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Application of glutathione to roots selectively inhibits cadmium transport from roots to shoots in oilseed rape

Glutathione is a tripeptide involved in various aspects of plant metabolism. This study investigated the effects of the reduced form of glutathione (GSH) applied to specific organs (source leaves, sink leaves, and roots) on cadmium (Cd) distribution and behaviour in the roots of oilseed rape plants (Brassica napus) cultured hydroponically. The translocation ratio of Cd from roots to shoots was significantly lower in plants that had root treatment of GSH than in control plants. GSH applied to roots reduced the Cd concentration in the symplast sap of root cells and inhibited root-to-shoot Cd translocation via xylem vessels significantly. GSH applied to roots also activated Cd efflux from root cells to the hydroponic solution. Inhibition of root-to-shoot translocation of Cd was visualized, and the activation of Cd efflux from root cells was also shown by using a positron-emitting tracer imaging system (PETIS). This study investigated a similar inhibitory effect on root-to-shoot translocation of Cd by the oxidized form of glutathione, GSSG. Inhibition of Cd accumulation by GSH was abolished by a low-temperature treatment. Root cells of plants exposed to GSH in the root zone had less Cd available for xylem loading by actively excluding Cd from the roots. Consequently, root-to-shoot translocation of Cd was suppressed and Cd accumulation in the shoot decreased.


Introduction
Cd is a toxic heavy metal that is released into the environment through human agricultural, industrial, and urban activities, resulting in Cd pollution of significant areas of farmland worldwide (McLaughlin et al., 1999). Cd causes serious human health problems when it enters the food chain (Obata and Umebayashi, 1997). To reduce Cd accumulation in crop plants, it is necessary to elucidate the mechanisms of Cd uptake and distribution. These mechanisms are not fully understood to date.
So far, various approaches have been followed to reduce Cd accumulation in crops. One of these attempts focuses on the removal of Cd from soils using plants, a technique called phytoremediation (Salt et al., 1998). However, it often takes many years to restore Cd-polluted soils by phytoremediation. It is necessary to establish novel, faster technologies to meet the demands of the market.
Previously, Nakamura et al. (2005) found that glutathione (GSH) concentrations in the phloem sap of oilseed rape plants increase under Cd treatment. GSH has a variety of physiological functions in removal of reactive oxygen species (Noctor and Foyer, 1998), heavy metal detoxification (Mendoza-Cózatl et al., 2005), and sugar metabolism (Ito et al., 2003). Additionally, GSH is a precursor of phytochelatins (PCs) (Rauser, 1995), which play important roles in vacuolar heavy metal sequestration (Salt and Rauser, 1995) and Cd transport from roots to shoots (Gong et al., 2003). These findings suggest that GSH might have a significant impact on the behaviour of Cd in plants. The Cd-dependent increase in GSH in the sieve tubes could be due to an increased requirement for GSH in sink tissues of Cd-treated plants. From this aspect, this study considered that GSH applied to specific parts of a plant might have significant effects on the behaviour of Cd throughout whole plants.
This study investigated the effects of applying GSH to roots, sink leaves, and source leaves of oilseed rape plants on Cd accumulation and Cd behaviour. Additionally, a positron-emitting tracer imaging system (PETIS) was employed to observe Cd movement in whole plants. PETIS enables the visualization of the transport and accumulation of positronemitters in the plant non invasively (Fujimaki, 2007). The present study is built on this study group's previous success in visualizing Cd movement in rice plants by monitoring 107 Cd as a positron emitter (Fujimaki et al., 2010). To demonstrate that glutathione is functioning via some physiological processes, but not by simple chemical binding between Cd and GSH, two experiments were performed. First, the effects of GSSG applied to roots on Cd accumulation in plants were investigated. Glutathione is generally known to have two different chemical forms: a reduced form (GSH) and an oxidized form (GSSG). GSSG lacks some ability to bind with Cd due to its molecular structure. Second, the effects of low-temperature treatment were examined. At low temperatures, only physiological processes should be suppressed in the roots.

Plant materials
Seeds of oilseed rape (Brassica napus L. cv. Nourin No. 16,Kaneko Seed,Gumma,Japan) were germinated in Petri dishes and then grown on floating nets in 1.5 l plastic containers with nutrient solution (modified Hoagland solution, described in Nakamura et al., 2008). After 1 week, seedling density was reduced to four seedlings per container. The containers were kept in a growth chamber at 24/18 °C 16/8 light/dark cycle (about 180 µmol m -2 s -1 ). Nutrient solutions were aerated continuously and renewed once a week. Four-week-old plants were treated with Cd and glutathione for 2 days (short-term) and 2 weeks (long-term). For the collection of xylem and phloem sap, plants were grown following the method of Nakamura et al. (2005).
Cd and glutathione treatment Plants were exposed to 0 or 10 µM Cd (CdCl 2 ) for 2 days (shortterm treatment) or 2 weeks (long-term treatment). GSH was applied to roots, source leaves, and sink leaves, while the plants were exposed to Cd. In the root treatment, 1 mM GSH and GSSG (final concentration) was added to the nutrient solution. In the leaf treatment, 20 mM GSH in a foliar application solution (10 mM MES-NaOH, pH 6.1, 0.01%, w/v, Triton X-100) was applied with a brush. The total amount of GSH applied to sink and source leaves was 2.5 µmol and 5.0 µmol, respectively, per application. GSH was applied to three sink or source leaves per plant, twice daily for 5 days before harvest. (Cd, Fe, Mn, and Zn) accumulation in plants After harvest, seedlings were divided into shoots and roots. The roots were washed in 0.1 M HNO 3 and deionized water, and were blotted with paper towels. Shoots and roots were dried at 105 °C for 24 h, weighed, and then digested in 6 ml of a 5:1 (v/v) mixture of HNO 3 and H 2 O 2 in a microwave oven (ETHOS1600, Milestone, Italy). Heavy metal concentrations in the digested solution were measured by an inductively coupled plasma atomic emission spectrometer (ICP-AES, IRIS Duo, Nippon Jarrell-Ash, Japan), and the heavy metal content of shoots and roots were calculated. The Cd translocation ratio indicates the ratio of Cd translocated to shoots compared to the Cd absorbed by plants.

Measurement of heavy metal
Collection of symplast sap, xylem sap, and phloem sap from oilseed rape plants and measurement of heavy metal concentrations in these saps After Cd and GSH treatment, root cell sap (symplast sap) was collected by centrifugation, following the method of Yu et al. (1999). Heavy metal concentrations (Cd, Fe, Mn, and Zn) in these cell saps were measured by an atomic absorption photometer (AA-6100, Shimadzu, Japan). After Cd and GSH treatment, xylem sap and phloem sap were collected following the method of Nakamura et al. (phloem sap 2005;xylem sap 2008). Heavy metal concentrations in xylem sap and phloem sap were also measured in the manner described above.

Effects of GSH on Cd efflux from root cells
Three-week-old oilseed rape plants were transferred to 1.5 l plastic containers (four plants per container) with a nutrient solution plus 10 µM CdCl 2 . After 24 h Cd treatment, plant roots were rinsed briefly in deionized water, followed by a desorption of apoplastic Cd in a desorbing solution containing 5 mM MES-KOH (pH 6.0), 1 mM K 2 HPO 4 , and 0.5 mM Ca(NO 3 ) 2 for 10 min. Then each plant was transferred to a light-shielding bottle with 0.6 ml nutrient solution for 24 h. After 24 h treatment, each nutrient solution was collected and root freshweights (FW) were measured. Cd concentration in solution was determined by an atomic absorption photometer (AA280Z, Varian, USA) and Cd efflux from roots was calculated.
HPLC analysis of GSH and phytochelatins (PC 2 ,PC 3 ,and PC 4 ) in the root tissues Two-week-old oilseed rape plants were treated with 10 µM CdCl 2, 1 mM GSH and 1 mM GSSG. After 2 days, plant roots were rinsed briefly in deionized water and a desorbing solution, as described above, for 10 min. Then root FWs were measured. About 0.3 g each sample was ground in liquid nitrogen using a mortar and pestle. GSH and PCs were extracted using 1.5 ml extract solution containing 1.5 mM diethylenetriamine pentaacetic acid and 1.5 mM Na 2 S 2 O 3 in 5.0% (w/v) 5-sulphosalicylic acid. The supernatant was collected after centrifugation at 15,000 g for 10 min at 4 °C. GSH and PC concentration in the supernatants were measured immediately using HPLC system (Class 10 vp system, Shimadzu, Kyoto, Japan). GSH and PCs were separated on a C18 column (LiChrospher 100 RP-18 e, 250 × 4 mm, particle size 5 µm, Merck Millipore, Darmstadt, Germany) using the method of de Knecht et al. (1994) with some modifications. The eluent was derivatized with 75 µM 5,5'-dithiobis(2-nitrobenzoic acid) in 50 mM potassium phosphate buffer (pH 7.6), which was added at a flow rate of 0.75 ml min -1 . Derivatized materials were detected with an ultraviolet detector (wavelength 412 nm). Identification of GSH and PCs was achieved by comparison of retention times with authentic standards. GSH was purchased from Wako Pure Chemical Industries (Osaka, Japan). Phytochelatin standards (PC 2 , PC 3 , and PC 4 ) were purchased from Bonac Corporation (Fukuoka, Japan).

107
Cd, a positron emitter with a half-life of 6.5 h, was produced as previously described (Ishioka et al., 2006;Fujimaki et al. 2010). Purified 107 Cd was dissolved in an appropriate volume of 0.5 mM CaCl 2 with or without 1 mM glutathione (GSH or GSSG). In these PETIS experiments, 2-week-old seedlings, grown hydroponically in a growth chamber under controlled growth conditions, were used. Test plants were set in an acrylic root box which was devised for PETIS experiments (Ishikawa et al., 2011). PETIS experiments were started by adding a total of 10 µM Cd including 107 Cd to 0.5 mM CaCl 2 solution. Images of the 107 Cd distribution were obtained every 4 minutes for 36 h.

Low-temperature treatment experiment
Four-week-old seedlings, cultivated hydroponically, were used in these experiments. Seedlings were pretreated in 0.5 mM CaC1 2 for 24 h before Cd treatment. The seedlings were treated with 10 µM Cd in 0.5 mM CaCl 2 or 10 µM Cd + 1 mM GSH in 0.5 mM CaCl 2 for 2 h at 2 or 24 °C. After harvest, the Cd content in shoots and roots was measured following the method described above.

Statistical analysis
Analytical data were analysed by Student's t-test at a significance level of 0.05.

Effects of GSH applied to different organs on Cd accumulation
To examine the effects of GSH applied to different organs on Cd accumulation, Cd content of oilseed rape plants were measured after 2 days (short-term treatment) and 2 weeks (long-term treatment) of Cd exposure. At the time of harvest, plants that had undergone the short-term treatment showed no obvious symptoms or changes in dryweight (DW) ( Supplementary Fig. S1, available at JXB online). However, plants treated with cadmium but without GSH for 2 weeks developed chlorosis and had a significant decrease in shoot dryweight ( Supplementary Fig. S1). In the short-term treatment, the Cd contents in shoots were about 0.4 µmol (g DW) -1 both in control plants and plants treated with GSH onto their leaves (Fig. 1A). In contrast, Cd content was roughly 4-fold lower in plants exposed to GSH in the root zone (Fig. 1A). In the long-term treatment, the Cd content in the shoots of control plants and plants that had received GSH on their leaves was about 1.2 and about 1.4 µmol (g DW) -1 , respectively (Fig. 1A). The shoot Cd content of plants exposed to GSH in the root zone was also roughly 4-fold lower (Fig. 1A). However, there was no significant difference in the Cd content of the roots in short-term and long-term treatments (Fig. 1B). Cd translocation ratios were calculated from these experimental results. The Cd translocation ratio was about 10% when GSH was applied to plant roots (Fig. 1C). This ratio was significantly lower than that of control plants and plants to which GSH was applied to their leaves (Fig. 1C). In the long-term treatment, these ratios from plants in which GSH was applied to leaves were about 1.5-fold higher (Fig. 1C). In these experiments, the Fe, Mn, and Zn contents of plants harvested after treatments were also measured ( Supplementary  Fig. S2). Compared with non-treated plants, the Fe, Mn, and Zn contents in shoots and roots of plants that had received GSH in the root zone were unaffected by the short-term Cd treatment. Compared with non-treated plants, Fe and Zn contents in the shoots of plants that were treated with GSH increased significantly under long-term Cd treatment. Compared with non-treated plants, the Fe, Mn, and Zn contents in roots of plants exposed to GSH also increased significantly under the long-term treatment.

Effects of GSH in the root zone on Cd behaviour
To understand more in detail the effects of GSH in the root zone on Cd behaviour throughout the entire plant, the Cd distribution in the root was investigated. In short-term treatment experiments, Cd concentration in the symplast sap collected from control plants was about 120 µM, whereas that from GSH-treated plants was reduced to about 30 µM ( Fig. 2A). A similar trend was observed in the long-term treatment ( Fig. 2A). In contrast, Fe, Mn, and Zn concentrations in the symplast sap did not differ significantly by these treatments (Supplementary Fig. S3). To examine the longdistance transport of Cd throughout the plant, xylem and phloem saps were collected from the Cd-treated plants as well as the Cd plus GSH-treated plants. The Cd concentration in the xylem sap from control plants was about 2.0 µM; this concentration was reduced to approximately one-half in GSH-treated plants (Fig. 2B). The concentrations of Fe, Mn, and Zn in the xylem sap were not affected by the GSH treatment ( Supplementary Fig. S3). Similarly, Cd concentration in the phloem sap decreased significantly in GSH-treated plants (Fig. 2C), whereas the levels of Fe, Mn, and Zn mostly remained constant; only Fe decreased somewhat after shortterm Cd treatment ( Supplementary Fig. S3).

HPLC analysis of GSH and PCs (PC 2 , PC 3 , and PC 4 ) in root tissues
GSH and PCs have effects on the radial transport of Cd in root tissues of plants (Sanità di Toppi and Gabbrielli, 1999). Therefore, the GSH and PC contents in root tissues were determined to investigate the effects of glutathione treatment. GSH and PC levels in root tissues are shown in Table 1. The GSH content was about 0.16 µmol (g FW) -1 in control plants. The GSH content in root tissues increased slightly in response to Cd and glutathione treatment. Biosynthesis of PCs was induced by Cd treatment. PC 2 , PC 3 , and PC 4 contents in root tissues were about 0.18, 0.27, and 0.18 µmol (g FW) -1 , respectively. However, glutathione treatment did not increase the PC content in root tissues; the PC content was slightly lower, but not significantly, when GSH and GSSG were applied to the roots.

Effect of GSH on Cd efflux from root cells
The effects of GSH in the root zone on Cd efflux from root cells were investigated (Fig. 3). Cd efflux from roots of control plants was about 120 nmol (g FW) -1 (Fig. 3). Meanwhile, that from GSH-treated plants was about 150 nmol (g FW) -1 (Fig. 3). Cd efflux from roots was activated significantly by GSH in the root zone.

Analysis of 107 Cd absorption, transport, and accumulation by PETIS
Cd movement in plants was analysed by PETIS. Fig. 4A shows a typical field of view; a representative time series of PETIS images is presented in Fig. 4B. It should be noted that all the 107 Cd values and intensities in the graphs and images from PETIS experiments in this paper have already been decay-corrected. That is, the figures directly indicate dynamics of the non-radioactive Cd. 107 Cd signals appeared in the shoot base region at 6 h after the addition of Cd to the root medium and accumulated gradually in control plants and GSSG-treated plants ( Fig. 4B; plants on the left and right in each image). In contrast, no 107 Cd signal appeared at 6 h in the shoots of plants additionally exposed to GSH ( Fig. 4B; plants on the centre in each image). In GSH-treated plants, 107 Cd appeared with a delay compared to control plants and plants exposed to GSSG, and the signal remained weaker throughout the period of observation (Fig. 4B). In GSSGtreated plants, 107 Cd signal also remained weaker than that of control plants and stronger than that of GSH-treated plants (Fig. 4B). In shoots of control plants and strong 107 Cd signals appeared at the node where petioles occurred. Animation movies of these images are also available in Supplementary Fig. S4. Fig. 5A C, and E show the regions of interest (ROI) to be analysed. Time courses of 107 Cd signals were established in the shoot (ROI-1), root (ROI-2) and solution (ROI-3) (Fig. 5B, D, F). In ROI-1 (plant shoots), 107 Cd signals in each plant increased gradually until about 10 h after 107 Cd feeding (Fig. 5B). After that, 107 Cd signals in the shoots of control plants increased more sharply than those in  Table 1. Contents of GSH and phytochelatins (PC 2 , PC 3 , and PC 4 ) in roots of oil seed rape plants exposed to Cd, GSH, and GSSG. Two-week-old seedlings were transferred to each treatment solution for 2 days. The GSH and PC contents of roots were calculated from results of HPLC analysis and their root freshweights as µmol (g FW) -1 . Data are means ± SE (n = 4). -, under detection limit. glutathione-treated plants (Fig. 5B). In ROI-2 (plant roots), 107 Cd accumulated following its addition to the root medium, but less rapidly with GSSG treatment than the control (Fig. 5D). There was no difference in the pattern of 107 Cd accumulation in control plant roots and GSH-treated plant roots (Fig. 5C). In these roots, 107 Cd signal increased continuously about 12 h after Cd feeding. Afterwards, 107 Cd signal reached equilibrium (Fig. 5D). In ROI-3 (0.5 mM CaCl 2 solution without or with GSSG), there was a significant difference in the pattern of 107 Cd decrease in the solution. In control plants and GSSG-treated plants, 107 Cd signal in the solution decreased continuously (Fig. 5F). However, 107 Cd signal in the solution with 1 mM GSH shifted to increase at 12-18 h and reached equilibrium after the initial decrease (Fig. 5F).

Effect of GSSG applied to roots on Cd accumulation
To examine the effects of GSSG applied to roots on Cd accumulation, the Cd contents were also measured in shoots and roots after 2 days (short-term treatment) and 2 weeks (longterm treatment) of Cd exposure. There were no differences in the dryweights of shoots and roots between GSH-treated plants and GSSG-treated plants (data not shown). In the short-term treatment, the Cd content in the shoots of GSHtreated plants was about 0.16 µmol (g DW) -1 (Fig. 6A). The shoot Cd content of plants exposed to GSSG in the root zone was roughly 2-fold higher (Fig. 6A). In the long-term treatment, the Cd content in the shoots of GSH-and GSSGtreated plants was about 0.40 and 0.33 µmol (g DW) -1 , respectively (Fig. 6A). Compared to control plants, the Cd contents in shoots decreased significantly after glutathione treatments (Fig. 6A). There was no significant difference in the root Cd content in GSH-treated plants and GSSG-treated plants in short-term and long-term treatments (Fig. 6B). In the shortterm treatment, Cd translocation ratios from GSSG-treated plants were a little higher, but not different significantly (Fig. 6C); however, in the long-term treatment, these ratios were nearly the same (Fig. 6C). No inhibitory effects of GSSG on the Fe, Mn, and Zn contents in shoots and roots were found in these experiments (data not shown).

Effects of GSH in the root zone on the Cd distribution at different temperatures
To investigate the effects of chemical interaction between Cd and GSH on Cd accumulation in plants in more detail, plants were treated with Cd and GSH at a low temperature. The shoot Cd content of control plants and GSH-treated plants at 24 °C was about 27 and 10 nmol (g DW) -1 , respectively (Fig. 7A).  All plants are exposed to 107 Cd in the root medium; plants on the centre and right were exposed additionally to GSH and GSSG, respectively.
There was no significant difference in the Cd content in shoots of control plants and GSH-treated plants at 2 °C (Fig. 7B). The root Cd content of control plants was significantly higher than that of GSH-treated plants at 2 and 24 °C (Fig. 7B). At 24 °C, Cd translocation ratios from the control plants were roughly 2-fold higher (Fig. 7C); however, at 2 °C, there was no  difference in these ratios (Fig. 7C). These results indicated that the inhibitory effects of Cd accumulation in the shoots were abolished by low-temperature treatments.

GSH applied to roots inhibits Cd translocation and accumulation
GSH applied to roots inhibited Cd translocation from roots to shoots and Cd accumulation in the shoot (Fig. 1A). On the other hand, GSH applied to leaves did not reduce Cd accumulation in shoots (Fig. 1A). On the contrary, Cd accumulation in the shoot in the long-term Cd treatment was slightly promoted by GSH application to leaves (Fig. 1A). Unlike the experimental results of Cd accumulation in shoots, GSH had no effect on Cd accumulation in roots, regardless of the type of application (Fig. 1B). GSH applied to roots had a significant effect on Cd translocation (Fig. 1C). From these experimental results it is concluded that GSH present in the root zone affects Cd translocation from roots to shoots.

Effects of GSH on Cd behaviour in plants
The Cd concentration in the symplast sap decreased significantly when GSH was present ( Fig. 2A). In these experiments, the Cd concentration in the xylem sap was also reduced significantly by GSH treatment (Fig. 2B), suggesting that the amount of Cd loaded into the xylem decreased, thereby reducing Cd accumulation in the shoots of GSHtreated plants. Cytosolic Cd concentrations in roots have a large effect on xylem loading of Cd (Uraguchi et al., 2009;Ishikawa et al., 2011;Ueno et al., 2011). GSH applied to roots might activate some processes in which the intracellular Cd concentration decreases in roots. The Cd concentration in the phloem sap showed a similar trend as that in the xylem sap (Fig. 2C). Thus, it is unlikely that the reduced Cd levels in the shoot observed in this study are due to Cd export from shoots via the phloem. How is GSH functioning to reduce the amount of Cd in the cytosol of root cells? Currently, two possibilities are seen. (1) Activation of Cd extrusion from root cells. ABC transporters can act as Cd extrusion pumps (Kim et al., 2007). On the basis of in vitro experiments, thiol compounds have been proposed to activate Cd efflux systems in cucumber root cells (Migocka et al., 2011), and it is conceivable that GSH might activate Cd extrusion in roots of intact oilseed rape plants.
(2) GSH could promote the sequestration of Cd in the vacuoles of root cells. Cd is detoxified by accumulation and storage in vacuoles (Mendoza-Cózatl et al., 2011). PC, which is synthesized from GSH, is involved in the detoxification process. GSH also may be directly involved in the sequestration of Cd (Li et al., 1997). To determine which of the two possible mechanisms are operative in living plants, this study tried to evaluate Cd efflux from and PC content in root cells.

Effect of GSH on Cd efflux from root cells
Cd efflux from root cells was significantly activated by GSH applied to roots (Fig. 3). In the brassica family, cation efflux transporters have been reported (Xu et al., 2009;Lang et al., 2011). Further studies are needed to identify and characterize Cd efflux transporters that are activated by GSH in the roots of oilseed rape plants.

GSH and PCs in root tissues
To investigate the effects of GSH and PCs on Cd radial transport, the GSH and PC contents in root tissues were measured. The experimental results indicated that the GSH content of root tissues increased slightly by glutathione treatment (Table 1). Synthesis of PCs was induced by Cd treatment. In contrast to the result with the GSH treatment, the PC content of root tissues decreased slightly by glutathione treatment (Table 1). That is, glutathione, applied to roots, does not seem to have any effect on increasing the PC content of root tissues. PCs are related to Cd translocation from roots to shoots (Gong et al., 2003). Therefore, a decrease in the PC levels in root tissues might trigger the inhibition of Cd translocation. A PC transporter, localized in the tonoplast membrane, was recently identified (Mendoza-Cózatl et al., 2010). There remains a possibility that sequestration of PCs into vacuoles is activated by GSH, exceeding the biosynthesis of PCs. It will be necessary to investigate the distribution of Cd, GSH, and PCs in root cells in detail in order to elucidate the molecular mechanisms for the inhibition of Cd translocation from roots to shoots.

Dynamic analysis of Cd translocation to shoots in intact plants
The PETIS method enabled visualization of Cd transport and accumulation in whole plants. The inhibitory effect of GSH treatment on Cd translocation from roots to shoots was clearly observed and provided hints at the effects of GSH on these processes (Fig. 4B). The 107 Cd signal accumulated in the nodes of oilseed rape plants (Fig. 4B). 107 Cd also accumulated significantly in the shoot bases of rice plants where nodes are assembled (Fujimaki et al., 2010). Therefore, it seems that the nodes, from which vascular tissues branch, play an important role in Cd distribution throughout the entire plant. During the initial 10 hours after 107 Cd feeding, there were no significant differences in the pattern of decrease of 107 Cd signals in the culture solution of control plants and however,107 Cd signals in the culture solution stopped decreasing and shifted to increasing only in the GSH-treated plants after that (Fig. 5F). Afterwards, the 107 Cd signal in this solution reached an equilibrium (Fig. 5F). These experimental results supported the notion that Cd extrusion from root cells was activated by GSH. In GSH-treated plants, a response time of about 10 hours was required to change the pattern of 107 Cd accumulation (Fig. 5B, D, F). This long response time supported the hypothesis that some physiological responses to GSH must occur in the roots or shoots of oilseed rape plants. These responses might have a special effect on Cd radial transport in roots of oilseed rape plants.

Effects of chemical binding between Cd and GSH on Cd behaviour in roots
GSH is known to bind Cd directly and form bis(glutathionato) cadmium (Cd-GS 2 ) (Nocito et al., 2007). This Cd-GS 2 complex strongly reduces Cd bioavailability. To verify that the reduced Cd accumulation in shoots was caused mainly by physiological functions of GSH and not by chemical binding between GSH and Cd, two experiments were performed. The first was an experiment using GSSG, which is formed by oxidizing GSH. Under these experimental conditions (oxidative conditions), GSSG in hydroponic solution is present without binding Cd. GSSG lacks the ability to bind Cd because GSSG lacks free thiol groups. However, GSSG applied to roots also had an inhibitory effect on Cd accumulation in the shoots (Fig. 6A). Additionally, the effects of GSSG increased as the treatment periods became longer (Fig. 6A). These results demonstrated that glutathione applied to roots triggered some physiological process in plant roots. The results of short-term treatment experiments indicated that there were some differences in the effects of GSH and GSSG on Cd behaviour (Fig. 6A). These differences were also confirmed by PETIS experiments. The 107 Cd accumulation pattern in roots differed depending on the presence of GSH or GSSG (Figs. 4 and 5). In future research, it will be necessary to elucidate how GSH and GSSG are metabolized and function in root cells. The second experiment was a low-temperature treatment experiment. In this experiment, a significant difference in the Cd translocation ratio observed at 24 °C disappeared at 2 °C (Fig. 7C). At 2 °C, only physiological processes in plant roots should be suppressed. These experimental results also indicated that GSH triggers some physiological processes. These experimental results demonstrated that inhibition of Cd translocation from roots to shoots is highly dependent on the physiological processes triggered by GSH rather than chemical binding between Cd and GSH.

Effects of GSH on other heavy metal behaviour in plants
ZIP family proteins, which are involved in Cd absorption by roots, are also related to the membrane transport of Fe, Mn, and Zn (Guerinot, 2000). Therefore, the present study examined the effects of GSH on the transport and accumulation of these heavy metals. Intriguingly, the presence of GSH in the root medium had no restrictive influence on the behaviour of other heavy metals in the plant ( Supplementary Fig. S2). Fe, Mn, and Zn concentrations in the symplast sap or xylem sap did not change with GSH treatment ( Supplementary  Fig. S3). These experimental results demonstrated that GSH, applied to roots, selectively inhibited Cd translocation from roots to shoots.
The work reported here demonstrated that GSH, applied to roots, activated Cd efflux from root cells to the outer culture and decreased the level of Cd in the cytosol of root cells, as well as Cd loading into the xylem. Consequently, the Cd content in the shoots selectively decreased about 4-fold lower than that of control plants. The future elucidation of the molecular mechanisms underlying Cd efflux from root cells will establish new agronomic practices without gene manipulation to realize farm products with low Cd contents.

Supplementary material
Supplementary data are available at JXB online. Supplementary Fig. S1. Dryweights of shoots and roots of oilseed rape plants harvested after various treatments. Supplementary Fig. S2. Fe, Mn, and Zn contents in shoots and roots from oilseed rape plants harvested after various treatments. Supplementary Fig. S3. Fe, Mn, and Zn concentrations in the symplast sap, xylem sap, and phloem sap collected from oilseed rape plants after various treatments. Supplementary Fig. S4. Animation movie of transport and accumulation of Cd in oilseed rape plants.