Zinc adsorption and desorption characteristics in root cell wall involving zinc hyperaccumulation in Sedum alfredii Hance

Zinc adsorption and desorption characteristics in root cell wall involving zinc hyperaccumulation in Sedum alfredii Hance first-author †E-mail:litq76@163.com;xyang@zju.edu.cn †E-mail:litq76@163.com;xyang@zju.edu.cn litq76@163.com corresp ‡Corresponding Author ‡Corresponding Author †E-mail:litq76@163.com;xyang@zju.edu.cn †E-mail:litq76@163.com;xyang@zju.edu.cn litq76@163.com Ministry of Education Key Laboratory of Environmental Remediation and Ecosystem Health, Zhejiang University, Hangzhou 310029, China Ministry of Education Key Laboratory of Environmental Remediation and Ecosystem Health, Zhejiang University, Hangzhou 310029, China Radiotracer techniques were employed to characterize 65 Zn adsorption and desorption in root-cell-wall of hyperaccumulating ecotype (HE) and non-hyperaccumulating ecotype (NHE) species of Sedum alfredii Hance. The results indicated that at the end of a 30 min short time radioisotope loading period, comparable amounts of 65 Zn were accumulated in the roots of the two ecotypes Sedum alfredii , whereas 2.1-fold more 65 Zn remains in NHE root after 45-min desorption. At the end of 60 min uptake period, no difference of 65 Zn accumulation was observed in undesorbed root-cell-wall of Sedum alfredii . However, 3.0-fold more 65 Zn accumulated in desorbed root-cell-wall of NHE. Zn 2+ binding in root-cell-wall preparations of NHE was greater than that in HE under high Zn 2+ concentration. All these results suggested that root-cell-wall of the two ecotypes Sedum alfredii had the same ability to adsorb Zn 2+ , whereas the desorption characteristics were different, and with most of 65 Zn binding on root of HE being available for loading into the xylem, as a result, more 65 Zn was translocated to the shoot. Adsorption Desorption Hyperaccumulator Sedum alfredii Hance Zn INTRODUCTION 1 Phytoremediation has emerged as an alternative technique for removing toxic metals from soil and offers the benefits of being in situ, cost-effective and environmentally sustainable (Raskin et al., 1997 1997 ; McGrath and Zhao, 2003 2003 ; Santos et al., 2006 2006 ; Bañuelos, 2006 2006 ). Plants with high metal uptake and accumulation capacity have been termed as hyperaccumulator species. An understanding of the physiological mechanism of metal tolerance and hyperaccumulation is very important for exploring new metal hyperaccumulating plant species that are tolerant to high levels of metal, have large biomass, extensive adaptation, and easy propagation (Ebbs and Kochian, 1997 1997 ; Salt et al., 1998 1998 ; Yang et al., 2005 2005 ). Sedum alfredii has been identified as a new Zn-hyperaccumulator plant native to China (Yang et al., 2002 2002 ). However, the mechanisms responsible for its Zn hyperaccumulation remain unknown. Root system is the main interface of material exchange between plants and their environment, and plays an important role in metal uptake and transport in plant (Marschner, 1995 1995 ; Fritioff and Greger, 2006 2006 ). In a previous study we reported that at the end of a 24-h radioisotope-loading period, comparable amounts of 65 Zn were accumulated in the roots of the two ecotypes of S . alfredii . However, after a long-term (48 h) efflux experiment, approximately 6.8-fold more 65 Zn was translocated from the root to the shoot of hyperaccumulating ecotype (HE), as compared with non-hyperaccumulating ecotype (NHE). However, during the same period, 3.7-fold more 65 Zn remained in NHE roots (Li et al., 2005 2005 ; Yang et al., 2006 2006 ). In both ecotypes roots, 65 Zn mainly located in the root cell wall (56%~64%) which had metal-sequestering properties (Volesky and Holan, 1995 1995 ). Thus we hypothesized that the difference of Zn biosorption on root-cell-wall may play an important role in controlling the difference of Zn translocation and storage in the shoot. In this study radiotracer techniques were employed to investigate the characteristics of Zn adsorption and desorption in the root of S . alfredii using hydroponically grown seedlings. The results should be helpful in elucidating the Zn uptake and transport mechanisms of this Zn hyperaccumulating S . alfredii . MATERIALS AND METHODS 1 Plant origins and culture 2 Plants were cultivated according to Li et al.( 2005 2005 ). Twenty-day old seedlings were used in all radiotracer studies. One day prior to the uptake experiment, seedlings roots were immersed in aerated pretreatment solution consisting of 2 mmol/L Mes-Tris buffer (pH 6.0) and 0.5 mmol/L CaCl 2 . 65 Zn desorption from roots of S . alfredii 2 Roots of HE and NHE S . alfredii were immersed in 3 L of aerated uptake solution containing 2 mmol/L Mes-Tris (pH 6.0), 0.5 mmol/L CaCl 2 , and 10 μmol/L 65 Zn (45 kBq/L). After 30 min, the radioactive uptake solution was replaced with 2 °C solution containing 5 mmol/L Mes-Tris (pH 6.0), 5 mmol/L CaCl 2 , and 100 μmol/L ZnCl 2 . At different times (0~45 min), seedlings were harvested and the roots were then blotted to remove adhering solution and the excised roots were oven-dried at 65 °C and weighed, and root radioactivity of 65 Zn was measured using a gamma detector (GR2519, Canberra Co., USA). The complete experiment was repeated four times. 65 Zn accumulation in roots of S . alfredii 2 Roots of HE and NHE S . alfredii were immersed in 3 L of aerated uptake solution containing 2 mmol/L Mes-Tris (pH 6.0), 0.5 mmol/L CaCl 2 , and 10 µmol/L 65 Zn 2+ (45 kBq/L). At different times (0~60 min), one plant of each ecotype was harvested and the roots were briefly rinsed in deionized water or/and desorbed for 20 min in 2 °C solution containing 5 mmol/L Mes-Tris (pH 6.0), 5 mmol/L CaCl 2 , 100 µmol/L ZnCl 2 and then separated from shoots. The excised roots were blotted, roots and shoots were oven-dried at 65 °C and weighed, and 65 Zn radioactivity was quantified. The complete experiment was repeated four times. 65 Zn adsorption in root-cell-wall preparations of S . alfredii 2 These root-cell-wall preparations were obtained by Hart’s method (Hart et al., 1992 1992 ) with some modification. Roots of intact HE and NHE S . alfredii were immersed in an MC (methanol/chloroform=2:1, v/v) solution for 3 d, then the root-cell-wall preparations were washed in a series of deionized water for 8 h. Roots of either intact or MC-treated seedlings were incubated in 1 L solution containing 2 mmol/L Mes-Tris (pH 6.0), 0.5 mmol/L CaCl 2 , and 10 μmol/L 65 Zn (45 kBq/L) for time periods between 1 and 45 min, and then either briefly rinsed in deionized water (undesorbed) or desorbed in 1 L of desorption solution for 20 min. Subsequently, root-cell-wall preparations were excised, blotted, oven-dried at 65 °C and weighed, and 65 Zn radioactivity was quantified. The complete experiment was repeated four times. Concentration-dependent kinetics of 65 Zn binding in root-cell-wall preparations of S . alfredii 2 MC-treated roots were incubated in 500 ml radiolabeled uptake solution (45 kBq/L 65 Zn, 0.5 mmol/L CaCl 2 , and 2 mmol/L Mes-Tris, pH 6.0) at concentrations of 1, 2.5, 5, 10, 25, 50, 100, 200 µmol/L Zn 2+ in Plexiglas uptake apparatus. After a 30-min uptake, uptake wells were refilled with 2 °C desorption solution. Following a 20-min desorption period, seedlings were harvested and their roots excised, blotted, oven-dried at 65 °C and weighed, and 65 Zn radioactivity was quantified. The experiment was randomly arranged with each treatment replicated for four times. RESULTS 1 Time course of 65 Zn desorption from roots of S . alfredii 2 At the end of a 30 min short time radioisotope-loading period, comparable amounts of 65 Zn were accumulated in the roots of the two S . alfredii ecotypes. The desorption curves were similar for the two S . alfredii ecotypes (Fig. 1 1 ). At the end of a 45-min desorption period in a solution containing high levels of Zn 2+ (100 µmol/L) and Ca 2+ (5 mmol/L), 87.1% and 72.9% of the 65 Zn accumulated in HE and NHE S . alfredii root during the uptake period was desorbed into the external solution respectively. 65 Zn release into the external solution was initially very rapid, and followed by a slower stage of desorption. There were 2.1-fold more 65 Zn remaining in NHE root after 45-min desorption, as compared with HE. However, during the 45-min desorption, about 1.9-fold more 65 Zn was accumulated in the shoot of HE, as compared with NHE, these results indicated that the HE has an extraordinary ability to transport 65 Zn to the shoot. Time-course of 65 Zn accumulation in roots of S . alfredii 2 65 Zn accumulation in undesorbed root of both S . alfredii ecotypes was biphasic, with the initial 10 min rapid phase followed by slower linear stage of accumulation over the subsequent 50 min (Fig. 2 2 ). At the end of 60 min uptake period, no difference of 65 Zn accumulation was observed in root of S . alfredii . In desorbed roots, however, Zn 2+ accumulation was linear, and the slope of the curve was similar to that of the slower phase for 65 Zn accumulation in the undesorbed roots. At the end of 60-min uptake, 2.0-fold more 65 Zn accumulated in NHE root as compared with HE. Time course of 65 Zn adsorption in root-cell-wall preparations of S . alfredii 2 65 Zn accumulation in both desorbed and undesorbed root-cell-wall of both ecotypes was linear (Fig. 3 3 ). At the end of 60 min uptake period, no difference of 65 Zn accumulation was observed in undesorbed root-cell-wall of both S . alfredii ecotypes. In desorbed root-cell-wall, however, 3.0-fold more 65 Zn accumulated in NHE as compared with HE at the end of 60-min uptake. Concentration-dependent kinetics of 65 Zn binding in root-cell-wall of S . alfredii 2 Concentration-dependent uptake kinetics for Zn 2+ in root-cell-wall preparations of S . alfredii was linear (Fig. 4 4 ). In both ecotypes root-cell-wall preparations, Zn 2+ accumulation increased with increasing Zn 2+ concentration. Zn 2+ binding in MC-treated roots of NHE was greater than that in HE under high Zn 2+ concentration ( > 100 µmol/L). DISCUSSION 1 Previous investigations showed that heavy metals bind to natural cellulosic materials (Bryant et al., 1992 1992 ; Shukla and Sakhardande, 1992 1992 ; Prasad and Freitas, 2000 2000 ). This phenomenon, known as biosorption, has attracted increasing research interests recently. Cell walls may represent a significant portion of the surface area exposed to both surface waters (Geesey et al., 1977 1977 ) and soil fluids (Mahmood and Rama, 1993 1993 ), the effects of cell walls on metal mobilities in these systems have been reported (de Lurdes et al., 1987 1987 ; Chen, 1997 1997 ; Zakir Hossain et al., 2006 2006 ), however the role in controlling metal translocation and storage in hyperaccumulator are still poorly understood. At the end of a 30 min short time radioisotope loading period, comparable amounts of 65 Zn were accumulated in the roots of the two S . alfredii ecotypes. However, at the end of a 45-min desorption period, there were 2.1-fold more 65 Zn remaining in NHE root (Fig. 1 1 ). In the time-course of 65 Zn accumulation in roots of S . alfredii , no difference of 65 Zn accumulation was observed in undesorbed root, whereas 2.0-fold more 65 Zn accumulated in desorbed root of NHE (Fig. 2 2 ). These results indicated that 65 Zn bind loosely to root of HE S . alfredii and could be desorbed easily. Desorbed Zn 2+ could have originated from the cell walls and/or from the cytosol via efflux across the plasma membrane back into the external solution. Radiotracer studies on Thlaspi caerulescens demonstrated that most Zn 2+ removed during desorption period originated from the cell wall (Lasat et al., 1996 1996 ). In this study, 65 Zn accumulation in undesorbed intact root of both S . alfredii ecotypes were biphasic (Fig. 2 2 ), the initial rapid phase presumably was due to diffusion into the free space and subsequent binding to root cell walls, the slower linear stage of accumulation over the subsequent 50 min was believed to represent transport across the plasma membrane. For root-cell-wall preparations, however, 65 Zn accumulation was monophasic and rapid, and was much higher in undesorbed root-cell-wall than that in undesorbed intact root, in contrast, less 65 Zn accumulated in desorbed MC-treated roots, compared with desorbed intact root (Fig. 3 3 ). These results support the hypothesis that in intact roots, most 65 Zn removed during desorption period originated from the cell wall. On the other hand, Zn 2+ accumulation in root-cell-wall preparations increased with increasing Zn 2+ concentration, and Zn 2+ binding in MC-treated roots of NHE was greater than that in HE under high Zn 2+ concentration (Fig. 4 4 ). These results suggested that the affinity of root cell wall of the two S . alfredii ecotypes for 65 Zn were different, and that most of this binding 65 Zn on root of HE are available for loading into the xylem, as a result, more 65 Zn was translocated to the shoot. 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Zinc compartmentation in root, transport into xylem, and absorption into leaf cells in the hyperaccumulating species of Sedum alfredii Hance AKM Zakir Hossain , H Koyama , T Hara . Growth and cell wall properties of two wheat cultivars differing in their sensitivity to aluminum stress . J Plant Physiol , 2006 . 163 ( 1 ): 39 - 47 . 16360802 DOI: 10.1016/j.jplph.2005.02.008 http://doi.org/10.1016/j.jplph.2005.02.008 . Growth and cell wall properties of two wheat cultivars differing in their sensitivity to aluminum stress 10.1631/jzus.2007.B0111 Project supported by the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT0536), the National Natural Science Foundation of China (No. 20277035), and the Postdoctoral Science Foundation of China (No. 2005038285) 2006-10-09 2006-12-26 2022-01-05 Copyright © 2007, Journal of Zhejiang University Science 2007 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 08