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Cadmium distribution in the root tissues of solanaceous plants with contrasting root-to-shoot Cd translocation efficiencies
Environmental and Experimental Botany 71 (2011) 198–206
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Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot
Cadmium distribution in the root tissues of solanaceous plants with contrasting root-to-shoot Cd translocation efficiencies Noriko Yamaguchi a,∗ , Shinsuke Mori a,1 , Koji Baba a , Saeko Kaburagi-Yada a,2 , Tomohito Arao a , Nobuyuki Kitajima b , Akiko Hokura c , Yasuko Terada d a
National Institute for Agro-environmental Sciences, 3-1-3 Kan-non-dai, Tsukuba 305-8604, Japan Fujita Co., 2025-1 Ono, Atsugi, Kanagawa 243-0125, Japan Tokyo Denki Univ., 2-2, Kandanishiki, Chiyoda, Tokyo 101-8457, Japan d SPring-8, JASRI, 1-1-1 Kouto, Sayo, Hyogo 679-5198, Japan b c
a r t i c l e
i n f o
Article history: Received 27 April 2010 Received in revised form 30 November 2010 Accepted 2 December 2010 Keywords: Cadmium Eggplants Root Solanum melongena Solanum torvum Synchrotron micro X-ray fluorescence spectrometry
a b s t r a c t Root-to-shoot cadmium (Cd) translocation in Solanum torvum is lower than that of the eggplant Solanum melongena; therefore, grafting S. melongena onto S. torvum rootstock can effectively reduce the Cd concentration in eggplant fruits. We hypothesized that Cd transport in S. torvum roots is restricted in the path between the epidermis and xylem vessel; hence, we investigated the Cd distribution in the roots at the micron-scale. Elemental maps of Cd, Zn and Fe accumulation in S. melongena and S. torvum root sections were obtained by synchrotron micro X-ray fluorescence spectrometry. The Cd was localized in both the stele and the epidermis of the S. melongena root cross sections regardless of the distance from the root apex. In S. torvum root sections taken at 30 and 40 mm above the root apex, a higher abundance of Cd was found within the cells of the endodermis and pericycle. The results suggested that the symplastic uptake and xylem loading of Cd in S. torvum roots were restricted, and thereby, the Cd that was unable to be loaded into the xylem accumulated in the endodermis and in the pericycle. Because symplastic uptake differs only slightly between the two species, the difference in xylem loading would explain the comparatively lower Cd concentration in S. torvum shoots. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The health risks associated with the long-term intake of Cd from food have been a cause of great concern. A field and market basket study in Japan revealed that the Cd concentration in 7% of 381 eggplant samples exceeded 0.05 mg kg−1 fresh weight, which is the maximum recommended level for fruiting vegetables set by CODEX Alimentarius Commissions of FAO/WHO (CODEX Alimentarius Commissions, 2005; Ministry of Agriculture Forestry and Fisheries of Japan, 2002). Thus, cultivation management systems that reduce Cd concentrations in eggplant are urgently required. Possible strategies for reducing Cd absorption by plants include the application of soil amendments to reduce the Cd con-
∗ Corresponding author. Tel.: +81 29 838 8315; fax: +81 29 838 8315. E-mail addresses: [email protected] (N. Yamaguchi), [email protected] (S. Mori), [email protected] (K. Baba), [email protected] (S. Kaburagi-Yada), [email protected] (T. Arao), [email protected] (N. Kitajima), [email protected] (Y. Terada). 1 Present address: National Agriculture and Food Research Organization, 6-12-1 Nishi-fukatsucho, Fukuyama, Hiroshima 721-8514, Japan. 2 Present address: National Agriculture and Food Research Organization, 3-1-1 Kan-non-dai, Tsukuba, Ibaraki 305-8517, Japan. 0098-8472/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2010.12.002
centration in the soil, and the selection of plant cultivars with a genetic tendency for low Cd uptake (Grant et al., 1998). For some fruiting vegetables, such as eggplant and tomato, grafting onto disease-resistant rootstock is a common cultivation method used for disease control. Takeda et al. (2007) found that grafting eggplant (Solanum melongena) onto Solanum torvum rootstock was effective in reducing Cd concentrations in the eggplant fruit by up to a quarter. Cd concentrations in the shoots and xylem sap of S. torvum were lower than those of S. melongena; this was assumed to be caused by either lower xylem loading or reduced Cd flux in S. torvum (Arao et al., 2008; Mori et al., 2009). Ions in the external soil solution enter the root through the epidermis and accumulate in the root cells or transverse cell layers in the cortex and the endodermis. The ions travel to the xylem vessel in the stele via the apoplastic and symplastic pathways. The Casparian strips prevent the ions from penetrating the endodermis through the apoplast and interrupt the flow of ions into the stele. Therefore, the ions must pass through the cell membrane via an energy-dependent process before they can be symplastically transported to the stele and into the xylem vessel (Assmann, 2006). The structural and/or physiological characteristics of roots affect the xylem loading of Cd and thereby determine root-to-shoot Cd translocation (Florijn and Van Beusichem, 1993; Seregin and
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Kozhevnikova, 2008; Zelko et al., 2008). Lux et al. (2004) suggested that the development of endodermal Casparian strips and suberin lamellae in Salix clones with high Cd accumulation occurred further from the root tip compared to clones with low Cd accumulation. Cd-tolerant Salix clones had a higher proportion of root apoplastic barriers between the other root tissues. There was less binding of Cd to the root cell wall of the hyperaccumulating Sedum alfredii ecotype compared to the root cell wall of the non-hyperaccumulating ecotype, whereas symplastic Cd uptake was doubled (Lu et al., 2008). Mori et al. (2009) compared the kinetics of Cd absorption in the roots of Solanum species with contrasting root-to-shoot Cd translocation using enriched Cd isotopes (113 Cd and 114 Cd), and estimated the difference in symplastic Cd uptake in the roots of the two species. They found that Cd absorption and Cd loading into the xylem were metabolically active and energy-dependent processes, and intercellular Cd absorption in S. melongena (higher root-toshoot translocation) was 1.5-fold higher than in S. torvum (lower root-to-shoot translocation). However, the difference in symplastic transport alone could not explain the fact that the Cd concentration of the xylem sap of S. melongena was several times higher than in S. torvum. In addition, although the xylem loading ability differed considerably between S. melongena and S. torvum, the Cd concentrations of the whole roots only showed a slight difference (Arao et al., 2008; Mori et al., 2009). Therefore, we hypothesized that the Cd unloaded into xylem accumulated between the epidermis and the xylem vessel in S. torvum roots because Cd transport to the xylem was restricted. This hypothesis was examined by determining the subcellular Cd distribution within the root tissues. Analytical techniques for investigating the Cd distribution at a cellular or subcellular resolution include the following: fluorescent microscopic observation using a molecular probes that specifically bind to Cd, such as BTC-5N (Kanthasamy et al., 1995; Shinmachi et al., 2003; Lu et al., 2008) and LeadmiumTM Green AM dye (Lu et al., 2008); an electron probe micro analyzer (EPMA, Biddappa and Chino, 1981; Shinmachi et al., 2003; Solís-Domínguez et al., 2007); micro proton induced X-ray emission (micro-PIXE; Vogel-Mikuˇs et al., 2008) and synchrotron micro X-ray fluorescence (SXRF) spectroscopy (Isaure et al., 2006; Fukuda et al., 2008). Among these techniques, SXRF has been a powerful tool yielding valuable information on metal homeostasis in plants (Punshon et al., 2009). The combined use of SXRF with micro-X-ray absorption near-edge structure (-XANES) provides information on the chemical forms of Cd observed at specific subcellular/cellular locations within the plant tissues (e.g., trichomes) as well as the Cd distribution in these tissues (Isaure et al., 2006; Fukuda et al., 2008). SXRF and X-ray absorption spectroscopy have been used to study elements that are present in larger concentrations in the plant tissues, such as essential elements and hazardous metals in hyperaccumulator plants. However, the improved sensitivity of SXRF in third generation synchrotron facilities has extended the potential applications of SXRF in investigating hazardous metals in non-hyperaccumulator plants, such as As and Cd in rice grains (Meharg et al., 2008; Lombi et al., 2009). Interpreting Cd distribution in the plant tissues by detecting the most intense Cd L␣1 (3.13 keV) or L1 (3.32 keV) lines is difficult because plants contain high concentrations of K as a macronutrient, generally at more than 10% weight, and the Cd L lines overlap with the potassium K␣ line (3.31 keV). Isaure et al. (2006) resolved this problem by exciting Cd below the absorption edge of K using a monochromatic X-ray of 3.55 keV. Vogel-Mikuˇs et al. (2008) and Fukuda et al. (2008) detected a Cd K␣ line (23.1 keV) that was free from elemental overlap using a proton beam of 3.0 MeV and a monochromatic high energy X-ray beam of 37 keV. In this study, we compared the micron-scale Cd distribution pattern in the root tissues of the eggplant S. melongena and the rootstock for eggplant cultivation S. torvum using SXRF at SPring-8, Japan Synchrotron Radiation Research Institute, Hyogo, Japan. The
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ability to focus optics in the high-energy X-ray region (Suzuki et al., 2007) enabled sensitive analyses of Cd in non-hyperaccumulator plants with the detection of the Cd K␣ line. Although investigating the Cd transport mechanism in crops is important from the viewpoint of food safety, SXRF has rarely been used for crop plants. In crop plants, Cd transport to the shoot is controlled by the roots via physiological phenomena, such as symplastic uptake and xylem loading. Our previous reports have suggested that the difference in the xylem loading of Cd in S. melongena and S. torvum was a critical factor in determining the rate of root-to-shoot Cd transport in these species (Mori et al., 2009). Our study aimed to elucidate the mechanism by which the transport of Cd into the xylem is restricted in S. torvum roots and to explain the mechanism responsible for reducing Cd concentration in eggplant fruits using S. torvum rootstock. 2. Materials and methods 2.1. Plant materials Seeds of S. melongena cv. Senryo 2 and S. torvum cv. Torubamubiga were germinated in moist perlite, and the seedlings were transferred to a nutrient solution containing the following (mol l−1 ): Ca(NO3 )2 , 5000; KNO3 , 3400; MgSO4 , 1000; NH4 H2 PO4 , 1000; H3 BO3 , 460; MnSO4 , 9.1; ZnSO4 , 0.76; CuSO4 , 0.31; NaMoO4 , 0.1; and FeSO4 , 53; pH 5.5. When the length of the main roots reached about 40 cm, plants were exposed to 1.33 mol l−1 CdCl2 in a fresh nutrient solution in a controlled environment chamber (25 ◦ C; 60–80% relative humidity) for 24 h or 7 d. After Cd treatment, the roots were rinsed with 500 mol l−1 Ca(NO3 )2 . 2.2. Sample preparation Cross sections that were 200 m thick were prepared from 5mm segments of the main root taken from 0 to 40 mm above the root apex (DTK-2000, Dosaka, Kyoto, Japan). Since the lateral roots had emerged, the root sections were not prepared using tissue more than 40 mm above the root apex. The root segments were pushed into a pinhole made in the center of a 4% agar block (10 mm in diameter and 10 mm in height) to prevent them from drying during the sectioning process. To decrease the possible Cd loss from the cut surfaces of the sections, the sections were not collected in water but carefully removed from the microtome blade using a brush. The fresh thin sections were immediately frozen on an aluminum block with a mirror surface pre-cooled in liquid nitrogen and then freeze-dried. The freeze-dried sections were placed on an acryl plate (4 cm × 4 cm with a 3-mm hole in the center) and were held in place using double-sided adhesive tape. After SXRF analyses, the freeze-dried sections were observed using a scanning electron microscope (SEM, JSM 5610LV, JEOL, Tokyo, Japan) under a low vacuum of 40 Pa and an acceleration voltage of 15 keV. To confirm the possible loss or redistribution of Cd due to the sectioning and freeze-drying processes, the Cd distribution pattern in the freeze-dried section was compared with that of the frozen-hydrated section. The 5-mm segments of the main root of S. torvum at 40 mm above the root apex after Cd treatment were immersed in a low-viscosity embedding substance, Cryo Mount I (Muto Pure Chemicals, Tokyo, Japan), and immediately frozen on an aluminum block that had been cooled using liquid nitrogen. The frozen section was cut into 50-m sections using a cryomicrotome (CM1850, Leica, Wetzlar, Germany). The sections were placed on acryl plates and covered with Mylar film® (SPC Science, Champlain, USA). The cut sections were stored in a freezer at −20 ◦ C until analysis.
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2.3. Elemental mapping by synchrotron micro X-ray fluorescence spectroscopy The two-dimensional distributions of Cd, Fe and Zn in the root cross sections were mapped using SXRF at an undulator beamline 37XU of SPring-8 (Terada et al., 2004). The incident beam was monochromatized using an Si(1 1 1) double-crystal monochromator. An incident X-ray beam of 30 keV [beam spot size: 2.0 m in the vertical direction (V) and 1.0 m in the horizontal direction (H)] was used for elemental mapping of Cd using a Cd K␣ line (23.11 keV). Fe and Zn were detected simultaneously with Cd. However, because the excitation efficiency of Zn and Fe is very low at an X-ray energy of 30 keV, the incident X-ray beam was set at 15 keV to measure the distributions of Fe and Zn in the root sections taken at 10 and 40 mm above the root apex. The focused beam size at an X-ray energy of 15 keV was 2.0 m (V) × 1.2 m (H). To avoid possible destruction due to irradiation with the high-energy X-ray beam (Lombi et al., in press), the same sections were not used for analyses under 15 and 30 keV. The incident X-ray beam was focused using a pair of aspherical mirrors in the Kirkpatrick–Baez configuration (Takeuchi and Suzuki, 2005; Suzuki et al., 2007). The fluorescent X-rays were detected using a Si(Li) solid-state detector and silicon drift detector for mapping with incident X-ray energies of 30 and 15 keV, respectively. The scan step size was 3 m, and the spectral acquisition time for each spot was 1 s for Cd and 0.3 s for Fe and Zn. The freeze-dried sections were analyzed at an ambient temperature, whereas the frozen-hydrated sections were analyzed under a cryogenic N2 gas stream (Cryojet XL, Oxford Instruments, UK) to keep them frozen. The fluorescence yield was normalized by the incident photon intensity (I0 ) to produce SXRF elemental maps. The SXRF analyses were repeated using sections taken from the different individual plants with the scan step size of 5 m to confirm reproducibility. Average fluorescence counts in the areas of the epidermis, the exodermis, the cortex, the endodermis and the stele were calculated using IgorPro 6.0.3.1 software (Wavemetrics, USA). The border of each tissue was detected by tracing an SXRF map or root section on its SEM image. In addition to the entire stele, average fluorescence counts in the pericycle were calculated separately because higher fluorescence counts of Cd were observed in the pericycle of S. torvum. The average fluorescence counts in the epidermis and exodermis could not be calculated separately because the epidermis and exodermis might have overlapped in some sections that were cut at an angle. We did not attempt to convert fluorescence counts into quantitative units because the purpose of our study was to compare Cd distribution patterns among root tissues between two solanaceous species. The relative amounts of same element contained in the root sections are comparable among samples when they were measured under the same conditions and normalized by I0 at the same beamtime.
2.4. Observation of the Casparian strips The 100-m thick root cross sections adjacent to the sections used for SXRF analyses were cut using the microtome and collected in deionized water. The fresh sections were stained with berberine hemisulfate aniline blue (Brundrett et al., 1988) and then observed using a fluorescent microscope (Axioskop2 plus, Zeiss, Oberkochen, Germany) equipped with an ultraviolet filter set (excitation filter BP 365, diachronic mirror FT 395, barrier filter LP 397, Zeiss). Other sections were immersed in a concentrated sulfuric acid drop placed on a glass slide and observed for dissolution or retention of the Casparian strips and suberin lamellae (Johansen, 1940) using a digital microscope (VHX-100, Keyence, Osaka, Japan).
2.5. Chemical analyses After sectioning, the rest of the roots and shoots were dried in an oven at 75 ◦ C and ground to a fine powder. The powder (0.2 g) was digested with HNO3 , HClO4 and HF in a Teflon beaker heated to 120 ◦ C. After digestion, the resulting solution was evaporated to near dryness, and the total volume was adjusted to 15 ml using 1% HNO3 . Concentrations of Cd, Fe and Zn were determined using an inductively coupled plasma optical emission spectrometer (VistaPro, Varian, CA, USA). 3. Results 3.1. Casparian strips Casparian strips were visible as small dots in the root sections stained with berberine hemisulfate-aniline blue (Brundrett et al., 1988). For both S. melongena and S. torvum, the root sections taken at less than 30 mm above the root apex showed little evidence of Casparian strips, whereas both the exodermis and endodermis of the root sections taken at 30 mm above the root apex possessed Casparian strips. Sections taken at 40 mm above the root apex were shown in supplementary data. 3.2. Cd distribution in the root sections The Cd concentrations in S. melongena shoots were about 4 times higher than those in S. torvum shoots (Fig. 1). The Cd concentrations in S. melongena roots were higher than those in S. torvum roots, although this difference was smaller than the difference between the shoots (Fig. 1). These trends were in accordance with our previous results (Arao et al., 2008; Mori et al., 2009). Cd was not detected by SXRF in the root sections taken at less than 10 mm above the root apex. Casparian strips were observed in the section 30 mm above the root apex; therefore, the root sections taken at 10, 30 and 40 mm above the root apex were chosen for SXRF analyses. The SEM images and SXRF elemental maps of Cd in S. melongena and S. torvum root sections taken at 10, 30 and 40 mm above the root apex after 24 h of Cd treatment are shown in Fig. 2. Cd was localized in the epidermis, exodermis and stele of S. melongena root sections taken 10 and 30 mm above the root apex (Fig. 2a and b). Cd was found in the stele of S. melongena root sections 40 mm from the root apex, but less Cd was found in the epidermis and exodermis (Fig. 2c). The average fluorescence count of Cd in the stele of S. melongena root sections was the lowest in the cortex and the highest in the stele (Fig. 3a). The distribution pattern of Cd in the root of S. torvum was different from that of S. melongena. Cd accumulated in the endodermis and pericycle and was also found in the epidermis and exodermis of S. torvum root sections taken at 30 and 40 mm above the root apex (Fig. 2e and f). The average fluorescence count of Cd was the lowest in the cortex and the highest in the pericycle (Fig. 3b) at 30 and 40 mm from the root apex. In comparison, the average fluorescence counts of Cd in each root tissue after treatment with Cd for 7 d are also shown in Fig. 3. The SXRF analyses of the root sections treated with Cd for 7 d were determined at different beamtimes from those treated with Cd for 24 h. It should be noted that direct comparison of the absolute value of the average fluorescence counts for the roots treated with Cd for 7 d with those for 24 h might be impractical despite normalization by I0 . In S. melongena, distribution patterns of Cd among the root tissues were almost the same regardless of the Cd treatment time (Fig. 3a). A higher ratio of Cd localized in the endodermis and pericycle of S. torvum roots treated with Cd for 7 d than in those treated with Cd for 24 h (Fig. 3b), suggesting that Cd continuously accumulated in the endodermis and pericycle.
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3.3. Comparison of Cd distribution in freeze-dried and frozen-hydrated sections The Cd distribution pattern in the freeze-dried sections was compared with that of the frozen-hydrated sections using S. torvum root sections taken at 40 mm above the root apex. Cd localization in the outermost layer and the center part of the root was observed in the frozen-hydrated sections, although this was less evident than in the freeze-dried sections because the thickness of the section was one-fourth that of the freeze-dried sections. Identifying the exact position of the tissues in the frozen-hydrated sections by SEM observation failed because the freeze-drying treatment of these sections after SXRF analysis caused shrinkage of the tissues. Higher fluorescence counts of Cd were observed in the outer most layer of the epidermis. The circular part where higher fluorescence counts were observed was either the endodermis or the pericycle. No significant differences were observed between Cd distributions of the frozen-hydrated and freeze-dried sections. Therefore, it is unlikely that freeze-drying treatment altered the Cd allocation pattern in the root tissues, at least at the tissue resolution level. The SXRF elemental map of Cd for frozen-hydrated S. torvum root cross sections taken at 40 mm above the root apex is given as supplementary data. 3.4. Distributions of Fe and Zn in the root sections with and without Cd treatment The Fe concentration in S. melongena shoots was higher than in S. torvum shoots regardless of the Cd treatment (Fig. 1). In contrast, the Zn concentration in S. melongena shoots was lower than in S.
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torvum shoots regardless of the Cd treatment (Fig. 1). In shoots, the Fe concentration was not affected by the Cd treatment, whereas the Zn concentration was decreased. The Fe concentration in the root was very high, amounting to 9.5 g kg−1 . Without Cd treatment, the Fe and Zn concentrations were higher in S. torvum roots than in the S. melongena roots. Cd treatment caused a decrease in the Fe concentration in the roots of both S. melongena and S. torvum and in the Zn concentration of S. torvum (Fig. 1). Fig. 4 shows the average fluorescence counts normalized by incident photon energy, I0 , in the epidermis, exodermis, cortex, endodermis, pericycle and stele for Fe and Zn in S. melongena and S. torvum root sections with and without Cd treatment. The SEM images and SXRF elemental maps are provided as supplementary data. The distribution patterns of Fe in the root sections taken at 10 mm and 40 mm above the root apex were not affected by the Cd treatment (Fig. 4a). In the root sections taken at 40 mm above the root apex, Fe was mostly localized in the epidermis and exodermis. Note that the average fluorescence counts in Fig. 4a are displayed as a log scale. Except for the epidermis and exodermis, the localization of Fe was not clearly observable. In accordance with Fe, the distribution pattern of Zn in the root sections among tissues was not affected by Cd treatment, except in root sections of S. melongena taken at 10 mm above the root apex in which a relatively higher ratio of Zn accumulated in the pericycle and stele (Fig. 4b). In the root section of S. melongena at 40 mm above the root apex, the highest average fluorescence counts of Zn were found in the epidermis and exodermis, regardless of Cd treatment. In the root of S. torvum, Zn was not localized in particular tissues. Without Cd treatment, the average fluorescence counts of Zn tended to be higher in the pericycle as well as in the entire
Fig. 1. Concentrations of Cd, Fe and Zn in the shoot and root of S. melongena and S. torvum with and without Cd treatments.
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Fig. 2. SEM images and SXRF elemental maps of Cd in Cd-treated S. melongena (a–c) and S. torvum (d–f) root cross sections taken at 10 (a and d), 30 (b and e) and 40 mm (c and f) above the root apex. Note that root cross sections taken at 30 mm above the root apex were analyzed at different beamtimes from the root sections taken at 10 and 40 mm above the root apex. Scale bars represent 100 m. ep, epidermis; ex, exodermis; en, endodermis; and pe, pericycle.
stele except in sections taken at 40 mm above the root apex of S. melongena. 4. Discussion Cd localization in the central vascular bundle of plant roots that exhibit higher rates of root-to-shoot Cd translocation has been observed for Cd hyperaccumulators, such as Arabidopsis thaliana (Isaure et al., 2006) and Echinochloa polystachya (Solís-Domínguez et al., 2007). In accordance with these results, the fluorescence counts of Cd were higher in the stele than in the cortex and the endodermis in S. melongena roots (Figs. 2a–c and 3a), which had higher rates of root-to-shoot Cd translocation. In contrast to the S. melongena root, higher amounts of Cd were found within the cells of the endodermis and pericycle of S. torvum root sections taken at 30 and 40 mm above the root apex (Figs. 2e, f, and 3b). These results mean that the radial flow of Cd was blocked in the endodermis and pericycle of the S. torvum root, whereas it was not restricted in the root of S. melongena. Apoplastic Cd transport to the stele can
be blocked in the endodermis due to the presence of the Casparian strips or suberized tissues. This would cause the accumulation of Cd in the endodermis of S. torvum (Figs. 2e, f, and 3b). Though endodermal Casparian strips had developed in S. melongena root sections taken at 30 and 40 mm above the root apex, Cd did not accumulate in the endodermis (Figs. 2b, c, and 3a). Lu et al. (2009) suggested that the symplastic pathway rather than the apoplastic bypass contributed greatly to the root uptake, xylem loading and translocation of Cd to the shoots of the hyperaccumulating ecotype S. alfredii, in comparison with the non-hyperaccumulating ecotype. In S. melongena roots with higher root-to-shoot Cd translocation, most of the Cd might have entered the cytoplasm and bypassed the Casparian strips on the endodermis via the symplastic pathway, thereby displaying less Cd accumulation in the endodermis. This is supported by the observation that the rates of symplastic Cd absorption by S. melongena roots were 1.5-fold faster than those of S. torvum roots (Mori et al., 2009). However, only a 1.5-fold difference in symplastic Cd transport is not enough to explain the several fold higher Cd concentration in the xylem sap of S. melongena than
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Fig. 3. Average fluorescence counts of Cd in epidermis and exodermis; cortex, endodermis, pericycle and stele in the root sections of S. melongena (a) and S. torvum (b). Root anatomy (c) showing ep, epidermis; ex, exodermis; en, endodermis; co, cortex; pe, pericycle; and st, stele. Note that pericycle is a composition of the stele.
in that of S. torvum. A difference in the ability and/or capacity of the transporter that loads Cd into the xylem was considered to be the most important factor determining the difference between Cd concentrations in the xylem saps (Mori et al., 2009). Yamaguchi et al. (2010) found that a xylem-loading citrate transporter, AtFRD3, was down-regulated during the process of Cd acclimation of S. torvum under mild Cd concentrations. It was interpreted that Cd that was unable to be loaded into the xylem accumulated in the pericycle of S. torvum root sections taken at 30 and 40 mm above the root apex due to its inability to transport Cd from the pericycle into the xylem. Although the Cd concentration in the bulk root, which was determined by ICP-MS after acid digestion, did not differ between the roots of S. torvum and S. melongena (Fig. 1), the fluorescence counts in the root sections of S. torvum were higher than for those of S. melongena except for sections taken at 10 mm above the root apex (Fig. 3). Our preliminary analyses of Cd distribution along the length of the main roots using laser ablation inductively coupled plasma mass spectrometry showed that Cd was evenly present from 0 to 80 mm above the root apex in S. melongena, whereas a higher ratio of Cd accumulated in the S. torvum root between 40 and 56 mm above the root apex. The graph showing Cd distribution along with the length of root is given as supplementary data. The variation of Cd concentration along the length of the root would explain the comparatively higher fluorescence count of Cd in the root sections of S. torvum at 30–40 mm above the root apex than in S. melongena. In addition, Cd in S. torvum might have accumulated in regions several centimeters above the root apex because Cd transport to the xylem was restricted.
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Fig. 4. Average fluorescence counts of Fe (a) and Zn (b) in epidermis, exodermis, cortex, endodermis, pericycle and stele in the root sections of S. melongena and S. torvum. ep, epidermis; ex, exodermis; en, endodermis; co, cortex; pe, pericycle; st, stele. Note that the pericycle is a composition of the stele.
In contrast to Cd, Zn was not localized in the endodermis or pericycle of S. melongena or S. torvum root sections regardless of the Cd treatment (Fig. 4b). This suggested that Zn was efficiently transported into the xylem. In dicots, Zn is transported into the xylem by heavy metal transporters HMA2 and HMA4 (Palmer and Guerinot, 2009). In the Cd hyperaccumulator Arabidopsis halleri, increased Zn concentrations restrict xylem loading of Cd. In addition, xylem loading of Cd could be competing with the loading of Zn (Ueno et al., 2008). In this study, Zn concentrations in the shoots were decreased by Cd treatment (Fig. 1), and the average fluorescence counts in the stele of the Cd treated root were lower than those without Cd treatment (Fig. 4b) in S. torvum. Although the Zn concentrations in the shoots of S. melongena were not changed due to the Cd treatment, the average fluorescence counts of Zn in the stele of S. melongena at 10 mm from the root apex was decreased due to the Cd treatment. Zn absorption in S. melongena and S. torvum probably competed with Cd absorption as was shown by De la Rosa et al. (2004). Ueno et al. (2008) suggested that Fe transporters may contribute to Cd absorption in A. halleri because Fe deficiency induced increased Cd absorption. The Fe concentration in the roots of S. torvum was decreased by Cd treatment, and a higher ratio of Fe in the root accumulated in the epidermis and exodermis (Fig. 4a). However, this could be caused by the deposition of divalent Fe in the solution culture as discussed below. Thus, the relationship between Fe and Cd absorption was difficult to interpret using our data. In addition to Cd localization in the stele of S. melongena root sections, Cd was also localized in the epidermis and exodermis of S. melongena root sections taken at 10 and 30 mm above the root apex. In S. torvum root sections, Cd localization was observed in
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Fig. 5. Relationships between X-ray fluorescence counts of Cd and Fe accumulation in the epidermis and exodermis of root sections determined at an incident X-ray energy of 30 keV. Cd-treated S. melongena (a–c) and S. torvum (d–f) root sections taken 10 (a and d), 30 (b and e) and 40 mm (c and f) above the root apex.
the endodermis and pericycle and in the epidermis and exodermis of root sections taken 30 mm above the root apex (Figs. 2 and 3). The relationships between X-ray fluorescence counts of Cd and Fe in the epidermis and exodermis of the root sections, both of which were determined at an incident X-ray energy of 30 keV, are shown in Fig. 5. At an incident X-ray energy of 30 keV, Fe was only detected in the epidermis and exodermis, although it was found in all tissues when determined at an incident X-ray energy of 15 keV (Fig. 4a). Since the sections used for SXRF mapping at an incident X-ray energy of 15 keV were different from those used for elemental mapping at an incident X-ray energy of 30 keV, a spatial comparison of Cd determined at 30 keV with Fe deter-
mined at 15 keV was impractical. Significant relationships were observed between fluorescence counts of Cd and Fe in the epidermis and exodermis of S. melongena root sections taken at 10 and 30 mm above the root apex and in those of S. torvum root sections taken at 30 mm above the root apex (Fig. 5). These samples, which showed a higher correlation between Cd and Fe, accumulated a higher ratio of Cd in the epidermis and exodermis (Figs. 2 and 3). The cell walls of the epidermis are the first barrier preventing Cd from entering the root; therefore, metals commonly accumulate in the epidermis (Seregin and Kozhevnikova, 2008). However, in our study, Fe hydroxide was deposited on the root surface because the nutrient solution contained Fe2+ , which easily oxidizes and pre-
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cipitates as Fe(OH)3 . The high Fe concentrations in Cd-treated S. melongena and S. torvum roots (Fig. 1) and the localization of Fe in the epidermis and exodermis (Fig. 4) implied the deposition of Fe hydroxide which could inhibit Cd absorption into the root since Fe hydroxide can adsorb Cd (Buekers et al., 2008). Therefore, we speculated that the Cd localization in the epidermis and/or exodermis might be an experimental artifact caused by the deposition of Fe hydroxide. In addition to the Casparian strips in the endodermis and exodermis, the cell walls of the cortex also play an important role in controlling apoplastic Cd transport (Seregin and Kozhevnikova, 2008; Radjala et al., 2009). In the roots of Zea mays L. and Noccaea caerulescens, adsorption of Cd in the root apoplast corresponded to 30–90% of the total root uptake (Radjala et al., 2009). Our SXRF mapping results suggested that Cd was present along the cell walls of the cortex (Fig. 2e and f) in S. torvum roots. S. torvum roots tended to be thicker than S. melongena roots, so apoplastic Cd transport in S. torvum could be delayed due to a greater adsorption function of the cell walls of the cortex compared to that of S. melongena. However, we cannot completely eliminate the possibility that the freeze-drying of the root sections induced the redistribution of intracellular Cd to the cell walls. 5. Conclusions SXRF mapping indicated that Cd accumulated in the endodermis and the pericycle in the root sections at 30 and 40 mm above the root apex for S. torvum, which has lower capacity for root-to-shoot translocation of Cd. In the root of S. melongena, which has a higher capacity for root-to-shoot Cd translocation, Cd was localized in the stele and accumulation of Cd in the endodermis or pericycle was not observed. The essential trace elements, Zn and Fe, also did not accumulate in the endodermis or pericycle. These results support the fact that Cd was efficiently transported into the xylem of the S. melongena root in a pathway similar to that of the essential trace elements, whereas Cd flow to xylem was restricted in the endodermis and pericycle of the root of S. torvum. The restricted flow of Cd into the xylem might be caused by a weaker ability or a shortage of the transporter required to efflux Cd into the xylem as was also suggested by Mori et al. (2009). Acknowledgements This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and was carried out with the approval of the SPring-8 Program Advisory Committee (2008A1087 and 2008B1791) and the support of NIMSCNN Nano Foundries and United Analysis Facilities (AC210003). We thank Prof. I. Nakai as well as Mses S. Mitsuo, S. Takada, W. Yamaoka and Mr. Y. Yoshii from the Tokyo University of Science for their assistance in SXRF analyses, Ms. M. Maeda from the Leica Microsystems for her support in the use of the cryomicrotome and Dr. S. Ishikawa from the National Institute for Agro-Environmental Sciences for constructive comments. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.envexpbot.2010.12.002. References Assmann, S.M., 2006. Solute transport. In: Taiz, L., Zeiger, E. (Eds.), Plant Physiology., 4th ed. Sinauer, Sunderland, UK, pp. 95–119. Arao, T., Takeda, H., Nishihara, E., 2008. Reduction of cadmium translocation from roots to shoots in eggplant (Solanum melongena) by grafting onto Solanum torvum rootstock. Soil Sci. Plant Nutr. 54, 555–559.
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Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot
Cadmium distribution in the root tissues of solanaceous plants with contrasting root-to-shoot Cd translocation efficiencies Noriko Yamaguchi a,∗ , Shinsuke Mori a,1 , Koji Baba a , Saeko Kaburagi-Yada a,2 , Tomohito Arao a , Nobuyuki Kitajima b , Akiko Hokura c , Yasuko Terada d a
National Institute for Agro-environmental Sciences, 3-1-3 Kan-non-dai, Tsukuba 305-8604, Japan Fujita Co., 2025-1 Ono, Atsugi, Kanagawa 243-0125, Japan Tokyo Denki Univ., 2-2, Kandanishiki, Chiyoda, Tokyo 101-8457, Japan d SPring-8, JASRI, 1-1-1 Kouto, Sayo, Hyogo 679-5198, Japan b c
a r t i c l e
i n f o
Article history: Received 27 April 2010 Received in revised form 30 November 2010 Accepted 2 December 2010 Keywords: Cadmium Eggplants Root Solanum melongena Solanum torvum Synchrotron micro X-ray fluorescence spectrometry
a b s t r a c t Root-to-shoot cadmium (Cd) translocation in Solanum torvum is lower than that of the eggplant Solanum melongena; therefore, grafting S. melongena onto S. torvum rootstock can effectively reduce the Cd concentration in eggplant fruits. We hypothesized that Cd transport in S. torvum roots is restricted in the path between the epidermis and xylem vessel; hence, we investigated the Cd distribution in the roots at the micron-scale. Elemental maps of Cd, Zn and Fe accumulation in S. melongena and S. torvum root sections were obtained by synchrotron micro X-ray fluorescence spectrometry. The Cd was localized in both the stele and the epidermis of the S. melongena root cross sections regardless of the distance from the root apex. In S. torvum root sections taken at 30 and 40 mm above the root apex, a higher abundance of Cd was found within the cells of the endodermis and pericycle. The results suggested that the symplastic uptake and xylem loading of Cd in S. torvum roots were restricted, and thereby, the Cd that was unable to be loaded into the xylem accumulated in the endodermis and in the pericycle. Because symplastic uptake differs only slightly between the two species, the difference in xylem loading would explain the comparatively lower Cd concentration in S. torvum shoots. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The health risks associated with the long-term intake of Cd from food have been a cause of great concern. A field and market basket study in Japan revealed that the Cd concentration in 7% of 381 eggplant samples exceeded 0.05 mg kg−1 fresh weight, which is the maximum recommended level for fruiting vegetables set by CODEX Alimentarius Commissions of FAO/WHO (CODEX Alimentarius Commissions, 2005; Ministry of Agriculture Forestry and Fisheries of Japan, 2002). Thus, cultivation management systems that reduce Cd concentrations in eggplant are urgently required. Possible strategies for reducing Cd absorption by plants include the application of soil amendments to reduce the Cd con-
∗ Corresponding author. Tel.: +81 29 838 8315; fax: +81 29 838 8315. E-mail addresses: [email protected] (N. Yamaguchi), [email protected] (S. Mori), [email protected] (K. Baba), [email protected] (S. Kaburagi-Yada), [email protected] (T. Arao), [email protected] (N. Kitajima), [email protected] (Y. Terada). 1 Present address: National Agriculture and Food Research Organization, 6-12-1 Nishi-fukatsucho, Fukuyama, Hiroshima 721-8514, Japan. 2 Present address: National Agriculture and Food Research Organization, 3-1-1 Kan-non-dai, Tsukuba, Ibaraki 305-8517, Japan. 0098-8472/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2010.12.002
centration in the soil, and the selection of plant cultivars with a genetic tendency for low Cd uptake (Grant et al., 1998). For some fruiting vegetables, such as eggplant and tomato, grafting onto disease-resistant rootstock is a common cultivation method used for disease control. Takeda et al. (2007) found that grafting eggplant (Solanum melongena) onto Solanum torvum rootstock was effective in reducing Cd concentrations in the eggplant fruit by up to a quarter. Cd concentrations in the shoots and xylem sap of S. torvum were lower than those of S. melongena; this was assumed to be caused by either lower xylem loading or reduced Cd flux in S. torvum (Arao et al., 2008; Mori et al., 2009). Ions in the external soil solution enter the root through the epidermis and accumulate in the root cells or transverse cell layers in the cortex and the endodermis. The ions travel to the xylem vessel in the stele via the apoplastic and symplastic pathways. The Casparian strips prevent the ions from penetrating the endodermis through the apoplast and interrupt the flow of ions into the stele. Therefore, the ions must pass through the cell membrane via an energy-dependent process before they can be symplastically transported to the stele and into the xylem vessel (Assmann, 2006). The structural and/or physiological characteristics of roots affect the xylem loading of Cd and thereby determine root-to-shoot Cd translocation (Florijn and Van Beusichem, 1993; Seregin and
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Kozhevnikova, 2008; Zelko et al., 2008). Lux et al. (2004) suggested that the development of endodermal Casparian strips and suberin lamellae in Salix clones with high Cd accumulation occurred further from the root tip compared to clones with low Cd accumulation. Cd-tolerant Salix clones had a higher proportion of root apoplastic barriers between the other root tissues. There was less binding of Cd to the root cell wall of the hyperaccumulating Sedum alfredii ecotype compared to the root cell wall of the non-hyperaccumulating ecotype, whereas symplastic Cd uptake was doubled (Lu et al., 2008). Mori et al. (2009) compared the kinetics of Cd absorption in the roots of Solanum species with contrasting root-to-shoot Cd translocation using enriched Cd isotopes (113 Cd and 114 Cd), and estimated the difference in symplastic Cd uptake in the roots of the two species. They found that Cd absorption and Cd loading into the xylem were metabolically active and energy-dependent processes, and intercellular Cd absorption in S. melongena (higher root-toshoot translocation) was 1.5-fold higher than in S. torvum (lower root-to-shoot translocation). However, the difference in symplastic transport alone could not explain the fact that the Cd concentration of the xylem sap of S. melongena was several times higher than in S. torvum. In addition, although the xylem loading ability differed considerably between S. melongena and S. torvum, the Cd concentrations of the whole roots only showed a slight difference (Arao et al., 2008; Mori et al., 2009). Therefore, we hypothesized that the Cd unloaded into xylem accumulated between the epidermis and the xylem vessel in S. torvum roots because Cd transport to the xylem was restricted. This hypothesis was examined by determining the subcellular Cd distribution within the root tissues. Analytical techniques for investigating the Cd distribution at a cellular or subcellular resolution include the following: fluorescent microscopic observation using a molecular probes that specifically bind to Cd, such as BTC-5N (Kanthasamy et al., 1995; Shinmachi et al., 2003; Lu et al., 2008) and LeadmiumTM Green AM dye (Lu et al., 2008); an electron probe micro analyzer (EPMA, Biddappa and Chino, 1981; Shinmachi et al., 2003; Solís-Domínguez et al., 2007); micro proton induced X-ray emission (micro-PIXE; Vogel-Mikuˇs et al., 2008) and synchrotron micro X-ray fluorescence (SXRF) spectroscopy (Isaure et al., 2006; Fukuda et al., 2008). Among these techniques, SXRF has been a powerful tool yielding valuable information on metal homeostasis in plants (Punshon et al., 2009). The combined use of SXRF with micro-X-ray absorption near-edge structure (-XANES) provides information on the chemical forms of Cd observed at specific subcellular/cellular locations within the plant tissues (e.g., trichomes) as well as the Cd distribution in these tissues (Isaure et al., 2006; Fukuda et al., 2008). SXRF and X-ray absorption spectroscopy have been used to study elements that are present in larger concentrations in the plant tissues, such as essential elements and hazardous metals in hyperaccumulator plants. However, the improved sensitivity of SXRF in third generation synchrotron facilities has extended the potential applications of SXRF in investigating hazardous metals in non-hyperaccumulator plants, such as As and Cd in rice grains (Meharg et al., 2008; Lombi et al., 2009). Interpreting Cd distribution in the plant tissues by detecting the most intense Cd L␣1 (3.13 keV) or L1 (3.32 keV) lines is difficult because plants contain high concentrations of K as a macronutrient, generally at more than 10% weight, and the Cd L lines overlap with the potassium K␣ line (3.31 keV). Isaure et al. (2006) resolved this problem by exciting Cd below the absorption edge of K using a monochromatic X-ray of 3.55 keV. Vogel-Mikuˇs et al. (2008) and Fukuda et al. (2008) detected a Cd K␣ line (23.1 keV) that was free from elemental overlap using a proton beam of 3.0 MeV and a monochromatic high energy X-ray beam of 37 keV. In this study, we compared the micron-scale Cd distribution pattern in the root tissues of the eggplant S. melongena and the rootstock for eggplant cultivation S. torvum using SXRF at SPring-8, Japan Synchrotron Radiation Research Institute, Hyogo, Japan. The
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ability to focus optics in the high-energy X-ray region (Suzuki et al., 2007) enabled sensitive analyses of Cd in non-hyperaccumulator plants with the detection of the Cd K␣ line. Although investigating the Cd transport mechanism in crops is important from the viewpoint of food safety, SXRF has rarely been used for crop plants. In crop plants, Cd transport to the shoot is controlled by the roots via physiological phenomena, such as symplastic uptake and xylem loading. Our previous reports have suggested that the difference in the xylem loading of Cd in S. melongena and S. torvum was a critical factor in determining the rate of root-to-shoot Cd transport in these species (Mori et al., 2009). Our study aimed to elucidate the mechanism by which the transport of Cd into the xylem is restricted in S. torvum roots and to explain the mechanism responsible for reducing Cd concentration in eggplant fruits using S. torvum rootstock. 2. Materials and methods 2.1. Plant materials Seeds of S. melongena cv. Senryo 2 and S. torvum cv. Torubamubiga were germinated in moist perlite, and the seedlings were transferred to a nutrient solution containing the following (mol l−1 ): Ca(NO3 )2 , 5000; KNO3 , 3400; MgSO4 , 1000; NH4 H2 PO4 , 1000; H3 BO3 , 460; MnSO4 , 9.1; ZnSO4 , 0.76; CuSO4 , 0.31; NaMoO4 , 0.1; and FeSO4 , 53; pH 5.5. When the length of the main roots reached about 40 cm, plants were exposed to 1.33 mol l−1 CdCl2 in a fresh nutrient solution in a controlled environment chamber (25 ◦ C; 60–80% relative humidity) for 24 h or 7 d. After Cd treatment, the roots were rinsed with 500 mol l−1 Ca(NO3 )2 . 2.2. Sample preparation Cross sections that were 200 m thick were prepared from 5mm segments of the main root taken from 0 to 40 mm above the root apex (DTK-2000, Dosaka, Kyoto, Japan). Since the lateral roots had emerged, the root sections were not prepared using tissue more than 40 mm above the root apex. The root segments were pushed into a pinhole made in the center of a 4% agar block (10 mm in diameter and 10 mm in height) to prevent them from drying during the sectioning process. To decrease the possible Cd loss from the cut surfaces of the sections, the sections were not collected in water but carefully removed from the microtome blade using a brush. The fresh thin sections were immediately frozen on an aluminum block with a mirror surface pre-cooled in liquid nitrogen and then freeze-dried. The freeze-dried sections were placed on an acryl plate (4 cm × 4 cm with a 3-mm hole in the center) and were held in place using double-sided adhesive tape. After SXRF analyses, the freeze-dried sections were observed using a scanning electron microscope (SEM, JSM 5610LV, JEOL, Tokyo, Japan) under a low vacuum of 40 Pa and an acceleration voltage of 15 keV. To confirm the possible loss or redistribution of Cd due to the sectioning and freeze-drying processes, the Cd distribution pattern in the freeze-dried section was compared with that of the frozen-hydrated section. The 5-mm segments of the main root of S. torvum at 40 mm above the root apex after Cd treatment were immersed in a low-viscosity embedding substance, Cryo Mount I (Muto Pure Chemicals, Tokyo, Japan), and immediately frozen on an aluminum block that had been cooled using liquid nitrogen. The frozen section was cut into 50-m sections using a cryomicrotome (CM1850, Leica, Wetzlar, Germany). The sections were placed on acryl plates and covered with Mylar film® (SPC Science, Champlain, USA). The cut sections were stored in a freezer at −20 ◦ C until analysis.
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2.3. Elemental mapping by synchrotron micro X-ray fluorescence spectroscopy The two-dimensional distributions of Cd, Fe and Zn in the root cross sections were mapped using SXRF at an undulator beamline 37XU of SPring-8 (Terada et al., 2004). The incident beam was monochromatized using an Si(1 1 1) double-crystal monochromator. An incident X-ray beam of 30 keV [beam spot size: 2.0 m in the vertical direction (V) and 1.0 m in the horizontal direction (H)] was used for elemental mapping of Cd using a Cd K␣ line (23.11 keV). Fe and Zn were detected simultaneously with Cd. However, because the excitation efficiency of Zn and Fe is very low at an X-ray energy of 30 keV, the incident X-ray beam was set at 15 keV to measure the distributions of Fe and Zn in the root sections taken at 10 and 40 mm above the root apex. The focused beam size at an X-ray energy of 15 keV was 2.0 m (V) × 1.2 m (H). To avoid possible destruction due to irradiation with the high-energy X-ray beam (Lombi et al., in press), the same sections were not used for analyses under 15 and 30 keV. The incident X-ray beam was focused using a pair of aspherical mirrors in the Kirkpatrick–Baez configuration (Takeuchi and Suzuki, 2005; Suzuki et al., 2007). The fluorescent X-rays were detected using a Si(Li) solid-state detector and silicon drift detector for mapping with incident X-ray energies of 30 and 15 keV, respectively. The scan step size was 3 m, and the spectral acquisition time for each spot was 1 s for Cd and 0.3 s for Fe and Zn. The freeze-dried sections were analyzed at an ambient temperature, whereas the frozen-hydrated sections were analyzed under a cryogenic N2 gas stream (Cryojet XL, Oxford Instruments, UK) to keep them frozen. The fluorescence yield was normalized by the incident photon intensity (I0 ) to produce SXRF elemental maps. The SXRF analyses were repeated using sections taken from the different individual plants with the scan step size of 5 m to confirm reproducibility. Average fluorescence counts in the areas of the epidermis, the exodermis, the cortex, the endodermis and the stele were calculated using IgorPro 6.0.3.1 software (Wavemetrics, USA). The border of each tissue was detected by tracing an SXRF map or root section on its SEM image. In addition to the entire stele, average fluorescence counts in the pericycle were calculated separately because higher fluorescence counts of Cd were observed in the pericycle of S. torvum. The average fluorescence counts in the epidermis and exodermis could not be calculated separately because the epidermis and exodermis might have overlapped in some sections that were cut at an angle. We did not attempt to convert fluorescence counts into quantitative units because the purpose of our study was to compare Cd distribution patterns among root tissues between two solanaceous species. The relative amounts of same element contained in the root sections are comparable among samples when they were measured under the same conditions and normalized by I0 at the same beamtime.
2.4. Observation of the Casparian strips The 100-m thick root cross sections adjacent to the sections used for SXRF analyses were cut using the microtome and collected in deionized water. The fresh sections were stained with berberine hemisulfate aniline blue (Brundrett et al., 1988) and then observed using a fluorescent microscope (Axioskop2 plus, Zeiss, Oberkochen, Germany) equipped with an ultraviolet filter set (excitation filter BP 365, diachronic mirror FT 395, barrier filter LP 397, Zeiss). Other sections were immersed in a concentrated sulfuric acid drop placed on a glass slide and observed for dissolution or retention of the Casparian strips and suberin lamellae (Johansen, 1940) using a digital microscope (VHX-100, Keyence, Osaka, Japan).
2.5. Chemical analyses After sectioning, the rest of the roots and shoots were dried in an oven at 75 ◦ C and ground to a fine powder. The powder (0.2 g) was digested with HNO3 , HClO4 and HF in a Teflon beaker heated to 120 ◦ C. After digestion, the resulting solution was evaporated to near dryness, and the total volume was adjusted to 15 ml using 1% HNO3 . Concentrations of Cd, Fe and Zn were determined using an inductively coupled plasma optical emission spectrometer (VistaPro, Varian, CA, USA). 3. Results 3.1. Casparian strips Casparian strips were visible as small dots in the root sections stained with berberine hemisulfate-aniline blue (Brundrett et al., 1988). For both S. melongena and S. torvum, the root sections taken at less than 30 mm above the root apex showed little evidence of Casparian strips, whereas both the exodermis and endodermis of the root sections taken at 30 mm above the root apex possessed Casparian strips. Sections taken at 40 mm above the root apex were shown in supplementary data. 3.2. Cd distribution in the root sections The Cd concentrations in S. melongena shoots were about 4 times higher than those in S. torvum shoots (Fig. 1). The Cd concentrations in S. melongena roots were higher than those in S. torvum roots, although this difference was smaller than the difference between the shoots (Fig. 1). These trends were in accordance with our previous results (Arao et al., 2008; Mori et al., 2009). Cd was not detected by SXRF in the root sections taken at less than 10 mm above the root apex. Casparian strips were observed in the section 30 mm above the root apex; therefore, the root sections taken at 10, 30 and 40 mm above the root apex were chosen for SXRF analyses. The SEM images and SXRF elemental maps of Cd in S. melongena and S. torvum root sections taken at 10, 30 and 40 mm above the root apex after 24 h of Cd treatment are shown in Fig. 2. Cd was localized in the epidermis, exodermis and stele of S. melongena root sections taken 10 and 30 mm above the root apex (Fig. 2a and b). Cd was found in the stele of S. melongena root sections 40 mm from the root apex, but less Cd was found in the epidermis and exodermis (Fig. 2c). The average fluorescence count of Cd in the stele of S. melongena root sections was the lowest in the cortex and the highest in the stele (Fig. 3a). The distribution pattern of Cd in the root of S. torvum was different from that of S. melongena. Cd accumulated in the endodermis and pericycle and was also found in the epidermis and exodermis of S. torvum root sections taken at 30 and 40 mm above the root apex (Fig. 2e and f). The average fluorescence count of Cd was the lowest in the cortex and the highest in the pericycle (Fig. 3b) at 30 and 40 mm from the root apex. In comparison, the average fluorescence counts of Cd in each root tissue after treatment with Cd for 7 d are also shown in Fig. 3. The SXRF analyses of the root sections treated with Cd for 7 d were determined at different beamtimes from those treated with Cd for 24 h. It should be noted that direct comparison of the absolute value of the average fluorescence counts for the roots treated with Cd for 7 d with those for 24 h might be impractical despite normalization by I0 . In S. melongena, distribution patterns of Cd among the root tissues were almost the same regardless of the Cd treatment time (Fig. 3a). A higher ratio of Cd localized in the endodermis and pericycle of S. torvum roots treated with Cd for 7 d than in those treated with Cd for 24 h (Fig. 3b), suggesting that Cd continuously accumulated in the endodermis and pericycle.
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3.3. Comparison of Cd distribution in freeze-dried and frozen-hydrated sections The Cd distribution pattern in the freeze-dried sections was compared with that of the frozen-hydrated sections using S. torvum root sections taken at 40 mm above the root apex. Cd localization in the outermost layer and the center part of the root was observed in the frozen-hydrated sections, although this was less evident than in the freeze-dried sections because the thickness of the section was one-fourth that of the freeze-dried sections. Identifying the exact position of the tissues in the frozen-hydrated sections by SEM observation failed because the freeze-drying treatment of these sections after SXRF analysis caused shrinkage of the tissues. Higher fluorescence counts of Cd were observed in the outer most layer of the epidermis. The circular part where higher fluorescence counts were observed was either the endodermis or the pericycle. No significant differences were observed between Cd distributions of the frozen-hydrated and freeze-dried sections. Therefore, it is unlikely that freeze-drying treatment altered the Cd allocation pattern in the root tissues, at least at the tissue resolution level. The SXRF elemental map of Cd for frozen-hydrated S. torvum root cross sections taken at 40 mm above the root apex is given as supplementary data. 3.4. Distributions of Fe and Zn in the root sections with and without Cd treatment The Fe concentration in S. melongena shoots was higher than in S. torvum shoots regardless of the Cd treatment (Fig. 1). In contrast, the Zn concentration in S. melongena shoots was lower than in S.
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torvum shoots regardless of the Cd treatment (Fig. 1). In shoots, the Fe concentration was not affected by the Cd treatment, whereas the Zn concentration was decreased. The Fe concentration in the root was very high, amounting to 9.5 g kg−1 . Without Cd treatment, the Fe and Zn concentrations were higher in S. torvum roots than in the S. melongena roots. Cd treatment caused a decrease in the Fe concentration in the roots of both S. melongena and S. torvum and in the Zn concentration of S. torvum (Fig. 1). Fig. 4 shows the average fluorescence counts normalized by incident photon energy, I0 , in the epidermis, exodermis, cortex, endodermis, pericycle and stele for Fe and Zn in S. melongena and S. torvum root sections with and without Cd treatment. The SEM images and SXRF elemental maps are provided as supplementary data. The distribution patterns of Fe in the root sections taken at 10 mm and 40 mm above the root apex were not affected by the Cd treatment (Fig. 4a). In the root sections taken at 40 mm above the root apex, Fe was mostly localized in the epidermis and exodermis. Note that the average fluorescence counts in Fig. 4a are displayed as a log scale. Except for the epidermis and exodermis, the localization of Fe was not clearly observable. In accordance with Fe, the distribution pattern of Zn in the root sections among tissues was not affected by Cd treatment, except in root sections of S. melongena taken at 10 mm above the root apex in which a relatively higher ratio of Zn accumulated in the pericycle and stele (Fig. 4b). In the root section of S. melongena at 40 mm above the root apex, the highest average fluorescence counts of Zn were found in the epidermis and exodermis, regardless of Cd treatment. In the root of S. torvum, Zn was not localized in particular tissues. Without Cd treatment, the average fluorescence counts of Zn tended to be higher in the pericycle as well as in the entire
Fig. 1. Concentrations of Cd, Fe and Zn in the shoot and root of S. melongena and S. torvum with and without Cd treatments.
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Fig. 2. SEM images and SXRF elemental maps of Cd in Cd-treated S. melongena (a–c) and S. torvum (d–f) root cross sections taken at 10 (a and d), 30 (b and e) and 40 mm (c and f) above the root apex. Note that root cross sections taken at 30 mm above the root apex were analyzed at different beamtimes from the root sections taken at 10 and 40 mm above the root apex. Scale bars represent 100 m. ep, epidermis; ex, exodermis; en, endodermis; and pe, pericycle.
stele except in sections taken at 40 mm above the root apex of S. melongena. 4. Discussion Cd localization in the central vascular bundle of plant roots that exhibit higher rates of root-to-shoot Cd translocation has been observed for Cd hyperaccumulators, such as Arabidopsis thaliana (Isaure et al., 2006) and Echinochloa polystachya (Solís-Domínguez et al., 2007). In accordance with these results, the fluorescence counts of Cd were higher in the stele than in the cortex and the endodermis in S. melongena roots (Figs. 2a–c and 3a), which had higher rates of root-to-shoot Cd translocation. In contrast to the S. melongena root, higher amounts of Cd were found within the cells of the endodermis and pericycle of S. torvum root sections taken at 30 and 40 mm above the root apex (Figs. 2e, f, and 3b). These results mean that the radial flow of Cd was blocked in the endodermis and pericycle of the S. torvum root, whereas it was not restricted in the root of S. melongena. Apoplastic Cd transport to the stele can
be blocked in the endodermis due to the presence of the Casparian strips or suberized tissues. This would cause the accumulation of Cd in the endodermis of S. torvum (Figs. 2e, f, and 3b). Though endodermal Casparian strips had developed in S. melongena root sections taken at 30 and 40 mm above the root apex, Cd did not accumulate in the endodermis (Figs. 2b, c, and 3a). Lu et al. (2009) suggested that the symplastic pathway rather than the apoplastic bypass contributed greatly to the root uptake, xylem loading and translocation of Cd to the shoots of the hyperaccumulating ecotype S. alfredii, in comparison with the non-hyperaccumulating ecotype. In S. melongena roots with higher root-to-shoot Cd translocation, most of the Cd might have entered the cytoplasm and bypassed the Casparian strips on the endodermis via the symplastic pathway, thereby displaying less Cd accumulation in the endodermis. This is supported by the observation that the rates of symplastic Cd absorption by S. melongena roots were 1.5-fold faster than those of S. torvum roots (Mori et al., 2009). However, only a 1.5-fold difference in symplastic Cd transport is not enough to explain the several fold higher Cd concentration in the xylem sap of S. melongena than
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Fig. 3. Average fluorescence counts of Cd in epidermis and exodermis; cortex, endodermis, pericycle and stele in the root sections of S. melongena (a) and S. torvum (b). Root anatomy (c) showing ep, epidermis; ex, exodermis; en, endodermis; co, cortex; pe, pericycle; and st, stele. Note that pericycle is a composition of the stele.
in that of S. torvum. A difference in the ability and/or capacity of the transporter that loads Cd into the xylem was considered to be the most important factor determining the difference between Cd concentrations in the xylem saps (Mori et al., 2009). Yamaguchi et al. (2010) found that a xylem-loading citrate transporter, AtFRD3, was down-regulated during the process of Cd acclimation of S. torvum under mild Cd concentrations. It was interpreted that Cd that was unable to be loaded into the xylem accumulated in the pericycle of S. torvum root sections taken at 30 and 40 mm above the root apex due to its inability to transport Cd from the pericycle into the xylem. Although the Cd concentration in the bulk root, which was determined by ICP-MS after acid digestion, did not differ between the roots of S. torvum and S. melongena (Fig. 1), the fluorescence counts in the root sections of S. torvum were higher than for those of S. melongena except for sections taken at 10 mm above the root apex (Fig. 3). Our preliminary analyses of Cd distribution along the length of the main roots using laser ablation inductively coupled plasma mass spectrometry showed that Cd was evenly present from 0 to 80 mm above the root apex in S. melongena, whereas a higher ratio of Cd accumulated in the S. torvum root between 40 and 56 mm above the root apex. The graph showing Cd distribution along with the length of root is given as supplementary data. The variation of Cd concentration along the length of the root would explain the comparatively higher fluorescence count of Cd in the root sections of S. torvum at 30–40 mm above the root apex than in S. melongena. In addition, Cd in S. torvum might have accumulated in regions several centimeters above the root apex because Cd transport to the xylem was restricted.
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Fig. 4. Average fluorescence counts of Fe (a) and Zn (b) in epidermis, exodermis, cortex, endodermis, pericycle and stele in the root sections of S. melongena and S. torvum. ep, epidermis; ex, exodermis; en, endodermis; co, cortex; pe, pericycle; st, stele. Note that the pericycle is a composition of the stele.
In contrast to Cd, Zn was not localized in the endodermis or pericycle of S. melongena or S. torvum root sections regardless of the Cd treatment (Fig. 4b). This suggested that Zn was efficiently transported into the xylem. In dicots, Zn is transported into the xylem by heavy metal transporters HMA2 and HMA4 (Palmer and Guerinot, 2009). In the Cd hyperaccumulator Arabidopsis halleri, increased Zn concentrations restrict xylem loading of Cd. In addition, xylem loading of Cd could be competing with the loading of Zn (Ueno et al., 2008). In this study, Zn concentrations in the shoots were decreased by Cd treatment (Fig. 1), and the average fluorescence counts in the stele of the Cd treated root were lower than those without Cd treatment (Fig. 4b) in S. torvum. Although the Zn concentrations in the shoots of S. melongena were not changed due to the Cd treatment, the average fluorescence counts of Zn in the stele of S. melongena at 10 mm from the root apex was decreased due to the Cd treatment. Zn absorption in S. melongena and S. torvum probably competed with Cd absorption as was shown by De la Rosa et al. (2004). Ueno et al. (2008) suggested that Fe transporters may contribute to Cd absorption in A. halleri because Fe deficiency induced increased Cd absorption. The Fe concentration in the roots of S. torvum was decreased by Cd treatment, and a higher ratio of Fe in the root accumulated in the epidermis and exodermis (Fig. 4a). However, this could be caused by the deposition of divalent Fe in the solution culture as discussed below. Thus, the relationship between Fe and Cd absorption was difficult to interpret using our data. In addition to Cd localization in the stele of S. melongena root sections, Cd was also localized in the epidermis and exodermis of S. melongena root sections taken at 10 and 30 mm above the root apex. In S. torvum root sections, Cd localization was observed in
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Fig. 5. Relationships between X-ray fluorescence counts of Cd and Fe accumulation in the epidermis and exodermis of root sections determined at an incident X-ray energy of 30 keV. Cd-treated S. melongena (a–c) and S. torvum (d–f) root sections taken 10 (a and d), 30 (b and e) and 40 mm (c and f) above the root apex.
the endodermis and pericycle and in the epidermis and exodermis of root sections taken 30 mm above the root apex (Figs. 2 and 3). The relationships between X-ray fluorescence counts of Cd and Fe in the epidermis and exodermis of the root sections, both of which were determined at an incident X-ray energy of 30 keV, are shown in Fig. 5. At an incident X-ray energy of 30 keV, Fe was only detected in the epidermis and exodermis, although it was found in all tissues when determined at an incident X-ray energy of 15 keV (Fig. 4a). Since the sections used for SXRF mapping at an incident X-ray energy of 15 keV were different from those used for elemental mapping at an incident X-ray energy of 30 keV, a spatial comparison of Cd determined at 30 keV with Fe deter-
mined at 15 keV was impractical. Significant relationships were observed between fluorescence counts of Cd and Fe in the epidermis and exodermis of S. melongena root sections taken at 10 and 30 mm above the root apex and in those of S. torvum root sections taken at 30 mm above the root apex (Fig. 5). These samples, which showed a higher correlation between Cd and Fe, accumulated a higher ratio of Cd in the epidermis and exodermis (Figs. 2 and 3). The cell walls of the epidermis are the first barrier preventing Cd from entering the root; therefore, metals commonly accumulate in the epidermis (Seregin and Kozhevnikova, 2008). However, in our study, Fe hydroxide was deposited on the root surface because the nutrient solution contained Fe2+ , which easily oxidizes and pre-
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cipitates as Fe(OH)3 . The high Fe concentrations in Cd-treated S. melongena and S. torvum roots (Fig. 1) and the localization of Fe in the epidermis and exodermis (Fig. 4) implied the deposition of Fe hydroxide which could inhibit Cd absorption into the root since Fe hydroxide can adsorb Cd (Buekers et al., 2008). Therefore, we speculated that the Cd localization in the epidermis and/or exodermis might be an experimental artifact caused by the deposition of Fe hydroxide. In addition to the Casparian strips in the endodermis and exodermis, the cell walls of the cortex also play an important role in controlling apoplastic Cd transport (Seregin and Kozhevnikova, 2008; Radjala et al., 2009). In the roots of Zea mays L. and Noccaea caerulescens, adsorption of Cd in the root apoplast corresponded to 30–90% of the total root uptake (Radjala et al., 2009). Our SXRF mapping results suggested that Cd was present along the cell walls of the cortex (Fig. 2e and f) in S. torvum roots. S. torvum roots tended to be thicker than S. melongena roots, so apoplastic Cd transport in S. torvum could be delayed due to a greater adsorption function of the cell walls of the cortex compared to that of S. melongena. However, we cannot completely eliminate the possibility that the freeze-drying of the root sections induced the redistribution of intracellular Cd to the cell walls. 5. Conclusions SXRF mapping indicated that Cd accumulated in the endodermis and the pericycle in the root sections at 30 and 40 mm above the root apex for S. torvum, which has lower capacity for root-to-shoot translocation of Cd. In the root of S. melongena, which has a higher capacity for root-to-shoot Cd translocation, Cd was localized in the stele and accumulation of Cd in the endodermis or pericycle was not observed. The essential trace elements, Zn and Fe, also did not accumulate in the endodermis or pericycle. These results support the fact that Cd was efficiently transported into the xylem of the S. melongena root in a pathway similar to that of the essential trace elements, whereas Cd flow to xylem was restricted in the endodermis and pericycle of the root of S. torvum. The restricted flow of Cd into the xylem might be caused by a weaker ability or a shortage of the transporter required to efflux Cd into the xylem as was also suggested by Mori et al. (2009). Acknowledgements This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and was carried out with the approval of the SPring-8 Program Advisory Committee (2008A1087 and 2008B1791) and the support of NIMSCNN Nano Foundries and United Analysis Facilities (AC210003). We thank Prof. I. Nakai as well as Mses S. Mitsuo, S. Takada, W. Yamaoka and Mr. Y. Yoshii from the Tokyo University of Science for their assistance in SXRF analyses, Ms. M. Maeda from the Leica Microsystems for her support in the use of the cryomicrotome and Dr. S. Ishikawa from the National Institute for Agro-Environmental Sciences for constructive comments. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.envexpbot.2010.12.002. References Assmann, S.M., 2006. Solute transport. In: Taiz, L., Zeiger, E. (Eds.), Plant Physiology., 4th ed. Sinauer, Sunderland, UK, pp. 95–119. Arao, T., Takeda, H., Nishihara, E., 2008. Reduction of cadmium translocation from roots to shoots in eggplant (Solanum melongena) by grafting onto Solanum torvum rootstock. Soil Sci. Plant Nutr. 54, 555–559.
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