The Nitrate-Inducible NAC Transcription Factor TaNAC2-5A Controls Nitrate Response and Increases Wheat Yield

Abstract

Nitrate is a major nitrogen resource for cereal crops; thus, understanding nitrate signaling in cereal crops is valuable for engineering crops with improved nitrogen use efficiency. Although several regulators have been identified in nitrate sensing and signaling in Arabidopsis (Arabidopsis thaliana), the equivalent information in cereals is missing. Here, we isolated a nitrate-inducible and cereal-specific NAM, ATAF, and CUC (NAC) transcription factor, TaNAC2-5A, from wheat (Triticum aestivum). A chromatin immunoprecipitation assay showed that TaNAC2-5A could directly bind to the promoter regions of the genes encoding nitrate transporter and glutamine synthetase. Overexpression of TaNAC2-5A in wheat enhanced root growth and nitrate influx rate and, hence, increased the root's ability to acquire nitrogen. Furthermore, we found that TaNAC2-5A-overexpressing transgenic wheat lines had higher grain yield and higher nitrogen accumulation in aerial parts and allocated more nitrogen in grains in a field experiment. These results suggest that TaNAC2-5A is involved in nitrate signaling and show that it is an exciting gene resource for breeding crops with more efficient use of fertilizer.

Figure 1.

Expression patterns of TaNAC2-5A in various wheat organs and in response to exogenous nitrate. A, TaNAC2-5A and TaNRT2.1-6B expression in response to nitrate in roots. Wheat seedlings deprived of N for 2 d were exposed to a nutrient solution containing 2 mm nitrate for the indicated times. B, Expression levels of TaNAC2-5A in shoots and roots of wheat seedlings grown in high N (HN) and low N (LN). Wheat plants were grown for 14 d in nutrient solutions that contained 2 mm nitrate (high N) or 0.2 mm nitrate (low N). C, Expression of TaNAC2-5A in different organs of wheat plants. The wheat plants were grown in a field experiment under high-N conditions, and the samples of different organs were collected 14 d after flowering. The relative expression levels of TaNAC2-5A were normalized to the expression of β-TUBULIN. Data are means ± se of three replicates.

Figure 2.

Expression levels of TaNAC2-5A in shoots and roots in wild-type (LC) and transgenic (OE1, OE2, and OE3) wheat. Wheat seedlings were grown for 14 d in a nutrient solution that contained 2 mm nitrate. The relative expression levels of TaNAC2-5A were normalized to the expression of β-TUBULIN. Data are means ± se of three replicates. Asterisks indicate that the difference between the means of the wild type and the transgenic lines was significant at the P < 0.05 (*) and P < 0.01 (**) levels.

Figure 3.

Plant growth and N accumulation of the wild type and the transgenic lines in the soil pot trial. A, Seedlings of the wild type (LC) and the transgenic lines (OE1, OE2, and OE3) grown under low-N and high-N conditions. B, Tiller number per plant. C, Shoot dry weight per plant. D, Root dry weight per plant. E, Shoot N concentration. F, Shoot N accumulation per plant. The wild type and the transgenic lines were grown for 30 d in soil supplied with 0 mg N kg⁻¹ soil (nil-N treatment), 10 mg N kg⁻¹ soil (low-N treatment), or 100 mg N kg⁻¹ soil (high-N treatment). Data are means ± se of four replications. Asterisks indicate that the difference between the means of the wild type and the transgenic lines was significant at the P < 0.05 (*) and P < 0.01 (**) levels.

Figure 4.

Expression levels of genes involved in nitrate uptake, translocation, and assimilation in wild-type (LC) and transgenic (OE1 and OE2) plants grown in hydroponic culture. A to E, Expression levels of TaNRT2.1-6B (A), TaNRT2.2-6D (B), TaNPF7.1-6D (C), TaNPF7.2-6B (D), and TaGS2 (E) in shoots and roots of the wild type and the transgenic lines. Wheat seedlings were grown for 14 d in a nutrient solution that contained 2 mm nitrate. Total RNA isolated from the shoots and roots was used in the quantitative real-time RT-PCR analysis. The relative expression levels were normalized to the expression of β-TUBULIN. Data are means ± se of three replicates. F, Nitrate influx rate of the wild type and the transgenic lines. Seedlings were pretreated with 1 mm nitrate and 0.2 mm nitrate and then used to measure nitrate influx rate at the root tip surface in measuring solutions that contained 1 mm nitrate (high N) and 0.2 mm nitrate (low N), respectively. Data are means ± se (n = 6). Asterisks indicate that the difference between the means of the wild type and the transgenic lines was significant at the P < 0.05 (*) and P < 0.01 (**) levels. ND, Not detectable.

Figure 5.

Subcellular localization and transcriptional activation activity of TaNAC2-5A. A, Nuclear localization of TaNAC2-5A in root tips of wheat seedlings. Seedlings 7 d after germination were used to stain TaNAC2 protein with immunofluorescence labeling. The primary antibody was a polyclonal antibody to TaNAC2, and the secondary antibody was a goat anti-rabbit fluorescein isothiocyanate (FITC)-conjugated antibody. Intracellular fluorescence was observed using a confocal microscope equipped with an argon laser (488 nm). Bar = 10 μm. B, Transactivation activity of TaNAC2-5A, TaNAC2-5AΔN, and TaNAC2-5AΔC in yeast cells. The TaNAC2-5A open reading frame, or the fragment of TaNAC2-5A encoding the N-terminal (TaNAC2-5AΔC) or C-terminal (TaNAC2-5AΔN) domain of TaNAC2-5A, was fused in frame with the GAL4 DNA-binding domain (BD) in the pGBKT7 vector. Empty pGBKT7 was used as a negative control. Ade, Adenine; SD, synthetic dropout.

Figure 6.

Binding abilities of TaNAC2 to the promoter fragments of TaNRT2.1-6B, TaNPF7.1-6D, and TaGS2-2A. A, Putative NAC recognition motifs in the promoters of TaNRT2.1-6B, TaNPF7.1-6D, and TaGS2-2A. B, Enrichment of the promoter regions in TaNRT2.1-6B, TaNPF7.1-6D, and TaGS2-2A recovered in ChIP assays performed with the TaNAC2 antibody using chromatin prepared from the wild-type Longchun 23. Two-week-old seedlings were used to perform a ChIP assay to precipitate TaNAC2-DNA complexes with TaNAC2 antibody (TaNAC2 AB) or without antibody (No AB) as a control. Enrichment of the promoter regions was determined by quantitative real-time PCR. The ChIP results were normalized to input DNA. Data are means ± se of three replicates. Asterisks indicate that the difference between the means of the TaNAC2-DNA complexes with and without antibody was significant at the P < 0.05 (*) and P < 0.01 (**) levels.

Figure 7.

Effects of TaNAC2-5A overexpression on root growth and the rate of nitrate uptake in Arabidopsis. A, Root images. B, Primary root length. C, Visible lateral root number. The seeds the wild type (Col) and TaNAC2-5A transgenic lines (OENAC-1 and OENAC-2) were germinated for 4 d on one-half-strength Murashige and Skoog medium and then transferred to agar medium containing either 6 mm KNO₃ (HN) or 0.2 mm KNO₃ (LN). After being grown vertically for 7 d, the root morphological parameters were measured. Data in B and C are presented as means ± se of at least 15 plants from three independent experiments. D and E, Nitrate influx rates at the root tip surface (D) and expression levels of nitrate transporters in whole seedlings (E). Seedlings were grown for 4 d on solid medium that contained 6 mm nitrate. The roots were used to measure nitrate flux rate in the measuring solution that contained 1 mm nitrate. Data are means ± se for six plants. Whole seedlings were used to analyze the expression of nitrate transporters. The relative expression levels were normalized to the expression of AtACTIN2 (AtACT2). Data are means ± se of three replicates. Asterisks indicate that the difference between the means of the wild type and the transgenic lines was significant at the P < 0.05 (*) and P < 0.01 (**) levels. F, Putative NAC recognition motifs in the promoter of AtNRT2.1. G, Enrichment of promoter regions of AtNRT2.1 recovered in ChIP assays performed with the TaNAC2 antibody using chromatin prepared from the TaNAC2-5A transgenic Arabidopsis plants. One-week-old seedlings were used to perform a ChIP assay to precipitate the TaNAC2-DNA complex with (TaNAC2 AB) or without (No AB) TaNAC2 antibody. Enrichment of the promoter regions of AtNRT2.1 was determined by quantitative real-time PCR. The ChIP results were normalized to input DNA. Data are means ± se of three replicates. Asterisks indicate that the difference between the means of the TaNAC2-DNA complexes with and without antibody was significant at the P < 0.01 (**) level.

Figure 8.

NAC gene expression in response to nitrate in different plant species. NAC gene expression is shown in response to nitrate treatment in the roots of wheat (A and H), rice (B and D), maize (C and E), soybean (F and G), and Arabidopsis (I and J). Seedlings were hydroponically grown with 2 mm (wheat and rice) or 5 mm (maize, soybean, and Arabidopsis) NH₄⁺ as the sole N source for 14 d and then treated with 2 mm (wheat and rice) or 5 mm (maize, soybean, and Arabidopsis) NO₃⁻ for the times indicated. Total RNA isolated from the roots was used in the quantitative real-time RT-PCR analysis. The relative expression levels were normalized to the expression of β-TUBULIN for wheat, OsACT for rice, ZmACT for maize, The reference gene of unknown function GmUNK1 for soybean, and AtACT2 for Arabidopsis. Data are means ± se of three replicates (two plants per replicate). Different letters indicate significant differences (P < 0.05).