⁵²Mn translocation in barley monitored using a positron-emitting tracer imaging system

Abstract

Until now, the real-time uptake and movement of manganese (Mn), an essential plant nutrient, has not been documented in plants. In this study, the real-time translocation of Mn in barley (Hordeum vulgare L. cv. Ehimehadaka no. 1) was visualized using the positron-emitting tracer ⁵²Mn and a positron-emitting tracer imaging system (PETIS). PETIS allowed the non-destructive monitoring of Mn translocation in barley under various conditions. In all cases, ⁵²Mn first accumulated in the discrimination center (DC) at the basal portion of the shoot, suggesting that this region may play an important role in Mn distribution in graminaceous plants. Manganese-deficient barley showed greater translocation of ⁵²Mn from roots to shoots than did Mn-sufficient barley, demonstrating that Mn deficiency causes enhanced Mn uptake and loading into vascular bundles. In contrast, the translocation of ⁵²Mn from roots to shoots was suppressed in Mn-excess barley. In these plants, the uptake of Mn may be suppressed or Mn may accumulate in the intercellular organelles of root cells, resulting in low rates of Mn translocation to shoots. In Mn-sufficient barley, the dark treatment did not suppress the translocation of ⁵²Mn to the youngest leaf, suggesting that the translocation of Mn to the youngest leaf is independent of the transpiration stream. When ⁵²Mn was supplied to the cut end of an expanded leaf, ⁵²Mn was transported to the DC within 27 min and then retranslocated to roots and other leaves. Our results show that the translocation of Mn from the roots to the DC depends passively on water flow, but actively on the Mn transporter(s).

Key words:

• ⁵²Mn
• Mn deficiency
• Mn excess
• positron-emitting tracer imaging system
• translocation

INTRODUCTION

Manganese (Mn) is an essential nutrient for plants and is required for a number of important cellular processes, including oxygen evolution during photosynthesis, detoxification of oxygen free radicals and CO₂ fixation during C₄ and Crassulacean acid metabolism (CAM) (Marschner 1995). In soil, Mn is often present in forms that are unavailable to plants, particularly in high-pH soils, in which equilibrium conditions favor the oxidation of the plant-available Mn²⁺ to the unavailable Mn⁴⁺ (Welch et al. 1991). When grown in such soils, crops may become Mn-deficient, resulting in reductions in growth and yield. In acidic and volcanic soils, Mn is reduced and, therefore, crop Mn toxicity is observed in many natural and agricultural systems (Carver and Ownby 1995; Foy et al. 1978).

Manganese is taken up by plants mainly in the form of Mn²⁺ ions (Fox and Guerinot 1998). Several genes have been reported to encode metal transporters that are able to transport Mn²⁺, such as OsNramp1, OsNramp3 (Belouchi et al. 1997), AtNramp1, AtNramp3, AtNramp4 (Thomine et al. 2000), IRT1 (Eide et al. 1996; Korshunova et al. 1999; Rogers et al. 2000) and AtOPT3 (Wintz et al. 2003). However, little is known about the translocation of Mn in intact plants after its absorption from roots. It has been proposed that Mn is transported to shoots via the xylem by the transpiration stream and root pressure, and then unloaded from the xylem into older leaves (Fisher 2000).

Recently, positron-emitting nuclides have been used in plants to study the distribution and translocation of ¹⁸F (Kume et al. 1997), [¹¹C]methionine (Bughio et al. 2001; Nakanishi et al. 1999), ¹¹C (Fujikake et al. 2003; Matsuhashi et al. 2005), ¹³NO⁻ ₃ (Hayashi et al. 1997; Matsunami et al. 1999; Ohtake et al. 2001), ¹³N (Kiyomiya et al. 2001b), H¹⁵ ₂O (Kiyomiya et al. 2001a; Mori et al. 2000; Nakanishi et al. 2002; Tsukamoto et al. 2004) and ⁵²Fe, ⁵²Mn and ⁶²Zn (Watanabe et al. 2001) using a positron-emitting tracer imaging system (PETIS). With this technique, γ-rays produced by positron-emitting nuclides are detected by scintillation detectors, allowing the real-time investigation of the movement of elements in intact plants. We have also examined the transport of ⁵⁹Fe in barley (Hordeum vulgare L. cv. Ehimehadaka no. 1) using ⁵⁹Fe(III)-epihydroxymugineic acid (epiHMA) and autoradiography detection (Mori 1998). To compare the translocation of ⁵²Mn with the translocation of ⁵⁹Fe and other positron-emitting nuclides, we initiated a study using barley plants. We have already reported the production of the metallic positron-emitting nuclides ⁵²Mn, ⁵²Fe and ⁶²Zn and their potential use in PETIS (Watanabe et al. 2001), but no physiological studies of metal translocation in plants have been reported. In the present study, we used PETIS to monitor the real-time translocation of ⁵²Mn in intact barley plants under various conditions for the first time, and calculated the velocity of ⁵²Mn translocation.

MATERIALS AND METHODS

Plant materials

Barley (Hordeum vulgare L. cv. Ehimehadaka no. 1) seeds were grown hydroponically in modified Kasugai's medium: 0.7 mmol L⁻¹ K₂SO₄, 0.1 mmol L⁻¹ KCl, 0.1 mmol L⁻¹ KH₂PO₄, 2.0 mmol L⁻¹ Ca(NO₃)₂, 0.5 mmol L⁻¹ MgSO₄, 10 µmol L⁻¹ H₃BO₃, 0.5 µmol L⁻¹ MnSO₄, 0.2 µmol L⁻¹ CuSO₄, 0.5 µmol L⁻¹ ZnSO₄, 0.01 µmol L⁻¹ (NH₄)Mo₆O₂₄, and 0.1 µmol L⁻¹ Fe-EDTA as described previously (Mori and Nishizawa 1987). Manganese-deficient plants were generated by culturing in a medium lacking Mn for 2 weeks prior to beginning the ⁵²Mn-absorption experiments. Manganese-excess plants were produced by culturing in medium containing 100 µmol L⁻¹ Mn for 1 week before beginning the absorption experiments. All absorption experiments were conducted approximately 3 weeks after germination, and the sizes and root weights of plants of both types were almost the same.

Production of ⁵²Mn

⁵²Mn (half-life 5.59 days) was produced by the ^natCr(p, xn)⁵²Mn reaction, in which a 1.5-mm-thick chromium foil (natural isotopic composition, 99.9% purity; Goodfellow Metals, Cambridge, UK) is bombarded with a 20-MeV proton beam (generated by the Takasaki Ion Accelerators for Advanced Radiation Application [TIARA] AVF cyclotron; Gunma, Japan; Watanabe et al. 2001). Approximately 5 MBq of ⁵²Mn was produced using a beam current of 1 µA for 2.4 h. ⁵²Mn was radiochemically separated from the target using the method described by Watanabe et al. (2001).

PETIS and PMPS

Plant samples were placed between two opposed 2-D block detectors consisting of Bi₄Ge₃O₁₂ scintillator arrays. The ⁵²Mn produced was used as a positron-emitting tracer. Two annihilation γ-rays emitted from the decaying positrons were detected simultaneously (Kume et al. 1997; Uchida et al. 2004). The original position of the annihilation was localized at the intersection of the object plane determined by a line connecting the two detection points on the detectors. The field of view was 143 mm × 215.6 mm, and the spatial resolution was 2.4 mm. After automatic correction for decay and relative detection efficiencies within the field of view, the resulting image was displayed on a monitor. After the imaging procedure was completed, the regions of interest (ROIs; Fig. 1c, Fig. 2c) within the data obtained were set and the radioactivity behavior over time within each ROI was extracted from the data. Two pairs of positron multi-probe system (PMPS) detectors were used for time-course tracer analysis of ⁵²Mn. Although the principles of PMPS are similar to those of PETIS, PMPS detectors directly measure the radioactivity time-course but bypass the imaging procedure.

Absorption and translocation of ⁵²Mn in plants

To study the absorption of ⁵²Mn by roots, the roots of a single plant were placed in a polyethylene bag containing 15 mL of 5× modified Kasugai's solution lacking Mn. To maintain the geometry, the plant and the bag were placed between two acrylic boards centered between the PETIS detectors in a chamber at 25°C, 65% humidity, and with a light density of 320 µmol m⁻² s⁻¹. ⁵²Mn²⁺ (250–900 kBq) in 1 mL water was added to the culture with gentle aeration for immediate mixing. The PETIS detectors were focused on the basal part of the shoot. After a 6–10 h trace analysis, the plant was removed from the polyethylene bag and the roots were gently washed for 1 min in 50 mL 5× modified Kasugai's solution. Next, the plant was exposed to a bio-imaging analyzer system (BAS) imaging plate (Imaging Plate BAS-MP2040S, Fujifilm, Tokyo, Japan) to obtain an autoradiographic image. After 30 min, the plate was scanned using a bio-imaging analyzer system (BAS-1500, Fujifilm, Tokyo, Japan). In the dark-treatment experiment, the plants were cut into parts after the PETIS and autoradiography analyses. The radioactivity of each part was then determined by γ-ray

Figure 1  (a,b) 52Mn translocation from the roots of (a) Mn-deficient and (b) Mn-sufficient (control) barley plants. Images were obtained by autoradiography. Rectangles show the area measured by the positron-emitting tracer imaging system (PETIS). DC, discrimination center; YL, youngest leaf. Scale bars are 4 cm. These images cannot be compared to each other. (c) Time-course of radioactivity in Mn-deficient (left) and control (right) barley as measured by PETIS. The images were taken at 15-min and 2-h intervals (0–120 min and 2–8 h, respectively). Arrowheads indicate the DC. Scale bars are 1 cm. (d) Time-course of radioactivity in the DC of Mn-deficient (purple dots) and control (orange dots) barley. The maximum measured radioactivity was defined as 100, and all measurements were converted relative to the maximum. (e) Time-course of radioactivity in the DC and at a position 3.41 cm above the DC in the main shoot of Mn-deficient barley (indicated as “shoot” in [c]). Arrows indicate the arrival time of 52Mn. The arrival time was defined as the time at which radioactivity began to be detected.

Figure 2  (a,b) 52Mn translocation from the roots of (a) Mn-excess and (b) control barley. Images were obtained by autoradiography. Rectangles show the area measured by the positron-emitting tracer imaging system (PETIS). Scale bars are 4 cm. These images cannot be compared to each other. (c) Time-course of radioactivity in Mn-excess (left) and control (right) barley as measured by PETIS. The images were taken at 1-h intervals. DC, discrimination center. Scale bars are 1 cm. (d) Time-course of radioactivity in the DC of Mn-excess (purple dots) and control (orange dots) barley. The maximum measured radioactivity was defined as 100, and all measurements were converted relative to the maximum.

spectrometry using an HPGe detector (crystal diameter 58.0 mm, length 67.3 mm) coupled to a MCA-7700 multichannel analyzer (Seiko EG & G Co., Matsudo, Japan). The peak analysis was carried out using Gamma Studio 2nd edition SP2 (Seiko EG & G Co.). The radioactivity of the ⁵²Mn was determined from the peak area at 935.52 keV. The effects of Mn-deficiency (14-day pretreatment), excess Mn (7-day pretreatment) and dark (1-h pretreatment) on ⁵²Mn absorption and translocation were examined.

To study the translocation of ⁵²Mn from a leaf, the second youngest leaf was used because this leaf is the longest. The leaf tip was cut in distilled water to prevent the introduction of air into the exposed leaf tissues. The cut end was then submerged in 3 mL of 5× modified Kasugai's solution lacking Mn in a vial. ⁵²Mn²⁺ (1.88 MBq) in 1 mL water was added to the vial, and the PMPS detectors were focused on the basal part of the shoot. After a 6-h trace analysis, autoradiography was carried out.

Each experiment was repeated at least three times to confirm the reproducibility of the results.

RESULTS

Mn²⁺ translocation in Mn-deficient and Mn-sufficient barley plants

To examine the effect of Mn deficiency on the absorption and translocation of Mn in intact plants, ⁵²Mn²⁺ was supplied to the roots of both Mn-deficient and Mn-sufficient (control) barley plants, and the translocation of ⁵²Mn was monitored using PETIS and autoradiography (Fig. 1). Autoradiography images showed that the discrimination center (DC), which is located in the basal part of the shoot, was strongly labeled in both Mn-deficient and control plants (Fig. 1a,b). In Mn-deficient barley, the youngest leaf was the most strongly labeled shoot (Fig. 1a). Figure 1c shows a montage of PETIS images. The radioactivity observed in images of shoots of Mn-deficient barley was higher than that of control barley shoots in a 10-h experiment, showing that more ⁵²Mn was translocated to shoots in Mn-deficient barley than in control barley. In Mn-deficient barley, an image of the DC (Fig. 1c, arrowhead) appeared within 75–90 min, and an image of the shoots appeared within 2–4 h (Fig. 1c). In contrast, in control barley, images of the DC and shoots appeared within 90–105 min and 4–6 h, respectively. The time-course of radioactivity in the DCs (indicated in Fig. 1c, squares) was extracted from the PETIS data (Fig. 1d,e). During the 10-h experiment, the ⁵²Mn counts in the DCs of Mn-deficient barley were higher than those in the DCs of control barley. This result shows that Mn accumulated in the DC in higher amounts in Mn-deficient barley than in control barley. The times of arrival of ⁵²Mn to the DC and the main part of the shoot (indicated as “shoot” in Fig. 1c, 3.41 cm above the DC) in Mn-deficient barley were calculated using the results of the time-course of ⁵²Mn radioactivity. The accumulation of ⁵²Mn in the DCs of Mn-deficient barley began at 42 min, and in the main shoot at 99 min (Fig. 1e, arrows). Therefore, the speed of ⁵²Mn translocation from the DCs to the main shoots of Mn-deficient barley was 0.060 cm min⁻¹ (= 3.41 cm/[99 − 42] min). It was not possible to calculate the ⁵²Mn translocation speed in control barley because of the very low levels of ⁵²Mn transport to shoots.

Mn²⁺ translocation in Mn-excess barley

⁵²Mn²⁺ was supplied to the roots of Mn-excess and Mn-sufficient (control) barley plants, and images of the shoots (indicated in Fig. 2a,b, rectangles) were captured. The montage images revealed the radioactivity of ⁵²Mn translocated to the shoots of Mn-excess and control barley in a 6-h experiment (Fig. 2c). The radioactivity recorded in images of shoots of Mn-excess barley was lower than that in control barley shoots. This result shows that Mn was translocated to the shoots in lower amounts in Mn-excess barley than in control barley under these experimental conditions. The time-course of ⁵²Mn radioactivity in the DCs (Fig. 2c, squares) of Mn-excess and control barley showed that the DCs of Mn-excess barley accumulated approximately 20% of the ⁵²Mn accumulated by the DCs of control plants in a 6-h experiment (Fig. 2d).

Effect of light on Mn translocation

The effect of light on ⁵²Mn translocation was examined by carrying out the same experiment in darkness. The time-course of ⁵²Mn radioactivity in the DC (Fig. 3a,b, squares) was extracted from the PETIS data (Fig. 3c). In a 10-h experiment, the ⁵²Mn counts in the DC were lower in darkness than in the light, showing that darkness suppressed ⁵²Mn translocation to the DC. The ⁵²Mn content of the youngest leaf (the fifth leaf) after 10 h of absorption was not suppressed in darkness, but the ⁵²Mn content of older leaves (fourth, third and second leaves) decreased (Fig. 3d).

Mn²⁺ translocation from a leaf

The translocation of Mn from a single leaf of an intact plant was examined by supplying ⁵²Mn²⁺ to the cut end of the second youngest leaf of an Mn-sufficient barley plant. ⁵²Mn was translocated from the cut end of the second-youngest leaf to the DC, other leaves and roots (Fig. 4a). The DC and root tips were particularly strongly labeled. The time-course of radioactivity in the DC revealed that accumulation of ⁵²Mn in the DC began at

Figure 3  (a,b) 52Mn translocation from the roots of control barley in (a) darkness and (b) light. Images were obtained by autoradiography. Leaf numbering: 4, the second-youngest leaf on the main shoot; 3, the third leaf; 2, the second leaf; 1, the first leaf. The youngest leaf, the fifth leaf, is present inside the leaf sheath. DC, discrimination center. Scale bars are 4 cm. (c) Time-course of radioactivity in the DC (as marked in [a,b]) of barley in darkness (•) and light (○). The maximum measured radioactivity was defined as 100, and all measurements were converted relative to the maximum. (d) 52Mn content in the DC and leaves of barley in darkness (▪) and light (□).

27 min (Fig. 4b).

DISCUSSION

Accumulation of minerals and metabolites in the DC of graminaceous plants

The time-course of ⁵²Mn translocation was traced in intact barley using PETIS, and the speed of ⁵²Mn translocation was then calculated. This is the first report on the real-time imaging of Mn uptake and translocation in intact plants. In all experiments, ⁵²Mn primarily accumulated in the DC, a region that contains the shoot meristem (Mori 1998). ⁵²Mn first accumulated in the DC and was then distributed to all plant tissues (Fig. 1c). A similar result was reported in wheat (Triticum aestivum cv. Aroona) using ⁵⁴Mn (Pearson and Rengel 1995). Similarly, we reported that radioactivity accumulated in the DCs of barley and rice and was then distributed to shoots, as determined in studies using ¹¹C-methionine (Bughio et al. 2001; Nakanishi et al. 1999), ¹³NH⁺ ₄ (Kiyomiya et al. 2001a) and H¹⁵ ₂O (Kiyomiya et al. 2001b; Mori et al. 2000; Nakanishi et al. 2002; Tsukamoto et al. 2004). Therefore, it is likely that not only Mn but also other minerals and metabolites accumulate in the DC after absorption from the roots of graminaceous plants. Taken together, these data suggest that the DC may play an important role in distributing minerals and metabolites in graminaceous plants.

The speed of ⁵²Mn translocation from the DC to shoots in barley was approximately 0.060 cm min⁻¹ (Fig. 1e). In other studies in which the translocation of other nutrients was examined in barley and rice, the transport rates from the DCs to shoots were 1 cm min⁻¹ (¹¹C-methionine; Bughio et al. 2001; Nakanishi et al. 1999), 8.6 cm min⁻¹ (¹³N-glutamine; Kiyomiya et al. 2001a) and 1.9–1.1 cm min⁻¹ (H¹⁵ ₂O; Kiyomiya et al. 2001b). Therefore, the speed of translocation of the substrate depends on the substrate itself. The translocation of ⁵²Mn from the DC to shoots in Mn-deficient barley (0.060 cm min⁻¹) was slower than the translocation of H¹⁵ ₂O, ¹¹C-methionine or ¹³N-glutamine, which suggests that substrates that are required for plant growth in large quantities are translocated more rapidly than those that are required in small quantities.

Effects of Mn status on Mn absorption and translocation from roots

More ⁵²Mn was translocated to shoots in Mn-deficient barley than in Mn-sufficient barley (Fig. 1c,d), suggesting that the demand for Mn affects its translocation. Similarly, after labeling a single wheat root with ⁵⁴Mn for 2 h, the main stem, leaves and tillers (other than the oldest leaf) of low-Mn plants received more ⁵⁴Mn than the control plants (Pearson and Rengel 1995). These results are consistent with our data. In addition, an image of the

Figure 4  (a) 52Mn translocation from a leaf tip (arrowhead) to other tissues in a control barley plant. Images were obtained by autoradiography. Leaf numbering: 4, the youngest leaf on the main shoot; 3, the third leaf; 2, the second leaf; 1-2, 1-1, younger and older leaf on the first tiller, respectively. Scale bar is 4 cm. (b) Time-course of radioactivity in the discrimination center (DC). The maximum measured radioactivity was defined as 100, and all measurements were converted relative to the maximum.

DC in Mn-deficient barley appeared within 75–90 min, whereas an image of the DC in control barley appeared within 90–105 min. This result shows that Mn deficiency enhanced the translocation of ⁵²Mn from the roots to the DC, suggesting that the response to Mn deficiency is regulated in the root system and that Mn deficiency causes enhanced Mn uptake and loading into vascular bundles. Several genes have been reported to encode metal transporters that are capable of transporting Mn, including OsNramp1, OsNramp3 (Belouchi et al. 1997), AtNramp1, AtNramp3, AtNramp4 (Thomine et al. 2000), IRT1 (Eide et al. 1996; Korshunova et al. 1999; Rogers et al. 2000) and AtOPT3 (Wintz et al. 2003). The expression of the putative oligopeptide transporter (OPT) AtOPT3 in roots was strongly induced by Mn deficiency and to some extent by Fe and Cu deficiency (Wintz et al. 2003). However, other Mn transporters did not appear to be regulated by external Mn concentrations. Our results show that the translocation of ⁵²Mn from roots to shoots in barley is enhanced by Mn deficiency, suggesting that Mn-transporter genes are induced in barley roots by Mn deficiency.

⁵²Mn was translocated preferentially to younger leaves in Mn-deficient barley (Fig. 1a), and the DC may play a critical role in this process. Similarly, when ⁵⁹Fe-epiHMA was supplied to barley roots, ⁵⁹Fe was also translocated preferentially to younger leaves (Mori 1998). Manganese may be translocated to younger leaves through a pathway similar to that of Fe. Although the expression of an Mn transporter in barley shoots has not been reported, a number of Mn or Mn-chelate complex transporters that are expressed in shoots of other plant species, including AtNramp3, AtNramp4 (Thomine et al. 2000), AtOPT3 (Stacey et al. 2002), MtZIP4, MtZIP7 (López-Millán et al. 2004), and OsYSL2 (Koike et al. 2004), have been reported. MtZIP4, MtZIP7 and OsYSL2 are not induced by Mn deficiency, and the expression of AtNramp3, AtNramp4 and AtOPT3 does not appear to be regulated by Mn deficiency. Genes encoding Mn transporters may be induced in the DC during Mn deficiency, and the resulting enhanced Mn transport may lead to the regulation of Mn distribution in plants.

In Mn-excess barley, less ⁵²Mn was translocated to shoots than in control barley (Fig. 2). Excess Fe and cadmium have been shown to inhibit Mn²⁺ accumulation (Hernandez et al. 1998; Roomizadeh and Karimian 1996). It has also been reported that transporters such as the Arabidopsis CAX2 (Hirschi et al. 2000), ECA1 (Wu et al. 2002) and ShMTP1 (Delhaize et al. 2003) pump Mn²⁺ into internal organelles to prevent the toxic effects of excess Mn²⁺ and are able to promote Mn tolerance under conditions of Mn toxicity. In the present study, radioactivity resulting from ⁵²Mn taken up into root cells of Mn-excess and control barley could not be measured because it is impossible to distinguish ⁵²Mn inside the cell from ⁵²Mn in the apoplastic space and cell wall. Therefore, it is possible that the absorption of ⁵²Mn is suppressed by excess Mn, or that more ⁵²Mn accumulates in root cells of Mn-excess barley than in the control.

Effects of light on Mn translocation

Interestingly, the translocation of ⁵²Mn to the youngest leaf was not suppressed in darkness (Fig. 3), whereas ⁵²Mn translocation to the older leaves was suppressed. We previously reported that the flow of H¹⁵ ₂O was immediately and completely stopped by a dark treatment during 30 min of absorption, suggesting that the transpiration stream in the xylem is completely inhibited in darkness (Kiyomiya et al. 2001b; Mori et al. 1999). This finding indicates that the transpiration stream has little effect on Mn translocation to the youngest leaf, but a strong effect on Mn translocation to older leaves. Tanner and Beevers (2001) reported that when transpiration was experimentally dissociated from the mineral supply of sunflowers (Helianthus annuus) grown in hydroculture, and mineral nutrients were provided only at night, the growth of the plants was similar to that of the control group, which received nutrients only during the day. They concluded that convective water transport in the xylem, brought about by root pressure, the resultant guttation called “growth water” and Münch's phloem counterflow, is sufficient for long-distance mineral supply and that transpiration is not required for this function. Similarly, the translocation of mineral nutrients, including Mn, to the youngest leaf appears to proceed regardless of the activity of the transpiration stream.

Mn²⁺ translocation from a barley leaf

When ⁵²Mn was supplied to the cut end of an expanded leaf, ⁵²Mn was transported to the DC within 25 min and then retranslocated to roots and other leaves (Fig. 4). In particular, ⁵²Mn was preferentially translocated to strong sink organs, such as younger leaves and root tips. Similarly, Vose (1963) reported that ⁵⁴Mn was translocated to the stem, the youngest leaf and the roots when applied as a drop to the fourth leaves of Mn-deficient oat plants whose flag leaves had emerged. This author also suggested that the root can function as a reservoir of available Mn. We reported the transport of ⁵⁹Fe in barley using ⁵⁹Fe(III)-epiHMA (Mori 1998). When ⁵⁹Fe-epiHMA was supplied to cut barley leaves, ⁵⁹Fe was translocated through the DC to other new chlorotic leaves and root tips within 45 min, as detected by autoradiography. The translocation of ⁵²Mn from shoots to roots showed similar characteristics to the transport of ⁵⁹Fe. It has been observed that many Mn²⁺ transporters have broad substrate specificities and some transporters are also capable of transporting not only Mn²⁺ but also Fe²⁺ and other nutrients (Pittman 2005). Therefore, the similar translocation of ⁵²Mn and ⁵⁹Fe from shoots to roots may be caused by transporters that are capable of transporting both Mn²⁺ and Fe²⁺. In contrast, we reported that ¹¹C-methionine is not translocated from the leaves to the roots of Fe-deficient barley (Nakanishi et al. 1999). These results indicate that not all minerals and metabolites are translocated from the leaves to the roots, suggesting that translocation from shoots to roots does not proceed through passive diffusion but is instead actively regulated, and the DC may contribute to this regulation.

ACKNOWLEDGMENTS

This research was supported by the Universities and Japan Atomic Energy Research Institute (JAERI) Joint Research Project. The Research Centre for Nuclear Science and Technology, University of Tokyo, is also acknowledged. We express our sincere regrets for the loss of S. Kiyomiya, the third author, who passed away on 16 August 2001.