BRAHMA represses STOP1-NRT1.1 module to control plant rhizosphere alkalization and acid stress adaptation

Domain: chemistry
Published: 2026-02-23
Editorial outcome: major_revision

Abstract
Covering 40–50% of world’s arable lands, acidic soils pose a major constraint on global crop productivity by severely restricting root development and nutrient acquisition. The Arabidopsis C2H2-type transcription factor STOP1 plays a fundamental role in mitigating acid stress by activating H+/NO3− symport via NRT1.1, thereby driving rhizosphere alkalinization to protect root growth and improving nitrogen use efficiency (NUE). However, the upstream regulation of this pH-responsive STOP1-NRT1.1 pathway remains poorly defined. Here, we identify the central SWI2/SNF2-type ATPase BRAHMA (BRM) as a key suppressor of the STOP1 pathway. BRM physically interacts with STOP1 and occupies the genomic region of NRT1.1, repressing the STOP1-dependent activation of NRT1.1 expression and consequently limiting NO3− uptake and rhizosphere alkalinization under chronic acidity. Genetic epistasis analysis using brm stop1 and brm nrt1.1 double mutants establish BRM as an upstream regulator of this signaling module. Notably, low pH rapidly triggers BRM degradation independent of NO3− availability, thereby relieving its repression on the STOP1-NRT1.1 pathway. This dynamic BRM disintegration enables robust induction of H+-coupled NO3− uptake, remodeling the rhizosphere pH landscape to foster optimal root growth under acidity. Collectively, our findings uncover the BRM-STOP1-NRT1.1 axis as a central regulatory module integrating NO3− acquisition with pH homeostasis, offering a dual-benefit strategy for enhancing crop resilience to acid soils and reducing fertilizer-driven acidification through improved NUE.

Introduction
Acidic soils occupy 40–50% of the world’s total arable lands, and their acidification pace is accelerating due to the widespread application of urea and ammonium-based fertilizers 1,2, posing a critical challenge to the sustainability of global agriculture. Acidic soils impose a vicious cycle: high proton (H+) concentrations in acidic soils severely limit crop productivity by inhibiting root growth and nutrient acquisition 3,4, while plants’ inability to efficiently utilize N fertilizers exacerbates soil acidification through residual H+ accumulation and also the risk of environmental pollution 5. Molecular breeding of plants to improve nitrogen acquisition and adaptation to acidic soils could be a cost-effective solution to these problems 6.

The C2H2-type transcription factor, SENSITIVE-TO-PROTON-RHIZOTOXICITY1 (STOP1), was identified as the first key regulator of plant H+ tolerance, with its disruption leading to over 70% inhibition of root growth under acidic conditions 4. Subsequent lines of evidence have suggested that STOP1 has pleiotropic roles in plant response to various environmental cues, including ammonium toxicity, Pi deficiency, N deficiency, Ca2+ deficiency, salt stress, drought, and hypoxia stresses 7–16. Furthermore, STOP1 is functional conserved across land plant species 17,18. Among critical STOP1-regulated modules, our previous study demonstrated that the STOP1-NITRATE TRANSPORTER 1.1 (NRT1.1) module emerges as a critical buffer in response to low pH: STOP1 activates NRT1.1-mediated H+/NO3− symport, alkalizing the rhizosphere to protect roots and enhance N use efficiency 5. However, the upstream mechanisms that underlie STOP1-enhanced plant adaptation to acidity remain unclear.

Soil acidification is a dynamically deteriorating process: even in moderately acidic soils (pH 5.0–5.5), repeated urea application can lower rhizosphere pH by 0.17–0.72 units over one growing season 1,19. If plant-induced H+ influx from root nutrient uptake could counteract (either fully or partially) H+ released during nitrification of ammonium-based fertilizers, this equilibrium could mitigate soil acidification, simultaneously benefiting agricultural ecosystem sustainability and crop productivity. In Arabidopsis, NRT1.1 serves as the major transporter for root NO3− uptake under adequate NO3− supplementation and acidic conditions 20,21. The NRT1.1-mediated H+/NO3− symport drives rhizosphere alkalinization to protect roots from acid toxicity and enhance nitrogen use efficiency (NUE) 5. Thus, adaptation to acidic stress requires plants to orchestrate both immediate stress responses and sustained transcriptional programming that stabilizes NRT1.1 expression to ensure sufficient NO3− uptake to neutralize protons derived from ammonium nitrification 22. It’s worth noting that STOP1-dependent H+ tolerance is functionally restricted by its post-translational modifications and dosage sensitivity, thereby stringently restricting its capacity to activate NRT1.1 transcription. Furthermore, STOP1 overexpression not only produces growth penalties but also fails to confer chronic stress adaptation, while its upstream regulators governing transcriptional activity under H+ stress are largely unknown. Chromatin remodeling, by enabling sustained transcriptional memory during prolonged stress 23, emerges as a promising solution to circumvent these limitations through epigenetic stabilization of stress-responsive genes.

In this study, we identified BRAHMA (BRM), a switch defective/sucrose non-fermentable (SWI/SNF) ATPase, as a key suppressor of the STOP1-mediated pathway. BRM directly occupies the genomic region of NRT1.1 and interacts with STOP1 to inhibit its trans-activation activity under acidity, thereby suppressing the STOP1-NRT1.1 module. Low pH rapidly triggers BRM degradation, relieving repression to augment H+-coupled nitrate influx and establish a rhizosphere pH optimum for root growth under acidic condition. Our findings may provide a strategy for simultaneously improving plant NUE to partially counter the H+ derived from nitrification of ammonium-based fertilizers and slowing soil acidification through the manipulation of the BRM-STOP1-NRT1.1 module.

Results
BRM physically interacts with STOP1
To identify potential regulators of STOP1 during H+ stress, we conducted a yeast two-hybrid (Y2H) screen using STOP1 as bait. Although pGBKT7-STOP1 exhibited weak autoactivation in the AH109 yeast strain, this background activity was effectively suppressed by 50 mM 3-amino-1,2,4-triazole (3-AT) (Supplementary Fig. 1). The screen yielded 75 positive colonies, corresponding to 23 putative STOP1-interacting proteins (Supplementary Data 1). Among these, BRAHMA (BRM), a core chromatin-remodeling subunit, was recovered in six independent clones, making it the most recurrent and therefore the highest-priority candidate among previously unreported STOP1 interactors. Region mapping assays showed that the N-terminal region of BRM (BRMn, amino acids 1 to 976) was both necessary and sufficient for their interaction (Fig. 1a–c). Since BRMn functions as a docking site for the recruitment of transcription factors 24, we employed this fragment, rather than full-length BRM, in subsequent biochemical assays. In Nicotiana benthamiana leaf BiFC experiments, co-expression of STOP1 and BRMn produced a robust nuclear fluorescence signal, confirming their interaction in planta (Fig. 1d). Furthermore, co-immunoprecipitation assay using Arabidopsis plants co-expressing pBRM:BRM-FLAG and pSTOP1:GFP-STOP1 (GFP-STOP1/BRM-FLAG) confirmed that GFP-STOP1 was specifically co-immunoprecipitated with BRM-FLAG after a 6-h low-pH and MG132 treatment (Fig. 1e). These results firmly establish that BRM interacts with STOP1 in the nucleus.

Lack of BRM enhances plant growth and NO3− acquisition under H+ stress
To investigate the role of BRM in response to acidic stress, we performed a phenotypical analysis of two hypomorphic alleles of BRM (brm-3 and brm-20) and a complementation line (pBRM:BRM-FLAG/brm-3, hereafter referred to as COM). Under low-pH conditions, loss of BRM function significantly enhanced root growth, with root elongation in brm-3 and brm-20 mutants increasing by approximately 60% compared with that of wild-type (Col-0) plants, while the complementation of BRM fully restored the root growth of brm-3 mutants (Fig. 2a, b). Even after prolonged exposure, brm-3 roots remained longer than those of Col-0 under acidic pH, whereas under neutral pH the brm-3 mutant displayed significantly shorter root elongation compared with Col-0 (Supplementary Fig. 2). These results indicate that BRM negatively regulates root tolerance to acidic stress.

Given that BRM degradation is crucial for maintaining root apical meristem integrity by alleviating DNA double-stand breaks (DSBs) induced under high-B conditions 25, we examined whether the enhanced H+ tolerance of brm-3 mutants is related to altered DSBs levels. However, the expression of DSBs-induced genes (GR1, RAD51, and BRCA1) was neither significantly induced by low-pH treatment nor affected by BRM or STOP1 activity (Supplementary Fig. 3). Consistently, 1-day low-pH treatment did not significantly alter WT root DSBs level, suggesting that plants tolerance to acidic stress is not closely related with DSBs. Furthermore, the DSBs level of the acid-tolerant brm-3 mutant showed similar level as that of the acid-sensitive stop1 mutant, though they were slightly suppressed when compared to Col-0 (Supplementary Fig. 4). These results indicate that BRM-regulated H+ tolerance is not primarily associated with its role in DNA damage repair. Given that STOP1-improved H+ tolerance and NUE are closely linked to enhanced root NO3− uptake 5 and that BRM physically interacts with STOP1 (Fig. 1), we speculated that BRM may regulates H+ tolerance in a NO3−-depended manner. To test this notion, we compared the effects of different concentrations of NO3− supplies (0.06 and 6 mM) on root elongation of Col-0, brm-3, and stop1 plants, with 1 mM NH4+ supplied as an additional nitrogen source. Under low-pH treatments with 0.06 mM NO3−, root growth of Col-0, brm-3, and stop1 mutants was comparable (Fig. 2c, d). In contrast, brm-3 mutants showed significantly enhanced root growth compared to Col-0 under low-pH treatments with 6 mM NO3−, while stop1 mutants exhibited reduced root elongation (Fig. 2c, d). These results suggest that BRM-regulated H+ tolerance depends on NO3− availability.

Notably, both brm-3 and brm-20 are weak alleles. While brm-3 harbors a T-DNA insertion that disrupts the bromodomain in the C terminus, brm-20 is predicted to result in a truncation of a small segment at the C terminus. These mutations do not appear to be essential for chromatin remodeling activity, but may affect BRM stability or its tethering to chromatin 26–28. To confirm the specific role of BRM in acid stress response, we further analyzed the phenotype of BRM-null mutant (brm-1), which is sterile and exhibits severe developmental defects 27,29,30. As shown in Fig. 2e, f, root elongation in the brm-1 mutant was largely insensitive to low-pH stress, in stark contrast to the significant inhibition observed in Col-0 plants. Moreover, decreasing the concentration of NO3− from 6 mM to 0.06 mM significantly inhibited root growth of Col-0 but barely affected that of brm-1 mutants under low-pH treatments, demonstrating that BRM-repressed H+ tolerance depends on NO3− availability.

Since NRT1.1 mediates the majority of root NO3− uptake under low-pH condition and the low pH-induced transcription of NRT1.1 is controlled by STOP1 5,21, we analyzed the expression of NRT1.1 in the roots of Col-0, brm-3, and stop1 mutants under different pH conditions with 6 mM NO3− supplementation. As expected, low pH-induced NRT1.1 expression was remarkably enhanced in roots of brm-3 and brm-1 mutants compared with that in Col-0 plants (Fig. 2g, h). To further investigate whether BRM and NRT1.1 function in the same pathway in response to H+ stress, we generated a brm nrt1.1 double mutant by crossing the nrt1.1-1 and brm-20 mutants (Supplementary Fig. 5). We were unable to obtain brm-3 nrt1.1 double mutant due to the severely reduced fertility of brm-3, as recently reported 31. In low-pH agar medium, brm nrt1.1 plants exhibited root growth comparable to that of the nrt1.1-1 single mutants (Fig. 2i, j), suggesting that NRT1.1 is epistatic to BRM in the regulation of H+ tolerance. Collectively, these results indicate that BRM inhibits H+ tolerance by repressing the low-pH-induced expression of NRT1.1.

Lack of BRM enhances STOP1-activated NRT1.1 expression and NRT1.1 chromatin accessibility
To elucidate how BRM regulates NRT1.1 expression in response to H+ stress, we first assessed whether BRM affects STOP1 protein accumulation, as the induced NRT1.1 expression by low pH is known to be STOP1-dependent 5. Under low pH condition, comparative analysis of GFP-STOP1 fluorescence in Col-0 versus brm-20 mutants (GFP-STOP1/brm-20) revealed no significant difference in STOP1 protein levels (Supplementary Fig. 6), suggesting that BRM does not repressed NRT1.1 expression by altering STOP1 protein accumulation. We next examined whether BRM modulates STOP1-dependent transcriptional activation of NRT1.1. Using a transient expression system in N. benthamiana leaves, we co-expressed STOP1 and BRM along with luciferase gene (LUC) reporter constructs driven by either the NRT1.1 promoter region (P1) or the 4th intron (P2), both containing STOP1-binding sites 5,14 (Fig. 3a). In addition, NLP7, another well-known transcription factor that activates NRT1.1 transcription 32, and the NRT1.1 promoter fragment (P3), which contain the binding regions of NLP7 but not STOP1 or BRM, were used as negative controls. As shown in Fig. 3b, c, BRM co-expression substantially decreased the STOP1-activated P1- and P2-derived LUC signals to an almost undetectable levels, suggesting that BRM indeed affects the activation of STOP1 on NRT1.1 expression. However, BRM co-expression had no significant impact on either the NLP7-activated P3-drived LUC activity driven by the P3 fragment or EV-resulted P3-mediated LUC activity (Supplementary Fig. 7). These results suggest that BRM specifically targets the STOP1-dependent activation of NRT1.1, rather than exerting a non-specific inhibitory effect on LUC expression. To assess whether this regulatory interaction occur in vivo, we carried out chromatin immunoprecipitation (ChIP) experiments. STOP1 enrichment at endogenous NRT1.1 chromatin regions was notably higher in brm-3 mutants than in Col-0 plants (Fig. 3d), confirming that BRM inhibits the binding of STOP1 to NRT1.1 genomic regions.

To further investigate BRM’s autonomous capacity to inhibit NRT1.1 expression, we analyzed publicly available datasets using WashU Epigenome Browser (https://epigenomegateway.wustl.edu/) and Plant Regulomics (https://plantregulomics.org/). This analysis revealed that BRM is associated with NRT1.1 genomic regions harboring STOP1-binding motifs (Fig. 3e; Supplementary Fig. 8). This association was further confirmed by ChIP-qPCR, which showed BRM enrichment at these sites (Fig. 3f). Together, these findings support the idea that BRM antagonistically regulates NRT1.1 expression by interfering with STOP1 activity. Given that BRM modulates gene expression by altering chromatin accessibility 33–35, we next assess the chromatin state of the NRT1.1 locus using FAIRE-qPCR. Consistent with the ChIP-qPCR results, chromatin accessibility at the P1-1 and P2-1 regions of NRT1.1 was significantly increased in brm mutants compared to Col-0, with a stronger effect observed at P2-1 (Fig. 3g). These results indicate that BRM represses NRT1.1 expression by limiting chromatin accessibility at STOP1-binding regions, thereby reducing STOP1 occupancy and inhibiting NRT1.1 activation.

In addition, previous studies have shown that BRM interacts with the histone deacetylase HDA6 to repress transposons under various stresses by modulating DNA methylation and H3 acetylation levels 26,36. Our data also implicate HDA6 in this regulatory circuit: loss of HDA6 function enhanced H+ tolerance and increased NRT1.1 expression in root tips—the zones most sensitive to acidic stress and the key sites where STOP1 acts to prevent H+ toxicity 5 (Fig. 3h–j). These findings suggest that BRM and HDA6 may cooperatively repress NRT1.1 through chromatin remodeling mechanisms that likely involve H3 acetylation and DNA methylation.

BRM acts genetically upstream of STOP1 in regulation of NO3− uptake and H+ tolerance
To clarify the genetic relationship between BRM and STOP1 in response to H+ stress, we generated a brm stop1 double mutant by crossing the brm-20 and stop1 mutants (Supplementary Fig. 9). When grown on low-pH agar medium, the brm stop1 double mutant displayed root growth similar to the stop1 mutant but notably shorter than that of Col-0 and brm mutant (Fig. 4a, b). In accord with the elevated NRT1.1 expression observed in roots of brm mutant, the brm plants also showed increased root NO3− uptake compared to Col-0 (Fig. 4c, d). However, both the enhancement of NRT1.1 expression and root NO3− uptake rate was completely abolished in the brm stop1 double mutant (Fig. 4c, d), indicating that the increased NRT1.1 expression and NO3− uptake in brm mutant requires functional STOP1. These results suggest that BRM exerts genetically upstream of STOP1 in regulating NO3− uptake and H+ tolerance under low pH conditions.

Lack of BRM enhances rhizosphere alkalization in response to H+ stress
Given the central role of the STOP1-NRT1.1 module in mediating H+-coupled NO3− uptake during acid stress, we hypothesized that targeted manipulation of BRM could promote a more favorable rhizosphere pH for root growth under low pH and curb N fertilizer-induced soil acidification. To test this, we first examined whether co-planting brm-3 and stop1 mutants could influence the root growth of stop1 mutants in low-pH agar media. Remarkably, when brm-3 and stop1 mutants were co-planted, stop1 root growth recovered to ~80% of brm-3 levels, versus only 57% in monoculture (Fig. 5a, b; Supplementary Fig. 10). Parallel experiments with Col-0 and brm-3 at pH 4.5 (accounting for inherent STOP1-NRT1.1 alkalinization capacity) similarly demonstrated improved Col-0 root growth in co-culture (Supplementary Fig. 10). These results suggest that loss of BRM function further promotes rhizosphere alkalization, thereby enhancing root growth in response to acidic stress.

To further assess the role of BRM in regulating rhizosphere pH, seedlings were grown in acidic agar medium with or without 0.75 mM Homo-PIPES, a pH buffer used to minimize pH fluctuations. In the buffered acidic media, brm-3 mutants lost their growth advantage, exhibiting root elongation comparable to that of Col-0 and stop1 (Fig. 5c, d). Conversely, in unbuffered acidic media, brm-3 mutants showed dramatically improved root growth and enhanced rhizosphere alkalization, as quantified by bromocresol purple staining and direct pH measurement after 6 h (Fig. 5e, f). In accordance with these results, the brm-1 mutant displayed significantly enhanced and rapid rhizosphere alkalization compared to Col-0 (Supplementary Fig. 11). Taken together, these results indicate that loss of BRM enhances rhizosphere pH, thereby increasing H+ tolerance and offering a promising strategy to mitigate soil acidification under acidic conditions.

H+ stress induces BRM degradation
We next investigated the regulatory dynamics of BRM in roots under H+ stress. While low-pH treatment did not significantly alter the transcript levels of BRM (Fig. 6a), it substantially reduced the nuclear accumulation of BRM-GFP in root epidermal cells of pBRM:BRM-GFP/brm-20 transgenic plants (Fig. 6b). This decrease occurred independently of NO3− availability (Supplementary Fig. 12), and was detected as early as 2 h after low-pH exposure, preceding the induction of GFP-STOP1, which became evident at 4 h (Fig. 6b, c). This temporal hierarchy establishes BRM destabilization as an early event in the acid stress response, positioning BRM upstream of STOP1 signaling. Furthermore, the low pH-repressed BRM accumulation was restored by the treatment with the 26S proteasome inhibitor MG132 (Fig. 6d–f), suggesting that H+ stress likely promotes BRM degradation via the proteasome-dependent proteolysis. To investigate whether this degradation requires SnRK2 kinases, which are known to phosphorylate and inactivate BRM upon ABA treatment 24, we assessed the growth of snrk2.2/2.3 and snrk2.6 mutants. However, these mutants exhibited root growth comparable to Col-0 under both neutral and low-pH conditions (Supplementary Fig. 13), ruling out SnRK2s participation in BRM-mediated H+ tolerance. Overall, these findings support a model in which low pH triggers proteasomal degradation of BRM, thereby enabling activation of the STOP1-NRT1.1 module to enhance H+ tolerance.

Methods
Plant material
The Arabidopsis thaliana plants used in this work were all from Columbian (Col-0) background. The various mutants were stop1 KO (SLAK_114108), nrt1.1-1 (SALK_097431), tfl2-1 (CS379655), axe1-5 36, brm-1 (chr2-1) 56. The brm-3 (SALK_088462), brm-20 (SALK_002500), pBRM:BRM-GFP/brm-20, and pBRM:BRM-FLAG/brm-3 transgenic line used in this study has been previously described 26. The brm stop1 and brm nrt1.1 double mutants were generated by crossing the brm-20 and stop1 mutants or nrt1.1-1 mutants, respectively, and homozygous lines were identified by PCR. The pSTOP1:GFP-STOP1/pBRM:BRM-FLAG (GFP-STOP1/BRM-FLAG) line was generated by crossing the pSTOP1:GFP-STOP1/stop1 (#B10) transgenic line 8 with pBRM:BRM-FLAG/brm-3, and the homozygous lines were selected in agar media containing basta and hygromycin. The primers used are shown in Supplementary Data 2.

Plant growth condition
Two days of stratification were conducted at 4 °C before sowing the seeds. Surface-sterilized Arabidopsis seeds were sown on basal agar medium containing KNO3 (6 mM), (NH4)2SO4 (1 mM), NaH2PO4 (1 mM), MgSO4 (500 μM), CaCl2 (1 mM), H3BO3 (10 μM), MnSO4 (0.5 μM), ZnSO4 (0.5 μM), CuSO4 (0.1 μM), (NH4)6Mo7O24 (0.1 μM), Fe-EDTA (50 μM), 1% agar (Sigma-Aldrich, A1296), 1% sucrose at pH 6.5 in a plant growth chamber (BPC600H, Fujian Jiupo Biotechnology, Co., Ltd.) at 22–23 °C under a 16-h light/8-h dark photoperiod cycle. Then, the 3-day-old plants were used for phenotype analysis in the above basal agar medium with an initial pH of either 4.7 or 6.5, and 0.75 mM Homo-PIPES were used for the buffering assay, as indicated in the figure legends. To analyze the effect of NO3− level on root growth in response to low pH, the media were supplemented with 0.06 or 6 mM KNO3, while the concentration of (NH4)2SO4 was reduced to 0.5 mM to avoid severe ammonium toxicity and the resulting differences in K concentrations were balanced by adjusting the K2SO4 concentration. To analyze the root growth of seedlings in response to low Pi, the Pi concentration in the media were reduced to 0.01 mM (low Pi). To analyze the root growth of seedlings in response to Al toxicity, seeds were directly sown on the control or Al treatment agar plates, prepared as previously described 57.

Yeast two-hybrid screen and assays
For screening, the full CDS of STOP1 was amplified and inserted into pGBKT7 using the EcorI and BamHI restriction sites resulting in a pGBKT7-STOP1 constructs. This gene construct was transformed into yeast AH109 cells. Transformants were selected on SD/-Trp medium (Takara), after which the autoactivation activity of the bait vector pGBKT7-STOP1 was tested by plating on SD/-Trp-His-Ade medium (Takara) supplemented with different concentrations of 3-amino-1,2,4-triazole (3-AT). Then, the pGBKT7-STOP1 bait vector was cotransformed with an Arabidopsis cDNA library into AH109 yeast competent cells according to the manufacturer’s instructions (Clontech). Transformed cells were plated on SD/-Trp-Leu-His-Ade medium supplemented with 50 mM 3-AT, following incubation on 30 °C. Plasmids were isolated from positive yeast clones for analysis of insert and sequencing.

For targeted Y2H assays, the coding region of BRM and truncated version of BRM was amplified and inserted into pGBKT7 using the EcorI and BamHI restriction sites. The full CDS of STOP1 was amplified and inserted into pGADT7 using the EcorI and BamHI restriction sites. Yeast AH109 cells were co-transformed with specific bait and prey constructs. Yeast growth was shown on SD/-Trp/-Leu and SD/-Trp/-Leu/-His/-Ade medium, following incubation on 30 °C. The primers used for generating the various clones are listed in Supplementary Data 2.

Bimolecular fluorescence complementation (BiFC) assays
The full CDS of STOP1 and coding region of truncated BRMn was individually inserted into p2YN and p2YC vectors 46 to fuse with the N-terminal or C-terminal fragment of YFP. The primers used to generate the various clones are listed in Supplementary Data 2. The different combinations of recombinant plastmids were co-infiltrated into young Nicotiana benthamiana leaves for 48 h. The YFP fluorescence was detected using a confocal laser-scanning microscope (LSM880; Zeiss) and indicated in green color.

Co-IP assay
To better reflect the authentic in vivo interaction status between BRM and STOP1, we specifically used the double-tagged genetic material (BRM-FLAG/GFP-STOP1) for Co-IP assays. However, under neutral-pH conditions, the abundance of GFP-STOP1 protein in this transgenic line is extremely low, making it technically challenging to capture the GFP-STOP1 protein through immunoprecipitation. To address this issue, 10-d-old pSTOP1:GFP-STOP1 and pSTOP1:GFP-STOP1/pBRM:BRM-FLAG with 6-h low-pH treatment to induce GFP-STOP1 protein accumulation and with MG132 to inhibit the degradation of BRM-FLAG protein. Then, seedlings were harvested for total protein extraction in RIPA lysis buffer (FD011; Fdbio Science Biotech Co., Ltd). After centrifugating at 13,000 × g for 10 min, 50 µL of supernatants were kept as input controls, whilst the rest was incubated with 30 µL of anti-FLAG magnetic beads for 2 h. After the beads were washed five times with TBS buffer, the proteins were eluted by adding 50 µL 2× SDS sampling buffer at 95 °C for 8 min. Then the eluted proteins and input controls were separated on 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The protein of interest was detected with α-GFP (1:10000; ABclonal) and α-FLAG (1:10000; ABclonal) antibodies.

Gene expression assay
Total RNA was extracted from plant roots using the FreeZol Reagent (R711-01; Vazyme Biotech Co., Ltd, Nanjing, PRC), and the first-strand cDNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan). The expression of the corresponding genes was determined using the SYBR Green Realtime PCR Master Mix (TOYOBO). The gene-specific primers used for quantitative PCR were listed in Supplementary Data 2. Expression levels of each mRNA were determined and normalized with the expression level of UBIQUITIN10.

Analysis of green fluorescent protein (GFP)
After different time of low-pH treatment, as indicated in figure legends, BRM-GFP and GFP-STOP1 expression in the roots of pBRM:BRM-GFP/brm-20 and pSTOP1:GFP-STOP1 transgenic plants was detected using a confocal laser-scanning microscope (LSM880; Zeiss). The excitation and emission wavelengths were 488 nm/500–530 nm for GFP protein. Images were analyzed using the ZEN 2012 Blue Edition and ImageJ 2X software.

Analysis of pH and visualization of rhizosphere alkalization
The 10-day-old Arabidopsis seedlings were pre-treated at pH 4.7 agar medium for 2 days before bromocresol purple staining and the pH value measurement. To visualize rhizosphere acidification, roots were transferred to a basal agar media with an initial pH of 4.9 and pH indicator bromocresol purple (0.008%, Sigma-Aldrich). The concentration of NaH2PO4 was reduced to 0.1 mM in the used agar media. Images were acquired 12 h after staining. The pH of the agar rooting medium was measured as previously described 58. Briefly, the rooting medium was collected and frozen at –20 °C for 4 h before thawed to free the aqueous phase from the agar. Then, the mixture was filtered at room temperature and the pH of the supernant was measured immediately using a pH electrode.

Chromatin immunoprecipitation (ChIP)-qPCR analysis
To assess the effect of BRM disruption on the binding ability of STOP1 to NRT1.1, protoplasts were isolated from the rosette leaves of 3-week-old WT or brm-3 plants, as described previously 59. The effector plasmids pHBT:STOP1-GFP and pHBT:GFP used in this study have been previously described 57. An effector or empty control plasmid (6 μg), and an internal control plasmid (pUBQ10:GUS; 2 μg) were co-transfected into 2 × 105 protoplasts using the polyethylene glycol (PEG)-mediated transformation method. At least 4 independent biological replicates were set for each transformation, and then the protoplasts were harvested together for the isolation of chromatin. The sheared DNA was immunoprecipitated with anti-FLAG magnetic beads overnight at 4 °C. In addition, ChIP assays were also carried out using 10-day-old Col-0 and pBRM:BRM-FLAG/brm-3 plants (approximately 2 g) and Pierce agarose ChIP Kit (26156; Thermo Fisher Scientific), according to the manufacturer’s instructions. The sheared DNA was immunoprecipitated with anti-FLAG magnetic beads overnight at 4 °C. The enrichment of specific DNA fragments in the immune precipitant was determined by qPCR analysis of the recovered DNA using specific primers (Supplementary Data 2). The fold enrichment represents the immune-precipitation efficiency in the transgenic plants normalized to that in the Col-0 plants.

Formaldehyde-assisted isolation of regulatory elements (FAIRE)-qPCR analysis
To assess the effect of BRM disruption on the open chromatin in the genome of NRT1.1, FAIRE was performed using the whole seedlings of 10-day-old WT, brm-3, and brm-20 plants, according to the protocol described previously with modifications 60,61. Briefly, chromatins are cross-linked by 3% (final vol) formaldehyde treatment, followed by shearing chromatin by sonication, and reversing crosslinking. Then, a phenol/chloroform/isoamyl alcohol (PCI) extraction is performed, separating protein-free genomic DNA fragments into the aqueous phase and histone-DNA complexes to the interphase. The enrichment of specific DNA fragments in the recovered DNA was determined by qPCR analysis using specific primers (Supplementary Data 2). The relative enrichment of each DNA sample was first calculated using the delta cycle threshold (2−ΔΔCt) method by calculating the ratio of FAIRE signal to input signal. Relative amplicon abundance represents the results in the mutants normalized to that in the Col-0 plants.

Transient expression assay
To performed transient expression assays in Nicotiana benthamiana leaves, three DNA segments, selected from NRT1.1 genomic region (P1 and P2) that containing the identified binding site of STOP1 5,14 and (P3) that containing the identified binding site of NLP7 32 but not STOP or BRM, were individually inserted into the reporter vector pGreen0800-LUC using XhoI and BamHI restriction sites and using ClonExpress Ultra One Step Cloning Kit V3 (C117-01; Vazyme Biotech Co., Ltd, Nanjing, PRC). The primers used to generate the various clones are listed in Supplementary Data 2. In dual-luciferase reporter assays, reporter and effector constructs (35S:STOP1 13 or 35S:NLP7) were co-transformed into young N. benthamiana leaves for 48 h. Firefly and Renilla luciferase activities were quantified using a dual-luciferase assay kit (Beyotime Biotechnology, China). Some infiltrated leaves were also sprayed with 1 mM luciferin (A600577; Sangon Biotech, Shanghai, China), after which the images of luminescence were captured using a cooled charge-coupled device (CCD) imaging apparatus (LB985; Berthold).

Measurement of NO3− uptake
For the assays of NO3− uptakes, 7-day-old seedlings were pre-treated in agar medium with a pH of 6.5 or 4.7, for 1 d. The plants were washed with 0.05 mM CaSO4 for 1 min and then transferred to a medium in which KNO3 was replaced with K15NO3 (atom% 15N, 99%) for 10 min under the same conditions used for pre-culturing seedlings. The plants were washed with 0.05 mM CaSO4 for 1 min, after which each plant was separated into shoots and roots, and the organs were dried at 65 °C for 3 d. The dried organs were weighed, and the roots and shoots of each plant were combined and ground into a fine powder. The samples were analyzed using an isotope ratio mass spectrometer (Isorime100; Elementar Analysensysteme, Hanau, Germany). The rate of 15NO3− uptake was calculated from the total 15N of the plant and the biomass of the roots.

Statistical analyses
Data were analyzed by one-way, two-way, or multi-way ANOVA with Tukey’s multiple comparison test.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Ye et al. entitled ‘Chromatin remodeler BRAHMA represses STOP1-NRT1.1 module to control plant rhizosphere alkalization and acid stress adaptation’ describes intriguing interplay between BRM- the central ATPase subunit of Arabidopsis BAS subclass of SWI/SNF chromatin remodelling complexes (CRCs) and the STOP1 a C2H2 zinc finger transcription factor in the co-regulation of NRT1.1 gene expression. This topic is of high interest given the potential involvement of BRM subunit of SWI/SNF chromatin remodelling complex in the control of plant rhizosphere alkalization and acid stress adaptation. The manuscript, however interesting, suffers from severe flaws and over interpretation causing that the conclusions drawn by authors are not fully supported by the data presented. The manuscript in its current form is premature.\nMajor commentsː\n1. The BRM ATPase is a central SWI2/SNF2-type ATPase, accordingly to existing knowledge, acting in the BAS subclass of SWI/SNF chromatin remodelling complexes, therefore the statement ‘Here we identify the SWI/SNF chromatin remodeler BRAHMA (BRM)…’ (lines 32-33) is strongly misleading. Moreover, the authors claim that they used in their study ‘BRM-null mutants’ (line 112) what is against the existing data coming from papers describing identification and characteristics of brm-3 and brm-20 mutant lines (see Hurtado et a., 2007, Zhang et al., 2017). These mutant lines are weak brm alleles with retained production of truncated BRM protein. Thus, the authors’ claims are far from the existing state-of-the-art in this field.\n2. The presented root phenotypes in control conditions for brm-3 are not fully matching the data presented in Archacki et a., 2013 and Yang et al., 2015 where the brm-3 roots were shortened and branching comparing to WT control. How authors explain such phenomenon? The brm-3 phenotype was less severe than exhibited by the true null brm-1 allele (Archacki et a., 2013 and Yang et al., 2015, Archacki et a., 2009). Given this fact, I strongly recommend the authors to include in their analyses presented in this manuscript the true null brm mutant(s) to prove their findings and avoid the interference of partially functional truncated BRM protein which may lead to wrong conclusions. It is especially important as the authors study root response and the root phenotype differs between weak and null brm mutants.\n3. The root length measurements presented on Fig. 2b and fig. 4b indicate discrepancies for plants cultivated in both control and low-pH conditions (even for Col-0 plants on Fig. 2b root length in pH 6.5 is 3.5 cm and in pH 4.7 is around 1.5 cm while in corresponding conditions the lengths are 2 cm for pH 6.5 and 1 cm for pH 4.7). The authors claim in figure legends that the seedlings age and treatments were the same and there were four replications of each experiment. The number of plants used in these experiments was very low, threfore I recommend use higher plants number. Furthermore, in the legend of Fig. 1e authors claim that the seedlings were treated with low pH and MG132 for one day, while in the description of the results of this experiment the 6-hours long low pH treatment is indicated (line 105). The Materials and methods section indicates again one-day long low pH treatment (line 336-337). Such discrepancies likely indicate existence of severe methodological and technical problems in used approaches.\n4. Authors indicate the interdependences between BRM and STOP1 in the transcripitonal control of NRT1.1 which is very interesting from mechanistic point of view, however the measurements of NRT1.1 expression level in brm/stop1 double mutant is missing. Such experiment would likely be very valuable to prove the proposed model.\n5. The proposed model is incomplete and the possible role of HDA6 in the NRT1.1 regulation should be elaborated further. Currently, some results related to HDA6 role in H+ tolerance and NRT1.1 expression control are presented on Supplementary figure 9 and described in the ‘Discussion’ section only, which is a very inappropriate way of data presentation.\nMinor comments\nThe axe 1-5 mutant line is not described in “Materials and methods’.", "reviewer_2": "The manuscript investigates how plants adapt to acidic soils, which cover nearly half of the world’s arable land and severely limit root growth and nutrient acquisition. The study focuses on the Arabidopsis transcription factor STOP1, which activates the nitrate transporter NRT1.1 to drive H⁺/NO₃⁻ symport, rhizosphere alkalinization, and improved nitrogen use efficiency. Although the role of the STOP1–NRT1.1 module in acid stress tolerance is established, its regulatory mechanism has remained poorly understood.\nThe authors propose that the chromatin remodeler BRM acts as a negative regulator of the STOP1–NRT1.1 pathway. They provide evidence that BRM interacts with STOP1 and represses NRT1.1 expression, thereby limiting nitrate uptake and rhizosphere alkalinization under acidic stress.\nHowever, I do not find clear novelty in this MS, and there is also insufficient supporting data to substantiate the authors’ claims. Therefore, I cannot recommend its publication in Nature Communications. Below are the major justifications for this decision.\n1.The interaction between STOP1 and BRM is already listed in the public protein–protein interaction database BioGRID .\n2.The authors claim that the STOP1–BRM interaction is involved in the transcriptional repression of NRT1.1, but there is no clear evidence demonstrating this.\n3.There is no doubt that the presence of BRM suppresses the STOP1-regulated NRT1.1 transcription under low-pH conditions. However, the biological significance of BRM as a repressor of the STOP1-NRT1.1 module remains unclear.\nThe following are my concerns regarding the manuscript\n1.L.95~: Please clarify how BRM was identified as a target of STOP1. In the manuscript (L.96), the authors state that “we performed a yeast two-hybrid (Y2H) screen using STOP1 as bait.” Does “Y2H screen” mean a Y2H screening experiment? If so, the authors should provide details of the experimental conditions. The authors should also include a list of the candidate proteins identified as potential STOP1-binding partners and explain the basis for selecting BRM among them.\n2.L.104~: The authors state that “GFP-STOP1 was specifically co-immunoprecipitated with BRM-FLAG after a 6-hour low-pH treatment,” whereas the caption of Fig. 1e describes this as a 1-day treatment. In addition, according to the figure legend, the samples used for the Co-IP analysis of STOP1 and BRM were treated with low pH with MG132. In this case, describing the result as an interaction occurring after low-pH treatment is inappropriate.\n3.L.120～: “These results indicate that BRM-regulated H⁺ tolerance is not primarily associated with its role in DNA damage repair.”\nIf this conclusion is intended, it would be more appropriate to directly compare DNA damage between Col-0 and brm, rather than only examining the expression of DNA damage–inducible genes.\n4. Fig.3b：The authors should clearly indicate which region of the leaf was used for transient BRM expression.\n5.Fig3c: Even in the co-expression of EV and BRM, LUC activity is significantly reduced. Therefore, the possibility that BRM expression suppresses LUC expression non-specifically, independent of STOP1, cannot be excluded.\n6.In the main text, Figures 3e and 3f are cited in reverse order. In addition, please clearly specify the ChIP-seq data in fig.3f, either in the figure legend or in the main text (including accession number, plant growth condition etc ).\n7.L.167~& L173~:\nThe authors state that BRM controls NRT1.1 transcription “under low-pH conditions” by interfering with STOP1 activity or accessibility to cis-elements. However, as far as I can see, none of the experiments in Fig. 3 were conducted under stress conditions. Which data demonstrate that BRM interferes with STOP1 under low-pH conditions?\n8.L177~: The authors state that “BRM acts upstream of STOP1 in regulation of NO₃⁻ uptake and H⁺ tolerance.” However, based on the author’s data presented, BRM and STOP1 appear to operate independently in a parallel relationship rather than in a hierarchical one. The authors should provide a more detailed explanation of the basis for claiming that BRM functions upstream of STOP1.\n9.Figure 6c does not represent quantitative data of nuclear fluorescence intensity of BRM-GFP. Please revise the figure.", "reviewer_3": "Ye et al. report a novel function of BRM (BRAHMA chromatin remodeler) in Arabidopsis, specifically in negatively regulating the STOP1-NRT1.1 pathway, which is one of the key mechanisms of low pH tolerance in this plant. Authors’ claims are well supported by experimental results. This is a significant finding that helps us understand and improve low pH tolerance in plants.\nMajor points\n- I’m wondering how to identify BRM? “we performed a yeast two-hybrid (Y2H) screen using STOP1 as a bait and uncovered BRAHMA (BRM),” (lines 95-96). However, there is no description of the screening; only the interaction between BRM and STOP1 is confirmed by Y2H (Fig. 1b,c).\n- Related to the above point, is this direct interaction between BRM and STOP1 really required, rather than interaction on the NRT1.1 genomic sequence? If the direct interaction is true, other downstream target genes of STOP1 must also be affected, not just NRT1.1, but this has not been verified here.\nMinor points\n- NUE (line 74) and SWI/SNF (line 84) are not defined before.\n- “co-immunoprecipitated with BRM-FLAG after a 6-hour low-pH treatment (Fig. 1e)” (line 105). Why was it only checked after low pH treatment?\n- “publicly available datasets using WashU Epigenome Browser and Plant Regulomics” (line 164). References or URLs are required.\n- Fig. 3b: The description may be incomplete. With or without BRM?"}, "author_response": "The following outlines our point-by-point responses (in blue) to reviewers’ comments:\n---------------------------------------------------------------------------------------------\nReviewer #1 (Remarks to the Author)\nThe manuscript by Ye et al. entitled ‘Chromatin remodeler BRAHMA represses\nSTOP1-NRT1.1 module to control plant rhizosphere alkalization and acid stress\nadaptation’ describes intriguing interplay between BRM- the central ATPase subunit of\nArabidopsis BAS subclass of SWI/SNF chromatin remodelling complexes (CRCs) and\nthe STOP1 a C2H2 zinc finger transcription factor in the co-regulation of NRT1.1 gene\nexpression. This topic is of high interest given the potential involvement of BRM\nsubunit of SWI/SNF chromatin remodelling complex in the control of plant rhizosphere\nalkalization and acid stress adaptation. The manuscript, however interesting, suffers\nfrom severe flaws and over interpretation causing that the conclusions drawn by authors\nare not fully supported by the data presented. The manuscript in its current form is\npremature.\nResponse: Thank you for your general assessment of the interest of our topic. We also\nappreciate you pointing out the concerns regarding the support of our conclusions. We\nhave thoroughly considered your comments and have performed a series of additional\nexperiments and in-depth data analyses to address the issues raised. These have been\nincorporated into a significantly revised manuscript. We are convinced that these\nrevisions significantly strengthen the study and provide robust support for our\nconclusions.\nMajor commentsː\n1. The BRM ATPase is a central SWI2/SNF2-type ATPase, accordingly to existing\nknowledge, acting in the BAS subclass of SWI/SNF chromatin remodelling complexes,\ntherefore the statement ‘Here we identify the SWI/SNF chromatin remodeler\nBRAHMA (BRM)…’ (lines 32-33) is strongly misleading. Moreover, the authors claim\nthat they used in their study ‘BRM-null mutants’ (line 112) what is against the existing\ndata coming from papers describing identification and characteristics of brm-3 and brm-\n20 mutant lines (see Hurtado et a., 2007, Zhang et al., 2017). These mutant lines are\nweak brm alleles with retained production of truncated BRM protein. Thus, the authors’\nclaims are far from the existing state-of-the-art in this field.\nResponse: We sincerely thank you for this critical correction and for pointing us to the\nessential references (Hurtado et al., 2007; Zhang et al., 2017). The reviewer is\nabsolutely right. We deeply apologize for our inaccurate description of the brm-3and\nbrm-20 alleles as 'null mutants'. We have now carefully studied the cited literature and\nconfirm that these are indeed weak alleles that produce truncated proteins, not true nulls.\nThis was a significant oversight on our part, and we are grateful for the opportunity to\ncorrect it.\nIn the revised manuscript, we have meticulously removed the term \"null mutant\"\nthroughout the text and have revised the relevant statement to ‘hypomorphic alleles’\nthroughout the manuscript. Furthermore, we have corrected the imprecise description\nof BRM to ‘switch defective/sucrose non-fermentable (SWI/SNF) ATPase’,\naligning\nwith the current consensus (Li et al., 2022).\n2. The presented root phenotypes in control conditions for brm-3 are not fully matching\nthe data presented in Archacki et a., 2013 and Yang et al., 2015 where the brm-3 roots\nwere shortened and branching comparing to WT control. How authors explain such\nphenomenon? The brm-3 phenotype was less severe than exhibited by the true null brm-\n1 allele (Archacki et a., 2013 and Yang et al., 2015, Archacki et a., 2009). Given this\nfact, I strongly recommend the authors to include in their analyses presented in this\nmanuscript the true null brm mutant(s) to prove their findings and avoid the interference\nof partially functional truncated BRM protein which may lead to wrong conclusions. It\nis especially important as the authors study root response and the root phenotype differs\nbetween weak and null brm mutants.\nResponse: Thank you for your insightful suggestions. In our previously presented\nphenotypes, the root elongation of the brm-3 mutant did show a slight reduction,\nthough\nnot statistically significant. This might be attributed to the use of very early-stage\nseedlings, as we observed that the growth difference between brm-3 and WT increases\nwith seedling age. To better illustrate this process, we further presented the phenotypes\n9 days after transplanting. It can be seen that under neutral-pH conditions, the root\nelongation of brm-3 mutant was significantly shorter than that of Col-0, while that of\nbrm-3 remains longer than that of WT under acidic-pH conditions (Supplementary Fig.\n2),\nconfirming that BRM indeed negatively regulates plant acid resistance. Furthermore,\nwe also noticed under the growth conditions of Li et al., (2022) and Lei et al., (2024),\nthe brm-3 mutant also exhibits a good but slightly shortened root growth when\ncompared with Col-0 plants, indicating that growth conditions significantly affect the\nphenotype of brm-3 mutants. These results, together with the further genotyping\nconfirmation of the brm-3 mutants we used are correct mutants, led us to speculate that\nthe phenotypic discrepancy between our brm-3 mutant and the materials in Archacki et\na., 2013 and Yang et al., 2015 is mainly due to differences in growth conditions and the\nage of seedlings we observed.\nFollowing your suggestion, we analyzed the phenotype of the BRM-null mutant\n(brm-1). As shown in Reviewer-only Fig. 1, low-pH treatment significantly inhibited\nroot elongation in Col-0, but had no significant effect on that of the brm-1 mutant,\nfurther supporting that BRM represses root tolerance to acidic stress. However,\ncompared to Col-0, the brm-1 mutant shows severe growth defects and is homozygous\nlethal, making it unsuitable for subsequent studies.\nSupplemental Fig 2. Phenotype of the brm-3 and brm-20 mutant after long-term\nlow-pH treatment. a, Genotyping the brm-3 mutant by PCR. The primers used for\ngenomic PCR are shown below the gel pictures. LBb1.3, left board T-DNA primer for\nSALK lines; LP, left genomic DNA primer; RP, right genomic DNA primer. b,c,\nRoot\ngrowth of Col-0, brm-3, brm-20, and pBRM:BRM-FLAG/brm-3 (COM) plants on\nneutral- and low-pH agar media. Three-day-old seedlings were transferred to the\nindicated media and grown for 9 days. Data are from 20 seedlings per line and condition.\nWhiskers indicate minimum and maximum values. Scale bar: 1 cm. Asterisks indicate\nstatistically significant differences (p < 0.05), as determined by one-way ANOVA\nfollowed by Tukey’s multiple comparisons test (ns, not significant).\n[FIGURE REDACTED]\nReviewer-only Fig. 1 The growth of Col-0 and chr2-1(brm-1) seedlings on neutral or\nlow-pH agar. Three-day-old seedlings were transferred to either neutral or low-pH\nmedium and grown for an additional 3 days. Box plots show mean ± SD (n = 20\nseedlings), with whiskers representing minimum and maximum values. Scale bar: 1 cm.\nAsterisks indicate statistically significant differences (p < 0.0001), as determined by\none-way ANOVA followed by Tukey’s multiple comparisons test (ns, not significant).\n3. The root length measurements presented on Fig. 2b and fig. 4b indicate discrepancies\nfor plants cultivated in both control and low-pH conditions (even for Col-0 plants on\nFig. 2b root length in pH 6.5 is 3.5 cm and in pH 4.7 is around 1.5 cm while in\ncorresponding conditions the lengths are 2 cm for pH 6.5 and 1 cm for pH 4.7). The\nauthors claim in figure legends that the seedlings age and treatments were the same and\nthere were four replications of each experiment. The number of plants used in these\nexperiments was very low, therefore I recommend use higher plants number.\nFurthermore, in the legend of Fig. 1e authors claim that the seedlings were treated with\nlow pH and MG132 for one day, while in the description of the results of this\nexperiment the 6-hours long low pH treatment is indicated (line 105). The Materials\nand methods section indicate again one-day long low pH treatment (line 336-337). Such\ndiscrepancies likely indicate existence of severe methodological and technical\nproblems in used approaches.\nResponse: Thank you for your valuable suggestions. Regarding the discrepancies in the\nroot elongation data you pointed out, we carefully reviewed the experimental\nprocedures and found that these differences should result from batch-to-batch variations\nin culture conditions and plant growth. Although we consistently described in the\nlegends that 3-day-old seedlings were used for transplanting, there were slight\ndifferences in initial root lengths among batches due to variations in seedling vigor.\nAdditionally, the seedlings in Fig. 2a were cultivated in a different growth chamber\nfrom that in Fig. 4a. Despite identical parameter settings, potential chamber effects\ncould contribute to phenotypic discrepancies. Therefore, to rule out this confounding\nfactor, we replicated the Fig. 2a experiment under the strictly controlled conditions of\nthe Fig. 4a chamber setup, which produced comparable growth phenotypes (Fig 2a).\nImportantly, despite such batch-to-batch fluctuations in absolute root length, the\nrelative root elongation of WT under low-pH conditions stably remained at 50–60% of\nthat under neutral-pH conditions, with significant differences observed within each\nexperimental group. These results confirm the stability and reliability of our treatment\nsystem and conclusions.\nRegarding the number of seedlings in the phenotypical analysis, although we used at\nleast 20 seedlings of a genotype in each experiment, we indeed only measured root\nelongation in a portion of the seedlings (less than 10) we observed. Following your\ncomments, we have included the rest seedlings used for phenotypic analysis, making\nthe number of seedlings per group increase to 20 in the revised manuscript.\nRegarding the conflicting annotations of treatment duration, we have realized that\nwe had made input errors in the figure legends and methods. We have confirmed that\nthe seedlings were treated with low pH and MG132 for 6 hours to induce the\naccumulation of STOP1-GFP and inhibit the degradation of BRM-FLAG. We therefore\nhave corrected all relevant descriptions throughout the manuscript.\n4. Authors indicate the interdependences between BRM and STOP1 in the\ntranscripitonal control of NRT1.1 which is very interesting from mechanistic point of\nview, however the measurements of NRT1.1 expression level in brm/stop1 double\nmutant is missing. Such experiment would likely be very valuable to prove the proposed\nmodel.\nResponse: Thank you for the good advice. Following this comment, we have detected\nthe expression of NRT1.1 in the suggested genotypes. When grown on low-pH agar\nmedium, the expression level of NRT1.1 in roots of the brm stop1 double mutant was\ncomparable to the stop1 mutants but notably lower than that of Col-0 and brm mutant\nunder low-pH conditions. This results further support our conclusion that the increased\nNRT1.1 expression in response to low pH in brm mutant requires functional STOP1.\nThe new data is present in new Fig. 4d.\nFig 4. STOP1 functions genetically downstream of BRM in response to low pH.\na,b, Root growth of Col-0, stop1, brm, and stop1 brm double mutants. Three-day-old\nseedlings were transferred to either neutral or low-pH medium and grown for an\nadditional 3 days. Box plots show mean ± SD (n = 20 seedlings), with whiskers\nrepresenting minimum and maximum values. Scale bar: 1 cm. c,d, Nitrate uptake rates\nand relative expression of NRT1.1 in Col-0, stop1, brm, and stop1 brm mutants under\nneutral and low-pH conditions. Data are shown as mean ± SD (n = 4 biological\nreplicates per genotype and condition). Different letters indicate statistically significant\ndifferences between means (p < 0.05), determined by Tukey’s multiple comparisons\ntest (one-way ANOVA in (b,d); two-way ANOVA in (c)).\n5. The proposed model is incomplete and the possible role of HDA6 in the NRT1.1\nregulation should be elaborated further. Currently, some results related to HDA6 role\nin H+ tolerance and NRT1.1 expression control are presented on Supplementary figure\n9 and described in the ‘Discussion’ section only, which is a very inappropriate way of\ndata presentation.\nResponse: We appreciate your insightful comments. As suggested, we have included\nthe HDA6 related data in the main figure of Results section instead of in the\nSupplementary figure. We have also integrated HDA6 into the updated regulatory\nmodel (new Fig. 6g). The revised model explicitly illustrates how BRM and HDA6\nmight cooperatively restrict the binding of STOP1 to the NRT1.1 promoter, thereby\nclarifying their synergistic role in repressing the STOP1-NRT1.1 module.\nMinor comments\nThe axe 1-5 mutant line is not described in “Materials and methods’.\nResponse: We have added details for axe1-5 mutants.\nReviewer #2 (Remarks to the Author):\nThe manuscript investigates how plants adapt to acidic soils, which cover nearly half\nof the world’s arable land and severely limit root growth and nutrient acquisition. The\nstudy focuses on the Arabidopsis transcription factor STOP1, which activates the\nnitrate transporter NRT1.1 to drive H⁺/NO₃⁻ symport, rhizosphere alkalinization, and\nimproved nitrogen use efficiency. Although the role of the STOP1–NRT1.1 module in\nacid stress tolerance is established, its regulatory mechanism has remained poorly\nunderstood. The authors propose that the chromatin remodeler BRM acts as a negative\nregulator of the STOP1–NRT1.1 pathway. They provide evidence that BRM interacts\nwith STOP1 and represses NRT1.1 expression, thereby limiting nitrate uptake and\nrhizosphere alkalinization under acidic stress. However, I do not find clear novelty in\nthis MS, and there is also insufficient supporting data to substantiate the authors’ claims.\nTherefore, I cannot recommend its publication in Nature Communications. Below are\nthe major justifications for this decision.\nResponse: Thank you for your thorough review and for raising important questions\nregarding the novelty and experimental support of our study. We appreciate the\nopportunity to clarify our work and have revised the manuscript accordingly to better\nhighlight its contributions. In response to your primary concern about novelty, we have\nnow more clearly articulated in the discussion how our work moves beyond the\nestablished STOP1-NRT1.1 module to identify BRM as a previously unknown\nepigenetic repressor that fine-tunes this critical pathway under acid stress. This\ndiscovery provides a novel mechanistic layer to plant acid tolerance. Furthermore, to\naddress the concern about data support, we have supplemented the manuscript with\nadditional experimental evidence and quantitative analyses (detailed in our point-by-\npoint responses below) that we believe now robustly substantiate our conclusions. We\nare confident that these revisions have significantly strengthened the manuscript and\nhope it now meets the high standards of Nature Communications.\nReviewer question: The interaction between STOP1 and BRM is already listed in the\npublic protein–protein interaction database BioGRID.\nResponse: Thank you for pointing out the listing of the STOP1-BRM interaction in the\nBioGRID database, which actually reinforces the reliability of our findings. We would\nlike to clarify that this database entry is solely based on preliminary high-throughput\nyeast two-hybrid (Y2H) screening results (BRM was used as a bait). As is common\nwith such high-throughput methods, a certain rate of false-positive interactions is\nexpected, and this particular entry still lacks subsequent validation or functional context.\nNotably, our study is the first to verify the authenticity of the STOP1-BRM interaction\nthrough both in vitro and in vivo assays, and also to elucidate its biological function in\nregulating plant tolerance to acidic stress: BRM physically interacts with STOP1 and\noccupies the genomic region of NRT1.1, repressing the STOP1-dependent activation of\nNRT1.1 expression and consequently limiting NO ⁻ uptake and rhizosphere alkalization\n3\nunder chronic acidity. In this way, our study bridges the critical gap between the\ndatabase-recorded predictive interaction and the uncharacterized functional mechanism,\nproviding novel insights into the molecular network governing plant adaptation to\nacidic environments. We have also incorporated this database entry in the revised\nmanuscript to clarify its relationship to our findings.\n2.The authors claim that the STOP1–BRM interaction is involved in the transcriptional\nrepression of NRT1.1, but there is no clear evidence demonstrating this.\nResponse: Thank you for this critical comment. Here we provided multiple lines of\nevidence demonstrating that BRM-STOP1 interaction regulates the transcriptional\nrepression of NRT1.1.\nFirstly, BRM physically interacts with STOP1 and occupies NRT1.1’s P1 and P2\nregions (Fig.1, 3). This dual regulation suppressed STOP1’s transcriptional activity and\nlimited its binding to NRT1.1 region, thus directly mediating NRT1.1 repression.\nNotably, BRM does not affect NLP7-mediated P3-LUC activation (Supplementary\nFig.7), suggesting that BRM specifically antagonizes STOP1-dependent activation\nof NRT1.1, rather than exerting a non-specific inhibitory effect on LUC expression.\nSecondly, we clarify the genetic analysis between BRM and STOP1 by using Col-0,\nbrm, stop1, and brm stop1 double mutants. In Fig 4, we have shown that the root growth\nof the brm stop1 double mutant was comparable to the stop1 single mutant but notably\nshorter than that of the WT and the brm single mutant, which rules out an additive or\nintermediate genetic interaction and demonstrates that STOP1 is epistatic to BRM in\nH⁺ tolerance. Consistently, the elevated NO₃⁻ uptake and NRT1.1 expression in brm\nmutants were completely abolished by STOP1 loss of function, as the brm stop1 double\nmutant displayed NO₃⁻ uptake and NRT1.1 expression level comparable to the stop1\nmutant (Fig. 4c, d). These results demonstrate that BRM exerts genetically upstream of\nSTOP1 in regulating H+ tolerance and NRT1.1 expression.\nFurthermore, we also showed that BRM-STOP1 interaction regulates multiple\nSTOP1 targets, further supporting a direct mechanistic role. As shown in Supplemental\nFig. 13, two canonical STOP1 targets, named ALMT1 and CIPK23 genes, were also\nsignificantly upregulated in brm mutants compared to Col-0, whose upregulation were\nabolished by STOP1 disruption. These results suggest that BRM could act as a global\nregulator of STOP1-mediated stress adaptation, further supporting the conclusion that\nthe direct BRM-STOP1 interaction is required for suppressing NRT1.1 expression,\nrather than solely via cis-acting regulation at NRT1.1. Collectively, these data provide\nclear evidence that BRM exerts dual regulation in plant adaptation to acidic stress—\nboth by physically interacting with STOP1 to suppress its transcriptional activation\ncapacity, and by occupying the NRT1.1 genomic region to directly repress its\ntranscription.\n3.There is no doubt that the presence of BRM suppresses the STOP1-regulated NRT1.1\ntranscription under low-pH conditions. However, the biological significance of BRM\nas a repressor of the STOP1-NRT1.1 module remains unclear.\nResponse: Thank you for your valuable comment. The biological significance of BRM\nas a repressor of the STOP1-NRT1.1 module is clarified as follows.\nBRM prevent detrimental effects caused by excessive STOP1 activation. Although\nSTOP1 is essential for stress responses, the sustained high activity of STOP1 are\ndetrimental to plant growth and development under normal conditions (Supplemental\nFig. 16). Through directly interacting with STOP1 to suppress its transcriptional\nactivity and occupying genomic regions of its target genes to limit chromatin\naccessibility, BRM effectively constrains both the intensity and breadth of STOP1\nactivity. This repression is critical to prevent the adverse impacts of aberrant STOP1\nactivation on plant growth and development.\nFurthermore, the BRM-STOP1-NRT1.1 module contributes to the balance between\ngrowth and stress responses, facilitating plant adaptation to complex environments.\nBRM establishes a low-activity baseline for the STOP1–NRT1.1 module, preventing\ninappropriate rhizosphere alkalinization and nitrate uptake under non-stress conditions.\nImportantly, low pH rapidly triggers BRM degradation (Fig.6), creating a dynamic\nactivation window in which STOP1–NRT1.1 can be robustly induced upon true acidic\nstress, therefore ensuring both stability and sensitivity of this pathway. Collectively,\nBRM functions as a critical upstream gatekeeper that enables precise and stress-\ndependent activation of the STOP1-NRT1.1 axis, integrating pH homeostasis with\nnitrate acquisition. More importantly, in natural environments, plants encounter\nmultiple fluctuating stresses, e.g., acid stress, aluminum stress, phosphate deficiency,\nammonium toxicity, and so on, while multiple stress-responsive pathways mediated by\nSTOP1 require flexible regulation. Given that BRM could act as a global regulator of\nSTOP1-mediated stress adaptation, such dynamic regulation of repression-activation\nmediated by BRM-STOP1 may also play an important role in plant adaptation to other\nstresses, which means BRM allows plants to maintain STOP1 activity at an optimal\nlevel under non-stress or mild stress conditions, ensuring normal growth; when stress\nintensifies, BRM’s inhibitory effect can be moderately alleviated, enabling precise\nactivation of STOP1-mediated stress adaptation pathways. We have integrated the\nabove logical elaborations into the revised manuscript.\nSupplemental Fig 16. Phenotype of the p35S:STOP1/Col-0 transgenic plants on\nnetural-pH media. Four-day-old seedlings were transferred to the netural-pH media\nand grown for 5 days. Data are from 20 seedlings per line and condition. Whiskers\nindicate minimum and maximum values. Scale bar: 1 cm. Asterisks indicate statistically\nsignificant differences (p < 0.05), as determined by one-way ANOVA followed by\nTukey’s multiple comparisons test.\nThe following are my concerns regarding the manuscript\n1.L.95~: Please clarify how BRM was identified as a target of STOP1. In the\nmanuscript (L.96), the authors state that “we performed a yeast two-hybrid (Y2H)\nscreen using STOP1 as bait.” Does “Y2H screen” mean a Y2H screening experiment?\nIf so, the authors should provide details of the experimental conditions. The authors\nshould also include a list of the candidate proteins identified as potential STOP1-\nbinding partners and explain the basis for selecting BRM among them.\nResponse: Thank you for your valuable comment. Here we clarify the key steps for\nidentifying BRM as a STOP1-interacting protein. To investigate potential regulator of\nSTOP1 in response to H+ stress, we generated the bait vector pGBKT7-STOP1 and first\ntested its autoactivation activity in AH109 yeast cells. Although weak autoactivation of\npGBKT7-STOP1 in AH109 yeast was observed, it was almost completely suppressed\nby 50 mM 3-amino-1,2,4-triazole (3-AT), confirming the suitability of this\nconcentration for subsequent screening (Supplementary Fig.1). Then, the pGBKT7-\nSTOP1 bait vector was cotransformed with an Arabidopsis cDNA library into AH109\nyeast competent cells according to the manufacturer’s instructions (Clontech).\nTransformed cells were plated on SD/-Trp-Leu-His-Ade medium supplemented with\n50 mM 3-AT. We obtained 75 positive clones after screening and identified a total of\n23 candidate interacting proteins, including MPK4, SIZ1 and other known/novel\ninteractors (Supplemental Dataset.S1). Among these, BRM was detected in 6\nindependent clones, representing the most frequent hit among unreported STOP1\ninteractors, which made it the highest-priority candidate. As per the suggestion from\nyou and another reviewer, we have supplemented detailed procedures in the revised\n‘Materials and Methods’ and ‘Results’ sections.\nSupplemental Fig 1. Detection of the autoactivation activity of pGBKT7-STOP1.\nThe pGBKT7-STOP1 construct was transformed into yeast AH109 cells and\ntransformants were selected on SD/-Trp medium. Then, the autoactivation activity of\npGBKT7-STOP1 was tested by plating transformants on SD/-Trp-His-Ade medium\nsupplemented with different concentrations of 3-amino-1,2,4-triazole (3-AT).\nRepresentative images were taken after 3 days incubation on 30 °C.\n2.L.104~: The authors state that “GFP-STOP1 was specifically co-immunoprecipitated\nwith BRM-FLAG after a 6-hour low-pH treatment,” whereas the caption of Fig. 1e\ndescribes this as a 1-day treatment. In addition, according to the figure legend, the\nsamples used for the Co-IP analysis of STOP1 and BRM were treated with low pH with\nMG132. In this case, describing the result as an interaction occurring after low-pH\ntreatment is inappropriate.\nResponse: Many thanks for pointing out this careless mistake in the text. We made\nconfusion in the description of the treatment time in the legend of Fig 1e as well as in\nthe Method section. The seedlings were treated with low pH and MG132 for 6 hours to\ninduce the accumulation of STOP1-GFP and inhibit the degradation of BRM-FALG.\nFurthermore, for easier understanding of this experimental design, we have clarified the\nnecessity of the 6-hour low-pH and MG132 treatment in the revised manuscript and\nrevised the description of the results to ‘after a 6-hour low-pH and MG132 treatment’.\n3.L.120~: “These results indicate that BRM-regulated H⁺ tolerance is not primarily\nassociated with its role in DNA damage repair.”\nIf this conclusion is intended, it would be more appropriate to directly compare DNA\ndamage between Col-0 and brm, rather than only examining the expression of DNA\ndamage–inducible genes.\nResponse: Thank you for your valuable suggestion. Following your recommendation,\nwe assessed DNA damage using the Comet assay. As shown in Supplemental Fig.4, 1-\nday low-pH treatment did not significantly alter WT root DSBs level, suggesting that\nplants tolerance to acidic stress is not closely related with DSBs. Furthermore, the DSBs\nlevel of the acid-tolerant brm-3 mutant showed similar level as that of the acid-sensitive\nstop1 mutant, though they were slightly suppressed when compared to Col-0. These\nresults further support our conclusion that BRM-regulated plant H⁺ tolerance is not\nprimarily associated with its role in DNA damage repair.\n25\n20\n15\n10\n5\n0\npH 6.5 pH 4.7\nSupplemental Fig 4. Levels of DSBs in root tips of Col-0, brm-3, and stop1 mutants\nunder control and low-pH conditions. Three-day-old seedlings were transferred to\nAND\nliaT\n%\nns ns Col-0\nbrm-3\n*\nstop1\neither neutral or low-pH medium and grown for an additional 1 days. Data represents\nmean ± SD (n = 50 nuclei). Asterisks indicate statistically significant differences (p <\n0.05), as determined by two-tailed Student’s t-tests (ns, not significant).\n4. Fig.3b: The authors should clearly indicate which region of the leaf was used for\ntransient BRM expression.\nResponse: Thank you so much for bringing that to our attention. We have labelled the\nregions in the figures.\n5.Fig3c: Even in the co-expression of EV and BRM, LUC activity is significantly\nreduced. Therefore, the possibility that BRM expression suppresses LUC expression\nnon-specifically, independent of STOP1, cannot be excluded.\nResponse: Thank you for this critical concern. To rule out the possibility of non-specific\nsuppression of LUC expression by BRM, we have supplemented a control experiment\nusing NLP7 (Zhao et al., 2018), another well-known transcription factor that\nactivates NRT1.1 transcription, and the NRT1.1 promoter fragment (P3), which\ncontains the binding regions of NLP7 but not STOP1 or BRM (Fig 3e). Transient assays\nin N. benthamiana leaves showed that BRM co-expression had no significant impact\non the NLP7-activated P3-drived LUC activity driven by the P3 fragment\n(Supplementary Fig.7). These results suggest that BRM specifically targets the STOP1-\ndependent activation of NRT1.1, rather than exerting a non-specific inhibitory effect on\nLUC expression. The possible reason why co-expression of EV and BRM significantly\nrepressed LUC activity driven by P1 and P2 fragments, is that BRM is directly\nassociated with the P1 and P2 regions of NRT1.1 promoter (Fig 3e, f) and it may reduce\nthe chromatin accessibility of these regions to inhibit their trans-activation by the\npotential STOP1 homologs in tobacco leaves. In line with this, co-expression of EV\nand BRM had no obvious effect on LUC activity driven by P3 fragment (Supplementary\nFig. 7). We have clarified this point in the revised manuscript.\nSupplemental Fig 7. The binding of NLP7 to the P3 regions of NRT1.1 is not\nregulated by BRM expression. a, Schematic representation of the effector and reporter\nconstructs used in the transient dual-luciferase assays. All constructs contain a NOS\nterminator (not shown). One NRT1.1 genomic fragments (-1600 to -1) were inserted\nupstream of the CaMV 35S minimal promoter (Mini; −46/0). b,c, Effect of BRM on\nNLP7 and STOP1 binding to the P3 regions of NRT1.1 assessed via transient dual-\nluciferase assays. The empty vector (EV, pCambia1300) was used as a negative control\n(set to 1). Effector and reporter constructs were co-transformed into N. benthamiana\nleaves for 48 h, after which the ratio of firefly to renilla luciferase activity was measured.\nData are presented as mean ± SD from four biological replicates. ‘ns’ denote non-\nsignificant differences as determined by two-tailed Student’s t-test (p < 0.05).\n6.In the main text, Figures 3e and 3f are cited in reverse order. In addition, please clearly\nspecify the ChIP-seq data in fig.3f, either in the figure legend or in the main text\n(including accession number, plant growth condition etc ).\nResponse: Thank you for your valuable comment. We have revised the order of Fig.3e\nand 3f in the revised manuscript. Furthermore, the BRM ChIP-seq data was\ndownloaded from GEO under accession no. GSM4233035 (SRR10768984) and the\nATAC-seq data were downloaded from GEO under accession no. GSM5799538. We\nhave added this point in the figure legend.\n7.L.167~& L173~:\nThe authors state that BRM controls NRT1.1 transcription “under low-pH conditions”\nby interfering with STOP1 activity or accessibility to cis-elements. However, as far as\nI can see, none of the experiments in Fig. 3 were conducted under stress conditions.\nWhich data demonstrate that BRM interferes with STOP1 under low-pH conditions?\nResponse: Thanks very much for pointing out it. To ensure the accurate presentation of\nthe conclusions in Fig. 3, we have revised the relevant descriptions to avoid\nemphasizing that this regulatory process occurs under acid stress conditions (e.g. the\nfindings demonstrate that BRM antagonistically regulates NRT1.1 expression by\ninterfering with STOP1 activity). In fact, the observation that BRM can inhibit STOP1\nfunction even under non-acid stress conditions further underscores the biological\nsignificance of the BRM-STOP1 module we aim to investigate (as detailed in the third\npoint above). The results in Fig. 3 demonstrate that BRM is not merely an acid stress-\nspecific suppressor, but a broad-spectrum suppressor of STOP1 activity. Since high\nlevels of active STOP1 are detrimental to plant growth and development, a suppressor\nlike BRM is essential for plants to control STOP1 activity, enabling them to maintain\na balance between growth and STOP1-mediated stress responses, thereby allowing\nthem to better adapt to changing environments. Moreover, the expression of STOP1 is\ngenerally not induced by various stresses, with exception of only hypoxia stress among\nthe reported stresses (Enomoto et al., 2019). Its stability and transcriptional activation\nactivity are more prominently reflected at the level of protein modification. The\ninvolvement of BRM provides a more broad-spectrum regulatory pathway for the\ntranscriptional activation regulation of STOP1.\n8.L177~: The authors state that “BRM acts upstream of STOP1 in regulation of NO₃⁻\nuptake and H⁺ tolerance.” However, based on the author’s data presented, BRM and\nSTOP1 appear to operate independently in a parallel relationship rather than in a\nhierarchical one. The authors should provide a more detailed explanation of the basis\nfor claiming that BRM functions upstream of STOP1.\nResponse: We appreciate this comment, and would like to clarify the genetic analysis\nbetween BRM and STOP1. In Fig 4, we have shown that the root growth of the\nbrmstop1 double mutant was comparable to the stop1 single mutant but notably shorter\nthan that of the WT and the brm single mutant (Fig. 4a, b), which rules out an additive or\nintermediate genetic interaction and demonstrates that STOP1 is epistatic to BRM in\nH⁺ tolerance. Furthermore, the elevated NO₃⁻ uptake and NRT1.1 expression in brm\nmutants were completely abolished by STOP1 loss of function, as the brmstop1 double\nmutant displayed NO₃⁻ uptake and NRT1.1 expression level comparable to the stop1\nmutant (Fig. 4c, d), suggesting that the increased NO₃⁻ uptake and NRT1.1 expression\nin response to low pH in brm mutant requires functional STOP1. Therefore, these\nresults demonstrate that BRM exerts genetically upstream of STOP1 in regulating H+\ntolerance. We have further clarified this genetic logic in the revised manuscript.\n9.Figure 6c does not represent quantitative data of nuclear fluorescence intensity of\nBRM-GFP. Please revise the figure.\nResponse: We appreciate this suggestion, and have revised this figure.\nReviewer #3 (Remarks to the Author)\nYe et al. report a novel function of BRM (BRAHMA chromatin remodeler) in\nArabidopsis, specifically in negatively regulating the STOP1-NRT1.1 pathway, which\nis one of the key mechanisms of low pH tolerance in this plant. Authors’ claims are\nwell supported by experimental results. This is a significant finding that helps us\nunderstand and improve low pH tolerance in plants.\nResponse: We sincerely appreciate your positive evaluation of our work.\nMajor points\n- I’m wondering how to identify BRM? “we performed a yeast two-hybrid (Y2H)\nscreen using STOP1 as a bait and uncovered BRAHMA (BRM),” (lines 95-96).\nHowever, there is no description of the screening; only the interaction between BRM\nand STOP1 is confirmed by Y2H (Fig. 1b,c).\nResponse: Thank you for your valuable comment. As the reviewer #2 also mentioned\nthe very similar question, please refer to our answer in Page 13.\n- Related to the above point, is this direct interaction between BRM and STOP1 really\nrequired, rather than interaction on the NRT1.1 genomic sequence? If the direct\ninteraction is true, other downstream target genes of STOP1 must also be affected, not\njust NRT1.1, but this has not been verified here.\nResponse: Thank you for this critical question, which has helped us clarify the breadth\nof BRM’s regulation of the STOP1 pathway. To address whether the direct BRM-\nSTOP1 interaction affects other STOP1 downstream targets beyond NRT1.1,\nwe\nperformed additional experiments and clarified the regulatory logic. We first examined\nthe expression of NRT1.1 and two well-characterized STOP1 target genes, ALMT1\nand CIPK23, in Col-0, brm, stop1, and brm stop1 double mutants. Consistent with\nNRT1.1, both ALMT1 and CIPK23 genes were significantly upregulated\nin brm mutants compared to Col-0 (Fig.4d; Supplemental Fig. 13), indicating that BRM\nbroadly represses the STOP1 pathway. Furthermore, we analyzed phenotypic responses\nof these mutants to aluminum (Al) stress and phosphate deficiency, both are regulated\nby STOP1. In contrast to the stop1 mutants, brm mutants exhibited enhanced Al\ntolerance and phosphate deficiency sensitivity, while the brm stop1 double mutants\nphenocopied stop1 mutants (Supplemental Fig. 14, 15). These results suggest that BRM\ncould act as a global regulator of STOP1-mediated stress adaptation.\nIn addition, the results that the upregulation of NRT1.1 in brm mutants, but not\nin brm stop1 double mutants (Fig. 4d), further support our conclusion that the direct\nBRM-STOP1 interaction is required for suppressing NRT1.1 expression, rather than\nsolely via cis-acting regulation at NRT1.1. Therefore, BRM exerts dual regulation in\nplant adaptation to acidic stress—both by physically interacting with STOP1 to\nsuppress its transcriptional activation capacity, and by occupying the NRT1.1 genomic\nregion to directly repress its transcription. Although our study mainly focused\non NRT1.1 because it is the key STOP1 target mediating plant tolerance to acidic stress\nand nitrogen use efficiency, we have included these new data in the revised manuscript,\nalong with a discussion of BRM’s global role in the STOP1 pathway, to clarify that the\ndirect BRM-STOP1 interaction impacts multiple downstream targets. These results\nstrengthen the conclusion that the direct BRM-STOP1 interaction is functionally\nrelevant and extends beyond NRT1.1 regulation.\nSupplemental Fig 13. BRM affects the expression of ALMT1 and CIPK23. Relative\nexpression of two key target genes of STOP1 in Col-0, brm, stop1, and brm stop1\nmutants under neutral and low-pH conditions. Data are shown as mean ± SD (n = 4\nbiological replicates per genotype and condition). Different letters indicate statistically\nsignificant differences between means (p < 0.05), determined by Tukey’s multiple\ncomparisons test (one-way ANOVA).\nSupplemental Fig 14. Phenotype of the Col-0, brm, stop1, and brm stop1 mutants\nunder P-sufficient and P-deficient conditions. a,b, Growth of seedlings grown on 1.0\nmM (CK) and 0.01 mM (low Pi) agar media for 6 days. Data are from 20 seedlings per\nline and condition. Whiskers indicate minimum and maximum values. Scale bar: 1 cm.\nDifferent letters indicate statistically significant differences between means (p < 0.05),\ndetermined by Tukey’s multiple comparisons test (one-way ANOVA).\nSupplemental Fig 15. Phenotype of the Col-0, brm, stop1, and stop1 brm mutants\nunder control and Al (1.25 mM) treatment. a,b, Root growth images (a) and their\nrelative quantification (b). Seeds were directly sown on the control or Al treatment agar\nplates and phenotype was observed 6 days after germination. The average length of\neach genotype was set to 1, and the relative root length was expressed as percentage\n(root length with Al treatment/root length without Al). Data are from 20 seedlings per\nline and condition. Whiskers indicate minimum and maximum values. Scale bar: 1 cm.\nAsterisks indicate statistically significant differences (*p < 0.05, ***p < 0.0001), as\ndetermined by one-way ANOVA followed by Tukey’s multiple comparisons test.\nMinor points\n- NUE (line 74) and SWI/SNF (line 84) are not defined before.\nResponse: We have made the necessary revision.\n- “co-immunoprecipitated with BRM-FLAG after a 6-hour low-pH treatment (Fig. 1e)”\n(line 105). Why was it only checked after low pH treatment?\nResponse: Thank you for your attention to our Co-IP experimental design. To better\nreflect the authentic in vivo interaction status between BRM and STOP1, we\nspecifically used the double-tagged genetic material (BRM-FLAG/GFP-STOP1) for\nCo-IP assays. However, under neutral-pH conditions, the abundance of GFP-STOP1\nprotein in this transgenic line is extremely low, making it technically challenging to\ncapture the GFP-STOP1 protein through immunoprecipitation. To address this issue,\nwe treated the plants with 6-hour low-pH treatment to induce GFP-STOP1 protein\naccumulation and also with MG132 to inhibit the degradation of BRM-FLAG protein.\nThis treatment allowed us to reliably detect the BRM-STOP1 complex via Co-IP. For\neasier understanding of this data, we have clarified the necessity of the 6-hour low-pH\nand MG132 treatment in the revised ‘Materials and Methods’ sections.\n- “publicly available datasets using WashU Epigenome Browser and Plant Regulomics”\n(line 164). References or URLs are required.\nResponse: Following your comment, we have added the URL of these two websites in\nthe revised manuscript.\n- Fig. 3b: The description may be incomplete. With or without BRM?\nResponse: Thank you so much for bringing that to our attention. We have made the\nnecessary revision."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised version of the manuscript by Ye et al. entitled ‘BRAHMA represses STOP1-NRT1.1 module to control plant rhizosphere alkalization and acid stress adaptation’ has been improved, however not all of my previous concerns have been properly addressed.\nMajor commentsː\nIn my previous assessment it has been requested that Authors should include in the manuscript the data from confirmatory experiments performed on the brm null mutant(s) to avoid the misleading conclusions coming from the use of mutant lines expressing truncated BRM protein.\nThe explanation justifying refusing of the use of brm null mutant line provided by Authors, namelyː ‘However, compared to Col-0, the brm-1 mutant shows severe growth defects and is homozygous lethal, making it unsuitable for subsequent studies.’ indicates fundamental lack of knowledge and is simply not logical and fully against the existing literature data showing that brm-1 homozygotes are viable but sterile. Given their explanations that the brm-1 is “homozygous lethal” how the Authors could present the data on figure called ‘Reviewer-only Fig. 1’ on brm-1 mutant plants? The scientific data should be presented in a very precise way, especially in such reputable journal.\nAuthors performed control experiment using brm-1 mutant plants and on figure ‘Reviewer-only Fig. 1’ they show that the low-pH conditions have no significant effect on the root growth of brm-1 mutant plants. This finding in very important because it shows that indeed in weak brm-3 and brm-20 alleles the BRM-dependent repression of response to low pH growth conditions is still partially retained whereas in brm-1 null mutant such repression is fully abolished. This finding is very important. Moreover, Authors introduced into the revised version of the manuscript a description of brm-3 and brm-20 mutant lines indicating the degree of truncation of BRM protein. Therefore, it would be very valuable for the manuscript quality if Authors present in the manuscript body the data for brm-1 and discuss which part of BRM protein may be crucial for the partially retained and fully abolished response to low pH conditions. Authors may attempt also to the analysis of BRM interactors (protein partners) in the context of truncated BRM proteins.\nTherefore, I insist to include the confirmatory data and their appropriate discussion to the manuscript."}, "author_response": "The following outlines our point-by-point responses (in blue) to editor’s and reviewers’\ncomments:\n---------------------------------------------------------------------------------------------\nReviewer #1 (Remarks to the Author)\nThe revised version of the manuscript by Ye et al. entitled ‘BRAHMA represses\nSTOP1-NRT1.1 module to control plant rhizosphere alkalization and acid stress\nadaptation’ has been improved, however not all of my previous concerns have been\nproperly addressed.\nResponse: We thank you for the careful and detailed comments regarding the use of\nBRM mutant alleles. We acknowledge that the inclusion and interpretation of the brm-1\nnull allele required clarification, and we appreciate the reviewer’s emphasis on rigor,\nwhich has helped to strengthen our conclusions. Below, we address each concern\nspecifically.\nMajor commentsː In my previous assessment it has been requested that Authors should\ninclude in the manuscript the data from confirmatory experiments performed on the\nbrm null mutant(s) to avoid the misleading conclusions coming from the use of mutant\nlines expressing truncated BRM protein. The explanation justifying refusing of the use\nof brm null mutant line provided by Authors, namelyː ‘However, compared to Col-0,\nthe brm-1 mutant shows severe growth defects and is homozygous lethal, making it\nunsuitable for subsequent studies.’ indicates fundamental lack of knowledge and is\nsimply not logical and fully against the existing literature data showing that brm-1\nhomozygotes are viable but sterile. Given their explanations that the brm-1 is\n“homozygous lethal” how the Authors could present the data on figure called\n‘Reviewer-only Fig. 1’ on brm-1 mutant plants? The scientific data should be presented\nin a very precise way, especially in such reputable journal.\nResponse: We sincerely thank you for pointing out this inaccuracy. We acknowledge\nthat our previous statement describing brm-1 as ‘homozygous lethal’ was incorrect, and\ndeeply apologize for this imprecise description. This was a significant oversight on our\npart, and we are grateful for the opportunity to correct it.\nIn the revised manuscript, we have corrected all descriptions of brm-1, explicitly\nstating that it is a sterile null allele that exhibits severe developmental defects. We also\nhave integrated the brm-1 data into the main manuscript, rather than restricting it to\nreviewer-only material.\nAuthors performed control experiment using brm-1 mutant plants and on figure\n‘Reviewer-only Fig. 1’ they show that the low-pH conditions have no significant effect\non the root growth of brm-1 mutant plants. This finding in very important because it\nshows that indeed in weak brm-3 and brm-20 alleles the BRM-dependent repression of\nresponse to low pH growth conditions is still partially retained whereas in brm-1 null\nmutant such repression is fully abolished. This finding is very important. Moreover,\nAuthors introduced into the revised version of the manuscript a description of brm-3\nand brm-20 mutant lines indicating the degree of truncation of BRM protein. Therefore,\nit would be very valuable for the manuscript quality if Authors present in the manuscript\nbody the data for brm-1 and discuss which part of BRM protein may be crucial for the\npartially retained and fully abolished response to low pH conditions. Authors may\nattempt also to the analysis of BRM interactors (protein partners) in the context of\ntruncated BRM proteins. Therefore, I insist to include the confirmatory data and their\nappropriate discussion to the manuscript.\nResponse: We sincerely appreciate your insightful comments regarding the use of\nBRM null mutant (brm-1), which have prompted us to perform critical confirmatory\nexperiments and to deepen our mechanistic discussion. In direct response to the\nsuggestion, we have incorporated comprehensive data obtained from brm-1 and have\nexpanded our interpretation of the phenotypic differences observed among the various\nbrm alleles.\nFollowing the suggestions from you and the Editor, we conducted detailed\nphenotypic analyses with the brm-1 mutant. As shown in Fig. 2e and 2f, root elongation\nin the brm-1 mutant was largely insensitive to low-pH stress, in stark contrast to the\nsignificant inhibition observed in wild-type (Col-0) plants. Moreover, decreasing the\nconcentration of NO ⁻ from 6 mM to 0.06 mM significantly inhibited root growth of\ndemonstrating that BRM-repressed H+ tolerance depends on NO ⁻ availability. At the\n3\nmolecular level, we have confirmed that the expression of NRT1.1 was remarkably\nincreased in the brm-1 mutant (versus Col-0) under low-pH condition (Fig. 2h).\nConsistent with these results, we have shown that the brm-1 mutant displayed\nsignificantly enhanced and rapid rhizosphere alkalinization compared to Col-0\n(Supplemental Fig.11). These findings collectively support our core conclusion that\nBRM inhibits H+ tolerance by repressing the low-pH-induced expression of NRT1.1.\nFig 2. Downregulation of BRM enhances plant tolerance to low pH and stimulates\nnitrate uptake under acidic conditions. e,f, Root growth of Col-0 and brm-1 mutants\non media containing different concentrations of nitrate. Three-day-old seedlings were\ntransferred to neutral- or low-pH media supplemented with either 0.06 mM or 6 mM\nKNO₃ and grown for 3 days. Potassium concentrations were balanced to 6 mM by\nadjusting K₂SO₄ levels. g, RT-qPCR analysis of NRT1.1 expression in the roots of Col-0\nand brm-1 mutants. Data represent means ± SD from four biological replicates. Scale\nbar: 1 cm. In box plots, whiskers indicate minimum and maximum values, and boxes\nrepresent means ± SD of 20 seedlings in (f). Different letters indicate statistically\nsignificant differences between means (p < 0.05), determined by Tukey’s multiple\ncomparisons test (one-way ANOVA in (h); multi-way ANOVA in (f)).\nSupplemental Fig 11. brm-1 mutants enhance rhizosphere alkalization under\nacidic stress. a, Visualization of rhizospheric alkalization using bromocresol purple\nstaining. Seedlings were grown for indicated time on basal agar medium with an initial\npH of 5.1. b, Measurement of pH in the rooting zone of Col-0 and brm-1.\nTen-day-old\nseedlings were pre-treated for 2 days on pH 4.7 agar before bromocresol purple staining\nand pH quantification. Data are shown as mean ± SD (n = 3 biological replicates).\nAsterisks denote significant differences as determined by two-tailed Student’s t-test (*p\n< 0.05).\nWe highly agree with your insightful comments that the phenotypic differences among\ndifferent brm alleles provide critical clues for dissecting BRM’s functional domains in\nacid stress adaptation. Actually, our Y2H and BiFC assays had previously revealed that\nthe N-terminus of BRM (BRMn, amino acids 1-976) is both necessary and sufficient\nfor its physical interaction with STOP1 (Fig.1a-d). This molecular finding provides a\nclear explanation for the phenotypic divergence among brm alleles. brm-3 harbors a T-\nDNA insertion disrupting the bromodomain in the C-terminus, while brm-20 undergoes\na small C-terminal truncation. Notably, both mutants retain the intact N-terminal\ndomain, thus maintaining the ability to interact with STOP1, which accounts for the\npartial retention of BRM’s repressive function on the STOP1-NRT1.1 module under\nlow pH conditions. In contrast, the brm-1 null mutant, which lacks the entire protein\nincluding the N-terminal STOP1-interaction domain, exhibits a complete loss of BRM-\nmediated repression, leading to the full acid-tolerant phenotype. This underscores that\nthe BRM N-terminus serves as the core domain for repressing the STOP1-NRT1.1\nmodule. Furthermore, previous studies have showed that deletion of the C-terminal\nbromodomain (disrupted in brm-3) and the small C-terminal segment (truncated in brm-\n20) seems not be essential for chromatin remodeling activity, but may affect BRM\nstability or its tethering to chromatin (Farrona et al., 2007; Zhang et al., 2017).\nTherefore, although these regions are not required for BRM-STOP1 interaction (Fig.1),\ntheir disruption does affect acid stress tolerance (Fig.2). These results, together with the\ndata that low pH promotes BRM degradation (Fig. 6b-e), suggest that BRM C-terminus\nalso play roles in modulating acid stress adaptation, maybe via fine-tuning BRM\nstability. Further studies are needed to elucidate the function and mechanisms of these\nfunctional domains in acid stress tolerance. We have incorporated this nuanced\ndiscussion into the revised manuscript, highlighting how the integration of genetic and\nmolecular interaction data elucidates the mechanistic basis of BRM's function as a\nchromatin-remodeling gatekeeper for acid stress adaptation."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The manuscript by Ye et al. has been improved. All my concerns were properly addressed."}, "author_response": ""}]

Decision letter