Stem and leaf sequestration of zinc at the cellular level in. the hyperaccumulator Sedum alfredii. Research

Transcription

1 Stem and leaf sequestration of zinc at the cellular level in Blackwell Publishing Ltd the hyperaccumulator Sedum alfredii Sheng-Ke Tian 1, Ling-Li Lu 1, Xiao-E Yang 1, John M. Labavitch 2, Yu-Ying Huang 3 and Patrick Brown 2 1 MOE Key Laboratory of Polluted Environment Remediation and Ecological Health, Zhejiang University, Huajiachi Campus, Hangzhou , China; 2 Department of Plant Sciences, University of California, Davis, CA, 95616, USA; 3 Institute of High Energy Physics, Chinese Academy of Sciences, Beijing , China Summary Author for correspondence: Xiaoe Yang Tel: Fax: xyang@zju.edu.cn, xyang571@yahoo.com Received: 23 September 2008 Accepted: 12 November 2008 New Phytologist (2009) 182: doi: /j x Key words: cellular distribution, hyperaccumulation, Sedum alfredii, synchrotron radiation X-ray fluorescence (SRXRF), zinc, Zinpyr-1. Sedum alfredii is a fast-growing, high-biomass zinc (Zn) hyperaccumulator native to China. Here, the characteristics of in vivo Zn distribution in stems and leaves of the hyperaccumulating (HE) and nonhyperaccumulating ecotypes (NHE) of S. alfredii were investigated by synchrotron radiation X-ray fluorescence (SRXRF) analysis, together with a Zn probe. Preferential Zn accumulation in leaf and stem epidermis was observed in both ecotypes, but to a much greater extent for HE. Epidermal Zn increased largely in leaves and stems of HE as exposure time was prolonged, while Zn saturation occurred relatively early in HE leaf mesophyll cells and stem vascular bundles. A second peak of Zn enrichment in stem and leaf vascular systems was shown in both ecotypes. However, the proportion of Zn accumulated in stem vascular bundles relative to other tissues was much greater for HE than for NHE. Leaf and stem distribution patterns of phosphorus (P) and sulphur (S) in the HE were very like that for Zn, while the calcium (Ca) distribution pattern was the reverse of that for Zn. No such relationship was observed in NHE. Our study mainly suggested that epidermal layers serve as important storage sites for accumulated Zn in the S. alfredii HE. Introduction Zinc (Zn) is an essential trace element for many physiological processes in plants and other organisms (Marschner, 1995), yet Zn can be toxic at elevated concentrations (Prasad & Hagemeyer, 1999). However, a rare class of plants designated hyperaccumulators (Brooks, 1998) can accumulate and tolerate extremely high concentrations of toxic elements in shoots; these include about 18 species of Zn hyperaccumulators (Reeves & Baker, 2000). The active accumulation of elements like Zn in plant shoots provides a promising approach for phytoremediation of contaminated soils (McGrath et al., 2002; Peuke & Rennenberg, 2005) and suggests the possibility of transferring the hyperaccumulation traits into crop species for use in biofortification (Broadley et al., 2007). Better knowledge of physiological and molecular mechanisms of element uptake from soils and accumulation by hyperaccumulators is needed to support efforts to exploit the enhanced metal uptake and sequestration capabilities in improved crop plants (Clemens et al., 2002). Plants themselves possess homeostatic mechanisms (Clemens et al., 2002; Hall, 2002) to maintain the normal concentrations of toxic elements in different plant tissues or organelles to minimize the damage from excess exposure. In recent years, the cellular and subcellular localization of heavy metals has been studied extensively in leaves of hyperaccumulators in order to identify the detoxification mechanisms that facilitate storage of excess foliar heavy metals. Zinc compartmentation has been investigated in the hyperaccumulators Arabidopsis halleri and Thlaspi caerulescens (Küpper et al., 1999, 2000; Frey et al., 2000; Zhao et al., 2000) and studies thus far have suggested that most heavy metals are accumulated in leaf epidermal and surface structures such as trichomes (Krämer et al., 1997; Mesjasz-Przybylowicz et al., 1997; Küpper et al., 1999, 2001; Frey et al., 2000; Robinson et al., 2003; Asemaneh et al., 2006; Hokura et al., 2006). Some studies indicated that leaf mesophyll cells played a dominant role in detoxification of excess Zn and Cd by A. halleri or T. caerulescens (Küpper et al., 2000; Zhao et al., 2000; Ma et al., 2005). However, very few studies have 116 New Phytologist (2009) 182: The Authors (2009) Journal compilation New Phytologist (2009)

2 117 investigated stem sequestration of metals at the cellular level, even though this may contribute to the heavy metal upward translocation and hyperaccumulation in plants. The hyperaccumulating ecotype (HE) of Sedum alfredii Hance, reported as a new Zn/Cd co-hyperaccumulating plant native to China (Yang et al., 2002, 2004), can accumulate 2.9% Zn in shoots without toxicity symptoms (Long et al., 2002; Yang et al., 2002, 2006), with the highest concentration of the Zn in stems. on S. alfredii root compartmentation revealed that approx 2.7-fold less Zn was stored in the vacuoles of root cells of the HE than in the nonhyperaccumulating ecotype (NHE). Thus, the Zn accumulated in roots of the HE was more available for xylem loading and subsequent translocation to shoots (Yang et al., 2005). By using leaf tissue fractionation, preferential distribution of Zn in the apoplast (cell wall space) of leaves and stems of the S. alfredii HE was also reported (Li et al., 2006). However, the detailed cellular and subcellular characteristics of stem and leaf sequestration of Zn in S. alfredii are unclear. Thus, the objectives of this study were to investigate the characteristics of Zn distribution in stems and leaves of both HE and NHE S. alfredii by using SRXRF microprobe analysis and the Zn-fluorophore, Zinpyr-1 (Sinclair et al., 2007; Hanikenne et al., 2008), and to use SRXRF microprobe analysis to examine the relationship of Zn cellular distribution patterns in stems and leaves with distribution patterns of other elements in these organs. Materials and Methods Plant culture Seedlings of the two Sedum alfredii Hance ecotypes were cultivated hydroponically. The HE S. alfredii was originally obtained from an old Pb/Zn mine area in Zhejiang Province, China, and the NHE from a tea plantation in Hangzhou, also in Zhejiang Province. Plants were chosen and grown in noncontaminated soils for several generations to minimize the internal metal contents, then uniform and healthy shoots were selected and cultivated in a basal nutrient solution containing 2 mm Ca 2+, 4 mm + NO, 1.6 mm K + 3, 0.1 mm H 2 HPO 2, 0.5 mm Mg 2+, 1.2 mm 2 4 SO, 0.1 mm Cl 4, 10 μm H 3 BO 3, 0.5 μm MnSO 4, 1 μm ZnSO 4, 0.2 μm CuSO 4, 0.01 μm (NH 4 ) 6 Mo 7 O 24, and 100 μm Fe EDTA. Nutrient solution ph was adjusted daily to 5.8 with 0.1 m NaOH or 0.1 m HCl. Nutrient solutions were aerated continuously and renewed every 3 d. Plants were grown under glasshouse conditions with natural light, day/night temperatures of 26/ 20 C, and day/night humidity of 70/85%. Measurement of Zn and other elements After pre-culturing for 14 d, HE and NHE seedlings were transferred to nutrient solutions containing different Zn concentrations. The treatments were a control (1 μm) and 25, 50, 100, 250, 500 μm Zn, supplied as the sulfate, with three replicates per treatment. Plants were harvested after 30 d. At harvest, roots were soaked in 20 mm Na 2 EDTA for 15 min to desorb putatively surface-adsorbed Zn. The harvested plants were separated into roots, stems and leaves and oven-dried at 65 C for 72 h. The dried plant materials were ground using a stainless steel mill and passed through a 0.25 mm sieve for analysis of Zn and other elements. Dry plant samples (0.1 g) of each treatment were digested with HNO 3 -HClO 4, and the digest was transferred to a 50 ml volumetric flask, made up to volume with water and filtered. Concentrations of Zn and other elements (i.e. K, Ca, Mn, Fe and Cu) in the filtrates were analyzed using inductively coupled plasma mass spectroscopy (Agilent 7500a, Agilent, Santa Clara, CA, USA). P content was analyzed by the molybdenum blue method after digestion with H 2 SO 4 -H 2 O 2 at 300 C, and normalized by dry weight. SRXRF analysis Elemental distributions in cross-sections of stems and leaves were determined by SRXRF microprobe at the XRF microprobe station in the Beijing Synchrotron Radiation Facility (BSRF), Beijing, China. Seedlings of S. alfredii were pre-cultured for 14 d before exposure to 100 μm Zn. Each treatment was done in triplicate. Stems and leaves were cut from plants after 7 or 30 d exposure to Zn and rinsed. The mid-transverse areas of stem and mature leaf samples at similar developmental stages were selected from both ecotypes for comparisons. Sections (40 μm thick) were cut with a cryotome (Leica, CM1850) at an ambient temperature of 20 C, and subsequently freeze-dried at 20 C for 3 d. The X-ray light source came from the 4W1B beam line at BSRF. This beam line can provide multi-chromatic X-rays (white light), with energy ranges from 3.5 to 35 kev. The electron energy in the storage ring is 2.2 GeV, with a current range from 70 to 128 ma. The XRF microprobe experimental station is 25 m away from the point of the light source. Since the emission angles of the light in the horizontal and vertical directions are only 1.0 and 0.1 mrad, respectively, this high collimation confers superior performance on the synchrotron radiation XRF microprobe. During the experiment, the spot size of the X-ray beam was μm 2, which is smaller than the size of most leaf and stem cells in Sedum. The acquisition time per spot was approximately 2 min. XRF spectra were collected using a PGT Si (Li) solid detector, positioned at 90 to the beam line, 7 mm from the target. The scanning points of the samples were selected and observed using a microscope. Ten or nine replicates were done for each of the scanning points in one specimen. The characteristics of the SRXRF apparatus at BSRF were reported in detail by Huang et al. (2001). All the SRXRF spectra were analyzed by AXIL. The Authors (2009) New Phytologist (2009) 182: Journal compilation New Phytologist (2009)

3 118 Fig. 1 Biomass of plant tissues (a, b) and concentrations of zinc (Zn) (c, d) in root (triangles), stem (circles) and leaf (squares) tissues of the hyperaccumulating (HE; a, c) and nonhyperaccumulating (NHE; b, d) Sedum alfredii. Plants were exposed to different Zn concentrations (0 500 μm) for 30 d. Note that the range of the y-axis values in (c) is five times that of (d). Data points and error bars represent means (n = 3) and SEs, respectively. DW, dry weight. A separate experiment of two-dimensional micro-x-ray fluorescence imaging of Zn in the HE leaf and stem crosssections was carried out on beam line 4A at the Photon Factory (PF), High Energy Accelerator Organization (KEK), Tsukuba, Japan. Sample preparation was as in the experiments described earlier with minor modification. Stem and leaf sections of 100 μm thickness were taken from HE S. alfredii seedlings exposed to 500 μm Zn for 30 d. Instruments and measurement conditions were almost the same as described by Hokura et al. (2006), with slight modification. The incident beam was monochromatized by a W/B4C double multilayer, and with a Si (111) double-crystal monchromator, and a spherical mirror coated with Rh was used for vertical focusing, with energy ranges from 14.2 to 16.5 kev. The electron energy in the storage ring was 2.5 GeV, with a current range from 300 to 420 ma, and the detector was Si (Li) SSD. The focused X-ray beam was adjusted by horizontal slits, and a beam size of μm 2 was obtained. The step size was set to 20 μm. Microscopic imaging of Zn in stem and leaf Microscopic imaging of Zn in stems and leaves of S. alfredii were conducted according to the method of Sinclair et al. (2007) and Hanikenne et al. (2008) with several modifications. After plants were grown in 100 μm Zn for 30 d, fresh stem and leaf slices were cut (thickness < 0.5 mm). Plant samples were then immersed in a 10 μm solution of the Zn fluorescent indicator Zinpyr-1 (Sigma, Allentown, PA, USA) in 10 mm MES buffer, ph 6.1. The samples immersed in the solution were vacuum-infiltrated for 10 min, and incubated in the same solution for a further 20 min (Gutierrez-Alcala et al., 2000). Samples were then rinsed in deionized water, immersed in 75 μm propidium iodide to stain cell walls and nuclei in cells, and rinsed again. Plant samples were kept in the dark during this procedure. After washing, a Leica DMR series fluorescent microscope equipped with a Chroma filter set (Chroma Technology, Rockingham, VT, USA) and CoolSNAP-HQ (Roper Scientific, Tucson, AZ, USA) were used to visualize the samples. Zinpyr-1 was visualized by using filters S484/15 for excitation and S517/30 for emission, and propidium iodide was visualized by using filters S550/15 for excitation and S620/30 for emission. Exposure times were uniform for all samples. All images were taken at 10 magnification. Images were pseudocolored with METAMORPH software (Universal Imaging, Downingtown, PA, USA). No autofluorescence was observed in roots of the two S. alfredii ecotypes. Statistical analysis of data All data were statistically analyzed using the SPSS package (version 11.0), analysis of variance (ANOVA) was performed on the data sets and the mean and SE of each treatment as well as LSD (P < 0.05 and P < 0.01) for each set of corresponding data were calculated. Results Plant growth and Zn concentrations While HE grew in all Zn treatments from 1 to 500 μm with no visible signs of stress (Fig. 1a), significant growth inhibition was observed for NHE after exposure to Zn concentrations > 100 μm for 30 d (Fig. 1b). In fact, growth of the HE appeared to be stimulated by the Zn addition, as the biomass of leaves, stems and roots in 100 μm Zn-treated New Phytologist (2009) 182: The Authors (2009) Journal compilation New Phytologist (2009)

4 119 Table 1 Effects of Zn treatments on the concentrations of Zn, K, Ca, Mg, P (mg g 1 DW), and Fe, Cu, Mn (μg g 1 DW) in leaf and stem tissues of hyperaccumulating (HE) and nonhyperaccumulating (NHE) Sedum alfredii Zn K Ca Mg P Fe Cu Mn Solution Zn (μm) Stem Leaf Stem Leaf Stem Leaf Stem Leaf Stem Leaf Stem Leaf Stem Leaf Stem Leaf HE Sig. a ** ** ns ns ns * ns ns ** * * ns * ns ns ns LSD P< LSD P< NHE Sig. a ** ** ** ** ** ** ** ** * ** * ns ns ns ns ns LSD P< LSD P< Plants were exposed to different Zn concentrations (0 500 μm) for 30 d. LSD 0.05 and LSD 0.01 represent the least significant differences (P < 0.05 and P < 0.01) among the corresponding groups of data (columns). a Significant levels: ns, not significant; *, P < 0.05; **, P < Data points represent means (n = 3). plants was much higher than in controls (Fig. 1a). At all Zn concentrations, there was no significant difference in HE and NHE root Zn concentrations. By contrast, there was great ecotypic variation in leaves and stems, as Zn in stems and leaves of the HE was up to 20 times higher than in the corresponding NHE leaf and stem tissues, depending on the Zn treatment (Fig. 1c,d). Preferential storage of Zn in shoots, especially the stems, rather than roots was indicated in HE plants (Fig. 1c), whereas Zn concentrations in both stems and leaves of the NHE were much lower than in roots (Fig. 1d). The concentrations of several other elements in the two S. alfredii ecotypes also showed differences in response to the Zn treatment (Table 1). In general, the post-zn-treatment concentrations were disturbed less in the HE than in the NHE. Zn exposure significantly decreased concentrations of K, Ca, and Mg in the stems and leaves of the NHE (P < 0.01 and P < 0.05, respectively), but had no such effects in the HE, except for a marked decrease of Ca in leaves (P < 0.05). P contents in both stems (P < 0.01) and leaves (P < 0.05) of the HE were significantly increased in parallel with increased Zn supply, except for the extremely high Zn treatment (500 μm). There was no consistent Zn treatment-related variation of P in stems and leaves of the NHE. SRXRF analysis of Zn and other elements The characteristic peaks of P, S, Cl, Ar, K, Ca, Mn, Fe, Ni, Cu and Zn in S. alfredii stems and leaves could be detected by SRXRF microprobe analysis. The main Zn Kα peak was located at 8.7 ev, with the secondary Zn Kβ peak at 9.6 ev (Fig. 2). A typical SRXRF microprobe spectrum is shown in Fig. 2. As light elements only emit X-rays with low energy, elements such as N, Mg and Na were difficult to detect using SRXRF. From the SRXRF microprobe peaks of P, S, Cl, K, Ca, Mn, Fe, Cu, Zn in stems and leaves of HE and NHE (data not shown), contents of these elements can be estimated (Supplementary material, Fig. S1). There was a significant linear correlation (P < 0.001) between the counts of elements in leaves and stems measured by SRXRF and the concentrations measured by ICP-MS (Fig. S2), demonstrating the accuracy and sensitivity of the SRXRF technique for trace elements analysis. Stem sequestration of Zn Clear differences in Zn distribution patterns across the stem with Zn exposure time were noted between the two ecotypes (Fig. 3). After 7 d treatment with 100 μm Zn, the highest Zn concentrations were located within the vascular bundle (scanning points 3 5) in the stem of HE, and decreased gradually from the vascular tissues, toward both the pith (1 2) and cortex (6 9) (Fig. 3b). As the exposure time increased to 30 d, Zn distribution in the epidermis (10) increased rapidly to reach the highest local concentration in treated stems. A significant enhancement of Zn in the cortex cells (7 9) was also observed (Fig. 3b). In stems of NHE, both The Authors (2009) New Phytologist (2009) 182: Journal compilation New Phytologist (2009)

5 120 stem cross-sections of the two ecotypes (Fig. 4). Here, the intensity of the fluorescence provides only a qualitative comparison, since the fluorescence response of the dye is usually not linear, but rather a sigmoidal function of the metal concentration. The results were consistent with those obtained by SRXRF analysis (Fig. 3), with intense fluorescence observed in the vascular tissues and epidermis for both ecotypes, but to a much greater extent in the HE (Fig. 4). Green fluorescence was much more intense in the stem cortex cells of the HE than in NHE stems (Fig. 4). Fig. 2 Typical synchrotron radiation X-ray fluorescence (SRXRF) microprobe spectrum in a stem cross-section of hyperaccumulating (HE) Sedum alfredii treated with 100 μm Zn for 7 d. The main Zn Kα peak is located at 8.7 ev, and the secondary Zn Kβ peak is at 9.6 ev. the preferential localization of Zn in the epidermis and the time-dependent increase of epidermal Zn also occurred (Fig. 3c). However, in contrast to the Zn distribution in HE stems, there was no obvious peak of Zn accumulation in the vascular bundle (3 5), pith or cortex cells of NHE, and the time-dependent increase of Zn in cortex cells, while detected, was less pronounced than in the HE (Fig. 3c). The proportion of Zn accumulated in stem vascular tissues relative to other tissues is much greater for HE than for NHE at both the 7 and 30 d Zn treatment times. If scanning point 4 (Fig. 3b,c) is taken as the vascular tissue reference point, and scanning point 10 as the epidermal layer reference point, the ratios of Zn content are approximately 3 : 1 and 3 : 4 (7 and 30 d times, respectively) for the HE stems and 1 : 2 and 1 : 3 for NHE stems. The use of the Zn-fluorophore, Zinpyr-1, revealed substantial differences of green fluorescence intensity across Leaf sequestration of Zn After 7 d treatment with 100 μm Zn, Zn distribution patterns in leaves of HE and NHE were quite similar, both showing little variation across the leaf sections from the upper to lower epidermis, although Zn contents (counts) were much higher in the HE (Fig. 5). However, when Zn exposure time was increased to 30 d, Zn localization to epidermal layers (scanning points 1 and 10) increased fourfold, but changed very little or even decreased slightly in leaf mesophyll cells (2 4, 6 9) of the HE (Fig. 5b). In contrast, Zn accumulation in mesophyll and epidermal cells was similar in the NHE after 30 d exposure (Fig. 5c). In microscopic images in which Zn distributions across leaf cross-sections are indicated by Zinpyr-1, preferential sequestration of Zn in epidermal and vascular tissues was consistently noted for both the NHE and HE (Fig. 6), but the effect was more pronounced in the HE (Fig. 6). Correlation of Zn distribution with other elements A significant and positive correlation between Zn, K and Mn distributions in HE and NHE stems and leaves was observed, especially in plants treated with Zn for extended times (Table 2). Moreover, there are positive correlations between Fig. 3 Zinc (Zn) distribution in the stem cross-sections of Sedum alfredii, as determined by synchrotron radiation X-ray fluorescence (SRXRF) microprobe analysis (unit: counts per 200s). Scanning points (a), from the stem center to its periphery, identify the x-axis values for the analyses reported in (b) and (c). Stems from the hyperaccumulating (HE; b) and nonhyperaccumulating (NHE; c) ecotypes exposed to 100 μm Zn for 7 d (triangles) and 30 d (circles), respectively, were determined. The scanning points in the stem section include the pith (1 2), vascular bundle (3 5), cortex (6 9) and epidermis (10), as shown in the microscopic image (a). Ten replicates were analyzed for each scanning point. Note the range of y-axis scale values in (b) is 20 times that of (c). Bar, 100 μm. Data points represent means from three individual plants. New Phytologist (2009) 182: The Authors (2009) Journal compilation New Phytologist (2009)

6 Fig. 4 Visualization of zinc (Zn) in stem cross-sections using the Zn-fluorophore, Zinpyr-1, in the hyperaccumulating (HE) and nonhyperaccumulating (NHE) Sedum alfredii. The sections were taken from the stems of seedlings treated with 100 μm Zn for 30 d. The bright green fluorescence indicates the binding of the dye with Zn; red fluorescence represents cell walls and nuclei marked by propidium iodide. All images were taken at 10 magnification. Ep, epidermis; Co, cortex; Pi, pith; Xy, xylem cells; Ph, phloem. Fig. 5 Zinc (Zn) distribution in the leaf cross-sections of Sedum alfredii determined by synchrotron radiation X-ray fluorescence (SRXRF) microprobe (unit: counts per 200s). Samples from plants of hyperaccumulating (HE; b) and nonhyperaccumulating ecotypes (NHE; c) exposed to 100 μm Zn for 7 d (triangles) and 30 d (circles), respectively, were examined. Scanning points (a) from 1 to 10 (upper to lower epidermis) provide the values for the x-axes in (b) and (c). The scanning points in the leaf section included upper (adaxial) epidermis (1), palisade mesophyll (2, 3, 4), vein (5, 6), spongy mesophyll (7, 8, 9), and lower (abaxial) epidermis (10), as shown in the microscopic images (a). Nine or 10 replications were analyzed for each scanning point. Note the range of y-axis scale values in (b) is 20 times that of (c). Bar, 100 μm. Data points represent means from three individual plants. The Authors (2009) Journal compilation New Phytologist (2009) New Phytologist (2009) 182:

7 122 Fig. 6 Visualization of zinc (Zn) in leaf cross-sections using the Zn-fluorophore, Zinpyr-1, in the hyperaccumulating (HE) and nonhyperaccumulating (NHE) Sedum alfredii. The sections were taken from the leaves of seedlings treated with 100 μm Zn for 30 d. The bright green fluorescence indicates the binding of the dye with Zn; red fluorescence represents cell walls and nuclei marked by propidium iodide. All images were taken at 10 magnification. UE, upper epidermis; LE, lower epidermis; SM, spongy mesophyll; PM, palisade mesophyll; V, vein. Table 2 The correlation coefficients between Zn and other elements distributed in the stem and leaf cross-sections of hyperaccumulating (HE) and nonhyperaccumulating (NHE) Sedum alfredii Samples P S Cl K Ca Mn Fe Cu HE Stem 7 d (N = 10) 0.71* 0.81** * d (N = 10) 0.87** 0.85** 0.97** 0.93** 0.97** 0.90** Leaf 7 d (N = 10) 0.65* 0.88** * d (N = 10) 0.75** ** 0.91** 0.93** 0.76** NHE Stem 7 d (N = 10) ** 0.66* d (N = 10) * 0.66* 0.71* Leaf 7 d (N = 9) d (N = 9) * ** 0.80** 0.19 Plants were exposed to 100 μm Zn for 7 and 30 d, respectively. Concentrations of Zn and other elements were determined by synchrotron radiation X-ray fluorescence (SRXRF) microprobe (unit: counts per 200s). N represents number of replications for each scanning point; 10 scanning points were analyzed in each stem or leaf sections as indicated in Figs 3 and 5, respectively. The paired-samples t-test was performed. **Correlation is significant at the 0.01 level (two-tailed); *correlation is significant at the 0.05 level (two-tailed). Data points represent means from three individual plants. Zn and P, S, and Cl distribution in the stems and leaves of the HE, but no such significant correlations were noted in the NHE (Table 2). However, Ca was negatively correlated with Zn in most HE samples, while this negative correlation was not noted in NHE (Table 2). μ-x-ray fluorescence maps of Zn and other elements in stems and leaves of HE exposed to 500 μm Zn seem to match these results (Fig. 7). The distribution trends of Zn in stems were consistent with the SRXRF results obtained from BSRF (Fig. 3), showing higher Zn distributed in the epidermal and vascular tissues (Fig. 7a). S, K, and P distribution patterns appeared to be similar to that of Zn in the stem section, while the Ca distribution pattern was the inverse of that for Zn (Fig. 7a). The concentrations of Fe and Mn across the stem section were relatively low. The μ-sxrf Zn map for the HE leaf sections also showed that consistently high Zn concentrations were distributed primarily to the upper epidermis with substantially less accumulated in the lower epidermis (Fig. 7b). The leaf palisade mesophyll cells, located near the upper epidermis, contained much more Zn than the spongy mesophyll cells. Similar distribution patterns of K, P, Mn, and Fe were observed, while Ca distribution again was shown to be the reverse of Zn distribution in leaf cross-sections (Fig. 7b). New Phytologist (2009) 182: The Authors (2009) Journal compilation New Phytologist (2009)

8 Fig. 7 μ-synchrotron X-ray fluorescence elemental maps for Zn, Ca, K, Fe, Mn, P and S of stem (a) and leaf (b) cross-sections of hyperaccumulating (HE) Sedum alfredii. Samples from plants of HE exposed to 500 μm Zn for 30 d were examined. The number of fluorescence yield counts was normalized by I0 and the dwell time. The red color, depicting elemental concentrations in each map, was scaled to the maximum value for each map. (a) E, epidermis; C, cortex; V, vascular tissues; P, pith. (b) UE, upper epidermis; LE, lower epidermis; SM, spongy mesophyll; PM, palisade mesophyll; V, vein. Discussion Greater tolerances of high concentrations of generally toxic metals, as well as hyperaccumulation of metals in aerial parts of the plants, are typical characteristics of hyperaccumulators The Authors (2009) Journal compilation New Phytologist (2009) (Krämer et al., 2000; Küpper et al., 2001; Asemaneh et al., 2006). These characteristics were confirmed for the HE S. alfredii in this study (Fig. 1, Table 1), and the results presented here are highly consistent with the results from previous studies (Yang et al., 2002, 2005). Successful New Phytologist (2009) 182:

9 124 detoxification of these excess metals probably requires sequestration in appropriate cellular compartments to accomplish permanent storage (Brooks, 1998). Therefore, investigation of Zn localization in stems and leaves was performed in HE and contrasted with that in NHE in order to reveal the possible mechanism of Zn hypertolerance. Mainly two techniques, SRXRF analysis and epifluorescence microscopy using the Zn-fluorophore, Zinpyr-1, were employed to determine Zn distribution in stems and leaves of S. alfredii; the former was more sensitive, specific and quantitative (Shi et al., 2004) and the latter provided higher resolution. The results obtained using SRXRF analysis of S. alfredii stems and leaves (Figs 3, 5), together with the μ-xrf maps of Zn (Fig. 7), agreed well with the microscopic fluorescence images of Zn in the same tissues (Figs 4, 6). These indicated the greatest Zn enrichment in epidermal layers, followed by a second peak of enrichment in stem and leaf vascular systems in both S. alfredii HE and NHE. A slight inconsistency of Zn localization in the leaf epidermis of HE was observed between SRXRF data (Fig. 5b) and μ-xrf map data (Fig. 7b). In leaf cross-sections from plants treated with 100 μm Zn for 30 d (Fig. 5b, SRXRF data), Zn in the upper epidermis was slightly higher than that in the lower epidermis, while in leaf cross-sections from plants treated with 500 μm for 30 d, Zn in the upper epidermis was much higher than in the lower epidermis (Fig. 7b, μ-xrf mapping data). In both methods the exposure time is constant (1 month), and leaves of a similar age were used. Thus the difference between the two results is mostly related to the difference in Zn exposure (100 vs 500 μm), which also caused an increase in whole-leaf [Zn] (Fig. 1). Here we used freeze-dried samples for SRXRF experiments (Figs 3, 5, 7) and fresh samples for Zinpyr-1 imaging experiment (Figs 4, 6); the consistent results demonstrated that there was no ion redistribution caused in the freeze-drying procedure. Moreover, the consistent reverse distribution of Ca with Zn in stem and leaf crosssections of HE plants (Table 2, Fig. 7) supported the efficacy of the sample preparation methodologies in this investigation. Preferential sequestration of heavy metals in leaf epidermis has been suggested as an important strategy of excess metal detoxification in hyperaccumulators (Frey et al., 2000; Küpper et al., 1999), presumably resulting in the protection of mesophyll cells and stomata against damage from the build-up of those toxicants, thus maintaining the functionality of these structures (Frey et al., 2000; Küpper et al., 2001). Results obtained in this study also indicated the greater preferential Zn accumulation in stem and leaf epidermal layers in both the HE S. alfredii and NHE, demonstrating that epidermal metal enrichment is not a unique characteristic of hyperaccumulators. However, epidermal sequestration contributes largely to Zn hyperaccumulation in shoots of the HE, most clearly after prolonged exposure time (30 d). As compared with the NHE, more epidermis-specific Zn was observed in the leaves of HE plants (Fig. 5). After treatment with 100 μm Zn for 30 d, the count ratio between epidermis and mesophyll for the leaves was approx. 6 in HE (Fig. 5b), but did not exceed 1.5 in NHE (Fig. 5c). Moreover, epidermal Zn increased fourfold in both stems and leaves of HE as Zn exposure time was increased more than fourfold (from 7 d to 30 d), suggesting an exposure time-dependent increase of epidermal Zn, while the data for HE leaf mesophyll cells and stem vascular bundles suggested that Zn saturation or oversaturation occurred relatively early. This suggested that epidermal layers serve as a large storage site for the excess metals, remaining unsaturated during 100 μm Zn exposure for at least 1 month, perhaps protecting leaf mesophyll and guard cells against Zn toxicity. The greater sequestration of Zn in the upper rather than the lower epidermis may also represent a strategy for protecting the stomatal apparatus. However, the contribution of other tissues in Zn accumulation in HE shoots should not be underestimated for the following reasons. Regardless of Zn exposure time, the metal count number in most other stem tissues was more than 20-fold higher in HE than in NHE, and Zn in cortex cells increased largely with time, suggesting that it could be an alternative to the epidermis as a Zn storage site in stems. Zn in HE leaf mesophyll cells was also much higher than in the NHE mesophyll, especially when plants were treated with 100 μm for 7 d. In another Zn hyperaccumulator, T. caerulescens, the mesophyll cells were also reported to tolerate up to at least 60 mm Zn in their sap, while in most plant species mesophyll Zn is probably < 1mm (Küpper et al., 1999). Therefore, leaf mesophyll and stem cortex cells also seem to play an important role in coping with the accumulated metal, with different cell and tissue types representing a large percentage of the total leaf and stem volume accumulating relatively high concentrations of Zn. In addition to epidermal enrichment, accumulation of Zn within the vascular bundles of stems and leaves was observed in both HE and NHE. To the best of our knowledge, very few studies have reported obvious enrichment of Zn within the vascular bundles such as that reported here. The presence of metals within vascular systems is thought to be associated with a primarily xylem mode of transport and delivery of metals to the leaves (McNear et al., 2005). The distribution pattern of Zn in the HE S. alfredii stem tissues seems to imply that a considerable amount of Zn has been transported via the xylem vessels. Zn xylem translocation was reported to be fivefold greater in T. caerulescens than in the nonaccumulator T. arvense (Lasat et al., 1998). Our data show that a larger proportion of Zn was localized to the stem vascular bundles of the HE, especially when the plants were treated with Zn for a relatively short time (7 d). In contrast, Zn in stems of NHE seems to be more easily lost from the conducting tissues, presumably to the cortex. Zn concentration in the xylem sap of the HE plants was 12-fold higher than that of New Phytologist (2009) 182: The Authors (2009) Journal compilation New Phytologist (2009)

10 125 the NHE when 100 μm external Zn solutions were applied (Tian et al., unpublished). These observations further support the HE s greater ability, relative to that of the NHE, to transport excess Zn upward (Yang et al., 2005) so that it can be distributed to appropriate shoot accumulation sites. Küpper et al. (2001) indicated that in the stems of Ni-hyperaccumulating Alyssum species, there was a peak of Ni in the unknown boundary cells between the cortical parenchyma and the vascular cylinder, in addition to the preferential distribution of Ni into epidermal cells. It is noteworthy that a large amount of Zn was located in the HE phloem cells, displayed as a discrete circle giving fairly bright green fluorescence between parenchyma and central cylinders in stem tissues, suggesting that considerable Zn may be transported by phloem and redistributed into plant tissues. In this study, phosphorus distribution was positively correlated with Zn in HE stems and leaves, while there was no such relationship between P and Zn in the seedlings of the NHE, and the simultaneously denser Zn and S distributions were also observed in the stem vascular tissues and epidermis of the HE plants (Table 2, Fig. 7). These results suggested the possibility that P and S may play a significant role in the HE s Zn hyperaccumulation. However, in T. caerulescens, it was reported that there is no correlation between leaf concentrations of Zn and S or P (Küpper et al., 2000, 2004). These authors argued that the plants should maintain a low P concentration or physically separate Zn from P in different leaf cells or different subcellular compartments to avoid coprecipitation of P with Zn. Thus, a detailed investigation is necessary to provide a clearer understanding of possible coupled or separate roles for these elements in the hyperaccumulator S. alfredii. Interestingly, in contrast to P and S, a consistent reverse distribution of Ca with Zn was observed in both stems and leaves of HE (Table 2, Fig. 7), suggesting a possibility of replacement of Ca by Zn in the plants. At present, however, a detailed understanding of the mechanisms of Zn speciation and transport in shoots of HE is still lacking. Further studies are necessary for a better understanding of Zn accumulation in shoots of the S. alfredii hyperaccumulator. Acknowledgements This study was financially supported by the National Science Foundation of China (# ), and Program for Changjiang Scholars and Innovative Team in University (#IRT0536). Part of this work was carried out with the approval of the Photon Factory Program Advisory Committee, Japan (proposal no. 2006G134). The authors sincerely thank Hongfei Lü and Wenrong Chen from Zhejiang Normal University, Dario Cantu from the University of California, Davis, and Hulin Hao from Zhejiang University for their great help in the experiments. References Asemaneh T, Ghaderian SM, Crawford SA, Marshall AT, Baker AJM Cellular and subcellular compartmentation of Ni in the Eurasian serpentine plants Alyssum bracteatum, Alyssum murale (Brassicaceae) and Cleome heratensis (Capparaceae). Planta 225: Broadley MR, White PJ, Hammond JP, Zelko I, Lux A Zn in plants. New Phytologist 173: Brooks RR Geobotany and hyperaccumulators. In: Brooks RR, ed. Plants that hyperaccumulate heavy metals. New York, NY, USA: CAB International, Clemens S, Palmgren MG, Krämer U A long way ahead: understanding and engineering plant metal accumulation. Trends in Plant Science 7: Frey B, Keller C, Zierold K, Schulin R Distribution of Zn in functionally different leaf epidermal cells of the hyperaccumulator Thlaspi caerulescens. Plant, Cell & Environment 23: Gutierrez-Alcala G, Gortor C, Meyer AJ, Fricker M, Vega JM, Romero LC Glutathione biosynthesis in Arabidopsis trichome cells. Proceedings of the National Academy of Sciences, USA 97: Hall JL Cellular mechanisms for heavy metal detoxification and tolerance. Journal of Experimental Botany 53: Hanikenne M, Talke IN, Haydon MJ, Lanz C, Nolte A, Motte P, Kroymann J, Weigel D, Krümer U Evolution of metal hyperaccumulation required cis-regulatory changes and triplication of HMA4. Nature 453: Hokura A, Onuma R, Terada Y, Kitajima N, Abe T, Saito H, Oshida S, Nakai I Arsenic distribution and speciation in an arsenic hyperaccumulator fern by X-ray spectrometry utilizing a synchrotron radiation source. Journal of Analytical Atomic Spectrometry 21: Huang YY, Lu JX, He RG, Zhao LM, Wang ZG, He W, Zhang YX Study of human bone tumor slice by SRXRF microprobe. Nuclear Instruments and Methods in Physics A 467/468: Krämer U, Grime GW, Smith JAC, Hawes CR, Baker AJM Micro-PIXE as a technique for studying nickel localization in leaves of the hyperaccumulator plant Alyssum lesbiacum. Nuclear Instruments and Methods in Physics B 130: Krämer U, Pickering IJ, Prince RC, Raskin I, Salt DE Subcellular localization and speciation of nickel in hyperaccumulator and nonaccumulator Thlaspi species. Plant Physiology 122: Küpper H, Lombi E, Zhao FJ, McGrath SP Cellular compartmentation of cadmium and zinc in relation to other elements in the hyperaccumulator Arabidopsis halleri. Planta 221: Küpper H, Lombi E, Zhao FJ, Wieshammer G, McGrath SP Cellular compartmentation of nickel in the hyperaccumulator Alyssum berolonii and Thlaspi caerulescens. Journal of Experimental Botany 52: Küpper H, Mijovilovich A, Meyer-Klaucke W, Kroneck PMH Tissue- and age-dependent differences in the complexation of cadmium and zinc in the cadmium/zinc hyperaccumulator Thlaspi caerulescens (Ganges Ecotype) revealed by X-ray absorption spectroscopy. Plant Physiology 134: Küpper H, Zhao FJ, McGrath SP Cellular compartmentation of zinc in leaves of the hyperaccumulator Thlaspi caerulescens. Plant Physiology 119: Lasat MM, Baker AJM, Kochian LV Altered Zn compartmentation in the root symplasm and stimulated Zn absorption into the leaf as mechanisms involved in Zn hyperaccumulation in Thlaspi caerulescens. Plant Physiology 118: Li TQ, Yang XE, Yang JY, He ZL Zn accumulation and subcellular distribution in the Zn hyperaccumulator Sedum alfredii Hance. Pedosphere 16: Long XX, Yang XE, Ye ZQ, Ni WZ, Shi WY Difference of uptake and accumulation of zinc in four species of Sedum. Acta Botanica Sinica 44: The Authors (2009) New Phytologist (2009) 182: Journal compilation New Phytologist (2009)

11 126 Ma JF, Ueno DFJ, Zhao FJ, McGrath SP Subcellular localisation of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotype of Thlaspi caerulescens. Planta 220: Marschner H Mineral nutrition of higher plants, 2nd edn. San Diego, CA, USA: Academic Press. McGrath SP, Zhao FJ, Lombi E Phytoremediation of metals, metalloids, and radionuclides. Advance in Agronomy 75: McNear DH Jr, Peltier E, Everhart J, Chaney RL, Sutton S, Newville M, Rivers M, Sparks DL Application of quantitative fluorescence and absorption-edge computed microtomography to image metal compartmentalization in Alyssum murale. Environmental Science & Technology 39: Mesjasz-Przybylowicz J, Przybylowicz WJ, Prozesky VM, Pineda CA Quantitative micro-pixe comparison of elemental distribution in Ni-hyperaccumulating and nonaccumulating genotypes of Senecio coronatus. Nuclear Instruments and Methods in Physics B 130: Peuke AD, Rennenberg H Phytoremediation. European Molecular Biology Organization 6: Prasad MNV, Hagemeyer J Heavy metal stress in plants: from molecules to ecosystems. Berlin, Germany: Springer. Reeves RD, Baker AJM Metal-accumulating plants. In: Raskin I, Ensley BD, eds. Phytoremediation of toxic metals: using plants to clean up the environment. New York, NY, USA: John Wiley, Robinson BH, Lombi E, Zhao FJ, McGrath SP Uptake and distribution of nickel and other metals in the hyperaccumulator Berkheya coddii. New Phytologist 158: Shi JY, Chen YX, Huang YY, He W SRXRF microprobe as a technique for studying elements distribution in Elsholtzia splendens. Micron 35: Sinclair SA, Sarah M, Sherson SM, Jarvis R, Camakaris J, Cobbett CS The use of the zinc-fluorophore, Zinpyr-1, in the study of zinc homeostasis in Arabidopsis roots. New Phytologist 174: Yang XE, Li TQ, Long XX, Xiong XH, He ZH, Stoffella PJ Dynamics of zinc uptake and accumulation in the hyperaccumulating and nonhyperaccumulating ecotypes of Sedum alfredii Hance. Plant and Soil 284: Yang XE, Li TQ, Yang JC, He ZH, Lu LL, Meng FH Zinc compartmentation in root, transport into xylem, and absorption into leaf cells in the hyperaccumulating species of Sedum alfredii Hance. Planta 224: Yang XE, Long XX, Ni WZ, Fu CX Sedum alfredii H: a new Zn hyperaccumulating plant first found in China. Chinese Science Bulletin 47: Yang XE, Long XX, Ye HB, He ZL, Calvert DV, Stoffella PJ Cadmium tolerance and hyperaccumulation in a new Zn-hyperaccumulating plant species (Sedum alfredii H.). Plant and Soil 259: Zhao FJ, Lombi E, Breedon T, McGrath SP Zinc hyperaccumulation and cellular distribution in Arabidopsis halleri. Plant, Cell & Environment 23: Supporting Information Additional supporting information may be found in the online version of this article. Fig. S1 Elemental (K, Ca, P, Fe, Cu, Mn, S, Cl) distributions in stem and leaf cross-sections of hyperaccumulating (HE) and nonhyperaccumulating ecotypes (NHE) of S. alfredii measured by SRXRF microprobe (unit: counts per 200s). Fig. S2 Correlations between count numbers of elements (K, Ca, P, Fe, Cu, Mn, Zn) in scanning points of the stem or leaf cross-sections, as measured by SRXRF, and concentrations of the elements in the corresponding stems and leaves of hyperaccumulating (HE) and nonhyperaccumulating ecotypes (NHE) of S. alfredii, as determined by ICP-MS. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. About New Phytologist New Phytologist is owned by a non-profit-making charitable trust dedicated to the promotion of plant science, facilitating projects from symposia to open access for our Tansley reviews. Complete information is available at Regular papers, Letters, reviews, Rapid reports and both Modelling/Theory and Methods papers are encouraged. We are committed to rapid processing, from online submission through to publication as-ready via Early View our average submission to decision time is just 29 days. Online-only colour is free, and essential print colour costs will be met if necessary. We also provide 25 offprints as well as a PDF for each article. For online summaries and ToC alerts, go to the website and click on Journal online. You can take out a personal subscription to the journal for a fraction of the institutional price. Rates start at 139 in Europe/$259 in the USA & Canada for the online edition (click on Subscribe at the website). If you have any questions, do get in touch with Central Office (newphytol@lancaster.ac.uk; tel ) or, for a local contact in North America, the US Office (newphytol@ornl.gov; tel ). New Phytologist (2009) 182: The Authors (2009) Journal compilation New Phytologist (2009)