## 4 Discussion  
In acidic Cambic soils at southeast Portugal, Mn toxicity has been described as inducing low productivity through reduced plant growth and increased oxidative stress [28; 35]. In a previous study, wheat nutritional status (Mg, Ca, P, and Mn) and antioxidant responses were assessed in an acidic soil with toxic Mn concentrations [28]. In these conditions, higher Mn levels in the soil promoted a decrease in nutrient uptake, a sharp increase in root and shoot Mn concentrations, an increased Mn translocation to shoot tissues, and a preferential accumulation of Mn at the shoot vacuoles, along with P and Mg. In the present work, the effects of toxic Mn concentrations in an acidic soil were followed, for the first time, for wheat Al, Fe, and Si uptake dynamics and subcellular redistribution. When no Mn was supplied to the acidic soil, Mn, Fe, or Al were detected in wheat shoots at high or toxic levels. In wheat, high concentrations are considered above 2.2 mg/kg for Al, 100 mg/kg for Mn, and 100 mg/kg for Fe [8]. Toxicity caused by individual metals is largely dependent on the concentrations of other metals and nutrients, but also on the susceptibility of wheat genotypes [3; 34; 38; 39; 40]. Due to this multi-factorial nature, limits of toxicity for these metals are generally not unanimously recognised in wheat, and several authors report different limits of toxicity [36; 41; 42; 43]. After soil Mn supplementation, a higher amount of Mn competed with Al, Fe, and Si for the negative charges of soil organic matter compounds, which probably led to their increased concentration in the soil solution [5; 43]. Wheat responded to the elevated bioavailable Mn concentrations with a reduction in Al, Fe, and Si uptake ratios. Root uptake was reduced while shoot concentrations were either slightly increased or unaltered (statistically insignificant changes). These elements seem to be transported outwards through the roots and/or translocated and stored in shoot tissues, along with Mn, to decrease their levels in the root system, in a similar, and apparently nonspecific, mechanism. In wheat, metal exclusion at the root system is a known mechanism to counter Al toxicity. Resistant plants often show an efflux of di- and tricarboxylic organic acids (e.g., citrate, malate, and oxalate) that chelate Al ions at the rhizosphere or form stable nontoxic complexes at the root surface [44]. The transporters responsible for these mechanisms were also found to permeate other relevant Al chelating anions [45]. Additionally, the release of carboxylates was determined to be a P-mobilising strategy in plants growing in soil with low P levels. This mechanism can be responsible for increased shoot metal levels since carboxylates can mobilise several micronutrients, including Fe, Mn, Cu, and Zn, alongside organic and inorganic P [46]. In the present work, similar mechanisms may be promoting increased shoot metal levels and/or element accumulation in the root apoplast, through e.g., metal/organic acid complexes. In wheat, Fe and Mn uptake and efflux appears to be linked. Increase in Fe levels reduced shoot and root dry weights; promoted Fe accumulation in the shoots; and reduced Mn, Ca, and Mg uptake and translocation, despite these responses being dependent on wheat genotype [47; 48]. Manganese, along with Fe and other metals, share common entry routes at the root system. These elements compete for root uptake through broad specificity metal transporters, whose position and abundance appear to be regulated by the plant to maintain an internal metal homeostasis [49; 50; 51; 52; 53]. Under metal toxicity, Si supplementation promotes a great number of stress mitigative mechanisms [54]. Subcellular redistribution of excessive Mn was observed in cucumber and cowpea. In cucumber, shoot Mn was redirected to the cell wall fraction reducing Mn toxicity stress in the symplast [55], and in cowpea, this mechanism was linked to a modification of cation binding capacity in cell wall components, leading to a co-precipitation of Mn and Si [56; 57].