Expression of the Nitrate Transporter Gene OsNRT1.1A/OsNPF6.3 Confers High Yield and Early Maturation in Rice

Abstract

Nitrogen (N) is a major driving force for crop yield improvement, but application of high levels of N delays flowering, prolonging maturation and thus increasing the risk of yield losses. Therefore, traits that enable utilization of high levels of N without delaying maturation will be highly desirable for crop breeding. Here, we show that OsNRT1.1A (OsNPF6.3), a member of the rice (Oryza sativa) nitrate transporter 1/peptide transporter family, is involved in regulating N utilization and flowering, providing a target to produce high yield and early maturation simultaneously. OsNRT.1A has functionally diverged from previously reported NRT1.1 genes in plants and functions in upregulating the expression of N utilization-related genes not only for nitrate but also for ammonium, as well as flowering-related genes. Relative to the wild type, osnrt1.1a mutants exhibited reduced N utilization and late flowering. By contrast, overexpression of OsNRT1.1A in rice greatly improved N utilization and grain yield, and maturation time was also significantly shortened. These effects were further confirmed in different rice backgrounds and also in Arabidopsis thaliana Our study paves a path for the use of a single gene to dramatically increase yield and shorten maturation time for crops, outcomes that promise to substantially increase world food security.

Figure 1.

OsNRT1.1A Displays Ammonium-Inducible Expression. (A) Phylogenetic analysis of amino acid sequences of AtNRT1.1 and OsNRT1.1A/B/C using maximum likelihood method by Mega 6.0. Bootstrap = 1000. (B) Expression analysis of OsNRT1.1A and OsNRT1.1B under different N sources (ammonium or nitrate) in roots of rice seedlings assessed by RNA-sequencing. RPKM, reads per kilobase per million mapped reads.

Figure 2.

GUS Staining of OsNRT1.1A_promoter:GUS Transgenic Plants. Tissues used for GUS staining include roots ([A] to [C]), culms (D), leaf sheaths (E), and leaf blades (F), showing cross sections in (B) to (F). (B) and (C) indicate the regions of root tip and root hair, respectively. Bars = 1 mm in (A), 0.3 mm in (B) and (C), and 1 mm in (D) to (F).

Figure 3.

OsNRT1.1A Predominantly Localizes to the Tonoplast. (A) The OsNRT1.1A-eGFP signal colocalizes with the tonoplast marker α-TIP (fused with RFP) in rice protoplasts. Bars = 20 µm. (B) Immunogold analysis of OsNRT1.1A-eGFP in 35S:OsNRT1.1A-eGFP transgenic rice and wild-type DJ (WT). Arrows indicate the immunodetection signal. PM, plasma membrane; V, vacuole. Bars = 100 nm.

Figure 4.

OsNRT1.1A Shows Nitrate Transport Activity in Vitro. Nitrate uptake assay in Xenopus laevis oocytes injected with water, OsNRT1.1A, and AtNRT1.1 using ¹⁵N-nitrate. AtNRT1.1 was used as the positive control. Twenty replicates were performed for each sample. Asterisks indicate the significant differences between water (negative control) and OsNRT1.1A as evaluated by Student’s t tests: **P < 0.01.

Figure 5.

OsNRT1.1A Displays Functional Divergence with OsNRT1.1B. (A) Growth of wild-type DJ (WT) and osnrt1.1a mutant seedlings under different N sources. (B) Growth of wild-type ZH11 (WT) and osnrt1.1b mutant seedlings under different N sources. NN, no N; A, ammonium (2 mM); N, nitrate (2 mM); AN, ammonium and nitrate (1 mM for each). Bars = 8 cm. (C) RT-qPCR-based expression analyses of genes involved in utilization of nitrate and ammonium in roots of wild-type DJ and osnrt1.1a mutant plants under normal hydroponic cultivation. (D) RT-qPCR-based nitrate induction assay in roots of osnrt1.1a mutant and osnrt1.1b mutant. Values are the means ±sd (n = 3). Asterisks indicate significant differences between wild-type plants and according mutants as evaluated by Student’s t tests: *P < 0.05 and **P < 0.01.

Figure 6.

Loss of Function of OsNRT1.1A Results in Severe Grain Yield Loss and Late Flowering. (A) Gross morphological phenotypes and panicle phenotypes of wild-type DJ (WT) and osnrt1.1a mutants grown in the field. Bars = 20 cm (gross morphological phenotypes) and 6 cm (panicle). (B) Grain yield per plant of wild-type DJ and osnrt1.1a mutant. (C) Days to flowering of wild-type DJ and osnrt1.1a mutant. (D) RT-qPCR-based expression analysis of flowering-promoting genes in leaves of wild-type DJ and osnrt1.1a mutant. Values are the means ± sd (10 replicates for grain yield per plant, 10 replicates for days to flowering, and 3 replicates for RT-qPCR). Asterisks indicate significant differences between wild-type DJ and osnrt1.1a mutant as evaluated by Student’s t tests: **P < 0.01.

Figure 7.

OsNRT1.1A Overexpression Activates N Utilization. (A) Growth of wild-type DJ (WT) and OsNRT1.1A-OE plants in long-term hydroponic culture under LN (400 μM) or HN (2 mM) conditions. Bars = 20 cm. (B) ¹⁵N accumulation assays in shoots of wild-type DJ and OsNRT1.1A-OE plants labeled with ¹⁵N-nitrate or ¹⁵N-ammonium. Values are the means ± sd (n = 4). (C) RT-qPCR-based expression analysis of N utilization genes in roots of wild-type DJ and OsNRT1.1A-OE plants. Numbers in table indicate the relative expression level, and the darker color represents the higher increased folds of gene expression in OsNRT1.1A-OE plants. Values are the means ± sd (n = 3). Asterisks indicate significant differences between wild-type DJ and OsNRT1.1A-OE plants as evaluated by one-way ANOVA with Tukey’s test: *P < 0.05 and **P < 0.01.

Figure 8.

OsNRT1.1A Promotes Nuclear Localization of NLPs. The effect of OsNRT1.1A on subcellular localization of OsNLP3/4-eGFP in rice protoplasts under nitrate-absent condition. OsNLP3/4-eGFP were cotransformed with 35S:RFP (red fluorescent protein) or 35S:OsNRT1.1A-RFP. Bars = 15 µm.

Figure 9.

OsNRT1.1A Overexpression Promotes Early Flowering and Grain Yield Improvement in Rice. (A) Growth of wild-type DJ (WT) and OsNRT1.1A-OE plants (OEnp-4) in the field at flowering (left) and grain-filling (right) stage. Bars = 50 cm. (B) The panicles of wild-type DJ and OsNRT1.1A-OE plants. Bar = 8 cm. (C) Grain yield per plant, actual yield per plot, NUE, and days to flowering of wild-type and OsNRT1.1A-OE plants under LN conditions in a field trial in Beijing (2015). (D) As in (C), for HN conditions in a field trial in Beijing (2015). Values in (C) and (D) are the means ± sd (18 replicates for grain yield per plant, 4 replicates for actual yield per plot and NUE, and 41 replicates for days to flowering). Asterisks indicate significant differences between wild-type and OsNRT1.1A-OE plants as evaluated by one-way ANOVA with Tukey’s test: **P < 0.01.

Figure 10.

OsNRT1.1A Overexpression in Arabidopsis Also Improves Seed Weight and Affects Flowering Time. (A) Growth of wild-type Arabidopsis (WT) and transgenic Arabidopsis overexpressing OsNRT1.1A (AtOE-4/12) at different growth stages. Bars = 5 cm (left) and 12 cm (middle and right). (B) RT-qPCR-based OsNRT1.1A expression, days to flowering, seed weight per plant, and biomass of wild-type Arabidopsis and overexpression plants. Values are the means ± sd (3 replicates for RT-qPCR analysis, 17 replicates for days to flowering, and 10 replicates for seed weight per plant and biomass). Asterisks indicate the significant differences between wild-type Arabidopsis and AtOE plants as evaluated by one way ANOVA with Tukey’s test: **P < 0.01.