Analysis of As and essential elements by ICP-AES
The concentrations of As, Ca, K, Mg, P, and S are shown in Fig. 1, and the concentrations of trace elements B, Cu, Fe, Mn, Mo, and Zn are shown in Fig. 2. The As-unexposed P. vittata accumulated 1.02 ± 0.56 mg As/kg. The concentrations of essential elements in the As-unexposed P. vittate were as follows: Ca, 7062 ± 1159 mg/kg; K, 8080 ± 721 mg/kg; Mg, 1964 ± 387 mg/kg; P, 7837 ± 2103 mg/kg; S, 7142 ± 2552 mg/kg; B, 19.0 ± 5.2 mg/kg; Cu, 5.17 ± 0.63 mg/kg; Fe, 68.2 ± 14.7 mg/kg; Mn, 28.0 ± 6.4 mg/kg; Mo, 1.30 ± 0.16 mg/kg; and Zn, 30.2 ± 7.04 mg/kg, which is consistent with the values of previous studies15. Conversely, the concentration of As in the As-exposed P. vittata was much higher, at 4563 ± 257 mg/kg. There was more browning of the outer periphery of pinnae under high As concentrations compared with the normal condition. The essential element concentrations in the As-exposed P. vittata were as follows: Ca, 6283 ± 208 mg/kg; K, 8779 ± 601 mg/kg; Mg, 2111 ± 286 mg/kg; P, 18591 ± 4582 mg/kg; S, 9962 ± 1964 mg/kg; B, 13.6 ± 4.8 mg/kg; Cu, 5.89 ± 1.09 mg/kg; Fe, 81.2 ± 20.5 mg/kg; Mn, 28.2 ± 6.7 mg/kg; Mo, 1.34 ± 0.18 mg/kg; and Zn, 37.8 ± 4.22 mg/kg. These concentrations were almost identical to those in the As-unexposed P. vittata. However, K, P, S, Fe, and Zn concentrations were higher in As-exposed P. vittata than unexposed P. vittata, whereas concentrations of Ca and B were lower. Similar increases and decreases in essential element concentrations with the accumulation of As have been reported in previous studies15. Yamazaki et al. reported a negative correlation between As and Ca concentrations in the pinnae of P. vittata, and proton-induced X-ray emission (PIXE) analysis showed that Ca concentrations decreased as As concentrations on the surface of pinnae increased16. In addition, Sugawara et al. reported a negative correlation of As with B, K, and P concentrations in pinnae when As exposure was increased in a stepwise manner15. In general, K is an indicator of plant activity, and it is well known that growth enhancement occurs in the presence of trace amounts of As in P. vittata. Therefore, the concentration of P in plants tended to increase due to the growth promotion associated with exposure to As. Moreover, S is a constituent element of low-molecular-weight thiols related to the detoxification of toxic substances in plants, which could explain the increase in S with increasing As. Based on these results, we decided to map four essential elements, P, S, Ca, and K, which were correlated with As and can be measured simultaneously with As in EPMA analysis.
Localized mapping of As, P, S, Ca, and K in the pinnae of P. vittata by EPMA
Frozen thin-layer sections of As-exposed and unexposed P. vittata pinnae analyzed by ICP-AES, as described above, were prepared using paraffin embedding and cryomicrotome, and elemental mapping was performed by EPMA. Figure 3 shows the results of the elemental mapping of pinnae of P. vittata prepared using the paraffin-embedding method, where the signals of elements such as Ca in the cell tissue were confirmed, but the signals of As and K were very weak or almost absent. It is generally believed that K is present in plant cells in a water-soluble ionic state at a concentration of 100 mM or more17; therefore, it is likely that K was eluted from the tissue during the sectioning process. These results suggest that As is not only present as arsenite but also accumulates in a water-soluble state without strong tissue binding. Figures 4 and 5 show the results of elemental mapping of As-exposed and unexposed pinnae of P. vittata, respectively. Analysis of the entire cross-section of the pinnae confirmed that even water-soluble elements were retained in the sample, unlike the sections prepared using the paraffin-embedding method, although frost was observed around the sample. The As signal was confirmed only in the As-exposed pinnae and not at all in the unexposed pinnae.
The behavior of As was compared with that of the four other essential elements measured. The S and P signals were strong in places where the As signal was also strong, indicating similar localization. However, no clear change in the behavior of Ca and K with the accumulation of As was observed. The results of Nano-SIMS and µ-XRF analyses of As-accumulated rice by Moore et al. showed that the localization sites for As and S were consistent and the signal intensity was positively correlated, whereas the localization sites for As and P were not consistent18. Thus, although the localization of S was consistent with the general mechanism of plants involved in the detoxification of As, the behavior of P may be unique to the fern. The cross-sectional analysis showed a tissue-level elemental mapping result consistent with the ICP-AES results as a trend of the entire pinnae. Therefore, in order to further analyze a narrower area, we decided to analyze two parts of the cross-section of the pinnae, the central part near the leaf vein and the periphery of the pinnae (Fig. 6). The accumulation mechanism of As is expected to be different in these two sites because the area around the leaf vein tends to be healthy while the area around the petiole tends to brown and die when exposed to As. Elemental mapping of As, Ca, K, S, and P in a narrow area near the pinnae vein of the As-exposed fern is shown in Fig. 7. It was confirmed that As was localized to the cell wall and not inside the cell. In addition, S was localized in the area where the As signal was relatively weak. However, the localization of P was completely different from that of As. The localization of K was relatively consistent with that of As, and Ca was localized near the epidermis of the pinnae. S and P were also present in the cells.
Elemental mapping of As, Ca, K, S, and P in a narrow area around the periphery of the pinnae of the As-exposed fern is shown in Fig. 8. The signal of As tended to be stronger in the vicinity of the leaf veins, consistent with the presence of As at the base and at the periphery of the leaf, as reported in previous studies. Consistent with the results of the analysis near the foliar veins, As was not localized near the epidermal tissue in the periphery, but in the fence-like or spongiform tissues. More interestingly, while As was localized near the cell wall in the vicinity of the veins, As was not clearly extracellularly localized in the periphery of the pinnae, but was distributed throughout the cell. The localizations of As, K, and S are partially consistent with each other, and the K signal was particularly strong. In addition, Ca was distributed throughout the apical region, which was very different from the distribution of As in the vicinity of the veins. The localization of P and As was quite different from the results of the analysis near the foliar veins, especially P, which did not localize near the epidermal tissue of the pinnae.
Comparison of the elemental mapping results in the narrow area near the leaf vein and near the periphery of the pinnae of P. vittata confirmed some major differences. First, we found that As was localized near the cell wall in the foliar vein, while it was present in the entire cell tissue in the periphery. As reported in previous studies, pinnae tend to die off from the periphery when As concentrations are high in P. vittata15. Therefore, it was expected that As localization around the veins, which are relatively healthy, would be different from that around the periphery. Although the periphery of the pinna of P. vittata was not dead, the presence of As in the cells even in this state suggested that death from the periphery occurs when As is not isolated from the cells for a long period of time. As and K tended to localize similarly in both the vicinity of the vein and the periphery, but the behavior of S, which tended to coincide with As, was different between the vicinity of the vein and the periphery. In the analysis near the fovea, where the cell condition is considered to be relatively healthy, S tends to be negatively correlated with the strength of the signal itself, although S is consistent with the localization of As. As mentioned above, small molecule thiols containing S as a constituent element are known to reduce toxicity by forming specific complexes with arsenite, which is the main chemical form of As present in P. vittata19. Therefore, although the As and S signals were expected to be positively correlated, they were in fact different. This is because, as mentioned earlier, As and S can be positively correlated in terms of both localization and abundance if only small thiols are used to detoxify As. However, these results suggest that P. vittata also has other mechanisms of detoxification. Alternatively, although the localization of As and S was consistent in the periphery, the overall low signal of S may be due to the high level of As present in the periphery. Since this trend has been observed in the vicinity of the leaf veins, the behavior of S in relation to As is, to some extent, consistent in pinnae. For Ca, the localized sites of As were completely different from those of As in the vicinity of the foliar veins and were observed only in the vicinity of epidermal cells, whereas the localized sites of As tended to coincide to some extent with those of As in the periphery. Ca plays a role in the pectin end of the cell wall in plants and is known as a constituent element of the cell wall17. Therefore, it is likely that Ca was localized near epidermal cells, which seemed to have a relatively strong structure. Conversely, at the periphery, Ca was relatively co-localized with As, K, and P. Considering that K is mainly present in the intracellular protoplasm and P is mainly present in nucleic acids and other intracellular substances, the structure of the cell itself is not maintained at the periphery of the cell, suggesting that the localization sites of elements with different properties are consistent. Therefore, it is thought that this situation is maintained for a long period of time, and eventually death from the periphery occurs.