GoKawiilPlant materials
The mutants nir2-1, nir2-2, gln2, gln3, gln4, gln5 and gln6 seeds were obtained from the mutant library (http://maizeems.qlnu.edu.cn/) and generated in the WT (B73 background). The ZmNIR2T1OE and ZmNIR2T2OE overexpression lines were generated in the WT (B73 background). The mutants nir1 and gln1 were created by Center for Crop Functional Genomics and Molecular Breeding of China Agricultural University and generated in the WT (ND101 background). The inbred lines and teosinte accessions were propagated by our own research team. The ZmNIR2T1OE-12 and ZmNIR2T1OE-25 overexpression lines were generated in the WT (YH7 background). All the maize genetic variants were grown in the experimental fields in Wenjiang, Sichuan Province (30.7° N, 103.8° E), Sanya, Hainan Province (18.2° N, 109.3° E) and Harbin, Heilongjiang Province (44.0° N, 125.4° E). Genomic accession numbers for the genes studied are ZmNIR1 (Zm00001eb255880), ZmNIR2 (Zm00001eb193660), ZmGLN1 (Zm00001eb432580), ZmGLN2 (Zm00001eb054990), ZmGLN3 (Zm00001eb399860), ZmGLN4 (Zm00001eb253820), ZmGLN5 (Zm00001eb190340) and ZmGLN6 (Zm00001eb009090).
Plant-growth conditions
Maize seeds were disinfected with 75% ethanol for 3 min, followed by four rinses with deionized water. Disinfected seeds were then soaked in saturated calcium sulfate solution for 5 h and subsequently placed on moist absorbent paper in a dark, humid environment at 28 °C to induce germination. After germination, seedlings were transferred to vermiculite and cultivated in a growth chamber with a 16-h light/8-h dark photoperiod at 28°C during the light periods and 22°C during the dark period. The relative humidity was 60% and the white-light illumination was 350 μmol m−2 s−1. Maize seedlings were cultivated in deionized water until they developed a single true leaf and a coleoptile, after which the seed coat and endosperm were removed. Plants were then treated with modified Hoagland nutrient solution containing varying concentrations of nitrogen (KCl: 0 mM N; LN: 0.04 mM N; NN: 4 mM N; HN: 8 mM N) for 11 days, after which physiological indices were measured.
Tobacco seeds were grown in a 1.5:1 mixture of vermiculite and substrate for 2 weeks. Subsequently, the seedlings were treated with modified Hoagland nutrient solution containing two nitrogen concentrations for 15 days, after which physiological indices were measured. The cultivation took place in a growth chamber with a 14 h light/10 h dark photoperiod (28 °C day/22 °C night), 60% relative humidity and 200 μmol m−2 s−1 white-light intensity.
Wheat seeds were soaked in deionized water at 4 °C for 12 h, then were placed in a dark environment at room temperature for 1 day. After germination, the seedlings were transferred to vermiculite and cultivated in a growth chamber with a 12 h light/12 h dark photoperiod (28 °C day/22 °C night), 50% relative humidity and 300 μmol m−2 s−1 white-light intensity. The plants were then treated with modified Hoagland nutrient solution containing two nitrogen concentrations for 11 days, after which physiological indices were measured.
Rice seeds were soaked in 0.5% hydrogen peroxide at 37 °C in darkness for 24 h, then transferred to deionized water for immersion until germination occurred, after which the seedlings were moved to a hydroponic cultivation system. The seedlings were transferred to vermiculite and cultivated in a growth chamber with a 12 h light/12 h dark photoperiod (25 °C day/20 °C night), 50% relative humidity, and 350 μmol m−2 s−1 white-light intensity. The plants were then treated with modified Hoagland nutrient solution containing two nitrogen concentrations for 21 days, after which physiological indices were measured.
Soybean seeds were surface-sterilized with 5% sodium hypochlorite for 10 min, followed by three rinses with distilled water. The sterilized seeds were germinated in a greenhouse using a 1.5:1 mixture of vermiculite and substrate. At the V1 stage, uniform and healthy seedlings were selected and transplanted into pots containing an autoclaved 1.5:1 mixture of vermiculite and substrate. The seedlings were transferred to vermiculite and cultivated in a growth chamber with a 16 h light/8 h dark photoperiod (25 °C day/22 °C night), 70% relative humidity and 300 μmol m−2 s−1 white-light intensity. Thereafter, plants were divided into groups and regularly supplied with the modified Hoagland nutrient solution prepared with two nitrogen concentrations. After 19 days of treatment, plant materials were harvested for subsequent physiological and biochemical analyses.
Transient expression in tobacco leaves
Transient expression assays were performed in Nicotiana benthamiana L. leaves by infiltrating with Agrobacterium tumefaciens (GV3101) harbouring the specified constructs. Following infiltration, plants were cultivated under white light (200 μmol m−2 s−1) for 48 h before analysis. To express plant proteins in tobacco leaves, pCAMBIA3100-eGFP and pCAMBIA2300-mCherry plasmids were used as the expression vectors. Except for the coding sequence of the PG-marker PSY3, which was cloned into KpnI/BamHI sites of pCAMBIA2300-mCherry, all other coding sequences for subcellular localization observation (including the full-length and various truncated versions of ZmNIRs, ZmGLNs and others) were inserted into pCAMBIA3100-eGFP using the same restriction sites. Gene sequence alignment was performed using SnapGene v6.0.2.
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