Genome-wide associated study identifies NAC42-activated nitrate transporter conferring high nitrogen use efficiency in rice

Abstract

Over-application of nitrogen fertilizer in fields has had a negative impact on both environment and human health. Domesticated rice varieties with high nitrogen use efficiency (NUE) reduce fertilizer for sustainable agriculture. Here, we perform genome-wide association analysis of a diverse rice population displaying extreme nitrogen-related phenotypes over three successive years in the field, and identify an elite haplotype of nitrate transporter OsNPF6.1^HapB that enhances nitrate uptake and confers high NUE by increasing yield under low nitrogen supply. OsNPF6.1^HapB differs in both the protein and promoter element with natural variations, which are differentially trans-activated by OsNAC42, a NUE-related transcription factor. The rare natural allele OsNPF6.1^HapB, derived from variation in wild rice and selected for enhancing both NUE and yield, has been lost in 90.3% of rice varieties due to the increased application of fertilizer. Our discovery highlights this NAC42-NPF6.1 signaling cascade as a strategy for high NUE and yield breeding in rice.

Fig. 1

Identification and functional validation of OsNPF6.1 on chromosome 1. a Local Manhattan plot (top) and LD heatmap (bottom) surrounding the peak on chromosome 1. Red dashed line indicates the candidate region for the peak. b Gene structure of OsNPF6.1 and DNA polymorphism in this gene. Green boxes and light blue box represent UTR and exon, respectively. c Comparative analyses of OsNPF6.1 between the low and high NUE haplotypes. NEPNR (normalized effective panicle number ratio)-2015 (left) and NEPNR-2016 (right) based on the haplotypes (Hap) of OsNPF6.1. Box edges represent the 0.25 quantile and 0.75 quantile with the median values shown by bold lines. Whiskers extend to data no more than 1.5 times the interquartile range, and remaining data are indicated by dots. Differences between the haplotypes were analyzed by Welch’s t test. d, e Comparison of the effective panicle numbers and yields per plant of Nip and complementary line (Nip pHapB::NPF6.1^HapB) in HN and LN, bar = 20 cm, n = 16, 6. f Two amino acids are deleted in Nip-cas. Red box indicates the missing amino acids. g, h Comparison of Nip and knock-out line (Nip-cas) plant heights and yields per plant under HN and LN conditions, bar = 20 cm, n = 16, 6. Data are presented as means ± SD. P values (versus the Nip) were calculated with Student’s t test. Nip Nipponbare. The source data underlying Figs. 1d, e, g, and h are provided as a Source Data file.

Fig. 2

Functional analysis of OsNPF6.1. a, b Comparison of the plant heights and yields per plant of NIL (HapA) and NIL (HapB), under HN (a) and LN (b) conditions, bar = 20 cm (the plants), 2 cm (the panicles and the seeds), n = 16, 6. c, d Nitrate influx rates in the roots. The root nitrate influx was measured in 0.25 mM (c) or 2.5 mM (d) ¹⁵NO₃⁻, n = 3. Data are presented as means ± SD. P values were calculated with Student’s t test. e, f Averages of the currents elicited under 0.5 mM (e) and 2.5 mM NO₃⁻ (f) in oocytes injected with complementary RNAs of the coding regions of two haplotypes (OsNPF6.1^HapA and OsNPF6.1^HapB) of OsNPF6.1 or water control. The oocytes were voltage-clamped at −60 mV, and the inward currents elicited with 0.5 mM or 2.5 mM NO₃⁻ at pH 5.5. Bars represent mean ± SEM, n = 7 (e), n = 7, 5, 5 (f). One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was used to analyze statistical significance (*P < 0.05 and **P < 0.01). g Concentration dependence of nitrate-elicited currents in a single injected oocyte. The oocyte was voltage-clamped at −60 mV, and the currents elicited by different concentrations of nitrate (0–20 mM) at pH 5.5, AtCHL1 as a positive control. Each bar represents the mean ± SEM, n = 4, 10, 10 (*P < 0.05 and **P < 0.01, Welch’s t test). h Current-to-voltage relationship for OsNPF6.1^HapA and OsNPF6.1^HapB. The curves presented were recorded from a single OsNPF6.1^HapA (blue) and OsNPF6.1^HapB (red)-injected oocyte treated with 10 mM nitrate at pH 5.5, and OsNPF6.1^HapA (light red) and OsNPF6.1^HapB (light blue) all at 10 mM and pH 7.4. The data in (e–h) obtained from independent oocytes from the same frog, and similar results were obtained from three different frogs. Source data are provided as a Source Data file.

Fig. 3

Agronomic traits enhanced by ectopic expression of OsNPF6.1. a, b Comparison of NJ11 and NJ11-OE plant heights under HN (a) and LN (b) conditions, bar = 20 cm (the plants), 2 cm (the panicles and the seeds), n = 16, 6. c, d Comparison of the effective panicle numbers at maturity of DJ and OsNPF6.1-D1 in HN (c) and LN (d) conditions, bar = 20 cm (the plants), 2 cm (the panicles and the seeds), n = 16, 5. Data are presented as means ± SD. P values (versus the NJ11 and DJ) were calculated with Student’s t test. NJ11, Nanjing11; DJ, Dongjin. Source data are provided as a Source Data file.

Fig. 4

Identification and functional validation of the transcription factor OsNAC42 on chromosome 9. a Local Manhattan plot (top) and LD heatmap (bottom) surrounding the peak on chromosome 9. Red dashed lines indicate the candidate region for the peak. b Comparative analyses of OsNAC42 between the low and high NUE haplotypes. Boxplots for NPHR (normalized plant height ratio)-2014 (top) and NPHR-2016 (bottom) based on the haplotypes (Hap) for OsNAC42. Box edges represent the 0.25 quantile and 0.75 quantile with the median values shown by bold lines. Whiskers extend to data no more than 1.5 times the interquartile range, and remaining data are indicated by dots. Differences between the haplotypes were analyzed by Welch’s t test. c Phenotype of WT (Zhonghua11, ZH11), Tilling mutant (osnac42) and heterozygote (H), bar = 20 cm. d qRT-PCR analysis of OsNPF6.1 of WT (Zhonghua11, ZH11), Tilling mutant (osnac42) and heterozygote (H). Three biological replicates were used for qRT-PCR. e OsNPF6.1 and OsNAC42 transcript levels in different tissues. OsActin1 was used as a control, n = 3. f OsNPF6.1 and OsNAC42 expression under HN treatment from 0 to 24 h in leaf (KCl as a control of LN representing nitrogen starvation), n = 3. Data are presented as means ± SD. P values (versus the ZH11) were calculated with Student’s t test. **P < 0.01. The source data underlying Fig. 4d–f are provided as a Source Data file.

Fig. 5

OsNPF6.1^HapB stably transactivated by OsNAC42 and rarely utilized in NUE improvement. a Schematic diagram of OsNAC42 tilling mutant (osnac42). ZH11, Zhonghua11. b ChIP assays by OsNAC42 and OsNAC42M on binding with DNA of the promoter region of OsNPF6.1^HapA. The fold enrichment was normalized against rice ubiquitin promoter, n = 3. Each bar represents the mean and SEM. **P < 0.01. c Schematic diagram of promoter haplotypes of OsNPF6.1 (OsNPF6.1^HapA and OsNPF6.1^HapB). The black triangles represent CACG motifs. Four CACG motif regions (A1, A2, B3, and B4) under triangles indicate the positions of the probes used in EMSA assays. d DNA binding activities of OsNAC42 and OsNAC42 mutant proteins on four CACG motif regions of the OsNPF6.1^HapA and OsNPF6.1^HapB promoters were tested by EMSA. Red arrows highlight B3 and B4 binding sites. (e) Transactivation activities of OsNAC42 and OsNAC42 mutant (OsNAC42M) on promoter of two OsNPF6.1 haplotypes (HapA-P and HapB-P). YFP, OsNAC42, and OsNAC42M proteins were used as effectors, n = 8. P < 0.05. f Haplotype network of the OsNPF6.1 gene. Each haplotype is separated by mutational changes, with hatches indicating differences between linked haplotypes. aro, aromatic; tej, temperate japonica; trj, tropical japonica; adx, admix; wild, O. rufipogon. g Geographical distribution of the OsNPF6.1 in O. rufipogon and O. sativa. The pie chart size is proportional to the number of accessions. h Nucleotide diversity across the OsNPF6.1 genomic region. Top: The 17 sampled loci (including OsNPF6.1) located in the genomic region around the OsNPF6.1 gene on chromosome 1. Middle: Nucleotide diversity pi of Hap X and B rice at the sampled loci. i Totally, 209 varieties harboring OsNPF6.1^HapB, accounting for only 9.7% with 97.3% in indica. j, k Yield per plot of OsNPF6.1-D1, OsNPF6.1^HapB overexpression line and OsNPF6.1^HapA knock-out line in HN and LN fields, n = 3. l NUE (grain yield—grain yield of zero-N plot (6 × 8)/N supply) of OsNPF6.1-D1, OsNPF6.1^HapB overexpression line and OsNPF6.1^HapA knock-out line, n = 3. Data are presented as means ± SD. DJ, Dongjin; NJ11, Nanjing11; Nip, Nipponbare. The source data underlying Fig. 5b, d, e, h, j–l are provided as a Source Data file.