Appropriate Supply of Ammonium Nitrogen and Ammonium Nitrate Reduces Cadmium Content in Rice Seedlings by Inhibiting Cadmium Uptake and Transport

doi:10.1016/j.rsci.2024.02.007

Abstract

Abstract:

Reasonable nitrogen (N) application is a promising strategy for reducing crop cadmium (Cd) toxicity. However, the specific form of N and the required amount that affect Cd tolerance and accumulation in rice remain unclear. This study explored the influence of different N-fertilizer forms (NH₄NO₃, NH₄Cl, and KNO₃) and dosages on Cd tolerance and uptake in Cd-stressed N-sensitive and N-insensitive indica rice accessions. The results indicated that the Cd tolerance of N-sensitive indica accessions is more robust than that of N-insensitive ones. Furthermore, the shoot Cd content and Cd translocation rate in both N-sensitive and N-insensitive indica accessions decreased with an appropriate supply of NH₄NO₃ and NH₄Cl, whereas they were comparable or slightly increased with increased KNO₃. Unfortunately, we did not find significant and regular differences in Cd accumulation or translocation between N-sensitive and N-insensitive rice accessions. Consistent with the reduction of shoot Cd content, the addition of NH₄NO₃ and NH₄Cl also inhibited the instantaneous root Cd²⁺ uptake. The expression changes of Cd transport-related genes under different N forms and dosages suggested that the decreased shoot Cd content, caused by the increased supply of NH₄NO₃ and NH₄Cl, is likely achieved by reducing the transcription of OsNRAMP1 and OsIRT1. In summary, our findings reveal that an appropriate supply of NH₄NO₃ and NH₄Cl could reduce Cd uptake and transport in rice seedlings, suggesting that rational N management could reduce the Cd risk in rice production.

Key words: Oryza sativa, cadmium, nitrogen, ammonium nitrate, ammonium chloride

Hu Yunchao, Yan Tiancai, Gao Zhenyu, Wang Tiankang, Lu Xueli, Yang Long, Shen Lan, Zhang Qiang, Hu Jiang, Ren Deyong, Zhang Guangheng, Zhu Li, Li Li, Zeng Dali, Qian Qian, Li Qing. Appropriate Supply of Ammonium Nitrogen and Ammonium Nitrate Reduces Cadmium Content in Rice Seedlings by Inhibiting Cadmium Uptake and Transport[J]. Rice Science, 2024, 31(5): 587-602.

Figures/Tables 8

Fig. 1. Effects of different NH4NO3 dosages on expression of cadmium (Cd) absorption and transport-related genes in roots and shoots of four indica rice accession based on RNA-seq data. Gradient color blocks represent log2-transformed FPKM values. The black blocks indicate that no FPKM value is available. FPKM, Fragments per kilo bases per million fragments.

Fig. 2. Phenotypic characterization of N-sensitive Yuefeng B (A-D) and N-insensitive Zaogui 1 (E-H) rice seedlings grown under different cadmium (Cd) stress conditions with three NH4NO3 levels. A, Growth phenotypes of Yuefeng B treated with varying levels of Cd and NH4NO3 cultured outdoors from July to September 2023 in Hangzhou, China. B and C, Root length (B, n = 8) and shoot length (C, n = 8) of Yuefeng B, as shown in panel A. D, Root-shoot ratio (root dry weight / shoot dry weight, n = 4) of Yuefeng B, as shown in panel A. E, Growth phenotypes of Zaogui 1 treated with varying levels of Cd and NH4NO3, cultured outdoors from July to September 2023 in Hangzhou, China. F and G, Root length (F, n = 8) and shoot length (G, n = 8) of Zaogui 1, as shown in panel E. H, Root-shoot ratio (root dry weight / shoot dry weight, n = 4) of Zaogui 1, as shown in panel E. In A and E, the roots in the right panels are cropped and combined. Scale bars are 5 cm. In B-D and F-H, data are Mean ± SD. Different lowercase letters above the bars indicate significant differences at the P < 0.05 level by Tukey’s test following a one-way analysis of variance.

Fig. 3. Effects of three NH4NO3 levels on capacity of antioxidant enzyme system in N-sensitive Yuefeng B and N-insensitive Zaogui 1 rice seedlings under cadmium (Cd) stress. A-F, H2O2 content (A), malondialdehyde (MDA) content (B), superoxide dismutase (SOD) activity (C), peroxidase (POD) activity (D), catalase (CAT) activity (E), and ascorbate peroxidase (APX) activity (F) per gram of fresh shoots under 5 μmol/L Cd treatment. Data are Mean ± SD (n = 3). Different lowercase letters above the bars indicate significant differences at the P < 0.05 level by Tukey’s test following a one-way analysis of variance.

Fig. 4. Effects of different nitrogen forms and dosages on growth parameters, and cadmium (Cd) and manganese (Mn) content in N-sensitive Yuefeng B and N-insensitive Zaogui 1 rice seedlings. A and B, Root-shoot ratio (root dry weight / shoot dry weight, n = 3) of Yuefeng B (A) and Zaogui 1 (B) in the presence and absence of Cd treatment. C, Cd sensitivity coefficient (n = 3) for Yuefeng B and Zaogui 1 under 5 μmol/L Cd treatment. D and E, Cd content in shoots (D, n = 4) and roots (E, n = 4) per gram dry weight of rice seedlings under 5 μmol/L Cd treatment. F, Cd translocation rate (n = 4) in Yuefeng B and Zaogui 1 under 5 μmol/L Cd treatment. G and H, Mn content in shoots (G, n = 4) and roots (H, n = 4) per gram dry weight of rice seedlings under 5 μmol/L Cd treatment. Data are Mean ± SD. Different lowercase letters above the bars indicate significant differences at the P < 0.05 level by Tukey’s test following a one-way analysis of variance.

Fig. 5. Effects of NH4NO3 and NH4Cl on instantaneous uptake of cadmium ions (Cd2+) in N-sensitive Yuefeng B rice seedlings. A, Location of net Cd2+ flux detection at the elongation zone of rice roots (500 μm from the root tip). B, Net Cd2+ influx in Yuefeng B roots over time under different instantaneous treatments with NH4NO3 or NH4Cl. Data are Mean ± SE (n = 10). C, Mean Cd2+ influx in Yuefeng B roots as shown in panel B. Data are Mean ± SD (n = 10). Different lowercase letters indicate significant differences at the P < 0.05 level by Tukey’s test following to a one-way analysis of variance.

Fig. 6. Effects of different nitrogen forms and dosages on expression of Cd transport-related genes in cadmium (Cd)-stressed N-sensitive Yuefeng B and N-insensitive Zaogui 1 rice seedlings. A-F, Effects of various N forms on OsNRAMP1 expression in roots (A), OsNRAMP1 expression in shoots (B), OsNRAMP5 expression in roots (C), OsNRAMP5 expression in shoots (D), OsIRT1 expression in roots (E), and OsIRT1 expression in shoots (F) under 5 μmol/L Cd treatment. Transcripts of Cd transport-related genes are quantified from four replicates using the 2−∆CT method with OsACT2 as the reference gene. Data are Mean ± SD (n = 4). Different lowercase letters above the bars indicate significant differences at the P < 0.05 level by Tukey’s test following to a one-way analysis of variance.

Fig. 7. Effects of three NH4NO3 levels on growth parameters and cadmium (Cd) concentration in nramp5 mutant and its wild type Huanghuazhan (HHZ). A, Mutation site in nramp5. B, Root-shoot ratio (root dry weight / shoot dry weight) of HHZ and nramp5 in the presence and absence of Cd. C, Cd sensitivity coefficient for HHZ and nramp5. D, Cd content per gram shoot or root dry weight under 5 μmol/L Cd treatment. E, Cd translocation rate under 5 μmol/L Cd treatment. Data are Mean ± SD (n = 4). Different lowercase letters above the bars indicate significant differences at the P < 0.05 level by Tukey’s test following a one-way analysis of variance.

Fig. 8. Schematic diagram illustrating possible mechanism, by which NH4NO3 and NH4+-N alleviate cadmium (Cd) content. Compared with the control or with an increased supply of NO3-, increasing the supply of NH4NO3 and NH4+ decreased the gene expression of OsNRAMP1 and OsIRT1, thereby inhibiting Cd uptake and transport in roots, and ultimately reducing the Cd content in shoots. The red upward arrow represents the transfer of Cd from the root to shoot. The black upward arrow indicates an increased N dosage. The size of oval represents the corresponding gene expression level. The thickness of the arrow on the transporter indicates the flux of Cd absorption and transport. The size of red circle in the root represents the relative concentration of Cd.

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