In vivo micro X-ray analysis utilizing synchrotron radiation of the gametophytes of three arsenic accumulating ferns, Pteris vittata L., Pteris cretica L. and Athyrium yokoscense, in different growth stages

Abstract

In vivo X-ray analysis utilizing synchrotron radiation was performed to investigate the distribution and oxidation state of arsenic in the gametophytes of two hyperaccumulators, Pteris vittata L. and Pteris cretica L., and an arsenic-accumulating fern, Athyrium yokoscense in the several growth stages from germination. The distribution of arsenic in P. vittata changed through the development of the plant tissues as follows. In two-week-old gametophyte, arsenic was mainly present along the rhizoid. In the one-month-old gametophyte with reproductive organs, arsenic was accumulating uniformly in the sheet of cells, except in the reproductive area. After fertilization, arsenic was observed in the aboveground part of the sporophyte structures. P. cretica and A. yokoscense showed different distributions, respectively. P. cretica showed an accumulation of arsenic in the reproductive area, in contrast to P. vittata, before fertilization, while arsenic was observed in the aboveground part of the sporophyte after fertilization. A. yokoscense showed an accumulation of arsenic along the rhizoids before fertilization, while it was present mainly along the roots of the sporophyte after fertilization. Reduced arsenic (As(III)) was observed in all stages and in all tissues of P. vittata gametophytes. Further, a reduction of arsenic was commonly observed among the three ferns, although arsenic was bounded to sulfur in A. yokoscense. These findings may be related to their own reproductive process or to detoxification mechanism. They provide basic information for the understanding of arsenic hyperaccumulation in these ferns, leading to further application of these gametophyte systems.

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1. Introduction

Pteris vittata L. is the first reported arsenic-hyperaccumulating fern.¹ It can accumulate up to 22 630 mg As kg⁻¹ dry weight in its aboveground biomass when grown in the soil containing as much as 1500 mg As kg⁻¹.¹ Since this ability is ideal for the remediation of contaminated soils, P. vittata has received much attention for its application to phytoremediation.^2,3 In fact, practical application of P. vittata for this purpose has already begun.

Arsenic is a nonessential element for plants, and inorganic As species in particular (= arsenate and arsenite) are highly phytotoxic. Arsenate acts as a phosphate analog and can disrupt phosphate metabolism, whereas arsenite reacts with the sulfhydryl groups of enzymes and tissue proteins, leading to inhibition of cellular functions and death.^4,5 Strikingly, most of the arsenic in P. vittata is present as the more toxic arsenite form, even though arsenate is the dominant form in soils or hydroponic solutions, indicating that a reduction of arsenic occurs in the plant body.^6–10 Furthermore, P. vittata efficiently transports arsenic from the roots to its shoots, stores more than 90% in its pinnae, and maintains a greater P/As ratio in its roots than non-hyperaccumulators.^11–17 Based on the previous reports, the reduction of arsenic and its distribution are considered to be important characteristics for arsenic hyperaccumulation in P. vittata.^9,11,16,17

However, the molecular mechanisms underlying the distribution and reduction of arsenic are still unknown. One of the reasons for this is the complexity of the biochemical systems in the sporophytes, which were used as samples in many previous studies. The sporophytes have a large biomass, a large genome size, slow perennial growth, and many developed tissues of various sizes. These factors lead to difficulties in treating the sporophytes as experimental materials in terms of their age and genetic homogeneity, or in interpreting the data in relation to the particular biochemical processes.

In contrast, the gametophytes, which are another generation in the lifecycle of the fern, are a simpler system than the sporophytes. This system has long been suggested as a useful model system to investigate plant biology because of its small size, simple structure, rapid growth rate, and haploid genome , which leads to an ease of culture and standardization of the experimental materials.^18–21 In the recent studies on arsenic hyperaccumulating ferns, this system has begun to be used for molecular biology experiments.^22–26 Some of P. vittata genes involved in arsenic accumulation have been successfully isolated using gametophytes,^23,24 showing the utility of this system.

In this study, we focused on the gametophyte system. While gametophytes are suitable for genetic approaches compared with the sporophytes, they also show several growth stages with or without structures during the growth process from germination. However, the distribution and species of arsenic in this system, i.e., how the distribution of arsenic changes in the plant body or when reduction of arsenic occurs in the development from germination, are still unknown. This would provide basic information on the growth process on an As-contaminated site, and would be useful for the characterization of arsenic hyperaccumulation in P. vittata.

Two arsenic-accumulating ferns with different characteristics were also studied to discuss the specific traits of P. vittata based on their comparison with P. vittata. Pteris cretica L. was one of these ferns, which is classified in the Pteris genus, and is also known as an arsenic hyperaccumulator with a dry weight concentration in the shoots and roots reaching up to 6000 mg As kg⁻¹ and 1000 mg As kg⁻¹, respectively.^16,27 This fern has a different reproductive process in its gametophytes from P. vittata, i.e., P. cretica usually shows nonsexual reproduction while P. vittata shows sexual reproduction. The other fern was Athyrium yokoscense, which has been known for a long time to accumulate several heavy metal elements, such as Cu, Zn, Pb, and Cd.^28–30 This fern has also been reported to be able to accumulate arsenic in its shoots and roots, reaching up to 900 mg As kg⁻¹ and 2200 mg As kg⁻¹, respectively, which is a different distribution of arsenic from the other two hyperaccumulators.³¹ The details of accumulation mechanisms of arsenic in these two ferns are not known, but it is expected that comparison with P. vittata may lead to a better understanding of arsenic hyperaccumulation in plants.

Highly sensitive methods with a high spatial resolution are required to analyze the distribution and chemical state of arsenic in such small gametophyte samples. X-ray fluorescence analysis utilizing synchrotron radiation (SR-XRF) was used in this study. This method enables us to analyze the plant samples in the living state after suitable sample preparation.^32–37 In our experiments, a new introduction of sample packing technique using phytoagar allowed for in vivo analysis over an extended time.

The aim of this study is to reveal the changes in distribution and chemical state of arsenic during the development of P. vittata gametophytes from germination, and to compare them with other two ferns that have a different reproductive process or distribution of arsenic from P. vittata. We discuss the relationship between the development of the plant tissues and behaviors of arsenic in P. vittata, and differences among the three ferns. Our investigation will provide basic information to reveal arsenic hyperaccumulation mechanisms in plants.

2. Experimental

2.1 The growth process of P. vittata gametophyte and focused three stages

Before the experiments, plant culture was conducted as described in subsection 2.2, and the development of P. vittata gametophytes was observed in detail. Fig. 1 shows photographs of the samples during the two month period from germination (Fig. 1(a)–(e)). In this study, we classified the growth process into the several growth stages, and the following three stages are focused on: (i) the infant stage without any particular structures, except for a tiny single-layered sheet of cells and several hair-like rhizoids after two weeks from germination (Stage 1, Fig. 1(b)), (ii) the one-month-old stage of a heart-shaped appearance, with a meristem and reproductive organs such as egg-forming archegonia and sperm-forming antheridia (Stage 2, before fertilization, Fig. 1(d)), and (iii) the two-month-old stage, with the structures of sporophyte such as roots, stems and fronds (Stage 3, after fertilization, Fig. 1(e)).

Fig. 1 The growth process of P. vittata gametophyte and focused three stages. (a): 7 days after germination, (b): with rhizoids and sheet of cells (14 days of growth, Stage 1), (c): with meristem (21 days of growth), (d): with reproductive tissues (1 month of growth, Stage 2), (e): after fertilization with the gametophyte and the sporophyte (after 2 months of growth, Stage 3).

2.2 Plant culture and arsenic treatment

Spores of P. vittata were collected from sporophyte plants obtained from the Fujita Co., Japan. Those of P. cretica and A. yokoscense were obtained from field collected sporophytes, the former from Tsukuba, Japan and the latter from Hanno, Japan. These spores were sterilized by soaking them in a solution containing 1% sodium hypochlorite for 15 min and then washing them three times in sterile water. The medium used for culturing gametophytes contained 1/2 strength Murashige and Skoog (MS), pH 6.5, which was solidified with 0.7% agar. Some of the gametophytes in Stage 2 were transferred to potting soil saturated with 1/10 strength MS liquid medium and were grown to Stage 3. These cultures were grown at 25 °C under 24 h fluorescent lighting. For the analysis, the gametophytes of P. vittata were grown to Stages 1, 2 and 3, and those of P. cretica and A. yokoscense were grown to Stages 2 and 3.

These gametophytes were exposed to arsenate solutions prepared by dissolving potassium arsenate (KH₂AsO₄) in deionized water. The concentration of the arsenic was 50 mg L⁻¹ for P. vittata and P. cretica and 1 mg L⁻¹ for A. yokoscense. These treatments were conducted by growing the gametophytes on filter papers soaked in the arsenate solutions for three days at 25 °C under continuous illumination.

2.3 Sample preparation for in vivoX-ray analysis

For XRF imaging, the gametophytes were embedded in 0.7% phytoagar and packed in 4 μm thick Mylar films® to prevent them from drying during the long measurement time. For the XANES analysis of Stages 1 and 2, some gametophytes were gathered and exposed to the X-ray beam in bulk because of their small size. In the analysis of Stage 3, gametophytes were packed individually in 4 μm thick Mylar film® and then subjected to XANES analysis because they were comparably large in size and easy to treat.

2.4 SR-XRF two-dimensional imaging

Two-dimensional X-ray fluorescence imaging was carried out at the BL-4A, in the Photon Factory (PF), High Energy Accelerator Research Organization (KEK), Tsukuba, Japan. The difference in size among the samples in each stage was so large that the following three optical systems were used to obtain the three beams: for imaging of Stage 1, the incident beam from the bending magnet was monochromatized using a W/B₄C double multilayer monochromator. The beam was focused by a K–B mirror system to the size of 4 μm (vertical, V) × 4 μm (horizontal, H). For Stage 2, a Si(111) double-crystal monochromator was used to obtain a monochromatic X-ray beam, the size of which was 30 μm (V) × 30 μm (H) focused by polycapillary. For Stage 3, the incident beam was monochromatized using a Si(111) double-crystal monochromator. Two-dimensional focusing was achieved by combining the sagittal horizontal focusing and vertical focusing mirror coated with Rh and adjusted to ca. 200 μm (V) × 200 μm (H) by slits. The X-ray energy was adjusted to 12.8 keV to excite the K line of arsenic and avoid the excitation of the L line of lead since they interfere with each other. The incident X-ray intensity (I₀) was measured using an ionization chamber filled with air. The arsenic Kα fluorescence was monitored by a Si(Li) solid-state detector placed at 90° to the incident beam to minimize scattering. The samples were mounted on an x-y sample stage and spatially rastered in the X-ray beam using pulse motors. The measurements were carried out under ambient conditions.

The integrated peak intensity of the XRF line of each element per pixel was normalized by I₀. Elemental mapping was obtained for the measurement area displayed using the 256 gradation color scale with red denoting the highest intensity and blue the lowest. The distance between the samples and the detector was fixed in a series of the measurement for each stage. In addition, the approximate quantity of arsenic per pixel was calibrated by recording the fluorescence intensity from standard solutions of arsenic (5, 10 and 100 mg L⁻¹) dropped on a filter paper with the same path length as the samples. The lowest limit of detection was calculated to be 0.397 ng/(4 × 4 μm²), 1.13 ng/(30 × 30 μm²), and 0.183 ng/(200 × 200 μm²), following the procedure done by Hokura et al. (2006).³²

2.5 As K-edge XANES analysis

The arsenic K-edge XANES spectra were measured at the BL-12C at PF, KEK, Japan utilizing the following optical system. A white X-ray from a bending magnet was monochromatized by a Si(111) double-crystal monochromator. A bent cylindrical mirror was used as the focusing optics. The beam size was adjusted to ca. 1 mm (V) × 1 mm (H) by slits. The intensity of I₀ was measured with a nitrogen-filled ionization chamber. The XANES spectra were measured in the fluorescence mode by monitoring the X-ray fluorescence intensity of the As Kα line (10 543 eV) with a 19-element Ge solid-state detector at 90° to the incident beam, taking care to maintain the count rate within the pseudolinear regime. A solar slit was placed in front of the detector to reduce scattering X-rays. The measurements were conducted under ambient conditions.

The As K-edge spectra were recorded from 11 850 to 11 890 eV, using a step size of 0.86 eV for the edge region. The chemicals of As₂O₃, KH₂AsO₄, and As₂S₃ (Wako Chemicals, Japan) were also measured as reference materials. As₂O₃ and As₂S₃ were used to identify the As–O and As–S bonds in plants based on the previous reports.³⁶ The X-ray energy was calibrated using As₂O₃.

XANES analysis was carried out by the REX2000 program, ver. 2.5 (Rigaku Co., Japan). Quantitative analysis of the XANES spectra was conducted by simulating the spectra of the samples using the As(III) (As₂O₃) and As(V) (KH₂AsO₄) within the range of 11 860–11 873 eV. The quality of the fit was given by the goodness of fit parameter R, defined by

R = Σ{χ_obs(E) − χ_cal(E)}²/Σ{χ_obs(E)}² (1)

Here, χ_obs(E) and χ_cal(E) are the experimental and calculated absorption coefficients at a given energy (E), respectively.

3. Results and discussion

3.1 SR-XRF imaging of P. vittata gametophytes in three different stages

Fig. 2(a)–(d) show the sample in Stage 1 and distributions of As, Fe, Zn. Rhizoids arose from the spores on the gametophyte (Fig. 2(a)). Arsenic accumulated along the spore and the rhizoids and only present at low concentrations in the cells (Fig. 2(b)). Iron showed a similar distribution to arsenic, as it was mainly present in the spores and rhizoids (Fig. 2(c)). Zinc was present uniformly along the sheet of cells (Fig. 2(d)).

Fig. 2 XRF imaging of P. vittata gametophytes. Measurement area is within the red square in the photograph. (a)–(d): Stage 1, (e)–(h): Stage 2, (i)–(l): Stage 3. SP: spore, RH: rhizoid, RA: reproductive area, GA: gametophyte, FS: frond of the sporophyte, RS: root of the sporophyte. Pixel size, pixel number and dwell time: 10 μm(V) × 10 μm(H), 35 (V) × 37 (H) pixels and 2 s/pixel ((b)–(d)); 30 μm(V) × 30 μm(H), 32 (V) × 42 (H) pixels and 5 s/pixel ((e)–(h)); 200 μm(V) × 200 μm(H), 66 (V) × 30 (H) pixels and 3 s/pixel ((j)–(l)). Point V1-6 were used for estimation of As amount. Correlation between As and Fe were analyzed in Area V1,2.

In Stage 2 (Fig. 2(e)–(h)), arsenic was accumulated uniformly in the sheet of cells, while it was almost absent from the specific area where rhizoids arose, i.e., the reproductive area (Fig. 2(f)). Archegonia were observed in this area (Fig. 2(e)) and arsenic was only slightly present around them (Fig. 2(f)). Arsenic was also accumulated as speckles around the spore, but except for these parts, it was almost absent in the reproductive area (Fig. 2(f)). On the other hand, zinc was localized along the rhizoids and reproductive area (Fig. 2(h)). Iron was present around the spore (Fig. 2(g)) as was the case in Stage 1 (Fig. 2(c)).

In Stage 3, after fertilization (Fig. 2(i)–(l)), arsenic was accumulated around the base of the sporophyte structures and was also slightly distributed toward the aboveground part (Fig. 2(j)). On the other hand, iron and zinc showed different patterns from arsenic. These elements were present around the base of the sporophyte structure and distributed slightly in the roots (Fig. 2(k) and (l)).

In previous reports, P. vittata gametophytes also showed a tolerance and a hyperaccumulation of arsenic to a similar extent as the sporophytes.²² The distribution of arsenic in the one-month-old gametophytes, corresponding to Stage 2 in the present study, has also been investigated previously using XAS imaging, and was found to accumulate uniformly in the sheet of cells, except for the specific area where the rhizoids and archegonia arose.³⁵ In the present study, a similar arsenic accumulation and distribution in the one-month-old gametophytes was observed.

In particular, the exclusion of arsenic from the reproductive area was found as a remarkable feature of Stage 2 (Fig. 2(f)). Arsenic toxicity is generally considered to be sequestered in vacuoles as tolerance at the cellular scale.^6,38 Similarly, the absence of arsenic (6.12 ng/pixel at Point V4) compared with the sheet of cells (85.8 ng/pixel at Point V3) may reflect a sequestration of arsenic from the reproduction process at the larger scale. That is, this distribution of arsenic may reflect a resistance mechanism, hence a tolerance, to arsenic toxicity around the reproductive area. The localization of zinc in the reproductive area revealed by our XRF imaging indicates a requirement for zinc, in contrast to arsenic, indirectly supporting this specific function in Stage 2.

In the infant stage, two weeks after germination (= Stage 1), the highest accumulation of arsenic was observed around the spore (Fig. 2(b), Point V1). At this point, the extent of arsenic accumulation (64.0 ng/pixel) was similar to that observed in the spore in Stage 2 (Fig. 2(f), 56.6 ng/pixel at Point V5), while the accumulation in the cells in Stage 1 (Fig. 2(b), 15.9 ng/pixel at Point V2) was less than those in Stage 2 (Fig. 2(f), 85.8 ng/pixel at Point V3). These observations imply that the ability to accumulate arsenic in the sheet of cells gradually develops from Stage 1 to Stage 2. In addition, a positive correlation between the concentration of arsenic and iron was shown around the spores in both stages (correlation coefficient, r² = 0.681 for Area V1 in Fig. 2(c), and r² = 0.915 for Area V2 in Fig. 2(g)). The reason for this is not clear from the present results alone.

On the other hand, after the fertilization stage (= Stage 3), most of the arsenic was accumulated at the base of the sporophytes (Fig. 2(j), 156 ng/pixel at the Point V6). The sporophytes of P. vittata in the previous reports showed an efficient transport of arsenic from the roots to shoots and maintained high P/As ratio in the roots. In the present study, only a slight distribution of arsenic in the sporophyte was observed (Fig. 2(j)). Thus, it was expected that the previously reported typical distribution pattern of arsenic in the sporophytes occurred gradually in this stage. Phosphorus, one of the key elements for the understanding of arsenic detoxification, is expected to be distributed toward the roots, though its fluorescence signal could not be detected due to the absorption by air in our in vivo analysis. Iron and zinc were also shown in the roots, in contrast to arsenic (Fig. 2(k) and (l)).

In addition to the previous report on arsenic in the one-month-old gametophyte,³⁵ much new information as to the distributions of As, Fe, Zn was revealed by our investigation of gametophytes in two more new stages. As discussed above, these elemental distributions were found to correspond to the development of typical structures, such as spores, rhizoids, reproductive organs, fronds and roots. This finding is interesting, because this series of information implies the development of an arsenic accumulation mechanism along with the growth process of P. vittata.

3.2 As K-edge XANES analysis of P. vittata gametophytes

The arsenic K-edge XANES spectra of samples of P. vittata and the reference materials are shown in Fig. 3. The spectra of the reference materials ((a): KH₂AsO₄ and (b): As₂O₃) showed clear chemical shifts, that is, the edge energy of the As(V) species was shifted higher by ca. 3 eV than that of As(III) species. This relationship was utilized to estimate the oxidation state of the arsenic in the gametophytes. The spectrum of a hydroponic solution prepared by dissolving KH₂AsO₄ in deionized water showed peaks at the same energy as As(V) (Fig. 3(a) and (c)). This result confirmed that the plant samples were exposed to As(V) during the cultural experiments. In contrast, As(III) was dominantly observed in all XANES spectra of the gametophyte samples (Fig. 3(d)–(g)). The fitting results, summarized in Table 1, show that above 86% of the arsenic was present as As(III) in all the stages.

Fig. 3 Arsenic K-edge XANES spectra of reference materials and the gametophytes of P. vittata.

Table 1 Fitting results of As K-edge XANES spectra of P. vittata gametophytes

Sample	As(III)	As(V)	R^a
a R was defined as eqn (1).
Stage 1	86%	14%	0.032
Stage 2	95%	5%	0.027
The gametophyte part of Stage 3	97%	3%	0.051
The sporophyte part of Stage 3	81%	19%	0.047

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These results clearly indicate that a reduction of the arsenic in P. vittata takes place, regardless of any of the specific structures developed through the growth process. Some previous reports have suggested that the reduction of arsenic mainly occurs in the fronds,^14,39 while others have suggested that the reduction mainly occurs in the roots.^40–42 Further, reduction of arsenic has also been reported in the one-month-old gametophytes with reproductive organs.^23,35 These inconsistencies in previous works are presumed to be due to the existence of many biochemical processes in their bodies, leading to the difficultly in determining the particular process where the reduction of arsenic takes place. On the other hand, our investigation of arsenic in the gametophytes with and without structures has enabled us to understand the relationship between specific structures and the behaviors of arsenic, which is a useful application of the gametophyte system. The reduction of arsenic observed in infant Stage 1, clearly indicates that reduction mechanism in infant stage is not directly related to the biochemical process occurring in the structures, such as the reproductive area on the gametophytes, or the roots, stems, and fronds of the sporophytes, but it may be a constitutive trait of each cell, because the infant gametophytes are composed of almost only a simple sheet of cells without any specific tissues.

Previous reports have suggested that arsenic hyperaccumulation by the Pteris species is a constitutive trait, with plants originating from As-contaminated and non-As-contaminated environments showing broadly similar hyperaccumulating abilities.²⁷ If the reduction of arsenic in the Pteris species is related to some process in the hyperaccumulation mechanism, then, the reduction of arsenic in the early stages, e.g., Stage 1, may imply that arsenic hyperaccumulation ability exists for the gametophytes as a basic function of each cell. This is reasonable, because detoxification of arsenic is necessary at least, even for infant cells, when germination occurs naturally on an As polluted site.

The reduction of arsenic was also observed in more developed gametophytes (= Stages 2 and 3) and in the sporophyte structures in the Stage 3 (Fig. 3(e)–(g)). However, it was difficult to know whether these reductions of arsenic are due to the same mechanism or not, because the biochemical systems increased in complexity through development of the plant body. Recently, several genes encoding an arsenate reductase have been found.^23,24 The existence of several genes may imply further enzymes or several pathways for the reduction of arsenic in plant bodies.^40,43 The significant amount of As(V) in Stage 1 (14%) and in the sporophyte in Stage 3 (19%) compared with the others (Table 1) may reflect different pathways of arsenic metabolism in each structure, although it is difficult to conclude from the present results alone. The complexity of the developed plant body can be a significant problem for the understanding of the basic molecular mechanisms.

However, we still suggest the utility of our approach, that is, the speciation of arsenic with the spatial information during the growth process, because it may be possible to determine the arsenic species through a particular biochemical process based on its expression in the growth process. This will give us insights into the understanding of whole developing plant system, as well as the next strategy for research of basic molecular mechanisms. Further analysis of developing gametophyte systems combined with biological data such as RNA or protein expression is expected for understanding the complex biochemical systems of arsenic hyperaccumulating plants.

3.3 SR-XRF imaging of P. cretica and A. yokoscense gametophytes

Fig. 4(a)–(h) show the samples and the distribution of As, Zn and Fe in P. cretica gametophytes in Stages 2 and 3. In Stage 2, arsenic was accumulated in the part where the sporopyhte would arise, compared to the sheet of cells (Fig. 4(b)). Iron was mainly present around the rhizoids (Fig. 4(c)), as in the case of P. vittata in Stage 2 (Fig. 2(g)). Zinc was also found extensively over this area (Fig. 4(d)). In Stage 3 for P. cretica, arsenic and zinc were distributed in both sporophytes and gametophytes (Fig. 4(f), and (h)). On the other hand, iron was mainly present around the rhizoids of the gametophyte part in Stage 3 (Fig. 4(g)), similar to Stage 2 (Fig. 4(c)).

Fig. 4 XRF imaging of the gametophytes of P. cretica and A yokoscense. Measurement area is within the red square in the photograph. P. cretica in Stage 2 ((a)–(d)) and Stage 3 ((e)–(h)). A. yokoscense in Stage 2 ((i)–(l)) and Stage 3((m)–(p)). RH: rhizoid, RA: reproductive area, GA: gametophyte, FS: frond of the sporophyte, RS: root of the sporophyte. Pixel size, pixel number and dwell time: 30 μm(V) × 30 μm(H), 55 (V) × 42 (H) pixels and 5 s/pixel ((b)–(d)); 200 μm(V) × 200 μm(H), 36 (V) × 58 (H) pixels and 5 s/pixel ((f)–(h)); 30 μm(V) × 30 μm(H), 36 (V) × 51 (H) pixels and 3 s/pixel ((j)–(l)); 200 μm(V) × 200 μm(H), 15 (V) × 57 (H) pixels and 5 s/pixel ((n)–(p)). Point C1,2 and A1,2 were used for the estimation of As amount. Correlation between As and Fe were analyzed in Area C and A.

Fig. 4(i)–(p) show the samples and distribution of As, Zn, and Fe in A. yokoscense in Stages 2 and 3. In Stage 2, most of the arsenic had accumulated along the rhizoids, while arsenic was only slightly observed along the shape of the cells (Fig. 4(j)). Iron was present along the rhizoids (Fig. 4(k)), similar to arsenic (Fig. 4(j)), and zinc was found extensively over the rhizoids and reproductive area (Fig. 4(l)). On the other hand, in Stage 3, these three elements were mainly present along the roots of the sporophyte (Fig. 4(n)–(p)).

Our observations provide the first information on the distribution of arsenic in the gametophytes of P. cretica and A. yokoscense. In particular, in Stage 2 of P. cretica, the accumulation of arsenic in the part where the sporophyte will arise (Fig. 4(b), 227 ng/pixel at Point C1) is remarkable, because this distribution contrasts with that of P. vittata, where arsenic was almost absent in the reproductive area (Fig. 2(f), 6.12 ng/pixel at Point V4). This distribution of arsenic clearly corresponds to their different reproductive processes: P. vittata shows sexual reproduction while P. cretica shows nonsexual reproduction, indicating that the exclusion of arsenic from the reproductive area is a characteristic of P. vittata. In other words, one-month-old gametophytes already show their own features, even though their sporophytes are classified into the same species as the Pteris genus. On the other hand, in Stage 3 of P. cretica, arsenic was distributed in the aboveground part of the sporophytes (Fig. 4(f), 144 ng/pixel at Point C2). This is consistent with previous reports on the accumulation of arsenic in the fronds of P. cretica sporophytes,²⁷i.e., the distribution pattern of arsenic typical of sporophyte is occurring at this stage, as for P. vittata.

In each stage, A. yokoscense clearly showed a different distribution of arsenic from the two hyperaccumulators. In Stage 3, arsenic was most concentrated at the roots of the sporophyte, although the intensity of the arsenic signal was comparably low (Fig. 4(n), 16.2 ng/pixel at Point A2). This result reflects the characteristics of the A. yokoscense sporophytes, which may be attributed to the unique function of their roots. The specific accumulation in the roots of A. yokoscense has been reported previously with regards to Cu, Zn, Pb, and Cd.^29,31,44 Although this fern is not a hyperaccumulator of arsenic, it can also accumulate up to 2200 mg As kg⁻¹ in its roots, compared with 900 mg As kg⁻¹ in its shoots.³¹ These previous reports support our observation of the relative accumulation of arsenic along the roots. In case of Stage 2 for A. yokoscense, observed accumulation of arsenic along the rhizoids (Fig. 4(j), 450 ng/pixel at Point A1) was also supported by previous reports. Kamachi et al. (2005)⁴⁵ suggested that gametophytes of A. yokoscense showing lead accumulation in the rhizoids have a possible resistance to lead toxicity through sequestration. They also pointed out that the rhizoids of the gametophytes are analogous to the roots of the sporophytes, functioning as organs of uptake and absorption. Therefore, the accumulation of arsenic in the rhizoids of A. yokoscense gametophytes may also be the result of a resistance mechanism attributed to the unique function of their rhizoids, as in the case of the roots of the sporophytes.

In addition, from our multielement XRF images, similar distributions of iron and zinc were revealed among the three ferns in Stage 2 (Fig. 2(g), (h), 4(c), (d), (k), and (l)), indicating that these elements may be required commonly among their gametophytes. In particular, the similar existence of iron along the rhizoids and its positive correlation with arsenic (r² = 0.915 for Area V2 in Fig. 2(g), r² = 0.839 for Area C in Fig. 4(c), and r² = 0.924 for Area A in Fig. 4(k)) is interesting, because it may possibly be related to the detoxification of arsenic in the rhizoids. Kamachi et al. (2005)⁴⁵ estimated that gametophytes of P. vittata, not only the gametophyte of A. yokoscense, possess detoxification mechanisms for lead toxicity in their rhizoids. There is no common information on the case of arsenic detoxification in the rhizoids of the three ferns, but it is reasonable that the rhizoids show resistance to toxic elements, because the rhizoids would be the first tissues to occur from their spores at a contaminated site. Further investigation into the relationship between arsenic and iron in the rhizoids would possibly lead to the detoxification of toxic elements occurred in the earliest processes after the germination.

3.4 As K-edge XANES analysis of P. cretica and A. yokoscense gametophytes

Fig. 5 shows the arsenic K-edge XANES spectra of the reference materials ((a): KH₂AsO₄, (b): As₂O₃, (c): As₂S₃) and gametophytes in Stage 2 of (d): P. cretica and (e): A. yokoskcense. It was found that the arsenic in both gametophytes was present as As(III) (Fig. 5(d) and (e)). In addition, a clear chemical shift was observed between the spectra of A. yokoscense and P. cretica (Fig. 5(e) and (d)), i.e., peak-top energy of A. yokoskece was 1 eV lower than that of P. cretica, which had the same energy as As₂S₃ (Fig. 5(c)), indicating the existence of As–S bonds in the arsenic species in the gametophytes of A. yokoscense.

Fig. 5 Arsenic K-edge XANES spectra of reference materials and gametophytes of P. cretica and A. yokoscense in Stage 2.

These are the first results on the speciation of arsenic in the gametophytes of P. crecita and A. yokoscense, which are an independent generation of their sporophytes. It was revealed that a reduction of arsenic occurred not only in P. vittata gametophytes but it was also common phenomena among all three gametophytes. On the other hand, the mechanism of reduction of arsenic in the gametophytes of the hyperaccumulators, P. vittata and P. cretica, is presumed to be different from that of the non-hyperaccumulator, A. yokoscense, based on the different arsenic species.

The reduction of arsenic is generally considered to be accompanied by the detoxification process in the plant cells,^46,47 although the molecular mechanism is still not clear. In particular, following complexation of arsenite with peptides, such as glutathione (GSH) and phytochelatins (PCs), is presumed to be an important mechanism of arsenic detoxification in As-nonhyperaccumulating plants.^46,47 Further, some researchers have pointed out the immobility of As–PCs or As–GSH, which leads to a lower ability for the upward transport of arsenic, because arsenic may be transported in the xylem sap in the form of oxyanions (= arsenite or arsenate) rather than in thiols complexed forms in both As-hyperaccumulating and non-hyperaccumulating plants.^40,41 Our results from A. yokoscense are consistent with these insights in the aspects of both tolerance and translocation. In Stage 2 before the development of a vascular system, the complexation of arsenic was observed as a detoxification mechanism (Fig. 5(e)), while after the development of a vascular system (Stage 3), arsenic was mainly stored in the roots (Fig. 4(n)), with less potential to be loaded into the xylem. One of the important findings in our study is that the complexation of arsenic takes place independent of the vascular system in the sporophyte.

On the other hand, in the arsenic detoxification of the two hyperaccumulators, P. vittata and P. cretica, complexation may not play a dominant role, even in the gametophyte, as in the case of sporophytes in previous reports,^6–10 because a higher tolerance is shown in these two gametophytes: they can grow in a solution of As 50 mg L⁻¹, whereas the gametophytes of A. yokoscense cannot survive in a solution of higher than As 5 mg L⁻¹ (data not shown). Moreover, in the translocation of arsenic after the development of the vascular system (Stage 3), its observed distribution to aboveground part of their sporophytes (Fig. 2(j) and 4(f)) is also consistent with the observation of inorganic arsenite species, which is in contrast to that obtained from A. yokoscense (Fig. 4(n)). Many previous studies have suggested that a lack of thiol coordination in the detoxification mechanism may be a common theme in plants that have evolved to hyperaccumulate metals, including Ni, Zn, Cd, Se, and As.^{35,38,48–50} Our results revealed that this is also true for the case of the gametophyte system of these three ferns, although the reason for arsenic tolerance in the two hyperaccumulators without complexation with –SH is still unknown.

Based on our observations of the differences in arsenic species and their distribution in the three ferns, it can be presumed that the different mechanisms in the arsenic reduction affect the distribution of arsenic in the plant body. We also suggest that an investigation into the difference in reduction mechanism of arsenic is one of the most important themes for understanding the mechanism of arsenic hyperaccumulation in the plants.

4. Summary

In vivo micro X-ray analysis was performed to investigate the distribution and oxidation state of the arsenic in the gametophytes of three different ferns, P. vittata, P. cretica and A. yokoscense, in the several growth stages from germination. It was found that the distribution of arsenic in P. vittata changed with the development of the plant tissues. The arsenic was mainly present along the rhizoids in the infant gametophytes in Stage 1. In Stage 2 with reproductive organs, the arsenic was accumulated uniformly in the sheet of cells except in the reproductive area. In Stage 3 after fertilization, arsenic was observed in the aboveground part of the sporophyte structures. These different distributions imply a development of accumulation mechanisms for arsenic in the plant bodies. On the other hand, the reduction of arsenic was observed regardless of particular structures, indicating that the reduction of arsenic in the infant gametophyte does not relate directly to a particular structure, such as reproductive organs, vascular system, or fronds.

The distribution of arsenic and its oxidation state in the gametophytes of P. cretica, and A. yokoscense has also been revealed for the first time. P. cretica showed an arsenic accumulation in the reproductive area, in contrast to P. vittata, while distribution of arsenic to the aboveground part of the sporophyte was observed after fertilization, as is the case for P. vittata. Interestingly, the contrast in distribution of arsenic between P. vittata and P. cretica in the reproductive area corresponds to their different reproductive processes, indicating that exclusion of arsenic from the reproductive area is a characteristic of the P. vittata gametophyte. A. yokoscense showed an accumulation of arsenic along the rhizoids before fertilization, while it was present along the roots of the sporophyte after fertilization. On the other hand, arsenic was bonded to sulfur only in the gametophytes of A. yokoscense, although a reduction of the arsenic was common in these three ferns. Based on the different arsenic species and their distribution, the mechanism of reduction is presumed to be different among the two hyperaccumulators and A. yokoscense, which may affect the translocation of arsenic from the roots to the shoots of the sporophytes.

This study provides basic information on the behaviors of arsenic during the growth process of each fern, where the observed differences in distribution and species of arsenic were related to their own individual characteristics, such as their reproductive process or detoxification mechanism. This information gives us insights into the next strategy in the understanding of the basic molecular mechanisms. Therefore, we conclude that our comparative study using in vivo micro X-ray analysis of developing gametophyte systems is a promising approach to understand the mechanism of arsenic hyperaccumulation in the plants.

Acknowledgements

We thank Dr Atsuo Iida (KEK) for setting the microbeam analysis system. We also appreciate the useful advice provided by Dr Yoshio Takahashi (Hiroshima University). This work was partly supported by a Grant-in-Aid for Scientific Research (No. 17350040) from the Ministry of Education, Science, Sports, and Culture, Japan. This work was carried out under approval of the Photon Factory Program Advisory Committee (Proposal Nos. 2004G332 and 2007G638).

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