Dynamic changes in the Cs distribution throughout rice plants during the ripening period, and effects of the soil-k level

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1 Plant Soil (218) 429: REGULAR ARTICLE Dynamic changes in the Cs distribution throughout rice plants during the ripening period, and effects of the soil-k level Junko Ishikawa & Shigeto Fujimura & Motohiko Kondo & Mari Murai-Hatano & Akitoshi Goto & Takuro Shinano Received: 13 March 218 /Accepted: 24 May 218 /Published online: 16 June 218 # Springer International Publishing AG, part of Springer Nature 218 Abstract Background and aims Radiocesium uptake and accumulation in crops has been studied in Japan since the March 211 nuclear power plant accident. However, few studies have reported how cesium (Cs) is distributed in aboveground parts and how it accumulates in edible parts. Here, we report the dynamics of Cs in aboveground parts of rice (Oryza sativa L.) plants throughout the cultivation period, and the effects of the amount of potassium (K) fertilizer applied to the soil. Methods We conducted two years of pot experiments with several soil-k levels and examined the K and Cs concentrations in each plant part throughout the cultivation period. Results During ripening, Cs accumulated most in the panicle neck. The ratio of the Cs concentration in brown rice to that in the straw was negatively correlated with the soil-k level, indicating that the proportion of Cs accumulated in the brown rice to that in the wholeaboveground parts increased at low soil K. During ripening, transport of Cs from belowground parts and translocation from leaf blades both seemed to increase at low soil K. Conclusion The Cs distribution in plant parts appears to be regulated dynamically during ripening by the soil-k level. Keywords Brown rice. Cesium. Distribution. Panicle neck. Potassium. Rice (Oryza sativa L.). Translocation Abbreviations AAS atomic absorption spectrophotometer Cs cesium DAT days after transplanting Junko Ishikawa and Shigeto Fujimura contributed equally to this work. Responsible Editor: Philip John White. Electronic supplementary material The online version of this article ( contains supplementary material, which is available to authorized users. J. Ishikawa (*): A. Goto NARO Institute of Crop Science, Kannondai, Tsukuba, Ibaraki , Japan junkoi@affrc.go.jp S. Fujimura : T. Shinano NARO Tohoku Agricultural Research Center, 5 Harajyuku-minami, Fukushima, Fukushima , Japan M. Kondo Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi , Japan M. Murai-Hatano NARO Tohoku Agricultural Research Center, 4 Akahira, Shimo-kuriyagawa, Morioka, Iwate 2-198, Japan

2 54 Plant Soil (218) 429: ICP-MS K N P Introduction inductively coupled plasma mass spectroscopy potassium nitrogen phosphorus Large amounts of radioactive nuclides were dispersed into the environment during and after the accident that occurred at the Tokyo Electric Power Company s Fukushima Daiichi Nuclear Power Plant in March 211. Among them, radiocesium ( 134 Cs and 137 Cs) created serious problems for agriculture in neighboring areas (Takata et al. 214). 137 Cs requires special attention because of its longer half-life (3.2 years) compared with 134 Cs (2.1 years). To reduce the radiocesium concentration in agricultural products, several countermeasures were conducted after the accident. Among them, topsoil removal and the application of potassium (K) fertilizer have been widely used in the contaminated areas (Fujimura et al. 213; Yamaguchi et al. 216). Brown rice (Oryza sativa) with a radiocesium concentration of >1 Bq kg 1, the limit for cereals defined by the Japanese government in 212 (MHLW 212), has not been produced since 215 ( jp/j/syouan/seisaku/radio_nuclide/radio_nuclide28. html). In the spring of 217, the area from which residents were excluded since the accident was reduced from 115 km 2 to 37 km 2. To reconstruct agriculture in the re-opened areas, researchers have begun to develop methods to evaluate the risk of radiocesium uptake and accumulation when plants are grown in a contaminated field. They are also seeking or developing cultivars that accumulate less radiocesium (Ishikawa et al. 217; Rai et al. 217). To support these efforts, it s necessary to improve our understanding of the mechanisms of radiocesium uptake, transport from the roots to aboveground parts, translocation among plant tissues, and accumulation in edible parts. Uptake of radiocesium from the soil is decreased by the application of K fertilizer, because K and Cs are taken up competitively by the plant roots (Shaw and Bell 1991). The competition between K and Cs suggests that Cs is transported by K transporters (Zhu and Smolders 2; White and Broadley 2). In Arabidopsis, a high-affinity K transporter (encoded by AtHAK5) and several voltageindependent cation channels (VICCs) were candidates for Cs transport by the roots (Qi et al. 28; Whiteetal. 21). Recently, several possible Cs transporters were reported in rice. A high-affinity K transporter (encoded by OsHAK1) appears to play a dominant role in Cs uptake by the roots, since dysfunction of this gene dramatically decreased Cs uptake (Nieves-Cordones et al. 217; Rai et al. 217). OsSOS2, a rice gene that is called as a saltover sensitive gene, appears to be a possible regulator for Cs uptake by roots at low-k conditions (Ishikawa et al. 217), as dysfunction of this gene significantly decreased Cs uptake by roots. Since the expression levels of several transporters, including OsHAK1 and OsAKT1, were downregulated in an OsSOS2 mutant, these transporters are candidates for the control of Cs uptake by roots (Ishikawa et al. 217). Despite these recent studies of the mechanism of Cs uptake from the soil by roots, little is known about the movement of Cs into the aboveground parts of rice after it has been taken up by the roots. For crop production, it s necessary to decrease the concentration of radiocesium in edible parts so as to minimize or prevent its consumption by humans and animals. In rice plants grown in pot experiments, Cs that accumulated in brown rice accounted for 15 to 2% of the total amount of Cs in the aboveground parts (Tsukada et al. 22). The proportion depended on the amounts of K applied to the soil: as the soil-k level decreased, the proportion increased (Fujimura et al. 214; Noboriet al. 214). Similar results were obtained for buckwheat grain (Kubo et al. 217). However, the mechanisms responsible for this phenomenon have not yet been confirmed. Among the aboveground parts of rice, Cs accumulates most in the straw (Tsukada et al. 22). However, accumulation in different parts of rice plants has not been fully determined. There are two possible sources of Cs accumulation in brown rice: direct transport from the roots through the internodes, nodes, and rachis, and translocation from other parts such as the leaf blades. The Cs concentration in leaf blades at maturity was higher in older leaf blades, whereas the K concentration was higher in younger leaf blades (Tsukada et al. 22). This result suggests that the rate of Cs translocation from older leaf blades may be slower than that of K translocation (Tsukada et al. 22). A hydroponic culture study with a continuous 137 Cs supply suggested that 137 Cs translocation from leaf blades might affect the increase in the 137 Cs concentration in brown rice (Nobori et al. 214). However, these results were based

3 Plant Soil (218) 429: only on samples at maturity. Precise analysis of the changes in the Cs concentration in each plant part throughout the growth period is needed to elucidate whether Cs translocation from other plant parts is truly related to Cs accumulation in brown rice. The aims of the present study were to elucidate the dynamics of Cs in the aboveground parts of rice throughout the growth period, and to clarify the effect of the soil-k level on the distribution of Cs in the plants. To achieve these aims, we conducted pot experiments with several soil-k levels. We determined the concentration of Cs in each plant part throughout the growth period, but with a particular focus on the ripening period from heading to maturity, when the weight of brown rice increases dramatically. On the basis of our results, we discuss the possible mechanisms of Cs accumulation in brown rice at different soil-k levels. Materials and methods Field experiment A field experiment was conducted in a paddy field at the Institute of Crop Science, NARO, in Tsukubamirai City, Ibaraki Prefecture, Japan, in 214 using Oryza sativa L. Koshihikari. Controlled-release fertilizer (equal parts LP4, LPs1 and LP14; JCAM Agri, Tokyo, Japan) was applied as a basal nitrogen dressing at a rate of 8.gofNm 2. Superphosphate was applied as basal phosphorus dressing at a rate of 3.5 g of P m 2.K fertilizer was not applied to suppress an inhibitory effect of K on Cs uptake by the roots. Seedlings were transplanted on 21 May 214 at 11.1 hills m 2. Each hill contained one plant. Plants were sampled at maturity (22 September) and divided into 15 parts: brown rice, husk, rachis, panicle neck (internode I), internode II, node I, node II, 1st leaf sheath, 2nd leaf sheath, flag leaf blade (1st leaf blade), 2nd leaf blade, lower leaves (below the 3rd leaf blades), lowest stem (up to 5 cm from the soil surface), lower stem (other stem parts), and dead leaf blades and leaf sheaths. They were dried for more than 48 h at 8 C in a ventilated oven. Potexperiment1 The pot experiment was conducted in a glasshouse at the Tohoku Agricultural Research Center, NARO, Fukushima Prefecture, Japan, in 215 and 216 using Koshihikari. Three seedlings at the third-leaf stage were transplanted into Wagner pots (2 cm 2,2-cm height) on 25 June 215 and 26 May 216. Each pot was filled with 3.5 kg of soil collected from a field at the Tohoku Agricultural Research Center, NARO, in the spring of 215. Ammonium sulfate, superphosphate, and potassium chloride were used as the N, P, and K fertilizers, respectively. They were applied as a basal dressing at.5 g of N and.2 g of P per pot and as a top dressing at.2 g of N per pot. Four rates of K application were used: in 215, these were,.11,.22, and.66 g K per pot. These values correspond to, 7.5, 15, and 45 mg K 2 O per 1 g air-dried soil; hereafter, we designate these four treatments as +, +7.5, +15, and + 45 mg K. In 216, we applied,.11,.22, and.44 g K in each pot. These values correspond to, 7.5, 15, and 3 mg K 2 O per 1 g of air-dried soil; hereafter, we designate these treatments as +, +7.5, +15, and + 3 mg K. The application rate of the highest K was changed from +45 mg K in 215 to +3 mg K in 216, because some inhibition of dry weight was observed in +45 mg K in 215 as mentioned in the Results, possibly due to inhibition of N uptake from soil by competition between potassium and ammonium ions when plants accumulated them in the roots. We used 1 pots for each K treatment in 215 and 15 in 216. Rice plants were sampled twice in 215: at heading (1 September, 68 days after transplanting: DAT) and at maturity (27 October, 124 DAT). They were sampled three times in 216: at panicle initiation (21 July, 56 DAT), heading (15 August, 81 DAT), and maturity (4 October, 131 DAT). Five pots were used for each sampling time (n = 5). The aboveground part of each plant was divided into 1 parts: brown rice, husk, rachis, upper internode (1st and 2nd), upper node (1st and 2nd), upper leaf blade (1st and 2nd), other leaf blades, upper leaf sheath (1st and 2nd), lowest stem (up to 4 cm from the soil surface), and lower stem (other stem parts). They were dried for more than 48 h at 8 C in a ventilated oven. Soil solution was collected periodically during the cultivation period by using soil moisture samplers (see section of Collection of soil solution in the Methods for details). Soil samples for exchangeable K and 133 Cs concentrations were collected several times during cultivation periods (see section of Exchangeable K and 133 Cs concentrations in the Methods for more details). The air temperature from transplanting to heading averaged 26.8 C in 215 and

4 56 Plant Soil (218) 429: C in 216. Temperatures from heading to maturity averaged 19.6 C in 215 and 23.6 C in 216. Potexperiment2 The pot experiment was conducted in 216 in the same glasshouse in pot experiment 1 using Koshihikari. Seedlings at the third-leaf stage were transplanted into Wagner pots (2 cm 2, 2-cm height) on 26 May 216 (at three plants per pot). Each pot was filled with 3.5 kg of soil collected from two paddy fields contaminated with radiocesium (soils A and B). The radiocesium concentrations in soils A and B were 378 and 177 Bq kg 1 dried soil, respectively. N and P were applied as in pot experiment 1. The rates of K application were,.7,.15,.22,.29,.37,.44, and.59 g per pot. These values correspond to, 5, 1, 15, 2, 25, 3, and4mgk 2 O per 1 g of air-dried soil. One pot was used for each K treatment. Rice plants were sampled at maturity (4 October). The aboveground part of each plant was divided into the brown rice, husk, and straw (all parts excluding brown rice, husk, and lowest stem up to4cmfromthesoilsurface).theyweredriedmore than 48 h at 8 C in a ventilated oven. Soil solution was collected for eight times during the cultivation period by using soil moisture samplers (see section of Collection of soil solution in the Methods for details). Collection of soil solution and determination of its K concentration Soil solution was collected using soil moisture samplers (resinous fiber-type) (DIK-31B, Daiki Rika Kogyo Co. Ltd., Kounosu, Japan). Sampler of 1 cm length was inserted in the soil at depth of between 1 and 11 cm below the surface. Soil solution was collected at 8, 27, 32, 47, 62, 92, and 16 DAT in 215 in pot experiment 1, and at 7, 13, 21, 28, 43, 57, 69, and 95 DAT in 216 in pot experiment 1 and in pot experiment 2. For each sampling time, soil solution was collected for 1 to 2 h. The K concentration in the soil solution was measured by means of inductively coupled plasma mass spectroscopy (Agilent 77 ICP-MS, Agilent, Technologies, Inc., Santa Clara, CA, USA). Exchangeable K and 133 Cs concentrations Soil samples were collected when the plants were sampled, as well as at 8 DAT in 215 and just before transplanting ( DAT) in 216. Sampled soil was airdried and passed through a 2.-mm sieve before measurements. The soil samples were extracted with 1 M ammonium acetate (soil:solution = 1:1 w/v). The K concentration in the solution was determined with an atomic absorption spectrophotometer (AAS; AA28FS, Agilent Technologies Japan, Ltd., Tokyo). The 133 Cs concentration was measured by means of ICP-MS (Agilent 77 ). Concentrations of 133 Cs, K, and other elements in the plant samples Plant samples were ground in a WSX-2 grinding mill (Fujiwara Scientific Co., Tokyo, Japan) and then ground in a milling machine (TI-1, Fujiwara Scientific Co.). We digested 45 mg of each sample in.45 ml of HNO 3. After digestion, 8.55 ml of water was added to the extract. The K concentration was measured with the AAS (Agilent AA28FS). The concentrations of 133 Cs and other elements (Na, Rb, Zn, Cd, As, Cu, Mg, Ca, Fe, and Mn) were measured by means of ICP-MS (Agilent 77 ). In the present work, the measured 133 Cs concentration in lowest stem in pot experiment 1 was not used for further analysis because contamination of soil increased the 133 Cs concentration in this part. We assumed that the 133 Cs concentration in the lowest stem was same as that in lower stem (other stem and leaf sheath), and calculated the 133 Cs amount of this part. 137 Cs concentrations in plant and soil samples The concentrations of 137 Cs were determined by using germanium detectors with multichannel analyzers (GC252-75SL and GCW U-ULB, Canberra Japan KK, Tokyo, Japan). The measurement results were decay-corrected to 1 October 216. Statistical analysis We compared the results among the K treatments in the pot experiment 1 by means of t-test or one-way ANOVA followed by multiple comparison (Ryan s method).we performed analysis of covariance (ANCOVA) to detect correlations for a series of individual pairs of observations in the pot experiment 2. All analyses were conducted in R v software (

5 Plant Soil (218) 429: Results Accumulation of 133 Cs in each part of the plant (field experiment) In this experiment, we determined which part of the plant accumulated the most Cs. We excluded the data for lowest stem (up to 5 cm from the soil surface), and dead leaf blades and sheaths from this analysis because they might be contaminated by soil. The concentration of Al in these plant samples was more than 5 times the value in the other parts (data not shown). Al is often used as indicator of soil contamination (Tsukada et al. 22). After we excluded the dead parts, the concentration of 133 Cs was highest in the panicle neck (Internode I) (Fig. 1). The accumulation of K and Rb was highest there too. The concentration of K in the panicle neck reached 5.5% of the dry weight. No other element had a high accumulation in the panicle neck (Fig. 1, Supp. Fig. S1). The concentrations of Zn and Cd were highest in nodes I and II. The concentration of As was highest in nodes I and II and the leaf blades. In contrast, the concentration of Cu was low in the nodes. The concentration of Na was highest in the lower stem and leaf sheath. Changes in K and 133 Cs level in soil during cultivation period (pot experiment 1) To clarify changes in the total uptake of Cs and K in the aboveground parts during the cultivation period, we conducted pot experiments in 215 and 216 using a paddy soil with four soil-k levels. The K treatments were successful in both years; those with the highest K produced the highest K concentrations in the soil solution throughout the cultivation period in both years (Fig. 2a, d). The K concentration decreased after transplanting and reached steady and low levels after 3 to 7 DAT, depending on the year and K treatment. The exchangeable K concentrations decreased after transplanting and reached their lowest levels toward the middle of the cultivation period in both years (Fig. 2b, e). Throughout the cultivation periods in both years, the exchangeable K concentration was highest in the Brown rice Husk Rachis Panicle neck (Internode I) Internode II Node I Node II Lower stem 1st leaf sheath 2nd leaf sheath Flag leaf (1st leaf blade) 2nd leaf blade Lower leaf blade Na (µmol/g DW) K (mmol/g DW) Rb ( µ mol/g DW) Cs (nmol/g DW) Brown rice Husk Rachis Panicle neck (Internode I) Internode II Node I Node II Lower stem 1st leaf sheath 2nd leaf sheath Flag leaf (1st leaf blade) 2nd leaf blade Lower leaf blade Zn ( µ mol/g DW) Cd (nmol/g DW) As (nmol/g DW) Cu (nmol/g DW) Fig. 1 Concentration of each element in various parts of the plants at maturity (214 field experiment). Values are means ± SE from three different plant samples. ND: Concentration was below the threshold of detection in the ICP-MS analysis ND ND

6 58 Plant Soil (218) 429: K conc. in soil soln. (µm) a mg K 18 b c mg K mg K mg K Days after transplanting Days after transplanting Days after transplanting Ex-K (mg K 2 O / 1 g soil) Ex- 133 Cs ( µ g Cs / 1 g soil) K conc. in soil soln. ( µ M) d mg K 18 e f mg K mg K 14 Panicle mg K 12 ini a on Panicle ini a on Panicle 6 6 ini a on 4 4 Ex-K (mg K 2 O / 1 g soil) 2 Ex- 133 Cs ( µ g Cs / 1 g soil) Days after transplanting Fig. 2 Changes in (a, d) K concentration in soil solution, (b, e) exchangeable K concentration, and (c, f) exchangeable 133 Cs concentration during the cultivation periods in (a c) 215 and Days after transplanting Days after transplanting (d f) 216 in pot experiment 1. Values are means ± SE from five pots. Arrows indicate growth stages treatment with the highest K, and was lowest in the lowest K treatment. In contrast to the exchangeable K concentration, the exchangeable 133 Cs concentration increased after transplanting (Fig. 2c, f). In 215, it reached its maximum at the heading stage and decreased slightly thereafter (Fig. 2c). In 216, it continued to increase throughout the cultivation period, most rapidly during the period from panicle initiation to heading (Fig. 2f). The exchangeable 133 Cs concentrations was highest in the + mg treatment, followed by the +7.5 and + 15 mg K treatments, and was lowest in the treatment with the highest K at transplanting and at the panicle initiation stage in 216, and at maturity in both years. Changes in total 133 Cs uptake during cultivation period (pot experiment 1) The dry weight of the total above ground parts per pot at maturity and the harvest index (i.e., the ratio of dry weight of brown rice to the total aboveground mass) were higher in 216 than in 215 in all K treatments (Supp. Table S1). The dry weight in the treatment with the highest K was slightly lower than that in the other treatments during the cultivation period in both 215 and 216 (Supp. Table S1). The total amount of 133 Cs in the aboveground parts increased from the panicle initiation stage to maturity in all K treatments in 216 (Fig. 3b, Table 1). The rate of increase in the amount of 133 Cs was greater from the panicle initiation stage to the heading stage than from the heading stage to maturity. At all sampling times in both years, the amount of 133 Cs was highest in the + mg K treatment, followed by the +7.5 mg and + 15 mg treatments, and was lowest in the treatment with the highest K (Table 1). The increase from the heading stage to maturity in 216 was largest in the + mg K treatment (a 45% increase), followed by the +7.5 and + 15 mg K treatments, and was lowest in the +3 mg K treatment (a 15% increase), even though the dry weight in all K treatments showed a similar increase during this period (by 49 to 59%; Fig. 3a, Supp. Table S1). Similar results were obtained in 215, but the increase during the ripening period was lower than that in 216 (3 to 19%; Table 1). The total amount of K in 216 increased from the panicle initiation stage to the heading stage, but then increased only slightly between heading and maturity in all K treatments (Fig. 3c, Table 1). From panicle initiation

7 Plant Soil (218) 429: Dry weight (g per pot) a + mg K +7.5 mg K +15 mg K +3 mg K Panicle ini a on 133 Cs amount (nmol per pot) b Panicle ini a on K amount (mmol per pot) c Panicle ini a on Day a er transplan ng Days a er transplan ng Days a er transplan ng Fig. 3 Changes of a dry weight, b 133 Cs amount, and c K amount in all aboveground parts of plants from panicle initiation to maturity. Results are from pot experiment 1 in 216. Values are means ± SE from five pots (each with three plants). Arrows indicate growth stages to maturity, the total amount of K was highest in the +15 mg K treatment, followed by the +3 and mg K treatments, and was lowest in the + mg K treatment, though the difference between the +15 and + 3 mg K treatments was not significant (Table 1). Similar results were obtained in 215. Changes in the 133 Cs distribution to each part of the plant during ripening (pot experiment 1) The ratio of the 133 Cs concentration in brown rice to that in straw increased with decreasing soil K in both years (Table 2). To clarify the reason, we analyzed the changes in the amount of 133 Cs and the concentration in each part of the plants from heading to maturity. In both 215 and 216, both the amount of 133 Cs and its concentration in each part increased significantly with decreasing soil K, both at heading and at maturity (Tables 1 and 2). We evaluated the changes in the amount of 133 Cs in each part of the plant during ripening by calculating the difference between the amounts at heading and maturity (Table 1). In the upper internodes (internodes I and II), the amount of 133 Cs increased greatly during ripening in all K treatments in both years. On the other hand, the amount in the lower stem, the upper leaf sheaths (1st and 2nd), upper leaf blades (1st and 2nd), and lower leaf blades (lower than 3rd) decreased in all K treatments in both years, except for the upper leaf sheath in the + mg K treatment in 216. In 215, the sum of the decreases in 133 Cs in all parts was greater than the increase in the whole-plant 133 Cs (in this work, we designate whole-aboveground as whole-plant) both in the + and + 45 mg K treatments (Table 1). In 216, in contrast, the sum was lower than the whole-plant increase in all K treatments except +3 mg K. We also evaluated the changes in the 133 Cs concentrations in each part during ripening using the ratio of the concentration at maturity to that at heading. During ripening, the whole-plant 133 Cs concentration decreased in both years, but the 133 Cs concentration in each part of the plant changed differently (Table 2). That in the upper internode increased in all K treatments in both years. The rate of the increase was greater in the + mg K treatment than in the +45 mg K treatment in 215. Similarly, the rate of increase was largest in the + mg K treatment in 216, followed by the +7.5 and + 15 mg K treatments, and was lowest in the +3 mg K treatment. The 133 Cs concentration also increased in the rachis in 215 and in the rachis in the + and mg K treatments in 216. In contrast, 133 Cs concentrations in the upper and lower leaf blades decreased in all K treatments in both years (Table 2). The rate of decrease wasgreaterinthe+mgktreatmentthaninthe +45 mg K treatments in 215 but not in 216. In 215, the 133 Cs concentration also decreased in the husk, upper nodes (nodes I and II), lower stem, and upper leaf sheath. In 216, it decreased in the husk in all K treatments, and decreased in the + mg K treatment but increased in the +15 and + 3 mg K treatments in the lower stem. Both the amount of K and its concentration in the lower stem, upper leaf sheath, and upper and lower leaf blades increased with increasing soil K at both heading and maturity, although some samples didn t show a significant difference (Tables 1 and 2). The amount of K increased in the upper internode and decreased in the leaf blades and leaf sheaths during ripening in all K treatments in both years (Table 1). The sum of the decreases in the amount of K in all parts was greater than the whole-plant K increment in all K treatments in

8 51 Plant Soil (218) 429: Table 1 Changes in amounts of 133 Cs and K from heading to maturity (pot experiment 1) Year 215 Cs amount (nmol per pot) K amount (mmol per pot) Plant part K applied at K applied at + mg +7.5 mg +15 mg +45 mg S 1 + mg +7.5 mg +15 mg +45 mg S Brown rice Maturity 26.1 a b 12.2 c 2.94 d *** 1.81 a 1.98 a 2.1 a 1.87 a ns Husk 16.1 NA 3 NA 2.27 ***.957 NA NA.885 ns Maturity 18.5 a 13.2 b 9.48 c 2.25 d *** 1.81 a 1.86 a 2.9 a 1.88 a ns Difference Rachis 2.59 NA NA.397 ***.175 NA NA.181 ns Maturity 4.33 a 2.78 b 2.1 c.498 d ***.285 a.29 a.37 a.314 a ns Difference Upper internode 6.61 NA NA.895 *** 1.74 NA NA 1.42 ns Maturity 28.3 a 17.5 b 9.48 c 2.51 d *** 4.94 a 4.64 ab 4.27 ab 3.95 a * Difference Upper node 1.35 NA NA.178 ***.267 NA NA.256 ns Maturity 1.8 a.83 b.432 c.112 d ***.135 a.167 a.141 a.126 a ns Difference Lower stem 33.6 NA NA 6.39 *** 5.57 NA NA 1.8 ** Maturity 32.3 a 33.3 ab 24.1 b 5.87 c *** 5.8 b 9.68 a 12.5 a 12.1 a *** Difference Upper leaf sheath 18.2 NA NA 2.83 *** 3.61 NA NA 3.34 ns Maturity 13.6 a 8.27 b 5.56 c 1.4 d *** 2.19 b 2.37 ab 2.62 a 2.33 ab * Difference Upper leaf blade 17. NA NA 2.65 *** 2.24 NA NA 2.19 ns Maturity 8.25 a 6.1 b 5.2 c 1.49 d *** 1.26 c 1.43 b 1.57 ab 1.71 a *** Difference Lower leaf blade 32. NA NA 3.72 *** 2.77 NA NA 4.14 ** Maturity 19.3 a 16. b 13.5 b 2.88 c *** 1.71 c 2.84 b 3.56 ab 3.81 a *** Difference Sum of decreases in all parts Straw 111 NA NA 17.1 *** 16.4 NA NA 22.4 * Maturity 17 a 84.7 b 6. c 14.8 d *** 15.6 b 21.4 a 25. a 24.4 a *** Difference Total 128 NA NA 19.3 *** 17.3 NA NA 23.3 * Maturity 152 a 116 b 81.7 c 2. d *** 19.2 b 25.2 a 29.2 a 28.1 a *** Difference Ratio

9 Plant Soil (218) 429: Table 1 (continued) Year 216 Cs amount (nmol per pot) K amount (mmol per pot) Plant part K applied at K applied at + mg +7.5 mg +15 mg +45 mg S + mg +7.5 mg +15 mg +45 mg S Brown rice Maturity 45.9 a 33.8 b 23.9 c 9.66 d *** 3.17 b 3.58 a 3.6 a 3.64 a ** Husk 15.3 a 11.9 b 8.79 c 4.53 d *** 1.22 b 1.27 b 1.4 a 1.42 a ** Maturity 21.9 a 16.1 b 11.6 c 4.76 d *** 2.67 a 2.62 a 2.91 a 2.96 a ns Difference Rachis 2.61 a 2.11 b 1.66 c.864 d ***.251 b.273 b.318 a.315 a *** Maturity 3.37 a 2.29 b 1.39 c.549 d ***.515 b.544 ab.556 ab.569 a * Difference Upper internode 5.96 a 4.31 b 2.49 c 1.43 c *** 1.59 a 1.57 a 1.23 a 1.39 a ns Maturity 26.3 a 18.5 b 11.8 c 4.81 d *** 6.5 a 6.96 a 6.56 a 6.13 a ns Difference Upper node.986 a.783 b.538 c.284 d ***.181 a.192 a.183 a.186 a ns Maturity 1.6 a 1.13 b.795 c.289 d ***.24 a.26 a.294 a.278 a ns Difference Lower stem 38.4 a 35. ab 29.3 b 11.4 c *** 7.12 c 13. b 16.6 a 15.2 ab *** Maturity 32.2 a 32.8 a 28.2 a 9.69 b *** 4.84 cd 9.19 c 13.2 b 13.4 a *** Difference Upper leaf sheath 15.4 a 13. b 1.5 c 4.95 d *** 4.7 b 4.32 ab 4.72 a 4.45 ab * Maturity 15.9 a 12.3 b 8.56 c 3.35 d *** 2.36 b 2.74 a 3.3 a 2.81 a *** Difference Upper leaf blade 14.3 a 12.5 b 9.1 c 4.56 d *** 2.65 b 2.93 ab 2.95 ab 3.19 a ** Maturity 1.4 a 7.61 b 5.62 c 2.39 d *** 2.24 b 2.25 b 2.29 b 2.59 a * Difference Lower leaf blade 27.5 a 22.3 b 15.9 c 5.71 d *** 4.12 c 6.6 b 7.2 a 5.75 b *** Maturity 17.5 a 14.8 a 1.9 b 3.31 c *** 2.15 c 3.55 b 4.64 a 3.79 b *** Difference Sum of decreases in all parts Straw 15 a 89.9 b 69.5 c 29.2 d *** 2. c 28.4 b 33.2 a 3.5 ab *** Maturity 17 a 89.3 b 67.4 c 24.4 d *** 18.8 c 25.5 b 3.5 a 29.6 ab *** Difference

10 512 Plant Soil (218) 429: Table 1 (continued) Year 216 Cs amount (nmol per pot) K amount (mmol per pot) Plant part K applied at K applied at + mg +7.5 mg +15 mg +45 mg S + mg +7.5 mg +15 mg +45 mg S Total Panicle Initiation 48.5 a 34.5 b 23.6 c 9.49 d *** 11. c 15.5 b 18.1 a 17. ab *** 121 a 12 b 78.3 c 33.7 d *** 21.2 c 29.6 b 34.6 a 31.9 ab *** Maturity 175 a 139 b 13 c 38.8 d *** 24.7 c 31.7 b 37.1 a 36.2 ab *** Difference Ratio Results of statistical analyses by t-test (for the results at the heading stage in 215) and ANOVA (for all other results). Significance: *P<.5, **P<.1, ***P<.1; ns, not significant 2 Values labeled with the same letter do not differ significantly between K treatments (ANOVA followed by Ryan's test) 3 Samples were not taken for the +7.5 and +15 mg K treatments at heading in Change from heading to maturity 5 Decreases are in bold-italic format 6 Sum of decreases from heading to maturity in all parts of the plants 7 Ratio of total amount at maturity to that at heading both years, except in the +45 mg K treatment in 215. The K concentrations in the upper internode, rachis, and husk increased during the ripening period, whereas those in the upper leaf sheath and the upper and lower leaf blades decreased in all K treatments in both years (Table 2). Effects of soil-k level on 137 Cs accumulation in brown rice with contaminated soils (pot experiment 2) We examined the effects of the soil-k level on the ratio of 137 Cs accumulation in the brown rice to that in the straw with eight levels of K application (from + to +45 mg K) using two 137 Cs-contaminated soils collected from different paddy fields in Fukushima Prefecture (soils A and B). The experiment was conducted successfully, except that the plants in the +3 mg K treatment with soil A failed to grow. Transfer factors (the ratio of the 137 Cs concentration in brown rice to that of soil) for the both soils decreased with increasing soil-k level. In this experiment, we used the average measured K concentration in the soil solution during the cultivation period as the soil-k level. Significant difference in regression lines was observed between the two soils (Fig. 4a). The ratio of the 137 Cs concentration in the brown rice to that in the straw was also negatively correlated with the soil-k level (Fig. 4b). The regression lines did not differ significantly between the two soils. A significant difference was not observed between the ratio of the 133 Cs concentration in brown rice to that in straw and the same ratio for 137 Cs (paired t-test, n =15, p =.47, Supplementary Fig. S2). Discussion Cs accumulated most in the panicle neck during ripening Despite intensive studies of Cs uptake by the roots, reports on the Cs distribution in aboveground parts have been limited (Kondo et al. 215; Nobori et al. 214). Here, we clarified the growth period dependent changes in the Cs distribution in each part of rice plants. We analyzed mainly 133 Cs to clarify the dynamics of radiocesium, since there appear to be no differences between 133 Cs and radiocesium in terms of their distribution among plant parts. We confirmed this by showing that the ratio of the Cs concentration in brown rice to straw did not differ significantly between 133 Cs and 137 Cs (Supp. Fig. S2). During ripening, Cs accumulated most in the panicle neck (the uppermost internode, internode I; Fig. 1). The accumulation increased further at low soil K (Table 2).

11 Plant Soil (218) 429: Table 2 Changes in 133 Cs and K concentrations from heading to maturity (pot experiment 1) Year 215 Cs concentration (nmol/g DW) K concentration (mmol/g DW) Plant part K applied at K applied at + mg +7.5 mg +15 mg +45 mg S 1 + mg +7.5 mg +15 mg +45 mg S Brown rice Maturity a2.82 b.542 c.146 d ***.872 a.916 a.936 a.922 a ns (Brown rice / straw) a.545 b.475 c.459 c ***.35 a.241 b.198 c.171 c *** Husk 3.21 NA 4 NA.496 ***.191 NA NA.199 ns Maturity 2.58 a 1.71 b 1.19 c.325 d ***.251 a.24 a.263 a.274 a ns Ratio Rachis 2.17 NA NA.355 ***.147 NA NA.167 ns Maturity 3.15 a 1.92 b 1.34 c.378 d ***.28 a.2 a.247 a.244 a ns Ratio Upper internode 2.53 NA NA.366 ***.666 NA NA.637 ns Maturity 4.7 a 2.89 b 1.8 c.517 d ***.823 a.768 a.823 a.826 a ns Ratio Upper node 2.88 NA NA.45 ***.571 NA NA.678 *** Maturity 2.86 a 2.6 b 1.33 c.37 d ***.36 a.429 a.442 a.425 a ns Ratio Lower stem 1.68 NA NA.393 ***.276 NA NA.686 *** Maturity 1.25 a 1.2 b.968 c.271 d ***.197 d.348 c.51 b.589 a *** Ratio Upper leaf sheath 2.97 NA NA.528 ***.589 NA NA.642 ns Maturity 2.4 a 1.25 b.842 c.232 d ***.328 b.359 ab.397 a.39 a ** Ratio Upper leaf blade 3.62 NA NA.625 ***.477 NA NA.534 *** Maturity 1.72 a 1.32 b 1.3 c.316 d ***.264 c.314 b.321 b.369 a *** Ratio Lowerleafblade 3.13 NA NA.492 ***.271 NA NA.552 *** Maturity 2.7 a 1.74 b 1.47 c.385 d ***.182 d.31 c.385 b.524 a *** Ratio Straw 2.46 NA NA.456 ***.361 NA NA.615 *** Maturity 1.98 a 1.51 b 1.14 c.319 d ***.288 d.382 c.475 b.544 a *** Ratio Total 2.53 NA NA.461 ***.344 NA NA.57 *** Maturity 1.85 a 1.35 b.982 c.271 d ***.234 d.296 c.35 b.389 a *** Ratio

12 514 Plant Soil (218) 429: Table 2 (continued) Year 216 Cs concentration (nmol/g DW) K concentration (mmol/g DW) Plant part K applied at K applied at + mg +7.5 mg +15 mg +45 mg S + mg +7.5 mg +15 mg +45 mg S Brown rice Maturity 1.18 a.852 b.566 c.241 d ***.818 b.898 ab.853 ab.912 a * (Brown rice / straw).668 a.59 b.54 bc.511 c ***.265 a.222 b.18 c.157 c *** Husk 2.79 a 2.13 b 1.57 c.778 d ***.222 a.227 a.252 a.246 a * Maturity 2.6 a 1.83 b 1.26 c.54 d ***.317 ab.296 b.317 ab.337 a * Ratio Rachis 1.92 a 1.48 b 1.12 c.564 d ***.184 b.192 ab.217 a.27 ab * Maturity 2.29 a 1.51 b.883 c.361 d ***.352 a.359 a.355 a.377 a ns Ratio Upper internode 1.86 a 1.4 b.921 c.468 d ***.498 a.58 a.457 a.455 a ns Maturity 4.58 a 3.29 b 2.1 c.899 d *** 1.14 a 1.24 a 1.16 a 1.15 a * Ratio Upper node 2.7 a 2.9 b 1.5 c.729 d ***.497 a.511 a.513 a.482 a ns Maturity b 1.47 c.592 d ***.415 b.471 b.543 a.572 a *** Ratio Lower stem 1.22 a 1.8 b.769 c.363 d ***.227 c.41 b.436 ab.492 a *** Maturity 1.11 a 1.8 a.95 b.418 c ***.169 d.31 c.423 b.585 a *** Ratio Upper leaf sheath 2.15 a 1.75 b 1.3 c.616 d ***.568 a.584 a.586 a.558 a ns Maturity 2.21 a 1.69 b 1.11 c.479 d ***.33 b.376 a.391 a.44 a ** Ratio Upper leaf blade 2.33 a 1.95 b 1.41 c.71 d ***.432 c.456 b.458 b.494 a *** Maturity 1.71 a 1.29 b.917 c.46 d ***.369 b.384 b.37 b.443 a ** Ratio Lower leaf blade 2.2 a 1.65 b 1.11 c.538 d ***.328 d.45 c.57 b.55 a *** Maturity 1.63 a 1.34 b.947 c.413 d ***.21 d.321 c.44 b.481 a *** Ratio Straw 1.69 a 1.39 b.971 c.475 d ***.321 c.438 b.465 ab.51 a *** Maturity 1.77 a 1.44 b 1.5 c.474 d ***.311 d.49 c.475 b.581 a *** Ratio

13 Plant Soil (218) 429: Table 2 (continued) Year 216 Cs concentration (nmol/g DW) K concentration (mmol/g DW) Plant part K applied at K applied at + mg +7.5 mg +15 mg +45 mg S + mg +7.5 mg +15 mg +45 mg S Total Panicle Initiation 1.83 a 1.3 b.879 c.469 d ***.419 d.589 c.684 b.861 a *** 1.78 a 1.45 b 1.1 c.51 d ***.313 c.421 b.449 ab.479 a *** Maturity 1.62 a 1.26 b.888 c.387 d ***.23 d.285 c.32 b.363 a *** Ratio Results of statistical analyses by t-test (for the results at the heading stage in 215) and ANOVA (for all other results). Significance: *P<.5, **P<.1, ***P<.1; ns, not significant 2 Values labeled with the same letter do not differ significantly between K treatments (ANOVA followed by Ryan's test) 3 Ratio of concentration in brown rice to that in straw 4 Samples were not taken for the +7.5 and +15 mg K treatments at heading in Ratio of concentration at maturity to that at heading 6 Decreases from heading to maturity are in bold-itanic format K and Rb also accumulated in the panicle neck (Fig. 1), maybe because they belong to the same alkali metal group. On the other hand, little Na, another alkali metal, accumulated in the panicle neck. Instead, the Na concentration was highest in lower parts of the plant (Fig. 1), suggesting that the translocation of toxic Na to the upper part of the plants is inhibited (Deinlein et al. 214). Zn and Cd accumulated strongly in the nodes, where the Cu concentration was low. These results agree well with previous reports (Yamaji and Ma 214), indicating that the allocation of each element is regulated differentlyinriceplants. Hitherto, no elements have been reported to accumulate most in the panicle neck. That being the case, why did Cs and K do so during ripening? Two factors may be involved. First, the panicle neck is a nexus of the transport routes from many parts of the plant toward the panicle; in addition to taking a direct route from the roots, Cs and K may therefore have accumulated there after translocation from other parts such as the leaf blades (discussed below). Second, Cs and K may not be easily transported from the panicle neck to the rachis. The panicle node (the ear-neck node), which connects the panicle neck and the rachis, may not be a barrier, because it doesn t have the developed node structure that has been described in other nodes such as nodes I and II (Hoshikawa 1989; Yamaji and Ma 214). Rather, it is possible that Cs and K might accumulate easily in some tissues in the panicle neck by means of an unknown mechanism. Recent studies in the Poaceae demonstrated the existence of possible transporters that play crucial roles in the translocation of several elements in aboveground parts (Yamaji and Ma 214, 217). Such transporters localized in the nodes play dominant roles in the regulation of translocation. Elements taken up from the root surface are released into the xylem and delivered to the upper parts of the plant mainly in the transpiration stream. Nodes function as hubs for elements by dividing the route into two directions: toward the adjacent upper leaf blade and toward the upper internode (Yamaji and Ma 214, 217). Nodes also control the re-translocation of elements from the leaf blades to other parts. Transporters specific to Si, Zn, Cs, Cu, and Mn were distributed differently in nodes in a cell-specific manner (Yamaji and Ma 214, 217). Differences in the localization pattern of each transporter may explain the different flow directions and different accumulations of each element in each part of the plant (Yamaji and Ma 214, 217). Until now, no candidate Cs transporters have been reported in aboveground parts. Future work on such transporters may reveal why Cs accumulated most in the panicle neck. Possible mechanisms involved in the increased Cs accumulation in brown rice at low soil K Our results clearly show that the ratio of Cs accumulation in the brown rice to that in the straw increased significantly with decreasing soil-k levels (Fig. 4b,

14 516 Plant Soil (218) 429: a Soil A Soil B been fully elucidated. Our results suggest two possible mechanisms: transport from belowground parts and translocation from aboveground parts. Transfer factor 137 Cs conc. ratio (brown rice / straw) Soil A y = -.432x +.35 Soil B y = -.15x r 2 =.82***, slope: *, intercept: *** b Avg. of K conc. in soil solution ( µ M) y = -.142x r 2 =.81*** Avg. of K conc. in soil solution (µm) Fig. 4 Relationships between soil-k level and a transfer factor and b ratio of 137 Cs concentration in brown rice to that in straw (pot experiment 2). Each point represents the result from one pot (each with three plants). Dashed line in a, regression line for soil A; solid black line in a, regression line for soil B. In b, regression line (solid black one) is based on the combined data from the two soils. r 2, coefficient of determination. Significance: *P <.5; ***P <.1 Table 2). This result agrees well with previous reports (Fujimura et al. 214; Noboriet al. 214). It is noteworthy that there were only small differences between the two soils derived from different paddy fields in terms of the relationship between the soil-k level and the ratio of 137 Cs accumulation in the brown rice to that in the straw (Fig. 4b), despite clearly different transfer factors for 137 Cs (Fig. 4a). This means that the proportion of Cs accumulated in the brown rice to that in the wholeaboveground parts may depend strongly on the soil-k level irrespective of the soil. Thus far, the precise mechanisms involved in the changes in the proportion hasn t (1) Transport from belowground parts In both years, the whole-plant Cs increased during ripening in all K treatments (Table 1). This increase indicates that Cs was transported from belowground to aboveground parts during this period, although it was not clear whether the transported Cs was newly taken up from the soil during ripening or it had already accumulated in the roots before heading. Hereafter, we designate this process as transport from belowground parts without distinguishing whether the Cs was derived from the soil or the roots. The rate of increase of the wholeplant Cs during ripening increased with decreasing soil K(Table 1), suggesting that transport from belowground parts may accelerate at low soil K. Most of the Cs transported from the belowground parts seemed to be transported directly to the brown rice through the internodes, nodes, and rachis, because the amount of Cs in the leaf blades, which is another target of Cs transport from belowground, decreased during ripening (Table 1). Therefore, the preferential transfer of Cs by the direct route from belowground parts might be one reason for the increased accumulation of Cs in brown rice at low soil K. Future works are needed to elucidate dynamics of Cs in whole plants including roots, for example, by using hydroponic culture to avoid direct contamination of soil particles in root tissue samples. We clearly demonstrated that low soil K increased the exchangeable Cs concentration in the soil (Figs. 3c, f). This indicates that the increased exchangeable Cs concentration at low soil K might increase the total uptake of Cs from the soil by the roots, resulting to the increased accumulation of Cs in brown rice. The exchangeable Cs concentration at low soil K also increased in the field and in pot experiments with buckwheat (Kubo et al. 215). Cs in the soil is selectively fixed at the edges of opened layers of clay minerals, which have been called frayed edge sites (FES) (Sawhney 1972). During the growth period, especially when the plants increase dramatically in weight, K uptake by the plants would lower the K concentration in the soil solution; as a result, Cs fixed in the FES of clay minerals would be released simultaneously with the release of nonexchangeable K from clay minerals (Gommers et al. 25; Thiry et al. 25). This mechanism may explain why the increase in the

15 Plant Soil (218) 429: exchangeable Cs concentration was higher at lower soil K. Further investigation of the effect of low soil K on changes in the plant-available Cs will be necessary to clarify this phenomenon. (2) Translocation from aboveground parts Both the amount and concentration of Cs in leaf blades decreased during ripening in all K treatments in both years (Tables 1 and 2), indicating that Cs is translocated from the leaf blades to other plant parts during this period. We can ignore the possibility of Cs leaching by rain because our pot experiment was conducted in a glasshouse. The contribution of translocation from aboveground parts to accumulation in brown rice seemed to differ between 215 and 216. In 215, the increase of the whole-plant Cs during ripening was similar to or slightly lower than the sum of the decreases in Cs in all parts of the plant (Table 1). This suggests that the amount of Cs translocated from aboveground parts was similar to the amount transported from belowground parts. Therefore, both translocation from aboveground parts and direct transport from belowground parts seem to have contributed equally to the accumulation in brown rice. The rates of decrease in Cs concentrations in the upper and lower leaf blades during ripening in 215 were greater in the + mg K treatment than in the +45 mg K treatment (Table 2). This suggests that translocation from the leaf blades might accelerate at low soil K, resulting in increased accumulation of Cs in brown rice, however future precise studies are needed to confirm this. In 216, on the other hand, the increase in wholeplant Cs was greater than the sum of the decreases in all parts in all K treatments except the +3 mg K treatment (Table 1). This suggests that the contribution of transport from belowground parts was greater than that of translocation from aboveground parts. The rate of the translocation of Cs from leaf blades during the ripening period in 216 was not dependent on the soil-k level (Table 2). We cannot explain the difference between the two years by our data. One reason might be a difference in climate conditions, since the experiments were conducted in different seasons. In fact, the conditions of air temperature were different between the two years as described in Materials and methods. This is supported by the fact that the total dry weight and the harvest index were both higher in 216 than in 215 (Supp. Table S1). Future studies are needed to elucidate the reasons for this difference. Low soil K promoted the translocation of Cs from the leaf blades in 215. Kondo et al. (215) showed that the stem K concentration was negatively correlated with the proportion of Cs accumulated in the brown rice to that in the whole-aboveground parts. They considered that the competitive effect of K and Cs may govern the transfer of Cs from one part of the plant to another. It is noteworthy that, as we found here, K and Cs had similar distributions among the plant parts: they both accumulated most in the panicle neck (Fig. 1) and were translocated from the leaf blades to other plant parts during ripening (Tables 1 and 2). These results suggest that K and Cs might be transferred via a similar transport mechanism in the aboveground parts, so competition between the two elements might occur. The K concentration in leaf blades was lower at low soil K than at high soil K both at heading stage and at maturity (Table 2). This difference might result in the soil-k level-dependent differences in Cs translocation from leaf blades to other parts of the plants, possibly due to competition between the two elements. Further studies are required to clarify the involvement of K and Cs competition during translocation between plant parts, including the translocation of Cs from the roots to the aboveground parts. The identification of possible Cs transporters localized in each part of the plants will uncover the mechanism involved in the increased accumulation of Cs in brown rice at low soil K. Conclusion We revealed the dynamics of Cs in aboveground parts of rice plants during ripening. One novel finding is that Cs accumulated most in the panicle neck. We also revealed two potential mechanisms for the increase in the proportion of Cs accumulated in brown rice to that in wholeplant at low soil K: first, the transport of Cs from belowground parts might increase at low soil K; Cs might be preferentially and directly transported to the brown rice through the internodes and nodes during ripening. Second, translocation of Cs from leaf blades might be promoted at low soil K. These results will contribute to evaluation of risks for radiocesium accumulation in edible parts when plants are grown in contaminated soil and also to the development of cultivars that will reduce the accumulation of radiocesium in edible parts.