DOI: 10.5061/dryad.18931zd4b
title: A DUF630 and 632 domains-containing protein, ZmNRL1, acts as a positive regulator of nitrogen stress response in maize

description: Nitrogen (N) is a key macronutrient whose availability often determines
 maize growth and productivity. Improving nitrogen use efficiency (NUE) is
 critical to increase maize yield while reducing N input, and more
 importantly, alleviating environmental pollution. However, only a few
 genes have been exploited for maize NUE improvement thus far. Here, we
 identified 44 candidate genes associated with NUE-related traits by
 performing a genome-wide association analysis in a maize natural
 population. We further found that the natural variations in ZmNRL1,
 encoding a DUF630 and DUF632 domains-containing protein, strongly
 associated with chlorophyll content under N starvation. Loss function of
 ZmNRL1 reduced nitrogen content and weakened plant growth under hydroponic
 and soil conditions, whereas overexpression of ZmNRL1 conferred better
 tolerance to N stress and elevated yields in transgenic maize and
 Arabidopsis. Comparative transcriptome analysis further revealed that
 ZmNRL1 has a broad impact on the expression of many N utilization and
 signaling genes. Moreover, we showed that ZmNRL1 anchored to the plasma
 membrane likely through the dual lipid modifications of myristoylation and
 palmitoylation. Thus, we propose that ZmNRL1 is a key regulator of the
 adaptation response to N limitation in maize and could be a potential
 target for breeding high-yield maize with enhanced NUE.
description: # A DUF630 and 632 domains-containing protein, ZmNRL1, acts as a positive
 regulator of nitrogen stress response in maize
 [https://doi.org/10.5061/dryad.18931zd4b](https://doi.org/10.5061/dryad.18931zd4b) ## Description of the data and file structure ### Fig.\_2.\_All\_sequence\_of\_ZmNRL1\_from\_43\_inbred\_lines.fasta. * Description: The resequencing of ZmNRL1 data of 43 maize inbred lines. ~4.6 kb genomic region was amplified using specific primers designed based on B73 genome sequences. ### Figure 1B.csv * Description: Box-plots of six NUE-related traits in a maize collection with different origins. Described in Materials and Methods: GWAS Analysis. * Variables: * Categories: TST: Tropical/subtropical; Mixed: mixed origin populations; NSS: non-stiff stalk; SS: stiff stalk. * "NA" in some cells indicates missing values. ### Figure 2A.csv * Description: Association study of ZmNRL1 with CC in a maize population. Described in Materials and Methods: ZmNRL1-based Association Analysis with CC. * Variables: * chrom: Maize Chromosomes * pos: Maize gene physical position. ### Figure3C\_D\_E.csv * Description: Figure3C: Leaf chlorophyll content and Chlorophyll degradation rate (%). Wildtype and zmnrl1 mutants (zmnrl1-e and zmnrl1-m) were treated for 11 days under different solutions containing different concentrations of KNO3 (0 mM, 0.15 mM or 15 mM), and the leaf chlorophyll content and degradation rate were scored and measured. Described in Materials and Methods: Experimental design and phenotypic data collection. * Description: Figure3D: The length of fourth leaf from Wildtype and zmnrl1 mutants (zmnrl1-e and zmnrl1-m) were measured after different solutions for 11 d. Described in Materials and Methods: Experimental design and phenotypic data collection. * Description:Figure3E: Total dry weight of wildtype and zmnrl1 mutants (zmnrl1-e and zmnrl1-m) were harvested and measured after different concentrations of KNO3 for 11 d. Described in Materials and Methods: Experimental design and phenotypic data collection. * Variables: * Genotype: different plant materials: wildtype (WT), zmnrl1-e (zmnrl1 EMS mutant), zmnrl1-m (zmnrl1 Mu insertion mutant). * Treatment: Hoagland solutions containing concentrations of KNO3 (0 mM, 0.15 mM, or 15 mM). * Chl_content: maize the second leaf chlorophyll content (SPAD values). * Total dry weight (g/plant): The dry weight of per plant. * "NA" in some cells indicates missing values. ### Figure4B\_C\_D\_E\_F.csv * Description: Under low-nitrate (0.15 mM NO3-) or high-nitrate (15 mM NO3-) conditions for treatment 6 d, the quantitative measurement of the leaf soluble protein contents (Figure 4B), quantitative measurement of nitrate contents (Figure 4C and D), quantitative measurement of the Nitrate Reductase enzyme activity (NR) of root and leaf tissues (Figure 4E and F) from Wild type, zmnrl1-e and zmnrl1-m plants. The methods as described in Materials and Methods, "Measurements of Nitrate, Protein Contents, and NR Enzyme Activity". * Variables: * Genotype: different plant materials: wildtype (WT), zmnrl1-e (zmnrl1 EMS mutant), zmnrl1-m (zmnrl1 Mu insertion mutant). * Treatment: Hoagland solutions containing concentrations of KNO3 (0.15 mM or 15 mM). * Nitrate content: root and leaf nitrate content under different solutions (0.15 mM and 15 mM N). * Nitrate reductase activity: root and leaf nitrate reductase activity under different solutions (0.15 mM and 15 mM N). * "NA" in some cells indicates missing values. ### Figure5C\_D\_E\_F\_G\_H\_I\_J\_K.csv * Description: ZmNRL1-overexpressing transgenic plants (ZmNRL1-OE19, ZmNRL1-OE111) and wildtype were grown hydroponically under different N conditions (0 mM, 0.15 mM, and 15 mM) for 11 d, chlorophyll content of the second leaf (Figure 5C) between ZmNRL1-OEs and wild type was scored. After treatment for 6 d under different N conditions, the fresh weight of root and above-ground tissues (Figure 5D and E), and dry weight per plant (Figure 5F) from ZmNRL1-OEs (ZmNRL1-OE19, ZmNRL1-OE111) and wild type were measured. The quantitative measurement of the leaf soluble protein contents (Figure 5G), the quantitative measurement of nitrate contents (Figure 5 H and I), and the quantitative measurement of the Nitrate Reductase enzyme activity (NR) of root and leaf tissues (Figure 5J and K) from ZmNRL1-OEs and wild type plants. * Variables: * Genotype: different plant materials: wildtype (WT), ZmNRL1-OE19 (ZmNRL1 overexpression line: OE19), ZmNRL1-OE111 (ZmNRL1 overexpression line: OE111). * Treatment: Hoagland solutions containing concentrations of KNO3 (0 mM, 0.15 mM or 15 mM). * Aboveground fresh weight: the above-ground fresh biomass weight per plant * Dry weight: The total dry weight of per plant. * Nitrate content: root and leaf nitrate content under different solutions (0.15 mM and 15 mM N). * Nitrate reductase activity: root and leaf nitrate reductase activity under different solutions (0.15 mM and 15 mM N). * "NA" in some cells indicates missing values. ### Figure6.csv * Description: qRT-PCR analysis of N-related genes in wild type, zmnrl1-m and ZmNRL1-OE111 plants. ZmActin was used as an internal control. There were two independent experiments. * Variables: * Genotype: different plant materials: wildtype (WT), zmnrl1-m (zmnrl1 Mu insertion mutant), ZmNRL1-OE111 (ZmNRL1 overexpression line: OE111). * Treatment: Hoagland solutions containing concentrations of KNO3 (0.15 mM or 15 mM). * ZmNRT2.1: Maize high-affinity NO3- transporter gene 2.1 relative expression * ZmNRT2.2: Maize high-affinity NO3- transporter gene 2.2 relative expression * ZmASN4: Maize asparagine synthetase 4 relative expression ### Figure7D.csv * Description: ZmNRL1-GFP and ZmNRL1(G2AC3S)-GFP were grown on MS medium for 7 d and then transplanted to low-N conditions for 8 d. G2AC3S indicates non-N myristoylated and non-S-acylated variant. The chlorophyll content of Wildtype and ZmNRL1-GFP or ZmNRL1(G2ACS3)-GFP lines was measured. * Variables: * Genotype: different plant materials: wildtype (WT), ZmNRL1-OE (ZmNRL1 overexpression lines in Arabidopsis, OE4 and OE6), ZmNRL1(G2AC3S)-OE (ZmNRL1 overexpression line in Arabidopsis, OE9 and OE14). * Treatment: MS liquid medium without nitrogen (-N) and +N. ### Figure8B\_C\_D\_E\_F\_G\_I\_K.csv * Description: V3-stage seedlings of wild type, zmnrl1-m, and ZmNRL1-OEs (OE19, OE111) were transplanted into different N-conditions, investigating adult plant traits. Aboveground and root fresh weights (g) of wild type, zmnrl1-m, and ZmNRL1-OEs (Figure 8B and C). (Figure 8D-G) measurment total nitrogen content in different tissues, in ear leaf(Figure 8D), Roots (Figure 8E), Stem (Figure 8F) and Kernel (Figure 8G). Under normal field conditions, comparisons of plant height and dry weight (Figure 8I), hundred-grain weight, and grain yield per plant (K) of wild type, zmnrl1-m, and ZmNRL1-OEs. * Variables: * Genotype: different plant materials: wildtype (WT), zmnrl1-m (zmnrl1 Mu insertion mutant), ZmNRL1-OE111 (ZmNRL1 overexpression line: OE111). * Aboveground/root fresh weight: the above-ground and root fresh biomass weight per plant under different N conditions (0.15 mM and 15 mM) * Total N: total nitrogen content in tissues, such as ear leaf, root, stem, and kernel under different N conditions (0.15 mM and 15 mM) * Dry weight: The total dry weight of per plant under normal growth conditions. * 100-kernel weight: kernel weight per hundred under normal growth conditions. * Grain yield per plant: The dry weight of whole kernels per plant under normal growth conditions. * Treatment: Hoagland solutions containing concentrations of KNO3 (0.15 mM or 15 mM). * "NA" in some cells indicates missing values. ### FigureS3B.csv * Description: Analysis of GWAS candidate genes relative expression. RNA-seq data were retrieved from McLoughlin et al., 2018. * Variables: * 2nd_leaf_W22_1/2/3+N: The second leaf of W22 background under high nitrogen conditions. * 2nd_leaf_W22_1/2/3-N: The second leaf of W22 background under low nitrogen conditions. * 4th_leaf_W22_1/2/3+N: The fourth leaf of W22 background under high nitrogen conditions. * 4th_leaf_W22_1/2/3-N: The fourth leaf of W22 background under low nitrogen conditions. ### FigureS5B\_D.csv * Description: qRT-PCR analysis of ZmNRL1 transcripts in various tissues from different stages (Figure S5B). 7-d-old seedlings grown on normal solutions were transferred to N-free solution for 2 d for qRT-PCR analysis (Figure S5D). * Variables: * Genotype: wildtype B73 * Tissues: Maize B73 different tissues under normal growth conditions. * Time: Nitrogen-free solution for 0 h or 48 h. * "NA" in some cells indicates missing values. ### FigureS7C\_D.csv * Description: Wildtype, zmnrl1-e, and zmnrl1-m mutants were grown on soil for 3 weeks and watered with Hoagland solutions containing 0.15 mM and 15 mM KNO3. The chlorophyll content and aboveground dry weight of wildtype and zmnrl1 mutants were measured. * Variables: * Genotype: different plant materials: wildtype (WT), zmnrl1-m (zmnrl1 Mu insertion mutant), zmnrl1-e (zmnrl1 EMS mutant). * Treatment: Hoagland solutions containing concentrations of KNO3 (0 mM, 0.15 mM, or 15 mM). * Dry weight: The total dry weight of per plant. ### FigureS9A\_E\_F.csv * Description: qRT-PCR analysis ZmNRL1 transcripts in maize ZmNRL1-OEs overexpression lines (Figure S9A). Wildtype, ZmNRL1-OE19, and ZmNRL1-OE111 plants were grown on soil for 3 weeks and watered with Hoagland solutions containing 0.15 mM and 15 mM KNO3. The aboveground and root dry weight of wildtype andZmNRL1-OEs plants were measured(Figure S9E,F). * Variables: * Genotype: different plant materials: wildtype (WT), ZmNRL1-OE19 (ZmNRL1 overexpression line: OE19), ZmNRL1-OE111 (ZmNRL1 overexpression line: OE111). * Treatment: Hoagland solutions containing concentrations of KNO3 (0 mM, 0.15 mM, or 15 mM). * Aboveground/root fresh weight: the above-ground and root fresh biomass weight per plant under different N conditions (0 mM, 0.15 mM, and 15 mM) ### FigureS12A\_D\_E\_F\_G\_H\_J\_K.csv * Description: qRT-PCR analysis ZmNRL1 transcripts in Arabidospsis overexpression lines (Figure S12A). All Arabidopsis lines (Wildtype, AtOE1, AtOE2) were grown on MS medium for 7 d and then transferred to 1/2 MS liquid medium contaning 0.1 mM KNO3 for 8 d. Measurements of souble protein content (Figure S12D), nitrate contents (Figure S12E,F), nitrate reductase activity (Figure S12G,H) of root and shoot tissues. (Figure S12J,K) Analysis of primary inflorescence height and shoot dry weight per plant under N-deficient conditions for 5 weeks. * Variables: * Genotype: different plant materials: wildtype (WT), AtOE1, AtOE2 (ZmNRL1 overexpression line in Arabidopsis). * Treatment: Hoagland solutions containing concentrations of KNO3 (0.1 mM or 10 mM). * Nitrate content: root and leaf nitrate content under different solutions (0.1 mM and 10 mM N). * Nitrate reductase activity: root and leaf nitrate reductase activity under different solutions (0.1 mM and 10 mM N). * Shoot dry weight: Arabidopsis shoot fresh biomass weight per plant. * "NA" in some cells indicates missing values. ### FigureS13B\_C\_D\_E.csv * Description: Arabidopsis overexpression lines and wild type under normal growth conditions. Analysis of primary inflorescence height (Figure S13B), total seed weight per plant (Figure S13C), and soluble protein contents of 5-week-old rosettes (Figure 13D). qRT-PCR analysis of the N-related genes relative expression level in wild type and Arabidopsis overexpression lines (AtOEs) under normal conditions (Figure S13E) * Variables: * WT: Arabidopsis wild-type materials * AtOE1, AtOE2: The primary inflorescence height of ZmNRL1 overexpression line in Arabidopsis. * Total seed weight per plant: seed dry weight per plant under normal growth conditions. * AtNRT1.1: Arabidopsis NO3- transporter gene 1.1 relative expression * AtNRT2.1: Arabidopsis NO3- transporter gene 2.1 relative expression * AtGln2: Arabidopsis Glutamin synthetase 2 relative expression * AtNIR1: Arabidopsis Nitrite reductase 1 relative expression * AtNIA1: Arabidopsis Nitrate reductase 1 relative expression * AtGlu1: Arabidopsis Glutamate synthase 1 relative expression * AtGln1;1: Arabidopsis Glutamin synthetase 1;1 relative expression * AtGln1;4: Arabidopsis Glutamin synthetase 1;4 relative expression ## Sharing/Access information Raw data used in the reanalysis in Figure S3B was derived from data from: McLoughlin F, Augustine RC, Marshall RS, Li FQ, Kirkpatrick LD, Otegui MS, Vierstra RD. 2018. Maize multi-omics reveal roles for autophagic recycling in proteome remodelling and lipid turnover. Nature Plants 4, 1056-1070.