Application of sodium chloride improves quinoa (Chenopodium quinoa Willd.) growth and cesium absorption under cesium stress

Application of sodium chloride improves quinoa (Chenopodium quinoa Willd.) growth and cesium absorption under cesium stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Application of sodium chloride improves quinoa (Chenopodium quinoa Willd.) growth and cesium absorption under cesium stress katsunori Isobe, Yuta Oku, Masao Higo This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8202702/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Quinoa contains epidermal bladder cells (leaf EBCs) on its surface that increase salt tolerance and facilitate the accumulation of unwanted substances. Additionally, cesium levels in the leaf EBCs are higher than in the leaf cells. Therefore, leaf EBCs are considered key for Cs absorption in quinoa. This study investigated whether, under Cs stress conditions 1) applying NaCl could restore quinoa growth and improve Cs absorption and 2) leaf EBCs maintained their Cs absorption capacity. K content in the leaf cells did not decrease upon CsCl treatment; therefore, CsCl-induced growth inhibition cannot be attributed to K deficiency. CsCl inhibited growth and this effect was rescued upon concomitant NaCl treatment. Cs content in the leaf cells was similar to or even higher than that when only CsCl was applied, suggesting that accumulated Cs in the leaf cells no longer inhibited growth or that Na had a growth-promoting effect. Phytoremediation of Cs can be achieved by applying NaCl, even when high soil Cs levels inhibit plant growth. Compared to leaf cells, Cs and K contents were higher in the leaf EBCs, regardless of CsCl treatment, indicating leaf EBC accumulation of these elements. Conversely, NaCl treatment led to higher Na content in the leaf cells than in the leaf EBCs, suggesting that when the internal Na content increases, quinoa does not actively transfer Na from the leaf cells to leaf EBCs. Therefore, leaf EBCs do not play a major role in salt tolerance in quinoa. Cesium Epidermal bladder cells Quinoa (Chenopodium quinoa Willd.) Sodium chloride Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction In Japan, a large amount of radiocesium (Cs-137) was released into the environment after the Fukushima Daiichi Nuclear Power Plant accident on March 11, 2011 (Chino et al. 2011; Fujii et al. 2014; Saravanan et al. 2025). Cesium does not easily diffuse into soil, and the half-life of Cs-137 is approximately 30 years; therefore, it remains in the soil for many years (Almgren and Isaksson 2006; Cao et al. 2022). The accumulation of Cs-137 in soil has long-term effects on human society, agriculture, and natural ecosystems (Ammar et al. 2024; Saravanan et al. 2025). Additionally, Cs is the most toxic element among the group I alkali metals (Sheahan et al. 1993; Adams et al. 2018; Hampton et al. 2004); even non-radioactive Cs, when accumulated in large amounts, can inhibit plant and organism growth. In Fukushima, the soil surface layer was removed to eliminate the Cs-137 accumulated due to the nuclear accident; however, this process is extremely costly and labor-intensive (Ito et al. 2012). Therefore, phytoremediation is considered an efficient method for extracting Cs from soil. However, phytoremediation of Cs is not yet practical because plants have a low capacity to absorb Cs (Tamaoki et al. 2016). Recent studies on the identification of proteins involved in Cs absorption and transport in plants have achieved significant progress in this area (Rai and Kawabata 2020). Phytoremediation becomes a sustainable solution for mitigating Cs contamination, notably for soil restoration and agricultural sustainability (Nurliati et al. 2024; Phang et al. 2024). Thus, we believe that plant-based Cs removal from soil will soon become a feasible technology. Quinoa (Chenopodium quinoa Willd.) is a pseudo-cereal native to South America with a high capacity for Cs absorption (Broadley et al. 1999; Isobe et al. 2019). Some quinoa cultivars grow to approximately 2 m tall and produce significant biomass among annual plants (Ali et al. 2020); furthermore, quinoa is a halophyte (Hussin et al. 2023). Applying an appropriate amount of NaCl enhances aboveground growth and increases Cs absorption (Isobe et al. 2019; Wada et al., Isobe et al. 2024). Quinoa contains epidermal bladder cells (leaf EBCs) on its surface, which are believed to increase salt tolerance and facilitate the accumulation of unwanted substances (Wada et al. 2020; Isobe et al. 2024; Palacios et al. 2024). Among the leaves, stems, and panicles, the leaves exhibit the highest Cs content, which remains stable after seed filling (Wada et al. 2020). Additionally, Cs levels in leaf EBCs are higher than those in leaf cells (Isobe et al. 2024). Therefore, leaf EBCs are considered key sites of Cs absorption in quinoa. However, excessive Cs accumulation in the soil may inhibit plant growth and Cs uptake due to toxicity. This study investigated whether applying NaCl could restore quinoa growth and enhance Cs absorption under Cs stress conditions. We also examined whether the leaf EBCs maintained their Cs absorption capacity under such stress. Materials and Methods Pot experiments were conducted in 2023 using the quinoa variety CICA-127. All experiments were performed in Wagner pots (1/5000a; diameter: 16 cm; height: 25 cm) filled with 2.5 kg of air-dried field soil (Andosol) of experimental field in Nihon University (Fujiswa-city, Kanagawa, Japan), 1.0 g of ammonium sulfate ((NH₄)₂SO₄; TORAY Ind., Tokyo, Japan), and 1.0 g of superphosphate (Katakura & Co-op Agri Corporation). In all experiments, samples of each pot served as one replicate, and a total of six replicates were prepared in experiments 1 and 3, and of five replicates were prepared in experiments 2 and 4. Experiment 1: Effects of the application of CsCl on the growth of above ground parts For the analysis of the effects of CsCl on the growth of the above-ground parts, five plots were prepared with different CsCl application rates. Overall, 0.3, 0.5, 0.7, and 1.0 g of CsCl (Wako Pure Chemical Industries, Tokyo, Japan) were applied, respectively; the control plot did not have CsCl. (NH₄)₂SO₄ and superphosphate were mixed equally with 2.5 kg of soil (Andosol), and CsCl was added to the top 5 cm of the soil; six pots were prepared per plot. Ten quinoa seeds per pot were sown on June 7, 2023, and after sowing, pots from all plots were placed randomly and independently in an artificial light-type air-conditioned room (KG-206, KOITO ELECTRIC INDUSTRIES LTD., Shizuoka, Japan). Day and night were set at 12 h each temperature was of 25 and 20 C, respectively. The seedlings were thinned to three plants per pot during the third leaf expansion stage. Weeds and diseases were controlled during the cultivation. Irrigation was performed upon observation of surface soil drying. On July 15, 2023, the plant height (length from the soil surface to the top) of quinoa plants in all plots was measured using a ruler, and the aboveground parts (stems and leaves) were cut at the soil surface. The fresh weights of the aboveground parts were measured using an electronic balance scale (GX-3000R, A&D Manufacturing Company Ltd., Ibaraki, Japan), and leaf areas were measured using a leaf area meter (LO-310, LO-COR Inc., Lincoln, NE, USA). The dry weight of above-ground parts was measured after oven drying the fresh samples at 80°C for 48 h. Dry leaf was ground to a powder using a blender (Force Mill FM-1, Osaka Chemical Co. Ltd., Osaka, Japan). To measure Cs content, 0.5 g of ground material was digested in 20.0 mL HClO4 (Kanto Chemical Co., Inc., Tokyo, Japan) for 3 h at 100°C using an acid digestion system, and Cs content was determined by atomic absorption spectrophotometry (iCE 3300 AAS Thermo Fisher Scientific, Waltham, MA, USA). The top 5 cm of the surface soil of each plot at sowing was air-dried to measure the total Cs content. After air-drying, the soil samples were sieved through a 2 mm mesh sieve for Cs analysis. Then, 1.0 g of soil samples was digested in 20.0 mL HClO4 for 3 h at 100°C using an acid digestion system, and Cs content was determined by atomic absorption spectrophotometry. Experiment 2: Effects of the application of CsCl on the Cs content of leaf cells and leaf EBCs For the analysis of the effects of CsCl application on the Cs content of leaf cells and leaf EBCs, three plots were prepared with different CsCl application rates. Overall, 0.3 and 0.6 g of CsCl were applied; the control plot did not have CsCl. (NH₄)₂SO₄ and superphosphate were mixed equally with 2.5 kg of soil (Andosol), and CsCl was added to the top 5 cm of the soil. A total of 50 pots were prepared per plot. Ten quinoa seeds per pot were sown on July 28, 2023, and after sowing, pots from all plots were placed randomly and independently in unheated vinyl (roof) and net (side) houses from sowing to sampling of the aboveground parts. On September 8, 2023, five pots from each plot were randomly selected for analysis of the growth of the aboveground parts. Plant height, fresh weight of aboveground parts, leaf area, and dry weight of aboveground parts were measured using the same method as previously described. The leaf EBCs on the leaves were collected from plants in 45 pots using a brush (Isobe et al. 2024). After leaf EBCs removal, leaf cells and leaf EBCs were dried at 80 ℃ for 48 h in a drying oven to analyze Cs and potassium (K) content. The dried leaf cells were ground into powder using a blender. To measure Cs and K content, 0.5 g of ground leaf and 0.2 g of dried leaf EBCs were digested in 20.0 mL HClO4 for 3 h at 100 ℃ using an acid digestion system, and Cs and K content was determined by atomic absorption spectrophotometry. The total Cs content of the soil at sowing was measured using the same method as previously described in Experiment 1. Experiment 3: Effects of the application of NaCl on the growth of above ground parts under Cs stress conditions For the analysis of the effects of NaCl application on the growth of above-ground parts under Cs stress conditions, six plots were prepared with different application rates of CsCl and NaCl. Overall, 0.5 g Cs/20 g Na and 0.7 g Cs/20 g Na were applied; the control plot did not have CsCl and NaCl. (NH₄)₂SO₄, superphosphate, and NaCl were mixed equally with 2.5 kg of soil (Andosol), and CsCl was added to the top 5 cm of the soil. Six pots were prepared for each plot. Ten quinoa seeds per pot were sown on July 21, 2023, and after sowing, pots from all plots were placed randomly and independently in an artificial light-type air-conditioned room. The day length and day and night temperatures were set at 12 h and 25 and 20°C, respectively. The seedlings were thinned to three plants per pot during the third leaf expansion stage. Weeds and diseases were controlled during the cultivation. Irrigation was performed upon observation of surface soil drying. On August 31, 2023, the aboveground parts were cut at the soil surface. Plant height, fresh weight of aboveground parts, leaf area, dry weight of aboveground parts, and Cs content of dried leaf cells were analyzed using the same methods as previously described. The Cs and Na contents of the soil at sowing were also analyzed using the same method as previously described. Experiment 4: Effects of the application of NaCl on the Cs content of leaf cells and leaf EBCs under Cs stress conditions For the analysis of the effects of NaCl application on the Cs content of leaf cells and leaf EBCs under Cs stress conditions, three plots were prepared with different application rates of CsCl and NaCl. Overall, 0.6 g Cs/20 g Na were applied; the control plot did not have CsCl and NaCl. (NH₄)₂SO₄ and superphosphate were mixed equally with 2.5 kg of soil (Andosol) and CsCl was added to the top 5 cm of the soil (Andosol). In all the plots, 100 pots were prepared. Ten quinoa seeds per pot were sown on September 2, 2023, and after sowing, pots from all plots were placed randomly and independently in unheated vinyl (roof) and net (side) houses from sowing to sampling of the aboveground parts. On October 31 and November 13, 2023, five pots from each plot was randomly selected for analysis of the growth of the aboveground parts. Plant height, fresh weight of aboveground parts, leaf area, and dry weight of aboveground parts were measured using the same methods as previously described. The leaf EBCs on the leaves were collected from plants in 45 pots using a brush on November 1 and 14, 2023. The Cs, Na, and K contents of the dried leaf cells and leaf EBCs were analyzed using the same methods as previously described. The Cs and Na contents of the soil at sowing were also analyzed using the same methods as previously described. Statistical analysis All values are expressed as averages and standard errors. Data were statistically analyzed, and significant differences at the 5% level among the plots were determined by multiple means test using Kaleida Graph ver. 4.0 software. Results Effects of CsCl application on the growth of the above ground parts The soil Cs content at sowing increased with the CsCl application rates; a significant difference was observed between all CsCl application plots and the control (Figs. 1 and 2). Conversely, plant height, leaf area, and the fresh and dry weights of the aboveground parts decreased with increasing CsCl application rates. A significant difference was observed in plant height at 0.5, 0.7, and 1.0 g Cs plots and the control. Regarding leaf area and fresh and dry weight of the aboveground parts, a significant difference was observed between 1.0 g Cs plots and the control (Table 1). A significant difference was observed in plant height, fresh and dry weights of the aboveground parts between the 0.6 g Cs plots and control (Table 2 and Fig. 3). In the 1.0 g Cs plot, it was not possible to collect a sufficient number of leaves required for Cs content analysis; In the 0.5 and 0.7 g Cs plots, the Cs content of the leaf cells significantly increased with the CsCl application rates compared with that of the control (Table 1). Effects of CsCl application on the Cs content of leaf cells and leaf EBCs The Cs content of the leaf cells and leaf EBCs increased with the CsCl application rates. A significant difference was observed in the Cs content of leaf cells and leaf EBCs between the 0.3 and 0.6 g Cs plots and that of the control. Cs content was significantly higher in leaf EBCs than that in leaf cells in all plots (Fig. 4). The K content of the leaf cells and leaf EBCs did not increase with increasing applications of CsCl, and it was significantly higher in leaf EBCs than that in leaf cells in all plots (Fig. 4). Effects of NaCl application on the growth of above ground parts under Cs stress conditions With only CsCl application, the Cs content of soil increased with the CsCl application rates; however, with the concomitant CsCl and NaCl application, the Cs content of soil did not increase. Soil Na content at sowing increased with NaCl applications. A significant difference was observed in the Na content of soil between all NaCl application plots and that of the control (Fig. 5). Moreover, a significant difference was observed in Na content of soil between the Cs 0.6g/Na 20g plot and that of the control and 0.6 g Cs plots (Fig. 6). Plant height, leaf area, and fresh and dry weights of the aboveground parts decreased only after CsCl application. A significant difference was observed in plant height between the 0.7 g Cs plots and that of the control. Conversely, plant height, leaf area, and the fresh and dry weights of the aboveground parts increased only with NaCl application. A significant difference was observed in leaf area and fresh weight of the aboveground parts between the 20 g Na plots and those of the control. Plant height, leaf area, and fresh and dry weights of the aboveground parts were increased by CsCl and NaCl application. A significant difference was observed in the fresh weight of above ground part between the 0.7 g Cs/20 g Na plots and that of the control. Even when CsCl was applied, the suppression of aboveground growth was not observed with the concomitant NaCl application. A significant difference was observed in plant height, leaf area, and fresh and dry weights of above ground parts between the Cs 0.7 g Cs/20 g Na plots and those of the 0.7 g Cs plot. A significant difference was observed in leaf area and fresh weight of the aboveground parts between the Cs 0.5 g Cs/20 g Na plots and the 0.5 g Cs plot (Table 3). The leaf Cs content increased with the application of CsCl. A significant difference was observed in the Cs content of leaf cells between all the CsCl application plots and that of control and 20 g Na plots (Table 3). The Na content of the leaf cells increased with NaCl application. A significant difference was observed in the Na content of the leaf cells between all NaCl application plots and that of control and no NaCl plots. The K content of leaf cells did not change with the application of CsCl or NaCl (Table 3). The growth of the aboveground parts was inhibited only by CsCl application. A significant difference was observed in leaf area and fresh and dry weights of the aboveground parts between the Cs 0.6 g Cs plots and those of the control. Even when CsCl was applied, the suppression of aboveground growth was not observed with the concomitant NaCl application. There were no significant differences in plant height, leaf area, and fresh and dry weights of above ground parts between the control and those of the Cs 0.6 g Cs/20 g Na plot. The growth of the aboveground parts was inhibited by CsCl application. A significant difference was observed in plant height and fresh and dry weights of above ground part between the 0.6g Cs plots and those of the control. Even when CsCl was applied, the aboveground growth was promoted by NaCl application. There were significant differences in leaf area and fresh weight of the aboveground parts between the control and those of the 0.6 g Cs and 0.6 g Cs/20 g Na plots (Table 4 and Fig. 7). Effects of NaCl application on the Cs content of leaf cells and leaf EBCs under Cs stress conditions The Cs content of the leaf cells and leaf EBCs increased with the CsCl application. Moreover, the Cs content in the leaf cells and leaf EBCs increased due to the application of NaCl. The Cs content of leaf EBCs was significantly higher than that of the leaf cells. Likewise, the Na content in the leaf cells and leaf EBCs increased with the NaCl application. There were significant differences in the Na content of leaf cells and leaf EBCs between those of the control and Cs 0.6 g Cs and 0.6 g Cs/20 g Na plots. the Na content of leaf EBCs was significantly higher than that of the leaf cells in the control and 0.6 g Cs plots. However, in the Cs 0.6 g Cs/20 g Na plot, the Na content of the leaf cells was significantly higher than that of the leaf EBCs. Moreover, the K content of the leaf cells increased with the CsCl application, unlike that of the leaf EBCs, which decreased with the CsCl application. There were significant differences in the K content of the leaf cells between the control and that of 0.6 g Cs and the 0.6 g Cs/20 g Na plots. The K content of leaf EBCs was significantly higher than that of the leaf cells (Table 5). Discussion Applying 0.5–0.6 g or more of CsCl (Fig. 1 , 2 , 5 , 6 ) to a 5000/a pot inhibited the above-ground growth of quinoa (Tables 1– 4 , Figs. 3 and 7 ). Plant growth inhibition results from a reduction in K absorption caused by Cs uptake (Adams et al. 2019). However, the inhibition of plant growth upon Cs uptake is not solely due to K deficiency, but also involves complex factors such as interference with K binding in proteins and disruption of the Cs/K ratio balance (Hampton et al. 2004; Le Lay et al. 2006; Adams et al. 2015; Adams et al. 2017). In this study, the K content in the leaf cells did not decrease even when CsCl was applied (Fig. 4 b, Tables 3 and 5 ). Thus, we suggest that the inhibition of quinoa growth caused by CsCl application is not due to K deficiency. Other possible factors for the inhibited growth of quinoa found in this study include Cs and Cl toxicity and increased osmotic pressure (Machado and Serralheiro 2017; Munns et al. 2019; Dubois and Inzé 2020; Lui et al. 2024). However, if Cs toxicity or increased osmotic pressure were the cause, quinoa growth would have been more severely inhibited when a large amount of NaCl was applied. As quinoa growth was not inhibited even when 20 g of NaCl per pot was applied (Tables 3 and 4 , Fig. 5 , 6 , 7 ), we believe that the growth inhibition of quinoa observed with the application of CsCl was not due to Cl or increased osmotic pressure. Thus, Cs primarily inhibited quinoa growth when CsCl was applied. The application of CsCl alone (Fig. 1 , 2 , 5 , and 6 ) inhibited the growth of aboveground parts (Tables 1– 4 ), which is thought to result from Cs absorbed into the plant (Fig. 4 a, Tables 1, 3 , and 5 ). However, the growth inhibition caused by CsCl was suppressed when NaCl was applied (Tables 3 and 4 ; Fig. 7 ). Therefore, the leaf CsCl content was expected to decrease when CsCl and NaCl were concomitantly applied (Figs. 5 and 7 ). However, the Cs content in the leaf cells was similar to or even higher than that when only CsCl was applied (Fig. 6 , Table 5 ). hence, although the leaf cells accumulated sufficient Cs to inhibit growth, the simultaneous application of NaCl prevented Cs-induced growth inhibition. This suggests that either the accumulated Cs in the leaf cells no longer inhibited growth or that Na had a growth-promoting effect. Previous studies on halophytes similar to quinoa, such as ice plants and Arabidopsis thaliana, have also reported that NaCl application promotes growth (Nakano and Watanabe 2018; Hongqiao et al. 2021; Eoh et al. 2024). Eoh et al. (2024) suggested that the reason for growth promotion in ice plants by NaCl was an increase in the accumulation of antioxidants, flavonoids, polyphenols, d-pinitol, and other substances. Hongqiao et al. (2021) also suggested that NaCl promotes sulfur absorption and the uptake of elements, such as Cl, Zn, and Cu in Arabidopsis thaliana, which in turn enhances photosynthesis and promotes plant growth. Furthermore, Subbarao et al. (2003) showed that when K is deficient, Na partially replaces the functions of K, such as osmotic pressure regulation in cells and enzyme activity. Since no K fertilizer was applied in any of the experiments in this study, it is possible that the increased quinoa growth with NaCl (Tables 3 and 4 ) was due to Na partially fulfilling the functions of K. However, the function of the absorbed NaCl within the quinoa plants remained unclear in the present study. Currently, the area of salt-accumulating land is expanding worldwide (Higashiyama et al. 2020), rendering the cultivation of halophytes essential for future food production. Quinoa, with its high salt tolerance, can be cultivated even in saline-alkali areas. Thus, it is necessary to clarify the mechanism by which NaCl application promotes quinoa growth. Additionally, it is believed that Cs phytoremediation using quinoa can be achieved by applying NaCl, even under conditions in which high soil Cs content inhibits plant growth. The leaf EBCs in quinoa are thought to accumulate waste products and growth-inhibiting substances to maintain the physiological functions of leaf cells and stems (Shabala et al. 2014; Kiani-Pouya et al. 2017). This study revealed that the accumulation of elements in leaf cells and leaf EBCs differed depending on the type. For example, the Cs and K contents were higher in the leaf EBCs than those in the leaf cells, regardless of the application of CsCl, indicating that these elements were actively accumulated in the leaf EBCs rather than in the leaf cells (Figs. 5 , 6 , Table 5 ). In contrast, the NaCl content was higher in leaf EBCs in the control condition and when only CsCl was applied. However, when NaCl was applied, Na content was higher in the leaf cells than in the leaf EBCs (Table 5 ). This result suggests that even when the internal Na content increases, quinoa does not actively transfer Na from the leaf cells to leaf EBCs. Moog et al. (2022) demonstrated that quinoa accumulates only trace amounts of Na in leaf EBCs, indicating that these cells do not play a major role in salt tolerance. Palacios et al. (2024) also reported that leaf EBCs do not accumulate to sequester excess Na from metabolically active cells. As mentioned, quinoa is a halophyte (Adolf et al. 2012; Kobayashi et al. 2025), and its growth is promoted by the application of Cs and NaCl (Saleem et al. 2017; Isobe et al. 2019). In the present study, the leaf area and shoot fresh weights were significantly higher in the NaCl-only plots than those in the control plots. Based on these findings, we believe that when quinoa absorbs large amounts of Na, it is retained it somatic cells and used to promote growth. In particular, because no K fertilizer was applied in this study, the absorbed Na may function similarly to K (Subbarao et al. 2003), thus promoting growth. In the present study, the NaCl application rate was set at 20 g per pot. It remains unclear whether the application of more NaCl induces stress and inhibits quinoa growth (Isobe et al., 2014; Isobe et al. 2019; Kobayashi et al. 2025). Therefore, future studies should clarify whether Na is actively transferred from leaf cells to leaf EBCs under Na-stress conditions. Conclusion The inhibition of quinoa growth by CsCl application was attributable to Cs rather than K deficiency. The growth inhibition caused by CsCl was suppressed when NaCl was applied. In addition, the Cs content in the leaf cells was similar to or even higher than that when only CsCl was applied. This suggests that either the accumulated Cs in the leaf cells no longer inhibited growth or that Na had a growth-promoting effect. It is believed that Cs phytoremediation in quinoa can be achieved by applying NaCl, even under conditions in which high Cs content in the soil inhibits plant growth. When NaCl was applied, Na content was higher in the leaf cells than in the leaf EBCs. This result suggests that even when the internal Na content increases, quinoa does not actively transfer Na from the leaf cells to leaf EBCs. Therefore, leaf EBCs do not play a major role in salt tolerance. Declarations Statements & Declarations Acknowledgements This research was supported by Ichimura Foundation for New Technology and College of Bioresource Sciences, Nihon University. Funding This research was conducted with the support of Ichimura Foundation for New Technology. We express our gratitude to the Ichimura Foundation. Author Contributions K. Isobe: Total coordination of the experiment. Y. Oku: Conducting the experiments 1 to 4. M. Higo: Statistical analysis. Ethical Approval This is not applicable Consent to Participate This is not applicable Consent to Publish This is not applicable Competing Interests The authors have no relevant financial or non-financial interests to disclose. Data Availability Statement Data available on request due to privacy/ethical restrictions. 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12:30:33","extension":"html","order_by":47,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":73069,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8202702/v1/efe309a2f32020f351035b56.html"},{"id":97702193,"identity":"2dac385f-ae30-410e-af46-6af427b0c235","added_by":"auto","created_at":"2025-12-08 12:30:31","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":69375,"visible":true,"origin":"","legend":"\u003cp\u003eTotal Cs content in the soil of each plot at sowing time.\u003c/p\u003e\n\u003cp\u003eValues followed by different letters are significantly different at \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, as determined by Tukey’s multiple range test\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8202702/v1/9da86c57322ef29b1c52bb8c.jpg"},{"id":97894905,"identity":"694c5017-eaf5-45aa-881c-0694b1fe85dc","added_by":"auto","created_at":"2025-12-10 15:33:11","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":54661,"visible":true,"origin":"","legend":"\u003cp\u003eTotal Cs content in the soil of each plot at sowing time.\u003c/p\u003e\n\u003cp\u003eValues followed by different letters are significantly different at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, as determined by Tukey’s multiple range test\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8202702/v1/49203dcdec3f202b2df8aa74.jpg"},{"id":97702194,"identity":"cb21fae3-b509-460e-bff0-a72ff0b3e683","added_by":"auto","created_at":"2025-12-08 12:30:31","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":106026,"visible":true,"origin":"","legend":"\u003cp\u003eQuinoa growth of each plot\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8202702/v1/55ff3dbf4bc97337c34f4b72.jpg"},{"id":97894661,"identity":"1a79bd3c-daf1-41fc-a024-66dc2088bb21","added_by":"auto","created_at":"2025-12-10 15:32:52","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":57542,"visible":true,"origin":"","legend":"\u003cp\u003eTotal Cs (a) and K (b) contents in leaf cells and epidermal bladder cells.\u003c/p\u003e\n\u003cp\u003eValues followed by different letters are significantly different at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, as determined by Tukey’s multiple range test.**,*, Statistically significant at \u003cem\u003eP\u003c/em\u003e≦0.01, and 0.05 by t-test, respectively\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8202702/v1/9a35b9ee47393af9fb6f0195.jpg"},{"id":97702196,"identity":"210eda60-19bf-4581-b4ea-d695301319e8","added_by":"auto","created_at":"2025-12-08 12:30:31","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":56544,"visible":true,"origin":"","legend":"\u003cp\u003eTotal Cs and Na contents in the soil at sowing time.\u003c/p\u003e\n\u003cp\u003eValues followed by different letters are significantly different at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, as determined by Tukey’s multiple range test\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8202702/v1/9d02a0a152cfb182b964f2d5.jpg"},{"id":97894767,"identity":"8ef94aac-b39a-431b-bccc-cf541a0a3cb4","added_by":"auto","created_at":"2025-12-10 15:33:00","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":33422,"visible":true,"origin":"","legend":"\u003cp\u003eTotal Cs and Na contents in the soil at sowing time.\u003c/p\u003e\n\u003cp\u003eValues followed by different letters are significantly different at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, according to Tukey’s multiple range test\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8202702/v1/631bd3db04f59a46d4269725.jpg"},{"id":97702202,"identity":"ad74bde4-2c77-411f-a97f-86e2609324cd","added_by":"auto","created_at":"2025-12-08 12:30:32","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":75483,"visible":true,"origin":"","legend":"\u003cp\u003eQuinoa growth of each plot with concomitant application of Na\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8202702/v1/a676386327b4640cb1489219.jpg"},{"id":101943401,"identity":"4116591a-67ab-4028-ad74-1b89737cff1f","added_by":"auto","created_at":"2026-02-05 09:41:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1192630,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8202702/v1/1ba16c2b-a668-40cb-b309-ba165b58c652.pdf"},{"id":97702199,"identity":"39587add-5b76-4d79-a3d3-f0e5d907f777","added_by":"auto","created_at":"2025-12-08 12:30:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":104963,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8202702/v1/53240b9fa8f8b0a29871dc8c.docx"}],"financialInterests":"","formattedTitle":"Application of sodium chloride improves quinoa (Chenopodium quinoa Willd.) growth and cesium absorption under cesium stress","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn Japan, a large amount of radiocesium (Cs-137) was released into the environment after the Fukushima Daiichi Nuclear Power Plant accident on March 11, 2011 (Chino et al. 2011; Fujii et al. 2014; Saravanan et al. 2025). Cesium does not easily diffuse into soil, and the half-life of Cs-137 is approximately 30 years; therefore, it remains in the soil for many years (Almgren and Isaksson 2006; Cao et al. 2022). The accumulation of Cs-137 in soil has long-term effects on human society, agriculture, and natural ecosystems (Ammar et al. 2024; Saravanan et al. 2025). Additionally, Cs is the most toxic element among the group I alkali metals (Sheahan et al. 1993; Adams et al. 2018; Hampton et al. 2004); even non-radioactive Cs, when accumulated in large amounts, can inhibit plant and organism growth. In Fukushima, the soil surface layer was removed to eliminate the Cs-137 accumulated due to the nuclear accident; however, this process is extremely costly and labor-intensive (Ito et al. 2012). Therefore, phytoremediation is considered an efficient method for extracting Cs from soil. However, phytoremediation of Cs is not yet practical because plants have a low capacity to absorb Cs (Tamaoki et al. 2016). Recent studies on the identification of proteins involved in Cs absorption and transport in plants have achieved significant progress in this area (Rai and Kawabata 2020). Phytoremediation becomes a sustainable solution for mitigating Cs contamination, notably for soil restoration and agricultural sustainability (Nurliati et al. 2024; Phang et al. 2024). Thus, we believe that plant-based Cs removal from soil will soon become a feasible technology.\u003c/p\u003e\u003cp\u003eQuinoa (Chenopodium quinoa Willd.) is a pseudo-cereal native to South America with a high capacity for Cs absorption (Broadley et al. 1999; Isobe et al. 2019). Some quinoa cultivars grow to approximately 2 m tall and produce significant biomass among annual plants (Ali et al. 2020); furthermore, quinoa is a halophyte (Hussin et al. 2023). Applying an appropriate amount of NaCl enhances aboveground growth and increases Cs absorption (Isobe et al. 2019; Wada et al., Isobe et al. 2024). Quinoa contains epidermal bladder cells (leaf EBCs) on its surface, which are believed to increase salt tolerance and facilitate the accumulation of unwanted substances (Wada et al. 2020; Isobe et al. 2024; Palacios et al. 2024). Among the leaves, stems, and panicles, the leaves exhibit the highest Cs content, which remains stable after seed filling (Wada et al. 2020). Additionally, Cs levels in leaf EBCs are higher than those in leaf cells (Isobe et al. 2024). Therefore, leaf EBCs are considered key sites of Cs absorption in quinoa. However, excessive Cs accumulation in the soil may inhibit plant growth and Cs uptake due to toxicity. This study investigated whether applying NaCl could restore quinoa growth and enhance Cs absorption under Cs stress conditions. We also examined whether the leaf EBCs maintained their Cs absorption capacity under such stress.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003ePot experiments were conducted in 2023 using the quinoa variety CICA-127. All experiments were performed in Wagner pots (1/5000a; diameter: 16 cm; height: 25 cm) filled with 2.5 kg of air-dried field soil (Andosol) of experimental field in Nihon University (Fujiswa-city, Kanagawa, Japan), 1.0 g of ammonium sulfate ((NH₄)₂SO₄; TORAY Ind., Tokyo, Japan), and 1.0 g of superphosphate (Katakura \u0026amp; Co-op Agri Corporation). In all experiments, samples of each pot served as one replicate, and a total of six replicates were prepared in experiments 1 and 3, and of five replicates were prepared in experiments 2 and 4.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eExperiment 1: Effects of the application of CsCl on the growth of above ground parts\u003c/h2\u003e\u003cp\u003eFor the analysis of the effects of CsCl on the growth of the above-ground parts, five plots were prepared with different CsCl application rates. Overall, 0.3, 0.5, 0.7, and 1.0 g of CsCl (Wako Pure Chemical Industries, Tokyo, Japan) were applied, respectively; the control plot did not have CsCl. (NH₄)₂SO₄ and superphosphate were mixed equally with 2.5 kg of soil (Andosol), and CsCl was added to the top 5 cm of the soil; six pots were prepared per plot. Ten quinoa seeds per pot were sown on June 7, 2023, and after sowing, pots from all plots were placed randomly and independently in an artificial light-type air-conditioned room (KG-206, KOITO ELECTRIC INDUSTRIES LTD., Shizuoka, Japan). Day and night were set at 12 h each temperature was of 25 and 20 C, respectively. The seedlings were thinned to three plants per pot during the third leaf expansion stage. Weeds and diseases were controlled during the cultivation. Irrigation was performed upon observation of surface soil drying.\u003c/p\u003e\u003cp\u003eOn July 15, 2023, the plant height (length from the soil surface to the top) of quinoa plants in all plots was measured using a ruler, and the aboveground parts (stems and leaves) were cut at the soil surface. The fresh weights of the aboveground parts were measured using an electronic balance scale (GX-3000R, A\u0026amp;D Manufacturing Company Ltd., Ibaraki, Japan), and leaf areas were measured using a leaf area meter (LO-310, LO-COR Inc., Lincoln, NE, USA). The dry weight of above-ground parts was measured after oven drying the fresh samples at 80\u0026deg;C for 48 h. Dry leaf was ground to a powder using a blender (Force Mill FM-1, Osaka Chemical Co. Ltd., Osaka, Japan). To measure Cs content, 0.5 g of ground material was digested in 20.0 mL HClO4 (Kanto Chemical Co., Inc., Tokyo, Japan) for 3 h at 100\u0026deg;C using an acid digestion system, and Cs content was determined by atomic absorption spectrophotometry (iCE 3300 AAS Thermo Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e\u003cp\u003eThe top 5 cm of the surface soil of each plot at sowing was air-dried to measure the total Cs content. After air-drying, the soil samples were sieved through a 2 mm mesh sieve for Cs analysis. Then, 1.0 g of soil samples was digested in 20.0 mL HClO4 for 3 h at 100\u0026deg;C using an acid digestion system, and Cs content was determined by atomic absorption spectrophotometry.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExperiment 2: Effects of the application of CsCl on the Cs content of leaf cells and leaf EBCs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor the analysis of the effects of CsCl application on the Cs content of leaf cells and leaf EBCs, three plots were prepared with different CsCl application rates. Overall, 0.3 and 0.6 g of CsCl were applied; the control plot did not have CsCl. (NH₄)₂SO₄ and superphosphate were mixed equally with 2.5 kg of soil (Andosol), and CsCl was added to the top 5 cm of the soil. A total of 50 pots were prepared per plot. Ten quinoa seeds per pot were sown on July 28, 2023, and after sowing, pots from all plots were placed randomly and independently in unheated vinyl (roof) and net (side) houses from sowing to sampling of the aboveground parts.\u003c/p\u003e\u003cp\u003eOn September 8, 2023, five pots from each plot were randomly selected for analysis of the growth of the aboveground parts. Plant height, fresh weight of aboveground parts, leaf area, and dry weight of aboveground parts were measured using the same method as previously described.\u003c/p\u003e\u003cp\u003eThe leaf EBCs on the leaves were collected from plants in 45 pots using a brush (Isobe et al. 2024). After leaf EBCs removal, leaf cells and leaf EBCs were dried at 80 ℃ for 48 h in a drying oven to analyze Cs and potassium (K) content. The dried leaf cells were ground into powder using a blender. To measure Cs and K content, 0.5 g of ground leaf and 0.2 g of dried leaf EBCs were digested in 20.0 mL HClO4 for 3 h at 100 ℃ using an acid digestion system, and Cs and K content was determined by atomic absorption spectrophotometry. The total Cs content of the soil at sowing was measured using the same method as previously described in Experiment 1.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExperiment 3: Effects of the application of NaCl on the growth of above ground parts under Cs stress conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor the analysis of the effects of NaCl application on the growth of above-ground parts under Cs stress conditions, six plots were prepared with different application rates of CsCl and NaCl. Overall, 0.5 g Cs/20 g Na and 0.7 g Cs/20 g Na were applied; the control plot did not have CsCl and NaCl. (NH₄)₂SO₄, superphosphate, and NaCl were mixed equally with 2.5 kg of soil (Andosol), and CsCl was added to the top 5 cm of the soil. Six pots were prepared for each plot. Ten quinoa seeds per pot were sown on July 21, 2023, and after sowing, pots from all plots were placed randomly and independently in an artificial light-type air-conditioned room. The day length and day and night temperatures were set at 12 h and 25 and 20\u0026deg;C, respectively. The seedlings were thinned to three plants per pot during the third leaf expansion stage. Weeds and diseases were controlled during the cultivation. Irrigation was performed upon observation of surface soil drying.\u003c/p\u003e\u003cp\u003eOn August 31, 2023, the aboveground parts were cut at the soil surface. Plant height, fresh weight of aboveground parts, leaf area, dry weight of aboveground parts, and Cs content of dried leaf cells were analyzed using the same methods as previously described. The Cs and Na contents of the soil at sowing were also analyzed using the same method as previously described.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExperiment 4: Effects of the application of NaCl on the Cs content of leaf cells and leaf EBCs under Cs stress conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor the analysis of the effects of NaCl application on the Cs content of leaf cells and leaf EBCs under Cs stress conditions, three plots were prepared with different application rates of CsCl and NaCl. Overall, 0.6 g Cs/20 g Na were applied; the control plot did not have CsCl and NaCl. (NH₄)₂SO₄ and superphosphate were mixed equally with 2.5 kg of soil (Andosol) and CsCl was added to the top 5 cm of the soil (Andosol). In all the plots, 100 pots were prepared. Ten quinoa seeds per pot were sown on September 2, 2023, and after sowing, pots from all plots were placed randomly and independently in unheated vinyl (roof) and net (side) houses from sowing to sampling of the aboveground parts. On October 31 and November 13, 2023, five pots from each plot was randomly selected for analysis of the growth of the aboveground parts. Plant height, fresh weight of aboveground parts, leaf area, and dry weight of aboveground parts were measured using the same methods as previously described. The leaf EBCs on the leaves were collected from plants in 45 pots using a brush on November 1 and 14, 2023. The Cs, Na, and K contents of the dried leaf cells and leaf EBCs were analyzed using the same methods as previously described. The Cs and Na contents of the soil at sowing were also analyzed using the same methods as previously described.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll values are expressed as averages and standard errors. Data were statistically analyzed, and significant differences at the 5% level among the plots were determined by multiple means test using Kaleida Graph ver. 4.0 software.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEffects of CsCl application on the growth of the above ground parts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe soil Cs content at sowing increased with the CsCl application rates; a significant difference was observed between all CsCl application plots and the control (Figs. 1 and 2). Conversely, plant height, leaf area, and the fresh and dry weights of the aboveground parts decreased with increasing CsCl application rates. A significant difference was observed in plant height at 0.5, 0.7, and 1.0 g Cs plots and the control. Regarding leaf area and fresh and dry weight of the aboveground parts, a significant difference was observed between 1.0 g Cs plots and the control (Table 1). A significant difference was observed in plant height, fresh and dry weights of the aboveground parts between the 0.6 g Cs plots and control (Table 2 and Fig. 3). In the 1.0 g Cs plot, it was not possible to collect a sufficient number of leaves required for Cs content analysis; In the 0.5 and 0.7 g Cs plots, the Cs content of the leaf cells significantly increased with the CsCl application rates compared with that of the control (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of CsCl application on the Cs content of leaf cells and leaf EBCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Cs content of the leaf cells and leaf EBCs increased with the CsCl application rates. A significant difference was observed in the Cs content of leaf cells and leaf EBCs between the 0.3 and 0.6 g Cs plots and that of the control. Cs content was significantly higher in leaf EBCs than that in leaf cells in all plots (Fig. 4). The K content of the leaf cells and leaf EBCs did not increase with increasing applications of CsCl, and it was significantly higher in leaf EBCs than that in leaf cells in all plots (Fig. 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of NaCl application on the growth of above ground parts under Cs stress conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWith only CsCl application, the Cs content of soil increased with the CsCl application rates; however, with the concomitant CsCl and NaCl application, the Cs content of soil did not increase. Soil Na content at sowing increased with NaCl applications. A significant difference was observed in the Na content of soil between all NaCl application plots and that of the control (Fig. 5). Moreover, a significant difference was observed in Na content of soil between the Cs 0.6g/Na 20g plot and that of the control and 0.6 g Cs plots (Fig. 6). Plant height, leaf area, and fresh and dry weights of the aboveground parts decreased only after CsCl application. A significant difference was observed in plant height between the 0.7 g Cs plots and that of the control. Conversely, plant height, leaf area, and the fresh and dry weights of the aboveground parts increased only with NaCl application. A significant difference was observed in leaf area and fresh weight of the aboveground parts between the 20 g Na plots and those of the control. Plant height, leaf area, and fresh and dry weights of the aboveground parts were increased by CsCl and NaCl application. A significant difference was observed in the fresh weight of above ground part between the 0.7 g Cs/20 g Na plots and that of the control. Even when CsCl was applied, the suppression of aboveground growth was not observed with the concomitant NaCl application. A significant difference was observed in plant height, leaf area, and fresh and dry weights of above ground parts between the Cs 0.7 g Cs/20 g Na plots and those of the 0.7 g Cs plot. A significant difference was observed in leaf area and fresh weight of the aboveground parts between the Cs 0.5 g Cs/20 g Na plots and the 0.5 g Cs plot (Table 3). The leaf Cs content increased with the application of CsCl. A significant difference was observed in the Cs content of leaf cells between all the CsCl application plots and that of control and 20 g Na plots (Table 3). The Na content of the leaf cells increased with NaCl application. A significant difference was observed in the Na content of the leaf cells between all NaCl application plots and that of control and no NaCl plots. The K content of leaf cells did not change with the application of CsCl or NaCl (Table 3).\u003c/p\u003e\n\u003cp\u003eThe growth of the aboveground parts was inhibited only by CsCl application. A significant difference was observed in leaf area and fresh and dry weights of the aboveground parts between the Cs 0.6 g Cs plots and those of the control. Even when CsCl was applied, the suppression of aboveground growth was not observed with the concomitant NaCl application. There were no significant differences in plant height, leaf area, and fresh and dry weights of above ground parts between the control and those of the Cs 0.6 g Cs/20 g Na plot. The growth of the aboveground parts was inhibited by CsCl application. A significant difference was observed in plant height and fresh and dry weights of above ground part between the 0.6g Cs plots and those of the control. Even when CsCl was applied, the aboveground growth was promoted by NaCl application. There were significant differences in leaf area and fresh weight of the aboveground parts between the control and those of the 0.6 g Cs and 0.6 g Cs/20 g Na plots (Table 4 and Fig. 7).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of NaCl application on the Cs content of leaf cells and leaf EBCs under Cs stress conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Cs content of the leaf cells and leaf EBCs increased with the CsCl application. Moreover, the Cs content in the leaf cells and leaf EBCs increased due to the application of NaCl. The Cs content of leaf EBCs was significantly higher than that of the leaf cells. Likewise, the Na content in the leaf cells and leaf EBCs increased with the NaCl application. There were significant differences in the Na content of leaf cells and leaf EBCs between those of the control and Cs 0.6 g Cs and 0.6 g Cs/20 g Na plots. the Na content of leaf EBCs was significantly higher than that of the leaf cells in the control and 0.6 g Cs plots. However, in the Cs 0.6 g Cs/20 g Na plot, the Na content of the leaf cells was significantly higher than that of the leaf EBCs. Moreover, the K content of the leaf cells increased with the CsCl application, unlike that of the leaf EBCs, which decreased with the CsCl application. There were significant differences in the K content of the leaf cells between the control and that of 0.6 g Cs and the 0.6 g Cs/20 g Na plots. The K content of leaf EBCs was significantly higher than that of the leaf cells (Table 5).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eApplying 0.5\u0026ndash;0.6 g or more of CsCl (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) to a 5000/a pot inhibited the above-ground growth of quinoa (Tables\u0026nbsp;1\u0026ndash;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Plant growth inhibition results from a reduction in K absorption caused by Cs uptake (Adams et al. 2019). However, the inhibition of plant growth upon Cs uptake is not solely due to K deficiency, but also involves complex factors such as interference with K binding in proteins and disruption of the Cs/K ratio balance (Hampton et al. 2004; Le Lay et al. 2006; Adams et al. 2015; Adams et al. 2017). In this study, the K content in the leaf cells did not decrease even when CsCl was applied (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Thus, we suggest that the inhibition of quinoa growth caused by CsCl application is not due to K deficiency. Other possible factors for the inhibited growth of quinoa found in this study include Cs and Cl toxicity and increased osmotic pressure (Machado and Serralheiro 2017; Munns et al. 2019; Dubois and Inz\u0026eacute; 2020; Lui et al. 2024). However, if Cs toxicity or increased osmotic pressure were the cause, quinoa growth would have been more severely inhibited when a large amount of NaCl was applied. As quinoa growth was not inhibited even when 20 g of NaCl per pot was applied (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), we believe that the growth inhibition of quinoa observed with the application of CsCl was not due to Cl or increased osmotic pressure. Thus, Cs primarily inhibited quinoa growth when CsCl was applied.\u003c/p\u003e\u003cp\u003eThe application of CsCl alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) inhibited the growth of aboveground parts (Tables\u0026nbsp;1\u0026ndash;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e4\u003c/span\u003e), which is thought to result from Cs absorbed into the plant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, Tables\u0026nbsp;1, \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e3\u003c/span\u003e, and \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, the growth inhibition caused by CsCl was suppressed when NaCl was applied (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Therefore, the leaf CsCl content was expected to decrease when CsCl and NaCl were concomitantly applied (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). However, the Cs content in the leaf cells was similar to or even higher than that when only CsCl was applied (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e5\u003c/span\u003e). hence, although the leaf cells accumulated sufficient Cs to inhibit growth, the simultaneous application of NaCl prevented Cs-induced growth inhibition. This suggests that either the accumulated Cs in the leaf cells no longer inhibited growth or that Na had a growth-promoting effect. Previous studies on halophytes similar to quinoa, such as ice plants and Arabidopsis thaliana, have also reported that NaCl application promotes growth (Nakano and Watanabe 2018; Hongqiao et al. 2021; Eoh et al. 2024). Eoh et al. (2024) suggested that the reason for growth promotion in ice plants by NaCl was an increase in the accumulation of antioxidants, flavonoids, polyphenols, d-pinitol, and other substances. Hongqiao et al. (2021) also suggested that NaCl promotes sulfur absorption and the uptake of elements, such as Cl, Zn, and Cu in Arabidopsis thaliana, which in turn enhances photosynthesis and promotes plant growth. Furthermore, Subbarao et al. (2003) showed that when K is deficient, Na partially replaces the functions of K, such as osmotic pressure regulation in cells and enzyme activity. Since no K fertilizer was applied in any of the experiments in this study, it is possible that the increased quinoa growth with NaCl (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e4\u003c/span\u003e) was due to Na partially fulfilling the functions of K. However, the function of the absorbed NaCl within the quinoa plants remained unclear in the present study. Currently, the area of salt-accumulating land is expanding worldwide (Higashiyama et al. 2020), rendering the cultivation of halophytes essential for future food production. Quinoa, with its high salt tolerance, can be cultivated even in saline-alkali areas. Thus, it is necessary to clarify the mechanism by which NaCl application promotes quinoa growth. Additionally, it is believed that Cs phytoremediation using quinoa can be achieved by applying NaCl, even under conditions in which high soil Cs content inhibits plant growth.\u003c/p\u003e\u003cp\u003eThe leaf EBCs in quinoa are thought to accumulate waste products and growth-inhibiting substances to maintain the physiological functions of leaf cells and stems (Shabala et al. 2014; Kiani-Pouya et al. 2017). This study revealed that the accumulation of elements in leaf cells and leaf EBCs differed depending on the type. For example, the Cs and K contents were higher in the leaf EBCs than those in the leaf cells, regardless of the application of CsCl, indicating that these elements were actively accumulated in the leaf EBCs rather than in the leaf cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In contrast, the NaCl content was higher in leaf EBCs in the control condition and when only CsCl was applied. However, when NaCl was applied, Na content was higher in the leaf cells than in the leaf EBCs (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This result suggests that even when the internal Na content increases, quinoa does not actively transfer Na from the leaf cells to leaf EBCs. Moog et al. (2022) demonstrated that quinoa accumulates only trace amounts of Na in leaf EBCs, indicating that these cells do not play a major role in salt tolerance. Palacios et al. (2024) also reported that leaf EBCs do not accumulate to sequester excess Na from metabolically active cells. As mentioned, quinoa is a halophyte (Adolf et al. 2012; Kobayashi et al. 2025), and its growth is promoted by the application of Cs and NaCl (Saleem et al. 2017; Isobe et al. 2019). In the present study, the leaf area and shoot fresh weights were significantly higher in the NaCl-only plots than those in the control plots. Based on these findings, we believe that when quinoa absorbs large amounts of Na, it is retained it somatic cells and used to promote growth. In particular, because no K fertilizer was applied in this study, the absorbed Na may function similarly to K (Subbarao et al. 2003), thus promoting growth. In the present study, the NaCl application rate was set at 20 g per pot. It remains unclear whether the application of more NaCl induces stress and inhibits quinoa growth (Isobe et al., 2014; Isobe et al. 2019; Kobayashi et al. 2025). Therefore, future studies should clarify whether Na is actively transferred from leaf cells to leaf EBCs under Na-stress conditions.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe inhibition of quinoa growth by CsCl application was attributable to Cs rather than K deficiency. The growth inhibition caused by CsCl was suppressed when NaCl was applied. In addition, the Cs content in the leaf cells was similar to or even higher than that when only CsCl was applied. This suggests that either the accumulated Cs in the leaf cells no longer inhibited growth or that Na had a growth-promoting effect. It is believed that Cs phytoremediation in quinoa can be achieved by applying NaCl, even under conditions in which high Cs content in the soil inhibits plant growth. When NaCl was applied, Na content was higher in the leaf cells than in the leaf EBCs. This result suggests that even when the internal Na content increases, quinoa does not actively transfer Na from the leaf cells to leaf EBCs. Therefore, leaf EBCs do not play a major role in salt tolerance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eStatements \u0026amp; Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by\u0026nbsp;Ichimura Foundation for New Technology\u0026nbsp;and College of Bioresource Sciences, Nihon University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was conducted with the support of Ichimura Foundation for New Technology. We express our gratitude to the Ichimura Foundation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK. Isobe: Total coordination of the experiment. Y. Oku: Conducting the experiments 1 to 4. M. Higo: Statistical analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis is not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis is not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis is not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData available on request due to privacy/ethical restrictions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdams E, Chaban V, Khandelia H, Shin R (2015) Selective chemical binding enhances cesium tolerance in plants through inhibition of cesium uptake. 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Cesium-insensitive mutants of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. The Plant Journal 3(5):647\u0026ndash;656\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSubbarao GV, Ito O, Berry WL, Wheeler RM (2003) Sodium-A functional plant nutrient. Crit Rev Plant Sci 22:391\u0026ndash;416\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTamaoki M, Yabe T, Furukawa J, Watanabe M, Ikeda K, Yasutani I, Nishizawa T (2016) Comparison of potentials of higher plants for phytoremediation of radioactive cesium from contaminated soil. Environ Control Biol 54:65\u0026ndash;69\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWada K, Takagi R, Horikoshi M, Higo M, Isobe K (2020) Effects of NaCl application on cesium accumulation in the aboveground parts of quinoa (\u003cem\u003eChenopodium quinoa\u003c/em\u003e Willd.). Water Air Soil Pollut 231:552\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cesium, Epidermal bladder cells, Quinoa (Chenopodium quinoa Willd.), Sodium chloride","lastPublishedDoi":"10.21203/rs.3.rs-8202702/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8202702/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eQuinoa contains epidermal bladder cells (leaf EBCs) on its surface that increase salt tolerance and facilitate the accumulation of unwanted substances. Additionally, cesium levels in the leaf EBCs are higher than in the leaf cells. Therefore, leaf EBCs are considered key for Cs absorption in quinoa. This study investigated whether, under Cs stress conditions 1) applying NaCl could restore quinoa growth and improve Cs absorption and 2) leaf EBCs maintained their Cs absorption capacity. K content in the leaf cells did not decrease upon CsCl treatment; therefore, CsCl-induced growth inhibition cannot be attributed to K deficiency. CsCl inhibited growth and this effect was rescued upon concomitant NaCl treatment. Cs content in the leaf cells was similar to or even higher than that when only CsCl was applied, suggesting that accumulated Cs in the leaf cells no longer inhibited growth or that Na had a growth-promoting effect. Phytoremediation of Cs can be achieved by applying NaCl, even when high soil Cs levels inhibit plant growth. Compared to leaf cells, Cs and K contents were higher in the leaf EBCs, regardless of CsCl treatment, indicating leaf EBC accumulation of these elements. Conversely, NaCl treatment led to higher Na content in the leaf cells than in the leaf EBCs, suggesting that when the internal Na content increases, quinoa does not actively transfer Na from the leaf cells to leaf EBCs. Therefore, leaf EBCs do not play a major role in salt tolerance in quinoa.\u003c/p\u003e","manuscriptTitle":"Application of sodium chloride improves quinoa (Chenopodium quinoa Willd.) growth and cesium absorption under cesium stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-08 12:30:27","doi":"10.21203/rs.3.rs-8202702/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a6268ae3-3c0d-44b0-8ccf-3da92a3212e8","owner":[],"postedDate":"December 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-21T18:53:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-08 12:30:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8202702","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8202702","identity":"rs-8202702","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}