Introduction

Aluminum (Al) ranks as the third, most common element in the Earth’s crust, following oxygen (O) and silicon (Si). It is estimated to comprise approximately 7% of the Earth’s total mass (Bhalerao and Prabhu, 2013). In soils with a neutral pH, aluminum is bound in silicates, phosphates, and oxides (Chowra et al. 2017). However, when the pH drops below 5.5, Al solubilized, and the phytotoxic forms like [Al(H2O)6]3+, AlOH2+, AL(OH)3 (for simplicity named Al3+) are released from the minerals (Rahman and Upadhyaya 2021). The toxic dose of Al differs among plant species – for some plants like barley, even 10µM of bioavailable Al3+ can cause major damage (Szurman-Zubrzycka et al. 2021), whereas treatment of rice plants with 20 times higher dose of Al did not inhibit the growth (Wang et al. 2023). However, the activity of Al3+ is dependent on various factors such as deficiencies of key nutrition elements (especially concentration of phosphorous), pH, and others (Kochian et al. 2004), that is why it is hard to compare the Al doses used in different experiments. Nonetheless, it is worth emphasizing that there are programs available (such as GEOCHEM-EZ; Shaff et al. 2010) which enable the computing of the theoretical bioavailable Al3+ dose in a medium,tailored to real factors such as temperature, pH or ingredient concentrations.

Globally, an estimation of 4000 million hectares of cultivated land exhibit low pH, where aluminum is the primary factor contributing to reduced crop yield. Over half of acidic soils are concentrated in the tropics and subtropics regions, posing a threat to food security in many developing countries that are localized in these regions (Bian et al. 2013; Kochian et al. 2015; Szurman-Zubrzycka et al. 2021). Most of the acidic soils are found in the regions of southwestern Asia, central Africa, and South America, as well as in Australia, the eastern part of North America, and some parts of Europe (Wei et al. 2021). It is essential to highlight anthropogenic influences, including using fertilizers containing ammonia and industrialization leading to acid rain, accelerating the soil acidification process (Hajiboland et al. 2022). Therefore, studying plant response mechanisms to this significant environmental stress is crucial. This knowledge may contribute to developing new plant varieties that will be more tolerant to the phytotoxic Al3+ ions. Among the plant kingdom, there is diversity between species in Al tolerance. One of the most well-known aluminum-tolerant plants is hydrangea (Hydrangea macrophylla), which is also an Al-hyperaccumulator. In acid soils, Al3+ creates complexes with organic acid ions like citrate or malate ions present in the rhizosphere, which, upon entering the root, are transported to the aboveground, less sensitive part of the plant (Chen et al. 2022). In hydrangeas, aluminum accumulation can reach approximately 5 mg Al g−1 in the dry weight of leaves, resulting in the characteristic blue color of the flower petals (Ma et al. 1997). Some species, like tea (Camellia sinensis), require aluminum for the roots and shoot growth. Tea is a typical aluminum hyperaccumulator in tropical areas with predominant acid soils (Sun et al. 2020). The Al tolerance differs among the crop species. In Al-sensitive rice variety treatment with 200 µM of Al significantly decreased root length, however the inhibition of elongation processes was not observed (Wang et al. 2023). Similar results were observed in maize, as treatment with 100 µM of aluminum did not halt the root elongation (Kong et al. 2014). Nevertheless, barley has been shown to be highly sensitive to Al stress among crop species. Research has demonstrated that plants grown in a medium with micromolar Al concentrations exhibit strong inhibition of the root system (Szurman-Zubrzycka et al. 2021). Due to the varied responses to this abiotic stress among cereals, the problem of Al toxicity is pivotal.

This review paper aims to explore and discuss various aspects of the mechanisms underlying plant tolerance to aluminum. Specifically, the focus will be put on clarifying the mechanisms regulating the expression of genes associated with Al response pathways. Furthermore, an evaluation will be undertaken to understand how post-transcriptional and post-translational modifications (PTMs) influence the activity of pivotal elements that regulate Al response mechanisms. Subsequent sections of this paper will attempt to outline future perspectives to advance our understanding of plant tolerance to aluminum.

The influence of Al 3+ ions on the plants

Al binding sites in plants

To better understand the undesirable effect of Al on plants, a comprehensive analysis of the binding sites for Al3+ ions is essential. Aluminum ions can bind to cellular elements in symplast and apoplast. The first and most vulnerable organ to aluminum toxicity is the root – particularly the root tips, where the Al accumulation level is the highest. Within the root apex, the transition zone is the most sensitive part. The visible effect of Al3+ ions toxicity is the reduction of cell division within the root meristem and inhibition of cell elongation processes, consequently leading to root growth inhibition (Sharma and Dubey 2007; Silva 2012; Berenji S et al. 2017; Szurman-Zubrzycka et al. 2019; Rahman and Upadhyaya 2021; Hajiboland et al. 2022). The cell wall is a critical component in the perception of Al. Approximately 90% of Al3+ ions accumulate in cell walls, with pectins being the primary site of Al binding due to their negatively charged carboxylic groups (Gupta et al. 2013; Hajiboland et al. 2022). Al binding to the cell wall induces disruptions in cell wall extensibility, porosity, and enzymatic activity, thus inhibits root growth. Additionally, Al3+ ions can displace Ca2+ cations from the cell wall (Silva 2012). At the cellular level, Al can also bind to the negatively charged phospholipids present in the bilayer of the plasma membrane, leading to depolarization and alteration in cell membrane fluidity, permeability, and integrity (Chandra and Keshavkant 2021; Ofoe et al. 2023). Similar to their impact on the cell wall, Al ions can cause displacement of Ca cations within the cell membrane (Silva 2012). Furthermore, Al interaction with the plasma membrane alters the function of the H+-ATPase, which affects the H+ gradient. Also, Al toxicity can modify the transport of critical cations like Ca2+, Mg2+, and K+ (Gupta et al. 2013). Another symptom of Al stress includes increased callose accumulation at the plasma membrane, potentially leading to inhibition of plasmodesmata transport (Rahman and Upadhyaya 2021). Long-term exposure of plants to the Al stress results in the disruption of cytoskeleton architecture. The organization of cytoskeleton elements is crucial, as this organelle plays a significant role in cell divisions and other processes. The presence of Al may directly disrupt cytoskeleton dynamics by interacting with microtubules and microfilaments. Moreover, Al indirectly disrupts the Ca2+ ions signaling pathway, which is crucial in stabilizing the cytoskeleton structure (Gupta et al. 2013; Singh et al. 2017).

Aluminum stress is one of the factors contributing to the increased production of ROS (Reactive Oxygen Species), resulting in the creation of oxidative stress. Studies on monocotyledonous plants, such as rice (Oryza sativa), reveal that Al3+ ions generate ROS like H2O2 or O2− ions. The increased ROS production leads to the creation of DNA damage, lipid peroxidation, or even cell death in root cells (Bera et al. 2019; Rahman and Upadhyaya 2021). The disturbance of ROS homeostasis endangers the proper functioning of organisms. However, plants develop an advanced antioxidant system whose main role is the elimination of reactive oxygen species. In the recovery of Al-dependent, ROS-mediated damage several enzymes like POD (Peroxidase), SOD (Superoxide Dismutase), CAT (Catalase), APX (Ascorbate Peroxidase), GR (Glutathione Reductase), GPX (Glutathione Peroxidase) take part, and non-enzymatic compounds like GSH (glutathione), ascorbate and proline also participate in ROS scavenging (You and Chanz 2015; Singh et al. 2016; Ranjan et al. 2021). Additionally, Al ions, which are not bound in the cell wall and cell membrane, can enter the cell and directly cause DNA damage by binding to the negatively charged phosphoric acid residues in DNA. This leads to DNA and chromatin structure disruption, affecting DNA replication processes by increasing the rigidity of the double helix (Gupta et al. 2013; Singh et al. 2017). It has been confirmed that under low pH conditions, aluminum disturbs the genomic integrity, causing the formation of single and double-strand breaks in the root meristem cells in barley (Hordeum vulgare), Arabidopsis (Arabidopsis thaliana) or onion (Allium cepa) (Mohan Murali Achary et al. 2008; Chen et al. 2019b; Szurman-Zubrzycka et al. 2019). Recent studies demonstrate that aluminum leads to the induction of the DDR (DNA Damage Response) pathway, which is highly specialized in detecting DNA damage (Szurman-Zubrzycka et al. 2019, 2023). In dividing tissues, DNA damage results in cell cycle inhibition to activate the repair mechanisms. Due to these actions, progeny cells do not inherit damage that could negatively affect their function (Pedroza-Garcia et al. 2022). After affective DNA damage repair, the cell cycle becomes unlocked. Depending on the phase of the cycle in which DNA damage occurred, the cell may be arrested in the S phase or in the G2 phase (Nisa et al. 2019). For example, in barley, exposure to Al caused the increase of cells in the G2/M phase (Jaskowiak et al. 2018). Another issue worth addressing when analyzing the problem of Al genotoxicity is the conditions necessary for the formation of toxic Al3+ ions. Low pH, at which aluminum becomes phytotoxic, is itself a factor inducing oxidative stress, leading to DNA damage. Considering all these factors, arable soils with low pH pose a significant challenge for modern agriculture (Mahapatra and Roy 2020; Szurman-Zubrzycka et al. 2023).

Aluminum resistance mechanisms

Exclusion of OAs to prevent entering the Al into the root

The best-known mechanism of Al tolerance is the Al exclusion mechanism (Fig. 1), whose main aim is to prevent from entering the Al3+ ions from the soil to the root cells (Kochian et al. 2015). The pivotal component of these actions is the efflux of OAs (Organic Acids), primarily malate, citrate, and oxalate, depending on the plant species (Ofoe et al. 2023).When OAs are present in the rhizosphere, they can form non-toxic complexes with Al ions. This occurs because OAs are naturally anionic and readily chelate Al ions and hence detoxify them (Ma et al. 2001). Differences exist in the type of excluded organic acids among the monocot and dicot plant species. For instance, Zea mays, Vigna umbellate and Oryza sativa release citrate (Maron et al. 2013; Liu et al. 2016; Yokosho et al. 2016a; Huang et al. 2019) Glycine max, Brassica napus, and Brassica oleracea exclude malate (Ligaba et al. 2006; Liang et al. 2013; Zhang et al. 2018) and Camelia sinensis release oxalate (Ofoe et al. 2023). However, species like Hordeum vulgare, Arabidopsis thaliana, and Secale cereale release malate and citrate (Furukawa et al. 2007; Collins et al. 2008; Huang et al. 2012). Fagopyrum esculentum releases both malate and oxalate (Zheng et al. 2005; Lei et al. 2017).

Fig. 1
figure 1

Mechanisms of Al tolerance. A) Exclusion of organic acids (OAs) ions into the rhizosphere facilitated by ALMTs and MATEs transporters. B) internal mechanisms of Al tolerance involve the transportation of Al ions (free or in complex form) to the cytoplasm through Nrat1 and NIPs transporters. Subsequently, Al3+:OAs complexes can be transported to the vacuole via the ALS1 transporter, where they are sequestrated and detoxified. Moreover, modification in cell wall properties plays a crucial role in plant Al tolerance. Pectin methylation enhances Al tolerance by reducing Al binding sites within the cell wall. Furthermore, the efflux of UDP-glucose, facilitated by the STAR1/2 transporter, mask Al binding sites. ABC transporters may take part in both, the transport of Al ions and in the efflux of UDP-glucose. Possibly AtALS3 is involved in the relocation of Al3+ through the phloem, and it may create a complex with AtSTAR1 to facilitate the UDP-glucose efflux. C) Role of transcription factors (TFs) in the regulation of Al. ALMTs (Al-Activated Malate Transporter), MATEs (Multidrug And Toxic Compound Extrusion), Nrat1 (Nramp aluminum transporter 1), NIPs (Nodulin 26-like Intrinsic Protein), AtALS3 (Aluminium Sensitive 3), ALS1 (Aluminium Sensitive 1), STAR1/2 (Sensitive to Aluminium Rhizotoxicity),STOP1 (Sensitive To Proton Rhizotoxicity 1), XTH (xyloglucan endotransglucosylase-hydrolase), ELP (Extensin-Like Protein), FRDL (Ferric Reductase Defective Like). This figure was created using BioRender.com

Full size image

Transporters of organic acid anions belong to the two families of cell membrane transporters – the first is ALMT (Al-Activated Malate Transporters), whose function is the extrusion of malate anions from root cells. The second one is a subgroup of proteins belonging to the large MATE (Multidrug And Toxic Compound Extrusion) family. Specifically, this subset includes OA/H+ antiport transporters, which participate in the primary transport of citrate to rhizosphere (Delhaize et al. 2012; Kochian et al. 2015). The first evidence that anion transporters participate in Al-tolerance was described in the last decade of the XX century. Ryan et al. (1997) identified a novel anion channel whose function was malate efflux from wheat roots treated with Al. Initially, the ALMT1 gene was described in wheat and was constantly expressed in the root apices of the Al-tolerance line (Sasaki et al. 2004). One of the first genes related to citrate efflux that belong to the MATE family was identified in barley. The HvAACT1 (Aluminium Activated Citrate Transporter 1) encodes an Al-activated citrate transporter, which participates in Al tolerance in barley (Furukawa et al. 2007). In the case of oxalate transport, the specific transporters proteins are still undiscovered (Ofoe et al. 2023).

Two patterns of organic acid exudation, dependent on the secretion time, are evident in the plant kingdom. The pattern I is characterized by the immediate release of OAs upon the presence of Al3+ in the soil (Yang et al. 2013). The main aim of pattern I is to protect the plant immediately after Al stress through the rapid activation of anion channels on the cell membrane (Yan et al. 2022). A plausible explanation of pattern I phenomena posits that Al interacts with pre-existing anion channels, leading to the activation of the OAs transport without the need for the induction of genes encoding transporters (Sade et al. 2016; Ofoe et al. 2023). Moreover, it has been suggested that protein phosphorylation may be involved in malate efflux from root apex in response to toxic Al3+ ions in wheat. It has been found that treatment with 200 µM of Al caused temporary activation of 48-kD MAP-like kinase, most likely as an early response to Al stress. Moreover, the application of K-252a—an inhibitor of protein kinases, suppress a 48-kD activity, which results in a decrease in malate efflux and an increase in Al accumulation in the root (Osawa and Matsumoto 2001). Buckwheat (Fagopyrum esculentum) serves as an excellent example of a species where pattern I is observed because oxalic acid secretion from an Al-tolerant cultivar is activated 30 min after Al treatment (Zheng et al. 2005). Furthermore, in the Al-resistant wheat cultivar Altas, malate efflux is observed already 5 min after adding Al to the medium (Osawa and Matsumoto 2001).

Pattern II is characterized by a delayed secretion of organic acids in response to the presence of Al in the rhizosphere. Organic acid efflux becomes active several hours after exposure to aluminum. It has been suggested that the activation of organic acid transporters requires the induction of the expression of genes encoding these proteins (Ma 2007). Pattern II of organic acid exclusions is observed in Arabidopsis. Liu and colleagues (Liu et al. 2009) documented that Al treatment resulted in the release of a significant amount of malate acid from Arabidopsis roots within 24 h. It is in line with the results for the relative expression level of the AtALMT1(Al-Activated Malate Transporter 1) gene, which encodes malate transporter because the expression was the highest after one day of Al treatment (Liu et al. 2009). Similarly, in maize (Zea mays) it is observed that Al triggered the release of citrate from roots in a way typical for Pattern II to enhance tolerance for this element (Du et al. 2021).

Internal mechanisms of Al tolerance in plants

Given the highly phytotoxic nature of Al3+ ions, it would be reasonable to assume that plants have evolved mechanisms to prevent these cations from entering their cells. However, the effectiveness of organic acid exclusion mechanisms is insufficient in detoxifying Al3+ ions in the rhizosphere, so plants had to develop intracellular mechanisms of response to the Al exposure.

Different types of proteins participate in Al tolerance mechanisms in plants. One of the most crucial proteins involved in intracellular mechanisms of Al detoxification is Nrat1 (Nramp aluminum transporter 1), belonging to the Nramp (Natural Resistance-Associated Macrophage Protein) family. Nrat1 is a transporter localized in the plasma membrane of all root tip cells, with the function of transporting only Al3+ ions from the cell wall to the cytoplasm. It has been documented that this protein is crucial for the Al tolerance mechanism in rice. A knock-out mutation in the Nrat1 gene resulted in a decreased Al uptake and increased binding of Al ions to the cell wall in this species (Xia et al. 2010; Ofoe et al. 2023). Once Al3+ ions are present in the cytoplasm of the root cells, they are sequestrated and detoxified. Additionally, aquaporins, such as the AtNIP1;2 transporters belonging to the NIP (Nodulin 26-like Intrinsic Protein) family and localized in the plasma membrane of root cells, are also involved in the influx of Al into the cell (Hajiboland et al. 2022). In Arabidopsis, AtNIP1;2 function is transporting of Al complexes with malate from the rhizosphere. The expression of the AtNIP1;2 gene is mainly observed in the root tips, and Al treatment enhances the expression of this gene. Mutation within the AtNIP1;2 gene results in aluminum accumulation in the root cell wall, reducing Al tolerance (Wang et al. 2017). Moreover, there are more members of the aquaporin family that are possibly related to Al tolerance. In Hydrangea macrophylla, aluminum concentration in the sepal is enlarged, which is connected with the existence of Al transport. Two novel proteins have been identified – a tonoplast-localized HmVALT1 (Vacuolar Al Transporter 1) and a membrane-localized HmPALT1 (Plasma membrane Al Transporter 1). Expression of HmVALT1 and HmPALT1 is high in sepal tissue, and overexpression of these genes in Arabidopsis influences the Al resistance (Negishi et al. 2012).

Aluminum can be present in the cytoplasm as free ions or complexes with organic acids or phenolic compounds. Toxic Al3+ cations are removed to the vacuole, where Al can be sequestrated and neutralized, is necessary. This intercompartment transport is crucial for eliminating the Al from the cytoplasm, where Al ions negatively influence the cell structures and their functioning. The key players in internal Al transport are transporters belonging to the large family of ABC (ATP-Binding Cassette) proteins. ABC transporters have been identified in various organisms, such as bacteria, plants, and humans, and are responsible for transporting a wide range of molecules across plasma membranes. The hydrolysis of the ATP molecule drives the energy for transport (Chauhan et al. 2021). ABC transporters in plants are built with two Transmembrane Domains (TMDs) anchored to the membrane and two cytosolic Nucleotide-Binding Domains (NBDs). ABC proteins can be divided into full-length and half-size ABC transporters depending on the number of genes encoding TMD-NBD subunits. A full-size ABC transporter occurs when a single gene encodes a protein consisting of two subunits. The second type of ABC transporters—a half-size are also built by NBD and TMD domains, but they are encoded by two genes (Kochian et al. 2015). Many ABC transporters were identified in plants, and their role in Al response was documented. The first evidence that ABC transporters participate in Al tolerance was documented in the last decade of the twentieth century. Larsen and colleagues (Larsen et al. 1996) identified several als (Al-sensitive) mutants in Arabidopsis. These mutants were hyper-sensitive to Al. From all identified mutants, als1 and als3 showed an extreme response to treatment with Al. These changes were unrelated to the known Al response pathways and suggested a previously unknown mechanism activated during Al stress that was disrupted in the mutants. Map-based cloning of the als3 mutant resulted in the discovery of the previous unknown gene ALS3 (Aluminium Sensitive 3), which encodes a half-size ABC protein (TMD), participating in the Al tolerance mechanism. The possible function of this protein is the redistribution of accumulated Al3+ ions from sensitive root tissues to less sensitive shoots to protect from the negative influence of Al (Larsen et al. 1996, 1997, 2005). Another ABC transporter that takes part in Al response is ALS1, which is localized in the tonoplast. For the first time, the ALS1 gene was identified in Arabidopsis using a map-based cloning method. The ALS1 encodes protein, which function is crucial for the proper transport of OA: AL complexes to the vacuole, where they are sequestered (Larsen et al. 2007; Kochian et al. 2015; Yan et al. 2022; Ofoe et al. 2023). Interestingly, the regulation of expression of ALS1 after Al treatment differs among the mono- and dicot plants. In rice, expression of OsALS1 is induced only by Al treatment in low pH conditions, compared to the treatment with other metals like Cd or La. Application of different doses of Al (10, 30, and 100 µM) caused a threefold increase in the OsALS1 relative expression level compared to the control plants (Huang et al. 2012). In Arabidopsis, the expression of AtALS1 was not induced after Al treatment (Zhu et al. 2013). In barley (Hordeum vulgare), there are two paralogs of the HvALS1 gene – HvALS1.1 and HvALS1.2 with 53.5% identity at the gene level (HvALS1 proteins are identical in 92%). RNAseq analysis performed on barley cultivar ‘Sebastian’ after Al treatment revealed that expression of both paralogs was up-regulated by Al treatment (Szurman-Zubrzycka et al. 2021).

Other ABC transporters that are reported in rice as related to Al tolerance are STAR1 (Sensitive to Aluminium Rhizotoxicity) and STAR2 (Huang et al. 2009). These proteins are crucial in cell wall modification due to Al presence (Delhaize et al. 2012). The function of STAR1/2 is the efflux of UDP-glucose to modify and mask Al3+ binding sites within the cell wall (Hajiboland et al. 2022). STAR proteins were initially discovered in rice. The OsSTAR1 gene encodes the NBD, while OsSTAR2 encodes the TMD of the ABC transporter. Mutations in these genes resulted in hypersensitivity to Al cations, with significantly higher inhibition of root growth after Al treatment compared to wild-type plants. The expression of OsSTAR1 and OsSTAR2 was observed primarily in the root cells and was induced after Al treatment (Huang et al. 2009). In Arabidopsis, the ortholog of OsSTAR1, known as AtSTAR1, is present. Plants with knock-out mutations of the AtSTAR1 showed similar phenotypes to rice mutants because the root length after Al treatment was strongly reduced compared to the WT. The expression pattern of AtSTAR1 differs from the rice ortholog—AtSTAR1 is expressed in the outer cell layers of the root tips and in the developing leaves. Moreover, the protein, which is a potential ortholog of OsSTAR2 in Arabidopsis is AtALS3, which may interact with AtSTAR1 and lead to the creation of the AtSTAR1-AtALS3 complex. However, Huang et al. (2010) showed that there are some incompatible issues. Expression of AtALS3 was induced by Al stress (Larsen et al. 2005), however the expression of the AtSTAR1 was not (Huang et al. 2010). Additionally, the AtALS3 is also expressed at the phloem, whereas the AtSTAR1 undergoes expression in different places (Larsen et al. 2005; Huang et al. 2010).

Furthermore, different transporter present in tonoplast also takes part in the internal detoxification of aluminum. Ferroportin (FPN) in mammals participates in iron efflux. In Arabidopsis the orthologs of mammals FPN were found – IREG1 (Iron Regulated 1)/ FPN1 and IREG2/FN2, with a role in iron and cobalt transport (Morrissey et al. 2009). Interestingly, studies performed on Fagopyrum esculentum showed that FeIRG1 is involved in Al tolerance in roots. Expression of FeIREG1 was greatly enhanced in roots after Al, and overexpression of FeIREG1 in Arabidopsis resulted in elevated Al tolerance (Yokosho et al. 2016b).

The cell wall is the primary site of Al binding. Drawing on empirical evidence, a plausible hypothesis suggests a correlation between aluminum tolerance and specific aspects of the cell wall structure, such as the degree of pectin methylation, the configuration of the xyloglucan-cellulose network or masking the Al binding sites within the cell wall.

The methylation status of pectins is regulated by the activity of the pectin methylesterases (PME) enzyme. The function of PME is the demethylation of highly methylated pectins, which results in exposure to free carboxylic groups. After treatment with aluminum, Al3+ cations can easily bind to the newly created, free, negatively charged carboxyl groups. It has been observed that Al-tolerant cultivars have higher levels of methylated pectins and lower PME activity. This relation influences the Al tolerance in various plants because pectin methylation reduces the likelihood of Al binding to these elements (Zhang et al. 2019a, b; Hajiboland et al. 2022). This correlation has been confirmed in studies performed on rice. Analysis of PME activity revealed that, after Al treatment, the PME activity was higher in the Al-sensitive cultivar than in the Al-tolerance cultivar. Moreover, the proportion of demethylated pectins and free pectic acid residues in the cell wall also was higher in the Al-sensitive cultivar (Yang et al. 2011a). Interestingly, in studies performed on Fagopyrum tataricum it was revealed that the general pectin content in the cell wall may vary between Al-tolerant and Al-sensitive cultivars. In the Al-tolerant cultivar of Fagopyrum the pectin content was significantly lower than in the sensitive cultivar. Furthermore, the activity of the PME enzyme was higher in the sensitive cultivar compared to the Al-tolerant genotype (Yang et al. 2011b). In barley, it was shown by Jaskowiak et al. (2019) that Al influenced cell differentiation and led to the inhibition of root growth. Moreover, based on research conducted using an immunocytochemical approach, it was shown that after Al exposure, the fluorescence intensity of esterified homogalacturonans and methyl-esterified homogalacturonans was higher compared to the untreated plants (Jaskowiak et al. 2019).

The formation of hemicellulose (xyloglucan) – cellulose network is mediated by two enzymes – xyloglucan endohydrolase (XEH) and xyloglucan endotransglucosylase (XET), both encoded by xyloglucan endotransglucosylase-hydrolase (XTH) genes. XEH and XET function is the cleavage and rejoining of xyloglucan chains during cell expansion (Zhang et al. 2019a). In Arabidopsis studies, Al stress inhibited XET activity within 30 min after exposure, as observed through in vivo localization. Moreover, the performed analysis showed that inhibition of XET activity is caused on the transcriptional level and is essential in inhibiting root growth after exposure to the Al treatment (Yang et al. 2011a). Mutants in the XTH15 gene in Arabidopsis showed increased aluminum resistance. Interestingly, the xth15 mutant showed a lower accumulation of Al3+ cations in the cytosol compared to the WT. Furthermore, the relative expression level of the ALS1 gene was higher in xth15 than in the WT (Zhu et al. 2013). These findings suggested that the relation between the structure of the cell wall and internal mechanisms of Al tolerance may play an essential role in response to the phytotoxic Al ions.

It is well known that Al treatment causes an increase in ROS production. It has been found that overexpression of the peroxidase gene AtPrx64 in tobacco resulted in less root growth reduction after Al treatment, compared to the control. Moreover, line with overexpression had reduced accumulation of Al and ROS (Wu et al. 2017). In soybean, the relative expression levels of seven genes encoding POD were upregulated in the tolerant variety compared to the sensitive one after Al treatment (Liu et al. 2021). Moreover, in maize, a ZmAT6 (Aluminum Tolerance 6) gene encodes a protein with an unknown function. It has been found that the overexpression line was tolerant to Al and had a lower content of ROS. Additionally, the activity of scavenging enzymes, especially SOD, was increased in this line (Du et al. 2020). These results showed that the role of ROS detoxification is important in the mediation of Al tolerance in plants.

Transcription factors – key players in response to Al stress

In diverse biological processes, the expression of crucial genes is regulated by various proteins known as transcription factors (TFs). In the case of response to Al stress in low pH conditions, the expression of critical genes, like AtALMT, AtMATE, and AtALS1, is also regulated at the transcriptional level. The most important regulator of gene expression in response to Al is STOP1 (Sensitivity to Proton Rhizotoxicity 1) transcription factor. The STOP1 belongs to the C2H2 Zn-finger family of TFs and participates in the response to both low pH and Al stress (Kochian et al. 2015). STOP1 was first identified in a study aimed at understanding plant response to the proton (H+) stress treatment, using the dicots model plant – Arabidopsis as an example. The stop1 mutant was hypersensitive to H+ rhizotoxicity, and the STOP1 gene was identified using positional cloning. Additionally, it is interesting to note that during the study of stop1, it was revealed that this mutant was hypersensitive only to Al treatment, not to treatment with other metals, such as cadmium, copper, or lanthanum (Iuchi et al. 2007). Sawaki et al. (2009) described that missense mutation in the nucleotide-binding domain of STOP1 protein results in the down-regulation of various genes connected to response to Al stress. In Al-treated stop1 mutant, expression of AtALMT1 and ALS3 was lower when compared to WT treated with Al. The AtSTOP1 homolog was also identified in rice – it is ART1 (Al Resistance Transcription factor) protein. Microarray analysis revealed that ART1 regulates the expression of 31 genes related to Al tolerance, like OsSTAR1 and OsSTAR2 (Yamaji et al. 2009). In 2011, Tsutsui et al. identified a cis-acting element present in the promoter regions of 29 genes, whose expression is dependent on ART1—GGN(T/g/a/C)V(C/A/g)S(C/G) (Tsutsui et al. 2011). A decade later, another research team identified a STOP1-binding motif within the promoter region of the AtALMT1 gene, and it is longer than that identified in rice and consists of 11 nucleotides—GGGGAGGCTCT (Tokizawa et al. 2021). Furthermore, in different plant species like rye, rice bean, wheat, sorghum, soybean, and cotton, STOP1 homologs have also been found to participate in Al response (Garcia-Oliveira et al. 2013; Fan et al. 2015; Huang et al. 2018; Wu et al. 2018a, b; Kundu et al. 2019; Silva-Navas et al. 2021).

The comparable studies on the functional analysis of the STOP1 transcription factor reveal the absence of a universal pattern for regulating STOP1 activity during aluminum stress. The induction of STOP1 gene expression is not consistently observed in the response to Al treatment across monocots and dicots. In rice bean, for instance, a change in the relative expression level of VuSTOP1 was noted as early as two hours after the induction of Al stress. Specifically, the expression of VuSTOP1 increased five times in the basal part of the root four hours after treatment with 25 µM of Al (Fan et al. 2015). Sweet sorghum (Sorghum bicolor) is another plant species in which Al induces the expression of the STOP1 gene. In sorghum, which possesses four STOP1-like proteins, the expression of four SbSTOP1 genes was induced in root apices after treatment with 15 µM of Al ions (Huang et al. 2018). However, opposite results were observed for AtSTOP1, where no induction of expression after treatment with various doses of Al or in solutions with different pH occurred (Iuchi et al. 2007). Similar results were achieved for OsART1 as treatment with 50 µM of Al did not induce OsART1 expression in either the root tip or the basal part of the root (Yamaji et al. 2009). The same results, as in rice and Arabidopsis, were observed in different crop species like barley, rye, and partially, in wheat (Garcia-Oliveira et al. 2013; Silva-Navas et al. 2021; Szurman-Zubrzycka et al. 2021). Garcia-Oliveira and colleagues working on hexaploid wheat conducted the characterization of three TaSTOP1 homoeologues—TaSTOP1-A, TaSTOP1-B, and TaSTOP1-D. Notably, the expression of two of these homoeologues – TaSTOP1-B and TaATOP1-D, was not induced after treatment with Al. However, intriguingly, the transcript level of TaSTOP1-A after treatment with Al3+ ions was significantly higher than under control conditions (Garcia-Oliveira et al. 2013). It can be assumed that induction of STOP1 gene expression in response to the Al stress is species-specific, and there is no universal pattern of activation of STOP1 expression among different plant species.

Except for STOP1, few studies describe other transcription factors that play a role in regulating Al-tolerance mechanisms. One of them is ANAC017, which belongs to a large family of transcription factors—NAC (NAM (No Apical Meristem), ATAF1/2 (Arabidopsis Transcription Activation Factor), and CUC (Cup-shaped Cotyledon). The NAC family is specific only for plants, and some of their members participate in regulating Al tolerance. Functional analysis of ANAC017 showed that this TF is a negative regulator of Al response in Arabidopsis. The loss-of-function mutant showed enhanced Al tolerance compared to the WT because anac017 accumulated less Al in roots and cell walls, and the root elongation process was not inhibited by Al as strongly as in the WT. Moreover, it was demonstrated through the yeast one-hybrid screening that ANAC017 interacts only with the XTH31 promoter region and induct its expression (Tao et al. 2022). Besides ANAC017, another NAC transcription factor possibly takes part in the regulation of Al response. SOG1 (Suppressor of Gamma Response) is a key protein in the DDR (DNA Damage Response) pathway that regulates the expression of genes participating in various biological processes like DNA repair mechanisms. In Arabidopsis, sog1-7 mutant, after treatment with different doses of Al, showed higher tolerance than WT. Compared to the wild type, the sog1-7 root length was greater after treatment with AlCl3 (Sjogren et al. 2015). Other transcription factors that participate in the regulation of Arabidopsis's response to the Al stress are AtHB7 and AtHB12, members of the HD-ZIP I (HomeoDomain Zipper I) subfamily. These transcription factors have characteristic DNA-binding homeodomain and Leucine Zipper motif, which participates in protein-dimer formation. HD-ZIPs are essential in developmental processes like vascular tissue or trichome development. Liu and colleagues showed that AtHB7 and AtHB12 during Al stress act as antagonists to control root growth via opposite regulation of processes related with cell wall capacity to bind Al ions. It has been shown that together with overexpression of the AtHB7 gene (35S:AtHB7), the reduction of root growth and accumulation of Al3+ ions within cell walls were lower after aluminium treatment compared to the control genotype. However, in the case of the AtHB12 gene, the overexpression line showed the opposite phenotype than 35S:AtHB7 because the root growth inhibition was elevated after Al stress, and these plants accumulated more Al ions in the cell wall than 35S:AtHB7. It is possible that this effect may arise from the regulation of the expression of different genes, which is crucial to determine the plant response to Al stress (Henriksson et al. 2005; Liu et al. 2020a, b). Several studies showed that different members of the WRKY family of TFs take part in the regulation Al response mechanisms in plants. The WRKY family is one of the largest family of transcription factors. They can act both as an activators or repressors of transcription in various biological processes, like abiotic stress responses, plant disease resistance, or embryogenesis (Bakshi and Oelmüller 2014). The study conducted by Li et al. (2020) showed on Arabidopsis plants, that the WRKY47 was involved in the regulation of Al tolerance. In the loss-of-function wrky47 mutant, the disruption of Al redistribution was evident, manifested by an increase in Al content within the symplast and decrease in apoplast. These results related to an interruption in Al binding within cell walls as a result of the reduction of hemicellulose content. Furthermore, it was documented that WRKY47 can regulate genes such as ELP (Extensin-Like Protein) and XTH17, which encode the proteins participating in the modification of cell wall proprieties (Li et al. 2020). Another protein that belongs to the WRKY family and participates in response to Al is WRKY46. In Arabidopsis, AtWRKY plays a role as a negative regulator of malate secretion through the direct inhibition of AtALMT1 expression. It has been shown that insertional mutant wrky46 exhibited greater tolerance to Al than WT with increased root malate secretion (Ding et al. 2013). In rice, it is also documented that WRKY protein takes part in regulating Al response. The OsWRKY22 positively regulates tolerance to Al via induction of the OsFRDL4 (Ferric Reductase Defective Like 4) gene expression. The OsFRDL4 encodes the citrate transporter and participates in the effective transport of citrate to the rhizosphere. Knock-out mutation of the OsWRKY22 gene resulted in the reduction of citrate efflux and, hence, increased Al-sensitivity. These results showed that in rice, besides OsART1, other TFs can regulate the expression of crucial Al response genes (Li et al. 2018).

Regulation of STOP1 activity is crucial for Al tolerance

As indicated earlier, STOP1 is the key transcription factor involved in Al response, and its expression (transcription) pattern differs between various plant species. However, the expression of genes can be regulated not only on the transcriptional level, but there are two additional levels of regulation – the mRNA might also be regulated post-transcriptionally, and encoded proteins are often regulated post-translationally. In the case of numerous plant species, the activity of STOP1 is regulated at the mRNA and/or protein level (Tab.1).

Table 1 Post-transcriptional and post-translational regulation of STOP1 activity
Full size table

Post-transcriptional regulation of STOP1 activity

During post-transcriptional regulation of STOP1, the mechanism of mRNA export through NPC (Nuclear Pore Complexes) is mediated via the nuclear protein complex—THO/TREX, which has been identified in many organisms, from yeast to plants and mammals. The TREX (Transcript Export) complex, which consists of the THO core, is involved in the processes that link transcription elongation with the maturation of the 3’ end of mRNA and, finally, mRNA transport from the nucleus to the cytoplasm (Luna et al. 2012). In yeast, the THO element is composed of Tho2, Hpr1, Mft1, and Thp2 subunits. THO's stability depends on proper interactions between these four subunits, and eliminating even one of them results in the demolition of the THO function (Luna et al. 2012; Oeffinger and Zenklusen 2012). In 2010, the first papers confirming that the THO/TREX complex exists in plants were published (Furumizu et al. 2010; Jauvion et al. 2010; Yelina et al. 2010). The structure of the THO/TREX complex in plants differs from yeast. In plants, the main subunits are HPR1/THO1/EMU, THO2, TEX1/THO3, THO5A/B,THO6 and THO7A/B (Furumizu et al. 2010; Jauvion et al. 2010; Yelina et al. 2010; Francisco-Mangilet et al. 2015; Guo et al. 2020). Guo and colleagues showed that HPR1 (Hyperrecombination protein 1), which is a core element of the THO/TREX complex, is responsible for the nucleocytoplasmic transport of STOP1 mRNA in Arabidopsis. It has been found that in the hpr1 mutant, the expression of Al resistance genes like AtALMT1, AtMATE, or AtALS3 was down-regulated. Moreover, plants have reduced tolerance to Al treatment—the root growth was strongly inhibited by aluminum stress than in the case of WT. The results showed that HPR1 is crucial for the effective export of STOP1 mRNA from the nucleus to the cytoplasm, where translation occurs, which enables the proper activity of STOP1 in the regulation of expression of down-stream Al-resistance genes (Guo et al. 2020).

Post-translational modifications are essential for the regulation of STOP1 activity

Post-translational modifications (PTMs) of proteins are important mechanisms regulating plant proteome in response to stressful conditions. They play an essential role in changing the protein structure. PTMs regulate miscellaneous cellular processes like signal transduction, protein–protein interactions, cell–cell interactions, and gene expression (Singh et al. 2022). PTMs are based on proteolytic cleavage followed by adding a modification group like acetyl, methyl, ubiquitin, SUMO, or phosphoryl groups to the particular aminoacids in the protein (Ramazi and Zahiri 2021). Various PTMs, such as ubiquitination, SUMOylation, and phosphorylation, are involved in the regulation of STOP1 activity.

Protein ubiquitination is the third, most abundant PTM process, and like other PTMs, is reversible. The primary function of this mechanism is the creation of a covalent bond between the C-terminal of the ubiquitin molecule and the N-terminal of the target protein. After this event, ubiquitinated protein is directed to degradation by the UPS (Ubiquitin 26S Proteasome System) (Ramazi and Zahiri 2021; Singh et al. 2022). Diverse classes of enzymes are involved in the process of ubiquitination. Attaching the ubiquitin molecule to the target protein is facilitated by a cascade of reactions involving the following steps: (1) ubiquitin-activation (mediated by UBA; E1 protein), (2) ubiquitin conjugation (mediated by UBC; E2), and (3) ubiquitin ligation (mediated by E3 ligases) (Singh et al. 2022). Many families of proteins have been distinguished among the E3 ligases. The best-known complex involved in ubiquitin ligation is the SCF (Skp1-CUL1-F-BOX). The core of the SCF complex is composed of four subunits: SKP1, Cullin1, RBX1, and F-box protein. Zhang and colleagues (2019) showed in that F-box protein RAE1 (Regulation of AtALMT1 Expression 1) regulates the STOP1 activity in Arabidopsis. The main function of the F-box proteins is interaction with target proteins that will be ubiquitinated and connection them with the ubiquitination complex. This study has proven that STOP1 degradation via 26-S proteasome ubiquitination is mediated by interactions of RAE1 with the STOP1 transcription factor. Moreover, it has been shown that RAE1 and STOP1 are functionally dependent via a negative feedback loop. Aluminum stress increases the accumulation of STOP1 protein, which can recognize and bind to the RAE1 promoter region. This results in positive regulation of RAE1 expression. Due to these actions, the RAE1 protein directly binds to the STOP1 in planta and promotes STOP1 degradation (Zhang et al. 2019b). In Arabidopsis, the homolog of the RAE1 protein has been found – RAH1 (RAE1 Homolog 1), which, together with RAE1, regulates the stability of STOP1 and, subsequently, Al resistance. RAH1, as an RAE1, can also directly bind to the STOP1 and promote STOP1 ubiquitination. However, the contribution of RAH1 and RAE1 in STOP1 degradation is unequal – these proteins maintain redundancy—RAE1 appears to be more important in the STOP1 degradation process. In the double mutant rah1rae1, the STOP1 accumulation level in plants grown in control conditions and treated with Al was similar. Furthermore, the rah1rae1 mutant showed reduced root length already in control conditions, without Al stress. These results suggested that when the Al stress is not present, RAH1 and RAE1 direct STOP1 into degradation to ensure normal root growth. However, during Al treatment, the STOP1 level increases due to the inhibition of STOP1 degradation. One of the possible explanations of this phenomenon is that under toxic conditions related to the presence of Al3+ ions, the structure of the STOP1 transcription factor may be changed, for example, via phosphorylation. This modification can prevent STOP1 degradation mediated by RAE1 and RAH1 and increase the level of STOP1 accumulation, which is crucial to activating the expression of genes participating in the response to the Al stress. Taken together, RAH1 and RAE1 in Arabidopsis play a crucial role in maintaining Al resistance via regulation of STOP1 accumulation (Fang et al. 2021).

Another PTM that is crucial for the proper functioning of STOP1 is SUMOylation. In the past, it was thought that the SUMOylation function was like ubiquitination—as a mediator in protein degradation. However, recent studies have shown that SUMOylation also activates some target proteins. The reversibility of the SUMOylation process is crucial in determining the status of the target protein, influencing whether it remains in an active form or becomes a target for degradation. During the SUMOylation, the C-terminal glycine residues of SUMO (Small Ubiquitin-like Modifier) molecules are attached to the lysine residues of the target protein. This reaction is mediated via an enzymatic complex. The SUMOylation process can be divided into three steps: (1) activation of SUMO molecule, (2) transport of activated SUMO molecule to conjugation complex, and (3) transfer of SUMO to the target protein. The first step is mediated by SAE (SUMO-Activating Enzyme Complex) or E1. The activation of the SUMO molecule is dependent on ATP. In Arabidopsis, the SAE complex is composed of two subunits – large subunit SAE2 and small subunit SAE1. The activated SUMO molecule is transferred to the core of the SUMOylation process – to the SCE (SUMO-Conjugating Enzymes) or E2. The primary function of SCE is creating a linkage (directly or indirectly) between active SUMO molecules and SUMO ligases E3, which participate in the attachment of the SUMO to the target protein. Moreover, proteins can be poly-SUMOylated, which is controlled by E4 ligases like PIAL1 or PIAL2. The reversibility of SUMOylation is mediated by DSPs (deSUMOylating proteases), which cleave the linkage between protein and SUMO. It has been suggested that plants have more DSPs than E3 ligases. This leads to the hypothesis that the removal of SUMO is more restricted and selective than the attachment to the target (Augustine and Vierstra 2018; Han et al. 2021; Sharma et al. 2021; Singh et al. 2022).

The knowledge of how STOP1 activity is regulated by SUMOylation and deSUMOylation is limited and provides a perspective for future research. Some studies provide insights into the roles of specific components of the SUMOylation pathway in the regulation of STOP1 activity. The ESD4 protease, for example, is involved in the deSUMOylation process of this transcription factor, which is increased in response to Al stress. Fang and colleagues (2020) found that in Arabidopsis STOP1 can be SUMOylated in three different K (Lys) residues – K40, K212, or K395, and ESD4 mediates the deSUMOylation of these sites. Consequently, the esd4 mutant, in which ESD4 protease was inactive, had an increased STOP1 SUMOylation level and, interestingly, it was found to be Al-tolerant because of an increased level of malate exudation in response to Al. Surprisingly, the level of STOP1 SUMOylation did not affect the STOP1 accumulation, whereas it affected the level of expression of STOP1 target genes, such as AtALMT1 or AtMATE. What was even more surprising, after Al treatment, the expression level of AtALMT1 was higher in the esd4 mutant than in WT (which is in line with esd4 being Al-tolerant) but, concomitantly, the expression of AtMATE was lower in the mutant than in WT treated with Al. The researchers proposed that mutation of ESD4 does not affect STOP1 abundance, however, an increased STOP1 SUMOylation level affects protein stability and has different effects on the association with the promoters of different target genes (Fang et al. 2020).

Another protein involved in SUMOylation is the SUMO E3 ligase SIZ1. This enzyme facilitates the attachment of SUMO molecules to the STOP1 in plants, primarily targeting the K40 and K212 residues. SIZ1 ligase is recognized as a negative regulator of Al tolerance in plants. A suggested signal transduction pathway in response to Al treatment involves SIZ1-STOP1-ALMT1, playing a crucial role. After the reception of the signal created in stress conditions related to the presence of phytotoxic Al3+ ions, the level of SIZ1 protein is decreased. This action (together with ESD4 being increased) downregulates the STOP1 SUMOylation process at K40 and K212 residues. It leads to increased ALMT1 expression, which is crucial for Al exclusion in the rhizosphere via malate efflux from the roots to the rhizosphere (Xu et al. 2021). Arabidopsis esd4 and siz1 mutants show similar Al-tolerant phenotypes. This is very surprising because these enzymes have opposite modes of action—SIZ1 facilitates SUMOylation, whereas ESD4 facilitates deSUMOylation. It can be speculated that ESD4 and SIZ1 may regulate STOP1 through the recognition of different target residues—it was shown that for both the target residues are K40 and K212, whereas ESD4 facilitates also the deSUMOylation of K395 (Fang et al. 2020; Xu et al. 2021). These findings demonstrate a complex and multifaceted impact of SUMOylation on STOP1 activity and function. Potentially, revealing the specifics of STOP1 regulation by SUMOylation could pave the way for developing Al tolerance in agricultural plants in the future.

Recent studies have revealed that phosphorylation plays a role in regulating STOP1 activity in the presence of Al stress. This PTM is one of the most extensively studied modifications and is crucial for better understanding how enzymes, membrane channels, and other proteins like transcription factors are regulated both in prokaryotes and eukaryotes. During phosphorylation mechanisms, the phosphate group is transferred to the receptor residues within the target molecule, and kinase enzymes mediate it. Mostly, the phosphorylation sites are S (Ser), T (Thr), Y (Tyr), and H (His) (Ramazi and Zahiri 2021). The MAPK (Mitogen-Activated Protein Kinase) cascade regulates plant signalling (intracellular or extracellular) and is crucial for regulating cell organization and gene expression in response to signals generated by external stimuli. In signal transduction mediated by MAPK cascades, three types of kinases take part: MAPKKKs (MAP Kinase Kinase Kinases; different name MEKKs), MKKs (MAP Kinase Kinases; different name MEKs) and MAPKs (MAP Kinases; different name MPK) (Jagodzik et al. 2018). In 2023, it was suggested that MAPKs cascade plays a role in positive regulation of aluminum resistance. The cascade involved MEKK1-MKK1/2-MPK4, which phosphorylate STOP1 and enhance response to Al. Aluminum stress causes an increase in MPK4 kinase activity, resulting in the phosphorylation of STOP1 at Thr386, Ser448, and Ser486. These actions disrupt STOP1’s interaction with RAE1, preventing its degradation. This enhances STOP1 stability and accumulation, positively regulating the expression of STOP1-dependent genes (Zhou et al. 2023).

Future perspectives on Al tolerance mechanisms in plants

In recent years, research efforts have been increasing to uncover new aluminum tolerance mechanisms. Currently, the main actions taken by farmers in the fight against aluminum toxicity involve limning to raise soil pH. Unfortunately, this method is not the optimal solution, because increase of soil pH is short-term and come with high costs. Considering that stress from phytotoxic Al3+ ions ranks as the second most significant abiotic stress (after a drought) in the acidic areas, continually improving our understanding of the potential solutions to this problem is crucial. This involves upgrading our knowledge and developing better tools to mitigate the negative effects of aluminum on plants.

Polyamines—a new player in the regulation of Al tolerance mechanisms

One of the potential solutions to mitigate the negative impact of Al is the use of polyamines. Polyamines (PAs) are organic polycations found in all organisms, characterized by aliphatic nitrogenous bases with two or more amino groups (Mustafavi et al. 2018; Chen et al. 2019a, b). PAs play a regulatory role in various biological processes in plants, including growth, development, and responses to biotic and abiotic stresses (Takahashi 2020). They regulate specific cellular processes such as cell division and expansion or nucleic acids and protein synthesis (Masson et al. 2017). Polyamines are spread throughout the plant cells, and they can be detected in different organelles like vacuoles, plastids, and mitochondria or can be present in the cell walls or cytoplasm (Mustafavi et al. 2018). PAs can exist in the organisms in different forms: (1) free (F-PAs);

(2) covalently conjugated (CC-PAs); and (3) non-covalently conjugated (NCC-PAs). The most common PAs form in plants is F-PAs, which can easily be translocated within cells due to their solubility in water (Mustafavi et al. 2018; Chen et al. 2019a). NCC-PAs function is associated with the regulation of enzyme activity, DNA replication, and regulation of gene transcription (Chen et al. 2019a, b). In higher organisms, the most common PAs are putrescine (Put) with two amino groups, spermidine (Spd) with three amino groups, and spermine (Spm) with four amino groups (Kaur-Sawhney et al. 2003). Put serves as a precursor in the biosynthesis of Spd and Spm and is the main polyamine involved in stress response. The manipulation of the polyamine metabolism has direct application in agriculture, serving as a useful technique in plant protection against various abiotic stresses or diseases (Tiburcio et al. 2014; Masson et al. 2017).

Putrescine's impact on the regulation of growth and development has been known for a long time. This compound primarily responds to abiotic stresses by scavenging the free radicals, modulation of ABA levels, preventing lipid peroxidation, and regulating cationic channel activity (González-Hernández et al. 2022). Furthermore, it has been observed that these properties can be effectively utilized to mitigate stress associated with phytotoxic Al3+ ions. The initial exploration of Put’s potential in alleviating Al toxicity dates back to 2016 when Yu and colleagues investigated its role in two wheat genotypes—one Al tolerant and another Al sensitive (Yu et al. 2016). It has been documented that Put (in different doses) successfully alleviated root growth inhibition induced by exposure to 30 µM of Al. It was evident that the alleviating effect was not correlated with the induction of expression of genes related to the secretion of organic acids like—TaALMT or TaMATE. Under Al stress conditions, ethylene level increased quickly. Putrescine treatment negatively affected ethylene biosynthesis, rendering it ineffective as a negative regulator of root elongation processes (Yu et al. 2016). Aluminum phytotoxicity is one of the factors contributing to the increase of ROS production, resulting in the creation of oxidative stress. In wheat, Al treatment caused oxidative stress, which led to oxidative damage like plasma damage in the root apex cells. Exogenous application of Put reduced oxidative damage in root cells by reducing H2O2 levels. During Al stress, polyamines like Spd are oxidized and are essential sources of H2O2. However, the application of Put can inhibit the oxidation process of Spd and alleviate the oxidative damage via the reduction of ROS accumulation and protect plasma membrane integrity (Yu et al. 2018). Also, it has been described that Al promotes the accumulation of bounded Put, and it was related to the increase of Al tolerance in wheat by maintaining the antioxidant capacity. The increase of bounded Put accumulation was directly proportional to Al exposure time in the Al-tolerant wheat genotype. Applying an inhibitor that stopped the accumulation of bounded Put led to a decrease in the root elongation process and an increase in callose production. Moreover, higher levels of accumulated, bounded Put maintains the redox homeostasis and mitigates the presence of oxidative damage. Treatment of Al tolerant wheat genotype with an inhibitor of bounded Put accumulation led to increased oxidative damage mediated by accumulation of ROS. These results suggested that the bounded Put form prevents ROS hyperaccumulation (Yu et al. 2019).

One of the Al binding sites is the cell wall. Changing the properties of the cell wall by Al leads to the inhibition of cell elongation. Modifications of cell wall structure may be considered as a mechanism of Al tolerance. One of the proposed modifications is an accumulation of hydroxycinnamic acids (HCAs) that alter the hemicellulose and lignin structure. The 4-coumarate: coenzyme A ligase (4CL4) regulates lignin biosynthesis. Mutation within the gene that encodes this enzyme led to an increased accumulation of lignin substrates – 4-coumaric acid and ferulic acid. The accumulation of lignin substrates led to reduced Al binding capacity to the cell wall and enhances the Al tolerance (Liu et al. 2020a).

Gao and coworkers (2023) described a novel pathway for regulating Al resistance in rice, wherein the ART1 transcription factor and bounded Put played a pivotal role. The study documented that exogenous application of bounded Put resulted in decreased Al binding within the cell wall of art1 and reversed the mutant Al-hypersensitivity. Furthermore, it was suggested that proper transformation of free form to bounded Put is crucial to Al resistance in rice. RNA-seq analysis revealed that the OsMYB30 gene, encoding the transcription factor, which acts as a negative regulator of Al tolerance, is controlled by ART1. The OsMYB30 induces the expression of the gene encoding the different 4-coumarate: coenzyme A ligase – Os4CL5, which can regulate the accumulation of PA and influence the Al capacity to cell wall binding. During Al stress, OsMYB30 expression can be inhibited through various mechanisms – directly via ART1 binding to the OsMYB30 promoter region or indirectly via reduction of H2O2 metabolism. The regulation of the relative expression level of the OsMYB30 gene is related to H2O2 presence, and the Put application regulates it through the increase of expression of peroxidase-encoding genes. These results showed that in plants, the novel Al resistance mechanism is present in which ART1, in a Put-mediated manner, regulates the repression of OsMYB30 expression (Gao et al. 2023). The research focused on using polyamines like Put to alleviate Al-induced stress symptoms is one of the prospects aimed at obtaining a powerful tool in the fight against the Al toxicity problem in acidic soils.

miRNAs – keys to plant tolerance against Al stress?

Over the past two decades, there has been extensive research into the role of microRNA (miRNA) in various biological processes. miRNAs, small RNA molecules (approximately 21–24 nucleotides long) are encoded by noncoding RNA genes called MIR genes located in intergenic regions (Xu and Chen 2023). Recent studies have documented that miRNAs play a role as post-transcriptional regulators of gene expression to control plant growth and development during stress conditions. miRNA can modulate gene expression through different mechanisms, including direct mRNA cleavage, translational repression, remodeling of chromatin, and DNA methylation (Shriram et al. 2016). The biogenesis of the miRNA is a multistep process that begins with the transcription of MIR genes by DNA-dependent RNA Polymerase II (Pol II) and forming a double-stranded stem-loop precursor of miRNA called pri-mRNA. The next step is recognition by processing a complex composed of DCLs (Dicer-like RNase III) endoribonuclease and two cofactors—SE (Serrate) and HYL1 (Hyponastic leaves 1) and activation of two nuclear processing steps. In these processes, DCL endoribonuclease mediates the cleavage of pri-miRNA, and SE HYL1 proteins control the efficiency and correctness of this mechanism. Then, the pri-miRNA precursor is converted to the miRNA/miRNA* duplex, which has to be methylated at the 3’ end by the HEN1 (Hua Enhancer 1) enzyme to protect it from RNA exonuclease-mediated degradation. The methylated miRNA/miRNA* structure is transported by plant homolog of the EXPO5 (Exportin 5) animal protein – HST (Hasty) from the nucleus to the cytoplasm. Afterward, the guide strand – miRNA is loaded to the AGO (Argonaute) protein to form the RISC (RNA- Induced Silencing Complex) complex. The miRNA* strand, also known as the passenger strand, must be removed and degraded. The formation of the RISC complex is crucial for target cleavage and/or translation inhibition. In RISC complex miRNA acts like a “navigator” to search the target through strand complementation and mediate the gene silencing (Chen 2005; Shriram et al. 2016; Wang et al. 2019; Zhang et al. 2022; Xu and Chen 2023).

In numerous studies conducted on various species of mono- and dicotyledonous plants, it has been discovered that miRNAs play a regulatory role in response to various abiotic stresses, for example, in heat (Ahmed et al. 2019; Tsai et al. 2023), drought stress (Eldem et al. 2012; Xia et al. 2020) or heavy metal stresses (Gao et al. 2019; Nie et al. 2021). Subsequently, the study has been performed to understand miRNAs' role in regulating the response to Al toxicity. Kong and coworkers (2014) performed a comprehensive analysis to elucidate the potential influence of miRNA in the response to the Al in Zea mays roots. Remarkably, the expression patterns of miRNAs in various types of maize roots have been observed to differ, and crown roots demonstrated the highest resistance to Al stress. The authors of this study suggest that these differential miRNA expressions likely play a pivotal role in responding to Al stress. Interestingly, they identified a potential target for zma-miR528 – a member of the MATE transporter family. Notably, in the crown roots, which exhibited the highest tolerance to Al stress, the expression level of zma-miR528 was found to be down-regulated after Al treatment. Moreover, the expression of the potential-miR528 target, which is a member of the MATE family, was the highest in crown roots after Al treatment (Kong et al. 2014). In barley, characterized as one of the most Al-sensitive crops among other plant species, it has been found that miR393 regulates Al response. miR393 is an RNA molecule present in various plants and it regulates the expression of genes that participate in auxin signaling, like TIR1 (Transport Inhibitor Response 1) or AFB (Auxin-signalling F-box). The miR393-mediated negative regulation of auxin signaling-related pathway is conserved across plants. Overexpression of miR393 in barley line demonstrated alleviated root growth inhibition after Al treatment, indicating its novel regulatory role in responding to Al treatment through the modulation of auxin signaling in roots. After 24 h of Al treatment, the relative expression level of miR393 was downregulated, whereas the expression of miR393 targets – HvAFB and HvTIR1 was upregulated. These findings suggest that inhibition of miR393 expression may be an early response to the Al treatment, resulting in a change of auxin signaling. Furthermore, exogenous application of NAA to the plants growing in an Al-containing medium reinforced root growth inhibition, affirming the role of auxin in the Al-induced reduction of root growth. The miR393-mediated alteration of auxin-signalling can effectively regulate root sensitivity during Al stress in barley. Additionally, the authors proposed that miR393 acts as a regulator of cell-wall-related genes to regulate root cell elongation under Al stress. The up-regulation of HvPMEI and HvEXP expression in line with miR393 overexpression may contribute to changes in cell elongation processes and cell wall modification, thereby leading to a reduction of Al uptake in root cells (Bai et al. 2017).

Another research, which confirmed the role of the miRNAs in regulating Al response, was performed for Tibetan wild barley known as XZ29, which was Al-tolerant. The authors identified 50 miRNAs governing the Al response in Tibetan wild barley in this study. Among these, 17 showed increased expression, while 24 miRNAs exhibited decreased expression. Notably, two novel miRNAs within this identified group exclusively participated in regulating Al tolerance in Tibetan wild barley. The first of these novel miRNAs, called PC-miR2, showed higher expression under Al stress and potentially regulates the expression of the gene encoding BZIP (Basic Leucine Zipper) transcription factor. The second, novel miRNA– PC-miR1 regulates the expression of a gene that encodes hexosyltransferase, which may contribute to pectin biosynthesis within the cell wall. The modulation of pectin content in cell walls significantly influences the Al tolerance in plants. The authors proposed that exclusive expression of PC-miR1 in Tibetan wild barley may be related to altering cell wall structure, leading to detoxification after Al stress. However, more precise analyses should be performed (Wu et al. 2018a). In addition to monocotyledonous plants such as maize or barley, the involvement of miRNA in response to Al stress has been identified in other plant species, including Medicago truncatula (Lu et al. 2023), or olive (Wu et al. 2023).

The research aimed at elucidating the role of miRNAs in the aluminum stress response holds promise. Numerous questions about the engagement of miRNAs in this context still need to be answered. Consequently, there is a need to intensify research to determine the precise role of these molecules in regulating biological processes during the Al stress response.

How can the DDR pathway help plants confer Al tolerance?

The integrity of the nuclear genome in all cells of eukaryotic organisms is constantly threatened. Genotoxic agents, such as ionizing radiation, UV radiation, chemical mutagenic compounds, or heavy metals, induce DNA damage. Aluminum is a mild genotoxic agent, and in many species like Arabidopsis or barley it is well documented that it leads to DNA damage (Rounds and Larsen 2008; Szurman-Zubrzycka et al. 2019) As a result of these events, a specialized mechanism called the DDR (DNA Damage Response) pathway, which is evolutionarily conserved, is activated (Yoshiyama 2016). The DDR pathway enables the detection of DNA damage and, after that, allows the activation of cell cycle checkpoints (which results in the stoppage of the cell cycle) and initiates the DNA repair mechanisms (Nisa et al. 2019).

In plants, the first step in the DDR pathway involves the recognition of DNA damage (Fig. 2). The key roles in this process play two kinases belonging to the PIKK (Phosphoinositide 3-Kinase-like Kinase) family – ATM (Ataxia Telangiectasia Mutated) and ATR (ATM and Rad3-Related) (Mahapatra and Roy 2020). The difference between these two proteins is the type of recognized DNA damage. The ATM protein recognizes the double-strand breaks, whereas the ATR kinase recognizes the single-strand breaks and various types of damages, leading to the creation of single-stranded DNA fragments, which are associated with the replication processes (Pedroza-Garcia et al. 2022). The mechanisms of detecting DNA damage by ATM and ATR kinases differ from each other. In the case of double-strand breaks, the crucial role plays the MNR complex (MRE11/RAD50/NSB1), composed of MRE1 (Meiotic Recombination 11), RAD50 (Radiation Sensitive Protein 50), and NBS1 (Nijmegen Breakage Syndrome 1). This complex recruits the ATM kinase to the site of double-strand breaks through the interaction of ATM kinase with NBS1 protein. As a result of this action, the ATM is activated through auto-phosphorylation (Yoshiyama et al. 2013; Pedroza-Garcia et al. 2022). In contrast, the ATR kinase is activated in a cascade of reactions. The first step of single-strand damage recognition is binding RPA (Replication Protein A) to the single strand. The RPA protein can coat this structure and protect the “overhang”. The following step includes the simultaneous recruitment of two protein complexes necessary for ATR activation – the ATRIP (ATR Interacting Protein) and RFC (Replication Factor C) with ‘9–1-1’ (RAD9-HYS1-RAD1) complex. The ATRIP complex is associated with inactive ATR kinase, while the RFC/9–1-1 complex is recruited to the single strand via interaction with DNA polymerase α. The result of these interactions is the activation of the ATR kinase (Cimini et al. 2019; Nisa et al. 2019; Pedroza-Garcia et al. 2022; Szurman-Zubrzycka et al. 2023).

Fig. 2
figure 2

DDR (DNA Damage Response) pathway in plants. The initial phase of the DDR pathway involves the recognition of DNA damage. For double-strand breaks (DSBs) the MNR complex assumes a crucial role, being recruited to the site of DSBs occurrence. After this action, the ATM kinase is recruited to the MNR complex, subsequently undergoing activation through autophosphorylation. In cases of single-strand breaks (SSBs), recognition is mediated by RPA, which binds to and coats the single strand upon identification. Furthermore, two complexes are recruited—ATRIP and RFC with 9–1-1. The inactive ATR kinase associates with the ATRIP complex, while the RFC/9–1-1 complex is recruited to the SSBs through interaction with DNA Polymerase α. These interactions lead to the activation of the ATR kinase. In the next step, activated ATM and ATR kinases phosphorylate the SQ motifs found in the SOG1 transcription factor, activating SOG1. Activated SOG1 regulates the expression of genes involved in diverse cellular processes, such as the regulation of cell cycle checkpoints, DNA repair mechanisms and cell death. ATM (Ataxia Telangiectasia Mutated), ATR (ATM and Rad3-Related), (MRE11/RAD50/NSB1), MRE1 (Meiotic Recombination 11, RAD50 (Radiation Sensitive Protein 50), and NBS1 (Nijmegen Breakage Syndrome 1), RPA (Replication Protein A), ATRIP (ATR Interacting Protein), RFC (Replication Factor C),‘9–1-1’ (RAD9-HYS1-RAD1), SOG1 (Suppressor of Gamma-Response 1). This figure was created with BioRender.com

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The primary regulatory factor of the DDR pathway in animals is the transcription factor p53. It plays a crucial role in regulating gene expression responsible for the activation of cell cycle checkpoints or initiating apoptosis processes if DNA damage is too extensive. Importantly, in plants, no homolog of p53 has been identified. However, there is a protein that exhibits similar functionality to animal p53 despite a lack of sequence similarity. The transcription factor SOG1 (Suppressor of Gamma-Response 1) is a functional homolog of p53 and belongs to the NAC family. The SOG1 contains the C-terminus transactivation domain with five serine-glutamine (SQ) motifs. After recognition of DNA damage, ATM and ATR kinases phosphorylate the SQ motifs, which results in the activation of SOG1. Activated SOG1 regulates the expression of target genes, which are involved in various processes: cell cycle regulation at the transition from G2 to M phase, DNA repair mechanisms, or programmed cell death induction (Nisa et al. 2019; Mahapatra and Roy 2021; Pedroza-Garcia et al. 2022). It has been demonstrated that SOG1 recognizes the motif CTT[N7]AAG, which has been identified in the promoter regions of approximately 50% of the genes regulated by this protein. Furthermore, based on in vitro and in vivo analysis, it has been shown that this motif is crucial for proper SOG1 binding to the promoter region (Bourbousse et al. 2018). In studies conducted on two models plants – Arabidopsis and rice, it has been demonstrated that SOG1 regulates the expression of hundreds of genes like RAD51, BRCA1, MYB3R3, or NAC44 (Takahashi et al. 2019; Bourbousse et al. 2018; Ryu et al. 2019; Nishizawa-Yokoi et al. 2023).

In 2008, Rounds and Larsen published a study aimed at identifying new factors involved in mechanisms of aluminum tolerance in plants (Rounds and Larsen 2008). Arabidopsis mutant – als3, which contains a mutation in the AtALS3 (Al Sensitive 3) gene encoding the ABC transporter was used. The als3 mutant exhibited hypersensitivity to aluminum due to a disruption in the mechanism of removing toxic Al ions from the roots to the shoot. Through suppressor mutagenesis, the authors identified a loss-of-function mutant in the ATR kinase-encoding gene, characterized by increased aluminum tolerance (compared to the wild type) (Rounds and Larsen 2008). These results suggested that the DDR pathway and mutants in key genes associated with it could represent an innovative approach to investigating plant tolerance to Al stress. This hypothesis was confirmed in numerous studies, demonstrating that mutants in genes such as AtSUV2 (Sensitive to UV 2) or AtSOG1 contribute to aluminum tolerance (Nezames et al. 2012; Sjogren et al. 2015; Sjogren and Larsen 2017). Furthermore, Szurman-Zubrzycka et al. (2019) have provided clear evidence supporting the universality of the DDR pathway’s involvement in aluminum tolerance across both monocotyledonous and dicotyledonous plants. Notably, in barley, it has been demonstrated that mutants in HvATR confer greater tolerance to aluminum stress than the parent variety (Szurman-Zubrzycka et al. 2019). However future analysis should be done, nevertheless the potential application of modifications in the DDR pathway holds great promise for future research and the development of crop plant cultivars that are tolerant to Al stress.

Conclusions

Aluminium (Al) is the third most abundant element in the Earth’s crust. In acid soils, which account for about 50% of cultivated lands, aluminum is present as phytotoxic Al3+ ions. They penetrate the roots, causing growth reduction via inhibition of the cell cycle and cell elongation process, leading to a reduction in yield.

For nearly three decades, intensive research has been conducted to understand the mechanisms of plant response to aluminum toxicity. Due to the growing global population, continuous technological advancement is essential to ensure a proper food supply to feed the world. Al toxicity significantly threatens food security, primarily in poorly developed and developing countries. Understanding fundamental Al tolerance mechanisms, such as the exclusion of organic acids, detoxification of Al3+ ions in the vacuole, or relocation of Al to less sensitive tissues, provides humanity with a foundation to combat the escalating problem associated with Al stress. However, there are concerns that these actions may prove insufficient for future generations.

Going beyond conventional Al tolerance mechanisms and identification of novel ones, such as the applications of polyamines or the role of miRNAs in regulating mechanisms connected with aluminum tolerance, as well as understanding the impact of the DDR pathway on processes leading to root growth inhibition, are crucial for deepening our knowledge of plant responses to the Al. Furthermore, recent modern technologies allow the investigation of the regulation of key protein activities, such as STOP1, in the Al response process. These opportunities offer the potential to create tools that will serve both present and future generations of scientists in combating damages caused by human activities, like the excessive use of nitrogen fertilizers or industrial processes that increase soil acidification. Through numerous studies performed on agronomically important plant species, we can expand our knowledge, supporting the development of new, more tolerant plant varieties.