A novel SlSTOP1–SlHAK5–cytosolic pH feedback loop drives dual adaptation to proton and aluminum toxicity in tomato

Abstract

Plants often experience aluminum (Al) toxicity in acidic soils, where the transcription factor SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) plays a pivotal role in regulating transcriptional responses to Al stress. While posttranscriptional regulation of STOP1 under Al toxicity has been extensively studied, the mechanisms linking Al stress signals to STOP1 protein stability remain unclear. In this study, we employed multiscale pH imaging and noninvasive microtest (NMT) techniques to demonstrate that Al stress induces cytosolic acidification in the root apex of tomato (Solanum lycopersicum), which promotes the accumulation of SlSTOP1. This finding suggests that cytosolic acidification serves as a critical intermediate connecting Al stress to SlSTOP1 stabilization. Comparative transcriptomic analysis revealed that a significant subset of Al-responsive genes, including the known Al-resistance gene SlHAK5, are coregulated by both Al stress and low pH. Further functional characterization showed that SlHAK5 not only contributes to Al resistance but also plays a key role in maintaining cytosolic pH homeostasis under Al stress. In Slhak5 mutants, the expression of Al-induced genes was dysregulated, concomitant with attenuated cytosolic acidification. Correspondingly, SlSTOP1 accumulation was significantly reduced in Slhak5 mutants compared to wild-type (AC) plants under Al stress, indicating that SlSTOP1-mediated SlHAK5 expression feedback regulates cytosolic acidification. Additionally, Slhak5 mutants exhibited heightened sensitivity to proton stress. Collectively, our findings uncover a novel regulatory circuit involving SlSTOP1 and SlHAK5, which modulates SlSTOP1 stability through cytosolic acidification, thereby enhancing plant adaptation to proton and Al toxicity.

Introduction

Aluminum (Al), ranking as the third most abundant element in the Earth's crust after oxygen and silicon, constitutes a significant environmental stressor in acidic soils [1]. When soil pH drops below 5.5, the dissolution of insoluble Al-containing minerals releases phytotoxic Al³⁺ ions into the soil solution, which predominantly target root apices to suppress primary root elongation and overall plant development [2, 3]. This pH-dependent Al³⁺ mobilization poses a critical limitation to agricultural productivity, particularly given that acidic soils occupy approximately 40% of the world’s arable land [4].

To counteract Al³⁺ toxicity, plants have evolved two principal resistance strategies: external exclusion and internal detoxification [5]. Among these, the external exclusion mechanism, predominantly mediated by Al-activated root exudation of organic acids such as malate, citrate, and oxalate to chelate toxic Al³⁺ ions in the rhizosphere, has been extensively documented as the most pivotal Al resistance mechanism across diverse plant species [6, 7]. A landmark breakthrough in this field was the identification of the Al-activated Malate Transporter 1 (ALMT1) gene from wheat through suppression subtractive cDNA library screening. This discovery not only unveiled the first bona fide Al resistance gene but also defined the ALMT protein family, a previously uncharacterized group of anion transporters [8]. Subsequently, map-based cloning approaches led to the isolation of citrate efflux transporters in barley (HvAACT1) and sorghum (SbMATE), both belonging to the multidrug and toxic compound extrusion (MATE) family [9, 10]. To date, extensive functional characterization of homologous genes across diverse plant species has further solidified the critical role of Al-activated organic acid (OA) secretion in plant Al resistance [7].

The C₂H₂-type zinc finger transcription factor STOP1 (SENSITIVE TO PROTON RHIZOTOXICITY1) was initially identified as a key regulator of proton (H⁺) and aluminum (Al) resistance mechanisms in Arabidopsis [11]. STOP1 controls the expression of multiple Al-tolerance genes, including ALMT1, MATE, and ALS3 (ALUMINUM SENSITIVE 3), thereby contributing to both external and internal Al detoxification [12]. Interestingly, Al stress enhances STOP1 protein accumulation in roots posttranscriptionally, rather than at the transcriptional level [13]. STOP1 stability is tightly regulated by ubiquitin-mediated proteasomal degradation. The F-box protein RAE1 (REGULATION OF ATALMT1 EXPRESSION 1) targets STOP1 for degradation via the 26S proteasome pathway. RAE1 forms a complex with RHD6 (ROOT HAIR DEFECTIVE6) and GL2 (GLABRA2) to coregulate ALMT1 expression [13, 14]. Additionally, SUMOylation of STOP1, mediated by Early in Short Days 4 (ESD4) and SIZ1, further modulates its activity [15, 16]. Recent studies have revealed that the MEKK1–MKK1/2-MPK4 kinase cascade enhances STOP1 phosphorylation and stability, promoting Al resistance [17]. Moreover, reactive oxygen species (ROS)-mediated oxidative modification has been implicated in STOP1 stability, adding another layer of regulation [18, 19].

Emerging evidence highlights the involvement of other factors in modulating STOP1 stability and nuclear localization. For instance, the phosphoinositide pathway, particularly PLC-mediated signaling, facilitates early STOP1 nuclear accumulation [20], suggesting that lipid-derived secondary messengers (e.g. IP₃, DAG) may prime STOP1 for transcriptional regulation. In align with Ca²⁺'s broader role in abiotic stress signaling, Ca²⁺ deprivation promotes STOP1 protein stability [21], implying that cytosolic Ca²⁺ fluxes suppress STOP1 under nonstress conditions. Under Pi deficiency, Fe availability (but not Pi deficiency alone) drives STOP1 nuclear accumulation [22]. Critically, low pH and Fe act synergistically, revealing a pH–Fe–STOP1 axis that integrates nutrient and stress signaling. In addition, NH₄⁺ uptake during Pi deficiency triggers rapid rhizosphere acidification, further amplifying STOP1 accumulation [23], linking nitrogen metabolism to Al resistance. Notably, it is well known that NO₃⁻ and K⁺ transport dynamically alter H⁺ homeostasis in root cells, potentially influencing STOP1 stability. We have previously demonstrated that the decreased citrate secretion in tomato Slhak5 mutant was associated with reduced SlFRDL1 expression, which contributes to the increased sensitivity of Slhak5 to Al stress, but the molecular mechanism remains unknown [24]. Considering HAK5 (high-affinity K⁺ transporter 5) is a H⁺/K⁺ cotransporter, it is possible that cytosolic H⁺ homeostasis is implicated in SlHAK5-mediated SlFRDL1 expression.

In the present study, we demonstrate that Al stress induces cytosolic acidification in tomato root apices, concomitant with reduced apoplastic acidification. This cytosolic acidification promotes the accumulation of SlSTOP1. Notably, both Al stress and low pH stress responses exhibit convergence, as evidenced by the overlapping expression patterns of Al resistance-related genes. Furthermore, we reveal that SlHAK5, a direct target of SlSTOP1, facilitates SlSTOP1 accumulation by regulating pH homeostasis, thereby establishing a SlSTOP1–SlHAK5–positive feedback loop that sustains SlSTOP1 levels. Our findings uncover a novel regulatory mechanism underlying plant adaptation to acidic soils, where H⁺ and Al stresses co-occur.

Results

Al³⁺ stress induces cytosolic acidification of tomato root apex

While STOP1 has been extensively characterized as a central regulator responsive to diverse abiotic stresses, emerging evidence suggests that cytosolic acidification may serve as an essential prerequisite for its activation [25]. Building upon our previous discovery that Al stress induces SlSTOP1 protein stabilization to transcriptionally activate at least 39 Al-responsive genes in tomato root apex [24], we postulated that Al-induced SlSTOP1 activation might be mechanistically associated with disruptions in H⁺ homeostasis. To investigate this hypothesis, we implemented a multiscale pH imaging approach to spatially resolve pH dynamics in both the rhizosphere and subcellular compartments of root apex cells.

Using 2,3,5-triphenyltetrazolium chloride (TTC)-based colorimetric staining [26], we observed markedly reduced red formazan precipitation in the apical transition zone (ATZ) of Al-treated roots compared to controls (Fig. 1A and B), suggesting impaired H⁺ extrusion capacity under Al stress. Complementary 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) ratiometric fluorescence imaging [27] revealed lower apoplastic acidification in Al-exposed roots (Fig. 1C and D). To determine whether these extracellular pH alterations were accompanied by intracellular changes, we employed the 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF) fluorescent probe [28] to monitor cytosolic pH in tomato root tips. Al treatment significantly increased fluorescence intensity, demonstrating cytoplasmic H⁺ accumulation (Fig. 1E and F). Furthermore, noninvasive microtest (NMT) technology revealed a net H⁺ influx at the distal transition zone of the root apex following Al treatment (Fig. 1G), consistent with the observed cytosolic acidification. Collectively, these findings demonstrate that Al stress induces significant cytosolic acidification in cells of tomato root apex.

Figure 1.

Al treatment induced cytosolic acidification of tomato root apex. (A, B) TTC staining of rhizosphere pH in AC root apex in response to Al treatment (A) and quantification of staining for A (B). (C, D) HPTS staining of apoplastic pH in AC root apex in response to Al treatment (A) and their quantification in C (D). (E, F) The BCECF fluorescent staining in AC root apex in response to Al treatment (A) and its statistical data (F). (G) H⁺ flux at the apical transition zone (ATZ) of the root apex with or without Al treatment. Seedlings with primary root length of 3 to 4 cm were subjected to 1/5 hoagland nutrient solution containing 0 (−Al) or 10 μM Al (+Al) for 6 h. After treatment, root apex was used for pH imaging and H⁺ flux measurement. Data are presented as the mean ± SD of six biological replicates per condition. Bar = 200 μm in (A), (C), and (E). Asterisks above the error bar indicate significant differences at P ≤ 0.05.

Low pH induces SlSTOP1 nuclear accumulation

The finding that Al stress acidifies cytoplasm of root apex cells prompted us to examine whether acidification activates SlSTOP1 protein accumulation, whereby activating transcription of Al resistance genes. To test it, we first determined whether SlSTOP1 accumulation is pH dependent by taking advantage of transgenic tomato lines carrying 35S:SlSTOP1:GFP construct. Compared to pH 5.0, a lower pH value of 4.5 (designated low pH stress hereafter) remarkably stimulated GFP signals, mirroring SlSTOP1 protein accumulation (Fig. 2A and B). For comparison, exposure of tomato roots to 10 μM Al was used as a positive control. Similar with low pH stress, Al treatment (pH 5.0) caused SlSTOP1 protein accumulation. We also determined SlSTOP1 protein accumulation under either Al treatment or low pH stress using antibodies raised against SlSTOP1 [29]. Obviously, SlSTOP1 protein was accumulated under Al treatment and low pH stress conditions, when compared to pH 5.0 (Fig. 2C). Furthermore, prolonged low pH treatment enhanced the nuclear accumulation of SlSTOP1 protein, demonstrating that acidic conditions promote SlSTOP1 translocation to the nucleus (Fig. S1).

Figure 2.

Low pH stress induced SlSTOP1 accumulation in tomato root apex. (A) SlSTOP1 accumulation by investigating GFP fluorescence intensity in 35S:SlSTOP1:GFP transgenic reporter lines. (B) Quantification of GFP fluorescence from recombinant SlSTOP1–GFP protein in root tips. The 35S:SlSTOP1:GFP transgenic reporter lines with primary root length of 3 to 4 cm were subjected to pH 5.0 (control), or 10 μM Al at pH 5.0 (+Al), or a lower pH of 4.5 for 6 h. GFP fluorescence was observed by fluorescence microscope. (C) Western blot analysis of SlSTOP1 accumulation. Tomato seedlings with primary root length of 3 to 4 cm were subjected to pH 5.0 (control), or 10 μM Al at pH 5.0 (+Al), or a lower pH of 4.5 for 6 h. Root tips (0–1 cm) were sampled for protein extraction. The same amount of total protein was then detected using antibodies raised against SlSTOP1 and Histone 3 (internal control). The different letters above the error bar indicate significant differences at P ≤ 0.05.

Low pH stress induces expression of Al-responsive genes

Since cytosolic acidification serves as an intermediate process during Al stress, we hypothesized that Al-responsive genes might be transcriptionally activated under low pH stress conditions. To test this hypothesis, we conducted comparative RNA-seq analyses between low pH stress and Al stress. When tomato roots were exposed to 10 μM Al (+Al) for 6 h, we identified 569 upregulated genes (Table S1). In contrast, 462 genes were induced by low pH stress during the same duration (Table S2). A Venn diagram analysis revealed 83 overlapping genes between the two treatments, indicating a convergence in their stress responses (Fig. 3A, Table S3). KEGG pathway enrichment analysis revealed that these genes were mainly related to signaling pathways, ABC transporters, RNA processing, and various metabolic processes (Fig. S2). Notably, this overlapping set included canonical Al-responsive genes such as SlFRDL1, SlFRDL2, SlFDH (formate dehydrogenase), SlAAE3-1 (acyl-activating enzyme 3-1), SlSTAR1 (sensitive to Al rhizotoxicity 1), and SlHAK5. Subsequent qRT-PCR analysis confirmed that these genes were indeed upregulated under low pH conditions (Fig. 3B–H).

Figure 3.

Response convergence between Al stress and low pH stress. (A) Venn diagram of overlapping genes between Al stress and low pH stress by comparative RNA-seq analysis. (B–H) qRT-PCR analysis of gene expression under Al stress or low pH stress. (B) SlFRDL1. (C) SlFRDL2. (D) SlFRDL3. (E) SlFDH. (F) SlAAE3-1. (G) SlSTAR1. (H) SlHAK5. Seedlings with primary root length of 3–4 cm were subjected to pH 5.0 (control), or 10 μM Al at pH 5.0 (+Al), or a lower pH of 4.5 for 6 h. Root apex (0–1 cm) were collected for RNA extraction. The expression level was calculated relative to ACTIN. Data are means ± SD (n = 3). Asterisks indicate significant differences at P ≤ 0.05.

Our previous work demonstrated that SlSTOP1 directly regulates the expression of 39 Al-responsive genes [24]. Interestingly, among 83 overlapping genes, there are 19 genes that are SlSTOP1 targets, suggesting the convergence of two stresses is connected to SlSTOP1 (Table S3). To further explore the connection between Al-activated SlSTOP1 and cytosolic acidification, we investigated SlHAK5, one of the targets of SlSTOP1, promoter activity under low pH stress. Using SlHAK5pro:GUS transgenic lines in the AC background, we observed enhanced GUS expression in roots following Al treatment (Fig. S3A). Further analysis of SlHAK5 expression under varying external pH levels revealed that its transcription was induced by low pH and intensified as pH decreased from 5.5 to 4.5 (Fig. S3B). Consistent with this, GUS activity was markedly stronger at pH 5.0 and 4.5 compared to pH 5.5 (Fig. S3C). Interestingly, when the pH of the Al treatment solution was adjusted to 5.5, GUS activity decreased significantly, suggesting that Al-induced SlHAK5 expression is pH-dependent (Fig. S3D). These findings collectively demonstrate that cytosolic acidification plays a critical role in SlHAK5 induction during Al stress.

SlHAK5 mediates cytosolic pH homeostasis under Al stress

Building on our prior findings that SlHAK5 positively regulates Al resistance by modulating SlFRDL1 expression and citrate secretion [24], we hypothesized that SlHAK5 may act as a molecular bridge connecting Al stress to cytosolic acidification. To investigate this, we systematically analyzed pH dynamics in both cytoplasmic and extracellular compartments of Slhak5 mutants under Al exposure.

Initial assessment of rhizosphere pH via TTC staining revealed no significant differences between wild-type (AC) and Slhak5 mutants under −Al conditions. While Al treatment inhibited TTC staining in root apex of AC plants, it induced markedly stronger TTC staining in both mutants (Fig. 4A and B), indicating enhanced H⁺ efflux into the rhizosphere or reduced H⁺ influx in Slhak5 mutants.

Figure 4.

The apoplastic and cytosolic pH in roots of AC plants and two Slhak5 mutants with or without Al treatment. (A) TTC staining of rhizosphere pH in roots of AC plants and two Slhak5 mutant with and without Al treatment. (B) Quantification of TTC staining shown in (A). (C) The BCECF fluorescence in roots of AC plants and two Slhak5 mutant without or with Al treatment. (D) Quantification of fluorescence intensity in (C). Seedlings with primary root length of 3–4 cm were subjected to 1/5 hoagland nutrient solution containing 0 (−Al) or 10 μM Al (+Al) for 6 h. After treatment, root apex was used for pH imaging. Data are presented as the mean ± SD of six biological replicates per condition in (D). Bar = 200 μm. The different letters above the error bar indicate significant differences at P ≤ 0.05.

BCECF-based cytosolic pH monitoring showed comparable baseline fluorescence intensities in AC and Slhak5 mutants under control conditions (pH 5.0, −Al; Fig. 4C and D). Upon Al treatment, all genotypes exhibited elevated fluorescence intensity (indicative of cytosolic acidification), but this response was significantly attenuated in Slhak5 mutants (Fig. 4C). Quantitative analysis confirmed these differential responses (Fig. 4D). The observed phenotype aligns with a dual role for SlHAK5 in both limiting excessive H⁺ efflux and facilitating H⁺ influx, thereby facilitating cytosolic acidification. This mechanism complements its previously characterized function in regulating citrate secretion, i.e. mediating SlSTOP1 accumulation to induce expression of genes. Similarly, we measured apoplastic and cytosolic pH in both AC and Slstop1 mutants under low pH conditions. Our results demonstrate that acidic treatment induced proton influx, leading to significant cytosolic acidification in AC root tips (Fig. S4A and B). In contrast, Slstop1 mutants exhibited impaired cytosolic acidification (Fig. S4C and D), mirroring the phenotype previously observed in Slhak5 mutants under Al stress.

SlHAK5 modulates the expression of Al-responsive genes

The finding that SlHAK5 plays a crucial role in maintaining cytosolic pH homeostasis under Al stress suggests its important involvement in Al stress responses. To further investigate this, we performed comparative RNA sequencing of root tips (0–1 cm) between wild-type AC plants and Slhak5 mutants. Under normal conditions (without Al treatment), only 65 differentially expressed genes (DEGs) were identified between AC and Slhak5 mutants (Table S4). However, under Al stress, this number increased dramatically to 2946 DEGs (Table S5). To identify Al-induced genes potentially regulated by SlHAK5, we first compared 569 Al-induced genes in AC plants with the 65 DEGs identified in the ‘AC −Al vs Slhak5 −Al’ comparison. This analysis revealed three overlapping genes: SlHAK5 itself, a gene encoding suberization-associated anionic peroxidase 2, and one uncharacterized gene (Fig. S5A and B). Since hydrogen peroxide (H₂O₂) acts as a signaling molecule and has been reported to degrade STOP1 via oxidative modification [19], we investigated whether H₂O₂ contributes to SlHAK5 induction under Al stress. To test this, we treated the AC and two Slhak5 mutants with exogenous H₂O₂ alongside Al and analyzed SlHAK5 expression. However, no significant difference was observed between treatments with or without H₂O₂ supplementation (Fig. S5C), indicating that SlHAK5 expression is independent of H₂O₂-mediated signaling pathways. Furthermore, we identified 143 overlapping genes by comparing the 569 Al-induced DEGs in AC plants with the 2946 DEGs between AC and Slhak5 mutants under Al treatment (Fig. S6). These genes could be classified into two distinct groups. One included 91 genes showing enhanced induction in Slhak5 mutants, suggesting negative regulation by SlHAK5 (Fig. S6A). The other group included 52 genes exhibiting reduced expression in Slhak5 mutants, indicating positive regulation by SlHAK5 (Fig. S6C). Gene ontology (GO) enrichment analysis revealed that the 91 SlHAK5-negatively regulated genes were significantly associated with the ‘lignin catabolic process’ pathway, while the 52 SlHAK5-positively regulated genes were predominantly enriched in the ‘ABC transporters’ pathway (Fig. S6B and D). These findings suggest that SlHAK5 plays a critical role in coordinating diverse metabolic processes in response to Al stress.

To further elucidate the role of SlHAK5 in Al stress responses, we examined its influence on the expression of canonical Al-responsive genes. We selected 16 genes homologous to known Al-resistance genes for expression analysis (Fig. 5A). Notably, the expression of several key genes including SlSTOP2, SlFRDL1, SlFRDL2, SlFRDL3, SlSTAR1, and SlALS1 was significantly suppressed in Slhak5 mutants compared to wild-type AC plants under Al stress. In contrast, SlALMT12, SlNIP1;1, and SlAAE3-1 exhibited upregulated expression in Slhak5 mutants under the same conditions. To validate these findings, we performed qRT-PCR analysis on selected four genes involved in different processes of Al detoxification. SlFRDL1 that mediates citrate secretion is involved in chelating Al at rhizosphere [24]. SlSTAR1 is homologous to rice OsSTAR1 and buckwheat FeSTAR1 that modifies cell wall to reduce Al binding [30, 31]. Both SlFDH and SlAAE3-1 are two enzymes associated with acetylation degradation of cytoplasmic oxalate, which may be critical for regulating normal cellular function [32, 33]. The qRT-PCR results confirmed the RNA-seq data, demonstrating strong consistency between the two methods (Fig. 5B–E). Our previous work revealed that SlHAK5 is a direct target of SlSTOP1, which plays a crucial role in citrate secretion and Al tolerance in tomato [24]. The observed downregulation of SlFRDL1 in Slhak5 mutants likely contributes to their reduced Al tolerance by impairing citrate efflux—a key mechanism for external Al detoxification. These findings further underscore the importance of SlHAK5 in regulating multiple Al-responsive pathways, mirroring its role in SlSTOP1 stability regulation.

Figure 5.

The influence of SlHAK5 mutation on the expression of canonical Al stress responsive genes. (A) Heatmap analysis of the canonical Al responsive genes between AC and Slhak5 mutant with or without Al treatment. The values in heatmap were FPKM in RNA-seq. (B–E) qRT-PCR analysis the selected four genes for SlFRDL1 (B), SlFDH (C), SlAAE3-1 (D), and SlSTAR1 (E). Seedlings with primary root length of 3–4 cm were subjected to 1/5 hoagland nutrient solution containing 0 (−Al) or 10 μM Al (+Al) for 6 h. Root apex (0–1 cm) were collected for RNA extraction. The expression level was calculated relative to ACTIN. Data are means ± SD (n = 3). The different letters above the error bar indicate significant differences at P ≤ 0.05.

SlHAK5 feedback regulates SlSTOP1 protein stability

SlSTOP1 regulates SlHAK5 expression in response to Al stress [24], which subsequently modulates the expression of Al-responsive genes (Fig. 5). This suggests a potential feedback loop in which SlHAK5 may regulate SlSTOP1 stability. To test this hypothesis, we conducted transient expression assays in leaf protoplasts derived from AC (wild type) and Slhak5 mutants. When pSAT–eGFP was expressed in both AC and Slhak5 protoplasts, no significant difference in GFP fluorescence intensity was observed (Fig. 6A and B). However, upon transient expression of the pSAT–SlSTOP1–eGFP fusion protein, GFP fluorescence localized exclusively to the nucleus, with markedly reduced intensity in Slhak5 mutant protoplasts compared to AC (Fig. 6A–C). Further analysis revealed that SlSTOP1 protein accumulation in the root apex under Al stress was significantly lower in both Slhak5 mutants than in AC (Fig. 6D). These findings demonstrate that SlHAK5 contributes to SlSTOP1 protein accumulation under Al stress, supporting the existence of a SlSTOP1–SlHAK5 positive feedback loop that maintains SlSTOP1 stability.

Figure 6.

SlHAK5 positively regulates SlSTOP1 protein stability and H⁺ tolerance. (A) Fluorescence of eGFP proteins in protoplasts of AC plants and Slhak5 mutant. (B) Fluorescence of SlSTOP1:GFP fusion proteins in protoplasts of AC plants and Slhak5 mutant. (C) The statistical data of fluorescence intensity in (A, B). (D) SlSTOP1 accumulation in AC and two Slhak5 mutant lines under Al treatment. AC and two Slhak5 mutant lines with primary root length of 3 to 4 cm were subjected to Al treatment for 6 h. 1 cm root tips were samples for protein extraction. The same amount of crude protein was then detected using antibodies raised against SlSTOP1 and Histone 3. (E) The phenotype of representative seedlings AC plants and Slhak5 mutant under low pH conditions. Bar = 1 cm. (F) The primary root elongation of AC and both Slhak5 mutants in (E). (G, H) SlFRDL1 and SlFRDL2 expression in AC plants and Slhak5 roots under low pH conditions. Tomato seedlings with primary root length of 3–4 cm were subjected to pH 5.5 or pH 4.5 for 2 days. Root length was measured before and after treatment. Data are means ± SD (n = 10). Root apex (0–1 cm) treated by low pH conditions for 6 h were collected for RNA extraction. The expression level was calculated relative to ACTIN. Data are means ± SD (n = 3). The different letters above the error bar indicate significant differences at P ≤ 0.05.

In addition to regulating Al resistance, SlSTOP1 has been shown to positively modulate H⁺ tolerance [24]. To further investigate the relationship between SlHAK5 function and SlSTOP1 protein stability, we assessed the role of SlHAK5 in H⁺ tolerance. We compared root elongation between wild-type AC plants and Slhak5 mutants under low pH stress conditions. At pH 5.5, no significant difference in root elongation was observed between AC plants and the Slhak5 mutants. However, under more acidic conditions, both Slhak5 mutants exhibited significantly reduced root elongation compared to AC plants, demonstrating enhanced H⁺ sensitivity in the mutant lines (Fig. 6E and F). These results provide supporting evidence that SlHAK5 contributes to the stabilization of SlSTOP1 under both H⁺ and Al³⁺ stress conditions.

Discussion

The transcription factor STOP1 plays a pivotal role in regulating the expression of aluminum (Al)-resistance genes in plants [7, 34]. While significant progress has been made in understanding the posttranscriptional regulation of STOP1, the mechanistic link between Al signal perception and STOP1 protein accumulation remains unclear. In this study, through the application of multiscale pH imaging techniques, we revealed that Al stress induces specific cytosolic acidification in the root apical region, concurrent with decreased acidification in both the apoplast and rhizosphere (Fig. 1). Importantly, our findings suggest that this cytosolic acidification likely facilitates SlSTOP1 accumulation, consequently activating downstream gene expression. This conclusion is substantiated by multiple lines of evidence. First, both Al stress and low pH conditions were found to promote SlSTOP1 accumulation (Fig. 2). Second, a considerable number of Al-responsive genes could also be induced by low pH stress (Fig. 3). Although the overlapping genes between these two treatments accounted for only 14.6% of total Al-responsive genes, key canonical Al-resistance genes, including direct targets of SlSTOP1 such as SlHAK5, were consistently upregulated by both stresses (Fig. S2). Further, it is hard to compare the signal intensity between low pH and Al stress. In fact, under our experimental condition, Al stress induced more abundant accumulation of SlSTOP1 protein than low pH-induced (Fig. 2). This may explain the expression induction of Al-responsive genes was less profound in low pH stress compared to Al stress (Fig. 3). Third, functional characterization demonstrated that SlHAK5 positively regulates Al resistance by modulating the expression of other Al resistance genes, including SlFRDL1 and SlFDH (Fig. 5). SlFRDL1, a member of the MATE-type citrate transporter family, has been shown to facilitate citrate efflux [32]. SlFDH, a mitochondrial-localized enzyme, catalyzes formate degradation while generating NADH, potentially contributing to ATP production during Al stress [33]. Notably, our results indicate that SlHAK5 regulates these Al resistance genes through its influence on cytosolic pH homeostasis and SlSTOP1 protein stability (Figs 4 and 6).

Our previous work demonstrated that functional disruption of SlHAK5 enhances Al sensitivity in mutants, concomitant with reduced citrate secretion [24]. Given the established role of cytosolic acidification in promoting SlSTOP1 accumulation, these findings strongly implicate SlHAK5 in modulating cytosolic pH dynamics. However, this raises a key mechanistic question: how does SlHAK5 regulate cytosolic pH homeostasis? As a plasma membrane-localized high-affinity K⁺ transporter, SlHAK5’s function in Al resistance appears independent of K⁺ deficiency [24]. Notably, experimental evidence from Arabidopsis and barley systems indicates that high-affinity K⁺ uptake occurs via H⁺-coupled transport, suggesting that AtHAK5 likely functions as a K⁺/H⁺ symporter in planta [35, 36]. Supporting this model, overexpression of OsHAK5 in rice leads to measurable alkalization of the growth medium [37]. Collectively, these observations support a mechanism whereby SlHAK5 mediates cytosolic pH regulation through H⁺ cotransport during Al stress (Fig. 7).

Figure 7.

Schematic illustration of proton or Al stress induced cytosolic acidification in root apex of tomato. When root tips perceive H⁺ or Al³⁺ signals, they might drive concomitant H⁺ influx through IAA-H⁺ symporters, or experience dysregulation of plasma membrane H⁺-ATPase activity for inhibition of proton pumps, leading to pronounced cytosolic acidification (dashed lines represent putative processes). Al³⁺- or H⁺-induced cytosolic acidification in the root apex triggers SlSTOP1 protein accumulation to activate H⁺ or Al³⁺ resistance genes including SlHAK5. In turn, the upregulated SlHAK5 expression promotes further cytosolic acidification through K⁺/H⁺ symport, thereby stabilizing SlSTOP1 itself.

Our findings align with and extend previous observations demonstrating that acidic conditions are essential for STOP1 activation and downstream gene expression. Several lines of evidence support this connection. First, experimental evidence showed that low pH alone can induce ALMT1 expression independently of aluminum stress, suggesting H⁺ sensitivity in STOP1-mediated signaling [25]. Second, in acid soils, STOP1 is constitutively active and regulates NRT1.1 expression, promoting rhizosphere alkalization as an adaptive response to low pH stress [38]. Third, under Pi-limiting conditions, STOP1-mediated ALMT1 expression drives malate secretion from root apices, facilitating Fe³⁺ chelation and root system remodeling—a process that operates preferentially in acidic environments [22, 39, 40]. Notably, this response is further enhanced by NH₄⁺, a proton-releasing nitrogen source [23]. While these studies collectively establish extracellular acidity as a prerequisite for STOP1 activation across multiple stress responses, our work provides novel insight by demonstrating that cytosolic acidification may serve as the unifying trigger for STOP1 stabilization and subsequent signaling (Fig. 7).

While this study does not fully elucidate the mechanisms underlying Al-induced cytosolic acidification, we propose two plausible explanations based on existing evidence (Fig. 7). The first explanation is auxin-mediated H⁺ influx. We found that the root apical transition zone exhibits the most pronounced Al-induced cytosolic acidification (Fig. 4C), coinciding with the region where Al triggers auxin (IAA) accumulation [41, 42]. IAA uptake is mediated by AUX/LAX transporters, which function as IAA-H⁺ symporters [43]. Thus, Al-induced IAA accumulation may drive concomitant H⁺ influx, consistent with our observation of enhanced H⁺ uptake in the transition zone (Fig. 1). The second explanation could be dysregulation of H⁺ pumps. Plasma membrane (PM) H⁺–ATPases extrude protons to establish electrochemical gradients, but Al may perturb their activity. For instance, Al induces hyperpolarization (increased H⁺–ATPase activity) in Al-resistant wheat but depolarization (inhibition of H⁺–ATPase activity) in Al-sensitive genotypes [44]. Similar variability occurs in soybean and faba bean [45–47]. Besides, tonoplast H⁺-PPases and H⁺-ATPases could also contribute to cytosolic pH shifts. A recent study on apple (Malus domestica) indicated that MdSTOP1 interacts with MdNAC2 to regulate MdNXH2 (SODIUM HYDROGEN EXCHANGER 2), which facilitates H⁺ efflux from the vacuole to the cytoplasm (cytosolic acidification) [48]. Given the interplay between auxin transport and PM H⁺-ATPase activity (e.g. auxin-triggered ATPase activation), we propose that both mechanisms synergize to induce cytosolic acidification under Al stress. Future studies should dissect their relative contributions.

In summary, our findings demonstrate that Al-induced cytosolic acidification in the root apex, particularly within the transition zone, triggers SlSTOP1 protein accumulation, leading to transcriptional activation of key genes involved in Al³⁺ and H⁺ tolerance. Crucially, we reveal a self-reinforcing regulatory mechanism whereby SlSTOP1 upregulates SlHAK5 expression, which in turn promotes further cytosolic acidification to stabilize SlSTOP1. This positive feedback loop represents an adaptive strategy that simultaneously enhances tomato's tolerance to both Al³⁺ toxicity and H⁺ stress (Fig. 7).

Materials and methods

Plant materials and treatment conditions

The tomato (Solanum lycopersicum) cultivar Ailsa Craig (AC), CRISPR/Cas9-based Slhak5 mutants and 35S:SlSTOP1:GFP transgenic reporter lines used in this study has been described in our previous study [24]. To construct the SlHAK5_Pro:GUS transgenic plants, a 2-kb promoter of SlHAK5 was cloned into pCAMBIA1300-NosP:HPTGUS Plus vector and transformed into AC plants via hygromycin selection to obtain homozygous transgenic plants. Seeds were surface sterilized by 10% NaClO (v/v) for 15 min. After washed with sterilized water thoroughly, the seeds were immersed in sterilized water overnight and then transferred to petri dishes containing 1/5 Hoagland nutrient solution (pH 5.5) and 0.8% agar with a 16-h day and 8-h night photoperiod. Temperature was 24°C at daytime and 22°C at night. Seedlings with primary root in 3 to 4 cm length were selected for treatments. All treatments were conducted in hydroponics as previous reported [19]. In brief, for Al resistance assay, seedlings were subjected to 1/5 Hoagland nutrient solution (pH 5.0) with (NH₄)H₂PO₄ concentration decreased to 10 μM either in the absence (−Al) or presence (+Al) of 10 μM Al for different treatment duration. For different pH stresses, seedlings were subjected to nutrient solution adjusted to pH 5.5, 5.0, or 4.5.

Quantitative real-time PCR and RNA-seq analysis

After treatment, root apices (0–1 cm) were collected for RNA extraction. Total RNA was extracted by RNAprep Pure Plant Kit and then transcribed into cDNA using PrimeScript RT Master Mix. SYBR Green serving as the detection dye was used to performed qPCR assay by a LightCycler 480 device. ACTIN was used as an internal control to normalize expression levels for each gene. The primer information is shown in Table S6. RNAseq was performed on the Illumina HiSeq platform with three biological replicates for each treatment, as described in a previous study [49].

pH imaging and measurement of net H⁺ flux in root tips

The pH indicator HPTS was used for apoplast pH staining [50]. The roots of AC and two Slhak5 mutants without or with Al treatment were incubated in 1 mM HPTS (pH 5) for 30 min and then rinsed with water three times before imaging. Images were collected at 510 nm with a plate reader (excitation wavelength = 460 nm). To assay intracellular pH in the root tips, the intracellular pH indicator BCECF-AM (Med Chem Express, Shanghai, China) was used. Root tips after Al treatments were incubated with 10 μM BCECF-AM for 1 h in darkness at 22°C and then rinsed with water before imaging. Images were taken at 535 to 550 nm emission after excitation at 488 nm by confocal microscope. The TTC method was used to qualitatively assess rhizosphere H⁺ according to [48].

The net H⁺ fluxes were measured by noninvasive microtest technology (NMT, Xuyue Beijing Science and Technology Company). The NMT system is NMT150S and the software is imFluxesV2.0 (Younger USA LLC, Amherst, MA 01002, USA). Briefly, about 3 to 4 cm primary roots of AC were treated without or with Al treatment for 6 h. Then, the H⁺ fluxes were measured using an H⁺-selective microelectrode with six biological replicates for each treatment.

Transient expression analysis in tomato leaf protoplast and GFP fluorescence observation

To investigate the SlSTOP1 protein accumulation in Slhak5 mutants, we extracted protoplasts from leaves of AC and Slhak5 mutants and then transiently expressed SlSTOP1 in these protoplasts. The ORFs of SlSTOP1 without ending codon were amplified and then cloned into pSAT6–eGFP vector. The protoplast transformation was performed according to the polyethylene glycol (PEG) method and then visualized the GFP fluorescence via fluorescence microscopy. Similarly, the GFP fluorescence was also observed in the root tips of 35S:SlSTOP1:GFP transgenic plants under low pH condition. The fluorescence intensity was subsequently quantified by using Image J software.

Western blot

To investigate SlSTOP1 protein accumulation in AC and Slhak5 mutants, total protein was extracted from the root tips of AC and Slhak5 mutants. The polyclonal antibody raised against STOP1 and anti-rabbit IgG HRP-conjugated antibody as the primary antibody and secondary antibody, respectively, were purchased from Abclonal (Wuhan, China). An anti-Histone 3 antibody (Sigma) was used to normalize the loading quantity of the proteins.

Statistical analysis

The data are presented as means ± standard deviation (SD) derived from four to six biological replicates across various assays. Statistical analyses were conducted using one-way or two-way ANOVA, with P ≤ 0.05 indicating significant differences between groups.

Author contributions

Huihui Zhu and Yuzhi Bai (Conceptualization, Investigation, Methodology, Data curation, Writing—original draft), Liqiong Jia and Feifei Liang (Methodology, Data curation, Software, Investigation), Liqiong Jia, Chao Li, and Wei Fan (Data curation), Zheng-An Yang and Jianli Yang (Writing—review & editing, Funding acquisition)

Data availability

RNA-Seq data is available as accession number PRJNA1052400 for AC and Slhak5 mutants in response to Al stress treatment, and PRJNA1255724 for low pH stress of AC plants in the NCBI SRA database (https://www.ncbi.nlm.nih.gov).

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.