Detailed list of references are provided in the Supplementary Text 2.

#### *5.1. Silicon Mediated Immobilization of Toxic Metal in the Soil*

Immobilization of toxic metals is the much simpler mechanism to explain Si-derived benefits. The toxic metal immobilization in the soil through Si application has been reported in several studies [21]. The immobilization took place either due to the increased soil pH or changing metal speciation in soil solution through the formation of silicate complexes. In rice, application of Si-rich amendments (fly ash and steel slag) was found to increase soil pH from 4.0 to 5.0–6.4, and decreased the phyto-availability of heavy metals by at least 60%, which further suppressed metal uptake [26]. Similarly, in banana, reduced uptake of Pb has been reported with Si application in Pb contaminated soil [34]. The decreased bio-availability of Pb in banana was found to be associated with significantly increased soil pH and decreased proportion of exchangeable Pb in soil [34].

In addition, Si helps by changing the speciation of metals from toxic to nontoxic form by the formation of silicate complexes in the soil solution. In Si-amended soil, mostly Cd was found in the form of oxides or adsorbed by Fe-Mn oxides [35]. It has been proposed that the bio-availability of Al to plants in the presence of Si can be limited by forming Al-Si complexes like hydroxyaluminosilicate (HAS) [36]. In aqueous solution, soluble silicate hydrolyzes and produces gelatinous metasilicic acid (H2SiO3) which can absorb heavy metals, or lead to deposition of heavy metals into their silicates in Si-rich soil [26], both of which reduce the metal concentration available for uptake. Zhang et al. [37] have shown that the Si application in Cr-contaminated soil can markedly decrease the amount of exchangeable Cr by accelerating the precipitation of organic matter bound Cr fraction. Similarly, Shim et al. [38] have observed the reduced metal mobility in Pb contaminated soil by the Si application. Subsequent investigation with X-ray diffraction analysis revealed the formation of insoluble Pb-silicate in the soil. Similarly, effects have been observed in the case of Cd and Zn contaminated soil where Si application accelerated the formation of more stable fractions of Cd and Zn [28].

#### *5.2. Stimulation of Antioxidant Defense System*

Heavy metal stress induces an excess formation of reactive oxygen species (ROS), which results in several metabolic disorders in crop plants [39,40]. In this context, the enzymatic and non-enzymatic antioxidant system stimulated by Si helps to lower oxidative stress by reducing the production of ROS. Alleviation of Mn toxicity by Si in cucumber was attributed to a significant reduction in lipid peroxidation (LPO) intensity caused by excess Mn, and to a significant increase in enzymatic (*Superoxide dismutase*, *ascorbate peroxidase*, and *glutathione reductase*), and non-enzymatic (ascorbate and glutathione) antioxidants [41]. Similarly, under Cd stress, Si application reduced the H2O2 and electrolytic leakage (EL) in *Solanum nigrum* [42]. Decreased contents of an oxidative stress related compound like malondialdehyde (MDA), H2O2 and EL were observed with Si application in plants under Cd [43], Zn [27], and Pb stress [44]. Thiobarbituric acid reactive substances (TBARS), a widely used marker for reactive oxygen contents, were found to be reduced significantly with Si supplementation in rice and maize plants grown under Cd stress condition [39]. Similarly, under Cd stress, the effect of Si supplementation on antioxidants has also been observed in many crop plants including maize [45], wheat [43], rice [23,46] and peanut [47]. Enhanced antioxidant enzyme activities as well as activities of non-enzymatic antioxidants like glutathione, non-protein thiols, and ascorbic acid have been reported in several plant species under heavy metal stress [48,49]. Si-mediated detoxification through stimulating enzymatic and non-enzymatic antioxidants has also been observed under Pb, Mn, Zn and Cu stress [20]. In light of the previous studies, application of Si appears to induce the antioxidant system in plants, thereby improving stress resistance, but it is still unclear if this results from a direct or indirect action of Si [50].

#### *5.3. Compartmentation within Plants*

Enhanced compartmentation of metal elements in plant tissues has been observed with Si supplementation in several studies. Williams and Vlamis [51] observed for the first time that the effect of Si in alleviating Mn toxicity in barley was not the result of a reduction in Mn concentration, but rather of improved compartmentation within the leaf tissues. Another level of compartmentation, mostly regulated through the translocation activity leading to the increased metal concentration in plant roots compared to shoots, has been widely reported with Si supplementation [52,53]. The Si treatment was observed to reduce transport of Zn from roots to shoots and increase the binding of Zn to the cell wall, thus decreasing Zn concentration in the rice shoots [53]. In wheat, Si application found to reduce Cd translocation from root to shoots and grains [54]. In rice, Shi et al. [29] observed a Si-decreased root to shoot translocation of Cd by 33%. Microscopic analysis performed by Shi et al. [29] clearly showed deposition of Cd near the endodermis and epidermis; however, a high amount of Si was found to be deposited near the endodermis than in the epidermis. The Si deposition at endodermis seems to play a crucial role in decreasing Cd translocation from the epidermis to the endodermis. In addition, with Si application in rice, decreased Cd accumulation in shoots was found to be associated with the increased compartmentalization of Cd in the root cell walls [23]. Considerable reduction in the symplasmic concentration of Cd and increase in the apoplastic concentration in maize grown with Si supplementation have been reported by Vaculik et al. [55]. Silicon was also observed to increase the Mn localization in the cell wall in cucumber [56] and cowpea [57] under Mn stress and in rice, under Zn stress [26]. Furthermore, in Si-treated plants, less Mn was located in the symplast (<10%) and more Mn was bound to the cell wall (>90%) compared to control plants (about 50% in each compartment) [58]. Hence, Si-mediated detoxification of heavy metals through their compartmentation into different plant tissues might be a key mechanism supporting the beneficial role of Si.

#### *5.4. Chelation Mediated Metal Toxicity Reduction with Silicon Application*

The Si-mediated heavy metal detoxification predominantly includes the chelation of metal by flavonoid-phenolics or organic acids. Increased phenol exudation up to 15 times has been observed in maize upon Si supplementation. The phenolic compounds like catechin and quercetin have high Al-chelating activity, which can alleviate Al toxicity in the root tip apoplast [10,59]. Barceló et al. [60] revealed a considerable increase of malic acid concentration upon Si application in maize under Al stress. The reduced Al toxicity was found to be correlated with the chelating of Al with malic acid. In bamboo, Si has also been observed to increase the concentration of Cu(I) S-ligands that chelate Cu as well as increase the Cu sequestration in a less toxic form [61]. Keller et al. [52] have observed reduction in translocation of Cu from root to shoot in wheat grown with Si supplementation. The altered translocation of Cu may be because of the increased proportion of citrate, malate, and aconitate in roots of wheat seedlings. Taken together, these studies sugges<sup>t</sup> that Si may indirectly promote the chelation of heavy metals in plants, thereby reducing their phytotoxicity.

Schaller et al. [62] recently reported that Si availability was significantly correlated to phosphorus (P) mobilization in Arctic soils. The results confirmed that the addition of Si significantly increases P mobilization by mobilizing Fe(II)-P phases from mineral surfaces as well as increases soil respiration in P deficient soils. The study highlights the Si as an important component regulating P mobilization in Arctic soils. Besides an important nutrient element, P supplementation also plays an important role in biochemical activities in the soil. In this regard, significant efforts have been performed to understand the effects of P mobilization on bioavailability of heavy metals [63]. A considerably high number of studies have shown the importance of P compounds to reduce bioavailability of heavy metals by immobilizing the metal ions in the soil [64]. Therefore, Si mediated P mobilization seems a valuable option for the sustainable managemen<sup>t</sup> of P availability as well as for the minimizing losses that occurred due to heavy metals in agro-ecosystems.

#### *5.5. Regulation of Gene Expression*

Phytochelatin synthesis represents one of the major heavy metal detoxification mechanisms in plants [65]. The Si-mediated mitigation of metal toxicity is also attributed to its role in altering gene expression. In Arabidopsis grown under Cu stress, Si supplementation was observed to stimulate

the genes governing the production of metallothioneins (MTs), a well-known chelating agen<sup>t</sup> [66]. Similarly, significantly downregulated expression of the genes encoding heavy metal transporters (*OsHMA2* and *OsHMA3*), and upregulation of the genes responsible for Si transport (*OsLSi1* and *OsLSi2*) has been observed with Si supplementation in rice [3]. Similarly, the enhanced expression of *phytochelatin synthase* 1 (*PCS1*) and decreased expression of the *metallothionein* gene (*MT1a*) was associated with Si supplementation in Arabidopsis under Cu stress [67]. Recently, Ma et al. [68] have shown that Si supply under Cd stress in rice upregulated the expression of *OsLsi1* (encoding for Si transport NIP-III (Nodulin 26-like intrinsic proteins-III) Aquaporin) and downregulated the expression of *Nramp5*, a gene involved in the Cd transport. However, many plant species particularly belonging to family *brassicaceae* [69], *solanaceae* [70], and *Linaceae* [71] does not carry any Si-transporter (NIP-III Aquaporin) and are known as poor Si accumulators. However, several reports suggesting Si derived benefits in such species make it more difficult to understand the molecular consequences [72,73]. So far, the Si-mediated mechanisms for the reduction of metal toxicity are less understood at the genetic and molecular levels. A better understanding of the gene expression dynamics involved in Si-driven alleviation of metal toxicity is necessary to properly decipher the molecular mechanism underlying this phenomenon. Still, limited efforts have been directed toward explaining Si-mediated transcriptomic changes in plants, and none of those were related to metal toxicity.

#### *5.6. Structural Alterations Related to Metal Stress Tolerance in Plants*

Morphological and anatomical features of crop plants ge<sup>t</sup> improved with Si supplementation that helps to overcome the adverse effects of heavy metal stress. Notable examples where increased plant height, root length, number of leaves and leaf size have been observed with Si application to plants under Cd, Zn and Pb stress [74,75]. Ali et al. [76] observed that Si + Cr treatments increased plant height, the number of tillers, root length, and leaf size of barley plants compared to plants treated with Cr only. Similarly, root length and shoot size were significantly increased with Si compared to without Si treatment [74]. Doncheva et al. [77] reported that the Si application increased the leaf-epidermal-layer thickness in maize plant under Mn stress. Silicon was also reported to increase xylem diameter, epidermis, mesophyll and the transverse area of collenchymas and mid-vein under Cd and Zn stress [28,78]. The accelerated development of the Casparian bands, suberin lamellae, and root vascular tissues was observed in maize with Cd + Si treatments [45,55]. Similarly, higher growth of suberin lamellae in the endodermis particularly near the root tips has been observed with Si application in rapeseed and Indian mustard grown under Cd stress [79]. In wheat, Greger et al. [80] reported the formation of apoplastic barriers in the endodermis closer to the root apex in the presence of Si in Cd-treated plants. Thus, structural alterations induced by Si under metal stress may explain the alleviation of heavy metal toxicity.

#### *5.7. Co-Precipitation of Metals by Silicon Application in Soil*

Many studies sugges<sup>t</sup> that the co-precipitation of metals by Si leads to the alleviation of heavy metal stress in plants. For example, Si treatment in plants under Al stress was suggested to form aluminosilicates or hydroxyaluminosilicates (or both) in the apoplast of the plant root apex leading to Al detoxification [22,60]. In *Minuartia verna* (Si-accumulating dicot), Si was reported to co-precipitate Zn as their silicates in the leaf epidermis cell wall [81]. Gu et al. [26] observed the co-precipitation of Si with Cd in the stem of rice that lowered heavy metal concentration in leaves. Similarly, Si was proposed to form a complex with Zn at the leaf surface of *Cardaminopsis halleri* [82]. Zhang et al. [23] observed synchronous accumulation of Si and Cd in the middle and border of phytoliths in rice shoots. However, there are some contrasting reports, such as ones by Rizwan et al. [83] and Keller et al. [52], where Cu and Cd were not found in the leaf phytoliths of wheat. These reports sugges<sup>t</sup> the possibility of a mechanism other than the co-precipitation involved in Cu detoxification in wheat plants. Similarly, Dresler et al. [84] report also support the notion since Si–Cd complexes were not observed in maize plants treated with Si under Cd stress.

#### **6. Approaches for Improving Silicon Accumulation in Crop Plants**

The beneficial effect of Si for mitigating the toxic effects of heavy metals is usually apparent in plants that accumulate high levels of Si in their shoots [53,85]. Silicon accumulation in plants depends upon the availability of silicic acid (H4SiO4) in the soil as well as the inherent capacity of the plant for Si uptake. Silicon availability in a soil can be improved by applying Si-rich fertilizers or by modifying soil properties, whereas genetic modification or selection can improve the inherent capacity a species to accumulate Si. The different approaches used for increasing Si accumulation in crop plants are presented in Figure 3 and are discussed in the following sections.

**Figure 3.** The different approaches that can be utilized for improving silicon accumulation to enhance metal stress tolerance in crop plants.

## *6.1. Silicon Fertilization*

The estimated amount of Si removed annually by different crops on a global scale is between 210 and 224 million tons [86]. Continuous and intensive cropping of Si-accumulator crops results in a significant reduction of plant-available Si in the soil [87]. Desplanques et al. [88] noted that five years of continuous cultivation of rice could exhaust most of the available Si from the soil. In addition, certain soils contain low levels of Si, particularly the plant-available form, and these soils include Oxisols, Ultisols, and Histosols as well as soils that are composed of a large fraction of quartz [89]. Silicon is absorbed by plants in the form of monosilicic acid (H4SiO4) and its concentration in the soil solution will determine the amount a plant accumulates [90]. Fertilization can rapidly increase the concentration of H4SiO4 in the soil and has become a standard practice in areas with intensive cropping systems, particularly for those soils that are inherently low in soluble silicon [91,92]. Several studies have shown a beneficial effect of an adequate supply of Si in growth medium not only for high Si-accumulating plants such as rice [46] and sugarcane but also for poor accumulators like tomato, cucumber, strawberry and orange [93,94] (Table 2). On the other hand, some studies have also shown that the fertilization regime can influence the amount of Si that will be available to the plant [94]. With respect to the alleviation of heavy metal tolerance, Ning et al. [95] and Jarosz et al. [93] observed less concentration of Zn and Cu in fruits of plants grown with Si-based fertilizer. However, most of the studies to date have been carried out in greenhouses, and there is a lack of data supporting the performance of Si-based fertilizers in large-scale field experiments.

#### *6.2. Modification of Soil Properties*

The abundance of Si in soils does not necessarily relate to the concentration of plant-available Si [91]. The concentration of H4SiO4 in the soil solution is influenced by many soil factors such as pH, temperature, weathering of soil, moisture, redox potential, amounts of clay, mineral, organic matter and Fe/Al oxides/hydroxides [86]. The solubility of both the crystalline and the amorphous silica is approximately constant between pH values 2 and 8.5 and increases rapidly at pH ~9. The soil pH also affects the formation of Si-complexes with other elements, for instance, the amount of monosilicic acids that is absorbed by Fe/Al oxides increases from pH 4 to pH 10. Kaczorek and Sommer [96] have revealed that, under the conditions of soil acidification, the number of free silica increases. Similarly, Höhn et al. [97] reported an increase of Si available in the soil with a decrease in pH. In this regard, the application of acid-producing fertilizer increases the concentration of H4SiO4 in the soil solution, whereas liming and high organic matter content results in a reduction in the concentration and mobility of the H4SiO4.

#### *6.3. Approaches for Genetic Gain*

Identification of two Si-transporters genes using low silicon (*Lsi*) rice mutants by Ma et al. [98,99] are the milestone discoveries that accelerated Si research many folds. One of the genes is a passive influx transporter (*OsLsi1*) belonging to an NIP group of the aquaporin family, which is responsible for the uptake of Si from soil into the root cells [15,100]. Several homologs of the *Lsi1* have been identified and functionally validated in different plant species (Table 3, Supplementary Text 1: Table S1). Another gene, *Lsi2*, encodes an active efflux transporter and belongs to the cation transporter family (Table 3; Figure S1). The information about the *Lsi1* and *Lsi2* was helpful to understand the uptake of Si from the root and subsequent trans-location from roots to aerial parts of the plant [101]. It also provided an opportunity to explore transgenic approaches for the enhancement of Si-uptake, particularly in poor accumulator plant species. In Arabidopsis, a well-known poor accumulator, heterologous expression of wheat Si-transporter (*TaLsi1*) showed increased Si absorption by several folds [102]. Similarly, functionally characterized Si-transporter (mostly with *Xenopus oocyte* assays) from different species could be utilized efficiently for the development of transgenic crop plants (Table S1). Recently, Deshmukh et al. [103] showed categorization of plant species as poor or high Si-accumulator based on the presence of *Lsi1* homolog. The interspecies variation can be sufficiently explained by the characterization of *Lsi1* homolog. Considerable interspecies variation for Si concentration has been reported, ranging from 0.1 to 10%, although these variations appear to be more limited at the intraspecies level [14]. For example, in sugarcane (*Saccharum officinarum*), the Si concentration in the shoots of different genotypes has been observed to range from 6.4 to 10.2 mg<sup>−</sup><sup>1</sup> [104]. Similarly, in a survey of about 400 cultivars of barley (*Hordeum vulgare*), the Si concentration in barley grain revealed a variation, ranging from 1.24 to 3.80 mg·g<sup>−</sup><sup>1</sup> in barley grains [105]. In rice, japonica rice cultivars usually accumulate more Si than indica rice cultivars [106]. Quantitative trait loci (QTLs) governing the intra-species variation have been identified in rice. However, these QTLs do not collocate with the major genes *Lsi1* and *Lsi2*, and the discovery of *Lsi1* and *Lsi2* enhanced molecular understanding of Si-uptake has limited use for crop improvement. The gene(s) responsible for the genotypic differences have ye<sup>t</sup> to be identified. Efforts towards the identification of such genes will help to develop cultivars with better Si uptake through breeding approaches.


**Table 3.** Influx/efflux Si transporters as well as their ortholog's identified in different crop species.

\* mutated version where removal of Valine at potion 140 in non-functional wildtype turn it into a functional Si-transporter; # Detailed list of references are provided in the Supplementary Text 2.

## **7. Conclusions**

Natural and anthropogenic activities have resulted in a higher concentration of toxic metals in the agricultural farmlands leading to the severe adverse effects on crop production and productivity as well as human health. In this regard, Si has emerged as a practical option to reduce phytotoxicity and accumulation of toxic metal in plants. Beneficial effects derived from Si have been reported in several studies and have been explained through different possible mechanisms, although more experiments are needed to validate them. Many studies argue that the use of Si-based fertilizers constitutes a promising approach to alleviate heavy metal toxicity and large-scale field studies should be undertaken to define with precision the extent of Si-derived benefits. At the same time, the enhancement of the genetic potential of plants to uptake Si would represent an interesting avenue to optimize their responses to Si. The recent advances in the field of genomics, computational biology, and high-throughput phenotyping will facilitate the deciphering of the genetic basis of Si accumulation in crop plants, which in turn should accelerate the development of varieties accumulating higher levels of Si. This strategy would fit very well in a program of sustainable agriculture to improve heavy metal tolerance in plants.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2223-7747/8/3/71/s1, **Figure S1:** Diagram showing the absorption of Si in rice plants. Silicon enters the exodermis in the form of silicic acid through specific influx transporter (Lsi1) and leaves into the cortex through active transporters (Lsi2). In the cortex, silicic acid moves apoplastically until it reaches the endodermis, where the silicic acid is loaded into the stele by Lsi1 and Lsi2 transporters. The solid green line shows the path of Si transport. **Table S1:** Details of silicon transporter genes from different plant species were validated using oocyte assay or transgenic approaches. **Supplementary Text 1:** Detailed discussion about the deficiency symptoms caused by different metals in plants, toxic effects of heavy metals on growth, development, and metabolism of different crop species. **Supplementary Text 2**: Details of references provided in Tables 1–3.

**Author Contributions:** J.A.B. and S.M.S. wrote the initial draft, P.S., D.B.N., D.K.T., P.K.D., A.U.S., H.S. and R.D. provided input and were involved in the compilation of the final draft.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors are thankful to the Department of Biotechnology, Government of India for the financial support in the form of the Ramalingaswami Fellowship to H.S. and R.D.

**Conflicts of Interest:** The authors declare no conflict of interest.
