Fe Absorption and Distribution of Imperata cylindrica (L.) P. Beauv. under Controlled Conditions

Iron toxicity is enhanced by low pH values, which cause Fe dissolution and increased bioavailability [5]. In low pH environments such as the Tinto River basin, plants are subdued to a strong abiotic stress, generating hydroxyl radicals that cause cellular damage. Nevertheless, Fe is essential for plant growth, photosynthesis and CO2 fixation, being usually a limiting factor in environments with higher pH values. Iron homeostasis is therefore a very dynamic and strictly regulated process, and different plant strategies to deal with the lack or excess of Fe in the substrate could be found [6,7].


Introduction
Imperata cylindrica (L.) P. Beauv. is a perennial helophyte grass with a highly developed and branched rhizome. This rhizomatous system can reach depths of more than 50 cm [1] and represents at least 60% of the total plant biomass [2]. The leaves are 40-90 cm long and 2 cm wide, growing grouped at the nodes of the rhizome.
I. cylindrica is native to Southeast Asia, Australia, China, Japan and the Philippines and widely distributed throughout the Mediterranean area. It is very resistant to droughts and floods, and it is found in a variety of soils, from strongly acidic to slightly alkaline.
In the south of the Iberian Peninsula it grows in riparian ecosystems, small streams, moist sandy banks and gullies, subdued to severe summer droughts. It could be found growing in the river banks of the Tinto River (Southwestern Iberian Peninsula, Spain) [3], an extremely acidic environment with high Fe and other metal concentrations in water and soils [4].
Iron toxicity is enhanced by low pH values, which cause Fe dissolution and increased bioavailability [5]. In low pH environments such as the Tinto River basin, plants are subdued to a strong abiotic stress, generating hydroxyl radicals that cause cellular damage. Nevertheless, Fe is essential for plant growth, photosynthesis and CO 2 fixation, being usually a limiting factor in environments with higher pH values. Iron homeostasis is therefore a very dynamic and strictly regulated process, and different plant strategies to deal with the lack or excess of Fe in the substrate could be found [6,7].
Previous studies in I. cylindrica using Inductive Coupled Plasma Mass Spectrometry (ICP-MS) reported Fe concentration values of about 1% of plant dry weight [8,9]. By means of Mössbauer spectrometry and X-ray diffraction, Rodríguez et al. [8] identified the iron content of I. cylindrica as ferrihydrite and jarosite at a proportion of 50%, respectively, within roots, rhizomes and leaves of the plant. Those biominerals have been observed in the cells of the epidermis; parenchyma and vascular bundles. Amils et al. [10] and Fuente et al. [11] interpret these particles as a result of degradation of ferritin protein metabolism, which stores in its core ferrihydrite, hematite and magnetite [12][13][14][15]. A more detailed localization of the subcellular distribution of ferritin using immunocytochemical techniques was described by Fuente et al. [11].
In this article we focus on the study of the Fe absorption capacities of I. cylindrica to reproduce its hyperacummulation under controlled conditions in the laboratory, locating places of Fe accumulation, and identifying the different structures of Fe compounds formed inside the organs of the plant.

Statistical analysis
Statistical analyses were carried out using Statistical release 6.0 (Statsoft Inc., Tulsa, USA). Means, medians, standard deviations, and standard errors were calculated. Data were log transformed after being tested for normality with the Shapiro-Wilk test (p>0.05). Different translocation coefficients (TC) were calculated as:

Elemental analysis (ICP-MS)
Leaf: The mean Fe concentration of the leaves at 0 days was 304.1 mg/kg d.w. From that point, the concentration increased significantly (p<0.05) along the experiment, reaching 4805 mg/kg d.w. at the final sampling (Table 1).

Rhizome:
At the beginning of the experiment (day 0), the Fe concentration of the rhizomes was 172.1 mg/kg d.w. After the first 15 days, the concentration reached 2081 mg/kg d.w. and remained without significant changes for 30 days. During the last fortnight of the treatment, the concentration increased until 7976 mg/kg d.w. at day 60.

Root:
The initial mean concentration of Fe in the roots was 193.3 mg/kg d.w. before the treatment. Fe concentration reached 8524 mg/kg d.w. at the first 15 days, and continued increasing during the experiment until values of 53886 mg/kg d.w. at the last day of the experiment.
There are no significant differences in the Fe concentrations measured before the treatment (day 0) between leaves, rhizomes and roots. The translocation coefficients (Table 2) indicate that, at the end of the experiment, the distribution of Fe concentration between organs was in the order: roots>rhizomes>leaves. Nevertheless, Fe concentration between rhizomes and leaves was not significantly different (p>0.05) along all the measured samples.

Scanning Electron Microscopy (SEM)
Plants grew during the experimental process, but the growth rate of the different organs differed. While there was leaf production, these were scarce and small, and did not reach their normal size in their natural habitat. However, root and rhizome were totally developed being the roots the organ with the highest rate of growth. The microscopic analysis of all the organs revealed that the tissues were well After a growth period of 12 months in the greenhouse, the resulting plants were removed, cleansed of all residues and transferred to a pure hydroponic culture in Hoagland solution (pH 5.1). This nutrient medium was enriched with 300 mg/Kg of Fe. To avoid Fe precipitation, the pH of the solution was lowered to 3 using H 2 SO 4 [1M]. The culture was maintained in the greenhouse with natural light and a controlled temperature of 30°C.
Samples were collected each fortnight (15,30,45 and 60 days) during two months, with a sample taken before treatment (0 days). Each sample consisted of three entire plants that were separated in root, rhizome and leaves, washed and frozen until analysis.

Elemental analysis
Plant samples were washed with distilled water, dried in an oven at 75°C for 24 h and powdered for homogenization. Small portions of dry plant tissues powder (500 mg) were digested at high pressure in a mixture of 8 ml of HNO 3 65% and 2 ml of H 2 O 2 30% in a MLS Ethos 1600 URM Milestone microwave digester, following the protocol described by Zuluaga et al. [16]. Aliquots of the different samples were analyzed by ICP-MS using an ELAN-6000 PE-Sciex (Toronto, Ontario, Canada) instrument. The ICP-MS technique used has an inherent error of 15%.

Microscopy analysis
Scanning Electron Microscopy (SEM -EDX): Three specimens of cultivated I. cylindrica for each sampling time were analyzed by SEM complemented with an Energy Dispersive X-ray analyzer (EDX) following the protocol for elemental analysis and metal localization study in plant material [8,10,11,17]. The organs and tissues analyzed were; roots (epidermis, parenchyma, endodermis and pith), rhizome (epidermis, cortex, central cylinder and pith) and leaves (cuticle, epidermis, trichomes, parenchyma and vascular bundles). Samples were mounted onto conductive graphite stubs and sputters then goldcoated in a BIO-RAD SC 502 apparatus to ensure electrical conductivity and prevent charging under electron beams. Samples were examined with a Hitachi S-3000N (Japan) SEM using an acceleration voltage of 20 kV and a working distance of 15 mm. Analyses were performed at room temperature. The qualitative element composition of samples was determined using an INCAx-sight with a Si-Li Detector (Oxford, England) with a detection limit of 10% of the main element.

Transmission Electron Microscopy (TEM -EDX):
Root and rhizome samples of three specimens of wild and cultivated I. cylindrica were analyzed by TEM. Those organs were selected because of their high Fe concentration. Small sections (1 mm 3 ) of roots and rhizomes from I. cylindrica were fixed in 4% paraformaldehyde and 2% glutaraldehyde in 0.05M cacodylate buffer (pH 7.4) for 2 hours at room temperature. Fixed samples were washed three times in the same buffer and postfixed with 1% OsO 4 in water for 30 min at room temperature in the dark. After three washes in bidistillated water samples were incubated with 2% aqueous Uranil Acetate for 1 h at room temperature, washed again and dehydrated in increasing concentrations of ethanol 30, 50 and 70% 20 min each, 90% 2 × 20 min and 100% 2 × 30 min at room temperature. Dehydration was completed with a mixture of ethanol: propylene oxide (1:1) for 10 min and pure propylene oxide 3 × 10 min. Infiltration of the resin was accomplished with propylene oxide:Epon (1:1) for 45 min and pure Epoxy (TAAB 812 Resin; TAAB Laboratories, Berkshire, England) resin over night at room temperature. Polymerization of infiltrated samples was done at 60°C for 2 days. Ultrathin sections of the samples were stained with uranyl acetate and lead citrate by standard . Near the end of the experiment, the number and density of those iron deposits were higher in the younger roots than in the roots that started growing at earlier stages of the treatment. Although the Fe observed accumulations were generally amorphous, some isolated particles showed a more defined structure (Figure 1 K).
In the endodermis cells between the root cortex and the vascular cylinder we have not detected any iron accumulation though some silicon deposits are observed in the cross section of old roots ( Figure  1 D and H). At the beginning of the experiment, the central cylinder and the vascular tissues were almost free from Fe particles, but those particles increased in number and size along the experiment, especially in the xylem vessels ( Leaf: Micrographs from leaf sections (Figure 3) also show the presence of Fe particles in the parenchyma around the vascular bundles, and the abaxial and adaxial epidermal cells (Figure 3 A, B and C). Those particles appear in less numbers than in other organs, but almost all of them are of lenticular or prismatic shape (Figure 3 D). There is also an increase in the appearance of Fe particles along the experiment. The EDX spectra show a composition mainly of Fe, S and K, with traces of P, Si, Ca and Mg in some of them. Figure 4 represents the mean values of Fe weight percentage obtained in the EDX spectra analysis over the different accumulations that we found in the tissues of roots, rhizomes and leaves. The analyses were separated in epidermis, cortex, and vascular cylinder Fe accumulations. Fe particles over epidermal cells of older roots show a higher Fe weight percentage than the same particles found in the youngest roots grown during the experiment, whilst the particles found inside the cortical tissues show a lower Fe weight percentage value (p<0.05). Mineral depositions found in the rhizomes and leaves have a similar Fe weight percentage in all analyzed tissues.

Transmission Electron Microscopy (TEM)
Root and rhizomes of wild and cultivated I. cylindrica were observed by TEM and a selection of representative micrographs can be found in Figure 5.       The root cross section allows identifying all the principal tissues in this organ and confirms their good state (Figure 5 A). The iron plaque previously observed by SEM is visible here in the outer side of the epidermal cells as a thick black discontinuous line (Figure 5 A). The cortex is composed of big cortical cells with fine cell walls which represent most of the volume of the root (Figure 5 A). These have a scarce number of organelles such as plastids, vacuoles, nuclei and mitochondria ( Figure 5 E and F). Most of these cells present a high proportion of black and long deposits grouped forming an almost continuous layer along the inner side of the cell wall (Figure 5 B). This electro-dense material can be found also covering the outer membrane of the organelles (Figure 5 E).
The cortex is followed by a layer of cells with thickened walls, the endodermis, which separates the cortex from the central cylinder. This is the only tissue free from the black particles (Figure 5 B). The central cylinder includes several types of cells as the phloem and xylem vessels alternated with parenchymatous cells. Here we also find the dark deposits covering the inner side of the cells walls and the outside of the organelles when present (Figure 5 C). These deposits are so abundant that even collapsed the pores and plasmodesmata that communicate cell to cell (Figure 5 D).
These long dark deposits are also visible in the rhizome with the same distribution (Figure 5 I). Moreover, we also observed darkened intercellular spaces ( Figure 5 F and H) composed of electro-dense particulate material (Figure 5 H).
All these observation carried out in cultivated material were corroborated in wild samples. An example is provided in Figure 5 J where a root cell wall of wild full grown I. cylindrica specimens shows a cover of long dark deposits which composition includes a high proportion of Fe as reveals the EDX analysis ( Figure 5 K). This layer is even thicker than that found in the experimental samples.

X-ray Difracction
XRD analysis spectra are represented in Figure 6. They indicate the presence of jarosite and Fe oxides in both roots and rhizomes of I. cylindrica cultivated during 60 days in Fe enriched Hoagland solution. The spectra also show the presence of opal crystals (SiO 2 ) possibly corresponding to the silica deposits observed in the endodermic cells, and some gypsum crystals (CaSO 4 ·H 2 O) in both roots and rhizomes.

Discussion
This is the first report of the production under laboratory controlled condition of the biomineralization of jarosite and Fe oxides and oxihydroxides by I. cylindrica. Though Fe accumulation is known in several species of Graminae growing in Tinto River (Phragmites australis, Panicum repens, Arundo donax) and species of other families (Typha angustifolia, Erica andevalensis) none of them reach Fe concentrations of I. cylindrica nor have been found to biomineralize jarosite [9,19].
After 60 days of treatment with the Fe enriched solution, high concentrations of Fe could be found inside I. cylindrica tissues. That concentration was higher in the roots and rhizomes than in the leaves, in consonance with the results obtained by Rodríguez et al. [8] in field grown individuals. Since the second fortnight of treatment, I. cylindrica reached Fe concentrations higher than the normal values given for vascular plants by Reeves and Baker [20]. In particular, root Fe concentration was over phytotoxic levels (1000-3000 mg/kg, [21]) since the first week, and after the 60 days of treatment reached concentration values that could be considered as hyperaccumulation. Rhizomes and leaves reached Fe concentrations over the phytotoxic levels in the last sampling day, but concentrations remained in range with values given for other non hyperaccumulator plants adapted to metalliferous environments.
During the experiment the plants grew. The organ that exhibited the fastest rate of growth was the root. The continuous production of roots and Fe accumulation in their surface (iron plaque) and tissues may be part of a protection strategy to cope with the excess of Fe and prevent a dangerous increase of Fe concentrations in rhizomes and leaves. The importance of root tissues in heavy metal retention and tolerance has been widely studied [22][23][24][25][26]. This behavior has been found in other plant species, such as Phragmites australis [27] and Cynodon dactylon [19], in which the Fe retention in roots is probably the most relevant strategy in rhizome protection against oxidative stress. Taggart et al. [28] also found an increase in the production of secondary roots in Typha latifolia under extreme conditions. However, leaves growth was slow, and they did not reach the usual size in natural conditions. This may indicate that the excess of Fe may affect somehow to the plant regular growth. Healthy plants but smaller have been observed in several species growing on Tinto River, when compared with those growing in other habitats less rich in metals [9]. The small plant size has been also emphasized as a feature of serpentine flora; another particular flora specialized in naturally metalliferous soils [29]. Nevertheless, the high concentrations of Fe found in rhizomes and leaves without any structural damage that could compromise its growth and functions, indicates that even when there is a high Fe root retention, these tissues should be highly tolerant to Fe excess, probably due to the existence of other simultaneous tolerance/detoxification mechanisms.
Though SEM-EDX technique has been proved as a good system to localize and analyze metals inside plant organs, the observation of massive biomineralization by TEM is not as usual, probably due in biomineralization plays an important role in this tolerance, reducing metal toxicity.
The ease of growth and reproduction of I. cylindrica, and its rapid development of a dense rhizomatous system, allow for a high production of biomass, which implies a higher immobilization of Fe. The fact that its rhizomatous system could reach deeps of 50 cm [36] also allows for a great stabilization of the soil and the decontamination of deeper soil layers impossible to reach with other gramineous species.
I. cylindrica is also tolerant to other numerous heavy metals such as Pb [37], Cu, Zn, Sn [38], Cr, Ni and Co [39], with an excluding strategy, and it have also been found colonizing highly polluted environments like Pb-Zn mines Zn [40,41]. These characteristics make I. cylindrica a species to take into account in contaminated soil stabilization programs, as it retains the highest metal concentrations in the roots, less accessible to feeding animals. Moreover, we have reproduced its hyperaccumulator behavior under controlled conditions, also achieving the bioformation of jarosite and Fe oxides in the laboratory. The control of these capabilities could be also useful for the employment of this species for the production of Fe biominerals and nanoparticles with diverse utilities (medicine, biomaterials, nanotechnology).
part to the inherent complications of sample preparation. The high Fe concentration in roots and rhizomes of I. cylindrica has allowed the visualization at cellular level of the biomineralization of this metal. In this regard, SEM and TEM combination has been useful to observe Fe accumulation and biomineralization and its distribution at tissular and cellular level.
The high Fe concentration of the roots was observed as an iron plaque in the epidermis by SEM and TEM. EDX analysis indicated a composition of Fe, S, K and P, suggesting the presence of the mineral jarosite that was confirmed by means of XRD. Therefore, we reproduced the formation of a similar iron plaque that the one formed by field individuals [8]. We should indicate though that iron plaques of different species have been intensively studied and has been found that their composition is pretty variable among species and even among ecotypes [30][31][32][33][34]. Iron plaques of other Tinto River Graminae such as Cynodon dactylon have high Fe concentrations, but no jarosite was found [19].
Fe deposits were observable by SEM in almost all the tissues composed of Fe, S and K inside the epidermal and cortical cells, and the xylem and phloem vessels in the vascular cylinder on roots and rhizomes. Those deposits appeared in form of isolated particles, grouped particles, and fully collapsed cells. The number and density of those deposits were higher in the younger roots. By other hand, the vascular tissues increased their Fe deposits as the increased the time of exposition. TEM images allow the detection of black and long deposits composed mainly by Fe and located in the inner side of the cell walls, surrounding the organelles and also in the intercellular space of almost all root tissues. These could be Fe oxides according to the XRD spectra. The presence of ferritin has been reported in roots, rhizomes and leaves of I. cylindrica by means of immunocytochemical analysis [11]. The degradation of this protein could derive in the formation of the iron oxides.
The endodermis was the only root tissue that was found to be free of Fe deposits. The endodermis is composed by cells with some part of their wall chemically modified in order to control the pass of water and ions to the central cylinder and therefore to the xylem and the rest of the organs. It seems that at the beginning it provides an efficient barrier, thus Fe depositions are almost inexistent in the central cylinder. But as the exposition to high Fe concentration in the solution carries on Fe deposits are more abundant in the central cylinder. TEM images showed the same deposits, with the same structure at both sides of the endodermis. Interestingly, endodermis cells remain free of Fe deposition, indicating their continuous activity transferring Fe to the central cylinder tissues where Fe can be transported or somehow retained leading to biomineralization. Abundant Fe deposits were found in root and rhizome xylem vessels by SEM technique, especially at the end of the experiment. This coincides with the field observations reported by Rodríguez et al. [8] who found pores of xylem elements totally collapsed by Fe biominerals. Previous observations on I. cylindrica roots showed silicon deposits on the proximal site of the endodermis cells [35]. Here, these were not visible in TEM because of the youth of the roots observed, though incipient silicon deposits were observed by SEM in older roots. Whether or not silicon and iron transport in the plant may be somehow related is unknown, but the fact is that whilst silicon is deposited in the endodermis cells, iron is not.
So even though I. cylindrica is a Fe hyperaccumulator, it presents a strategy of Fe retention in roots, and a regulation over the Fe translocation to aerial parts and reserve tissues (in the rhizome). Nevertheless all of the plant aerial and underground tissues have and tolerate a high Fe concentration. Our study suggests that internal