions significantly reduce the nitrate content in roots³¹. A marked reduction in soil water content hinders the nitrate uptake due to reduced root surface area in contact with soil pore water. Thus, nitrate uptake is significantly influenced by the constant fluctuations of the water content in the soil. Rain is always falling on the land somewhere, increasing the water content in the soil. Conversely, insolation causes the moisture in the soil to evaporate, decreasing the water content. In such a temporally variable environment, the CEPD-TGA system plays an important role in maintaining adequate nitrate uptake to meet N demand of the aboveground tissues. Plants, just like animals, would likely be unable to survive in the challenging natural environment without eating plenty when they can.

Methods

Growth conditions

Arabidopsis ecotype Col or Nössen plants were used as the WT control depending on the mutant ecotype. Surface-sterilized seeds were sown on B5 medium (1% sucrose) solidified using 1.5% agar in 13 × 10 cm plastic plates (18 seeds × 3 rows/plate) and grown vertically at 22 °C under continuous light at an intensity of 80 µmol·m^–2·s^–1. After 7 d, seedlings were transferred to modified Murashige-Skoog medium solidified using 1.5% agar in 13 × 10 cm plastic plates (12 seedlings/plate) and grown vertically at 22 °C with continuous light. Modified Murashige-Skoog medium was prepared for different NO₃^– concentrations. For the 10 mM NO₃^– medium, 10 mM KNO₃ was added as the sole source of N and K, along with half-strength concentrations of the other elements and 0.5% sucrose, adjusted to pH 5.7 with KOH. For the 3 mM NO₃^– medium, 3 mM KNO₃ and 7 mM KCl were added. For the 1 mM NO₃^– medium, 1 mM KNO₃ and 9 mM KCl were added. For the 0.3 mM NO₃^– medium, 0.3 mM KNO₃ and 9.7 mM KCl were added. For 0.1 mM NO₃^– medium, 0.1 mM KNO₃ and 9.9 mM KCl were added. For the N-depleted medium, 10 mM KCl was added solely as the K source. For the 50 mM or 120 mM NO₃^– medium, 50 mM or 120 mM KNO₃ was added solely as the N source, respectively.

Mutant plants

The tga1,4 double mutants were obtained from the SALK T-DNA mutant collection (tga1-1, SALK_028212; tga4-1, SALK_127923). This tga1,4 (Col) mutant was backcrossed three times to wild-type Nössen to generate the tga1,4 (Nos) mutant. The triple mutants cepd1-1 cepd2-1 cepdl2-1 (Nössen background) were described previously⁶. The ecotypes Col and Nos exhibit relatively similar responses to nitrate deficiency, such as lateral root elongation, among many natural variations³². The grxs1 mutant was also obtained from the SALK T-DNA mutant collection (SALK_032946). The grxs2 mutant, the grxs6 mutant, and the grxs3/4/5/7/8 quintuple mutants were generated using a CRISPR/Cas9 system. The U6.26 promoter and guide RNA sequences were cloned into pKIR1.0³³ and transformed into Col-0. Screening of transformants was performed by PCR and sequencing, revealing an 11-bp deletion and a 1-bp insertion in GrxS2, a 13-bp deletion in GrxS6, a 1-bp insertion in GrxS3, a 6650-bp deletion in GrxS4/5/7 (At1g15670-At1g15690), and a 6-bp deletion with a 2-bp insertion in GrxS8. The CRISPR/Cas9 construct was then removed to ensure genetic stability. The tpl tpr1 tpr4 mutant was obtained from the Arabidopsis Biological Resource Center (CS72353). Primers and gRNA sequences are listed in Supplementary Data 5.

Transgenic plants

For co-immunoprecipitation using GFP-GrxS5, the 2-kb 5’-upstream region of the GrxS5 gene, the cDNA fragment of the GrxS5 coding region, and the cDNA fragment of GFP were amplified by PCR. These three fragments were then cloned by translational fusion using a four-component ligation into the binary vector pCAMBIA1300-BASTA using a NEBuilder HiFi DNA Assembly Kit. The resulting GFP-GrxS5 construct was then introduced into wild-type Col. For the expression of GFP as a positive control of co-immunoprecipitation, the 2-kb 5’-upstream region of the NRT2.1 gene and the cDNA fragment of GFP were amplified using PCR. These two fragments were then cloned by translational fusion using three-component ligation into the binary vector pCAMBIA1300-BASTA. The resultant NRT2.1pro:GFP construct was introduced into wild-type Nössen. In addition, the previously described GFP-CEPDL2/cepd1,2 cepdl2 plants (Nössen background) were used⁶. For overexpression analysis of CEPDL2 or GrxS5 in the tga1,4 mutants, cDNA fragments of the CEPDL2 or GrxS5 coding regions were cloned downstream of the CaMV 35 S promoter in pCAMBIA1300-BASTA. These constructs were introduced into either wild-type Col or the tga1,4 (Col) mutant. For TGA1-GFP analysis, the full-length TGA1 genomic fragment containing the 2-kb promoter region and the GFP-coding region were ligated in-frame in this order into the binary vector pCAMBIA1300-BASTA. The resulting TGA1-GFP construct was introduced into either wild-type Nössen or the cepd1,2 cepdl2 triple mutant. For overexpression analysis of GrxS1 to GrxS8 in wild-type Col, cDNA fragments corresponding to the open reading frames of each gene were cloned downstream of the CaMV 35 S promoter in pBI121. For estradiol-inducible expression of GrxS5 in GFP-CEPDL2/cepd1,2 cepdl2 plants, a cDNA of GrxS5 was obtained by RT-PCR using total RNA isolated from Arabidopsis leaves. The resulting fragment was cloned into the estradiol-inducible XVE binary vector pER8³⁴ to obtain XVE:GrxS5/pER8. The CEPDL2pro:GFP-CEPDL2 was further cloned into XVE:GrxS5/pER8 digested with Kpn I. The final construct was introduced into cepd1,2 cepdl2 plants to obtain the XVE:GrxS5/GFP-CEPDL2 plants. For the estradiol-mediated expression of GrxS5, the roots of 14-day-old XVE:GrxS5/GFP-CEPDL2 plants were treated with β-estradiol for 18 h by directly adding 2 mL of 50 μM β-estradiol solution onto the transgenic plants on the plates. A list of primers used in this study is shown in Supplementary Data 5.

Co-immunoprecipitation

Surface-sterilized seeds of GFP-CEPDL2/cepd1,2 cepdl2, GFP-GrxS5, NRT2.1pro:GFP or XVE:GrxS5/GFP-CEPDL2 plants were sown on B5 medium (1% sucrose) solidified using 1.2% agar in 13 × 10 cm plastic plates (18 seeds × 3 rows/plate) and grown vertically at 22 °C under continuous light at an intensity of 80 µmol·m^–2·s^–1. After 7 d, the seedlings were transferred to 10 mM NO₃^– medium and grown vertically at 22 °C under continuous light. Roots of 14-day-old plants (≈500 mg) were frozen in liquid nitrogen and ground to a fine powder using a Multi-beads shocker (Yasui Kikai Corp., Japan). Total proteins were extracted by mixing 500 mg of the ground tissue with 2.0 mL of extraction buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, cOmplete Mini EDTA-free Protease Inhibitor Cocktail [Roche, 1 tablet per 10 mL]), followed by brief sonication in an ultrasonic bath for 5 s. After incubation on ice for 30 min, the homogenate was then centrifuged for 15 min at 10,000 × g at 4 °C, and the supernatant was transferred to a new tube. Anti-GFP antibody (ab290, Abcam) was crosslinked with magnetic Dynabeads Protein G (Invitrogen) at 0.5 μg/μL beads (50% slurry) with bis(sulfosuccinimidyl)suberate following the manufacturer’s instructions. The supernatant was then incubated with 25 μL of antibody-beads (50% slurry) at 4 °C for 2 h. After immunoprecipitation, beads were washed with wash buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Triton X-100), followed by two additional washes with wash buffer that did not contain the detergent (50 mM Tris-HCl, 150 mM NaCl).

Protein digestion and nano LC-MS/MS data acquisition

The immunoprecipitated beads were suspended in 20 μL of digestion buffer (8 M urea, 250 mM Tris-HCl [pH 8.5]), reduced with 25 mM tris(2-carboxyethyl)phosphine (TCEP) at 37 °C for 15 min and alkylated using 25 mM iodoacetamide at 37 °C for 30 min in the dark, both with shaking at 1200 rpm. Proteins bound to beads were directly digested with 0.1 μg Lys-C (FUJIFILM Wako, Japan) at 37 °C for 3 h with shaking at 1000 rpm. After dilution to a urea concentration of 2 M with 50 mM Tris-HCl (pH 8.5) followed by the addition of 1 mM CaCl₂, the Lys-C digest was further digested with 0.1 μg trypsin (Promega) at 37 °C overnight with shaking at 1000 rpm. Digestion was terminated by adding 5 μL of 20% trifluoroacetic acid (TFA), and the doubly digested Lys-C/trypsin peptides were desalted using a GL-Tip SDB (GL Science) according to the manufacturer’s instructions. Nano-LC-MS/MS analysis was performed using an EASY-nLC 1200 LC system (Thermo Fisher Scientific) connected to an Orbitrap Exploris 480 hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific). Desalted samples were dissolved in 200 μL of 2% acetonitrile (0.1% TFA) and 7.5-μL aliquots of the supernatant were used for LC-MS/MS analysis. Samples were loaded in direct injection mode and separated on a nano-HPLC capillary column (Aurora column [75 μm I.D. × 250 mm], IonOpticks,) with a gradient of 3–32% acetonitrile (containing 0.1% formic acid) over 90 min at a flow rate of 300 nl/min. The Orbitrap Exploris 480 mass spectrometer was operated in data-dependent acquisition mode with dynamic exclusion enabled (20 s). MS/MS scans used higher-energy collisional dissociation with normalized collision energy set at 30. The MS/MS raw files were processed and analyzed with Proteome Discoverer 2.5 (Thermo Fisher Scientific) using the SEQUEST HT algorithm and searching against the TAIR10 Arabidopsis protein database. The identification of a protein depended on detecting at least one peptide unique to the protein. The protein abundance was defined as the sum of all unique peptide abundances. CoIP-MS data obtained from GFP-CEPDL2/cepd1,2 cepdl2 and NRT2.1pro:GFP plants (Nössen background) were also analyzed against the Arabidopsis Nössen protein database for verification³⁵. Protein abundance was normalized relative to the bait protein abundance for each immunoprecipitation, and volcano plots were generated based on the calculated protein abundance³⁶. Any missing values were imputed using a random selection from the bottom 5% of all protein abundances.

Yeast two-hybrid assay

Yeast two-hybrid screening was performed using the Matchmaker Gold Yeast Two-Hybrid System (Clontech). The cDNA fragments corresponding to CEPDL2 and GrxS5 were subcloned into the pGBKT7 vector, which was subsequently introduced into the Y187 yeast strain. The cDNA fragments for TGA1 were subcloned into the pGADT7 vector and then transformed into the Y2H Gold yeast strain. After the mating process between the Y187 strain expressing CEPDL2 or GrxS5, and the Y2H Gold strain expressing TGA1, resulting zygotes were selected by culturing on SD/−Leu/−Trp/−Ade/−His/X-α-Gal plates for 5 days at 28 °C. The list of primers is shown in Supplementary Data 5.

Real-time RT-qPCR

Total RNA was extracted from roots or shoots using an RNeasy kit (Qiagen). First-strand cDNA synthesis was conducted from 0.5 μg of total RNA using the Superscript IV VILO Master Mix (Thermo Fisher Scientific) according to the manufacturer’s instructions. Primers and Taqman probes were designed using the Probe Finder software available from the Universal Probe Library (UPL) Assay Design Center (Roche). All PCR reactions were performed using a StepOne System (Applied Biosystems). The constitutively expressed EF-1α was used as a reference gene for normalization of quantitative RT-PCR data. Details on primers and Taqman probes are listed in Supplementary Data 5.

Root imaging

Cell outlines were stained with 50 µg/mL propidium iodide for 2 min and observed under a confocal laser-scanning microscope (Olympus FV3000) with helium-neon laser excitation at 543 nm. GFP images were collected with argon laser excitation at 488 nm.

ChIP assay

The roots of 16-day-old TGA1–GFP plants grown on 10 mM NO₃^– medium (~900 mg) were fixed in 30 mL of 1% formaldehyde under vacuum for 5 cycles of 3 min. Cross-linking was quenched in 2 M glycine under vacuum for 3 min. Quenched samples were washed twice with 30 mL of cold 1× PBS on ice, dried briefly on paper towels, frozen in liquid nitrogen and then stored at −80 °C. Frozen samples were ground to a fine powder using a Multi-beads shocker and dissolved in 3.5 mL of nuclei extraction buffer (10 mM Tris-HCl [pH 8.0], 0.25 M sucrose, 10 mM MgCl₂, 40 mM β-mercaptoethanol, and protease inhibitor cocktail). Samples were then filtered through two layers of Miracloth (Calbiochem) and centrifuged for 5 min at 11,000 rpm at 4 °C. The pellets were resuspended in 75 μL of nuclei lysis buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 1% SDS). After incubation on ice for 30 min, samples were mixed with 625 μL of ChIP dilution buffer without Triton (16.7 mM Tris-HCl [pH 8.0], 167 mM NaCl, 1.2 mM EDTA, and 0.01% SDS). Chromatin samples were sonicated for 100 cycles of 15 s ON/45 s OFF using a Bioruptor UCD-250 (Cosmo Bio) to produce DNA fragments followed by the addition of 200 μL of ChIP dilution buffer with Triton (16.7 mM Tris-HCl [pH 8.0], 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, and 1.1% Triton X-100) was added. After undergoing centrifugation twice for 5 min at 15,000 rpm at 4 °C, the supernatant was transferred to a new tube. A total of 900 μL of solubilized sample was incubated with 25 μL of beads crosslinked with anti-GFP antibody or rabbit IgG isotype control (ab37415, Abcam) at 4 °C for 16 h, while an 18 μL aliquot was used as the input control. Beads were then washed twice with 1 mL of low-salt wash buffer (20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 2 mM EDTA, 0.1% SDS, and 1% Triton X-100), twice with 1 mL of high salt wash buffer (20 mM Tris-HCl [pH 8.0], 500 mM NaCl, 2 mM EDTA, 0.1% SDS, and 1% Triton X-100), twice with 1 mL of LiCl wash buffer (10 mM Tris-HCl [pH 8.0], 0.25 M LiCl, 1 mM EDTA, 1% sodium deoxycholate, and 1% Nonidet P-40), and twice with 1 mL of TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA). After washing, the beads were resuspended in 100 μL of elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 1% SDS) and incubated at 65 °C for 30 min. Then, 82 μL of elution buffer was added to 18 μL of the input control sample. Both supernatant and input samples were mixed with 6 μL of 5 M NaCl and incubated at 65 °C overnight to reverse crosslinks. ChIP samples were mixed with 550 μL of PB Buffer and purified using the QIAquick PCR Purification Kit (Qiagen) following the manufacturer’s instructions.

Construction of Illumina sequencing libraries and sequencing of ChIP DNA

ChIP-seq libraries were constructed from 100 ng of DNA samples using the NEB Ultra II DNA Library Prep Kit for Illumina (New England BioLabs) according to the manufacturer’s instructions. The amount of cDNA was determined by the QuantiFluor dsDNA System (Promega). All ChIP-seq libraries were sequenced as 81-bp single-end reads using a NextSeq550 system (Illumina Inc.).

Analysis of ChIP-Seq data

Reads were mapped to the TAIR10 Arabidopsis genome database using Bowtie2 using default parameters³⁷. The Sequence Alignment/Map (SAM) file generated by Bowtie2 was converted to a Binary Alignment/Map (BAM) format file by SAMtools³⁸. To visualize the mapped reads, a Tiled Data File (TDF) file was generated from the BAM file using the igvtools package in the Integrative Genome Browser (IGV)³⁹. ChIP-seq peaks were called by comparing the IP (Anti-GFP) with the Input (Rabbit IgG-Isotype Control) using Model-based Analysis of ChIP-Seq (MACS2) with the “-q 0.01” parameter (q value < 0.01)⁴⁰. The peaks were annotated with the nearest gene using the ChIPpeakAnno package in the Bioconductor suite and in R^41,42. Gene Ontology (GO) analysis of the set of 1105 genes was performed using PANTHER⁴³ on The Arabidopsis Information Resource (TAIR) website (https://www.arabidopsis.org/tools/go_term_enrichment.jsp). Sequences corresponding to the identified peaks were extracted from the Arabidopsis thaliana genome as a FASTA file using Bedtools⁴⁴. To identify potential TGA1 binding motifs, the extracted FASTA files were subjected to using MEME (Multiple EM for Motif Elicitation)-ChIP with default parameter settings (–meme-minw 6–meme-maxw 10)⁴⁵. The distributions of the generated motifs were then visualized using a density plot.

Plasmid construction for dual-luciferase reporter assay

A luciferase reporter plasmid was generated by replacing the GAL4-TATA region between the Hind III and Nco I sites in the pGAL4-TATA-LUC vector⁴⁶ with a 3 kb promoter region of CEPH obtained by PCR. For the preparation of effector plasmids, the 430T12 vector⁴⁶ (35S-P:Ω:GAL4DBD:NOS-T) was linearized by inverse PCR to exclude the GAL4 DNA binding domain. The linearized vector fragment was then ligated with cDNA fragments of GFP-CEPDL2 or GFP-GrxS5 using the NEBuilder HiFi DNA Assembly Kit to obtain effector plasmids. Both the pGAL4-TATA-LUC and 430T12 vectors were generously provided by N. Mitsuda. The list of primers is shown in Supplementary Data 5.

Isolation of Arabidopsis mesophyll protoplasts

Protoplasts were isolated from 20 to 24 leaves of 3-week-old Nössen or tga1,4 plants grown under 12 h light/12 h dark conditions using the “Tape-Arabidopsis Sandwich” method^47,48. The Scotch 3 M tape was applied to both the upper and lower epidermal surfaces of the leaves. Subsequently, the tape on the lower epidermal surface was carefully removed to peel off the lower epidermal cell layer. The peeled leaves, still attached to the 3 M tape, were then transferred to a 20 mL of enzyme solution (1% cellulase Onozuka R10 (Yakult, Japan), 0.25% macerozyme R10 (Yakult), 400 mM mannitol, 10 mM CaCl₂, 20 mM KCl, 10 mM β-mercaptoethanol and 20 mM MES-KOH [pH 5.7]) and incubated with gentle shaking at 60 rpm for 60 min at room temperature. The released protoplasts were filtered through a sterile 70 μm nylon mesh (FALCON) and transferred to 50 mL tubes containing 20 mL of W5 solution (150 mM NaCl, 125 mM CaCl₂, 5 mM KCl, 2 mM MES-KOH [pH 5.7]). Protoplasts were centrifuged at 100 × g for 10 min, washed twice with 20 mL of W5 solution, and then incubated on ice for 30 min. Subsequently, the protoplasts were recentrifuged and resuspended in MMg solution (400 mM mannitol, 15 mM MgCl₂, and 4 mM MES-KOH [pH 5.7]) to achieve a final cell density of 2 × 10⁵ cells/mL.

Dual-luciferase reporter assay in Arabidopsis mesophyll protoplasts

The 35 S:rLUC (Renilla luciferase) plasmid (kindly gifted by N. Nakamichi) and respective effectors were co-transformed with the CEPHpro:LUC reporter. A total of 100 μL protoplasts (2 × 10⁴ cells) were transferred to a 1.5 mL tube containing 10 μL DNA (CEPHpro:LUC 2.5 µg, Effector 1 µg, 35 S:rLUC 0.5 µg). Transformations were carried out by adding 210 μL of polyethylene glycol (PEG) solution (40% PEG-4000, 200 mM mannitol, 100 mM CaCl₂) and gently mixed. After incubating at room temperature for 10 min, the transformed protoplast suspension was washed three times with 500 µL of W5 solution. Finally, 500 µL of W5 solution containing 1 mM KNO₃ was added, followed by incubation at 22 °C for 18 h under continuous light. The dual-luciferase activity of transformed protoplasts was quantified using a Dual-Luciferase Reporter Assay System (Promega). The transformed protoplasts were lysed in 100 μL passive lysis buffer. Then, 20 μL of cell lysate was added to each side wall of a flat-bottomed 96-well plate and thoroughly mixed with 100 μL of LAR II buffer to measure firefly luciferase activity. The luminescence of each well was measured using a VICTOR Nivo multimode plate reader (PerkinElmer). Subsequently, 100 μL of Stop & Glo Reagent for Renilla luciferase activity was added to the mixture as an internal control and the luminescence was measured again. The reporter activity value was normalized to the internal control activity.

ChIP-qPCR analysis

The roots of 16-day-old TGA1–GFP/Nössen and TGA1–GFP/cepd1,2 cepdl2 plants grown on 10 mM NO₃^– medium (~900 mg) were used for the ChIP-qPCR analysis. Chromatin samples were sonicated using a Bioruptor UCD-250 for 30 cycles of 15 s ON/45 s OFF to obtain longer DNA fragments than those typically used for ChIP-seq analysis. Real-time RT-qPCR was carried out on a StepOne System (Applied Biosystems) using the THUNDERBIRD Next SYBR qPCR Mix (Toyobo), 10-fold diluted purified DNAs, and gene-specific primers. The list of primers used is shown in Supplementary Data 5.

Promoter β-glucuronidase (GUS) analysis

For GUS reporter-aided analysis of GrxS1 to GrxS8 promoter activity, the upstream 2.0 kb promoter regions of the genes were amplified by genomic PCR and cloned by translational fusion in frame with the GUS coding sequence into the binary vector pBI101. The list of primers used is shown in Supplementary Data 5. GUS activity was visualized using a standard protocol with X-Gluc as the substrate. For leaf sectioning, leaves were fixed in FAA solution (3.7% formaldehyde, 5% acetic acid and 50% ethanol), dehydrated through a graded ethanol series, and embedded in Technovit 7100 resin (Heraeus Kulzer, Germany) following the manufacturer’s instructions. Sections were cut to a thickness of 5 μm using a rotary microtome (RM2235, Leica), counter-stained with 0.05% Nile red, mounted with Entellan (Merck) and observed under a standard light microscope (BX60, Olympus).

Nitrate treatment of the detached shoots

Shoots of 12-day-old WT (Col) seedlings that had been grown on 10 mM NO₃^– medium were cut below the cotyledons and transferred to N-depleted (0 mM NO₃^–) liquid medium for N-starvation. After 24 h, the shoots were transferred to 10 mM NO₃^– liquid medium or N-depleted liquid medium as control for 3 h.

RNA-seq analysis

Total RNA was extracted from the roots of 14-day-old Col and GFP-GrxS5 plants grown on 10 mM NO₃⁻ using an RNeasy Mini Kit (QIAGEN). A total of 1 μg of RNA was used for mRNA purification using a NEBNext Oligo d (T) 25 magnetic isolation module (New England Biolabs), followed by first strand cDNA synthesis using a NEBNext Ultra II RNA Library Prep Kit for Illumina according to the manufacturer’s instructions. Samples were then ligated with NEBNext multiplex oligo adapter kits for barcoding. The amount of cDNA was quantified on an Agilent 4150 TapeStation System. The resulting cDNA libraries were sequenced on an Illumina NextSeq 550 with single-end 81-bp sequencing. Reads were mapped to the Arabidopsis TAIR10 reference genome using BaseSpace software (Illumina, https://basespace.illumina.com). Pairwise comparisons between samples were performed with the EdgeR package (Degust, https://degust.erc.monash.edu)⁴⁹. Genes with a q < 0.05 and absolute fold change <0.7 were identified as differentially expressed genes. Data shown are the mean of at least three biologically independent RNA-seq datasets.

Root ¹⁵N influx

Vertically grown 14-day-old plants were sequentially transferred to 0.1 mM CaSO₄ for 1 min and then to modified Murashige-Skoog medium containing 0.2 mM or 10 mM ¹⁵NO₃^– as the N source for 10 min. At the end of ¹⁵N labeling, the roots were washed for 1 min in 0.1 mM CaSO₄ and separated from the shoots. The roots were then lyophilized in vacuo and analyzed for total N and ¹⁵N content using an elemental analysis–isotope ratio mass spectrometry system (Flash EA1112-DELTA V PLUS ConFlo III system, Thermo Fisher Scientific).

Survival rate under high nitrate condition

Surface-sterilized WT (Col) and grxs1-s8 mutant seeds were sown on B5 medium (1% sucrose) solidified using 1.2% agar and grown at 22 °C under continuous light. After 5 d, the seedlings were transferred to a medium containing 120 mM NO₃^– and further grown for 9 d at 22 °C under continuous light. The survivability of the plants was assessed based on the presence of 4 or more green leaves, which indicated survival under high nitrate conditions, while seedlings that appeared bleached were categorized as dead.

Yeast three-hybrid assay

Yeast three-hybrid screening was performed using the Matchmaker Gold yeast two-hybrid system and the pBridge vector (Clontech). The cDNA fragments encoding TGA1 were subcloned into the multiple cloning site 1 (MCS1) of the pBridge vector for expression of the GAL4 DNA-binding domain (BD) fusion. Concurrently, cDNA fragments for CEPDL2 or GrxS5 were subcloned into the MCS2 of the pBridge vector. Each of these vectors was then introduced into the Y2H Gold yeast strain. Additionally, the cDNA fragment for TPL was subcloned into the pGADT7 vector to facilitate expression of the GAL4 AD fusion, and transformed into the Y187 yeast strain. After the mating between the Y2H Gold strain expressing TGA1 + CEPDL2 or TGA1+GrxS5 and the Y187 strain expressing TPL, the resulting zygotes were selected by culturing on SD/−Leu/−Trp/−Met/−His/+X-α-Gal plates for 5 days at 28 °C. Details of the primers are listed in Supplementary Data 5.

Hydroponic culture system

In a hydroponic system, modified Murashige-Skoog liquid medium without sucrose was used to prevent bacterial growth. Thirteen-day-old Arabidopsis seedlings were transplanted into the system. The roots of each seedling were carefully threaded through pre-drilled apertures (5 mm I.D.) in a Styrofoam plate, which was then positioned above a 6-well culture plate filled with 1 mM NO₃^– liquid medium. The plant roots were fully immersed in the medium and subjected to a 24-h pre-incubation period. Both WT and tga1,4 plants were cultured under various nitrogen conditions: continuous nitrogen-sufficient (1 mM NO₃^–), continuous low nitrogen (0.1 mM NO₃^–), or fluctuating nitrogen conditions. The latter involved alternating periods of 18 h of nitrogen-deficient (0 mM NO₃^–) with 6 h of nitrogen sufficiency (1 mM NO₃^–) over a period of 7 d.

Nitrate content

The concentration of NO₃^– ions in tissues was quantified using an ion chromatography system (Dionex Aquion, Thermo Fisher Scientific). The shoots and roots of 14-day-old plants were powdered in liquid nitrogen and mixed with 1 mL of water to extract nitrate. Following centrifugation, the crude tissue extract was diluted 10-fold with water, and 25-μL aliquots were analyzed using a Dionex IonPac AS22 column (Thermo Fisher Scientific; 4 mm i.d. × 250 mm) over 15 min. The mobile phase eluent, composed of 4.5 mM Na₂CO₃ and 1.4 mM NaHCO₃, was used at a flow rate of 1.2 mL/min at 30 °C. Separation of ions was monitored by a conductivity detector equipped with a Dionex AERS 500 suppressor unit (Thermo Fisher Scientific).

Stable isotope metabolic labeling with ¹⁵N

For quantitative proteomic analyses, WT (Nössen) and tga1,4 (Nos) seedlings were reciprocally labeled with light (normal) ¹⁴N or heavy ¹⁵N via metabolic incorporation. We designated pairs of ¹⁴N-labeled WT/¹⁵N-labeled tga1,4 seedlings as forward labeling, and pairs of ¹⁵N WT/¹⁴N tga1,4 seedlings as reciprocal labeling. Surface-sterilized WT and tga1,4 seeds were sown on B5 medium containing either ¹⁴N- or ¹⁵N (1% sucrose) solidified with 1.5% agar in 13 × 10 cm plastic plates (18 seeds × 3 rows/plate) and then cultivated vertically under continuous light at 22 °C for 7 d. In the ¹⁵N B5 medium, conventional nitrogen salts, K¹⁴NO₃ and (¹⁴NH₄)₂SO₄, were substituted with their heavy nitrogen isotropic counterparts, K¹⁵NO₃ and (¹⁵NH₄)₂SO₄, respectively. After 7 d, the seedlings were transferred to 3 mM NO₃^– medium (containing either K¹⁴NO₃ or K¹⁵NO₃) solidified using 1.5% purified agar in 13 × 10 cm plastic plates (12 seedlings/plate), and further cultivated vertically under continuous light at 22 °C for 7 d. Subsequently, these plants were transplanted into a hydroponic system where they underwent alternating cycles of 18 h of N-deficiency (0 mM NO₃^–) and 6 h of N-sufficiency (1 mM NO₃^–) for 2 d. For forward labeled samples, ¹⁴N-labeled WT roots were combined at a 1:1 fresh weight ratio with ¹⁵N-labeled tga1,4 roots. Similarly, for the reciprocal labeling, ¹⁵N-labeled WT roots were combined at a 1:1 ratio with ¹⁴N-labeled tga1,4. The combined root tissues (0.5–1.0 g) were then frozen in liquid nitrogen and ground to a fine powder using a Multi-beads shocker. Microsomal membranes were extracted by mixing 500 mg of ground tissue with 2.5 mL of extraction buffer (25 mM Tris-HCl [pH 7.0], 10 mM MgCl₂, 2 mM DTT, 2 µM leupeptin, 2 mM PMSF, 250 mM sucrose, and 1× PhosSTOP phosphatase inhibitors [Roche]). The homogenate was centrifuged at 10,000 × g for 15 min at 4 °C, and the resulting supernatant was further centrifuged at 100,000 × g for 30 min at 4 °C to precipitate the microsomal membranes. The pellets were solubilized in digestion buffer (8 M urea, 250 mM Tris-HCl [pH8.5]) to a final protein concentration of 2.0 mg/mL. The protein concentration was determined using a Bradford protein assay kit. Protein digestion and nano LC-MS/MS data acquisition methods are described above.

Statistics and reproducibility

All statistical analyses were performed using Prism software (version 8; GraphPad). No statistical method was used to predetermine sample size. No data were excluded from the analysis. Samples were grown under the same conditions and randomly allocated in the growth chamber. Experimental plant material was collected randomly without any bias. Investigators were not blinded to allocation during the experiments and outcome assessment.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The RNA-seq data have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession number GSE240738. The raw mass spectrometry data have been deposited in the ProteomeXchange Consortium via the jPOST partner repository under accession number PXD044498 [https://repository.jpostdb.org/entry/JPST002285]. The source data underlying Figs. 1–5 and Supplementary Figs. 1–3 are provided as a Source Data file. The Arabidopsis lines generated in this study are available from the corresponding author upon request. Source data are provided with this paper.

References

Lejay, L. et al. Molecular and functional regulation of two NO₃^– uptake systems by N- and C-status of Arabidopsis plants. Plant J. 18, 509–519 (1999).
Article CAS PubMed Google Scholar
Gansel, X., Munos, S., Tillard, P. & Gojon, A. Differential regulation of the NO₃^– and NH₄⁺ transporter genes AtNrt2.1 and AtAmt1.1 in Arabidopsis: relation with long-distance and local controls by N status of the plant. Plant J. 26, 143–155 (2001).
Article CAS PubMed Google Scholar
Ohkubo, Y., Tanaka, M., Tabata, R., Ogawa-Ohnishi, M. & Matsubayashi, Y. Shoot-to-root mobile polypeptides involved in systemic regulation of nitrogen acquisition. Nat. Plants 3, 17029 (2017).
Article CAS PubMed Google Scholar
Ohyama, K., Ogawa, M. & Matsubayashi, Y. Identification of a biologically active, small, secreted peptide in Arabidopsis by in silico gene screening, followed by LC-MS-based structure analysis. Plant J. 55, 152–160 (2008).
Article CAS PubMed Google Scholar
Tabata, R. et al. Perception of root-derived peptides by shoot LRR-RKs mediates systemic N-demand signaling. Science 346, 343–346 (2014).
Article ADS CAS PubMed Google Scholar
Ota, R., Ohkubo, Y., Yamashita, Y., Ogawa-Ohnishi, M. & Matsubayashi, Y. Shoot-to-root mobile CEPD-like 2 integrates shoot nitrogen status to systemically regulate nitrate uptake in Arabidopsis. Nat. Commun. 11, 641 (2020).
Article ADS CAS PubMed PubMed Central Google Scholar
Ohkubo, Y., Kuwata, K. & Matsubayashi, Y. A type 2C protein phosphatase activates high-affinity nitrate uptake by dephosphorylating NRT2.1. Nat. Plants 7, 310–316 (2021).
Article CAS PubMed Google Scholar
Rouhier, N., Couturier, J. & Jacquot, J. P. Genome-wide analysis of plant glutaredoxin systems. J. Exp. Bot. 57, 1685–1696 (2006).
Article CAS PubMed Google Scholar
Gutsche, N., Thurow, C., Zachgo, S. & Gatz, C. Plant-specific CC-type glutaredoxins: functions in developmental processes and stress responses. Biol. Chem. 396, 495–509 (2015).
Article CAS PubMed Google Scholar
Xing, S., Rosso, M. G. & Zachgo, S. ROXY1, a member of the plant glutaredoxin family, is required for petal development in Arabidopsis thaliana. Development 132, 1555–1565 (2005).
Article CAS PubMed Google Scholar
La Camera, S. et al. The glutaredoxin ATGRXS13 is required to facilitate Botrytis cinerea infection of Arabidopsis thaliana plants. Plant J. 68, 507–519 (2011).
Article PubMed Google Scholar
Laporte, D. et al. Glutaredoxin GRXS13 plays a key role in protection against photooxidative stress in Arabidopsis. J. Exp. Bot. 63, 503–515 (2012).
Article CAS PubMed Google Scholar
Patterson, K. et al. Nitrate-regulated glutaredoxins control Arabidopsis primary root growth. Plant Physiol. 170, 989–999 (2016).
Article CAS PubMed Google Scholar
Walters, L. A. & Escobar, M. A. The AtGRXS3/4/5/7/8 glutaredoxin gene cluster on Arabidopsis thaliana chromosome 4 is coordinately regulated by nitrate and appears to control primary root growth. Plant Signal Behav. 11, e1171450 (2016).
Article PubMed PubMed Central Google Scholar
Jung, J. Y., Ahn, J. H. & Schachtman, D. P. CC-type glutaredoxins mediate plant response and signaling under nitrate starvation in Arabidopsis. BMC Plant Biol. 18, 281 (2018).
Article CAS PubMed PubMed Central Google Scholar
Ehrary, A. et al. Glutaredoxin AtGRXS8 represses transcriptional and developmental responses to nitrate in Arabidopsis thaliana roots. Plant Direct 4, e00227 (2020).
Article CAS PubMed PubMed Central Google Scholar
Liu, Z. & Karmarkar, V. Groucho/Tup1 family co-repressors in plant development. Trends Plant Sci. 13, 137–144 (2008).
Article CAS PubMed Google Scholar
Ndamukong, I. et al. SA-inducible Arabidopsis glutaredoxin interacts with TGA factors and suppresses JA-responsive PDF1.2 transcription. Plant J. 50, 128–139 (2007).
Article CAS PubMed Google Scholar
Li, S. et al. Nuclear activity of ROXY1, a glutaredoxin interacting with TGA factors, is required for petal development in Arabidopsis thaliana. Plant Cell 21, 429–441 (2009).
Article CAS PubMed PubMed Central Google Scholar
Murmu, J. et al. Arabidopsis basic leucine-zipper transcription factors TGA9 and TGA10 interact with floral glutaredoxins ROXY1 and ROXY2 and are redundantly required for anther development. Plant Physiol. 154, 1492–1504 (2010).
Article CAS PubMed PubMed Central Google Scholar
Li, S., Gutsche, N. & Zachgo, S. The ROXY1 C-terminal L**LL motif is essential for the interaction with TGA transcription factors. Plant Physiol. 157, 2056–2068 (2011).
Article CAS PubMed PubMed Central Google Scholar
Zander, M., Chen, S., Imkampe, J., Thurow, C. & Gatz, C. Repression of the Arabidopsis thaliana jasmonic acid/ethylene-induced defense pathway by TGA-interacting glutaredoxins depends on their C-terminal ALWL motif. Mol. Plant 5, 831–840 (2012).
Article CAS PubMed Google Scholar
Li, N. et al. TGACG-BINDING FACTORs (TGAs) and TGA-interacting CC-type glutaredoxins modulate hyponastic growth in Arabidopsis thaliana. N. Phytol. 221, 1906–1918 (2019).
Article CAS Google Scholar
Uhrig, J. F. et al. CC-type glutaredoxins recruit the transcriptional co-repressor TOPLESS to TGA-dependent target promoters in Arabidopsis thaliana. Biochim Biophys. Acta Gene Regul. Mech. 1860, 218–226 (2017).
Article CAS PubMed Google Scholar
Alvarez, J. M. et al. Systems approach identifies TGA1 and TGA4 transcription factors as important regulatory components of the nitrate response of Arabidopsis thaliana roots. Plant J. 80, 1–13 (2014).
Article CAS PubMed Google Scholar
Swift, J., Alvarez, J. M., Araus, V., Gutierrez, R. A. & Coruzzi, G. M. Nutrient dose-responsive transcriptome changes driven by Michaelis-Menten kinetics underlie plant growth rates. Proc. Natl. Acad. Sci. USA 117, 12531–12540 (2020).
Article ADS CAS PubMed PubMed Central Google Scholar
Canales, J., Contreras-Lopez, O., Alvarez, J. M. & Gutierrez, R. A. Nitrate induction of root hair density is mediated by TGA1/TGA4 and CPC transcription factors in Arabidopsis thaliana. Plant J. 92, 305–316 (2017).
Article CAS PubMed Google Scholar
Wirth, J. et al. Regulation of root nitrate uptake at the NRT2.1 protein level in Arabidopsis thaliana. J. Biol. Chem. 282, 23541–23552 (2007).
Article CAS PubMed Google Scholar
Lin, S. H. et al. Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport. Plant Cell 20, 2514–2528 (2008).
Article CAS PubMed PubMed Central Google Scholar
Yeshno, E., Arnon, S. & Dahan, O. Real-time monitoring of nitrate in soils as a key for optimization of agricultural productivity and prevention of groundwater pollution. Hydrol. Earth Syst. Sc. 23, 3997–4010 (2019).
Article ADS CAS Google Scholar
Gloser, V. et al. Early changes in nitrate uptake and assimilation under drought in relation to transpiration. Front Plant Sci. 11, 602065 (2020).
Article PubMed PubMed Central Google Scholar
Katz, E. et al. Genetic variation underlying differential ammonium and nitrate responses in Arabidopsis thaliana. Plant Cell 34, 4696–4713 (2022).
Article PubMed PubMed Central Google Scholar
Tsutsui, H. & Higashiyama, T. pKAMA-ITACHI Vectors for highly efficient CRISPR/Cas9-mediated gene knockout in Arabidopsis thaliana. Plant Cell Physiol. 58, 46–56 (2017).
CAS PubMed Google Scholar
Zuo, J., Niu, Q. W. & Chua, N. H. Technical advance: an estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J. 24, 265–273 (2000).
Article CAS PubMed Google Scholar
Gan, X. et al. Multiple reference genomes and transcriptomes for Arabidopsis thaliana. Nature 477, 419–423 (2011).
Article ADS CAS PubMed PubMed Central Google Scholar
Mullan, K. A. et al. ggVolcanoR: a Shiny app for customizable visualization of differential expression datasets. Comput. Struct. Biotechnol. J. 19, 5735–5740 (2021).
Article CAS PubMed PubMed Central Google Scholar
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
Article CAS PubMed PubMed Central Google Scholar
Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Article PubMed PubMed Central Google Scholar
Thorvaldsdottir, H., Robinson, J. T. & Mesirov, J. P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief. Bioinform. 14, 178–192 (2013).
Article CAS PubMed Google Scholar
Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).
Article PubMed PubMed Central Google Scholar
Zhu, L. J. et al. ChIPpeakAnno: a Bioconductor package to annotate ChIP-seq and ChIP-chip data. BMC Bioinforma. 11, 237 (2010).
Article Google Scholar
Zhu, L. J. Integrative analysis of ChIP-chip and ChIP-seq dataset. Methods Mol. Biol. 1067, 105–124 (2013).
Article CAS PubMed Google Scholar
Mi, H., Muruganujan, A., Ebert, D., Huang, X. & Thomas, P. D. PANTHER version 14: more genomes, a new PANTHER GO-slim and improvements in enrichment analysis tools. Nucleic Acids Res. 47, D419–D426 (2019).
Article CAS PubMed Google Scholar
Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).
Article CAS PubMed PubMed Central Google Scholar
Machanick, P. & Bailey, T. L. MEME-ChIP: motif analysis of large DNA datasets. Bioinformatics 27, 1696–1697 (2011).
Article CAS PubMed PubMed Central Google Scholar
Fujimoto, S. Y., Ohta, M., Usui, A., Shinshi, H. & Ohme-Takagi, M. Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression. Plant Cell 12, 393–404 (2000).
CAS PubMed PubMed Central Google Scholar
Wu, F. H. et al. Tape-Arabidopsis Sandwich—a simpler Arabidopsis protoplast isolation method. Plant Methods 5, 16 (2009).
Article PubMed PubMed Central Google Scholar
Yoo, S. D., Cho, Y. H. & Sheen, J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protoc. 2, 1565–1572 (2007).
Article CAS PubMed Google Scholar
Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).
Article CAS PubMed Google Scholar

Download references

Acknowledgements

This research was supported by a Grant-in-Aid for Scientific Research (S) (No. 23H05477 to Y.M.), Grant-in-Aid for Transformative Research Areas (A) (No. 20H05907 to Y.M.), Grant-in-Aid for JSPS Fellows (No. 22KJ1616 to Y.O.) and Grant-in-Aid for Early-Career Scientists (No. 24KJ18137 to Y.O.) from the Japan Society for the Promotion of Science.

Author information

These authors contributed equally: Ryutaro Kobayashi, Yuri Ohkubo.

Authors and Affiliations

Graduate School of Science, Nagoya University, Nagoya, Japan
Ryutaro Kobayashi, Yuri Ohkubo, Mai Izumi, Ryosuke Ota, Keiko Yamada, Yoko Hayashi, Yasuko Yamashita, Saki Noda, Mari Ogawa-Ohnishi & Yoshikatsu Matsubayashi

Authors

Ryutaro Kobayashi
View author publications
You can also search for this author in PubMed Google Scholar
Yuri Ohkubo
View author publications
You can also search for this author in PubMed Google Scholar
Mai Izumi
View author publications
You can also search for this author in PubMed Google Scholar
Ryosuke Ota
View author publications
You can also search for this author in PubMed Google Scholar
Keiko Yamada
View author publications
You can also search for this author in PubMed Google Scholar
Yoko Hayashi
View author publications
You can also search for this author in PubMed Google Scholar
Yasuko Yamashita
View author publications
You can also search for this author in PubMed Google Scholar
Saki Noda
View author publications
You can also search for this author in PubMed Google Scholar
Mari Ogawa-Ohnishi
View author publications
You can also search for this author in PubMed Google Scholar
Yoshikatsu Matsubayashi
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

Y.M. conceived this project, and Y.M., R.K., Y.O. and M.O.-O. designed the experiments. All authors performed the experiments and interpreted the results. Y.M. and Y.O. wrote the manuscript.

Corresponding author

Correspondence to Yoshikatsu Matsubayashi.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. A peer review file is available.

Additional information

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

Supplementary information

Supplementary Information

Peer Review File

Description of additional supplementary files

Supplementary Data 1

Supplementary Data 2

Supplementary Data 3

Supplementary Data 4

Supplementary Data 5

Reporting Summary

Source data

Source Data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Cite this article

Kobayashi, R., Ohkubo, Y., Izumi, M. et al. Integration of shoot-derived polypeptide signals by root TGA transcription factors is essential for survival under fluctuating nitrogen environments. Nat Commun 15, 6903 (2024). https://doi.org/10.1038/s41467-024-51091-5

Download citation

Received: 16 October 2023
Accepted: 28 July 2024
Published: 23 August 2024
DOI: https://doi.org/10.1038/s41467-024-51091-5

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.