Visual symptoms of manganese stress include reduced fresh weight, as well as chlorosis and necrotic spots on the leaves (Millaleo et al. 2010; Schmidt et al. 2016). In the tested cultivars, no significant color changes were observed on the leaf surface following MnNPs application, but the decrease of the seedling fresh weight suggested that the applied NP concentrations, especially the higher one (250 mg/mL), may initiate some toxic reactions. However, these stress effects of NPs did not result from mechanical damage to the leaf tissues, as evidenced by a lack of differences in electrolyte leakage between the control and treated objects. A rise in electrolyte leakage is usually indicated as one of the major factors responsible for increased cell membrane permeability of plants growing under different stresses (Tabaei-Aghdaei 2000). Relative electrolyte leakage (in comparison with total ion content) exceeding 50% informs of the irreversible damage to cell membranes. In our study, no significant electrolyte leakage was found, and we concluded that MnNPs treatment did not lead to drastic destruction of the plasmalemma.
As shown in spectrometric studies, an increase of Mn level in the leaves by about 20–45 times in relation to the control, suggests the penetration of NPs into the internal structures of the leaf. Interestingly, greater content of Mn (at the highest dose of NPs) was noted for cv. Alibi, for which the administration of Mn ions via the root system to leaves resulted in lower absorption of this element than in cv. Nimfa (Sieprawska et al. 2021). A drastic increase in Mn accumulation in the presence of Mn particles was previously demonstrated by Ghosh et al. (2019) in Physcomitrella patens. The possibility that the nanoparticles enter the cells via the same transporters as other ions was suggested in many studies. The knowledge on Mn ion transporters is limited, and only a few transporters involved in Mn uptake by roots have been identified up to now (Alejandro et al. 2020). The properties of Mn transporters in the plasma membrane of leaf cells have not been clearly described so far, however, Sasaki et al. (2011) and Peris-Peris et al. (2017) suggested these transporters exist also in leaf organelles.
The changes we observed in the structural and biochemical properties of leaf membranes may suggest the possibility of MnNPs interactions with lipid fractions of the membranes, which may affect their stiffness and in consequence activation of the transmembrane protein transporters. Fatty acid saturation of the membrane building lipids is the main factor responsible for the membrane stiffness. On the other hand, the changes in lipid saturation were considered the effects of the membrane modification to protect cells against toxicity of stress factors (Rudolphi-Szydło et al. 2020), or the signs of partial destruction of these structures upon stress (Liang et al. 2020). One of the determinants of oxidative stress is the increase of MDA concentration, informing of the reduction of fatty acid unsaturation. The rise of lipid saturation is triggered by ROS at the sites of double bonds in unsaturated fatty acids (Rudolphi-Skórska et al. 2017). For the tested cultivars, a progressive increase in MDA levels was observed as NPs doses increased, but the change was not as significant as in the case of other stressors, where even a 20% rise in MDA concentration was reported (Ahanger et al. 2020; Abdelaal et al. 2020). Therefore, it can be concluded that foliar administration of NPs initiated oxidative stress but the effects of this stress were relatively small, and greater in cv. Alibi than cv. Nimfa. The activation of antioxidant enzymes confirmed the occurrence of stress conditions. SODs, CAT and POX are responsible for the inactivation of excessive ROS (Hasanuzzaman et al. 2020). Activation of these enzymes may reduce possible ROS reactions with intracellular biomolecules leading to their destruction. The enhancement of SOD activity following the application of MnNPs could be also due to more abundant presence of Mn in the cells, as shown for MnSOD after treatment with Mn ions (Skórka et al. 2020).
Higher activity of SODs (enzymes transforming peroxide radical into hydrogen peroxide), found in cv. Alibi than in cv. Nimfa may have suggested greater ROS production in cv. Alibi. However, both CAT and POX, responsible for deactivation of hydrogen peroxide, showed lower activity in cv. Alibi. Such an effect might be related to lower efficiency of H2O2 removal from the cells of this cultivar and might indirectly indicate its greater sensitivity to stress. Progressive growth of starch content in cv. Alibi initiated by MnNPs application confirmed the occurrence of oxidative stress in this cultivar, exacerbated along with growing concentration of NPs. Enhanced accumulation of starch in response to various stress factors was demonstrated in many studies as a disorder of photosynthesis (Filek et al. 2015; Sieprawska et al. 2021). Interestingly, in cv. Nimfa starch level rise noted at 125 MnNPs was maintained at higher MnNPs dose.
Disorders of photosynthesis were also indicated by a decrease in chlorophyll a to b ratio. Baldisserotto et al. (2007) suggested that reduced chlorophyll content may be due to Mn oxidation in chloroplasts initiated by excessive production of ROS. Disruption of photosynthesis efficiency and a drop in chlorophyll content under excessive Mn was shown by Liu et al. (2010). In the tested cultivars, the changes in chlorophyll content were rather small, as confirmed by lack of leaf chlorosis. However, they indicated the possibility of disturbances in chloroplast functioning evoked by the presence of the nanoparticles. Experiments conducted on the chloroplasts isolated from the leaves of plants subjected to NPs showed changes in the physicochemical properties of their membranes. An increase of the chloroplast surface charge toward positive was detected by measuring the electrokinetic potential. The potential shifted toward less negative values after NPs treatment. A stronger effect visible for cv. Alibi chloroplasts even at the lower NPs dose indicated greater modifications of cv. Alibi membranes. Such modifications could diminish the affinity of cationic substances to more positively (less negatively) charged surface and reduce the effectiveness of Mn ion absorption. A similar direction of changes in the electrokinetic potential of chloroplast surface was shown under Cd stress (Filek et al. 2009).
The modifications in the membrane lipid structure initiated by MnNPs were investigated in detail in the experiments carried out for individual lipid membrane fractions isolated from the leaves. An increase in Alim area per a lipid molecule in the PL (in relation to the control) was due to enhanced fatty acid unsaturation of this fraction and may be interpreted as a factor increasing the membrane fluidity (Rudolphi-Szydło et al. 2020). PLs constitute the main lipid fraction of the plasmalemma and for the tested wheat cultivars they accounted for about 50% of the pool of lipids extracted from the membranes. Enhanced fluidity of PL fraction may lead to greater exposure of the hydrophobic part of the lipids (increasing the distance between polar groups) in order to reduce the possibility of adsorption of ionic components on non-polar parts of the membranes. Such effects initiated by MnNPs treatment, more pronounced in cv. Nimfa, may suggest that in this cultivar the studied NPs activated the protective mechanisms to a higher degree than in cv. Alibi. The changes in membrane stiffness under the treatment with MnNPs were also evidenced by the values of πcol and Cs-1 parameters, suggesting greater "decompression" of the lipids of PL fraction in cv. Nimfa. The strongest effects (Cs) shown after the administration of 250 mg/mL NPs may indicate that this concentration activated the defense mechanisms to a much greater extent than 125 NPs. For the studied galactolipids, the reduction of Alim values (relative to the control) may have designated an increase in lipid saturation of this fraction, stimulated by NP treatment. In general, the changes in galactolipids represent the reaction of chloroplast membranes, as these lipids are the main lipid fractions in plastids (Hölzl and Dörmann 2019). The polarity of MGDG and DGDG fractions results mainly from the presence of sugar groups. The decrease of Alim, together with the rise of πcol and Cs-1 upon NPs treatment, represented a trend toward increasing lipid stiffness (Alim) and membrane stability (parameters π and Cs-1). These effects were especially visible in DGDG fraction of both cultivars, however, some differences between the tested cultivars were ascribed to the extent of changes between MGDG and DGDG fraction. This proved the cultivar specificity in response to NPs, which was also confirmed in the analysis of other biochemical parameters mentioned above. In addition, it indicated that chloroplasts were the organelles that determined the cultivar response to NPs.
Studies carried out for selected human cell lines demonstrated the possibility of direct interaction of MnNPs with membrane lipids in intact cells. Human cell membranes are composed mainly of phospholipids (Lyberg et al. 1983; Chabot et al. 1989; Berkovic et al. 1997; Ecker et al. 2009), so it was possible to assess the degree to which the applied concentrations of NPs exerted a stressful effect on these structures and led to their disruption. The experimental setup included four types of human cell lines: U-937, HL-60, HUT-78, and COLO 720L. Selected cell types (U-937 and HL-60) after differentiation become monocytes, macrophages, and granulocytes (Harris and Ralph 1985). As cells widely distributed in blood and tissues, they make the first contact with "foreign" agents, such as for example nanoparticles. It is assumed that the body "response" to a toxic agent depends on these cell types, as they are the fastest to reach the place of contact with the toxic agent. Next, the cells of the acquired response, T and B lymphocytes (represented by HUT-78 and COLO 720L lines), together with companion cells, are engaged in the defense of the organism. The selected cell lines allowed us to carry out tests on the cells of human immune system, as the ones most exposed to contacts with toxic agents. Cell viability analysis based on mitochondrial activity (via mitochondrial dehydrogenase activity), made it possible to characterize sensitivity of the tested cells to MnNPs at the concentrations lower and the same as those used in plants. The most sensitive line was U937, in which the dose of 15.625 NPs caused a 10% drop in viability. The dose of 250 mg/mL NPs, the highest one applied to the plants, significantly reduced human cell viability, at a similar percent for all the tested lines. To check to what extent MnNPs treatment resulted in direct damage to the cell structure in the tested lines, LDH analysis was performed. It demonstrated electrolyte leakage from the cells caused by interrupted continuity of the plasmalemma. In comparison with the action of other nanoparticles (Ag, Zn), this effect was rather small (Barbasz et al. 2015; Oćwieja and Barbasz 2020; Czyżowska et al. 2021). This may be related to other processes of oxidative disintegration of MnNPs and to their size as compared with Ag and ZnNPs when dissolved in the cellular environment. Moreover, the examined nanoparticles showed significantly lower immunogenicity than silver nanoparticles (Barbasz et al. 2017). Despite similarities in viability at 250 mg/mL NPs, some differences between the tested lines were noticed also when analyzing LD50. Its value was higher for the innate response cells (U937 and HL-60), suggesting that those cell lines were less sensitive to the toxic effects of manganese nanoparticles.