Aluminum resistance in plants: A critical review focusing on STOP1

Abstract

Aluminum (Al) toxicity poses a significant challenge for plant production on acidic soils, which constitute approximately 30% of the world’s ice-free land. To combat Al toxicity, plants have evolved both external and internal detoxification mechanisms. The zinc-finger transcription factor STOP1 (SENSITIVE TO PROTON RHIZOTOXICITY 1) plays a critical and conserved role in Al resistance by inducing genes involved in both external exclusion and internal detoxification mechanisms. Recent studies have uncovered multiple layers of post-transcriptional regulation of STOP1 and have elucidated mechanisms by which plants sense Al and activate signaling cascades that regulate STOP1 function. This review offers a comprehensive overview of the mechanisms through which STOP1 and its homologs confer Al resistance in plants, with a particular focus on Arabidopsis thaliana and rice. Additionally, we discuss recent advances and future perspectives in understanding the post-transcriptional regulation of STOP1, as well as the Al sensing and signaling pathways upstream of STOP1.

Aluminum (Al) toxicity poses a major challenge for plant growth on acidic soils, which cover approximately 30% of the world’s ice-free land. The transcription factor STOP1 plays a critical and conserved role in conferring Al resistance. This review explores the mechanisms by which STOP1 and its homologs mediate Al resistance, with a focus on its post-transcriptional regulation and the associated Al sensing and signaling pathways.

Introduction

Aluminum (Al) constitutes roughly 8% of the Earth’s crust and is the most abundant metallic element after oxygen and silicon (Driscoll and Schecher, 1988; Ma, 2007). In neutral and alkaline soils, Al exists as harmless insoluble aluminosilicates or oxides. However, acidic soils with a pH of 5.5 or below promote the solubilization of Al into its toxic trivalent form (Al³⁺). Al³⁺ toxicity targets multiple parts of the roots, including the cell wall, plasma membrane (PM), and symplastic components (Kochian, 1995; Rengel and Reid, 1997; Ma, 2007). Consequently, the Al toxicity of acidic soils is a pervasive issue, affecting approximately 30% of the global ice-free land and half of all potentially arable lands (von Uexkull and Mutert, 1995).

To mitigate the toxic effects of Al, plants have evolved two principal resistance strategies, exclusion and tolerance, also known as external and internal detoxification, respectively (Ma et al., 2001; Kochian et al., 2004, 2015). The secretion of organic acids, such as malate, citrate, and oxalate, is the most thoroughly documented and crucial mechanism for Al exclusion in plants (Ma et al., 2001; Ryan et al., 2001; Kochian et al., 2004). In response to Al toxicity, root tips release these organic acid anions into the rhizosphere, where they chelate Al³⁺ ions, preventing them from infiltrating and damaging the root system, with the root apex being the most Al-sensitive site. Various plant species use different organic acids for Al detoxification. For instance, wheat and oilseed rape release both malate and citrate to counter Al toxicity (Delhaize et al., 1993; Ligaba et al., 2004; Ryan et al., 2009), whereas maize and soybean mainly use citrate, though they may also exude malate as a defense (Pellet et al., 1995; Jorge and Arruda, 1997; Yang et al., 2000; Liao et al., 2006). Buckwheat, on the other hand, primarily secretes oxalate to detoxify Al (Ma et al., 1997a; Zheng et al., 1998), and it can also use citrate under Al stress (Lei et al., 2017).

Tolerance mechanisms, initially identified in Al hyperaccumulators like hydrangea, buckwheat, and tea, involve the internal sequestration of Al. These plants can accumulate Al in their shoots at concentrations exceeding 1000 mg/kg (Ma et al., 2001; Kochian et al., 2015). Within the root and leaf symplast, Al is stored in vacuoles in non-toxic forms, such as Al–citrate in hydrangea and Al–oxalate in buckwheat (Ma et al., 1997b, 1998; Ma and Hiradate, 2000; Shen et al., 2002). Recent research has revealed that non-accumulating plants, such as Arabidopsis thaliana and rice, also detoxify Al internally by absorption and sequestration in vacuoles (Larsen et al., 2007; Xia et al., 2010; Huang et al., 2012; Wang et al., 2017; Fan et al., 2024). In addition, plants can enhance Al resistance by modifying cell wall structures using enzymes such as expansins, endo-β-1,4-glucanases, and pectin methylesterases (Kochian et al., 2015). The degree of pectin methylation regulated by pectin methylesterases has been shown to influence Al resistance in maize and rice (Eticha et al., 2005; Yang et al., 2008). In addition, hemicelluloses can actively modify their structure in response to Al to reduce Al toxicity, and have recently been found to be as critical as pectin for Al binding in Arabidopsis thaliana and rice (Yang et al., 2011; Liu et al., 2020; Zhu et al., 2012).

The C2H2 zinc-finger transcription factor STOP1 (SENSITIVE TO PROTON RHIZOTOXICITY 1) and its homologs play a pivotal role in plant Al resistance by regulating both exclusion and tolerance mechanisms (Iuchi et al., 2007; Yamaji et al., 2009; Ohyama et al., 2013). This review offers a comprehensive overview of the roles of STOP1 and its homologs in Al detoxification, with a particular focus on Arabidopsis thaliana and rice, and explores the recent advancements and future directions in understanding the post-transcriptional regulation, Al sensing, and upstream signaling pathways of STOP1.

The roles of STOP1 and its homologs in regulating external and internal Al resistance

Arabidopsis thaliana

The stop1 mutant was originally discovered through a forward genetic screen targeting Arabidopsis thaliana mutants with reduced root growth under acidic conditions (Iuchi et al., 2007). Subsequent work showed that it is highly sensitive to Al toxicity. Physiological assessments have shown that STOP1 is crucial for regulating malate secretion from roots (Iuchi et al., 2007), which is a key mechanism of Al resistance in Arabidopsis. Malate exudation is the primary means of Al resistance, although citrate exudation also contributes to Al resistance (Hoekenga et al., 2003; Liu et al., 2009).

In wheat, the first transporter implicated in the secretion of malate was named ALMT1 (AL-ACTIVATED MALATE TRANSPORTER 1), representing a novel family of anion transporters (Sasaki et al., 2004). Following this discovery, two research teams identified citrate transporters, HvAACT1 in barley and SbMATE in sorghum, which belong to the multidrug and toxin extrusion (MATE) family (Furukawa et al., 2007; Magalhaes et al., 2007). Building on sequence homology and functional studies, two Arabidopsis homologs were identified: the PM-localized AtALMT1 is responsible for malate secretion, and AtMATE is responsible for citrate secretion (Hoekenga et al., 2006; Liu et al., 2009).

AtALMT1 expression is significantly reduced in the stop1 mutant, often to nearly undetectable levels (Iuchi et al., 2007; Sawaki et al., 2009). This suggests that STOP1 is a crucial regulator of AtALMT1 expression, mediating malate secretion and Al resistance. Although the role of secreted malate in external Al resistance is well established, recent findings indicate that secreted malate can also facilitate Al uptake as part of an internal detoxification mechanism (Wang et al., 2017; Fan et al., 2024). Malate chelates Al³⁺ ions to form Al–malate complexes, which are taken up by root cells via NIP1;1 and NIP1;2, which are PM transporters in the nodulin 26-like intrinsic protein (NIP) subfamily (Wang et al., 2017; Fan et al., 2024). The Al–malate complexes are presumably sequestered into vacuoles for internal Al detoxification, facilitated by the tonoplast-localized bacterial-type ATP-binding cassette (ABC) transporter complex AtSTAR1/ALS3 (ALUMINUM SENSITIVE 3) (Fan et al., 2024) (Figure 1). The gene STAR1 (SENSITIVE TO ALUMINUM RHIZOTOXICITY 1), encoding a nucleotide-binding domain, was first identified in rice and found to interact with STAR2, an ortholog of the Arabidopsis transmembrane domain protein ALS3, to form a functional ABC transporter (Larsen et al., 2005; Huang et al., 2009, 2010). In rice, the ATSTAR/ALS3 complex is proposed to modify the cell wall to confer Al resistance (Huang et al., 2009). However, in Arabidopsis, the complex is localized to the tonoplast (Dong et al., 2017), implying a role in internal Al detoxification. Supporting this hypothesis, alterations in Al uptake correspond to changes in Al sensitivity in Atstar1 and/or als3 mutants (Fan et al., 2024). STOP1 also positively regulates the expression of ALS3 (Sawaki et al., 2009) (Figure 1), which positively influences the accumulation of the AtSTAR1 protein (Fan et al., 2024). This suggests that STOP1 enhances internal Al detoxification by inducing the expression and assembly of the AtSTAR1/ALS3 complex.

Figure 1.

Mechanisms of Al resistance regulated by STOP1 in Arabidopsis thaliana and ART1 in rice.

The zinc-finger transcription factors STOP1 and ART1 are central to Al resistance in Arabidopsis thaliana and rice respectively. STOP1 regulates the expression of multiple Al-resistance genes, including AtALMT1, AtMATE, ALS3, AHA2, SAUR55, GDH1, and GDH2. AtALMT1 and AtMATE encode plasma membrane (PM) anion transporters that mediate the exudation of malate and citrate, respectively, aiding in the process of chelating and detoxifying Al ions (Al³⁺). The Al–malate complex may also be taken into the cells by the PM-localized NIP1;1 and NIP1;2 transporters, then sequestered into vacuoles by the tonoplast-localized ABC transporter complex AtSTAR1/ALS3 for internal Al detoxification. SAUR55 enhances PM H⁺-ATPase activity, potentially by inhibiting the phosphatases PP2C.D2 and PP2C.D5. The increased activity, supported by the upregulation of both SAUR55 and AHA2, promotes malate exudation. Additionally, the mitochondrial enzymes GDH1 and GDH2 are involved in pH regulation, which likely contributes to Al resistance by preventing cellular acidosis.

In rice, ART1 induces the expression of Al-resistance genes, including STAR1, STAR2, OsALS1, NRAT1, OsFRDL4, OsCDT3, and OsMGT1. STAR1 and STAR2 form an ABC transporter complex at vesicle membranes, which is hypothesized to transport UDP-glucose or Al into vesicles. These vesicles are secreted through the cell wall via exocytosis for modification and Al detoxification or delivered to vacuoles for Al sequestration and detoxification. The PM-localized NRAT1 facilitates Al uptake into cells, where it is subsequently sequestered in vacuoles by the ABC transporter OsALS1. The PP2C.D phosphatase SAL1 inhibits PM H⁺-ATPase activity, thereby reducing NRAT1-mediated Al uptake. The MATE family protein OsFRDL4 mediates citrate exudation, aiding in the process of Al chelation and detoxification. The Mg transporter OsMGT1 enhances Mg uptake, which mitigates internal Al toxicity, whereas the PM-localized small cysteine-rich peptide OsCDT3 is thought to prevent Al entry into root cells by binding to Al. ALS3 and STAR2, as well as AtMATE and OsFRDL4, are homologous genes targeted by STOP1 in Arabidopsis thaliana and ART1 in rice, respectively. The question marks indicate unconfirmed substrates or processes.

In addition to inducing AtALMT1 expression to stimulate malate secretion, STOP1 directly activates the expression of SAUR55 (SMALL AUXIN UP RNA55) and AHA2, which encodes a PM H⁺-ATPase, further facilitating malate exudation (Agrahari et al., 2024) (Figure 1). Members of the SAUR family inhibit PP2C.D phosphatases, which in turn activate PM H⁺-ATPase activity (Spartz et al., 2014). In particular, SAUR55 has been shown to enhance PM H⁺-ATPase activity, potentially by interacting with and inhibiting the phosphatases PP2C.D2 and PP2C.D5 (Agrahari et al., 2024). PM H⁺-ATPase activity has been linked to increased malate secretion in Arabidopsis (Zhang et al., 2019a; Xie et al., 2023). Consequently, STOP1 promotes malate secretion not only by inducing AtALMT1 expression but also by augmenting PM H⁺-ATPase functionality (Figure 1).

In addition to facilitating malate secretion, STOP1 aids Al exclusion via other methods, such as inducing AtMATE expression to promote citrate exudation (Liu et al., 2009). Furthermore, STOP1 modulates Al tolerance by regulating GDH1 (GLUTAMATE DEHYDROGENASE 1) and GDH2 expression (Enomoto et al., 2019; Tokizawa et al., 2021) (Figure 1). These enzymes are involved in pH-regulating metabolism and may help prevent cellular acidosis (Hirata et al., 2003; Bouche and Fromm, 2004), suggesting that STOP1 may contribute to the maintenance of cellular pH homeostasis as an additional defense against Al toxicity.

STOP1 has a homologous protein in Arabidopsis, called STOP2 (Iuchi et al., 2007; Kobayashi et al., 2014). However, STOP2 expression is significantly lower than that of STOP1 (Kobayashi et al., 2014). Overexpressing STOP2 using the cauliflower mosaic virus 35S promoter or the STOP1 promoter can partially rescue the stop1 mutant’s sensitivity to low pH but not to Al toxicity (Kobayashi et al., 2014). This suggests that STOP2 may not be involved in the regulation of Al resistance. Nevertheless, to definitively determine whether STOP2 has a role in low pH or Al resistance, it will be necessary to generate a stop2 single mutant and/or a stop1stop2 double mutant and evaluate their sensitivity to these conditions.

Rice

Through a forward genetic screen of Al-sensitive mutants, Yamaji et al. (2009) identified a mutant termed art1 (sensitive to Al rhizotoxicity), which exhibits heightened sensitivity to Al toxicity. Subsequent gene cloning revealed ART1 as a C2H2 zinc-finger transcription factor and a homolog of the Arabidopsis STOP1 protein (Yamaji et al., 2009). Functional analysis showed that ART1 orchestrates Al resistance both externally and internally by inducing a series of Al-resistance genes, including STAR1, STAR2, OsALS1, NRAT1 (NRAMP ALUMINUM TRANSPORTER 1), OsFRDL4, OsCDT3, and OsMGT1 (Yamaji et al., 2009) (Figure 1). As mentioned earlier, STAR1, a nucleotide-binding domain protein, interacts with the transmembrane domain protein STAR2, which is the ortholog of Arabidopsis ALS3, to form an active ABC transporter complex. STAR1 and STAR2 are proposed to localize to vesicle membranes and transport UDP-glucose into vesicles. The UDP-glucose is then secreted to the cell wall via exocytosis for modification and Al detoxification (Huang et al., 2009). However, the precise mechanism of STAR1/2-mediated cell wall modification remains elusive, and to date, there is no genetic evidence confirming UDP-glucose as a substrate for STAR1/2. The ability of OsSTAR1 to rescue the Al-sensitive phenotype of Atstar1 in Arabidopsis (Huang et al., 2010) suggests that OsSTAR1/2 may be involved in internal Al detoxification in rice, although this requires further investigation (Figure 1). Notably, ART1 robustly induces the expression of both STAR1 and STAR2 in rice, whereas in Arabidopsis, STOP1 only induces ALS3 expression, with no impact on AtSTAR1 (Huang et al., 2009; Sawaki et al., 2009). Furthermore, ART1 induces the expression of OsALS1, which encodes a tonoplast-localized half-size ABC transporter that sequesters Al into vacuoles (Figure 1), whereas the Arabidopsis homolog, ALS1, is not regulated by STOP1 either (Larsen et al., 2007; Sawaki et al., 2009; Huang et al., 2012).

NRAT1, a member of the natural resistance-associated macrophage protein family, localizes to the PM and facilitates the uptake of ionized Al (Xia et al., 2010). Phylogenetic analysis and structural modeling indicate that NRAT1 has homologs in monocots such as maize and sorghum, but not in the dicot Arabidopsis (Lu et al., 2018). Mutation of NRAT1 reduces Al uptake, leading to increased Al accumulation in the cell wall and heightened Al sensitivity. Conversely, overexpression of NRAT1 enhances Al uptake and Al sensitivity in rice plants (Xia et al., 2011). Mutation of SAL1 (SENSITIVE TO ALUMINUM 1), which encodes a PP2C.D phosphatase, increases PM H⁺-ATPase activity and Al uptake, resulting in increased sensitivity to internal Al toxicity (Xie et al., 2023) (Figure 1). Interestingly, knockout of NRAT1 rescues the Al-sensitive phenotype of the sal1 mutant, emphasizing the importance of balancing Al accumulation between the cell wall and symplasm for Al resistance in rice (Xie et al., 2023). The simultaneous induction of NRAT1 and OsALS1 by ART1 suggests a coordinated mechanism of Al uptake and vacuolar sequestration for internal Al detoxification (Figure 1).ART1 also upregulates OsMGT1, which encodes a magnesium (Mg) transporter, enhancing Mg uptake and potentially mitigating internal Al toxicity (Chen et al., 2012) (Figure 1).

Furthermore, ART1 facilitates external Al detoxification by modulating the expression of OsFRDL4 and OsCDT3. OsFRDL4, a MATE family citrate transporter, is responsible for root citrate exudation in response to Al toxicity (Yokosho et al., 2011). Although citrate exudation contributes significantly less to Al resistance in rice than in other species (Ma et al., 2002; Yokosho et al., 2011), OsFRDL4-mediated citrate exudation still enhances Al resistance. OsCDT3 encodes a PM-localized small cysteine-rich peptide that may prevent Al entry into root cells by binding to Al (Xia et al., 2013) (Figure 1).

The rice genome contains six homologs of the Arabidopsis STOP1 protein, one of which is ART1 (Yamaji et al., 2009). Among these homologs, ART2 has recently been identified as being regulated by ART1 and appears to play a smaller role in Al resistance than ART1 (Che et al., 2018), though its precise contribution remains unclear. The potential involvement of other ART1 homologs in Al resistance remains an open question. In contrast to the Arabidopsis stop1 mutant, which is highly sensitive to low pH (Iuchi et al., 2007), the rice art1 mutant shows no change in low-pH sensitivity (Yamaji et al., 2009). This suggests that the functions of ART1 and STOP1 may not be entirely conserved between rice and Arabidopsis. Notably, the closest homolog of STOP1 in the rice genome is not ART1 but rather another protein, Os01g0871200 (Yamaji et al., 2009), which we designate as ART1B. The potential role of ART1B in regulating low-pH tolerance, whether independently or in conjunction with other homologs, remains unknown and warrants further investigation.

Other plant species

STOP1 homologs have been identified in various plant species, including tobacco, soybean, sorghum, rice bean, tomato, and Physcomitrella patens, based on sequence homology (Ohyama et al., 2013; Fan et al., 2015; Huang et al., 2018; Wu et al., 2018; Zhou et al., 2018; Zhang et al., 2022). Only the homologs from tobacco, tomato, and Physcomitrella patens have been functionally characterized in vivo, and all have been shown to play critical roles in Al resistance (Ohyama et al., 2013; Zhang et al., 2022). In tobacco, knockdown of NtSTOP1 reduces the expression of NtMATE and NtALS3, resulting in decreased citrate exudation and reduced Al resistance (Ohyama et al., 2013). Similarly, knockout of SlSTOP1 in tomato reduces the expression of SlMATE3 and SlALS3, which also reduces Al resistance (Zhang et al., 2022). These findings suggest that the regulation of MATE and ALS3 homologs by STOP1 homologs is a conserved mechanism across plants. Although knockout of PpSTOP1 in Physcomitrella patens results in increased Al sensitivity, its impact on gene expression and organic acid exudation remains to be determined (Ohyama et al., 2013). Collectively, these findings indicate that STOP1 and its homologs play a conserved role in regulating Al resistance in plants.

Post-transcriptional regulation of STOP1

STOP1 mRNA expression is not affected by Al or low pH; however, STOP1’s downstream genes are strongly induced by Al (Iuchi et al., 2007; Sawaki et al., 2009). Recent studies have shown that Al, low pH, and iron toxicity promote the accumulation of STOP1 protein through post-transcriptional mechanisms (Zhang et al., 2019b; Godon et al., 2019; Tian et al., 2021). To investigate the post-transcriptional regulation of STOP1, our research group conducted a forward genetic screen using an ethyl methanesulfonate-mutagenized population of the ALMT1 promoter-driven luciferase (LUC) reporter (pALMT1:LUC) line, which is directly controlled by STOP1. This identified a series of rae mutants (regulation of ALMT1 expression) with altered LUC signals (Zhang et al., 2019b). Gene cloning and functional analysis of these RAE genes revealed multiple levels of post-transcriptional regulation of STOP1 (Figure 2).

Figure 2.

Post-transcriptional regulation of STOP1.

The zinc-finger transcription factor STOP1 is regulated at multiple post-transcriptional levels. RAE3/HPR1, a core subunit of the THO/TREX complex, controls the nucleocytoplasmic export of STOP1 mRNA, thereby influencing STOP1 accumulation. The F-box proteins RAE1 and RAH1 mediate STOP1 ubiquitination and degradation, whereas the SUMO E3 ligase SIZ1 and SUMO protease RAE5 (ESD4) facilitate STOP1 SUMOylation and deSUMOylation respectively, affecting its activity and/or stability. Al triggers the MEKK1–MKK1/2–MPK4 signaling cascade, which promotes STOP1 phosphorylation and protein accumulation. MPK3/6 may also contribute to the regulation of STOP1 phosphorylation. Additionally, Al induces hydrogen peroxide (H₂O₂) production, which promotes STOP1 oxidation and degradation, whereas the thioredoxin TRX1 catalyzes STOP1 reduction, protecting it from oxidative damage. It is also suggested that Al is detected by ALR1, which increases RBOHD phosphorylation and activity, thereby inhibiting RAE1-mediated STOP1 degradation. AtSTAR1, the nucleotide-binding protein of the AtSTAR1/ALS3 ABC transporter complex, interacts with and promotes STOP1 degradation. In tomato, the putative zinc-finger transcription factor SlSZP1 interacts with SlSTOP1, shielding it from degradation by the F-box protein SlRAE1. The question marks indicate unconfirmed processes.

RAE3 encodes HPR1, a core subunit of the THO/TREX (TRanscription and EXport) complex (Guo et al., 2020), which is involved in transcription, mRNA processing, and export (Katahira, 2012; Luna et al., 2012; Oeffinger and Zenklusen, 2012). Guo et al. (2020) demonstrated that RAE3/HPR1 regulates the nucleocytoplasmic export of STOP1 mRNA (Figure 2). Mutation of RAE3/HPR1 impairs STOP1 mRNA export, leading to reduced STOP1 protein accumulation and decreased Al resistance (Guo et al., 2020).RAE2/TEX1, which influences STOP1 accumulation and Al resistance, is also a core subunit of the THO/TREX complex (Zhu et al., 2021). Unlike RAE3/HPR3, RAE2/TEX1 does not affect STOP1 mRNA export, despite reduced STOP1 protein levels in the rae2/tex1 mutant (Zhu et al., 2021). The mechanism by which RAE2/TEX1 regulates STOP1 protein accumulation requires further investigation.

RAE1 encodes an F-box protein (Zhang et al., 2019b) that serves as a key component of the SKP1-CUL1-F-box ubiquitin ligase (E3) complex (Lechner et al., 2006; Callis, 2014). F-box proteins are known to selectively interact with target proteins, facilitating their delivery to the E3 complex for ubiquitination (Choi et al., 2014). Zhang et al., 2019a, Zhang et al., 2019b demonstrated that RAE1 directly interacts with STOP1, promoting its ubiquitination and subsequent degradation via the ubiquitin-26S proteasome pathway (Figure 2). Conversely, STOP1 binds to the RAE1 promoter to enhance its expression, establishing a negative feedback loop between STOP1 and RAE1 (Zhang et al., 2019b). Additionally, RAH1, the only homolog of RAE1, has a partially redundant role with RAE1 in mediating STOP1 ubiquitination and degradation (Fang et al., 2021b) (Figure 2). These findings highlight the importance of dynamic STOP1 regulation in balancing Al resistance and normal plant growth (Fang et al., 2021b).

RAE5 encodes the SUMO protease ESD4 (Fang et al., 2020), one of eight ubiquitin-like proteases (ULPs) identified in Arabidopsis (Castro et al., 2018). ULPs influence SUMOylation levels through their involvement in processing SUMO precursors and the deSUMOylation of target substrates (Murtas et al., 2003). RAE5/ESD4, but not other ULPs, specifically interacts with and deSUMOylates STOP1 (Fang et al., 2020). Mutation of ESD4 increases STOP1 SUMOylation, enhancing its association with the ALMT1 promoter, resulting in elevated ALMT1 expression and improved Al resistance (Fang et al., 2020). Notably, Al treatment promotes STOP1 deSUMOylation, likely through the Al-induced accumulation of RAE5/ESD4. This reduction in SUMOylation may facilitate the degradation of excess STOP1, allowing plants to modulate Al resistance responses when Al stress is alleviated. Furthermore, Fang et al. (2020) identified three mono-SUMOylated sites on STOP1 (K40, K212, and K395), which influence its stability and/or activity. Additionally, the SUMO E3 ligase SIZ1 mediates STOP1 SUMOylation (Fang et al., 2021a; Mercier et al., 2021; Xu et al., 2021) (Figure 2).

Beyond ubiquitin and SUMO modifications, Zhou et al. (2023) recently identified phosphorylation as crucial for STOP1 stability. They demonstrated that Al induces the kinase activity of MPK4, which phosphorylates STOP1 at Thr386, Ser448, and Ser486. This disrupts STOP1’s interaction with RAE1, enhancing Al resistance via increased STOP1 accumulation and the expression of STOP1-downstream genes (Zhou et al., 2023). Additionally, the upstream kinases MEKK1, MKK1, and MKK2 positively regulate STOP1 phosphorylation and accumulation, indicating that Al promotes STOP1 accumulation partially through the MEKK1–MKK1/2–MPK4 signaling cascade (Zhou et al., 2023) (Figure 2). Conversely, Liu et al. (2024) suggested that MPK3/6 also phosphorylates and stabilizes STOP1, regulating the root stem cell niche through negatively modulating auxin signaling in root tips. Given the contrasting roles of the MEKK1–MKK1/2–MPK4 and MKK4/5–MPK3/6 pathways in plant immunity and cold tolerance (Petersen et al., 2000; Asai et al., 2002; Teige et al., 2004; Suarez-Rodriguez et al., 2007; Gao et al., 2008; Qiu et al., 2008; Furuya et al., 2013; Meng and Zhang, 2013; Zhao et al., 2017), the cooperative regulation of STOP1 phosphorylation and accumulation by both MPK4 and MPK3/6 seems unlikely. Liu et al. (2024) used an NA-PP1 inducible knockout line, MPK3SR (genotype mpk3 mpk6 pMPK3:MPK3^TG), to assess the impact of mpk3 mpk6 double mutations on STOP1 accumulation, whereas Zhou et al. (2023) used the NA-PP1 inducible knockout line MPK6SR (genotype mpk3 mpk6 pMPK6:MPK6^YG) to investigate the impact of double mutations on the expression of STOP1-regulated genes. Liu et al. (2024) found that, with or without the use of NA-PP1 and the synthetic auxin naphthalene acetic acid, STOP1 accumulation increased in the MPK3SR line compared to wild type, whereas Zhou et al. (2023) reported no differences in the expression of STOP1-regulated genes between wild type and the MPK6SR line treated with or without NA-PP1 and Al. The reasons for these contradictory conclusions are unclear; one possibility is the use of different lines or experimental conditions. Future research should clarify the roles of MPK4 and MPK3/6 in regulating STOP1 accumulation.

More recently, Wei et al. (2024) found that STOP1 is subject to oxidative modification by H₂O₂ (Figure 2). The rae6 mutant showed reduced STOP1 accumulation and Al resistance. RAE6 encodes a mitochondria-localized pentatricopeptide repeat protein involved in mitochondrial nad5 splicing (Wei et al., 2024). This indicates that the rae6 mutation impairs complex I activity in the mitochondrial electron transport chain due to defective nad5 splicing, leading to H₂O₂ accumulation. Increased H₂O₂ levels promote the oxidation of STOP1 at residues C8, C27, and C185, enhancing its interaction with RAE1 and facilitating STOP1 degradation (Wei et al., 2024). Conversely, the thioredoxin TRX1 interacts with STOP1, catalyzing its reduction and promoting STOP1 accumulation and Al resistance (Figure 2).

In addition to post-translational modifications, recent studies have shown that STOP1 accumulation is negatively regulated by the tonoplast-localized AtSTAR1/ALS3 complex (Dong et al., 2017). Fan et al. (2024) demonstrated that the nucleotide-binding domain protein AtSTAR1 can also localize to the nucleus, interacting with and promoting STOP1 degradation, and the transmembrane domain protein ALS3 indirectly regulates STOP1 by modulating AtSTAR1 levels. STOP1 degradation by AtSTAR1 appears to occur via a pathway independent of RAE1/RAH1 (Fan et al., 2024), which warrants further investigation. Interestingly, Zhang et al. (2022) identified a new regulatory mechanism for SlSTOP1, the tomato homolog of Arabidopsis STOP1. They found that SlSTOP1 interacts with the putative zinc-finger transcription factor SlSZP1, which protects it from degradation by the F-box protein SlRAE1 (Zhang et al., 2022). Mutation of SlSZP1 reduced SlSTOP1 accumulation and increased Al sensitivity in tomato (Figure 2). Future research should investigate whether this SlSZP1-mediated protection of SlSTOP1 is conserved across other plant species.

Al sensing and STOP1 upstream signaling

Al³⁺ is one of the strongest Lewis acids, characterized by a high ionic index and low covalent interactions (Poschenrieder et al., 2008). These properties allow Al³⁺ to strongly interact with oxygen groups on organic molecules, leading to phytotoxic effects on cellular targets such as the cell wall, PM, and intracellular components (Ma, 2007). To mitigate Al toxicity, plants have developed mechanisms to detect the presence of Al³⁺ at different subcellular locations. Members of the ALMT1 subgroup, including TaALMT1, AtALMT1, and BnALMT1 from wheat, Arabidopsis, and rape, respectively, function as sensors for extracellular Al³⁺, which induces their transport activity (Sasaki et al., 2004; Hoekenga et al., 2006; Ligaba et al., 2006; Pineros et al., 2008; Zhang et al., 2008). Structural analysis by Wang et al., (2022a) revealed that Al³⁺ binds to three acidic amino acids of AtALMT1 (Asp49, Glu156, and Asp160), inducing conformational changes that open its extracellular gate and enhance malate transport (Wang et al., 2022a). Although ALMT1 is involved in sensing Al³⁺, it does not appear to participate in Al signaling.

Recently, Ding et al. (2024) identified the first plant Al ion sensor involved in Al signaling and proposed that the cytoplasmic kinase domain of the LRR (Leucine Rich Repeat) receptor-like kinase ALR1/PSKR1 is involved in Al ion binding and sensing. However, Al³⁺ concentration in the cytosol is negligible due to the high cytosolic pH (7–8), so it is unclear how ALR1 senses Al ions in this environment. One hypothesis is that ALR1 may bind to Al–hydrate species. The authors also proposed that ALR1 recruits the BAK1 co-receptor kinase to phosphorylate RBOHD (Respiratory Burst Oxidase Homolog), increasing the generation of reactive oxygen species (ROS). In turn, ROS oxidize RAE1 at the C364 site, inhibiting RAE1-mediated STOP1 degradation and increasing STOP1 accumulation (Ding et al., 2024). However, another recent study showed that H₂O₂, a primary ROS, negatively regulates STOP1 accumulation by promoting its oxidation and enhancing its interaction with RAE1 (Wei et al., 2024). In contrast to Ding et al. (2024), who reported significantly reduced Al resistance and STOP1 accumulation in rbohD mutants, Wei et al. (2024) found slightly increased Al resistance and STOP1 accumulation. Furthermore, Wei et al. assessed Al resistance and STOP1 accumulation using the rae6 mutant and CAT2 overexpression lines, which produce increased and decreased H₂O₂ levels, respectively. They found that Al resistance and STOP1 accumulation were reduced in the rae6 mutant and increased in CAT2 overexpression lines. Notably, introducing the rbohD mutation or CAT2 overexpression into the rae6 mutant background rescued the reduced Al resistance and STOP1 accumulation. These findings from Wei et al. (2024) strongly suggest that H₂O₂ reduces both STOP1 accumulation and Al resistance. The reasons for the contradictory conclusions in these two studies remain unclear; one possibility is that different experimental conditions were used. Future research should clarify the role of H₂O₂ in regulating STOP1 accumulation.

Regarding the phosphorylation and stabilization of STOP1 by the MEKK1–MKK2–MPK4 signaling cascade, the upstream mechanism by which plants sense Al to activate this cascade remains unknown (Zhou et al., 2023). In plant immunity, MAPK cascades transmit signals from receptor-like kinases to downstream targets (Pitzschke et al., 2009). Future studies should elucidate whether ALR1/PSKR1 and/or other receptor-like kinases participate in Al signaling upstream of the MEKK1–MKK2–MPK4 cascade. Additionally, pharmacological assays suggest that the phosphatidylinositol-specific phospholipase C (PI-PLC) pathway may have a potential role in Al signaling upstream of STOP1 (Wu et al., 2019; Tokizawa et al., 2021). However, conflicting reports suggest that Al may inhibit PI-PLC activity in vitro (Jones and Kochian, 1995) and reduce phosphatidic acid formation in Coffea arabica suspension cells, potentially through PI-PLC inhibition (Ramos-Diaz et al., 2007). Further research is needed to clarify how plants sense Al to regulate the PI-PLC pathway and influence STOP1 accumulation.

Concluding remarks and perspectives

Recent studies have highlighted the critical role of the zinc-finger transcription factor STOP1 in abiotic stress responses, significantly enhancing plant resilience to various environmental challenges. STOP1 is crucial not only in responding to Al toxicity but also in mitigating the effects of other stresses such as low pH, phosphate deficiency-induced iron toxicity, and deficiencies in potassium, nitrogen, boron, and oxygen (Balzergue et al., 2017; Mora-Macias et al., 2017; Enomoto et al., 2019; Sadhukhan et al., 2021; Wang et al., 2022b; Tokizawa et al., 2023; Zhang et al., 2024). Additionally, STOP1 has been implicated in the regulation of tolerance to salinity and excess ammonium levels (Sadhukhan et al., 2019, 2021; Tian et al., 2021). Among these stresses, Al toxicity remains a significant constraint on plant growth in acidic soils. STOP1 and its homologs play a critical and conserved role in regulating Al resistance across plant species.

Recent research has demonstrated that STOP1 and its homologs regulate Al resistance both externally and internally by inducing the expression of a suite of Al-resistance genes (Iuchi et al., 2007; Sawaki et al., 2009; Yamaji et al., 2009; Ohyama et al., 2013). In particular, MATE and ALS3 have emerged as common targets regulated by STOP1 and its homologs across various plant species, suggesting that STOP1-regulated mechanisms involving organic acid exudation and Al sequestration into vacuoles are widely conserved in plants. Despite these conserved mechanisms, there appears to be a divergence in the Al resistance strategies used by STOP1 and its homologs in dicotyledonous and monocotyledonous plants. In dicots, such as Arabidopsis, STOP1 predominantly facilitates Al resistance through organic acid exudation. However, in monocots such as rice, ART1 (a STOP1 homolog) is more prominent in internal Al sequestration. Moreover, monocots tend to have a greater number of STOP1 homologs than dicots, yet the closest homolog of Arabidopsis STOP1 in monocots does not seem to play a significant role in Al resistance. STOP1’s functional divergence between dicots and monocots warrants further investigation. Understanding these differences could offer new insights into the evolution of Al resistance mechanisms in plants and may inform breeding strategies aimed at enhancing Al resistance in crops.

Research has also revealed that STOP1 is regulated at multiple post-transcriptional levels, including nucleocytoplasmic mRNA export, and by post-translational modifications, such as ubiquitination, SUMOylation, phosphorylation, and oxidation. Al facilitates STOP1 accumulation, partly by activating the MEKK1–MKK1/2–MPK4 signaling cascade, which phosphorylates STOP1 at Thr386, Ser448, and Ser486 (Zhou et al., 2023). However, the MPK4-targeted phosphorylation sites in STOP1 are not conserved in rice ART1. Additionally, unlike STOP1, which regulates the expression of its downstream genes under both control and Al stress conditions, ART1 induces target gene expression specifically in the presence of Al (Iuchi et al., 2007; Sawaki et al., 2009; Yamaji et al., 2009). These findings suggest that Al regulates ART1 function in rice through mechanisms distinct from those in Arabidopsis STOP1, warranting further investigation.

Although ALR1 has been identified as the first Al ion sensor, its potential role in regulating STOP1 function through the RBOHD-mediated RAE1 pathway remains unconfirmed. It is established that Al triggers STOP1 accumulation by inducing the MEKK1–MKK1/2–MPK4 cascade, but the mechanism by which plants sense Al and transmit this signal to the MEKK1–MKK1/2–MPK4 pathway is still unclear. This connection could be mediated by receptor-like kinases, however, the identity and mechanism of any specific receptor-like kinase involved in Al sensing have yet to be determined. Additionally, STOP1 was recently reported to confer tolerance to calcium (Ca) deprivation by activating the expression of CCX1, which facilitates the flux of calcium ions (Ca²⁺) from the endoplasmic reticulum to the cytosol. Notably, Ca deprivation can promote STOP1 accumulation (Tian et al., 2024). Given the critical role of calcium ions as a second messenger in plant stress responses, exploring whether and how Ca²⁺ signaling contributes to Al-induced STOP1 accumulation would be an interesting avenue for future research.

Funding

This work was supported by the National Natural Science Foundation of China (grant no. 32170261 to C.-F.H.).

Acknowledgments

No conflict of interest is declared.

Author contributions

C.-F.H. designed the study and drafted the manuscript. Y.M. generated the figures and reviewed the manuscript.

Published: December 2, 2024