Plant material and growth conditions
The rice (Oryza sativa L.) variety Kitaake (WT) was used in this study. The homozygous osnia1 and osnia3 knockout mutant lines (Kitaake background; generated by CRISPR/Cas9), an OsNia3 overexpression (OE) line, and an osnia3 mutant line with restored OsNia3 expression (RG) were identified and screened in Jiangxi Agricultural University. The specific mutation for each line is shown in Table S1. In the OE and RG lines, exogenous OsNIA3 was constitutively expressed using the CamV35S promoter in both WT and osnia3 knockout mutant plants. Construction of the carrier and vector transformation was undertaken in the laboratory of Wan Jianmin, Institute of Crop Science, Chinese Academy of Agricultural Sciences.
Experiments were performed at the Key Laboratory of Crop Physiology, Ecology, and Genetics Breeding of Jiangxi Agricultural University and Rice Research Institute of Jiangxi Academy of Agriculture, Nanchang, Jiangxi, P.R. China. A hydroponic experiment was used to analyze the expression characteristics of the OsNia3 gene and the effects of the osnia3 and osnia1 mutations on N utilization and the responses of the two mutant lines to different types of N in rice seedlings. Seedlings were cultured in a hydroponic nutrient solution prepared according to the formula of the International Rice Research Institute. After 5 days of growth, rice seedlings were cultured in nutrient solution with potassium nitrate, ammonium nitrate, or ammonium sulfate as N sources (the N content was identical in the three nutrient solutions; the ammonium: N concentration ratio was 0:1:2, respectively) (Table S2). Three repetitions were performed for the different forms of N application. Samples were taken after 15 days of culture in nutrient solution. Two rows (16 plants) were randomly mixed for each sampling and 3 samples were repeatedly obtained per condition for storage at −80 °C. In the phenotype identification test, the materials were planted in the field with conventional fertilization.
Phylogenetic analysis and comparison of gene structures and protein sequences
AtNia1/2 and OsNia1/2/3 gene sequences were blasted against the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Primers were designed for PCR amplification and the amplification products was sequenced by Tsingke Biotechnology Co., Ltd (Wuhan, China). Phylogenetic analysis of the amino acid sequences of AtNIA1/2 and OsNIA1/2/3 was undertaken in Mega 6.0 using a maximum likelihood method. The OsNIA1 and OsNIA3 protein sequences were compared using Geneious software (Biomatters Ltd, Auckland, New Zealand). All primers were designed using primer-BLAST at NCBI (https://www.ncbi.nlm.nih.gov/tools/primer- blast/) (Table S2).
Nitrogen reductase (NR) activity
NR activity was determined according to the method described by Lea et al. (2006). Three samples were tested for each treatment group in each replicate experiment. Leaves (0.5 g) were homogenized in 4 mL of 0.1 M HEPES-KOH (pH 7.5), 3% (w/v) PVP, 1 mM EDTA, and 7 mM Cys. The assay mixture (2 mL total volume) contained 50 mM HEPES-KOH (pH 7.5), 100 mM NADH, and 5 mM KNO3 with 2 mM EDTA. Activity was measured in crude extracts by determining NO2− formation following the addition of 1% (w/v) sulfanilamide and 0.2% (w/v) N-(1-naphthyl)ethylenediamine dihydrochloride in 3 M HCl. The NR activation state (% active NR) can be defined as NR activity assayed in the presence of Mg2+ (and 14-3-3 proteins) as a percentage of NR activity measured in the presence of EDTA and reflects how much of the enzyme is in the non-phosphorylated, active form. Assays were run at 25 °C.
Nitrate, ammonium, and free amino acid contents
Rice leaves (0.5 g) were weighed and added to 10 mL of double-distilled water. Nitrate, ammonium, and amino acids were extracted using boiling water for 30 min. Three samples were tested for each treatment group in each replicate experiment. The nitrate content in rice leaves was determined using the nitrosalicylic acid colorimetric method (Cataldo et al. 1975). The ammonium content in rice leaves was determined using indophenol blue colorimetry (Hachiya et al. 2012). The amino acid content in rice leaves was determined using the ninhydrin method (Xu et al. 2017).
Chlorophyll was extracted from shoots with 80% (v/v) acetone after which the chlorophyll content was determined spectrophotometrically (Lattanzio et al. 2009). Three samples were tested for each treatment group in each replicate experiment.
RNA extraction, cDNA preparation, and qPCR
Total RNA was isolated from the leaves of seedlings using a Takara MiniBEST Plant RNA Extraction Kit (Takara, Beijing, China). Approximately 0.5 μg of total RNA was reverse-transcribed to cDNA using PrimeScript RT Master Mix (Takara). qPCR was performed on a CFX96 Real-Time PCR Detection System (Bio-Rad, California, USA) using SYBR Premix Ex Taq II (Takara). Data were analyzed using Opticon Monitor Software (Bio-Rad). Three technical replicates from one of three biological replicates were performed for each gene. Rice Ubiquitin1 (LOC4327162) was used as the internal reference gene. The expression of the following genes related to N metabolism and photoperiod regulation network for rice heading (flowering) was evaluated: OsNia1 (LOC4345795), OsNia2 (LOC4330867), OsNia3 (LOC4345798), OsNiR (LOC4330835), OsNRT 2.1 (LOC4328051), OsNRT 2.3a (LOC4324249), OsGS (LOC4337272), OsFDGS (LOC4344164), OsNGS1 (LOC4324398), OsNGS2 (LOC4339561), OsAMT1.1 (LOC4336365), OsEhd1 (LOC107276289), OsHd3a (LOC4340185), OsGhd7 (LOC107276161), OsMADS51 (LOC4342744), OsGI (LOC4325329), OsRFT1 (LOC4340184), and OsHd1 (LOC4340429). All primers were designed using primer-BLAST at NCBI (https://www.ncbi.nlm.nih.gov/tools/primer- blast/) (Table S2).
A 2-kb promoter region of OsNia3 was amplified from Kitaake genomic DNA and cloned into pCAMBIA1305.1-GFP to generate OsNia3promoter:GUS and the resulting vector was transformed into Kitaake. For GUS staining, root, leaf sheath, leaf blade, stem, and stem node tissues of OsNia3promoter:GUS transgenic rice were immersed in X-Gluc (5-bromo-4-chloro-3-indolyl-b-d- glucuronic acid/cyclohexyl ammonium salt) staining solution containing 100 mM sodium phosphate (pH 7.0), 0.1% Triton X-100, 10 mM EDTA (pH 8.0), 2% DMSO, 0.1% X-Gluc, 1 mM K3[Fe(CN)6], 1 mM K4[Fe(CN)6]·3H2O, and 5% methanol (Jefferson 1989). After staining for 1 h at 37 °C, the samples were dehydrated using an ethanol series (70%, 85%, 95%, and 100%) to remove the chlorophyll. The stained tissues were observed under an Olympus SZX16 stereomicroscope (Olympus, Tokyo, Japan) and imaged using a Nikon D700 digital camera (Nikon, Tokyo, Japan).
To investigate the subcellular localization of OsNIA3, 35S:OsNia3-GFP fusion constructs were generated by inserting the open reading frame of OsNia3 into the pCAMBIA1390-35S:GFP vector. Plasmids were extracted and purified using the Plasmid Midi Kit (No. 12143) (Qiagen, Hilden, Germany) following the manufacturer’s manual. The plasmid was sent to Biorun Biotechnology Co., Ltd (Wuhan, China) for protoplast transformation of green seedlings of rice. Confocal microscopy was used for observation of subcellular localization with chloroplast autofluorescence (red) serving as the control.
Means and standard errors of the means were calculated from independent samples in Microsoft Excel 2007 (Microsoft, Washington, USA). SPSS Statistics 22 (IBM SPSS Inc, New York, USA) was used for statistical and correlation analysis and the least significant difference (LSD) method was used to determine whether differences between means were significant. P-values <0.05 were considered significant.