Aluminum (Al) toxicity represents one of the main yield-limiting factors for crops in acid soils (von Uexküll and Mutert, 1995). Under acidic conditions, large and toxic amounts of Al³⁺ become available to plants, thereby affecting a wide range of physical, cellular, and molecular processes, with a consequent reduction in plant growth (Kochian et al., 2005; Mora et al., 2006; Cartes et al., 2010, 2012; Ryan and Delhaize, 2010; Singh et al., 2017). Alterations in the structure and/or functions of cell wall components (Horst et al., 2010), plasma membrane properties (Yamamoto et al., 2001), nutrient homeostasis (Delhaize and Ryan, 1995; Gupta et al., 2013; Singh et al., 2017), and signal transduction pathways (Matsumoto, 2000; Ma et al., 2002; Sivaguru et al., 2003; Goodwin and Sutter, 2009) can be induced as a consequence of Al binding to numerous cell sites. In most plant species, reactive oxygen species (ROS) production can also be induced by Al toxicity (Kochian et al., 2005), leading to oxidative damage of biomolecules and biological membranes (Yamamoto et al., 2001, 2002, 2003; Singh et al., 2017).

To cope with the deleterious effects of Al, plant species have developed diverse mechanisms, which are generally associated with Al exclusion (also referred to as avoidance or resistance) and/or internal tolerance mechanisms (e.g., Barcelo and Poschenrieder, 2002; Kochian et al., 2005; Poschenrieder et al., 2008). Briefly, exclusion mechanisms involve the root exudation of organic acid anions and/or phenolic compounds, which bind Al³⁺ and limit its uptake into the cytosol. Tolerance mechanisms comprise internal detoxification by forming Al complexes with organic substances in the cytosol, compartmentalization in the vacuole, and enhanced scavenging of ROS (e.g., Barcelo and Poschenrieder, 2002; Kochian et al., 2005; Poschenrieder et al., 2008). Molecular approaches have revealed that Al resistance in several plant species is regulated by genes encoding membrane transporter proteins involved in the efflux of organic acid anions, including members of the ALMT (aluminum-activated malate transporters) and MATE (multidrug and toxic compound extrusion) families (Sasaki et al., 2004; Furukawa et al., 2007; Ryan et al., 2011). In addition, a bacterial-type ATP binding cassette (ABC) transporter (Huang et al., 2009) and antioxidant defense genes (e.g., Milla et al., 2002; Goodwin and Sutter, 2009; Du et al., 2010; Panda and Matsumoto, 2010) have also been implicated in Al tolerance in plants.

Over the last decades, silicon (Si) has become a focus of increasing interest in plant science, since it is considered as a beneficial element for plant growth, particularly under conditions of biotic and abiotic stress (Ma, 2004; Liang et al., 2007; Guntzer et al., 2012; Ma and Yamaji, 2015). To date, several pieces of evidence have indicated that most of the beneficial effects of Si depend on the differential ability of plants to take up Si. Recently, it has been reported that Si accumulation is ascribed to an efficient uptake system mediated by both channel-type and efflux transporters, which perform coordinated functions for effective Si transport from soil to roots and its subsequent distribution within the plants (e.g., Ma et al., 2006, 2007; Yamaji et al., 2008, 2012; Chiba et al., 2009; Mitani et al., 2009a,b, 2011a,b; Yamaji and Ma, 2009; Grégoire et al., 2012; Montpetit et al., 2012; Deshmukh et al., 2013; Ma and Yamaji, 2015). Overall, these transporters appear to be keys features that enable plants to gain an advantage from Si uptake. Nevertheless, the regulation of Si transporters under stress conditions remains poorly understood.

The significant role of Si in the toxicity associated with metals, including manganese (Mn), iron (Fe), cadmium (Cd), arsenic (As), chromium (Cr), copper (Cu), lead (Pb), zinc (Zn), and Al, has been widely reported (Li et al., 2012; Vaculík et al., 2012; Adrees et al., 2015; Liang et al., 2015; Pontigo et al., 2015; Tripathi et al., 2015, 2016). On the basis of the current evidence, Si can regulate plant resistance and/or tolerance to metal toxicity by either external (ex planta) or internal (in planta) mechanisms (Cocker et al., 1998a; Adrees et al., 2015; Liang et al., 2015; Pontigo et al., 2015; Tripathi et al., 2016). In this regard, it has been proposed that the alleviation of Al stress by Si in plants can mainly be explained by the following events: (i) Si-induced increase in solution pH (Li et al., 1996; Cocker et al., 1998a), (ii) formation of Al-Si complexes in the growth media (Barcelo et al., 1993; Baylis et al., 1994; Ma et al., 1997; Cocker et al., 1998a) or/and within the plant (Corrales et al., 1997; Cocker et al., 1998b; Britez et al., 2002; Zsoldos et al., 2003; Wang et al., 2004; Prabagar et al., 2011), (iii) exudation of organic acid anions and phenolic compounds (Barcelo et al., 1993; Cocker et al., 1998b; Kidd et al., 2001), and (iv) increase in the chlorophyll and carotenoid contents of leaves (Singh et al., 2011). Activation of the plant antioxidant system has also been reported in response to Si supply under Al stress (Shahnaz et al., 2011; Shen et al., 2014; Tripathi et al., 2016). However, to our knowledge, there is a dearth of reports regarding the molecular aspects of the effect of Si on the genes involved in antioxidant defense.

Perennial ryegrass (Lolium perenne L.) is a temperate pasture species supporting forage-based intensive dairy and beef production systems in many parts of the world. Due to elevated yields and high nutritional value, ryegrass has become one of the most commonly cultivated species in the permanent pastures of Southern Chile. Nevertheless, large areas of these pastures are sown on acidic soils, which exhibit elevated availability of toxic Al⁺³, thereby limiting their yield and quality (Mora et al., 2006). Furthermore, our previous studies have demonstrated that toxic levels of Al induced oxidative damage and activated antioxidant enzymes in ryegrass roots, including peroxidase (POD), ascorbate peroxidase (APX), and superoxide dismutase (SOD) (Cartes et al., 2010, 2012). In an attempt to identify new alternatives to alleviate the deleterious effects produced by Al on ryegrass, we aimed in this study to investigate the effect of Si on the modulation of Si/Al uptake and the antioxidant performance of ryegrass plants subjected to Al toxicity.

Although several previous studies have reported that Si provide beneficial effects on plants subjected to Al stress, the mechanisms underlying these responses have remained poorly understood. Moreover, only a few studies have examined the effect of Si-mediated amelioration of Al toxicity in terms of the regulation of Al and Si uptake systems (e.g., Britez et al., 2002; Wang et al., 2004; Dorneles et al., 2016) and plant antioxidant performance (e.g., Shahnaz et al., 2011; Shen et al., 2014). Likewise, to date, the effect of Si on Al stress in ryegrass, a forage species belonging to Si-accumulator plants (Jarvis, 1987; Nanayakkara et al., 2008), has yet to be addressed.

The high level of toxic Al in acid soils is an important limiting factor for plant production (Mora et al., 2006). In our study, the exposure of plants to 0.2 mM Al significantly increased Al accumulation, mainly in the roots (Table 2), with a consequent reduction of approximately 28.5% in root dry matter production (Table 2). These results are consistent with our previous findings for ryegrass (Cartes et al., 2010), since it is well known that Al toxicity involves the rapid inhibition of root growth (e.g., Matsumoto, 2000; Kochian et al., 2005; Horst et al., 2010; Singh et al., 2017). The role played by Si in promoting plant growth under Al toxicity has been widely accepted (e.g., Hara et al., 1999; Singh et al., 2011; Shen et al., 2014; Tripathi et al., 2016). Correspondingly, Si application to Al-treated plants significantly reduced the Al concentration in ryegrass (Table 2) and improved root dry weight by at least 51% (Table 2). A slight reduction in Si concentration in plant tissues was also found when plants were simultaneously supplied with Al and Si (Table 2). Moreover, our results revealed a negative correlation between Si and Al uptake in plants treated with Al and Si, whereas Si concentration and dry matter production were positively related (Table 3). The reduction in Al and Si uptake might be attributed to the formation of biologically inactive aluminosilcate (Al-Si) complexes in the growth media, thus lowering Al availability (Barcelo et al., 1993; Baylis et al., 1994; Ma et al., 1997; Cocker et al., 1998a), with the consequent enhancement of root growth. Nevertheless, the formation of Al-Si inside plant tissues could also be involved in the growth-promoting effect of Si under Al stress (Hodson and Sangster, 1993; Cocker et al., 1998b; Wang et al., 2004). Indeed, it has been demonstrated that Al toxicity may be decreased by co-deposition of Al and Si in the root epidermal walls of sorghum (Hodson and Sangster, 1993). Similarly, Cocker et al. (1998b) and Wang et al. (2004) have also suggested that formation of Al-Si complexes in the root apoplast of wheat and maize is a possible mechanism for Al detoxification in plants.

Although all plants contain Si in their tissues, the concentration of this element varies greatly among species, in a range from 0.1 to 10% on a dry weight basis (Epstein, 1999; Ma and Takahashi, 2002), which is indicative of the fact that the benefits of Si to plants grown under stress can also be highly variable. Recent studies have shown that Si accumulation in plants is controlled by influx and efflux Si transporters that could be involved in the differential Si-induced responses to cope with different plant stress (e.g., Ma et al., 2006, 2007; Yamaji et al., 2008, 2012; Chiba et al., 2009; Mitani et al., 2009a,b, 2011a,b; Yamaji and Ma, 2009; Grégoire et al., 2012; Montpetit et al., 2012; Deshmukh et al., 2013; Ma and Yamaji, 2015). To further investigate the effect of Si uptake on ryegrass subjected to Al stress, we assessed the gene expression of two Si transporters (Lsi1 and Lsi2) in plants with different Al and Si supply (Figures 1A,B). Lsi1 is a channel-type transporter belonging to aquaporin Nodulin26-like intrinsic protein (NIP) III subfamily (Ma et al., 2006), whereas Lsi2 is an Si efflux transporter belonging to the family of putative anion transporters (Ma et al., 2007). Efficient coupling of Lsi1 with Lsi2 controls the uptake of Si in species such as rice, barley, and maize (Ma et al., 2006, 2007; Chiba et al., 2009; Mitani et al., 2009a,b). Our study showed that in plants cultivated without Al, the mRNA expression levels of both LpLsi1 and LpLsi2 were down-regulated in plants supplied with Si (Figures 1A,B). Some studies have shown that the accumulation of Lsi1 mRNA in maize (ZmLsi1), barley (HvLsi1), and wheat (TaLsi1) is not affected by the addition of Si (Chiba et al., 2009; Mitani et al., 2009a; Montpetit et al., 2012). Nevertheless, Ma et al. (2006, 2007) found that the gene expression of both OsLsi1 and OsLsi2 was decreased by approximately 25% in rice, as a consequence of continuous Si application. A similar expression pattern has been detected for Lsi1 in maize (ZmLsi1) (Bokor et al., 2014) as well as for Lsi2 in barley (HvLsi2) (Mitani et al., 2009b) and maize (Bokor et al., 2014). Moreover, a recent study has stated that the Si-induced down-regulation of Si transporter genes is controlled by Si accumulation in the shoots of rice (Mitani et al., 2016).

At present, there is little information on the effect of any plant stress on the transcriptional regulation of Si transporters genes. Bokor et al. (2014) observed that Si supply down-regulated the expression of ZmLsi1 and ZmLsi2 in the roots of maize subjected to excess zinc (Zn). By contrast, it has been reported that Si increased the expression level of OsLsi1 and OsLsi2 under conditions of cadmium (Cd) and copper (Cu) toxicity in rice plants (Kim et al., 2014). Likewise, Vulavala et al. (2016) found that a putative Si transporter in potato (StLsi1) was up-regulated in response to Si and drought stress. Interestingly, we found that the transcript levels of both LpLsi1 and LpLsi2 were significantly down-regulated by Al supply, but up-regulated by 5.4-fold (LpLsi1) and 2.5-fold (LpLsi2) when Al was added in combination with Si (Figures 1A,B). Compared with plants cultivated with Si alone, the reduction in Si concentration in plants simultaneously supplied with Al and Si (Table 2), could be responsible for the up-regulation of LpLsi1 and LpLsi2 (Figures 1A,B). This behavior might indicate an increased requirement for Si in ryegrass in order to cope with Al-induced toxicity. Further studies are needed to confirm this assumption.

As a possible alternative mechanism of Si-mediated Al detoxification in plants, enhancement of the antioxidant defense system has also been proposed (Shahnaz et al., 2011; Shen et al., 2014; Liang et al., 2015; Tripathi et al., 2016). As stated above, Al toxicity can lead to the generation of reactive oxygen species (ROS), such as superoxide radicals (O₂^•-), hydroxyl radicals (^•OH), and hydrogen peroxide (H₂O₂) molecules, which cause oxidative damage to plant cells (e.g., Yamamoto et al., 2001, 2002, 2003; Kochian et al., 2005; Singh et al., 2017). In agreement with previous reports (Cartes et al., 2010, 2012), our results show that 0.2 mM Al increased lipid peroxidation in ryegrass (Figures 2A,B), confirming that oxidative stress occurs under Al supply. Nevertheless, 2 mM Si significantly diminished Al-induced lipid peroxidation by approximately 32 and 28% in shoots and roots, respectively (Figures 2A,B). Moreover, a negative correlation between Si concentration and lipid peroxidation was detected in Al-treated plants (Table 3). Consistent with our findings, Shen et al. (2014) observed a noticeable decrease in lipid peroxidation attributable to Si in peanut grown under Al excess. Similarly, there is increasing evidence showing that oxidative damage to biological membranes decreases as a consequence of Si application to plants subjected to different environmental stresses (e.g., Liang et al., 2003; Zhu et al., 2004; Shi et al., 2005; Gunes et al., 2007, 2008; Li et al., 2012; Khoshgoftarmanesh et al., 2014; Kim et al., 2014; Habibi, 2015; Zia-ur-Rehman et al., 2016).

Whereas Al toxicity enhanced plant phenols concentration (Figures 3A,B) and augmented the activities of antioxidant enzymes (Figures 4A–H), Si application induced differential responses in the antioxidant system of Al-stressed plants (Figures 3A,B, 4A–H). It has been suggested that Si may enhance Al tolerance by increasing the production of phenolic compounds with Al-chelating ability (Kidd et al., 2001; Shahnaz et al., 2011). Furthermore, it has been reported that Si uptake by plants subjected to certain stresses can lead to increased production of phenolics with antioxidant and/or structural function (Fleck et al., 2010, 2015; Song et al., 2016). Likewise, enzymes and genes involved in the biosynthesis of either soluble phenolics (e.g., flavonoids) or structural polyphenols (e.g., lignin) have also been shown to be induced by Si (Liang et al., 2007; Shetty et al., 2011; Zhang et al., 2013; Song et al., 2016). Here, we found that Si addition (mainly at the highest dose) increased the total phenol concentration in plants treated with Al and Si (Figures 3A,B), and that there was a negative relationship between phenols concentration and lipid peroxidation (Table 3). Thus, the enhanced phenols accumulation triggered by Si may have contributed to the amelioration of Al-induced oxidative stress in ryegrass.

Differential changes in the activity of antioxidant enzymes, as a consequence of Al and Si treatments, were also observed. SOD constitutes the first line of defense in the enzymatic antioxidant responses by catalyzing the dismutation of O₂^•- to H₂O₂ and O₂ (Takahashi and Asada, 1983; Alscher et al., 2002). Our results indicate that the highest Si dose decreased SOD activity in plants subjected to Al stress (Figures 4A,B), as supported by the negative correlation between SOD activity and Si concentration (Table 3). Likewise, differential gene expression of SOD isoforms occurred in plants exposed to Al and Si (Figures 5A–F). The major differences were detected in the roots at the highest Si level, which induced a significant decrease in expression of the LpCu/Zn-SOD and LpMn-SOD genes in plants grown under Al toxicity (Figures 5D,F). A similar expression pattern was observed for the LpMn-SOD gene in shoots, which was down-regulated by at least 2.2-fold in combined Al-Si treatments (Figure 5E). The decrease in either SOD activity (Figures 4A,B) or the gene expression pattern of SOD isoforms (Figures 5A,D,F) coincided with a significant reduction in lipid peroxidation at the highest Si dose (Figures 2A,B), denoting that 2 mM Si can diminish the requirement for SOD enzyme in Al-treated plants.

It is noteworthy that the antioxidant enzymes responsible for H₂O₂ scavenging (CAT, POD, and APX) were activated by Si in the roots of Al-stressed plants (Figures 4D,F,H). Moreover, a direct correlation between Si concentration and the activities of CAT, POD, and APX was found in the roots of plants treated with Al and Si (Table 3). The activation of these enzymes was accompanied by a noticeable decrease in lipid peroxidation (Figures 2A,B), with a consequent reduction in the oxidative damage of biological membranes induced by Al.

We also detected an apparent increase in intracellular H₂O₂ production in shoot protoplasts of plants simultaneously supplied with Al and Si (Figures 6A,B). It is remarkable that there is so little information available regarding the role of Si in H₂O₂ generation under either biotic or abiotic stress conditions. In this context, the only study that has examined the relationship between Si and H₂O₂ production in plants subjected to Al toxicity (Lima et al., 2016) showed an opposite trend when compared with our results. Nevertheless, under freezing stress, Habibi (2015) detected an increase in H₂O₂ levels induced by Si in pistachio plants, which is consistent with the findings of the present study. This significant increase in H₂O₂ production might be related to the reduction in POD activity observed in the shoots of plants simultaneously treated with Al and Si (Figure 4E). Indeed, H₂O₂ plays a dual role in vascular plants by either inducing oxidative damage or acting as signaling molecule in several physiological processes, including senescence (Peng et al., 2005), photorespiration and photosynthesis (Noctor and Foyer, 1998), and growth and development (Foreman et al., 2003). H₂O₂ also functions as a second messenger that modulates the expression of antioxidant enzymes and stress responses (Apel and Hirt, 2004). Accordingly, further work should focus on the mechanisms underlying the Si modulation of H₂O₂ production under Al stress.

Finally, taken together, our findings provide the first biochemical and molecular evidence that Si counteracts the negative effects of Al by modulating Al and Si uptake as well as enzymatic and non-enzymatic antioxidant responses in ryegrass plants.

SP and PC conceived the idea and wrote the manuscript. SP performed all the experiments and PC supervised the research. AG-M and HJ contributed to evaluation and discussion regarding aspects of the study related to gene expression analyses. KG assisted with management and analysis of the flow cytometry and laser scanning CLSM data. MM contributed to discussion on aspects associated with the influence of Si on plants subjected to Al toxicity. All authors contributed to the discussion and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.