OsAMT1.1 Expression by Nitrate-Inducible Promoter of OsNAR2.1 Increases Nitrogen Use Efficiency and Rice Yield

doi:10.1016/j.rsci.2023.03.006

Abstract

Abstract:

Nitrate (NO₃^-) and ammonium (NH₄⁺) are two main inorganic nitrogen (N) sources during crop growth. Here, we enhanced the expression of OsAMT1.1, which encodes a NH₄⁺ transporter, using the NO₃^--inducible promoter of OsNAR2.1 and an ubiquitin promoter in transgenic rice plants. Under field condition of 120 kg /hm² N, agronomic N use efficiency, N recovery efficiency and N transport efficiency, and grain yield of the pOsNAR2.1:OsAMT1.1 transgenic lines were increased compared with those of the wild type (WT) and the pUbi:OsAMT1.1 transgenic plants. Under 2.0 mmol/L NO₃^- + 0.5 mmol/L NH₄⁺ and 0.5 mmol/L NO₃^- + 2.0 mmol/L NH₄⁺ conditions of hydroponic culture, compared with the WT, both biomass and total N content were increased in the pOsNAR2.1:OsAMT1.1 transgenic lines. However, biomass was significantly reduced in pUbi:OsAMT1.1 transgenic plants under 0.5 mmol/L NO₃^- + 2.0 mmol/L NH₄⁺ condition. The lines expressing pOsNAR2.1:OsAMT1.1 exhibited increased OsAMT1.1 expression and ¹⁵NH₄⁺ influx in roots under both 2.0 mmol/L NO₃^- + 0.5 mmol/L NH₄⁺ and 0.5 mmol/L NO₃^- + 2.0 mmol/L NH₄⁺ conditions. Our study showed that expression of OsAMT1.1 can be promoted when driven by the OsNAR2.1 promoter, especially under high-level nitrate condition, leading to enhancement of NH₄⁺ uptake, N use efficiency and grain yield.

Key words: OsAMT1.1, OsNAR2.1 promoter, nitrogen use efficiency, yield, rice

Jiang Hongzhen, Wang Yamei, Lai Liuru, Liu Xintong, Miao Changjian, Liu Ruifang, Li Xiaoyun, Tan Jinfang, Gao Zhenyu, Chen Jingguang. OsAMT1.1 Expression by Nitrate-Inducible Promoter of OsNAR2.1 Increases Nitrogen Use Efficiency and Rice Yield[J]. Rice Science, 2023, 30(3): 222-234.

Figures/Tables 8

Fig. 1. Effects of nitrogen levels on transcriptional expression of OsAMT1.1 and OsNAR2.1 in rice roots. A, Expression of OsAMT1.1. B, Expression of OsNAR2.1.Nipponbare seedlings were cultured in the IRRI solution containing 1.0 mmol/L NH4NO3 for three weeks and shifted to different N supply levels for one additional week. LN, 0.5 mmol/L NO3-; HN, 2.5 mmol/L NO3-; HNLA, 2.0 mmol/L NO3- + 0.5 mmol/L NH4+; LNHA, 0.5 mmol/L NO3- + 2.0 mmol/L NH4+; HA, 2.5 mmol/L NH4+. Actin1 gene of rice was used as an internal control. Data are Mean ± SE (n = 3). Different lowercase letters above the bars indicate significant differences at the 0.05 level.

Fig. 2. Characterization of pOsNAR2.1: OsAMT1.1 and pUbi:OsAMT1.1 transgenic lines (T3 generation). A, Gross morphology of wild type (WT), pOsNAR2.1:OsAMT1.1 (pN2 and pN5) and pUbi:OsAMT1.1 (pU2 and pU4) transgenic lines. Scale bar, 10 cm. B and C, Endogenous OsAMT1.1 expression in root (B) and leaf blade I (C). Actin1 gene of rice was used as an internal control. Data are Mean ± SE (n = 3).D, Grain yield and dry weight per plant. Mean dry weight values referred to all the biomass aboveground, including grain yield. Data are Mean ± SE (n = 4). Different lowercase letters above the bars for the same trait indicate significant differences at the 0.05 level.

Fig. 3. Comparison of agronomic traits in transgenic lines (T3 generation). A, Plant height. B, Total tiller number per plant. C, Effective tiller number per plant. D, Grain weight per panicle. E, Panicle length. F, Seed-setting rate. G, Grain number per panicle. H, 1000-grain weight. WT, Wild type; pN2 and pN5 are pOsNAR2.1:OsAMT1.1 transgenic lines while pU2 and pU4 are pUbi:OsAMT1.1 transgenic lines.Data are Mean ± SE (n = 4). Different lowercase letters above the bars indicate significant differences at the 0.05 level.

Fig. 4. Biomass and nitrogen (N) content in different parts of transgenic lines at flowering and maturity stages in fields. A and B, WT and T4 generation transgenic plants in the field. C-E, Dry weight (C), total N concentration (D) and total N content per plant (E) in different parts of transgenic lines and WT at the flowering stage. F-H, Dry weight (F), total N concentration (G) and total N content (H) in different parts of transgenic lines and WT at the maturity stage. WT, Wild type; pN2 and pN5 are pOsNAR2.1:OsAMT1.1 transgenic lines while pU2 and pU4 are pUbi:OsAMT1.1 transgenic lines.Data are Mean ± SE (n = 4). Different lowercase letters above the bars indicate significant differences at the 0.05 level.

Fig. 5. Biomass and nitrogen (N) accumulation of transgenic lines (T4 generation) in fields. A, Dry matter at the flowering stage. B, Dry matter at the maturity stage. C, Grain yield. D, Total N accumulation at the flowering stage. E, Total N accumulation at the maturity stage. F, Grain N accumulation at the maturity stage. WT, Wild type; pN2 and pN5 are pOsNAR2.1:OsAMT1.1 transgenic lines while pU2 and pU4 are pUbi:OsAMT1.1 transgenic lines.Data are Mean ± SE (n = 4). Different lowercase letters above the bars indicate significant differences at the 0.05 level.

Fig. 6. Comparison of nitrogen (N) use efficiency between WT and transgenic lines (T4 generation). A, Agronomic N use efficiency. B, N recovery efficiency. C, Physiological N use efficiency. D, N harvest index. E, Dry matter translocation efficiency. F, N translocation efficiency. WT, Wild type; pN2 and pN5 are pOsNAR2.1:OsAMT1.1 transgenic lines while pU2 and pU4 are pUbi:OsAMT1.1 transgenic lines.Data are Mean ± SE (n = 4). Different lowercase letters above the bars indicate significant differences at the 0.05 level.

Fig. 7. Comparison of growth and total nitrogen (N) content of transgenic lines at different N supply levels. A-C, Dry weights of seedlings treated with 0.5 mmol/L NO3- (A), 2.0 mmol/L NO3- + 0.5 mmol/L NH4+ (B), and 0.5 mmol/L NO3- + 2.0 mmol/L NH4+ (C).D-F, Total N concentration in roots and shoots in the pN lines, pU lines and WT under 0.5 mmol/L NO3- (D), 2.0 mmol/L NO3- + 0.5 mmol/L NH4+ (E), and 0.5 mmol/L NO3- + 2.0 mmol/L NH4+ (F). G-I, Total N content of roots and shoots, under 0.5 mmol/L NO3- (G), 2.0 mmol/L NO3- + 0.5 mmol/L NH4+ (H), and 0.5 mmol/L NO3- + 2.0 mmol/L NH4+ (I). WT, Wild type; pN2 and pN5 are pOsNAR2.1:OsAMT1.1 transgenic lines while pU2 and pU4 are pUbi:OsAMT1.1 transgenic lines.WT and transgenic rice seedlings were cultured in the IRRI solution containing 1.0 mmol/L NO3- for one week and then in different forms of N for two additional weeks.Data are Mean ± SE (n = 4). Different lowercase letters above the bars indicate significant differences at the 0.05 level.

Fig. 8. NH4+ and NO3- influx rates in transgenic lines using 15N enriched sources. A-C, 15NO3- influx rate (A), 15NH4+ influx rate (B) and ratio of 15NH4+ to 15NO3- influx (C) at 2.0 mmol/L 15NO3- + 0.5 mmol/L NH4+.D-F, 15NO3- influx rate (D), 15NH4+ influx rate (E) and ratio of 15NH4+ to 15NO3- influx (F) at 0.5 mmol/L 15NO3- + 2.0 mmol/L NH4+. Wild type (WT) and transgenic seedlings were grown in 1.0 mmol/L NO3- for three weeks and N starved for 3 d. Data are Mean ± SE (n = 4). Different lowercase letters above the bars indicate significant differences at P ≤ 0.05.

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